MicroRNA 21 Blocks Apoptosis in Mouse Periovulatory Granulosa Cells1

MicroRNAs (miRNAs) play important roles in many developmental processes, including cell differentiation and apoptosis. Transition of proliferative ovarian granulosa cells to terminally differentiated luteal cells in response to the ovulatory surge of luteinizing hormone (LH) involves rapid and pronounced changes in cellular morphology and function. MicroRNA 21 (miR-21, official symbol Mir21) is one of three highly LH-induced miRNAs in murine granulosa cells, and here we examine the function and temporal expression of Mir21 within granulosa cells as they transition to luteal cells. Granulosa cells were transfected with blocking (2′-O-methyl) and locked nucleic acid (LNA-21) oligonucleotides, and mature Mir21 expression decreased to one ninth and one twenty-seventh of its basal expression, respectively. LNA-21 depletion of Mir21 activity in cultured granulosa cells induced apoptosis. In vivo, follicular granulosa cells exhibit a decrease in cleaved caspase 3, a hallmark of apoptosis, 6 h after the LH/human chorionic gonadotropin surge, coincident with the highest expression of mature Mir21. To examine whether Mir21 is involved in regulation of apoptosis in vivo, mice were treated with a phospho thioate-modified LNA-21 oligonucleotide, and granulosa cell apoptosis was examined. Apoptosis increased in LNA-21-treated ovaries, and ovulation rate decreased in LNA-21-treated ovaries, compared with their contralateral controls. We have examined a number of Mir21 apoptotic target transcripts identified in other systems; currently, none of these appear to play a role in the induction of ovarian granulosa cell apoptosis. This study is the first to implicate the antiapoptotic Mir21 (an oncogenic miRNA) as playing a clear physiologic role in normal tissue function

RUNX2 Transcription Factor Regulates Gene Expression in Luteinizing Granulosa Cells of Rat Ovaries

The LH surge promotes terminal differentiation of follicular cells to become luteal cells. RUNX2 has been shown to play an important role in cell differentiation, but the regulation of Runx2 expression and its function in the ovary remain to be determined. The present study examined 1) the expression profile of Runx2 and its partner CBFβ during the periovulatory period, 2) regulatory mechanisms of Runx2 expression, and 3) its potential function in the ovary. Runx2 expression was induced in periovulatory granulosa cells of human and rodent ovaries. RUNX2 and core binding factor-β (CBFβ) proteins in nuclear extracts and RUNX2 binding to a consensus binding sequence increased after human chorionic gonadotropin (hCG) administration. This in vivo up-regulation of Runx2 expression was recapitulated in vitro in preovulatory granulosa cells by stimulation with hCG. The hCG-induced Runx2 expression was reduced by antiprogestin (RU486) and EGF-receptor tyrosine kinase inhibitor (AG1478), indicating the involvement of EGF-signaling and progesterone-mediated pathways. We also found that in the C/EBPβ knockout mouse ovary, Runx2 expression was reduced, indicating C/EBPβ-mediated expression. Next, the function of RUNX2 was investigated by suppressing Runx2 expression by small interfering RNA in vitro. Runx2 knockdown resulted in reduced levels of mRNA for Rgc32, Ptgds, Fabp6, Mmp13, and Abcb1a genes. Chromatin immunoprecipitation analysis demonstrated the binding of RUNX2 in the promoter region of these genes, suggesting that these genes are direct downstream targets of RUNX2. Collectively, the present data indicate that the LH surge-induced RUNX2 is involved in various aspects of luteal function by directly regulating the expression of diverse luteal genes