Abstract
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.
Highlights
Ovulation is a multi-gene, multi-step process involving complex signaling pathways, which facilitates synchronization of oocyte maturation and cumulus expansion with that of follicular rupture
Some of the important signaling pathways through which luteinizing hormone (LH) brings about ovulatory events are cAMP/Protein Kinase A (PKA) pathway, Mitogen-activated protein kinase 3/1 (Mapk3/1; ERK1/2) pathway and phosphatidylinositide 3-kinases (PI3K) pathway [1,2,3,4]
In the present study we show that transient pharmacological inhibition of Mapk3/1 activity using a single dose of the Map2k inhibitor, PD0325901 during preovulatory follicle maturation resulted in anovulation
Summary
Ovulation is a multi-gene, multi-step process involving complex signaling pathways, which facilitates synchronization of oocyte maturation and cumulus expansion with that of follicular rupture. It is unequivocal that preovulatory luteinizing hormone (LH) surge initiates these processes through remarkable changes in gene expression program of granulosa cells within ovulating follicles. Granulosa cells from Mapk3/1 KO mice showed altered expression of hundreds of LH regulated genes [5], but which transcription factors act as mediators of their signals have not been completely identified [6]. It was reported that 19% of the LH-driven genes were regulated in granulosa cells of both Mapk3/1 and Cebpa/b conditional KO mice at 4h hCG [6]. This indicates that the rest 81% Mapk3/1-dependent genes are regulated by transcription factors other than Cebpa/b, which are yet to be identified
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