Faculty Bibliography

Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.

Embryonic stem cells (ESCs) provide a valuable in vitro model for testing toxicity of chemicals and environmental contaminants including cigarette smoke. Mouse ESCs were acutely or chronically exposed to smoke components, cigarette smoke condensate (CSC), or cadmium, an abundant component of CSC, and then evaluated for their self-renewal, apoptosis, DNA damage and telomere function. Acute exposure of ESCs to high dose of CSC or cadmium increased DNA damage and apoptosis. Yet, ESCs exhibited a remarkable capacity to recover following absence of exposure. Chronic exposure of ESCs to low dose of CSC or cadmium resulted in shorter telomeres and DNA damage. Together, acute exposure of ESCs to CSC or cadmium causes immediate cell death and reduces pluripotency, while chronic exposure of ESCs to CSC or cadmium leads to DNA damage and telomere shortening. Notably, a sub-proportion of ESCs during passages is selected to resist to smoke-induced oxidative damage to telomeres.

Accumulation of double-stranded DNA breaks and inhibition of DNA repair contributes to reproductive aging by diminishing ovarian reserves in mice and women.

Female germline or oogonial stem cells transiently residing in fetal ovaries are analogous to the spermatogonial stem cells or germline stem cells (GSCs) in adult testes where GSCs and meiosis continuously renew. Oocytes can be generated in vitro from embryonic stem cells and induced pluripotent stem cells, but the existence of GSCs and neo-oogenesis in adult mammalian ovaries is less clear. Preliminary findings of GSCs and neo-oogenesis in mice and humans have not been consistently reproducible. Monkeys provide the most relevant model of human ovarian biology. We searched for GSCs and neo-meiosis in ovaries of adult monkeys at various ages, and compared them with GSCs from adult monkey testis, which are characterized by cytoplasmic staining for the germ cell marker DAZL and nuclear expression of the proliferative markers PCNA and KI67, and pluripotency-associated genes LIN28 and SOX2, and lack of nuclear LAMIN A, a marker for cell differentiation. Early meiocytes undergo homologous pairing at prophase I distinguished by synaptonemal complex lateral filaments with telomere perinuclear distribution. By exhaustive searching using comprehensive experimental approaches, we show that proliferative GSCs and neo-meiocytes by these specific criteria were undetectable in adult mouse and monkey ovaries. However, we found proliferative nongermline somatic stem cells that do not express LAMIN A and germ cell markers in the adult ovaries, notably in the cortex and granulosa cells of growing follicles. These data support the paradigm that adult ovaries do not undergo germ cell renewal, which may contribute significantly to ovarian senescence that occurs with age. Stem Cells 2013;31:2538-2550.

In this Journal in 1972, 100 leaders in obstetrics and gynecology published a compelling statement that recognized the legalization of abortion in several states and anticipated the 1973 Supreme Court decision in Roe v Wade. They projected the numbers of legal abortions that likely would be required by women in the United States and described the role of the teaching hospital in meeting that responsibility. They wrote to express their concern for women's health in a new legal and medical era of reproductive control and to define the responsibilities of academic obstetrician-gynecologists. Forty years later, 100 professors examine the statement of their predecessors in light of medical advances and legal changes and suggest a further course of action for obstetrician gynecologists.

Abstract Background: There have been recent reports that lactational history is associated with long-term women's health benefits. Most of these studies are epidemiological. If particular cardiometabolic changes that occur during lactation ultimately influence women's health later is unknown. Methods: Seventy-one healthy women participated in a prospective postpartum study that provided an opportunity to study anthropometric, endocrine, immune, and behavioral variables across time. Variables studied were heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), C-reactive protein, body mass index (BMI), perceived stress, and hormones. A cohort of women without a change in breastfeeding (N=22) or formula feeding (N=23) group membership for 5 months was used for analysis of effects of feeding status. The data were analyzed using factorial repeated measures analysis of variance and analysis of covariance. Results: SBP and HR declined across the postpartum and were significantly lower in breastfeeding compared to formula feeding mothers (p<0.05). These differences remained statistically significant when BMI was added to the model. Other covariates of income, stress, marital status, and ethnicity were not significantly associated with these variables over time. DBP was also lower, but the significance was reduced by the addition of BMI as a covariate. Stress also was lower in breastfeeders, but this effect was reduced by the addition of income as a covariate. Conclusions: These data suggest that there are important physiological differences in women during months of breastfeeding. These may have roles in influencing or programming later risks for a number of midlife diseases.

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.

STUDY QUESTION: Does resveratrol counteract age-associated infertility in a mouse model of reproductive aging? SUMMARY ANSWER: Long-term-oral administration of resveratrol protects against the reduction of fertility with reproductive aging in mice. WHAT IS KNOWN ALREADY: Loss of oocytes and follicles and reduced oocyte quality contribute to age-associated ovarian aging and infertility. Accumulation of free radicals with age leads to DNA mutations, protein damage, telomere shortening, apoptosis and accelerated ovarian aging. Increasing evidence shows that resveratrol, enriched in certain foods, for example red grapes and wine, has anti-tumor and anti-aging effects on somatic tissues by influencing various signaling pathways, including anti-oxidation, as well as activating Sirt1 and telomerase. We investigated the potential of resveratrol to stave off ovarian aging in the inbred C57/BL6 mouse model. STUDY DESIGN, SIZE, DURATION: Young C57/BL6 females (aged 2-3 months) were fed with resveratrol added to drinking water at 30 mg/l (providing approximately 7.0 mg/kg/day) for 6 or 12 months, and the fertility and ovarian functions were compared among mice treated with or without resveratrol, and young mice served as reproductive controls. Experiments were repeated three times, with an average of 25 females randomly allocated to each treatment group for each repeat. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reproductive performance of female mice was determined by litter size, ovarian follicles and oocyte quantity and quality, and compared with age-matched controls. The impact of resveratrol on telomeres and telomerase activity, and expression of genes associated with cell senescence also was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Young mice fed with resveratrol for 12 months retained the capacity to reproduce, while age-matched controls produced no pups. Consistently, mice fed with resveratrol for 12 months exhibited a larger follicle pool than controls (P < 0.05). Furthermore, telomerase activity, telomere length and age-related gene expression in ovaries of mice fed with resveratrol resembled those of young mice, but differed (P < 0.05) from those of age-matched old mice. Resveratrol improved (P < 0.05) the number and quality of oocytes, as evidenced by spindle morphology and chromosome alignment. Also, resveratrol affected embryo development in vitro in a dose-dependent manner. LIMITATIONS, REASONS FOR CAUTION: The doses of resveratrol and the experimental conditions used by different research groups have varied considerably, and the dosage influences both the effectiveness and toxicity of resveratrol. Fine-tuning the dosage of resveratrol likely will optimize its anti-aging effects on ovarian function. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide a proof of principle of the fertility-sparing effect of resveratrol in female mice. Although depletion of the ovarian reserve of high-quality oocytes also contributes to increased infertility with reproductive aging in women, the data obtained using a mouse model may not extrapolate directly to human reproduction, and more extensive research is needed if any clinic trials are to be attempted. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by MOST of China National Basic Research Program (grant number: 2010CB94500 and 2012CB911200). The authors have no competing interests to declare.

BACKGROUND Ovarian aging is associated with declining numbers and quality of oocytes and follicles. Oxidative stress by reactive oxygen species (ROS) contributes to somatic aging in general, and also has been implicated in reproductive aging. Telomere shortening is also involved in aging, and telomeres are particularly susceptible to ROS-induced damage. Previously, we have shown that antioxidant N-acetyl-l-cysteine (NAC) effectively rescues oocytes and embryos from ROS-induced telomere shortening and apoptosis in vitro. Using mice as models, we tested the hypothesis that reducing oxidative stress by NAC might prevent or delay ovarian aging in vivo. METHODS Initially, young females were treated with NAC in drinking water for 2 months and the quality of fertilized oocytes and early embryo development were evaluated. Next, young mice 1-1½ months old were treated for 1 year with NAC added in drinking water, and their fertility was analyzed starting at 6 months, as indicated by litter size, oocyte number and quality. The ovaries were also examined for telomere activity and length and the expression of selected genes related to aging and DNA damage. RESULTS Short-term treatment of mice for 2 months with NAC demonstrated that NAC improved the quality of fertilized oocytes and early embryo development. Mice treated with a long-term low concentration (0.1 mM) of NAC had increased litter sizes at the ages of 7-10 months compared with age-matched controls without NAC treatment. NAC also increased the quality of the oocytes from these older mice. Moreover, the expression of sirtuins was increased, telomerase activity was higher and telomere length was longer in the ovaries of mice treated with NAC compared with those of the control group. CONCLUSIONS These data suggest that appropriate treatment with the antioxidant NAC postpones the process of oocyte aging in mice.