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.
We recently reported that a targeted disruption of the gene encoding the aromatic hydrocarbon receptor (AHR) in mice reduces fetal oocyte apoptosis, leading to a 2-fold increase in the number of primordial follicles endowed at birth. Although the identity of the natural ligand(s) for the AHR remains to be unequivocally established, these findings indicate that the level of AHR function is an important physiological determinant of how many oocytes will succumb to apoptosis during development of the fetal ovaries. Furthermore, the AHR is a well established receptor for polycyclic aromatic hydrocarbons (PAHs), a class of ubiquitous environmental chemicals known to cause the death of female germ cells in fetal life. Given the possibility that the AHR serves as a key mediator of fetal oocyte death under both physiological and pathological situations, this study was conducted to more fully examine the impact of PAH-AHR interaction on fetal ovarian germ cells. In addition, experiments were designed to begin identification of the mechanism(s) by which ligand activation of the AHR induces prenatal oocyte depletion after transplacental exposure of fetuses to PAHs in vivo. Embryonic d 13.5 murine fetal ovaries cultured in the presence of PAHs exhibited a high level of germ cell loss via apoptosis that was prevented by the selective AHR antagonist, alpha-napthoflavone (ANF). Immunohistochemical analysis revealed an accumulation of Bax protein in germ cells of fetal ovaries exposed to PAHs before the onset of apoptosis, whereas cotreatment with ANF inhibited the induction of Bax expression. The functional importance of increased Bax expression to the cytotoxic response was confirmed by findings that fetal ovarian germ cell loss caused by in utero exposure of wild-type female fetuses to PAHs was not observed in Bax-deficient female fetuses exposed in parallel. We conclude that a central role exists for the AHR in transducing the actions of PAHs in fetal ovarian germ cells, and that the proapoptotic Bcl-2 family member, Bax, is a required mediator of PAH-induced oocyte loss in female fetuses exposed to PAHs in utero.
Objective: Adrenomedullin (AM) has diverse functions and is expressed in a variety of tissues. This study was conducted to investigate the expression of AM in the human ovary and its effect on progesterone production by human granulosa lutein cells. Design and Methods: Follicular fluid and blood samples were obtained at the time of oocyte retrieval from patients undergoing in vitro-fertilization cycles. Concentrations of AM in follicular fluid and plasma were measured by RIA. Granulosa cells were isolated from follicular fluid and expression of AM mRNA was examined by RT-PCR. Granulosa lutein cells were cultured in vitro and secretion of AM by those cells was determined by immunoprecipitation followed by PAGE. Immunohistochemical staining with human ovaries was carried out, using a specific antibody to AM. Furthermore, the effect of AM on progesterone production by cultured granulosa lutein cells was studied.Results: Concentrations of AM in follicular fluid collected just before ovulation were significantly higher than those in the plasma (P<0.01). AM mRNA was expressed in granulosa cells at the preovulatory stage. Cultured granulosa lutein cells secreted immunoreactive AM. With immunohistochemical staining, it was revealed that AM was most abundantly expressed in granulosa lutein cells at the midluteal phase. No appreciable staining for AM was observed in granulosa cells in primordial and preantral follicles, whereas immunolocalization of AM was noted in granulosa cells of dominant follicles although it was not as prominent as in granulosa lutein cells at the midluteal phase. Furthermore, addition of AM to cultured granulosa lutein cells augmented progesterone secretion in a dose-dependent manner. Conclusions: These results suggest that AM is transcribed and secreted in human granulosa lutein cells as a local factor to enhance progesterone production by those cells.
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