X-linked lymphocyte regulated gene 5c-like (Xlr5c-like) is a novel target of progesterone action in granulosa cells of periovulatory rat ovaries

Abstract

Progesterone (P4), acting through its nuclear receptor (PGR), plays an essential role in ovulation by mediating the expression of genes involved in ovulation and/or luteal formation. To identify ovulatory specific PGR-regulated genes, a preliminary microarray analysis was performed using rat granulosa cells treated with hCG ± RU486 (PGR antagonist). The transcript most highly down-regulated by RU486 was an EST (Expressed Sequence Tag) sequence (gb: BI289578.1) that matches with predicted sequence for Xlr5c-like mRNA. Since nothing is known about Xlr5c-like, we first characterized the expression pattern of Xlr5c-like mRNA in the rat ovary. The level of mRNA for Xlr5c-like is transiently up-regulated in granulosa cells of periovulatory follicles after hCG stimulation in PMSG-primed rat ovaries. The transient induction of Xlr5c-like mRNA was mimicked by hCG treatment in cultured granulosa cells from preovulatory ovaries. We further demonstrated that the LH-activated PKA, MEK, PI3K, and p38 signaling is involved in the increase in Xlr5c-like mRNA. The increase in Xlr5c-like mRNA was abolished by RU486. The inhibitory effect of RU486 was reversed by MPA (synthetic progestin), but not by dexamethasone (synthetic glucocorticoid). Furthermore, mutation of SP1/SP3 and PGR response element sites in the promoter region of Xlr5c-like decreased Xlr5c-like reporter activity. RU486 also inhibited Xlr5c-like reporter activity. ChIP assay verified the binding of PGR and SP3 to the Xlr5c-like promoter in periovulatory granulosa cells. Functionally, siRNA-mediated Xlr5c-like knockdown in granulosa cell cultures resulted in reduced levels of mRNA for Snap25, Cxcr4, and Adamts1. Recombinant Xlr5c-like protein expressed using an adenoviral approach was localized predominantly to the nucleus and to a lesser extent to the cytoplasm of rat granulosa cells. In conclusion, this is the first report showing the spatiotemporally regulated expression of Xlr5c-like mRNA by hCG in rat periovulatory ovaries. P4/PGR mediates the LH-induced increase in Xlr5c-like mRNA. In turn, Xlr5c-like is involved in regulating the expression of specific ovulatory genes such as Snap25, Cxcr4, and Adamts1, possibly acting in the nucleus of periovulatory granulosa cells.

The expression of CXCR4 is induced by the luteinizing hormone surge and mediated by progesterone receptors in human preovulatory granulosa cells†

“…Indeed, we found that the levels of both mRNA and protein for CXCR4 were increased by hCG treatment in cultured cells, indicating that hCG-initiated signaling pathways are involved in the upregulation of CXCR4 expression in granulosa cells of human preovulatory follicles. These data are in agreement with previous findings in other species; hCG or forskolin (an activator of adenylate cyclase) increased the levels of mRNA for Cxcr4 in rodent as well as bovine granulosa cell cultures [8,11,29], respectively. Among the LH-induced mediators in preovulatory granulosa cells, the PGR is known to play a critical role in ovulation by regulating the expression of specific ovulatory genes [28].…”

“…in the level of Cxcr4 mRNA in rat and bovine granulosa cell cultures [8,29]. In this study, we first confirmed that cultured human granulosa cells increased the production of progesterone and PGR expression in response to hCG stimulation.…”

“…It is well known that progesterone (P4) and progesterone receptor (PGR) play an essential role in ovulation by regulating the expression of specific genes in ovulatory follicles (reviewed in [28]). Previous studies have shown that P4/PGR plays a role in the upregulation of Cxcr4 mRNA expression in granulosa cells of rats and cows [8,29]. To verify that our cultured cells increase P4 production and PGR expression in response to hCG treatment, we measured the concentration of P4 in culture media and the levels of mRNA for PGR.…”

Genetic and Hormonal Regulation of Egg Formation in the Oviduct of Laying Hens

“…Progesterone along with its cognate receptor regulates the female fertility [41,42]. Progesterone is mainly produced by the granulosa cells of the larger follicles (F1-F3).…”

Expression of follistatin is associated with egg formation in the oviduct of laying hens

“…The gene expression was analyzed using qPCR as described previously (Mishra, Park, Wilson, & Joe, Park, Wilson & Jo, 2015). Specific primer pairs for the detection of each gene were designed using the NCBI Primer‐Blast tool (Table 1).…”