Abstract
The 78-kilodalton glucose-regulated protein (GRP78) is a molecular chaperone that assists in protein assembly, folding, and translocation. Recently, our laboratory reported that GRP78 regulates the expression of luteinizing hormone-human chorionic gonadotropin receptor (LHR) in the early stage of corpus luteum formation. In this study, we investigated whether microRNAs (miRNAs), which post-transcriptionally regulate mRNA, are involved in the regulation mechanism of GRP78 in the ovary. A miRNA microarray was performed to analyze the overall miRNA expression profile, and the results indicated that 44 miRNAs were expressed highly after ovulation was induced. The results from a bio-informative database analysis and in vitro granulosa cell culture studies led us to focus on rno-miR-376a for further analysis. In both in vivo and in vitro studies, rno-miR-376a levels increased 12 h after human chorionic gonadotropin (hCG) administration. To elucidate whether rno-miR-376a induced mRNA destabilization or translational repression of GRP78, rno-miR-376a was transfected into cultured granulosa cells, resulting in decreased GPR78 protein levels without an alteration in GRP78 mRNA levels. To confirm that rno-miR-376a binds to GRP78 mRNA, we cloned the 3′-end of GRP78 mRNA (nucleotides 2439–2459) into a reporter vector that contained a Renilla luciferase coding region upstream of the cloning site. The luciferase assays revealed that rno-miR-376a bound to the 3′-end of GRP78 mRNA. From these data, we conclude that rno-miR-376a potentially negatively regulates GRP78 protein expression through translational repression at an early stage transition from the follicular phase to luteinization.
Highlights
In females, ovulation—the release of a mature and fertilizable oocyte—is an essential process in the establishment of pregnancy
MiRNA expression in rat ovaries In both in vivo and in vitro experiments, we previously demonstrated that GRP78 mRNA levels peaked while luteinizing hormone-human chorionic gonadotropin receptor (LHR) mRNA was downregulated by human chorionic gonadotropin (hCG) and that the increase of LHR protein levels was dependent on the increment of GRP78 protein [10]
Our recent finding suggests that GRP78 is involved in restoring LHR expression following the down-regulation induced by ovulation [10]
Summary
Ovulation—the release of a mature and fertilizable oocyte—is an essential process in the establishment of pregnancy. Ovulation provokes a dramatic transformation of ovulated follicle to form the corpus luteum, which in turn induces synthesis of progesterone in order to sustain pregnancy. This indicates that granulosa cells are under stress, which has led to ovulation being considered an inflammation-like phenomenon [4,5]. Our laboratory determined that the 78-kilodalton glucose-regulated protein (GRP78), an ER-associated molecular chaperone that assists in proper protein folding to execute primary protein maturation in the ER [8,9], is involved in the recovery of LHR after its down-regulation [10]. GRP78 is known be an important molecule for the upregulation of LHR, the precise mechanism underlying the regulation of GRP78 expression in the ovary has not been fully elucidated
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