Faculty Bibliography

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

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

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

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

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

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

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

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

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