Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a FACS-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically-active cells that exhibit a gene expression profile consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and spontaneously generate 35–50 µm oocytes, as determined by morphology, gene expression and attainment of haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, like adult mice, possess rare mitotically-active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

These results suggest accumulation of BPA in early fetuses and significant exposure during the prenatal period, which must be considered in evaluating the potential for human exposure to endocrine-disrupting chemicals.

Abstract. This study was performed to investigate the serum levels of bisphenol A (BPA), an endocrine disruptor, in women with ovarian dysfunction and obesity. Fasting serum samples were obtained from 19 non-obese and 7 obese women with normal menstrual cycles: 7 patients with hyperprolactinemia, 21 patients with hypothalamic amenorrhea, and 13 non-obese and 6 obese patients with polycystic ovary syndrome (PCOS). BPA was measured by an enzyme-linked immunosorbent assay. BPA was detected in all human sera. Serum BPA concentrations were significantly higher in both non-obese and obese women with polycystic ovary syndrome (1.05 ± 0.10 ng/ml, 1.17 ± 0.16 ng/ml; p<0.05, respectively) and obese normal women (1.04 ± 0.09 ng/ml, p<0.05) compared with those in non-obese normal women (0.71 ± 0.09 ng/ ml). There was no difference among women with hyperprolactinemia, women with hypothalamic amenorrhea, and nonobese normal women. There were significant positive correlations between serum BPA and total testosterone (r = 0.391, p<0.001), free testosterone (r = 0.504, p<0.001), androstenedione (r = 0.684, p<0.001), and DHEAS (r = 0.514, p<0.001) concentrations in all subjects. These findings show that there is a strong relationship between serum BPA and androgen concentrations, speculatively due to the effect of androgen on the metabolism of BPA.

Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at ∼1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

Abstract.Bisphenol A (BPA), a monomer of plastic used in consumer products, is abundant in the environment and enters the body by ingestion or adsorption.We developed a cell based transcription assay system using a reporter gene under the transcriptional control of estrogen receptor a (ERa) as well as ER~3 and performed chloramphenicol acetyltransferase (CAT) assay on HeLa cells transfected with either human ERa cDNA or ER/I cDNA to characterize the estrogenic effect of BPA. Estrogenic activity of BPA was detectable at a concentration of 10-9 M and the activity increased in a dose dependent manner between concentrations of 10_9 M and 10-6 M of BPA for both ERa and ER. The estrogenic activity of 17 j3-estradiol at a concentration of 10-g M was almost compatible with that of BPA at the concentration of 10-6 M of BPA for ERa as well as ER. CAT activity was significantly decreased when cells expressing ERa were incubated with 10-6 M of BPA and 10-8 M of 17j3-estradiol while the activity was essentially the same for ER~3 in the same condition, indicating that BPA exhibits only agonistic action for ERJ3 whereas it has dual actions as an agonist and antagonist of estrogen for ERa. These results indicates that BPA exerts its effects in ER subtype specific way, thus suggesting that the mode of action of endocrine disruptors are more complex than thought.

An age-dependent acceleration of apoptosis occurs in female germ cells (oocytes), and this requires communication between the oocyte and its surrounding somatic (cumulus) cells. Here we show in aged mice that ceramide is translocated from cumulus cells into the adjacent oocyte and induces germ cell apoptosis that can be prevented by sphingosine-1-phosphate. Trafficking of ceramide requires gap junction-dependent communication between the cumulus cells and the oocyte as well as intact lipid rafts. Further, the occurrence of the elevated incidence of apoptosis in oocytes of aged females is concomitant with an enhanced sensitivity of the oocyte to a spike in cytosolic ceramide levels, as well as increased bax mRNA and Bax protein levels. Thus, the force driving the age-related increase in female germ cell death is multifactorial, but changes in the intercellular trafficking of ceramide, along with hypersensitivity of oocytes to ceramide, are key factors in this process.

The menopausal transition in human females, which is driven by a loss of cyclic ovarian function, occurs around age 50 and is thought to underlie the emergence of an array of health problems in aging women. Although mice do not undergo a true menopause, female mice exhibit ovarian failure long before death because of chronological age and subsequently develop many of the same age-associated health complications observed in postmenopausal women. Here we show in mice that inactivation of the proapoptotic Bax gene, which sustains ovarian lifespan into advanced age, extends fertile potential and minimizes many age-related health problems, including bone and muscle loss, excess fat deposition, alopecia, cataracts, deafness, increased anxiety, and selective attention deficit. Further, ovariectomy studies show that the health benefits gained by aged females from Bax deficiency reflect a complex interplay between ovary-dependent and -independent pathways. Importantly, and contrary to popular belief, prolongation of ovarian function into advanced age by Bax deficiency did not lead to an increase in tumor incidence. Thus, the development of methods for postponing ovarian failure at menopause may represent an attractive option for improving the quality of life in aging females.aging ͉ apoptosis ͉ menopause ͉ ovary A lthough the process of aging has been attributed in part to increased apoptosis in various tissues (1), animal models lacking cell death-regulatory genes are rarely subjected to longitudinal aging studies. In females, one of the first organ systems to fail with age is the reproductive axis, which in humans is a principal contributing factor to the onset of menopause (2). Ovarian failure, whether natural (age-related) or induced as a consequence of pathological insults, is driven by depletion of ovarian follicles, the hormonally active support structures that house oocytes, through apoptosis (3). As a consequence, dramatic changes in the endocrine activity of the female gonads ensue, which are thought to underlie the emergence of a spectrum of physiological and psychological health complications in aging females (2). Although it has been shown that aged female mice transplanted with ovaries of young donors live longer than nontransplanted control or ovariectomized females (4), it remains unclear whether postmenopausal health complications arise as a direct result of ovarian failure or simply reflect the aging process.Despite the fact that mice do not undergo a true menopause, female mice exhaust their follicle pool long before death because of chronological age (5), similar to that seen in humans (6). Further, aging female mice exhibit many of the same health complications associated with postmenopausal life in women (see below). In a previous study, we reported that oocyte and follicle loss in female mice lacking the proapoptotic Bax protein is attenuated, leading to a dramatic prolongation of ovarian function into very advanced age (7). To better understand the consequences of Bax deficiency and sustained ovar...

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