Disruption of follistatin by RNAi increases apoptosis, arrests S-phase of cell cycle and decreases estradiol production in bovine granulosa cells.
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.
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
Zhenlu
Z
Chong
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
Ping
P
Dong
Department of Bio sciences, COMSATS Institute of Information Technology, Sahiwal 57000, Pakistan.
Hasan
H
Riaz
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
Lei
L
Shi
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
Xue
X
Yu
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
Ying
Y
Cheng
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China. Electronic address: yangliguo2006@qq.com.
Liguo
L
Yang
D028341:Activins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002417:Cattle / Q000502:physiology*; D002478:Cells, Cultured; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D038681:Follistatin / Q000235:genetics / Q000378:metabolism*; D006107:Granulosa Cells / Q000378:metabolism; D010957:Plasmids; D011374:Progesterone / Q000378:metabolism; D034622:RNA Interference; D059807:S Phase Cell Cycle Checkpoints / Q000502:physiology*
D038681:Follistatin; D028341:Activins; D011374:Progesterone; D004958:Estradiol
Apoptosis; Cell cycle; Follistatin; Granulosa cells; RNAi