RNAi-mediated knockdown of <i>INHBB</i> increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Abstract

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.

Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients

“…TGF-β2 is a member of retinoblastoma protein and regulates the proliferation of CCs, and CAV1 is regulated by LH, and its decreased expression is associated with the deregulation of granulosa cell differentiation43. In mice, downregulation of INHβB is shown to significantly increase apoptosis and inhibit steroidogenesis in granulosa cells44. And INHβB is overexpressed in PCOS CCs, which could have increased the proliferation and steroidogenesis of CCs.…”

Runx3 transcription factor regulates ovarian functions and ovulation in female mice

“…Inhibin and activin, both produced in granulosa cells, stimulate and inhibit androgen synthesis in theca cells, respectively [25, 26]. Interestingly, a recent report suggests that Inhbb , which is one of the inhibin subunit genes, is involved in the regulation of Cyp11a1 expression in mouse ovaries [27]. IGF1, produced in granulosa cells, regulates androgen production through stimulation of Cyp11a1 and Hsd3b1 expression [23, 28, 29].…”

The role of Melatonin receptor MTNR1A in the action of Melatonin on bovine granulosa cells

“…Granulosa cells (GCs) are critical for normal ovarian function, and synthesize an array of factors required for follicle development. The major role of GCs include production of sex steroids and a number of growth factors thought to interact with the oocyte during its development (M'baye, Hua, Khan, & Yang, ). The proliferation, differentiation, and apoptosis of GCs dictate the growth and maturation of oocytes within the follicle (Chowdhury, Thomas, Zeleznik, & Thompson, ) as well as the follicle itself, both before birth and during the reproductive period (Denkova et al, ).…”