Mechanistic target of rapamycin (MTOR) signaling during ovulation in mice.

A complex network of endocrine/paracrine signals regulates granulosa-cell function in ovarian follicles. Mechanistic target of rapamycin (MTOR) has recently emerged as a master intracellular integrator of extracellular signals and nutrient availability. The objectives of the present study were to characterize the expression pattern and kinase activity of MTOR during follicular and corpus luteum development, and to examine how inhibition of MTOR kinase activity affects preovulatory maturation of ovarian follicles. MTOR expression was constitutive throughout follicular and corpus luteum development. Gonadotropins induced MTOR kinase activity in the ovary, which was inhibited by rapamycin treatment (10 µg/g body weight, intraperitoneal injection). Inhibition of human chorionic gonadotropin (hCG)-induced MTOR activity during preovulatory follicle maturation did not change key events of ovulation. Granulosa cells of rapamycin-treated mice showed reduced MTOR kinase activity at 1 and 4 hr post-hCG and overexpression of hCG-induced ovulation genes at 4 hr post-hCG. Overexpression of these ovulatory genes was associated with hyper-activation of extracellular signal-regulated kinase 1/2 (ERK1/2), which occurred in response to inhibition of MTOR with rapamycin and suggested that MTOR may function as a negative regulator of the mitogen-activated protein kinase (MAPK) pathway. Indeed, simultaneous inhibition of MTOR and ERK1/2 activities during preovulatory follicle maturation caused anovulation. Inhibition of hCG-induced ERK1/2 activity alone suppressed MTOR kinase activity, indicating that MAPK pathway is upstream of MTOR. Thus, normal ovulation appears to be a result of complex interactions between MTOR and MAPK signaling pathways in granulosa cells of ovulating follicles in mice.

Role of leptin receptors in granulosa cells during ovulation.

Leptin is an important hormone influencing reproductive function. However, the mechanisms underpinning the role of leptin in the regulation of reproduction remain to be completely deciphered. In this study, our objective is to understand the mechanisms regulating the expression of leptin receptor (Lepr) and its role in ovarian granulosa cells during ovulation. First, granulosa cells were collected from superovulated mice to profile mRNA expression of Lepr isoforms (LeprA and LeprB) throughout follicular development. Expression of LeprA and LeprB was dramatically induced in the granulosa cells of ovulating follicles at 4 h after human chorionic gonadotropin (hCG) treatment. Relative abundance of both mRNA and protein of CCAAT/enhancer-binding protein ß (Cebpß) increased in granulosa cells from 1 to 7 h post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpß to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpß, which led us to hypothesise that Lepr may play a role during ovulation. To test this hypothesis, we used a recently developed pegylated superactive mouse leptin antagonist (PEG-SMLA) to inhibit Lepr signalling during ovulation. I.p. administration of PEG-SMLA (10 µg/g) to superovulated mice reduced ovulation rate by 65% compared with control treatment. Although the maturation stage of the ovulated oocytes remained unaltered, ovulation genes Ptgs2 and Has2 were downregulated in PEG-SMLA-treated mice compared with control mice. These results demonstrate that Lepr is dramatically induced in the granulosa cells of ovulating follicles and this induction of Lepr expression requires the transcription factor Cebpß. Lepr plays a critical role in the process of ovulation by regulating, at least in part, the expression of the important genes involved in the preovulatory maturation of follicles.