Regulation Mechanism Of Follistatin On Bovine Granulosa Cells

Follistatin (FST), known as activin-binding protein, regulates the folliculogenesis of the female mammal animals. However, few work has done in study the function of follistatin as an autocrine glycoprotein in regulating the activities of granulosa cells and the development of follicles. In order to understanding the regulatory function of the follistatin to the bovine granulosa cells, two FST RNA interfere plasmids were constructed in the present study. Follistatin gene expressions were partially silenced at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors in bovine granulosa cells (bGCs). After transfecting the bGCs with the FST RN Ai vectors,(1) the cell cycle was determined by flow cytometry. The cell had shown the tendency in arresting in G1phase, furthermore, the protein expression level of p21, a potent cyclin-dependent kinase inhibitor, was up-regulated significantly.(2) the cell apoptosis was determined by flow cytometry. Both the early and late phase of cell apoptosis were increased, these results were further verified by determining the expressions of apoptosis related genes in mRNA and protein levels. The expressions of caspase-3and Bcl2-associated X protein (BAX) were up-regulated and the B-cell leukemia/lymphoma2(Bcl-2) were down-regulated significantly.(3) the steriodogenesis of the bGCs were measured by ELISA. We found that the estradiol (E2) production was significantly suppressed while the progesterone production did not change.(4) the mRNA expressions of all the activin receptor types â…  (ACVR1) and â…¡ (ACVR2Aå'ŒACVR2B) and inhibin a subunit (INHA) in bGCs were up-regulated significantly, however, the expressions of both the inhibin β subunits (INHBA and INHBB) did not change.These findings suggested the direct cross talk of follistatin, and caspase3-dependent apoptosis through Bcl-2/Bax gene family in the development of bGCs. In addition, up regulation of activin receptors and intrusion in the cell cycle phases and the steriodogenesis were further involved in this regulation.