David Keefe, M.D.

Association of telomere length with authentic pluripotency of ES/iPS cells
Huang, Junjiu; Wang, Fang; Okuka, Maja; Liu, Na; Ji, Guangzhen; Ye, Xiaoying; Zuo, Bingfeng; Li, Minshu; Liang, Ping; Ge, William W; Tsibris, John Cm; Keefe, David L; Liu, Lin
2011 May;21(5):779-792, Cell research
Telomerase and telomeres are important for indefinite replication of stem cells. Recently, telomeres of somatic cells were found to be reprogrammed to elongate in induced pluripotent stem cells (iPSCs). The role of telomeres in developmental pluripotency in vivo of embryonic stem cells (ESCs) or iPSCs, however, has not been directly addressed. We show that ESCs with long telomeres exhibit authentic developmental pluripotency, as evidenced by generation of complete ESC pups as well as germline-competent chimeras, the most stringent tests available in rodents. ESCs with short telomeres show reduced teratoma formation and chimera production, and fail to generate complete ESC pups. Telomere lengths are highly correlated (r > 0.8) with the developmental pluripotency of ESCs. Short telomeres decrease the proliferative rate or capacity of ESCs, alter the expression of genes related to telomere epigenetics, down-regulate genes important for embryogenesis and disrupt germ cell differentiation. Moreover, iPSCs with longer telomeres generate chimeras with higher efficiency than those with short telomeres. Our data show that functional telomeres are essential for the developmental pluripotency of ESCs/iPSCs and suggest that telomere length may provide a valuable marker to evaluate stem cell pluripotency, particularly when the stringent tests are not feasible
— id: 133642, year: 2011, vol: 21, page: 779, stat: Journal Article,

Germline competency of parthenogenetic embryonic stem cells from immature oocytes of adult mouse ovary
Liu, Zhong; Hu, Zhe; Pan, Xinghua; Li, Minshu; Togun, Taiwo A; Tuck, David; Pelizzola, Mattia; Huang, Junjiu; Ye, Xiaoying; Yin, Yu; Liu, Mengyuan; Li, Chao; Chen, Zhisheng; Wang, Fang; Zhou, Lingjun; Chen, Lingyi; Keefe, David L; Liu, Lin
2011 Apr 1;20(7):1339-1352, Human molecular genetics
Parthenogenetic embryonic stem cells (pESCs) have been generated in several mammalian species from parthenogenetic embryos that would otherwise die around mid-gestation. However, previous reports suggest that pESCs derived from in vivo ovulated (IVO) mature oocytes show limited pluripotency, as evidenced by low chimera production, high tissue preference and especially deficiency in germline competence, a critical test for genetic integrity and pluripotency of ESCs. Here, we report efficient generation of germline-competent pESC lines (named as IVM pESCs) from parthenogenetic embryos developed from immature oocytes of adult mouse ovaries following in vitro maturation (IVM) and artificial activation. In contrast, pESCs derived from IVO oocytes show defective germline competence, consistent with previous reports. Further, IVM pESCs resemble more ESCs from fertilized embryos (fESCs) than do IVO pESCs on genome-wide DNA methylation and global protein profiles. In addition, IVM pESCs express higher levels of Blimp1, Lin28 and Stella, relative to fESCs, and in their embryoid bodies following differentiation. This may indicate differences in differentiation potentially to the germline. The mechanisms for acquisition of pluripotency and germline competency of IVM pESCs from immature oocytes remain to be determined
— id: 133643, year: 2011, vol: 20, page: 1339, stat: Journal Article,

Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients
Agarwal, Suneet; Loh, Yuin-Han; McLoughlin, Erin M; Huang, Junjiu; Park, In-Hyun; Miller, Justine D; Huo, Hongguang; Okuka, Maja; Dos Reis, Rosana Maria; Loewer, Sabine; Ng, Huck-Hui; Keefe, David L; Goldman, Frederick D; Klingelhutz, Aloysius J; Liu, Lin; Daley, George Q
2010 Mar 11;464(7286):292-296, Nature
Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients
— id: 133647, year: 2010, vol: 464, page: 292, stat: Journal Article,

Telomere susceptibility to cigarette smoke-induced oxidative damage and chromosomal instability of mouse embryos in vitro
Huang, Junjiu; Okuka, Maja; McLean, Mark; Keefe, David L; Liu, Lin
2010 Jun 15;48(12):1663-1676, Free radical biology & medicine
Cigarette smoke is associated with high risk of lung, cardiovascular, and degenerative diseases, reduced fertility, and possibly the health of newborns. Cigarette smoke contains many components and exerts its genotoxicity in part by generating reactive oxidative stress. Telomeres consist of repeated 'G' rich sequences and associated proteins located at the chromosomal ends that maintain chromosomal integrity. We tested the hypothesis that telomere shortening and dysfunction are implicated in smoke associated oxidative damage and chromosomal instability using early mouse embryos in vitro and short-telomere mouse model. Mouse embryos exposed to smoke components, cigarette smoke condensate (CSC) at the concentration of 0.02 mg/ml continuously or 0.1mg/ml for 20 h, or cadmium at 5-100 microM, exhibited increased oxidative stress and telomere shortening and loss, associated with chromosomal instability, apoptosis, and compromised embryo cleavage and development. Remarkably, reduction of oxidative stress by an antioxidant N-acetyl-L-cysteine (NAC) greatly reduced these toxicities. Notably, cadmium led to more severe oxidative damage and telomere dysfunction, which could be more effectively rescued by antioxidant treatment, than did CSC. Moreover, short telomeres predisposed embryos to smoke component-induced oxidative damage. These data further extend our understanding of mechanisms underlying smoke-induced oxidative damage to include telomere dysfunction and chromosomal instability
— id: 133645, year: 2010, vol: 48, page: 1663, stat: Journal Article,

Generation of pluripotent stem cells from eggs of aging mice
Huang, Junjiu; Okuka, Maja; Wang, Fang; Zuo, Bingfeng; Liang, Ping; Kalmbach, Keri; Liu, Lin; Keefe, David L
2010 Apr;9(2):113-125, Aging Cell
Oocytes can reprogram genomes to form embryonic stem (ES) cells. Although ES cells largely escape senescence, oocytes themselves do senesce in the ovaries of most mammals. It remains to be determined whether ES cells can be established using eggs from old females, which exhibit reproductive senescence. We attempted to produce pluripotent stem cell lines from artificial activation of eggs (also called pES) from reproductive aged mice, to determine whether maternal aging affects pES cell production and pluripotency. We show that pES cell lines were generated with high efficiency from reproductive aged (old) mice, although parthenogenetic embryos from these mice produced fewer ES clones by initial two passages. Further, pES cell lines generated from old mice showed telomere length, expression of pluripotency molecular markers (Oct4, Nanog, SSEA1), alkaline phosphatase activity, teratoma formation and chimera production similar to young mice. Notably, DNA damage was reduced in pES cells from old mice compared to their progenitor parthenogenetic blastocysts, and did not differ from that of pES cells from young mice. Also, global gene expression differed only minimally between pES cells from young and old mice, in contrast to marked differences in gene expression in eggs from young and old mice. These data demonstrate that eggs from old mice can generate pluripotent stem cells, and suggest that the isolation and in vitro culture of ES cells must select cells with high levels of DNA and telomere integrity, and/or with capacity to repair DNA and telomeres
— id: 133646, year: 2010, vol: 9, page: 113, stat: Journal Article,

Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis
Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin
2010 Dec;2(6):333-344, Journal of molecular cell biology
Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos
— id: 133644, year: 2010, vol: 2, page: 333, stat: Journal Article,

Maintenance of blood-pressure-lowering effect following a missed dose of aliskiren, irbesartan or ramipril: results of a randomized, double-blind study
Palatini, P; Jung, W; Shlyakhto, E; Botha, J; Bush, C; Keefe, D L
2010 Feb;24(2):93-103, Journal of human hypertension
Most patients inadvertently miss an occasional dose of antihypertensive therapy, and hence drugs that provide sustained blood-pressure (BP) reduction beyond the 24-h dosing interval are desirable. The primary objective of this study was to compare the 24-h mean ambulatory BP reductions from baseline after a simulated missed dose of the direct renin inhibitor aliskiren, irbesartan or ramipril. In this double-blind study, 654 hypertensive patients (24-h mean ambulatory diastolic BP (MADBP) >/=85 mm Hg) were randomized 1:1:1 to once-daily aliskiren 150 mg, irbesartan 150 mg or ramipril 5 mg. Doses were doubled after 2 weeks. At day 42, patients were again randomized equally within each group to receive 1 day of placebo ('missed dose') on either day 42 or day 49. Patients with a successful 24-h ambulatory BP measurement at baseline and on day 42/49 were included in the analyses. The 24-h mean ambulatory systolic BP (MASBP)/MADBP reductions from baseline after a missed dose of aliskiren 300 mg (9.3/7.0 mm Hg) were similar to irbesartan 300 mg (9.5/7.3 mm Hg) and significantly larger than ramipril 10 mg (7.1/5.0 mm Hg, P</=0.008). Loss of BP-lowering effect with aliskiren in the 24 h after a missed dose (1.0/0.7 mm Hg for 24-48-h vs 0-24-h MASBP/MADBP) was significantly lower than with irbesartan (3.6/2.2 mm Hg, P<0.01) or ramipril (4.0/2.6, P<0.0001). This equates to maintenance of 91/91% of the MASBP/MADBP-lowering effect with aliskiren, greater than irbesartan (73/77%) or ramipril (64/65%). The incidence of adverse events was similar across treatments (32.9-36.0%), although ramipril treatment was associated with an increased incidence of cough (ramipril, 6.1%; aliskiren, 0.5%; irbesartan, 1.8%). Aliskiren 300 mg provided a sustained BP-lowering effect beyond the 24-h dosing interval, with a significantly smaller loss of BP-lowering effect in the 24-48 h period after dose than irbesartan 300 mg or ramipril 10 mg.Journal of Human Hypertension advance online publication, 21 May 2009; doi:10.1038/jhh.2009.38
— id: 101969, year: 2010, vol: 24, page: 93, stat: Journal Article,

Frozen hope: fertility preservation for women with cancer
Quinn, Gwendolyn P; Vadaparampil, Susan T; Jacobsen, Paul B; Knapp, Caprice; Keefe, David L; Bell, Geri E
2010 Mar-Apr;55(2):175-180, Journal of Midwifery & Women's Health
Young women diagnosed with cancer have the option of preserving their fertility by using assisted reproductive technology (ART) techniques prior to undergoing cancer treatment. This article presents a composite case of a young woman with cancer who had many unanswered emotional and ethical questions about her future as a parent. Fertility preservation techniques, including preimplantation genetic diagnosis (PGD), and related patient education are described. Current literature regarding reproductive counseling for cancer survivors is reviewed. Resources for providing psychosocial support for decisions about fertility preservation are lagging behind the rapid pace of scientific advancements in cancer treatment and ART. As more young women are surviving cancer and taking steps to preserve fertility, there is great need for the provision of psychologic support services and the establishment of ethical guidelines to aid them on this path. Women's health care providers can provide support to cancer survivors facing fertility and parenting issues by becoming knowledgeable about the long-term aspects of decision making and developing educational materials and guidelines for these patients
— id: 144118, year: 2010, vol: 55, page: 175, stat: Journal Article,

Birth of Parthenote Mice Directly from Parthenogenetic Embryonic Stem Cells
Chen, Zhisheng; Liu, Zhong; Huang, Junjiu; Amano, Tomokazu; Li, Chao; Cao, Shanbo; Wu, Chao; Liu, Bodu; Zhou, Lingjun; Carter, Mark G; Keefe, David L; Yang, Xiangzhong; Liu, Lin
2009 Sep;27(9):2136-2145, Stem cells
Mammalian parthenogenetic embryos are not viable and die due to defects in placental development and genomic imprinting. Parthenogenetic embryonic stem cells (pESC) derived from parthenogenetic embryos might advance regenerative medicine by avoiding immuno-rejection. However, previous reports suggest that pESC may fail to differentiate and contribute to some organs in chimeras, including muscle and pancreas, and it remains unclear whether pESC themselves can form all tissue types in the body. We found that derivation of pESC is more efficient than of fESC, in association with reduced MAPK signaling in parthenogenetic embryos and their ICM outgrowth. Furthermore, in vitro culture modifies the expression of imprinted genes in pESC and these cells, being functionally indistinguishable from fertilized embryo-derived ESCs, can contribute to all organs in chimeras. Even more surprisingly, our study shows that live parthenote pups were produced from pESC via tetraploid embryo complementation, which contributes to placenta development. This is the first demonstration that pESCs are capable of full-term development, and can differentiate into all cell types and functional organs in the body
— id: 101968, year: 2009, vol: 27, page: 2136, stat: Journal Article,

Effects of cigarette smoke on fertilization and embryo development in vivo
Huang, Junjiu; Okuka, Maja; McLean, Mark; Keefe, David L; Liu, Lin
2009 Oct;92(4):1456-1465, Fertility & sterility
OBJECTIVE: To determine the effects of smoking on eggs and subsequent embryo development by maternal exposure to cigarette smoke. DESIGN: Mice were exposed to cigarette smoke or cigarette smoke condensate (CSC) for 4 weeks and then examined for development and telomere function of embryos in vitro after fertilization. In addition, the effects of continuous smoke on embryo development and telomere length were determined by treating mice for 4 weeks, followed by continous exposure to cigarette smoke or CSC after fertilization. SETTING: Laboratory study. ANIMAL(S): CD1 mice. INTERVENTION(S): Mice were exposured to cigarette smoke or CSC. MAIN OUTCOME MEASURE(S): The percentage (rate) of blastocyst development, quality of embryos assessed by total cell number, apoptosis, Oct4 expression (a molecular marker of embryonic stem cells), telomere length and loss, and chromosomal instability were compared between smoke- and CSC- treated mice and sham-treated mice. RESULT(S): Mice exposed to cigarette smoke or CSC for 4 weeks exhibited increased egg fragmentation or delayed fertilization, thus reducing development to blastocysts in vitro. Fragmented eggs showed increased reactive oxygen species. Mice exposed to smoke or CSC showed increased apoptosis and altered expression of Oct4 in developed embryos. The effects of smoke or CSC on embryo development showed a dose-dependent relationship to exposure time. Exposure to smoke or CSC beginning 4 weeks before fertilzation altered expression of Oct4 and increased apoptosis in blastocysts. Notably, the rate of abnormal embryos significantly increased in the smoke and CSC groups. Smoke and CSC shortened telomeres in embryos, but their telomere shortening was not enough to induce major chromosome abnormalities in mice, which have unusually long telomeres. CONCLUSION(S): Together, the whole animal exposure model shows that cigarette smoke induces oxidative stress, telomere shortening, and apoptosis, and compromises embryo development in vivo
— id: 101972, year: 2009, vol: 92, page: 1456, stat: Journal Article,

Telomeres and reproductive aging
Keefe, David L; Liu, Lin
2009 ;21(1):10-14, Reproduction, fertility & development
Infertility, miscarriage and aneuploid offspring increase with age in women, and meiotic dysfunction underlies reproductive aging. How aging disrupts meiotic function in women remains unclear, but as women increasingly delay having children, solving this problem becomes an urgent priority. Telomeres consist of a (TTAGGG)(n) repeated sequence and associated proteins at chromosome ends, mediate aging in mitotic cells and may also mediate aging during meiosis. Telomeres shorten both during DNA replication and from the response to oxidative DNA damage. Oocytes do not divide in adult mammals, but their precursors do replicate during fetal oogenesis; eggs ovulated from older females have traversed more mitotic cell cycles before entering meiosis during fetal oogenesis than eggs ovulated from younger females. Telomeres also would be expected to shorten from inefficient DNA repair of oxidative damage, because the interval between fetal oogenesis and ovulation is exceptionally prolonged in women. We have tested the hypothesis that telomere shortening disrupts meiosis by shortening telomeres experimentally in mice, which normally do not exhibit age-related meiotic dysfunction. Interestingly, mouse telomeres are much longer than human telomeres, but genetic or pharmacological shortening of mouse telomeres recapitulates in mice the human reproductive aging phenotype as the mouse telomeres reach the length of telomeres from older women. These observations led us to propose a telomere theory of reproductive aging. Moreover, chronological oxidative stress increases with reproductive aging, leading to DNA damage preferentially at (TTAGGG)(n) repeats. Finally, if telomeres shorten with aging, how do they reset across generations? Telomerase could not play a significant role in telomere elongation during early development, because this enzyme is not active until the blastocyst stage, well after the stage when telomere elongation takes place. Rather, telomeres lengthen during the early cell cycles of development by a novel mechanism involving recombination and sister chromatid exchange. Telomere dysfunction resulting from oxidative stress, a DNA damage response or aberrant telomere recombination may contribute to reproductive aging-associated meiotic defects, miscarriage and infertility
— id: 101971, year: 2009, vol: 21, page: 10, stat: Journal Article,

Correlation of expression and methylation of imprinted genes with pluripotency of parthenogenetic embryonic stem cells
Li, Chao; Chen, Zhisheng; Liu, Zhong; Huang, Junjiu; Zhang, Wei; Zhou, Lingjun; Keefe, David L; Liu, Lin
2009 Jun 15;18(12):2177-2187, Human molecular genetics
Mammalian parthenogenetic embryos (pE) are not viable due to placental deficiency, presumably resulting from lack of paternally expressed imprinted genes. Pluripotent parthenogenetic embryonic stem (pES) cells derived from pE could advance regenerative medicine by avoiding immuno-rejection and ethical roadblocks. We attempted to explore the epigenetic status of imprinted genes in the generation of pES cells from parthenogenetic blastocysts, and its relationship to pluripotency of pES cells. Pluripotency was evaluated for developmental and differentiation potential in vivo, based on contributions of pES cells to chimeras and development to day 9.5 of pES fetuses complemented by tetraploid embryos (TEC). Consistently, pE and fetuses failed to express paternally expressed imprinted genes, but pES cells expressed those genes in a pattern resembling that of fertilized embryos (fE) and fertilized embryonic stem (fES) cells derived from fE. Like fE and fES cells, but unlike pE or fetuses, pES cells and pES cell-fetuses complemented by TEC exhibited balanced methylation of Snrpn, Peg1 and U2af1-rs1. Coincidently, global methylation increased in pE but decreased in pES cells, further suggesting dramatic epigenetic reprogramming occurred during isolation and culture of pES cells. Moreover, we identified decreased methylation of Igf2r, Snrpn, and especially U2af1-rs1, in association with increased contributions of pES cells to chimeras. Our data show that in vitro culture changes epigenetic status of imprinted genes during isolation of pES cells from their progenitor embryos and that increased expression of U2af1-rs1 and Snrpn and decreased expression of Igf2r correlate with pluripotency of pES cells
— id: 101970, year: 2009, vol: 18, page: 2177, stat: Journal Article,

Physician referral for fertility preservation in oncology patients: a national study of practice behaviors
Quinn, Gwendolyn P; Vadaparampil, Susan T; Lee, Ji-Hyun; Jacobsen, Paul B; Bepler, Gerold; Lancaster, Johnathan; Keefe, David L; Albrecht, Terrance L
2009 Dec 10;27(35):5952-5957, Journal of clinical oncology
PURPOSE: Cancer survival rates are improving, and the focus is moving toward quality survival. Fertility is a key aspect of quality of life for cancer patients of childbearing age. Although cancer treatment may impair fertility, some patients may benefit from referral to a specialist before treatment. However, the majority of studies examining patient recall of discussion and referral for fertility preservation (FP) show that less than half receive this information. This study examined the referral practices of oncologists in the United States. METHODS: This study examined oncologists' referral practice patterns for FP among US physicians using the American Medical Association Physician Masterfile database. A 53-item survey was administered via mail and Internet to a stratified random sample of US physicians. RESULTS: Forty-seven percent of respondents routinely refer cancer patients of childbearing age to a reproductive endocrinologist. Referrals were more likely among female physicians (P = .004), those with favorable attitudes (P = .043), and those whose patients routinely ask about FP (odds ratio = 2.09; 95% CI, 1.31 to 3.33). CONCLUSION: Less than half of US physicians are following the guidelines from the American Society of Clinical Oncology, which suggest that all patients of childbearing age should be informed about FP
— id: 144119, year: 2009, vol: 27, page: 5952, stat: Journal Article,

Trace element concentrations in follicular fluid of small follicles differ from those in blood serum, and may represent long-term exposure
Silberstein, Tali; Saphier, Oshra; Paz-Tal, Ofra; Gonzalez, Liliana; Keefe, David L; Trimarchi, James R
2009 May;91(5):1771-1774, Fertility & sterility
OBJECTIVE: To determine the levels of elements in follicular fluid (FF) of patients undergoing IVF and evaluate the relationship between the concentration of elements in FF, follicular volume, and blood. DESIGN: Prospective blinded study. SETTING: University-based IVF center. PATIENT(S): Follicular fluid/blood samples from 6/3 patients, respectively, undergoing IVF. INTERVENTION(S): Single follicular aspirations of 33 follicles were performed. Blood samples ( approximately 5 mL) were drawn at the time of oocyte retrieval from 3/6 patients only. The concentrations 26 elements were measured by inductively coupled plasma mass spectroscopy. MAIN OUTCOME MEASURE(S): Trace elements concentrations in follicular fluid and blood. RESULT(S): [1] Calcium and magnesium were the most abundant, followed by Cu, Zn, Fe, Cr, Rb. The elements V, Sr, Se, B, As, Pb, Al, Mo, Mn, and Cs were found in trace amounts. The elements Li, Be, Ag, Cd, Ba, Ti, Bi, U were not detected. [2] Element concentrations in small follicles frequently differed from those of large follicles. [3] Element concentrations in large follicles more closely resembled those in blood. CONCLUSION(S): Concentrations of elements in FF of small follicles can differ from those of large follicles in the same woman and from those of blood serum. When follicles grow they become filled with fluid of an elemental composition similar to blood. Concentrations of elements in small follicles may represent longer term element exposure, whereas those of growing follicles represents the coincident blood concentrations
— id: 101974, year: 2009, vol: 91, page: 1771, stat: Journal Article,

Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation
Huang, Junjiu; Deng, Kai; Wu, Haojia; Liu, Zhong; Chen, Zhisheng; Cao, Shanbo; Zhou, Lingjun; Ye, Xiaoying; Keefe, David L; Liu, Lin
2008 Jul;26(7):1883-1890, Stem cells
The conventional method for producing embryonic stem (ES) cell-derived knockout or transgenic mice involves injection of ES cells into normal, diploid blastocysts followed by several rounds of breeding of resultant chimeras and thus is a time-consuming and inefficient procedure. F0 ES cell pups can also be derived directly from tetraploid embryo complementation, which requires fusion of two-cell embryos. Recently, F0 ES cell pups have been produced by injection of ES cells into eight-cell embryos using a laser-assisted micromanipulation system. We report a simple method for producing F0 ES cell germline-competent mice by piezo injection of ES cells into four- or eight-cell embryos. The efficiency of producing live, transgenic mice by this method is higher than that with the tetraploid blastocyst complementation method. This efficient and economical technique for directly producing F0 ES cell offspring can be applicable in many laboratories for creating genetically manipulated mice using ES cell technology and also for stringent testing of the developmental potency of new ES cell or other types of pluripotent stem cell lines
— id: 101973, year: 2008, vol: 26, page: 1883, stat: Journal Article,

Defective cohesin is associated with age-dependent misaligned chromosomes in oocytes
Liu, Lin; Keefe, David L
2008 Jan;16(1):103-112, Reproductive biomedicine online
Aneuploidy often results from chromosome misalignment at metaphases. Oocytes from senescence-accelerated mice (SAM) exhibit increased chromosome misalignment with age, which originates from nuclear factors. This work sought to further characterize the underlying defects of chromosome misalignments. Using immunofluorescence microscopy with specific antibodies, several specific components associated with spindles or chromosomes, including centrosomes, centromeres and cohesin complex were examined. No obvious differences were found in the distribution of centrosome focus at the spindle pole of oocytes from young and aged SAM, regardless of chromosome alignments, although cytoplasmic centrosome foci were significantly reduced in aged SAM (P < 0.0001). Oocytes from both young and aged SAM exhibited centromere-associated protein-E (CENP-E) at centromeres of all chromosomes, including misaligned chromosomes from aged SAM, demonstrating that CENP-E did not contribute to chromosome misalignments. Notably, both meiotic cohesin proteins located between sister chromatids, REC8 (recombinant 8), STAG3 (stromal antigen 3) and SMC1beta, were remarkably reduced in oocytes from aged SAM. Further, degradation of the cohesin was even more obvious in SAM than in hybrid F1 mice with age, which may explain why SAM are vulnerable to aneuploidy. This natural ageing mouse model shows that defective cohesin coincides with increased incidence of chromosome misalignment and precocious separations of sister chromatids
— id: 101975, year: 2008, vol: 16, page: 103, stat: Journal Article,

Telomeres and meiosis in health and disease
Keefe, D L
2007 Jan;64(2):115-116, Cellular & molecular life sciences: CMLS
— id: 101979, year: 2007, vol: 64, page: 115, stat: Journal Article,

Telomeres and aging-related meiotic dysfunction in women
Keefe, D L; Liu, L; Marquard, K
2007 Jan;64(2):139-143, Cellular & molecular life sciences: CMLS
Meiotic dysfunction increasingly afflicts women as they age, resulting in infertility, miscarriage and handicapped offspring. How aging disrupts meiotic function in women remains unclear, but as women increasingly delay childbearing, this issue becomes urgent. Telomeres, which mediate aging in mitotic cells, may also mediate aging during meiosis. Telomeres shorten during DNA replication. In mammals, oocytes remain quiescent, but their precursors replicated during fetal oogenesis. Moreover, eggs ovulated from older women entered meiosis later during fetal oogenesis than eggs ovulated when younger, and therefore underwent more replications. Telomeres also shorten from reactive oxygen, which triggers a DNA repair response, so the prolonged interval between fetal oogenesis and ovulation in some women would further shorten telomeres. Mice normally do not exhibit age-related meiotic dysfunction (interestingly, their telomeres are manyfold longer than telomeres in women), but genetic or pharmacologic shortening of mouse telomeres recapitulates the reproductive aging phenotype of women. This has led to a telomere theory of age-related meiotic dysfunction in women, and underlined the importance to human health of a mechanistic understanding of telomeres and meiosis
— id: 101980, year: 2007, vol: 64, page: 139, stat: Journal Article,

Telomere lengthening early in development
Liu, Lin; Bailey, Susan M; Okuka, Maja; Munoz, Purificacion; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L
2007 Dec;9(12):1436-1441, Nature cell biology
Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism
— id: 101976, year: 2007, vol: 9, page: 1436, stat: Journal Article,

Nuclear transfer methods to study aging
Liu, Lin; Keefe, David L
2007 ;371:191-207, Methods in molecular biology
Maternal age affects oocyte quality and early embryo development. Aberrant meiosis of oocytes and compromised early embryo development from older females could originate from defects in the nucleus, the cytoplasm, or both. Nuclear transfer has been used for decades as a tool to study nuclear-cytoplasmic interactions in early embryos, and has uncovered genomic imprinting, nuclear reprogramming, and produced animal clones. Here, we describe the technique for investigating nuclear-cyoplasmic interactions in oocytes and zygotes in female reproductive aging. Nuclear transfer can be performed efficiently and effects of the technique itself on meiosis and early embryo development are minimal as long as care is taken to minimize insult to oocytes or embryos. This protocol first focuses on use of nuclear transfer to study nucleus versus cytoplasmic origin in agingassociated meiosis defects in oocytes at the germinal vesicle (GV) stage. Then, nuclear transfer is used at the zygote stage to study nuclear and cytoplasmic abnormality and apoptosis in early development
— id: 101977, year: 2007, vol: 371, page: 191, stat: Journal Article,

Germline stem cells and neo-oogenesis in the adult human ovary
Liu, Yifei; Wu, Chao; Lyu, Qifeng; Yang, Dongzi; Albertini, David F; Keefe, David L; Liu, Lin
2007 Jun 1;306(1):112-120, Developmental biology (Orlando)
It remains unclear whether neo-oogenesis occurs in postnatal ovaries of mammals, based on studies in mice. We thought to test whether adult human ovaries contain germline stem cells (GSCs) and undergo neo-oogenesis. Rather than using genetic manipulation which is unethical in humans, we took the approach of analyzing the expression of meiotic marker genes and genes for germ cell proliferation, which are required for neo-oogenesis, in adult human ovaries covering an age range from 28 to 53 years old, compared to testis and fetal ovaries served as positive controls. We show that active meiosis, neo-oogenesis and GSCs are unlikely to exist in normal, adult, human ovaries. No early meiotic-specific or oogenesis-associated mRNAs for SPO11, PRDM9, SCP1, TERT and NOBOX were detectable in adult human ovaries using RT-PCR, compared to fetal ovary and adult testis controls. These findings are further corroborated by the absence of early meiocytes and proliferating germ cells in adult human ovarian cortex probed with markers for meiosis (SCP3), oogonium (OCT3/4, c-KIT), and cell cycle progression (Ki-67, PCNA), in contrast to fetal ovary controls. If postnatal oogenesis is confirmed in mice, then this species would represent an exception to the rule that neo-oogenesis does not occur in adults
— id: 101978, year: 2007, vol: 306, page: 112, stat: Journal Article,

Fate of centrosomes following somatic cell nuclear transfer (SCNT) in bovine oocytes
Dai, Yunping; Wang, Lili; Wang, Haiping; Liu, Ying; Li, Ning; Lyu, Qifeng; Keefe, David L; Albertini, David F; Liu, Lin
2006 Jun;131(6):1051-1061, Reproduction
Cloning mammalians by somatic cell nuclear transfer (SCNT) remains inefficient. A majority of clones produced by SCNT fail to develop properly and of those which do survive, some exhibit early aging, premature death, tumors, and other pathologies associated with aneuploidy. Alterations of centrosomes are linked to aberrant cell cycle progression, aneuploidy, and tumorigenesis in many cell types. It remains to be determined how centrosomes are remodeled in cloned bovine embryos. We show that abnormalities in either distribution and/or number of centrosomes were evident in approximately 50% of reconstructed embryos following SCNT. Moreover, centrosome abnormalities and failed 'pronuclear' migration which manifested during the first cell cycle coincided with errors in spindle morphogenesis, chromosome alignment, and cytokinesis. By contrast, nuclear mitotic apparatus protein (NuMA) exhibited normal expression patterns at metaphase spindle poles and in 'pronucleus' during interphase. The defects in centrosome remodeling and 'pronuclear' migration could lead to chromosome instability and developmental failures associated with embryo production by SCNT. Addressing these fundamental problems may enhance production of normal clones
— id: 101983, year: 2006, vol: 131, page: 1051, stat: Journal Article,

The telomere theory of reproductive senescence in women
Keefe, David L; Marquard, Kerri; Liu, Lin
2006 Jun;18(3):280-285, Current opinion in obstetrics & gynecology
PURPOSE OF REVIEW: A unifying theory of reproductive aging, based on telomere shortening, is proposed. RECENT FINDINGS: Telomere shortening may mediate both 'hits' involved in reproductive aging, that is late exit from the fetal production line and long interval to ovulation in the adult. SUMMARY: As women age egg dysfunction increases, with meiotic nondisjunction, embryonic arrest, apoptosis, and miscarriage. Egg dysfunction results from two 'hits' - reduced formation of chiasmata during fetal oogenesis, and accumulation of reactive oxygen damage during the prolonged interval until ovulation. Late exit from a production line during oogenesis presumably contributes to the first hit. The later insult also involves meiotic spindle abnormalities. Telomeres, repetitive sequences of DNA, cap chromosome ends and dissipate during divisions. Oocytes do not divide, but oogonia do, and telomerase, the enzyme responsible for maintaining telomere length, is inefficient, and remains inactive in oocytes and embryos until blastocyst stage. Reactive oxygen also shortens telomeres, so the prolonged interval between birth and ovulation would further shorten telomeres from chronic exposure to reactive oxygen. In support of this theory, experimental shortening of telomeres in mice produced a phenotype similar to reproductive aging in women, with abnormal chiasmata, spindles, cell cycles, apoptosis, and genomic instability, and telomere length in human eggs correlated with in-vitro fertilization outcome
— id: 101982, year: 2006, vol: 18, page: 280, stat: Journal Article,

Arsenite induces aberrations in meiosis that can be prevented by coadministration of N-acetylcysteine in mice
Navarro, Paula A A S; Liu, Lin; Ferriani, Rui A; Keefe, David L
2006 Apr;85 Suppl 1:1187-1194, Fertility & sterility
OBJECTIVE: To evaluate in vitro effects of arsenite and of arsenite plus N-acetylcysteine on mouse oocyte meiosis. DESIGN: Morphological study using mouse oocytes submitted to in vitro maturation (IVM). SETTING: Laboratory of reproductive biology. ANIMAL(S): Six-week-old CD-1 mice superovulated with pregnant mare serum gonadotropin. INTERVENTION(S): During IVM, mouse oocytes were exposed to arsenite alone or to arsenite plus N-acetylcysteine. MAIN OUTCOME MEASURE(S): Meiotic anomalies were assessed using immunofluorescence microscopy and PolScope (Cambridge Research and Instrumentation, Boston, MA) imaging. RESULT(S): In vitro arsenite administration produced dose-dependent and time-dependent meiotic anomalies, characterized by spindle disruption or chromosome misalignment. After 12-14 hours of IVM, exposure to 2 microg/mL of arsenite for 12-14 hours or to 8 microg/mL of arsenite for 2 hours arrested oocyte maturation at the germinal vesicle or germinal-vesicle breakdown stage. Exposure to 4 microg/mL of arsenite for 2 hours arrested oocyte maturation at metaphase I stage in 95% of exposed oocytes (80% exhibiting abnormalities) after 12-14 hours in IVM. After 12-14 hours in IVM, of the oocytes exposed to 2 microg/mL of arsenite for 2 hours, only 15% reached the meiosis II stage (5% exhibiting abnormalities). After 15-17 hours in IVM, however, of the oocytes exposed to 2 microg/mL of arsenite for 2 hours, 65.2% reached the meiosis II stage (43.5% exhibiting abnormalities). Co-administration of N-acetylcysteine prevented the arsenite-induced meiotic abnormalities and the delayed IVM. CONCLUSION(S): In vitro arsenite exposure caused meiotic abnormalities that were prevented by co-administration of N-acetylcysteine, suggesting that arsenite-induced meiotic aberrations are mediated by reactive oxygen species
— id: 101984, year: 2006, vol: 85 Suppl 1, page: 1187, stat: Journal Article,

Mullerian inhibiting substance levels at the time of HCG administration in IVF cycles predict both ovarian reserve and embryo morphology
Silberstein, T; MacLaughlin, D T; Shai, I; Trimarchi, J R; Lambert-Messerlian, G; Seifer, D B; Keefe, D L; Blazar, A S
2006 Jan;21(1):159-163, Human reproduction
BACKGROUND: Pre-antral and early antral follicles secrete Mullerian inhibiting substance (MIS), suggesting that MIS may directly reflect ovarian reserve. Since little is known about how ovarian reserve affects oocyte quality, we attempt here to assess the predictive value of MIS on embryo morphology and IVF outcome. To do so, we measured MIS at the time of HCG administration 36 h prior to oocyte retrieval. METHODS: A total of 257 patients undergoing IVF were prospectively recruited. We measured MIS levels by enzyme-linked immunosorbent assay at the time of HCG, and compared the MIS values to day 3 FSH levels in the prediction of embryo morphology and IVF outcome. RESULTS: The distribution of MIS levels was skewed, with a median of 2.7 ng/ml (range 0 to 28.5 ng/ml). MIS values at the time of HCG administration inversely correlated with basal FSH levels (P = 0.002), and both correlated significantly with patient age, number of mature follicles, number of oocytes retrieved and serum estradiol levels. MIS levels correlated significantly with a greater number of 6-cell embryos and better embryo morphology score, while basal FSH levels did not correlate with these outcome variables. MIS levels > or =2.7 ng/ml portended improved oocyte quality as reflected in a higher implantation rate (P = 0.001) and a trend toward a better clinical pregnancy rate (P = 0.084). CONCLUSIONS: MIS levels seem to predict not only ovarian reserve, but also embryo morphology. Measurement of MIS at the time of HCG administration may, therefore, in the future improve management of patients undergoing treatments with assisted reproductive technology
— id: 101988, year: 2006, vol: 21, page: 159, stat: Journal Article,

Lead concentrates in ovarian follicle compromises pregnancy
Silberstein, Tali; Saphier, Oshra; Paz-Tal, Ofra; Trimarchi, James R; Gonzalez, Liliana; Keefe, David L
2006 ;20(3):205-207, Journal of trace elements in medicine & biology
Following absorption, lead can concentrate in bodily compartments where it disrupts cellular processes and can result in detrimental health consequences. The concentration and impact of lead within follicular fluid has not been characterized and we used inductively coupled plasma mass spectroscopy (ICP-MS) to determine lead levels in blood and follicular fluid from nine patients undergoing in vitro fertilization (IVF) treatment. Lead levels within follicular fluid were found to be significantly higher in non-pregnant patients compared to pregnant patients suggesting that elevated concentrations of the environmental toxicant lead adversely affect female reproduction
— id: 101981, year: 2006, vol: 20, page: 205, stat: Journal Article,

Assessing the quality of oocytes derived from in vitro maturation: are we looking under the lamppost?
Trimarchi, James Robert; Keefe, David L
2006 Apr;85(4):839-840, Fertility & sterility
Oocytes are complex cells comprising many cellular systems, each of which is essential for proper oocyte function. Methods to assess the diverse and critical cellular systems in oocytes derived from in vitro maturation are badly needed
— id: 101985, year: 2006, vol: 85, page: 839, stat: Journal Article,

Telomere length predicts embryo fragmentation after in vitro fertilization in women--toward a telomere theory of reproductive aging in women
Keefe, David L; Franco, Sonia; Liu, Lin; Trimarchi, James; Cao, Benning; Weitzen, Sherry; Agarwal, Shoba; Blasco, Maria A
2005 Apr;192(4):1256-1260, American journal of obstetrics & gynecology
OBJECTIVE: Telomeres are DNA repeats which cap and protect chromosome ends, facilitate homologue pairing and chiasmata formation during early meiosis, and shorten with cell division and exposure to reactive oxygen to mediate aging. Early germ cells contain telomerase, a reverse transcriptase which adds telomeres to 3-prime DNA ends, but telomerase activity declines in oocytes, fixing telomere length earlier during development. Experimentally induced telomere shortening in mice disrupts meiosis, impairs chiasmata formation, halts embryonic cell cycles, and promotes apoptosis in embryos, a phenotype which mimics reproductive senescence in women. Ethical constraints limit study of human embryos to nondestructive assays, such as morphologic evaluation under transmission optics, but cytoplasmic fragmentation is a reliable marker of apoptosis. STUDY DESIGN: Study design consisted of observational study of effect of telomere length in human eggs on cytoplasmic fragmentation, and on other morphologic features of preimplantation embryos. To test the hypothesis that telomere shortening triggers apoptosis in human embryos, we evaluated telomere length as a predictor of cytoplasmic fragmentation in embryos from women undergoing in vitro fertilization. RESULTS: Telomere length negatively predicted fragmentation in day 3 preimplantation embryos, after controlling for patient age and basal follicle stimulating hormone level. Telomere length did not predict other features of preimplantation embryo morphology. CONCLUSION: The finding that telomere length in human eggs predicts cytoplasmic fragmentation in embryos provides evidence that telomere shortening induces apoptosis in human preimplantation embryos, consistent with a telomere theory of reproductive senescence in women
— id: 101989, year: 2005, vol: 192, page: 1256, stat: Journal Article,

New approaches to assisted reproductive technologies
Keefe, David L; Parry, John P
2005 Nov;23(4):301-308, Seminars in reproductive medicine
Egg infertility remains the greatest challenge in the treatment of the infertile couple. As women increasingly delay attempts at childbearing, egg infertility has become more prevalent. Attempts to overcome egg infertility by superovulation and in vitro fertilization have produced an epidemic of multiple gestations, itself a major public health concern. The pathophysiology of egg infertility arises from chromosomal nondisjunction. Cytogenetic analyses of polar bodies and/or blastomeres currently provide the most powerful predictors of egg infertility. Approaches that label all chromosomes (spectral karyotyping and comparative genomic hybridization), or identify predisposition to aneuploidy (spindle imaging, telomere length measurement) are on the horizon. For the foreseeable future, the treatment of egg infertility will be limited to egg donation for severe cases and transfer of the most viable embryos for milder cases. Oocyte reconstitution not only lacks evidence of clinical efficacy, but also biological credibility, given that growing evidence supports the primacy of chromosomes themselves in meiotic nondisjunction
— id: 101986, year: 2005, vol: 23, page: 301, stat: Journal Article,

Noninvasive imaging of spindle dynamics during mammalian oocyte activation
Navarro, Paula A A S; Liu, Lin; Trimarchi, James R; Ferriani, Rui A; Keefe, David L
2005 Apr;83 Suppl 1:1197-1205, Fertility & sterility
OBJECTIVE: To develop a method to evaluate spindle dynamics in living oocytes and in karyoplasts during the initial stages of activation and after pharmacological disruption of cytoskeleton. DESIGN: Morphological study using a novel microscope. SETTING: Translational research laboratory at marine biological laboratory. ANIMAL(S): Six-week-old CD-1 or B6C3F1 mice superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). INTERVENTION(S): Spindles of living oocytes and karyoplasts were imaged at 5-10 minute intervals using the Pol-Scope during the initial stages of oocyte activation and after pharmacological disruption of cytoskeleton. MAIN OUTCOME MEASURE(S): Assessment of spindle dynamics using Pol-Scope imaging. RESULT(S): During oocyte activation, spindle mid-region birefringence increased, followed by spindle rotation and second polar body extrusion in both intact oocytes and karyoplasts. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate failed to induce spindle activation in 60% of living oocytes and caused spindle disruption in some oocytes. Inhibition of PKC by a myristoylated PKC pseudosubstrate inhibited metaphase II release in most oocytes evaluated (86.7%). Cytochalasin D inhibited only spindle rotation and separation. Nocodazole disrupted spindles in less than 5 minutes after administration. CONCLUSION(S): Pol-Scope imaging allows investigation at near real time of spindle dynamics during activation of living oocytes. Spindles also showed evidence of activation even in karyoplasts. The procedure may be useful for detecting functional spindle aberrations in living oocytes. Further studies are needed to determine whether spindle dynamics predict clinical outcome
— id: 101990, year: 2005, vol: 83 Suppl 1, page: 1197, stat: Journal Article,

Pregnancy outcome in in vitro fertilization decreases to a plateau with repeated cycles
Silberstein, Tali; Trimarchi, James R; Gonzalez, Liliana; Keefe, David L; Blazar, Andrew S
2005 Oct;84(4):1043-1045, Fertility & sterility
Clinical pregnancy rate (CPR) and implantation rate (IR) in 1,177 patients who had 1,788 fresh, nondonor, nonPGD IVF cycles were highest in cycle 1, significantly declined in cycle 2, and reached a plateau for cycles 3-5 at a rate lower than in cycle 2. In patients >38 years of age CPR and IR in cycles 1 and 2 were significantly lower than in younger patients, but there was no decline in CPR or IR with advancing IVF attempts
— id: 101987, year: 2005, vol: 84, page: 1043, stat: Journal Article,

Responses to a saline load in gonadotropin-releasing hormone antagonist-pretreated premenopausal women receiving progesterone or estradiol-progesterone therapy
Stachenfeld, Nina S; Keefe, David L; Taylor, Hugh S
2005 Jan;90(1):386-394, Journal of clinical endocrinology & metabolism
The effects of estradiol (E(2)) and progesterone (P(4)) on fluid and sodium regulation may have important clinical implications with respect to cardiovascular and renal disease as well as reproductive syndromes such as preeclampsia and ovarian hyperstimulation syndrome. We tested the hypothesis that sodium excretion is reduced in response to a sodium load during combined P(4)-E(2) treatment, but P(4) administration alone has little effect on sodium regulation. Fifteen women (22 +/- 2 yr) used a GnRH antagonist to suppress endogenous E(2) and P(4) for 9 d; for d 4-9, eight subjects used P(4) (200 mg/d), and seven subjects used P(4) with E(2) (two E(2) patches, 0.1 mg/d each). On d 3 and 9, isotonic saline (0.9% NaCl) was infused [120 min at 0.1 ml/kg body weight (BW).min], followed by 120 min of rest. Compared with GnRH antagonist alone, P([P4]) increased from 1.6 +/- 0.8 to 9.4 +/- 2.3 ng/ml (5.1 +/- 2.5 to 29.9 +/- 7.3 nmol/liter, P < 0.05) in the P(4) treated group, with no change in P([E2]). In the P(4)-E(2) treated group P([P4]) increased from 1.6 +/- 0.5 to 6.7 +/- 0.6 ng/ml (5.1 +/- 1.6 to 21.3 +/- 1.6 nmol/liter, P < 0.05 and P([E2]) increased from 17.9 +/- 6.3 to 200 +/- 41 pg/ml (65.7 +/- 23 to 734.6 +/- 150.0 pmol/liter, P < 0.05). Before isotonic saline infusion, renal sodium and water excretion were similar under all conditions, but during isotonic saline infusion, cumulative sodium excretion was lower in the P(4)-E(2) treated women (34.1 +/- 5.1 mEq) compared with GnRH antagonist (50.2 +/- 11.4 mEq). Sodium excretion was unaffected by P(4) treatment (48.0 +/- 8.2 and 41.2 +/- 5.1 mEq, for GnRH antagonist and P(4)). Compared with GnRH antagonist alone, P(4)-E(2) treatment increased distal sodium reabsorption and transiently decreased proximal sodium reabsorption, whereas P(4) treatment did not alter either distal or proximal sodium reabsorption. Before isotonic saline infusion, the plasma aldosterone (Ald) concentration was greater during P(4) treatment (153 +/- 25 pg/ml; 3883 +/- 1102 pmol/liter) and P(4)-E(2) treatment (242 +/- 47 pg/ml; 6373 +/- 1390 pmol/liter) than during their respective GnRH antagonist alone treatments [96 +/- 13 and 148 +/- 47 pg/ml (2598 +/- 475 and 3284 +/- 973 pmol/liter) for P(4) and combined P(4)-E(2), respectively]. Compared with GnRH antagonist alone treatments, preisotonic saline infusion plasma renin activity was greater only with P(4)-E(2) treatment, whereas the plasma atrial natriuretic peptide concentration was lower only with P(4) treatment. Isotonic saline infusion suppressed plasma Ald under all conditions, but decreased plasma renin activity only with P(4)-E(2) treatment (average decrease, 1.3 +/- 0.5 ng/ml angiotensin I.h; P < 0.05). In summary, we found that P(4)-E(2) treatment decreased sodium excretion via either renin-angiotensin-Ald system stimulation or direct effects on kidney tubules. P(4) treatment at these plasma concentrations had no independent effect on the renal response to acute sodium loading. These data suggest that E(2) is the more powerful reproductive hormone involved in sodium retention relative to P(4), and that estrogen-induced up-regulation of P(4) receptors is required for the effects of P(4) on sodium regulation
— id: 101993, year: 2005, vol: 90, page: 386, stat: Journal Article,

Direct visual and circadian pathways target neuroendocrine cells in primates
Abizaid, Alfonso; Horvath, Balazs; Keefe, David L; Leranth, Csaba; Horvath, Tamas L
2004 Nov;20(10):2767-2776, European journal of neuroscience
The effect of light on neuroendocrine functions is thought to be mediated through retinal inputs to the circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). The present studies were conducted to provide experimental evidence for this signaling modality in non-human primates. In the St. Kitts vervet monkey, anterograde tracing of SCN efferents revealed a monosynaptic pathway between the circadian clock and hypothalamic neurons producing luteinizing hormone-releasing hormone (LHRH). Using a variety of tracing techniques, direct retinal input was found to be abundant in the SCN and in other hypothalamic sites. Strikingly, in hypothalamic areas other than the SCN, primary visual afferents established direct contacts with neuroendocrine cells including those producing LHRH and dopamine, neurons that are the hypothalamic regulators of pituitary gonadotrops and prolactin. Thus, our data reveal for the first time in primates that light stimuli can reach the hypothalamo-pituitary-gonadal axis, directly providing a pathway independent of but parallel to that of the circadian clock for the photic modulation of hormone release
— id: 101991, year: 2004, vol: 20, page: 2767, stat: Journal Article,

Serum estradiol positively predicts outcomes in patients undergoing in vitro fertilization
Blazar, Andrew S; Hogan, Joseph W; Frankfurter, David; Hackett, Richard; Keefe, David L
2004 Jun;81(6):1707-1709, Fertility & sterility
In patients undergoing in vitro fertilization, the presence of higher E(2) levels at the time of hCG administration predict a greater likelihood of ongoing pregnancy
— id: 101996, year: 2004, vol: 81, page: 1707, stat: Journal Article,

Middle to lower uterine segment embryo transfer improves implantation and pregnancy rates compared with fundal embryo transfer
Frankfurter, David; Trimarchi, James B; Silva, Celso P; Keefe, David L
2004 May;81(5):1273-1277, Fertility & sterility
OBJECTIVE: To assess differences in pregnancy and implantation rates as a function of the embryo placement. DESIGN: Prospective cohort study. SETTING: A tertiary care center. SUBJECT(S): All fresh, nondonor IVF cycles performed in 2001. INTERVENTION(S): Alteration in embryo transfer (ET) target location from the fundal region to the middle to lower uterine segment. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (sonographic sac evidence/number of transfer cycles), implantation rate (number of sacs/number of embryos transferred), patient age, peak E(2), and fertilization rate. RESULT(S): A total of 393 fundal and 273 lower to middle uterine segment ETs were performed. The pregnancy (PR), implantation, and birth rates were significantly higher after a middle to lower uterine segment ET compared with fundal ET (39.6% vs. 31.2%; 21% vs. 14%; and 34.1% vs. 26.2%, respectively). Groups did not differ regarding patient age, basal FSH, peak E(2), number of intracytoplasmic sperm injection (ICSI) cycles, fertilization rate, embryo quality, or number of embryos transferred. CONCLUSION(S): Both PR and implantation rates are favorably affected by directing embryo placement to the lower to middle uterine segment. By some unknown mechanism, it appears that this endometrial location provides a more favorable region for embryo deposition
— id: 101998, year: 2004, vol: 81, page: 1273, stat: Journal Article,

Monozygotic pregnancies from transfers of zona-free blastocysts
Frankfurter, David; Trimarchi, James; Hackett, Rick; Meng, Li; Keefe, David
2004 Aug;82(2):483-485, Fertility & sterility
— id: 144321, year: 2004, vol: 82, page: 483, stat: Journal Article,

Effect of ploidy and parental genome composition on expression of Oct-4 protein in mouse embryos
Liu, Lin; Czerwiec, Eva; Keefe, David L
2004 Jul;4(4):433-441, Gene expression patterns
The transcription factor Oct-4 is expressed in germ cells and also is considered as a marker for pluripotency of stem cells. We first examined dynamics of Oct-4 protein expression during preimplantation development using both Western blot analysis, and immunofluorescence staining. We show that intact Oct-4 protein is not detected in either ovulated mature oocytes, or in zygotes and 2-4-cell embryos, which are the only known totipotent cell types in mammals. This finding is unexpected, since Oct-4 has been proposed to play a role in the control of totipotency. The results suggest that Oct-4 is not indispensable for fertilization and early cleavage. Rather, expression of Oct-4 protein is first detected in the nuclei of 8-16 cell morula, increases in early blastocysts, and declines in late blastocysts, in which most Oct-4 protein is confined to the inner cell mass (ICM) region, consistent with previous findings. We further compared Oct-4 protein expression in diploid and tetraploid blastocysts derived from normal fertilization or parthenogenesis, as well as expression in diploid androgenetic blastocysts. Expression levels and localization of Oct-4 protein are similar in both diploid and tetraploid early blastocysts, regardless of whether blastocysts are derived from fertilization or parthenogenesis. Androgenetic diploid blastocysts also express similar levels of Oct-4. Late blastocysts generated by both fertilization and parthenogenesis show a similar pattern of Oct-4 expression, suggesting that paternal genome activation is not required for Oct-4 expression. Expression of Oct-4 protein does not differ between diploid and tetraploid embryos, indicating that tetraploidy does not influence Oct-4 expression. Thus, expression of Oct-4 protein is initiated at morula stage in preimplantation embryos and completely controlled by a mechanism activated in oocytes. Downregulation of Oct-4 expression coincides with differentiation of trophectoderm. Similar profiles of Oct-4 expression observed in embryos with different ploidy and genome composition, are suggestive of Oct-4 being necessary but not sufficient for developmental potency
— id: 101997, year: 2004, vol: 4, page: 433, stat: Journal Article,

Telomerase deficiency impairs differentiation of mesenchymal stem cells
Liu, Lin; DiGirolamo, Carla M; Navarro, Paula A A S; Blasco, Maria A; Keefe, David L
2004 Mar 10;294(1):1-8, Experimental cell research
Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR(-/-)) and wild-type (WT) mice. The MSCs isolated from mTR(-/-) mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR(-/-)MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation
— id: 102001, year: 2004, vol: 294, page: 1, stat: Journal Article,

Irregular telomeres impair meiotic synapsis and recombination in mice
Liu, Lin; Franco, Sonia; Spyropoulos, Barbara; Moens, Peter B; Blasco, Maria A; Keefe, David L
2004 Apr 27;101(17):6496-6501, Proceedings of the National Academy of Sciences of the United States of America
Telomere shortening can lead to chromosome instability, replicative senescence, and apoptosis in both somatic and male germ cells. To study roles for mammalian telomeres in homologous pairing and recombination, we characterized effects of telomere shortening on spermatogenesis and oogenesis in late-generation telomerase-deficient mice. We show that shortened telomeres of late-generation telomerase-deficient mice impair meiotic synapsis and decrease recombination, in particular, in females. In response to telomere shortening, male germ cells mostly undergo apoptosis, whereas female germ cells preferentially arrest in early meiosis, suggesting sexually dimorphic surveillance mechanisms for telomere dysfunction during meiosis in mice. Further, meiocytes of late-generation telomerase-deficient females with shortened telomeres, bred with early-generation males harboring relatively long telomeres, exhibit severely impaired chromosome pairing and synapsis and reduced meiotic recombination. These findings imply that functional telomeres are important in mammalian meiotic synapsis and recombination
— id: 101999, year: 2004, vol: 101, page: 6496, stat: Journal Article,

Nuclear origin of aging-associated meiotic defects in senescence-accelerated mice
Liu, Lin; Keefe, David L
2004 Nov;71(5):1724-1729, Biology of reproduction
Factors of both cytoplasmic and nuclear origin regulate metaphase chromosome alignment and spindle checkpoint during mitosis. Most aneuploidies associated with maternal aging are believed to derive from nondisjunction and meiotic errors, such as aberrations in spindle formation and chromosome alignment at meiosis I. Senescence-accelerated mice (SAM) exhibit aging-associated meiotic defects, specifically chromosome misalignments at meiosis I and II that resemble those found in human female aging. How maternal aging disrupts meiosis remains largely unexplained. Using germinal vesicle nuclear transfer, we found that aging-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model. Cytoplasm of young hybrid B6C3F1 mouse oocytes could partly rescue aging-associated meiotic chromosome misalignment, whereas cytoplasm of young SAM was ineffective in preventing the meiotic defects of old SAM oocytes, which is indicative of a deficiency of SAM oocyte cytoplasm. Our results demonstrate that both nuclear and cytoplasmic factors contribute to the meiotic defects of the old SAM oocytes and that the nuclear compartment plays the predominant role in the etiology of aging-related meiotic defects
— id: 101994, year: 2004, vol: 71, page: 1724, stat: Journal Article,

In vivo effects of arsenite on meiosis, preimplantation development, and apoptosis in the mouse
Navarro, Paula A A S; Liu, Lin; Keefe, David L
2004 Apr;70(4):980-985, Biology of reproduction
Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including metabolic abnormalities accompanied by growth inhibition and carcinogenesis. Much of the toxicity of arsenic is known to stem from its uncoupling effects on mitochondria. Because previously we had shown that mitochondrial dysfunction can disrupt oocyte and embryo development, we investigated effects of arsenite on meiotic progression and early embryo development in mice. Six-week-old CD-1 mice were treated with 0 (solvent as control), 8 mg/kg (a dose previously established in mice as the maternal no-observed-adverse-effect level), and 16 mg/kg doses of sodium arsenite every 2 days for a total of seven i.p. injections ver a period of 14 days. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment, was significantly increased in arsenite-treated groups (25% after 8 mg/kg and 62.5% after 16 mg/kg), compared to normal metaphase II in control oocytes. Further, arsenite treatment significantly decreased cleavage rates of zygotes at 24 h, morula formation at 72 h, and development to blastocysts at 96 h in a dose-dependent manner. The total cell number in developed blastocysts did not differ significantly between the 8 mg/kg arsenite treatment and control groups, but was significantly reduced in the 16 mg/kg arsenite treatment group. Moreover, the percentage of apoptotic nuclei was significantly increased in blastocysts following 16 mg/kg arsenite treatment. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis
— id: 102002, year: 2004, vol: 70, page: 980, stat: Journal Article,

Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos
Pelletier, Cory; Keefe, David L; Trimarchi, James R
2004 Mar;81 Suppl 1:850-856, Fertility & sterility
OBJECTIVE: To characterize the architecture of the zona pellucida in living human eggs and embryos, noninvasively with the PolScope, a digital polarizing light microscope. DESIGN: The PolScope was used to examine zonae pellucida of living human eggs and embryos. SETTING: Academic IVF clinic. PATIENT(S): Patients undergoing fresh, nondonor infertility treatment who underwent egg aspiration, fertilization by intracytoplasmic sperm injection or traditional IVF, and cleavage-stage embryo transfer (day 3). INTERVENTION(S): The PolScope imaged the zona of eggs before intracytoplasmic sperm injection and in cleavage-stage embryos before transfer. MAIN OUTCOME MEASURE(S): Thickness and retardance of three zona layers were measured from eight quadrants. Mean and variance in thickness and retardance were calculated for individual eggs and embryos, between eggs and embryos of a cohort, and across the sample patient population. RESULT(S): Cleavage-stage (day 3) embryos have thinner zonae (15.2 +/- 2.9 microm) than both immature (20.4 +/- 2.4 microm) and mature (19.5 +/- 2.2 microm) eggs. The zona of embryos is thinner, primarily owing to thinning of the outer layer. The thicker the zona layer, the greater its retardance. Considerable variation exists in the thickness and retardance of zona layers around individual eggs and embryos and between members of a cohort. The zona of eggs and embryos from different patients differ in thickness, retardance, and variation. CONCLUSION(S): Thickness and organization of zonae pellucida of human eggs and embryos varies considerably and can be quantitatively imaged with the PolScope
— id: 102000, year: 2004, vol: 81 Suppl 1, page: 850, stat: Journal Article,

Cannulation of a resistant internal os with the malleable outer sheath of a coaxial soft embryo transfer catheter does not affect in vitro fertilization-embryo transfer outcome
Silberstein, Tali; Weitzen, Sherry; Frankfurter, David; Trimarchi, James R; Keefe, David L; Plosker, Shayne M
2004 Nov;82(5):1402-1406, Fertility & sterility
OBJECTIVE: To assess the impact of cannulation of a resistant cervical os with the outer malleable sheath of a double-lumen, soft ET catheter on IVF-ET outcomes. DESIGN: Retrospective cohort study. SETTING: University-based IVF center. PATIENT(S): One hundred forty-two patients undergoing 142 ETs. INTERVENTION(S): Trial ultrasound-guided ET at all transfers, leaving the malleable outer sheath in situ when the soft inner catheter could not negotiate the internal os. MAIN OUTCOME MEASURE(S): Implantation rate and clinical pregnancy rate. RESULT(S): In 102 ETs (71.8%), the soft inner sheath easily negotiated the internal os (group 1). Forty ETs (28.2%) required cannulation of resistant internal ora with the outer sheath of the trial catheter (group 2). Implantation rates (35% vs. 32% in groups 1 and 2, respectively) and clinical pregnancy rates (50% vs. 45%) were not significantly different between groups. Blood was present on the transfer catheter after ET more frequently in group 2 than in group 1 (55% vs. 15%); however, neither the implantation rate nor the clinical pregnancy rate were affected by the presence of blood. CONCLUSION(S): Cannulation of a resistant internal os by the malleable outer sheath and blood on the transfer catheter after ET do not have an adverse effect on implantation rate or clinical pregnancy rate
— id: 101992, year: 2004, vol: 82, page: 1402, stat: Journal Article,

Overheating is detrimental to meiotic spindles within in vitro matured human oocytes
Sun, Xiao-Fang; Wang, Wei-Hua; Keefe, David L
2004 Feb;12(1):65-70, Zygote
The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24-36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 degrees C for up to 20 min. After heating, oocytes were returned to 37 degrees C and then imaged for another 20 min at 37 degrees C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 degrees C during 40 min of examination and their maximum retardance (1.72-1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 degrees C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 degrees C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 degrees C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 degrees C and 40 degrees C, respectively. Heating to 40 degrees C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 degrees C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 degrees C is crucial for normal spindle morphology
— id: 101995, year: 2004, vol: 12, page: 65, stat: Journal Article,

Improved endometrial assessment using cyclin E and p27
Dubowy, Rebecca L; Feinberg, Ronald F; Keefe, David L; Doncel, Gustavo F; Williams, Shaun C; McSweet, Juliette C; Kliman, Harvey J
2003 Jul;80(1):146-156, Fertility & sterility
OBJECTIVE: To evaluate endometrial expression of cyclin E and p27 in fertile and infertile women. DESIGN: Retrospective clinical study. SETTING: University medical center and private practice. PATIENT(S): Thirty-three fertile volunteers, 83 women seeking infertility treatment, and 23 women undergoing mock cycles. INTERVENTION(S): Endometrial biopsy. MAIN OUTCOME MEASURE(S): Cyclin E and p27 immunohistochemistry. RESULT(S): Glandular cyclin E and p27 expression dramatically changed in intensity and subcellular localization throughout the menstrual cycle. In normal control biopsies, glandular cyclin E progressed from the basal to the lateral cytoplasm (midproliferative phase) to the nucleus (days 18 to 19) and was absent in biopsies after day 20. First appearing on days 17 to 19, p27 was found only in the nuclei. Cyclin E was more frequently seen after day 20 in infertility patients. In the hyperstimulated cycles, staining for cycle E in proliferative samples was more intense than in the natural cycles, but p27 staining was unchanged. CONCLUSION(S): Cyclin E and p27 may be clinically useful markers of development in the endometrium. As cell cycle regulators, cyclins reveal underlying biochemical processes driving endometrial progression and may partly represent the means by which estrogen and progesterone regulate this dynamic tissue
— id: 102005, year: 2003, vol: 80, page: 146, stat: Journal Article,

The transfer point is a novel measure of embryo placement
Frankfurter, David; Silva, Celso P; Mota, Francisco; Trimarchi, James B; Keefe, David L
2003 Jun;79(6):1416-1421, Fertility & sterility
OBJECTIVE: To study the relationship between IVF-ET pregnancy outcomes and measures of embryo placement. DESIGN: Case-control study. SETTING: Tertiary care center. PATIENT(S): Twenty-three patients who underwent two ultrasonography-guided ETs, of which one resulted in a clinical pregnancy and the other did not. MAIN OUTCOME MEASURES: Point of embryo placement normalized to the endometrial cavity length (the transfer point), distance from the point of embryo placement to the uterine fundus, time required for ET, contact with the uterine fundus, and evidence of trauma. Videotaped ETs were quantitatively analyzed. RESULT(S): From February 1, 2000, to March 31, 2001, videotaped ETs from 23 pairs of pregnant and nonpregnant cycles were identified. Embryo placement was more shallow in pregnancy cycles than in nonpregnancy cycles. The groups did not differ in the absolute distance of embryo placement to the fundus, ovarian stimulation, or other features of the ET. CONCLUSION(S): The transfer point may serve as a better marker of embryo position than does the absolute distance to the uterine fundus
— id: 102006, year: 2003, vol: 79, page: 1416, stat: Journal Article,

Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF
Keefe, David; Liu, Lin; Wang, Wei; Silva, Celso
2003 Jul-Aug;7(1):24-29, Reproductive biomedicine online
Meiotic spindles tether the chromosomes of oocytes and have been found to be structurally abnormal in older women. Conventional methods to image the meiotic spindle, such as immunostaining or transmission electron microscopy, require prior fixation, so they cannot be used clinically, and their utility in developmental studies is limited. Spindles can also be imaged non-invasively based on their birefringence, an inherent optical property of highly ordered molecules, such as microtubules, as they are illuminated with polarized light. Polarized light microscopy has been gainfully applied to embryology for decades, but recently a digital, orientation-independent polarized light microscope, the polscope, has demonstrated the exquisite sensitivity needed to image the low levels of birefringence exhibited by mammalian spindles. Its use of nearly circularly polarized light also produces orientation-independent measures of spindle birefringence, thus providing a method to quantify spindle architecture in living oocytes. The safety and utility of polscope imaging has been demonstrated in mammalian oocytes, including those from women undergoing ICSI. Spindle imaging with the polscope provides structural information closely related to the more invasive immunostaining method, and also enables study of the dynamic architecture of spindles. Profound effects of cooling on meiotic spindles have also been shown, and polscope imaging has been used to optimize thermodynamic stability of oocytes during ICSI. It has been shown that embryos derived from oocytes with normal, intact meiotic spindles exhibit superior development after fertilization and in-vitro culture. The mechanisms underlying age-related disruption of meiotic spindles in women remain unclear, but may relate to factors residing within the chromosomes themselves, since mice engineered to shorten their telomeres exhibit structurally abnormal spindles in their oocytes, and their embryos undergo cell cycle arrest and apoptosis, a phenotype remarkably similar to that observed in oocytes and embryos from older women. A time-lapse video of a mouse oocyte imaged by polscope may be purchased for viewing on the internet at www.rbmonline.com/Article/824 (free to web subscribers)
— id: 133648, year: 2003, vol: 7, page: 24, stat: Journal Article,

Oxidative stress contributes to arsenic-induced telomere attrition, chromosome instability, and apoptosis
Liu, Lin; Trimarchi, James R; Navarro, Paula; Blasco, Maria A; Keefe, David L
2003 Aug 22;278(34):31998-32004, Journal of biological chemistry
The environmental contaminant arsenic causes cancer, developmental retardation, and other degenerative diseases and, thus, is a serious health concern worldwide. Paradoxically, arsenic also may serve as an anti-tumor therapy, although the mechanisms of its antineoplastic effects remain unclear. Arsenic exerts its toxicity in part by generating reactive oxygen species. We show that arsenic-induced oxidative stress promotes telomere attrition, chromosome end-to-end fusions, and apoptotic cell death. An antioxidant, N-acetylcysteine, effectively prevents arsenic-induced oxidative stress, telomere erosion, chromosome instability, and apoptosis, suggesting that increasing the intracellular antioxidant level may have preventive or therapeutic effects in arsenic-induced chromosome instability and genotoxicity. Embryos with shortened telomeres from late generation telomerase-deficient mice exhibit increased sensitivity to arsenic-induced oxidative damage, suggesting that telomere attrition mediates arsenic-induced apoptosis. Unexpectedly, arsenite did not cause chromosome end-to-end fusions in telomerase RNA knockout mouse embryos despite progressively damaged telomeres and disrupting embryo viability. Together, these findings may explain why arsenic can initiate oxidative stress and telomere erosion, leading to apoptosis and anti-tumor therapy on the one hand and chromosome instability and carcinogenesis on the other
— id: 102007, year: 2003, vol: 278, page: 31998, stat: Journal Article,

Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals
Pepperell, John R; Porterfield, D Marshall; Keefe, David L; Behrman, Harold R; Smith, Peter J S
2003 Sep;285(3):C642-C651, American journal of physiology. Cell physiology
In luteal cells, prostaglandin (PG)F2a mobilizes intracellular calcium concentration ([Ca]i), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca]i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca]i and ROS, respectively. Menadione, a generator of intracellular superoxide radical (O2-), PGF2a, and calcium ionophore were shown to increase [Ca]i and stimulate intracellular ROS. With calcium ionophore and PGF2a, but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 +/- 20.3 fmol x cm-1 x s-1 (mean +/- SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 +/- 4.9 fmol x cm-1 x s-1; n = 5, P < 0.05). PGF2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF2a-stimulated luteal cells may occur through other signaling pathways
— id: 102008, year: 2003, vol: 285, page: C642, stat: Journal Article,

Oestrogen effects on urine concentrating response in young women
Stachenfeld, Nina S; Taylor, Hugh S; Leone, Cheryl A; Keefe, David L
2003 Nov 1;552(Pt 3):869-880, Journal of physiology
Oestrogen lowers the plasma osmotic threshold for arginine vasopressin (AVP) release but without commensurate changes in renal concentrating response, suggesting oestrogen (OE2) may lower renal sensitivity to AVP. Ten women (23 +/- 1 years) received a gonadotropin releasing hormone analogue (GnRHa), leuprolide acetate, to suppress OE2 for 35 days, and then added OE2 (two patches each delivering 0.1 mg day-1) on days 32-35. On days 28 and 35 we tested blood and renal water and sodium (Na+) regulation during stepwise 60 min AVP infusions (10, 35, 100, 150 and 200 microu (kg body weight)-1 Pitressin). Plasma OE2 concentration increased from 19 +/- 4 to 152 +/- 3 pg ml(-1) and plasma progesterone concentration was unchanged (1.0 +/- 0.4 and 0.7 +/- 0.1 ng ml(-1)) for GnRHa and OE2 administration, respectively. Standard log plots of plasma AVP concentration ([AVP]P) vs. urine osmolality (OsmU) were fitted to a sigmoidal curve, and EC50 was determined by non-linear regression curve fitting of concentration-response data. OsmU rose exponentially during AVP infusions, but hormone treatments did not affect EC50 (3.3 +/- 0.07 and 3.1 +/- 0.6 pg ml(-1), for GnRHa and OE2, respectively). However, the urine osmolality increase was greater within the physiological range (approximately 2.5-3.4 pg ml(-1) [AVP]P) during OE2 treatment. Throughout most of the AVP infusion, the rate of clearance of AVP from plasma (PCRAVP) was increased during OE2 (45.5 ml (kg body weight)(-1) min(-1)) compared to GnRHa administration (33.1 ml (kg body weight)(-1) min(-1); mean for the 100-200 microu (kg body weight)(-1) infusion rates). The rate of renal free water clearance (CH2O) was similar between hormone treatments. Sodium excretion fell during OE2 administration due to greater distal tubular sodium reabsorption. Despite more rapid PCRAVP, renal concentrating response to graded AVP infusions was unaffected by oestrogen treatment suggesting oestrogen does not affect overall renal sensitivity to AVP. However, OE2 may increase renal fluid retention within a physiological range of AVP
— id: 102003, year: 2003, vol: 552, page: 869, stat: Journal Article,

Sex hormones and neural mechanisms
Keefe, David L
2002 Oct;31(5):401-403, Archives of sexual behaviour
Sex steroids play important and diverse roles in the regulation of structure and function of the central nervous system. Early in life, steroids shape the structure of sensitive areas of the brain, especially those involved in the control of reproductive behavior and ovarian function. Original studies demonstrating organizing effects of steroids on the brain were carried out in rodents, but more recently these studies have been extended to primates, including humans. Throughout life, sex steroids regulate neural function by influencing steroid receptor-bearing neurons and by influencing neurons via steroid receptor-independent mechanisms. Sex steroid receptors have been identified in the brain, especially in the phylogenetically ancient structures that regulate reproductive behavior. Sex steroids that affect neural function can originate peripherally from the brain and/or adrenal gland, and can be synthesized within the brain itself. A number of neurally active progestogens and androgens are synthesized de novo in the brain, and estrogens can be converted within the brain from androgens by the enzyme aromatase. Thus, ovarian and central nervous system sex steroids play important roles in regulating reproductive behavior by regulating neural structure and function
— id: 102012, year: 2002, vol: 31, page: 401, stat: Journal Article,

Requirement of functional telomeres for metaphase chromosome alignments and integrity of meiotic spindles
Liu, Lin; Blasco, Maria A; Keefe, David L
2002 Mar;3(3):230-234, EMBO reports
Telomerase deficiency in the mouse eventually leads to loss of telomeric repeats from chromosome ends and to end-to-end chromosome fusions, which result in defects in highly proliferative tissues. We show that telomere dysfunction resulting from telomerase deficiency leads to disruption of functional meiotic spindles and misalignment of chromosomes during meiotic division of oocytes in late-generation (G4) mice. However, oocytes from first-generation (G1) mice lacking telomerase showed no appreciable telomere dysfunction and exhibited chromosome alignment at the metaphase plates of meiotic spindles, in a manner similar to that of wild-type mouse oocytes. These findings suggest that telomerase does not directly influence chromosome alignment and spindle integrity. Rather, functional telomeres may be involved in mediating metaphase chromosome alignment and maintaining functional spindles during meiotic division
— id: 102017, year: 2002, vol: 3, page: 230, stat: Journal Article,

An essential role for functional telomeres in mouse germ cells during fertilization and early development
Liu, Lin; Blasco, Maria; Trimarchi, James; Keefe, David
2002 Sep 1;249(1):74-84, Developmental biology (Orlando)
Late generations of telomerase-null (TR(-/-)) mice exhibit progressive defects in highly proliferative tissues and organs and decreased fertility, ultimately leading to sterility. To determine effects of telomerase deficiency on germ cells, we investigated the cleavage and preimplantation development of embryos derived from both in vivo and in vitro fertilization of TR(-/-) or wild-type (TR(+/+)) sperm with either TR(-/-) or TR(+/+) oocytes. Consistently, fertilization of TR(-/-) oocytes with either TR(+/+) or TR(-/-) sperm, and TR(-/-) sperm with TR(+/+) oocytes, resulted in aberrant cleavage and development, in contrast to the normal cleavage and development of TR(+/+) oocytes fertilized by TR(+/+) sperm. Many (>50%) of the fertilized TR(-/-) eggs developed only one pronucleus, coincident with increased incidence of cytofragmentation, in contrast to the normal formation of two pronuclei and equal cleavage of wild-type embryos. These results suggest that both TR(-/-) sperm and oocytes contribute to defective fertilization and cleavage. We further found that a subset (7-9%) of telomeres was undetectable at the ends of some metaphase I chromosomes from TR(-/-) spermatocytes and oocytes, indicating that meiotic germ cells lacking telomerase ultimately resulted in telomere shortening and loss. Dysfunction of meiotic telomeres may contribute to aberrant fertilization of gametes and lead to abnormal cleavage of embryos, implying an important role of functional telomeres for germ cells undergoing fertilization and early cleavage development
— id: 133649, year: 2002, vol: 249, page: 74, stat: Journal Article,

Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice
Liu, Lin; Keefe, David L
2002 Oct;17(10):2678-2685, Human reproduction
BACKGROUND: The senescence-accelerated mouse (SAM) has been shown to exhibit ageing-associated mitochondrial dysfunction and oxidative stress, and early decline in fertility. METHODS: We compared meiotic progression of germinal vesicle oocytes between young (2-3 months) and old (10-14 months) SAM mice using triple immunostaining and fluorescence microscopy as well as Pol-Scope imaging. RESULTS: At 8-9 h of in-vitro maturation (IVM), most young SAM oocytes (86%, 32/37) were at meiosis I (MI) stage, with chromosomes aligned in the mid-region of MI spindles, whereas disrupted MI spindles and/or chromosome misalignments (45%, 18/40) and a few oocytes (20%, 8/40) with abnormal MII spindles were found in old SAM oocytes. At 15-17 h of IVM, old SAM oocytes, despite errors at MI stage, extruded a first polar body at an incidence of 88% (n = 85), which did not differ from that (92%, n = 106) of young SAM oocytes. However, oocytes from old SAM (64%, 32/50) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to normal MII in most young SAM oocytes (87%, 65/75), showing chromosome alignment at the metaphase plate of MII spindles. Moreover, Pol-Scope imaging non-invasively detected disrupted or absent visible spindles and possibly aberrant chromosome alignment. CONCLUSIONS: Spindle disruption and/or chromosome misalignments at both MI and MII are associated with maternal ageing in the SAM mouse. Our findings also suggest that meiotic division lacks a competent checkpoint for spindle integrity and chromosome alignment during reproductive ageing-associated oocyte senescence
— id: 102011, year: 2002, vol: 17, page: 2678, stat: Journal Article,

Haploidy but not parthenogenetic activation leads to increased incidence of apoptosis in mouse embryos
Liu, Lin; Trimarchi, James R; Keefe, David L
2002 Jan;66(1):204-210, Biology of reproduction
Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase II oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome
— id: 102019, year: 2002, vol: 66, page: 204, stat: Journal Article,

Checkpoint for DNA integrity at the first mitosis after oocyte activation
Liu, Lin; Trimarchi, James R; Smith, Peter J S; Keefe, David L
2002 Jun;62(2):277-288, Molecular reproduction & development
Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed
— id: 102015, year: 2002, vol: 62, page: 277, stat: Journal Article,

Mitochondrial dysfunction leads to telomere attrition and genomic instability
Liu, Lin; Trimarchi, James R; Smith, Peter J S; Keefe, David L
2002 Oct;1(1):40-46, Aging Cell
Mitochondrial dysfunction and oxidative stress have been implicated in cellular senescence, apoptosis, aging and aging-associated pathologies. Telomere shortening and genomic instability have also been associated with replicative senescence, aging and cancer. Here we show that mitochondrial dysfunction leads to telomere attrition, telomere loss, and chromosome fusion and breakage, accompanied by apoptosis. An antioxidant prevented telomere loss and genomic instability in cells with dysfunctional mitochondria, suggesting that reactive oxygen species are mediators linking mitochondrial dysfunction and genomic instability. Further, nuclear transfer protected genomes from telomere dysfunction and promoted cell survival by reconstitution with functional mitochondria. This work links mitochondrial dysfunction and genomic instability and may provide new therapeutic strategies to combat certain mitochondrial and aging-associated pathologies
— id: 102004, year: 2002, vol: 1, page: 40, stat: Journal Article,

Estrogen effects on osmotic regulation of AVP and fluid balance
Stachenfeld, Nina S; Keefe, David L
2002 Oct;283(4):E711-E721, American journal of physiology. Endocrinology & metabolism
To determine estrogen effects on osmotic regulation of arginine vasopressin (AVP) and body fluids, we suppressed endogenous estrogen and progesterone using the gonadotropin-releasing hormone (GnRH) analog leuprolide acetate (GnRHa). Subjects were assigned to one of two groups: 1) GnRHa alone, then GnRHa + estrogen (E, n = 9, 25 +/- 1 yr); 2) GnRHa alone, then GnRHa + estrogen with progesterone (E/P, n = 6, 26 +/- 3). During GnRHa alone and with hormone treatment, we compared AVP and body fluid regulatory responses to 3% NaCl infusion (HSI, 120 min, 0.1 ml. min(-1). kg body wt(-1)), drinking (30 min, 15 ml/kg body wt), and recovery (60 min of seated rest). Plasma [E(2)] increased from 23.9 to 275.3 pg/ml with hormone treatments. Plasma [P(4)] increased from 0.6 to 5.7 ng/ml during E/P and was unchanged (0.4 to 0.6 ng/ml) during E. Compared with GnRHa alone, E reduced osmotic AVP release threshold (275 +/- 4 to 271 +/- 4 mosmol/kg, P < 0.05), and E/P reduced the AVP increase in response during HSI (6.0 +/- 1.3 to 4.2 +/- 0.6 pg/ml at the end of HSI), but free water clearance was unaffected in either group. Relative to GnRHa, pre-HSI plasma renin activity (PRA) was greater during E (0.8 +/- 0.1 vs. 1.2 +/- 0.2 ng ANG I. ml(-1). h(-1)) but not after HSI or recovery. PRA was greater than GnRHa during E/P at baseline (1.1 +/- 0.2 vs. 2.5 +/- 0.6) and after HSI (0.6 +/- 0.1 vs. 1.1 +/- 1.1) and recovery (0.5 +/- 0.1 vs. 1.3 +/- 0.2 ng ANG I. ml(-1). h(-1)). Baseline fractional excretion of sodium was unaffected by E or E/P but was attenuated by the end of recovery for both E (3.3 +/- 0.6 vs. 2.4 +/- 0.4%) and E/P (2.8 +/- 0.4 vs 1.7 +/- 0.4%, GnRHa alone and with hormone treatment, respectively). Fluid retention increased with both hormone treatments. Renal sensitivity to AVP may be lower during E due to intrarenal effects on water and sodium excretion. E/P increased sodium retention and renin-angiotensin-aldosterone stimulation
— id: 102013, year: 2002, vol: 283, page: E711, stat: Journal Article,

Safe and cost effective use of alteplase for the clearance of occluded central venous access devices
Timoney, J P; Malkin, M G; Leone, D M; Groeger, J S; Heaney, M L; Keefe, D L; Klang, M; Lucarelli, C D; Muller, R J; Eng, S L; Connor, M; Small, T N; Brown, A E; Saltz, L B
2002 Apr 1;20(7):1918-1922, Journal of clinical oncology
PURPOSE: To determine whether cryopreserved solutions of the thrombolytic agent alteplase could be used as a safe, effective, and economically reasonable alternative to urokinase in patients presenting with occluded central venous access devices (CVADs). MATERIALS AND METHODS: Alteplase has been reported as an efficacious alternative to urokinase for treatment of occluded CVADs. However, the practicality of using alteplase as the thrombolytic of choice for this indication remained conjectural. To make this approach economically feasible, alteplase was diluted to 1 mg/mL and 2.5-mL aliquots were stored at -20 degrees C until use. A need to confirm that the cryopreserving and thawing of the reconstituted solution did not compromise the safety and efficacy reported from prior trials was recognized. A quality assessment initiative was undertaken to concurrently monitor the safety and efficacy of this approach. Patients presenting with occluded CVADs received a sufficient volume of the thawed alteplase solution to fill the occluded catheter(s). Data, including efficacy, adverse reactions, dwell time, and catheter type, were collected over a 5-month period. RESULTS: One hundred twenty-one patients accounting for 168 attempted clearances were assessable for safety and efficacy. One hundred thirty-six (81%) of the 168 catheter clearance attempts resulted in successful catheter clearance (95% confidence interval, 74% to 86%). No adverse events were reported. CONCLUSION: Cryopreserved 1-mg/mL aliquots of alteplase are safe and effective in the clearance of occluded CVADs when stored at -20 degrees C for 30 days. The ability to cryopreserve alteplase aliquots makes it an economically reasonable alternative to urokinase in the setting of CVAD occlusion
— id: 102016, year: 2002, vol: 20, page: 1918, stat: Journal Article,

Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation
Trimarchi, James R; Liu, Lin; Smith, Peter J S; Keefe, David L
2002 Mar;282(3):C588-C594, American journal of physiology. Cell physiology
Cell shrinkage is an incipient hallmark of apoptosis and is accompanied by potassium release that decreases the concentration of intracellular potassium and regulates apoptotic progression. The plasma membrane K+ channel recruited during apoptosis has not been characterized despite its importance as a potential therapeutic target. Here we provide evidence that two-pore domain K+ (K(2P)) channels underlie K+ efflux during apoptotic volume decreases (AVD) in mouse embryos. These K(2P) channels are inhibited by quinine but are not blocked by an array of pharmacological agents that antagonize other K+ channels. The K(2P) channels are uniquely suited to participate in the early phases of apoptosis because they are not modulated by common intracellular messengers such as calcium, ATP, and arachidonic acid, transmembrane voltage, or the cytoskeleton. A K+ channel with similar biophysical properties coordinates regulatory volume decreases (RVD) triggered by changing osmotic conditions. We propose that K(2P) channels are the pathway by which K+ effluxes during AVD and RVD and that apoptosis co-opts mechanisms more routinely employed for homeostatic cell volume regulation
— id: 102018, year: 2002, vol: 282, page: C588, stat: Journal Article,

Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope
Wang, Wei Hua; Keefe, David L
2002 Nov;78(5):1077-1081, Fertility & sterility
OBJECTIVE: To examine whether spindle morphologic features imaged with the LC-PolScope (Cambridge Research and Instrumentation, Woburn, MA) in living human oocytes matured in vitro can be used to predict chromosome configuration and select oocytes with normal chromosomes. DESIGN: Morphological study. SETTING: Academic IVF clinic. PATIENT(S): Women undergoing oocyte retrieval for ICSI treatment. INTERVENTION(S): Oocytes were examined after in vitro maturation. MAIN OUTCOME MEASURE(S): The study examined meiotic spindle morphologic features and chromosome alignments. RESULT(S): After culture for 22 to 24 hours, 77.1% of oocytes reached metaphase II stage, with 51.9% of oocytes showing birefringent spindles. Confocal microscopy revealed that 71% of oocytes with the birefringent spindles had normal chromosome alignment, and 29% of oocytes with birefringent spindles and all oocytes without birefringent spindles had abnormal microtubule organization and abnormal chromosome alignment. CONCLUSION(S): The spindle images obtained with the PolScope in living human oocytes are coordinate with those in fixed oocytes as imaged by confocal microscopy. Spindle images with the PolScope can be applied to human in vitro fertilization to help predict chromosomally normal oocytes for insemination
— id: 102009, year: 2002, vol: 78, page: 1077, stat: Journal Article,

Spindle observation in living mammalian oocytes with the polarization microscope and its practical use
Wang, Wei-Hua; Keefe, David L
2002 ;4(3):269-276, Cloning & stem cells
The meiotic spindle is crucial for normal chromosome alignment and separation of maternal chromosomes during meiosis. Conventional methods to image spindles rely on fixation and transmission electron microscope or immunofluorescence staining and fluorescence microscope, so they provide limited value to studies of spindle dynamics and human clinical in vitro fertilization. A new orientation-independent polarized light microscope, the LC Polscope, was used to examine the bi-refringent spindles in living mammalian oocytes. It was found that spindles could be imaged with the Polscope in living oocytes in all mammals so far examined, including hamster, mouse, cattle, human, and rat. The first polar body did not accurately predict the spindle location in most metaphase II oocytes. Intracytoplasmic sperm injection (ICSI) could be performed by monitoring spindle position. Studies in humans indicated that, aftr ICSI, higher fertilization and embryonic developmental rates could be achieved in oocytes with than without bi-refringent spindles. Because spindles in most mammalian oocytes are extremely sensitive to slight changes in temperature, maintenance of temperature at 37 degrees C is crucial for normal spindle function. As chromosomes#10; are usually associated with microtubule fibers in the spindles, the position of chromosomes could be indirectly located by imaging spindles. Removing spindles under the Polscope can achieve an enucleation#10; efficiency rate of 100% in mouse oocytes. The Polscope can also be used to examine the spindle dynamics, detect spindle morphology, predict chromosome misalignment, and perform spindle transfer
— id: 102010, year: 2002, vol: 4, page: 269, stat: Journal Article,

Rigorous thermal control during intracytoplasmic sperm injection stabilizes the meiotic spindle and improves fertilization and pregnancy rates
Wang, Wei-Hua; Meng, Li; Hackett, Richard J; Oldenbourg, Rudolf; Keefe, David L
2002 Jun;77(6):1274-1277, Fertility & sterility
OBJECTIVE: To examine the effects of different thermodynamic control systems on the temperature stability of human eggs during in vitro manipulation, with the integrity of meiotic spindles imaged using the LC-PolScope (Cambridge Research & Instrumentation, Inc., Woburn, MA). DESIGN: We performed intracytoplasmic sperm injection (ICSI) and/or imaging of eggs with the temperature regulated by three different systems: thermostated coverslip (system 1), thermostated coverslip combined with objective heater (system 2), and conventional stage warmer (system 3). SETTING: Academic in vitro fertilization clinic. PATIENT(S): Oocytes were aspirated from stimulated ovaries of patients undergoing oocyte retrieval for ICSI. INTERVENTION(S): Measurement of temperature regulation in media surrounding eggs during in vitro manipulation and imaging. MAIN OUTCOME MEASURE(S): Rate of oocytes with spindles, fertilization rates, and clinical pregnancy rates after ICSI. RESULT(S): We imaged spindles in more oocytes with system 2 (81.2%) than with system 1 (61.4%). Spindles could not be imaged for system 3 because of technical limitations. Fertilization rates were significantly higher when oocytes were imaged and used for ICSI with system 2 (78.8%) than with system 1 (56.7%) or system 3 (64.0%). Most importantly, a significantly higher clinical pregnancy rate was observed when oocytes were manipulated with system 2 (51.7%) than with system 1 (25.0%) or system 3 (23.1%). No differences were found in average ages, number of previous cycles, number of eggs, or day 3 FSH or E2 levels among groups. CONCLUSION(S): Imaging meiotic spindles with the PolScope provides an intracellular thermostat during ICSI. Rigorous thermal control during ICSI stabilized spindles and increased the fertilization and clinical pregnancy rates achieved after ICSI. The presence of birefringent spindles in living human eggs served as a monitor for in vitro conditions
— id: 102014, year: 2002, vol: 77, page: 1274, stat: Journal Article,

Circulatory shock
Bogolioubov, A; Keefe, D L; Groeger, J S
2001 Jul;17(3):697-719, Critical care clinics
Patients with malignancy may present with acute circulatory compromise requiring ICU monitoring and care. The clinician must be familiar with a multiplicity of acute and chronic medical conditions common to the general population and also with conditions directly related to cancer or therapy thereof
— id: 102026, year: 2001, vol: 17, page: 697, stat: Journal Article,

Progesterone does not alter osmotic regulation of AVP
Calzone, W L; Silva, C; Keefe, D L; Stachenfeld, N S
2001 Dec;281(6):R2011-R2020, American journal of physiology. Regulatory, integrative, & comparative physiology
To test the hypothesis that progesterone, independent of estrogen, decreases the plasma osmotic threshold for arginine vasopressin (AVP) release and thirst onset, we compared AVP and thirst responses to hypertonic saline infusion (HSI) during administration of oral contraceptives (OCs) containing progesterone (OCP) with responses to infusion of OCs containing progesterone and estrogen (OCEP). Eight women (29 +/- 2 yr) were infused with 3% NaCl (120 min, 0.1 ml. kg body wt(-1). min(-1)) and consumed fluid (90 min, 15 ml/kg body wt) in the early follicular and midluteal phases of a 28-day menstrual cycle and also after 4 wk of OCP and after 4 wk of OCEP in a randomized crossover design. Baseline plasma osmolality (P(osm)) was lower in the luteal phase (280 +/- 1 mosmol/kgH(2)O) and during OCEP (283 +/- 1 mosmol/kgH(2)O) than in the follicular phase (286 +/- 1 mosmol/kgH(2)O, P < 0.05) but was unaffected by OCP (284 +/- 1 mosmol/kgH(2)O). P(osm) remained lower in the follicular phase than in the luteal phase and with OCEP throughout the first 50 min of HSI. The mean abscissal plasma AVP concentration-P(osm) intercept was unaffected by OCP (267 +/- 1 mosmol/kgH(2)O) but was greater in the follicular phase (273 +/- 2 mosmol/kgH(2)O) than in the luteal phase (266 +/- 4 mosmol/kgH(2)O) and with OCEP (268 +/- 2 mosmol/kgH(2)O, P < 0.05). There were no differences in osmotic thresholds for thirst onset across experimental days. Despite the lower osmotic threshold for AVP release during the luteal phase and with OCEP, fluid balance, renal free water clearance, and Na(+) regulation during HSI were unaffected by menstrual phase or OC treatment, indicating a lower osmotic operating point for body water balance. OCP did not affect osmotic AVP regulation, suggesting that progesterone does not affect osmotic fluid regulation through a mechanism independent of estrogen
— id: 102021, year: 2001, vol: 281, page: R2011, stat: Journal Article,

Anthracycline-induced cardiomyopathy
Keefe, D L
2001 Aug;28(4 Suppl 12):2-7, Seminars in oncology
The highly active chemotherapeutic agents doxorubicin, duanorubicin, idarubicin, epirubicin, and mitoxantrone are also associated with acute, largely reversible cardiotoxic effects and a dose-related cardiomyopathy. This cardiomyopathy is characterized by minimal left ventricular enlargement and global systolic dysfunction, usually with associated mild to moderate mitral insufficiency. Historically, this was characterized by myocardial biopsy and radionuclide angiography. More recently, echocardiography has become the most widely available and cost-efficient tool for diagnosis. The precise mechanism of this toxicity has not been fully defined. However, the maximum tolerated cumulative dose can by increased by reducing peak drug levels and concurrent administration of the iron chelator, dexrazoxane. Because anthracycline-induced cardiomyopathy is largely irreversible and cumulative, prevention is the preferred strategy. Monitoring by assessment of left ventricular function by the most reproducible method available as patients approach potentially toxic doses can substantially reduce toxicity. Stress studies before major procedures such as bone marrow or stem cell transplants may be of benefit. This syndrome responds well to conventional therapy for congestive heart failure with angiotensin-converting enzyme inhibitors, digoxin, and diuretics. The beta-blocker carvedilol is often associated with significant improvement in ejection fraction and symptoms and spironolactone is well tolerated and often of benefit. The long-term outlook of the syndrome is much better than previously reported because of advances in therapy and prevention
— id: 102025, year: 2001, vol: 28, page: 2, stat: Journal Article,

Mitochondrial modulation of calcium signaling at the initiation of development
Liu, L; Hammar, K; Smith, P J; Inoue, S; Keefe, D L
2001 Dec;30(6):423-433, Cell calcium
Fertilization triggers cytosolic Ca(2+) oscillations that activate mammalian eggs and initiate development. Extensive evidence demonstrates that Ca(2+) is released from endoplasmic reticulum stores; however, less is known about how the increased Ca(2+) is restored to its resting level, forming the Ca(2+) oscillations. We investigated whether mitochondria also play a role in activation-associated Ca(2+) signaling. Mitochondrial dysfunction induced by the mitochondrial uncoupler FCCP or antimycin A disrupted cytosolic Ca(2+) oscillations, resulting in sustained increase in cytosolic Ca(2+), followed by apoptotic cell death. This suggests that functional mitochondria may participate in sequestering the released Ca(2+), contributing to cytosolic Ca(2+) oscillations and preventing cell death. By centrifugation, mouse eggs were stratified and separated into fractions containing both endoplasmic reticulum and mitochondria and fractions containing endoplasmic reticulum with no mitochondria. The former showed Ca(2+) oscillations by activation, whereas the latter exhibited sustained elevation in cytosolic Ca(2+) but no Ca(2+) oscillations, suggesting that mitochondria take up released cytosolic Ca(2+). Further, using Rhod-2 for detection of mitochondrial Ca(2+), we found that mitochondria exhibited Ca(2+) oscillations, the frequency of which was not different from that of cytosolic Ca(2+) oscillations, indicating that mitochondria are involved in Ca(2+) signaling during egg activation. Therefore, we propose that mitochondria play a crucial role in Ca(2+) signaling that mediates egg activation and development, and apoptotic cell death
— id: 102020, year: 2001, vol: 30, page: 423, stat: Journal Article,

Estrogen and progesterone effects on transcapillary fluid dynamics
Stachenfeld, N S; Keefe, D L; Palter, S F
2001 Oct;281(4):R1319-R1329, American journal of physiology. Regulatory, integrative, & comparative physiology
The purpose of this study was to determine estrogen (E(2)) and progesterone (P(4)) effects on atrial natriuretic peptide (ANP) control of plasma volume (PV) and transcapillary fluid dynamics. To this end, we suppressed reproductive function in 12 women (age 21-35 yr) using a gonadotropin releasing-hormone (GnRH) analog (leuprolide acetate) for 5 wk. During the 5th week, the women either received 4 days of E(2) administration (17beta-estradiol, transdermal patch, 0.1 mg/day) or 4 days of E(2) with P(4) administration (vaginal gel, 90 mg P(4) twice per day). At the end of the 4th and 5th week of GnRH analog and hormone administration, we determined PV (Evans blue dye) and changes in PV and forearm capillary filtration coefficient (CFC) during a 120-min infusion of ANP (5 ng x kg body wt(-1) x min(-1)). Preinfusion PV was estimated from Evans blue dye measurement taken over the last 30 min of infusion based on changes in hematocrit. E(2) treatment did not affect preinfusion PV relative to GnRH analog alone (45.3 +/- 3.1 vs. 45.4 +/- 3.1 ml/kg). During ANP infusion CFC was greater during E(2) treatment compared with GnRH analog alone (6.5 +/- 1.4 vs. 4.9 +/- 1.4 microl. 100 g(-1) x min(-1) mmHg(-1), P < 0.05). The %PV loss during ANP infusion was similar for E(2) and GnRH analog-alone treatments (-0.8 +/- 0.2 and -1.0 +/- 0.2 ml/kg, respectively), indicating the change in CFC had little systemic effect on ANP-related changes in PV. Estimated baseline PV was reduced by E(2)-P(4) treatment. During ANP infusion CFC was approximately 30% lower during E(2)-P(4) (6.0 +/- 0.5 vs. 4.3 +/- 4.3 microl. 100 g(-1) x min(-1) mm Hg(-1), P < 0.05), and the PV loss during ANP infusion was attenuated (-0.9 +/- 0.2 and -0.2 +/- 0.2 ml/kg for GnRH analog-alone and E(2)-P(4) treatments, respectively). Thus the E(2)-P(4) treatment lowered CFC and reduced PV loss during ANP infusion
— id: 102024, year: 2001, vol: 281, page: R1319, stat: Journal Article,

Sex differences in osmotic regulation of AVP and renal sodium handling
Stachenfeld, N S; Splenser, A E; Calzone, W L; Taylor, M P; Keefe, D L
2001 Oct;91(4):1893-1901, Journal of applied physiology (Bethesda)
To determine sex differences in osmoregulation of arginine vasopressin (AVP) and body water, we studied eight men (24 +/- 1 yr) and eight women (29 +/- 2 yr) during 3% NaCl infusion [hypertonic saline infusion (HSI); 120 min, 0.1 ml. kg body wt(-1). min(-1)]. Subjects then drank 15 ml/kg body wt over 30 min followed by 60 min of rest. Women were studied in the early follicular (F; 16.1 +/- 2.8 pg/ml plasma 17beta-estradiol and 0.6 +/- 0.1 ng/ml plasma progesterone) and midluteal (L; 80.6 +/- 11.4 pg/ml plasma 17beta-estradiol and 12.7 +/- 0.7 ng/ml plasma progesterone) menstrual phases. Basal plasma osmolality was higher in F (286 +/- 1 mosmol/kgH(2)O) and in men (289 +/- 1 mosmol/kgH(2)O) compared with L (280 +/- 1 mosmol/kgH(2)O, P < 0.05). Neither menstrual phase nor gender affected basal plasma AVP concentration (P([AVP]); 1.7 +/- 4, 1.9 +/- 0.4, and 2.2 +/- 0.5 pg/ml for F, L, and men, respectively). The plasma osmolality threshold for AVP release was lowest in L (x-intercept, 263 +/- 3 mosmol/kgH(2)O, P < 0.05) compared with F (273 +/- 2 mosmol/kgH(2)O) and men (270 +/- 4 mosmol/kgH(2)O) during HSI. Men had greater P([AVP])-plasma osmolality slopes (i.e., sensitivity) compared with F and L (slopes = 0.14 +/- 0.04, 0.09 +/- 0.01, and 0.24 +/- 0.07 for F, L, and men, respectively, P < 0.05). Despite similar Na+-regulating hormone responses, men excreted less Na+ during HSI (0.7 +/- 0.1, 0.7 +/- 0.1, and 0.5 +/- 0.1 meq/kg body wt for F, L, and men, respectively, P < 0.05). Furthermore, men had greater systolic blood pressure (119 +/- 5, 119 +/- 5, and 132 +/- 3 mmHg for F, L, and men, respectively, P < 0.05) than F and L. Our data indicate greater sensitivity in P([AVP]) response to changes in plasma osmolality as the primary difference between men and women during HSI. In men, this greater sensitivity was associated with an increase in systolic blood pressure and pulse pressure during HSI, most likely due to a shift in the pressure-natriuresis curve
— id: 102023, year: 2001, vol: 91, page: 1893, stat: Journal Article,

Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination
Wang, W H; Meng, L; Hackett, R J; Keefe, D L
2001 Jul;16(7):1464-1468, Human reproduction
BACKGROUND: Birefringent spindles imaged with the Polscope can predict fertilization rates after intracytoplasmic sperm injection (ICSI). The present study examined the development of human oocytes with or without birefringent spindles, imaged with the Polscope before ICSI. METHODS: Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocyte retrieval for ICSI. Spindles were imaged with the Polscope combined with a computerized image analysis system. After imaging and ICSI, oocytes with or without spindles were cultured separately for examination of fertilization and embryo development. A total of 1544 oocytes from 136 cycles were examined with the Polscope and inseminated by ICSI. RESULTS: Spindles were imaged in 82% of oocytes. After ICSI, more oocytes (P < 0.05) with spindles (69.4%) fertilized normally, forming 2 pronuclei, than oocytes without spindles (62.9%). At day 3, more oocytes (P < 0.01) with spindles (66.3%) developed to 4-11 cell stages than oocytes without spindles (55.4%). Significantly more (P < 0.001) oocytes with spindles developed to morula and blastocyst by day 5 (51.1 versus 30.3%) and day 6 (53.2 versus 29.3%) compared with oocytes without spindles. CONCLUSIONS: The results indicate that the presence of a birefringent spindle in human oocytes can predict not only higher fertilization rate, but also higher embryo developmental competence
— id: 102027, year: 2001, vol: 16, page: 1464, stat: Journal Article,

Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy
Wang, W H; Meng, L; Hackett, R J; Odenbourg, R; Keefe, D L
2001 Nov;16(11):2374-2378, Human reproduction
BACKGROUND: Spindles are formed from microtubules and are exquisitely sensitive to changes in temperature. An orientation-independent polarized light microscope, the Polscope, can be used to image spindles in living oocytes allowing analysis of spindle kinetics in the living state. This study examined the effects of cooling on spindle disassembly in living human oocytes and spindle recovery after rewarming. METHODS: Oocytes were imaged continuously with the Polscope during cooling and rewarming. The quantity of microtubules in the spindles was measured by its birefringence using the Polscope. RESULTS: Spindles had completely disassembled by 5 min after cooling and recovered by 20 min after rewarming to 37 degrees C if rewarming started soon after the oocyte's temperature dropped to room temperature. However, when oocytes were cooled and kept at 33, 28 or 25 degrees C for 10 min and then warmed, it was found that warming allowed 5/5, 2/5 and 0/5 oocytes of the spindles to recover respectively. CONCLUSIONS: These results indicate that human meiotic spindles are exquisitely sensitive to alterations in temperature. The maintenance of temperature at 37 degrees C during in-vitro manipulation is important for spindle integrity and, therefore, is likely to be important for normal fertilization and subsequent embryo development
— id: 102022, year: 2001, vol: 16, page: 2374, stat: Journal Article,

The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes
Wang, W H; Meng, L; Hackett, R J; Odenbourg, R; Keefe, D L
2001 Feb;75(2):348-353, Fertility & sterility
OBJECTIVE: To image spindles in living human oocytes and to examine the relation between spindles and fertilization after ICSI. DESIGN: The LC polscope was used to examine spindles in an observational study of living oocytes. SETTING: Academic IVF clinic. PATIENT(S): Women being treated for infertility. INTERVENTION(S): Oocytes retrieved from patients for infertility treatment were examined before ICSI. Aged, unfertilized oocytes after IVF or ICSI were examined with polscope and confocal microscopes to compare the two methods. MAIN OUTCOME MEASURE(S): Spindle structure in living oocytes and fertilization after ICSI. RESULT(S): Spindles could be imaged in 61.4% of oocytes. More oocytes with spindles than oocytes without spindles fertilized normally after ICSI (61.8% vs. 44.2%). Spindles in most aged oocytes were partially or completely disassembled, and only a few microtubules around the chromosomes or dispersed microtubules in the cytoplasm were observed. Confocal images of immunostained spindles were almost identical to polscope images of spindle birefringence. CONCLUSION(S): Spindles in living human oocytes can be imaged by using the polscope. A birefringent spindle in human oocytes may clinically predict the quality and age of oocytes. This method also can be used to monitor spindle position during ICSI
— id: 102029, year: 2001, vol: 75, page: 348, stat: Journal Article,

Bioavailability and bioequivalence: average, population and/or individual
Colburn, W A; Keefe, D L
2000 Jun;40(6):559-560, Journal of clinical pharmacology
— id: 102031, year: 2000, vol: 40, page: 559, stat: Journal Article,

Cardiovascular emergencies in the cancer patient
Keefe, D L
2000 Jun;27(3):244-255, Seminars in oncology
Cardiovascular emergencies in oncology patients include all of the usual cardiac problems, as well as complications of cancer and its therapy. Pericardial effusions and tamponade, cardiac masses, and extrinsic compression of the heart and great vessels by tumor masses, or fluid collections may all occur. Certain tumors may secrete mediators that are directly toxic to the heart; for example, catecholamines are secreted by pheochromocytomas and serotonin is secreted by carcinoid tumors. Tumors can also cause arrhythmias due to the mediators they secret or to direct mechanical irritation of the heart or pericardium. Cancer therapy is also associated with cardiac emergencies. Perioperative myocardial ischemia or infarction, as well as arrhythmias, may complicate surgery. Pericardial effusions and tamponade can follow surgery, radiation, or chemotherapy. Chemotherapy with anthracyclines, mitoxantrone, and trastuzumab may prompt acute and chronic heart failure. 5-Fluorouracil causes coronary spasm in some patients, leading to angina, myocardial infarction, arrhythmias, and/or sudden death. Cyclophosphamide, particularly in high doses, may produce acute myopericarditis. Radiation may cause acute pericardial disease and late sequelae such as myocardial infarction, acute valvular insufficiency, or effusive constrictive pericarditis. Endocarditis also occurs in cancer patients in association with vascular access devices and immune compromise. This review will discuss each of these complications of cancer and its therapy
— id: 102032, year: 2000, vol: 27, page: 244, stat: Journal Article,

Clinical pharmacology of telmisartan:
Keefe, D L
2000 Dec;40(12 Pt 1):1311-1311, Journal of clinical pharmacology
— id: 102028, year: 2000, vol: 40, page: 1311, stat: Journal Article,

Cytoplasm mediates both development and oxidation-induced apoptotic cell death in mouse zygotes
Liu, L; Keefe, D L
2000 Jun;62(6):1828-1834, Biology of reproduction
Eggs must be the major locus of reproductive aging in women, because donation of eggs from younger to middle-aged women abrogates the effects of age on fertility. Oxidative stress, mitochondrial dysfunction, and apoptosis are associated with senescence. To develop an animal model of egg senescence, we treated mouse zygotes with 175 microM H(2)O(2) that induced mitochondrial dysfunction and developmental arrest, followed by delayed cell death, consistent with apoptosis. We reconstructed zygotes with nuclei and cytoplasm from treated or untreated zygotes, then followed development and apoptotic cell death in the reconstituted embryos. Pronuclear exchange between untreated, normal zygotes served as nuclear transfer controls. Rates of cleavage and development to morula and blastocysts were significantly lower (P<0.01) in zygotes reconstituted from untreated pronuclei and H(2)O(2)-stressed cytoplasts than those of nuclear transfer controls. Instead, the arrested, reconstituted zygotes displayed TUNEL staining at a similar rate to that of H(2)O(2)-treated controls, suggesting that apoptotic potential could be transferred cytoplasmically. On the other hand, rates of cleavage and development to morula and blastocyst of the reconstituted zygotes, derived from stressed pronuclei and untreated cytoplasm, were significantly increased (P<0.05), compared to those of H(2)O(2)-treated, control zygotes, indicating that healthy cytoplasm could partly rescue pronuclei from oxidative stress. Although oxidation stressed both nuclei and cytoplasm, cytoplasm was more sensitive than nuclei to oxidative stress. It is suggested that cytoplasm, most likely mitochondria, plays a central role in mediating both development and apoptotic cell death induced by oxidative stress in mouse zygotes
— id: 102036, year: 2000, vol: 62, page: 1828, stat: Journal Article,

A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes
Liu, L; Oldenbourg, R; Trimarchi, J R; Keefe, D L
2000 Feb;18(2):223-225, Nature biotechnology
Factors affecting the efficiency of animal cloning remain to be elucidated. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA, which may disrupt functions of the cytoplast, especially mitochondria. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes. Not only might evaluation of the aspirated karyoplast portion inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts
— id: 102039, year: 2000, vol: 18, page: 223, stat: Journal Article,

Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes
Liu, L; Trimarchi, J R; Keefe, D L
2000 Jun;62(6):1745-1753, Biology of reproduction
Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are 'immature,' in contrast to 'mature' mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 microM for 15 min) hydrogen peroxide (H(2)O(2)) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H(2)O(2) induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress
— id: 102037, year: 2000, vol: 62, page: 1745, stat: Journal Article,

Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes
Liu, L; Trimarchi, J R; Oldenbourg, R; Keefe, D L
2000 Jul;63(1):251-258, Biology of reproduction
The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase II oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies
— id: 102033, year: 2000, vol: 63, page: 251, stat: Journal Article,

Estrogen modifies the temperature effects of progesterone
Stachenfeld, N S; Silva, C; Keefe, D L
2000 May;88(5):1643-1649, Journal of applied physiology (Bethesda)
To test the hypothesis that progestin-mediated increases in resting core temperature and the core temperature threshold for sweating onset are counteracted by estrogen, we studied eight women (24 +/- 2 yr) at 27 degrees C rest, during 20 min of passive heating (35 degrees C), and during 40 min of exercise at 35 degrees C. Subjects were tested four times, during the early follicular and midluteal menstrual phases, after 4 wk of combined estradiol-norethindrone (progestin) oral contraceptive administration (OC E+P), and after 4 wk of progestin-only oral contraceptive administration (OC P). The order of the OC P and OC E+P were randomized. Baseline esophageal temperature (T(es)) at 27 degrees C was higher (P < 0.05) in the luteal phase (37.08 +/- 0.21 degrees C) and in OC P (37.60 +/- 0.31 degrees C) but not during OC E+P (37.04 +/- 0.23 degrees C) compared with the follicular phase (36.66 +/- 0.21 degrees C). T(es) remained above follicular phase levels throughout passive heating and exercise during OC P, whereas T(es) in the luteal phase was greater than in the follicular phase throughout exercise (P < 0.05). The T(es) threshold for sweating was also greater in the luteal phase (38.02 +/- 0.28 degrees C) and OC P (38.07 +/- 0.17 degrees C) compared with the follicular phase (37.32 +/- 0.11 degrees C) and OC E+P (37.46 +/- 0.18 degrees C). Progestin administration raised the T(es) threshold for sweating during OC P, but this effect was not present when estrogen was administered with progestin, suggesting that estrogen modifies progestin-related changes in temperature regulation. These data are also consistent with previous findings that estrogen lowers the thermoregulatory operating point
— id: 102038, year: 2000, vol: 88, page: 1643, stat: Journal Article,

A non-invasive method for measuring preimplantation embryo physiology
Trimarchi, J R; Liu, L; Porterfield, D M; Smith, P J; Keefe, D L
2000 Feb;8(1):15-24, Zygote
The physiology of the early embryo may be indicative of embryo vitality and therefore methods for non-invasively monitoring physiological parameters from embryos could improve preimplantation diagnoses. The self-referencing electrophysiological technique is capable of non-invasive measurement of the physiology of individual cells by monitoring the movement of ions and molecules between the cell and the surrounding media. Here we use this technique to monitor gradients of calcium, potassium, oxygen and hydrogen peroxide around individual mouse preimplantation embryos. The calcium-sensitive electrode in self-referencing mode identified a region of elevated calcium concentration (approximately 0.25 pmol) surrounding each embryo. The calcium gradient surrounding embryos was relatively steep, such that the region of elevated calcium extended into the medium only 4 microns from the embryo. By contrast, using an oxygen-sensitive electrode an extensive gradient of reduced dissolved oxygen concentration was measured surrounding the embryo and extended tens of micrometres into the medium. A gradient of neither potassium nor hydrogen peroxide was observed around unperturbed embryos. We also demonstrate that monitoring the physiology of embryos using the self-referencing technique does not compromise their subsequent development. Blastocyts studied with the self-referencing technique implanted and developed to term at the same frequency as did unexamined, control embryos. Therefore, the self-referencing electrode provides a valuable non-invasive technique for studying the physiology and pathophysiology of individual embryos without hindering their subsequent development
— id: 102034, year: 2000, vol: 8, page: 15, stat: Journal Article,

Oxidative phosphorylation-dependent and -independent oxygen consumption by individual preimplantation mouse embryos
Trimarchi, J R; Liu, L; Porterfield, D M; Smith, P J; Keefe, D L
2000 Jun;62(6):1866-1874, Biology of reproduction
The self-referencing electrode technique was employed to noninvasively measure gradients of dissolved oxygen in the medium immediately surrounding developing mouse embryos and, thereby, characterized changes in oxygen consumption and utilization during development. A gradient of depleted oxygen surrounded each embryo and could be detected >50 microm from the embryo. Blastocysts depleted the surrounding medium of 0.6+/-0.1 microM of oxygen, whereas early cleavage stage embryos depleted the medium of only 0.3+/-0.1 microM of oxygen, suggesting a twofold increase in oxygen consumption at the blastocyst stage. Mitochondrial oxidative phosphorylation (OXPHOS) accounted for 60-70% of the oxygen consumed by blastocysts, while it accounted for only 30% of the total oxygen consumed by cleavage-stage embryos. The amount of oxygen consumed by non-OXPHOS mechanisms remained relatively constant throughout preimplantation development. By contrast, the amount of oxygen consumed by OXPHOS in blastocysts is greater than that consumed by OXPHOS in cleavage-stage embryos. The amount of oxygen consumed by one-cell embryos was modulated by the absence of pyruvate from the culture medium. Treatment of one-cell embryos and blastocysts with diamide, an agent known to induce cell death in embryos, resulted in a decline in oxygen consumption, such that the medium surrounding dying embryos was not as depleted of oxygen as that surrounding untreated control embryos. Together these results validate the self-referencing electrode technique for analyzing oxygen consumption and utilization by preimplantation embryos and demonstrate that changes in oxygen consumption accompany important physiological events, such as development, response to medium metabolites, or cell death
— id: 102035, year: 2000, vol: 62, page: 1866, stat: Journal Article,

Noninvasive measurement of potassium efflux as an early indicator of cell death in mouse embryos
Trimarchi, J R; Liu, L; Smith, P J; Keefe, D L
2000 Sep;63(3):851-857, Biology of reproduction
Programmed cell death (apoptosis) occurs in nearly all cell types examined, including mammalian oocytes and embryos, where it may underlie some forms of infertility in humans. Although the molecular machinery participating in apoptosis have been intensely investigated, the accompanying physiological changes have not received similar attention. In this study, a novel electrophysiology technique has been employed to monitor real-time perturbations in the physiology of mouse embryos undergoing apoptosis evoked by hydrogen peroxide, diamide, and staurosporine. Despite differences in their mode of action, these agents evoked a similar early change in cellular physiology; namely, a pronounced, transient, potassium efflux through tetraethylammonium-sensitive potassium channels accompanied by cell shrinkage. Mouse zygotes exposed to 200 microM H(2)O(2) exhibited potassium efflux that elevated the potassium concentration of the media surrounding embryos by 1.4 +/- 0.1 microM. Pretreatment with tetraethylammonium inhibited this increase (0.2 +/- 0.1 microM). Our results indicate that potassium efflux through potassium channels and concurrent cell shrinkage are early indicators of cell death in embryos and that noninvasive measurements of potassium pathophysiology may identify embryos undergoing cell death prior to the manifestation of other morphological or molecular hallmarks of cell death
— id: 102030, year: 2000, vol: 63, page: 851, stat: Journal Article,

Hormone replacement therapy may alleviate sleep apnea in menopausal women: a pilot study
Keefe, D L; Watson, R; Naftolin, F
1999 Fall;6(3):196-200, Menopause
OBJECTIVE: The incidence of sleep apnea syndrome (SAS) in women increases after menopause. Progestins alone do not alleviate SAS in menopausal women. However, progestins may require concomitant estrogen administration and estrogen alone may stimulate breathing during sleep. To test these hypotheses, we studied the effects of estrogen alone and estrogen combined with progestin on SAS in menopausal women, using a prospective, cross-over, inception cohort study. DESIGN: In this pilot study, five women who developed SAS after menopause underwent 2 nights of polysomnography to obtain a baseline, then returned for polysomnography after 3-4 weeks of taking micronized 17 beta-estradiol (E2) and after 10-12 days of taking E2 combined with medroxyprogesterone acetate (E2 + P). Sleep stages were scored according to Rechtshaffen and Kales, frequency and length of apneas were recorded for each subject each night, and the data were analyzed by Student's t test. RESULTS: E2 and E2 + P both reduced the Respiratory Distress Index. E2 also raised the lowest oxygen desaturation associated with apneic episodes. Total minutes of rapid eye movement sleep increased, and the number of waking episodes decreased when the women were taking E2 and E2 + P, as previously reported. CONCLUSIONS: Within 1 month after initiating E2 or E2 + P, SAS was reduced in all patients. The Respiratory Distress Index decreased by 25%, and the addition of progestin brought the SAS reduction to 50% in this pilot study. A randomized study in a large group of patients is justified by the findings of this study. Because SAS increases the risk of cardiovascular disease and fatal accidents, the amelioration of SAS by sex steroid hormones could have significant implications for the health of menopausal women
— id: 102041, year: 1999, vol: 6, page: 196, stat: Journal Article,

Thiol oxidation-induced embryonic cell death in mice is prevented by the antioxidant dithiothreitol
Liu, L; Trimarchi, J R; Keefe, D L
1999 Oct;61(4):1162-1169, Biology of reproduction
The oxidation of cellular sulfhydryl (SH) groups has been implicated in the induction of apoptosis in various types of cells and in the disturbance of the meiotic spindle of murine oocytes during aging. The objective of this study was to determine whether the SH-specific oxidant diamide could inhibit embryo development and induce cell death, and whether the antioxidant dithiothreitol (DTT) could counteract such effects. Exposure of mouse zygotes to diamide for 3 h at 25 or 50 microM (but not 12.5 microM) resulted in cell cycle arrest and cell death with evidence of apoptosis. At higher concentrations (100 or 200 microM), diamide induced necrosis as evidenced by propidium iodide-positive pronuclei within 24 h of treatment. Simultaneous addition of DTT at equimolar concentration prevented these effects. However, when DTT was added later, it was no longer protective. DTT also effectively protected against the thiol-oxidative damage caused by diamide in blastocysts. These results suggest that altering thiol-redox status in zygotes and blastocysts may result in cell cycle arrest, apoptosis, and/or cell death
— id: 102040, year: 1999, vol: 61, page: 1162, stat: Journal Article,

Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova
Pepperell, J R; Kommineni, K; Buradagunta, S; Smith, P J; Keefe, D L
1999 May;60(5):1137-1143, Biology of reproduction
Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i
— id: 102044, year: 1999, vol: 60, page: 1137, stat: Journal Article,

Microinjection of mitochondria into zygotes creates a model for studying the inheritance of mitochondrial DNA during preimplantation development
Rinaudo, P; Niven-Fairchild, T; Buradagunta, S; Massobrio, M; Revelli, A; Keefe, D L
1999 May;71(5):912-918, Fertility & sterility
OBJECTIVE: To determine the effect of mutant mitochondria on preimplantation embryo development and of preimplantation embryo development on the survival of mutant mitochondrial DNA. DESIGN: Laboratory research. SETTING: Academic research laboratory. PATIENT(S): None. INTERVENTION(S): Mutant and wild-type mitochondria, fractionated from tissue obtained from a patient with MELAS syndrome, a mitochondrial disease, were microinjected into mouse zygotes. Control zygotes received either no injection or sham injection. MAIN OUTCOME MEASURE(S): Preimplantation embryo development and survival of mutant mitochondrial DNA as determined by polymerase chain reaction analysis. RESULT(S): After microinjection into zygotes, the MELAS mutation could be identified by polymerase chain reaction until the hatched blastocyst stage of embryo development. The survival of MELAS-injected zygotes, observed for 4 days after injection, did not differ from the survival of zygotes injected with wild-type mitochondria or from the survival of uninjected or sham-injected controls. CONCLUSION(S): It appears that preimplantation embryo development does not screen out mitochondrial DNA mutations introduced into fertilized oocytes, and low levels of mutant mitochondrial DNA do not disrupt early embryo development
— id: 102043, year: 1999, vol: 71, page: 912, stat: Journal Article,

The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes
Silva, C P; Kommineni, K; Oldenbourg, R; Keefe, D L
1999 Apr;71(4):719-721, Fertility & sterility
OBJECTIVE: To evaluate how well polar body location predicts spindle localization and to examine spindle morphology. DESIGN: Randomized, controlled animal study. SETTING: University-affiliated research laboratory. ANIMAL(S): Mature, female golden hamsters. INTERVENTION(S): After superovulation with pregnant mare serum gonadotropin and hCG, metaphase II oocytes were obtained and imaged under digital polarization microscopy. MAIN OUTCOME MEASURE(S): Identify the meiotic spindle in living, unfixed hamster oocytes and determine spindle location relative to the polar body. RESULT(S): Spindles were imaged in 30 oocytes and only in 5 of them could the polar body predict the spindle localization. In the remaining oocytes, the spindles presented a random distribution within the cytoplasm. CONCLUSION(S): These data show that the polar body location is not an accurate predictor for meiotic spindle location in mammalian oocytes
— id: 102045, year: 1999, vol: 71, page: 719, stat: Journal Article,

Physiological variability of fluid-regulation hormones in young women
Stachenfeld, N S; DiPietro, L; Kokoszka, C A; Silva, C; Keefe, D L; Nadel, E R
1999 Mar;86(3):1092-1096, Journal of applied physiology (Bethesda)
We tested the physiological reliability of plasma renin activity (PRA) and plasma concentrations of arginine vasopressin (P[AVP]), aldosterone (P[ALD]), and atrial natriuretic peptide (P[ANP]) in the early follicular phase and midluteal phases over the course of two menstrual cycles (n = 9 women, ages 25 +/- 1 yr). The reliability (Cronbach's alpha >/=0.80) of these hormones within a given phase of the cycle was tested 1) at rest, 2) after 2.5 h of dehydrating exercise, and 3) during a rehydration period. The mean hormone concentrations were similar within both the early follicular and midluteal phase tests; and the mean concentrations of P[ALD] and PRA for the three test conditions were significantly greater during the midluteal compared with the early follicular phase. Although Cronbach's alpha for resting and recovery P[ANP] were high (0.80 and 0.87, respectively), the resting and rehydration values for P[AVP], P[ALD], and PRA were variable between trials for the follicular (alpha from 0.49 to 0.55) and the luteal phase (alpha from 0.25 to 0. 66). Physiological reliability was better after dehydration for P[AVP] and PRA but remained low for P[ALD]. Although resting and recovery P[AVP], P[ALD], and PRA were not consistent within a given menstrual phase, the differences in the concentrations of these hormones between the different menstrual phases far exceeded the variability within the phases, indicating that the low within-phase reliability does not prevent the detection of menstrual phase-related differences in these hormonal variables
— id: 102046, year: 1999, vol: 86, page: 1092, stat: Journal Article,

Effects of oral contraceptives on body fluid regulation
Stachenfeld, N S; Silva, C; Keefe, D L; Kokoszka, C A; Nadel, E R
1999 Sep;87(3):1016-1025, Journal of applied physiology (Bethesda)
To test the hypothesis that estrogen reduces the operating point for osmoregulation of arginine vasopressin (AVP), thirst, and body water balance, we studied nine women (25 +/- 1 yr) during 150 min of dehydrating exercise followed by 180 min of ad libitum rehydration. Subjects were tested six different times, during the early-follicular (twice) and midluteal (twice) menstrual phases and after 4 wk of combined [estradiol-norethindrone (progestin), OC E + P] and 4 wk of norethindrone (progestin only, OC P) oral contraceptive administration, in a randomized crossover design. Basal plasma osmolality (P(osm)) was lower in the luteal phase (281 +/- 1 mosmol/kgH(2)O, combined means, P < 0.05), OC E + P (281 +/- 1 mosmol/kgH(2)O, P < 0.05), and OC P (282 +/- 1 mosmol/kgH(2)O, P < 0. 05) than in the follicular phase (286 +/- 1 mosmol/kgH(2)O, combined means). High plasma estradiol concentration lowered the P(osm) threshold for AVP release during the luteal phase and during OC E + P [x-intercepts, 282 +/- 2, 278 +/- 2, 276 +/- 2, and 280 +/- 2 mosmol/kgH(2)O, for follicular, luteal (combined means), OC E + P, and OC P, respectively; P < 0.05, luteal phase and OC E + P vs. follicular phase] during exercise dehydration, and 17beta-estradiol administration lowered the P(osm) threshold for thirst stimulation [x-intercepts, 280 +/- 2, 279 +/- 2, 276 +/- 2, and 280 +/- 2 mosmol/kgH(2)O for follicular, luteal, OC E + P, and OC P, respectively; P < 0.05, OC E + P vs. follicular phase], without affecting body fluid balance. When plasma 17beta-estradiol concentration was high, P(osm) was low throughout rest, exercise, and rehydration, but plasma arginine vasopressin concentration, thirst, and body fluid retention were unchanged, indicating a lowering of the osmotic operating point for body fluid regulation
— id: 102042, year: 1999, vol: 87, page: 1016, stat: Journal Article,

Do attitudes toward disclosure in donor oocyte recipients predict the use of anonymous versus directed donation?
Greenfeld, D A; Greenfeld, D G; Mazure, C M; Keefe, D L; Olive, D L
1998 Dec;70(6):1009-1014, Fertility & sterility
OBJECTIVE: To compare the demographic and psychological characteristics of oocyte recipients and to determine whether the issue of disclosure about the use of a donor is a correlate of the decision to use an anonymous or directed donor. DESIGN: Cross-sectional study. SETTING: University teaching hospital. PATIENT(S): Ninety consecutive recipients of donated oocytes (64 of whom used anonymous donors and 26 of whom used directed donors). INTERVENTION(S): Pretreatment psychosocial evaluation. MAIN OUTCOME MEASURE(S): Recipient opinions and attitudes regarding the choice of donor type and disclosure to others as determined through a semistructured interview. RESULT(S): There were no statistically significant differences with regard to demographic characteristics between recipients who used anonymous and directed donors. There were statistically significant differences between the groups with regard to the issue of disclosure. Recipients who used directed donors were more likely to have told others about using an oocyte donor and were more likely to indicate that they intended to inform the child about the nature of his or her conception. CONCLUSION(S): Oocyte recipients who use known donors differ significantly from those who use anonymous donors with regard to the issue of disclosure to others. Further studies are needed to determine the causal direction of this relation
— id: 102047, year: 1998, vol: 70, page: 1009, stat: Journal Article,

Lack of increased cardiac toxicity with sequential doxorubicin and paclitaxel
Hudis C; Riccio L; Seidman A; Baselga J; Currie V; Fennelly D; Gilewski T; Lebwohl D; Moynahan M; Raptis G; Surbone A; Sklarin N; Yao TJ; Keefe D; Norton L
1998 ;16(2):67-71, Cancer investigation
— id: 38037, year: 1998, vol: 16, page: 67, stat: Journal Article,

Reproductive aging is an evolutionarily programmed strategy that no longer provides adaptive value
Keefe, D L
1998 Aug;70(2):204-206, Fertility & sterility
— id: 102049, year: 1998, vol: 70, page: 204, stat: Journal Article,

Characterization of oxygen and calcium fluxes from early mouse embryos and oocytes
Porterfield, D M; Trimarchi, J R; Keefe, D L; Smith, P J
1998 Oct;195(2):208-209, Biological bulletin
— id: 102048, year: 1998, vol: 195, page: 208, stat: Journal Article,

Aging and infertility in women
Keefe, D L
1997 Dec;80(12):403-405, Medicine & health, Rhode Island
— id: 102050, year: 1997, vol: 80, page: 403, stat: Journal Article,

Dissociation between intracellular calcium elevation and development of human oocytes treated with calcium ionophore
Rinaudo, P; Pepperell, J R; Buradgunta, S; Massobrio, M; Keefe, D L
1997 Dec;68(6):1086-1092, Fertility & sterility
OBJECTIVE: To develop an acceptable model system to study calcium activation of human oocytes. DESIGN: Study of oocyte development and intracellular calcium [Ca]i dynamics of activated oocytes. SETTING: Research center affiliated with infertility service. MAIN OUTCOME MEASURE: Morphologic evidence of meiotic maturation and cell division under high-power Hoffman optics with an inverted microscope. Meiotic maturation was determined by the number of polar bodies or the presence of a pronucleus, and cell division was determined by evidence of a cleavage furrow or presence of blastomeres. To monitor the effect of calcium ionophore on [Ca]i levels, oocytes were incubated with fura-2 (2 microM) for 30 minutes and [Ca]i was determined by rationing the emission fluorescence (510-nm long-pass filter) during simultaneous excitation at 340 and 380 nm with a microspectrofluorimeter. RESULT(S): All oocytes loaded with fura-2 and then exposed to ionophore exhibited an isolated elevation of [Ca]i, followed by prompt return to baseline levels. None of the oocytes showed signs of cleavage or of meiotic maturation after treatment with calcium ionophore. CONCLUSION(S): Human oocytes activated with calcium ionophore A23187 or ionomycin exhibited elevated [Ca]i but remained resistant to subsequent meiotic maturation and cleavage. Our results differ from some reports of parthenogenetic activation of human oocytes. These differences may result from different activation protocols or culture conditions. Because none of the 126 oocytes cleaved after the activation protocols used in these experiments, this approach should provide an ethically acceptable model system to study calcium dynamics in human oocytes
— id: 102051, year: 1997, vol: 68, page: 1086, stat: Journal Article,

Who is responsible for clinical pharmacology?
Colburn, W A; Keefe, D L
1996 May;36(5):383-385, Journal of clinical pharmacology
— id: 102054, year: 1996, vol: 36, page: 383, stat: Journal Article,

Transvaginal uterine cervical dilation with fluoroscopic guidance: preliminary results in patients with infertility
Dickey, K W; Zreik, T G; Hsia, H C; Eschelman, D J; Keefe, D L; Olive, D L; Pollak, J S; Rosenblatt, M; Glickman, M G
1996 Aug;200(2):497-503, Radiology
PURPOSE: To assess efficacy of uterine cervical dilation performed with fluoroscopic guidance to treat patients with infertility who have cervical stenosis, false channels within the endocervical canal, or both. MATERIALS AND METHODS: Fifteen patients in whom infertility was diagnosed were referred because the uterine lumen could not be accessed. Three of the patients had endometriosis. With fluoroscopic guidance, the cervix was cannulated and the endocervical canal was dilated with an angioplasty balloon or with dilators. Five patients underwent simultaneous fallopian tube recanalization. Five of 15 patients who underwent dilation subsequently underwent in vitro fertilization for embryo transfer (IVF-ET) or intrauterine insemination. RESULTS: Four patients became pregnant. Of those four, one underwent IVF-ET and one underwent intrauterine insemination. Two patients became pregnant spontaneously. In the five patients who underwent IVF-ET or intrauterine insemination and in the remaining eight patients, the cervix could be easily cannulated up to 7 months after dilation. CONCLUSION: Dilation of the uterine cervix may provide options for treatment in selected patients with infertility. The effect of dilation on patients with other sequelae of cervical obstruction such as endometriosis remains uncertain
— id: 102053, year: 1996, vol: 200, page: 497, stat: Journal Article,

Dependency on progesterone in woman with self-diagnosed premenstrual syndrome
Keefe, D L; Sarrel, P
1996 Apr 27;347(9009):1182-1182, Lancet
— id: 102055, year: 1996, vol: 347, page: 1182, stat: Journal Article,

Fluoroscopically Guided Cervical Dilatation in Patients with Infertility
Zreik TG; Dickey KW; Keefe DL; Glickman MG; Olive DL
1996 Aug;3(4, Supplement):S56-S56, Journal of the American Association of Gynecologic Laparoscopists
Uterine cervical stenosis of either congenital or acquired etiology is a contributing factor in fertility. In such patients it is technically difficult to traverse the tortuous or stenotic cervical canal, precluding diagnostic procedures such as endometrial biopsy and hysterosalpingography, as well as therapies such as in vitro fertilization and embryo transfer (IVF-ET) or insemination. The standard method of dilatation with successively larger dilators may be difficult and traumatic, causing false channels or perforation of the uterus. Fifteen patients were referred for cervical dilatation because of inability to gain access to the uterine lumen. Under fluoroscopic guidance, the cervix was cannulated and the endocervical canal dilated with an angioplasty balloon. Five women had simultaneous fallopian tube recanalization. Only one woman had mild postoperative vaginal bleeding that subsided spontaneously at 48 hours. No patients experienced pain requiring narcotics, and no infections occurred. Five women conceived, one after IVF-ET, two with intrauterine inseminations, and two spontaneously. In those who did not conceive, the cervix was easily cannulated after the procedure. Cervical dilatation may provide options for treatment that would otherwise not be available to a select group of infertile women with cervical stenosis
— id: 102052, year: 1996, vol: 3, page: S56, stat: Journal Article,

Similarities and differences between anonymous and directed candidates for oocyte donation
Greenfeld, D A; Mazure, C M; Olive, D L; Keefe, D L
1995 Feb;12(2):118-122, Journal of assisted reproduction & genetics
PURPOSE: To compare anonymous and directed candidates for oocyte donation within a single program. METHODS: 75 consecutive candidates for oocyte donation (49 anonymous and 26 directed) were studied using a semistructured interview to collect data on demographics, psychosocial history, motivation, and candidate views on disclosure to a potential child. RESULTS: Donor groups were similar with regard to race, religion and education. Anonymous donors (mean age 27.33 years, SD 4.17) were significantly younger than directed donors (mean age 37.54 years, SD 4.94), (t = -4.83, df = 73, p < 0.001). Marital duration was significantly longer for directed donors (6.92 years, SD 5.64) versus (2.57 years, SD 3.96) for anonymous donors (t = -3.50, df = 38.47, p = .001). Forty-one percent of anonymous and 69% of directed donors had previous pregnancies (X2 = 4.60, p < .05). A greater percentage of anonymous donors (34.7%) felt that the potential child should be informed of his or her origins, compared to 19% of directed donors, but this difference fell short of statistical significance. CONCLUSIONS: There were more similarities than differences among groups of potential donors with the exception of age, marital status, and previous pregnancies. Differing views about disclosure were suggested in both groups with anonymous donors tending to opt for disclosure
— id: 102057, year: 1995, vol: 12, page: 118, stat: Journal Article,

Mitochondrial deoxyribonucleic acid deletions in oocytes and reproductive aging in women
Keefe, D L; Niven-Fairchild, T; Powell, S; Buradagunta, S
1995 Sep;64(3):577-583, Fertility & sterility
OBJECTIVE: To determine whether oocytes from women harbor deletions in mitochondrial DNA (mtDNA) and whether deleted mtDNA is more common in oocytes from older women than oocytes from younger women. DESIGN: A polymerase chain reaction (PCR)-based strategy, which depends on deletions approximating otherwise widely separated primers to demonstrate mtDNA deletions in individual oocytes, was used. SETTING: Yale In Vitro Fertilization Clinic and Laboratory at Yale University School of Medicine. MAIN OUTCOME MEASURES: Primers flanked a region of the mitochondrial genome in which long direct repeated sequence predispose to deletions. The primers identified the 0.5-kb 'common' deletion. Deleted mtDNA was represented by a 0.5-kb band when primers separated by 5 kb were used. Control reactions used primers that amplify mtDNA outside the deletion hotspot. Positive controls included brain and/or muscle from aged individuals, and negative controls included fetal tissue and DNA-free blanks. Nested primers confirmed the specificity of the deleted product. RESULTS: Unfertilized oocytes, muscle, and brain tissue contained PCR products consistent with deleted mtDNA. Fetal tissue lacked the mtDNA deletion product. Deleted mtDNA was detected in single oocytes. Oocytes from older women were more likely to contain deleted mtDNA than oocytes from younger women. CONCLUSION: Deleted mtDNA in unfertilized oocytes may serve as a marker of oocyte senescence
— id: 102056, year: 1995, vol: 64, page: 577, stat: Journal Article,

Estrogen mustard induces cell cycle arrest of human epithelial ovarian cancer cell lines
Nathan, J D; Keefe, D L; Weinstein, M A; Chen, Z; Naftolin, F
1994 Jan-Mar;1(1):97-103, Journal of the Society for Gynecologic Investigation
OBJECTIVE: Pharmacologic disruption of microtubule function may provide effective therapy for advanced epithelial ovarian cancer, as has been observed in clinical trials using taxol. However, the limited availability of taxol and taxol's side effects emphasize a need to develop alternative antimicrotubule agents. Estramustine (EM) inhibits binding of microtubule-associated proteins (MAPs) to microtubules, promotes microtubule disassembly, disrupts spindle formation, and induces metaphase arrest in human prostate carcinoma and glioma cells in culture. We studied the effect of EM on DNA synthesis and on the cell cycle in four human ovarian carcinoma cell lines and examined the cell lines for evidence of MAP-like immunoreactivity. METHODS: The effect of EM on DNA synthesis and on the cell cycle was determined using [3H]thymidine incorporation assays and flow cytometry, respectively. Microtubule-associated protein-like immunoreactivity was determined using monoclonal antibodies directed against MAP 1A, MAP 1B, and MAP 2(2A + 2B) for Western analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: We demonstrated a dose-dependent inhibitory response to EM in BIXLER, DK2NMA, and SKOV3. BIX3 showed a dose-dependent inhibitory response to EM concentrations from 25 micrograms/mL to 100 micrograms/mL, but a stimulatory response at 10 micrograms/mL. Estramustine inhibited exponentially growing cells by causing mitotic arrest with subsequent accumulation of cells in G2/M phase of the cell cycle in all four cell lines. We found MAP 1A, MAP 1B, and MAP 2-like immunoreactivity in all four cells lines studied. CONCLUSIONS: These results are consistent with a MAP-microtubule mechanism of action for EM in ovarian carcinoma cells and provide reason to conduct further study of EM for potential use in the treatment of human epithelial ovarian cancer
— id: 102058, year: 1994, vol: 1, page: 97, stat: Journal Article,

INCREASING HYPOTHALAMIC ARCUATE NUCLEUS GLIAL PEROXIDASE-ACTIVITY IN AGING FEMALE RATS IS REDUCED BY AN ANTIESTROGEN AND A GONADOTROPIN-RELEASING-HORMONE AGONIST
SEIFER, DB; ROAPENA, L; KEEFE, DL; ZHANG, HP; GOODMAN, S; JONES, EE; NAFTOLIN, F
1994 ;1(2):83-90, Menopause
Progressive arcuate glial peroxidase activity is associated with reproductive aging of the female rat. We tested the hypothesis that age-related increase of glial peroxidase activity within the arcuate nucleus and posterior periventricular area is due to endogenous estrogen and could be reduced by an antiestrogen, keoxiphene, or a gonadotropin-releasing hormone super agonist, goserelin acetate (Zoladex), or both. At 3 months of age, 4-5-day regularly cycling female rats were divided into four groups. One group served as a baseline 3-month-old control and was killed by perfusion-fixation followed by serial sectioning of the hypothalamus and staining with 3,3'-diaminobenzidine tetrahydrochloride (DAB). Other groups consisted of animals receiving keoxiphene, goserelin acetate depot (Zoladex), or sham implants for 6 months. Two months after removal of the implants, at 11.25 months of age, the animals were killed by perfusion-fixation, and serial hypothalamic sections were stained with DAB. Sections from each of the four groups were examined in a fixed-size standard test area using a computerized image analyzer to assess the surface density of the DAB reaction. Overall, periventricular peroxidase reaction of the 11.25-month keoxiphene group showed a 63.5% increase in surface density of DAB reaction over the 3-month control group (5,834.6 +/- 101.0 mu m(2) versus 3,566.2 +/- 173.6 mu m(2)), whereas the 11.25-month Zoladex group showed a 42.4% increase in surface density of DAB reaction over the 3-month control group (5,078.5 +/- 114.6 mu m(2) versus 3,566.2 +/- 173.6 mu m(2)). This was in sharp contrast to the 11.25 month sham control group, which showed a 94% increase over the 3-month control group (6,926.6 +/- 121.3 mu m(2) versus 3,566.2 +/- 173.6 mu m(2)), p < 0.0001. The differences in increase of the surface density of DAB in zone of reaction in keoxiphene-treated and Zoladex-treated rats were not equally distributed throughout the periventricular brain: the greatest difference was noted in the anterior arcuate (p < 0.0001), followed by the posterior arcuate (p < 0.001), with no difference noted in the posterior periventricular area. These results demonstrate than an antiestrogen and functional hypogonadotropic hypogonadism induced by a gonadotropin-releasing hormone agonist reduce the increase in hypothalamic glial peroxidase activity in the arcuate nucleus of maturing (aging) female rats but not in the posterior periventricular area. They support the hypothesis that estrogen secreted during the ovarian cycle contributes causally to hypothalamic aging in the female rat and indicate the presence of an estrogen-sensitive anterior region and an estrogen-insensitive posterior periventricular zone of age-dependent glial reaction. The failure of blockade by Zoladex further indicates that hypothalamic aging in the posterior periventricular area may be indifferent to the presence of the ovary. $$:
— id: 103013, year: 1994, vol: 1, page: 83, stat: Journal Article,

Neuron-glial Interactions In The Estrogen-sensitive Zone Of Reaction Of The Rat Hypothalamus
Keefe, DL; Moore, TC; Stein, M; Naftolin, F
Local systems in reproduction New York : Raven Press, 1993,
— id: 5188, year: 1993, vol: , page: 33, stat: Chapter,

Estramustine and estrone analogs rapidly and reversibly inhibit deoxyribonucleic acid synthesis and alter morphology in cultured human glioblastoma cells
Piepmeier, J M; Keefe, D L; Weinstein, M A; Yoshida, D; Zielinski, J; Lin, T T; Chen, Z; Naftolin, F
1993 Mar;32(3):422-430, Neurosurgery
Estramustine is an estradiol-based agent that has been shown to accumulate in human glioma cells, resulting in a concentration-dependent alteration in cell size and shape within minutes and an inhibition of proliferation over 3 to 6 days. We evaluated human glioblastoma cultures with [3H]thymidine incorporation assays to determine estramustine's early effects on deoxyribonucleic acid synthesis in these tumors. Because estramustine shares a common structural motif with other antimicrotubule drugs, we synthesized four A-ring conjugates of estrone that contained a carbamate moiety but lacked nitrogen mustard. These analogs were examined by [3H]thymidine incorporation and compared with vinblastine. Greater than 70% inhibition of [3H]thymidine incorporation occurred within 1 hour of treatment with estramustine at 10(-5) mol/L, which increased to 80% inhibition at 4 hours. Ethyl carbamate JE208 was nearly as effective as estramustine in inhibiting deoxyribonucleic acid synthesis, and both were more effective than vinblastine. The inhibitory effects of estramustine and estrone analogs were reversible; vinblastine was not reversible. Although estramustine and JE208 induced similar antiproliferative and morphological changes in glioblastoma cells that persisted for at least 4 days, there was a modest recovery of morphology and thymidine incorporation with JE208 after prolonged treatment. The common findings with estramustine and JE208 suggest that these agents may have a similar mechanism of action and form the basis for the investigation of new agents that may rapidly and reversibly inhibit glioblastoma
— id: 102059, year: 1993, vol: 32, page: 422, stat: Journal Article,

Quantification of hormone pulsatility via an approximate entropy algorithm
Pincus, S M; Keefe, D L
1992 May;262(5 Pt 1):E741-E754, American journal of physiology
Approximate entropy (ApEn) is a recently developed formula to quantify the amount of regularity in data. We examine the potential applicability of ApEn to clinical endocrinology to quantify pulsatility in hormone secretion data. We evaluate the role of ApEn as a complementary statistic to widely employed pulse-detection algorithms, represented herein by ULTRA, via the analysis of two different classes of models that generate episodic data. We conclude that ApEn is able to discern subtle system changes and to provide insights separate from those given by ULTRA. ApEn evaluates subordinate as well as peak behavior and often provides a direct measure of feedback between subsystems. ApEn generally can distinguish systems given 180 data points and an intra-assay coefficient of variation of 8%. This suggests ApEn as applicable to clinical hormone secretion data within the foreseeable future. Additionally, the models analyzed and extant clinical data are both consistent with episodic, not periodic, normative physiology
— id: 102061, year: 1992, vol: 262, page: E741, stat: Journal Article,

Estrogen-effects On The Organization Of The Rat Brain Astrocytes Within The Hypothalamic Arcuate Nucleus Contain Estrogen-sensitive Peroxidase Activity And May Mediate Synaptic Plasticity In The Rat
Keefe DL; Michelson DS; Naftolin F
The new biology of steroid hormones New York : Raven Press, 1991,
— id: 5820, year: 1991, vol: , page: 265, stat: Chapter,

Astrocytes within the hypothalamic arcuate nucleus contain estrogen-sensitive peroxidase, bind fluorescein-conjugated estradiol, and may mediate synaptic plasticity in the rat
Keefe, D L; Michelson, D S; Lee, S H; Naftolin, F
1991 Apr;164(4):959-966, American journal of obstetrics & gynecology
Estrogen treatment induces synaptic plasticity accompanied by damaged structures and aggregates of peroxidase in astrocytes in the hypothalamic arcuate nucleus of the rat. Synaptic plasticity also occurs within the arcuate nucleus after physiologic surges of estrogen. Although the function of estrogen-induced peroxidase is unclear at present, in other systems peroxidase can generate free radicals by catalyzing the oxidation of some molecules, including estrogen. Because free radicals underlie remodeling in a number of tissues, estrogen-induced free radicals could mediate synaptic remodeling within the arcuate nucleus. Although they contain estrogen-inducible peroxidase, astrocytes do not contain estrogen receptors as measured by conventional techniques, suggesting that estrogen-inducible peroxidase arises from some novel mechanism. Estrogen could induce peroxidase within receptor-deficient astrocytes by binding to receptors in neurons and stimulating the release of some factor that interacts with astrocytes. Alternatively, estrogen could act directly on astrocytes in the absence of estrogen receptors. Although astrocytes in the hypothalamus of the rat do not contain classical nuclear estrogen receptors, they do bind fluorescein-conjugated estradiol in extranuclear sites. The distribution of fluorescein-conjugated estradiol binding within the hypothalamus overlaps that of peroxidase-rich astrocytes, and double labeling reveals many cells with the stellate morphology of astrocytes, containing both peroxidase and fluorescein-conjugated estradiol binding. However, because peroxidase and fluorescein-conjugated estradiol always occupy different compartments of the cell, the fluorescein-conjugated estradiol is not binding to peroxidase
— id: 102063, year: 1991, vol: 164, page: 959, stat: Journal Article,

A cholinergic antagonist, mecamylamine, blocks the phase-shifting effects of light on the circadian rhythm of locomotor activity in the golden hamster
Keefe, D L; Earnest, D J; Nelson, D; Takahashi, J S; Turek, F W
1987 Feb 17;403(2):308-312, Brain research
Despite the well known role of the light-dark cycle in the entrainment of circadian rhythms, very little is known about the neurochemical events that mediate the effects of light on the mammalian circadian clock. Recent anatomical and pharmacological data support the hypothesis that acetylcholine may be involved in relaying light-dark information from the retina to, or within, the circadian clock of rodents. If acetylcholine is required for this response, it should be possible to block the phase-shifting effects of a light pulse by blocking cholinergic neurotransmission. To test this possibility, hamsters free-running in constant darkness received an intraventricular injection of the anticholinergic drug, mecamylamine (450 micrograms), 10 min before being exposed to a 5-min pulse of light known to induce sub-maximal phase shifts in the circadian rhythm of wheel-running behavior. Compared to vehicle-injected control animals, mecamylamine treatment blocked or reduced both the phase-advancing and phase-delaying effects of light. These results support the hypothesis that acetylcholine is involved in mediating the phase-shifting effects of light on the mammalian circadian clock
— id: 102064, year: 1987, vol: 403, page: 308, stat: Journal Article,

Circadian time keeping processes in mammalian reproduction
Keefe, D L; Turek, F W
1985 ;7:346-400, Oxford reviews of reproductive biology
— id: 102066, year: 1985, vol: 7, page: 346, stat: Journal Article,