TY - JOUR
T1 - Disruption of follistatin by RNAi increases apoptosis, arrests S-phase of cell cycle and decreases estradiol production in bovine granulosa cells
AU - Chong, Zhenlu
AU - Dong, Ping
AU - Riaz, Hasan
AU - Shi, Lei
AU - Yu, Xue
AU - Cheng, Ying
AU - Yang, Liguo
N1 - Publisher Copyright:
© 2015 Elsevier B.V.
PY - 2015/4/1
Y1 - 2015/4/1
N2 - 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.
AB - 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.
KW - Apoptosis
KW - Cell cycle
KW - Follistatin
KW - Granulosa cells
KW - RNAi
UR - http://www.scopus.com/inward/record.url?scp=84926167419&partnerID=8YFLogxK
U2 - 10.1016/j.anireprosci.2015.02.003
DO - 10.1016/j.anireprosci.2015.02.003
M3 - Article
C2 - 25728901
AN - SCOPUS:84926167419
SN - 0378-4320
VL - 155
SP - 80
EP - 88
JO - Animal Reproduction Science
JF - Animal Reproduction Science
ER -