Disruption of follistatin by RNAi increases apoptosis, arrests S-phase of cell cycle and decreases estradiol production in bovine granulosa cells

• Published in: Animal reproduction science Vol. 155; pp. 80 - 88
• Main Authors: Chong, Zhenlu, Dong, Ping, Riaz, Hasan, Shi, Lei, Yu, Xue, Cheng, Ying, Yang, Liguo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2015
• Subjects: Activins - metabolism; Animals; Apoptosis; Apoptosis - physiology; B-lymphocytes; caspase-3; cattle; Cattle - physiology; Cell cycle; Cells, Cultured; culture media; estradiol; Estradiol - metabolism; Female; follicle-stimulating hormone; Follistatin; Follistatin - genetics; Follistatin - metabolism; genes; Granulosa cells; Granulosa Cells - metabolism; inhibin; leukemia; lymphoma; messenger RNA; Plasmids; progesterone; Progesterone - metabolism; receptors; RNA Interference; RNAi; S Phase Cell Cycle Checkpoints - physiology; secretion; steroidogenesis; transcription (genetics); transfection; translation (genetics); Granulosa cells; RNAi; Follistatin; Cell cycle; Apoptosis
• ISSN: 0378-4320; 1873-2232
• DOI: 10.1016/j.anireprosci.2015.02.003

•Knockdown of FST arrested granulosa cells at S-phase cell cycle.•Follistatin may participate in caspase3-dependent apoptosis through Bcl2/Bax gene family.•Knockdown of FST suppressed estradiol level without affecting progesterone production. Follistatin (FST), a local regulator of gonadal functions is a powerful inhibitor of follicle stimulating hormone (FSH) secretion. In the present study, the expression of FST was partially silenced at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors in bovine granulosa cells (bGCs). The results showed that transfection with FST-1 and FST-2 vectors significantly down-regulated mRNA and protein expressions of follistatin by 51% (P=0.0093) and 72% (P=0.0078) respectively. After down-regulation of FST in bGCs, cell cycle was arrested at S-phase (9.2±0.6 vs 12.5±0.2, P=0.0055), and apoptosis was significantly (21.3±2.7 vs 13.9±2.5, P=0.0051) increased. These findings were further verified by down-regulation of protein level of B-cell leukemia/lymphoma 2 (Bcl2, P=0.0423), and up-regulation of caspase-3 (P=0.0362), p21 (P=0.0067) and mRNA levels of Bcl2-associated X protein (Bax, P=0.041). Knockdown of FST in bGCs significantly increased activin A concentration in culture medium, while level of estradiol (E2) was suppressed without affecting progesterone production. In addition, mRNA levels of all activin receptor subtypes [activin receptor types I (ACRI) and II (ACRIIA and ACRIIB)] and inhibin α-subunit were augmented (P<0.05) without altering both inhibin β-subunits. These findings suggest that follistatin may participate in caspase3-dependent apoptosis through Bcl2/Bax gene family in bovine GCs, whereas, activin and its receptors are associated with its regulation. Activin-induced up-regulation of inhibin-α subunit in bGCs seems to be involved in the regulation of steroidogenesis.