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Abolition of the LH-induced ERK1/2 pathway leads to dramatic changes in gene expression in granulosa cells, subsequently abrogating ovulation. Here we explored whether sustained ERK1/2 signaling beyond immediate-early hours of the LH surge is important for ovulation in mice. First, we examined the effect of inhibition of ERK1/2 activity at 4 h after hCG stimulation on ovulation in superovulated immature mice. Treatment with the ERK1/2 pathway inhibitor PD0325901 at 4 h post-hCG disrupted follicular rupture without altering cumulus expansion, oocyte meiotic maturation and luteinization. Profiling the expression pattern of genes of the RSK family of ERK1/2 signal mediators revealed that RSK3, but not other isoforms, was induced by hCG treatment. Further, RSK3-knockout mice were sub-fertile with reduced ovulation rate and smaller litter size compared to WT mice. Given that PD0325901 inhibits all mediators of ERK1/2 signaling, we chose to evaluate the gene expression underlying deficient follicular rupture in ERK1/2 inhibited mice. We found that inhibition of ERK1/2 signaling at 4 h post-hCG resulted in an imbalance in the expression of genes involved in extracellular matrix degradation and leukocyte infiltration necessary for follicular rupture. In conclusion, our data demonstrate that sustained ERK1/2 signaling during ovulation is not required for cumulus expansion, oocyte meiotic maturation and luteinization, but is required for follicular rupture.

Successful ovulation requires the actions of gonadotropins along with those mediated by growth factors binding to their receptor tyrosine kinases (RTKs). There are several growth factors such as epidermal growth factor family ligands and interleukins that play a role during ovulation initiated by the preovulatory surge of luteinizing hormone (LH). The aim of this project was to analyze growth factor signaling pathways induced by LH in mouse granulosa cells. Immature female mice were treated with equine chorionic gonadotropin (eCG) followed 48 hr later by human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. We performed protein array analysis where we identified higher phosphorylation of insulin-like growth factor 1 receptor (IGF1R), the fibroblast growth factor receptor 2 (FGFR2) and ephrin receptor B1 (EPHB1) in granulosa cells at 4 hr post-hCG compared to 0 hr hCG (p < 0.05). We report both a significant increase in transcript abundance (p < 0.05) and the phosphorylation level (p < 0.05) of the IGF1R in granulosa cells at hCG4h. The mRNA abundance of the Fgfr2 and Ephb1 receptors remained unaltered upon hCG treatment. Nonetheless, transcript abundance of the fibroblast growth factor 2 (Fgf2) ligand was elevated at hCG4h (p < 0.01). Based on these results we conclude that the preovulatory LH surge activates signaling pathways of IGF1R through increase in the expression of the Igf1r gene in granulosa cells of ovulating follicles in mice. The LH surge also appears to activate FGFR2 IIIc and EPHB1 signaling, although further investigation is required., (© 2018 Wiley Periodicals, Inc.)

Mitogen-activated protein kinase 3/1 (Mapk3/1) pathway is critical for LH signal transduction during ovulation. However, the mechanisms remain incompletely understood. We hypothesized that Mapk pathway regulates ovulation through transcriptional regulation of ovulatory genes. To test this hypothesis we used immature mice superovulated with equine and human chorionic gonadotropins (eCG and hCG) and PD0325901, to inhibit hCG-induced Mapk3/1 activity. Mice received either the inhibitor PD0325901 (25 μg/g, i.p.) or vehicle at 2h before hCG stimulation. Administration of the inhibitor abolished Mapk3/1 phosphorylation in granulosa cells. While vehicle-treated mice ovulated normally, there were no ovulations in inhibitor-treated mice. First, we analyzed gene expression in granulosa cells at 0h, 1h and 4h post-hCG. There was expected hCG-driven increase in mRNA abundance of many ovulation-related genes including Ptgs2 in vehicle-treated granulosa cells, but not (P<0.05) in inhibitor-treated group. There was also reduced mRNA and protein abundance of the transcription factor, early growth response 1 (Egr1) in inhibitor-treated granulosa cells. We then used GRMO2 cell-line to test if Egr1 is recruited to promoter of Ptgs2 followed by chromatin immunoprecipitation with either Egr1 or control antibody. Enrichment of the promoter regions in immunoprecipitants of Egr1 antibody indicated that Egr1 binds to the Ptgs2 promoter. We then knocked down Egr1 expression in mouse primary granulosa cells using siRNA technology. Treatment with Egr1-siRNA inhibited Egr1 transcript accumulation, which was associated with reduced expression of Ptgs2 when compared to control-siRNA treated granulosa cells. These data demonstrate that transient inhibition of LH-stimulated MAPK3/1 activity abrogates ovulation in mice. We conclude that Mapk3/1 regulates ovulation, at least in part, through Egr1 and its target gene, Ptgs2 in granulosa cells of ovulating follicles in mice.

The fatty acid binding protein 6 (Fabp6) is commonly regarded as a bile acid binding protein found in the distal portion of the small intestine and has been shown to be important in maintaining bile acid homeostasis. Previous studies have also reported the presence of Fabp6 in human, rat and fish ovaries, but the significance of Fabp6 in this organ is largely unknown. Therefore, we surveyed murine ovaries for Fabp6 gene expression and evaluated its role in ovarian function using mice with whole body Fabp6 deficiency. Here we show that the Fabp6 gene is expressed in granulosa and luteal cells of the mouse ovary. Treatment with gonadotropins stimulated Fabp6 gene expression in large antral follicles. The ovulation rate in response to superovulatory treatment in Fabp6-deficient mice was markedly decreased compared to wildtype (C57BL/6) mice. The results of this study suggest that expression of Fabp6 gene in granulosa cells serves an important and previously unrecognized function in fertility.

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., (© 2014 Wiley Periodicals, Inc.)

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 β (Cebpβ) increased in granulosa cells from 1 to 7 h post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpβ to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpβ, 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 Cebpβ. 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.