中国农业科学2012，Vol.45Issue(2)：391-398,8.DOI:10.3864/j.issn.0578-1752.2012.02.023
Kisspeptin-10对无血清培养鸡F1级卵泡颗粒细胞孕酮分泌的影响及机制研究
Effect and Mechanism of Kisspeptin-10 on Progesterone Secretion in Granulosa Cells of Chicken F1 Follicles Cultured in Serum-Free Medium
摘要
Abstract
[Objective] The objective of this study was to investigate the effect and mechanism of Kisspeptin-10 (Kp-10) on progesterone (P4) secretion in cultured granulose cells of chicken follicles. [Method] Two-hundred-day ISA laying hens were sacrificed before the expected time to ovulation. The follicles (Fi) were collected and the granulosa cells were isolated and cultured in serum medium for one day, and then cultured in serum-free medium. After one day stabilization in serum-free medium, the granulosa cells were treated with different concentrations of Kp-10 alone or in combination with U73122, EGTA and/or Ca2+ for 24 h, respectively. The media was collected for measuring P4 by radioimmunoassay (RIA). [Result] The cells were determined to be kisspeptin-positive expression with specific antibody against Kp-10. After 24 h treatment, Kp-10 significantly increased the viability of granulosa cells as well as P4 secretion (P<0.05). U73122 (PLC inhibitor) at 0.5 umolL-1 and 2 umol-L-1 blocked the effect of Kp-10 on stimulating P4 secretion markedly (P<0.05), while 2 umolL"' U73122 had no effect on P4 secretion. Verapamil (thecalcium channel blocker) suppressed P4 secretion in a dose-dependent manner, reaching the statistical significance at 100 umol-L'1 dosage (P<0.05). Under low dosage of Verapamil (1 umol-L1) background, 100 nmol-L-1 Kp-10 can still significantly increase P4 secretion (P<0.05). However, under higher dosage of Verapamil (10 or 100 umol-L'1), 100 nmol-L"1 Kp-10 could not reverse the significant decrease of P4 secretion by Verapamil in medium of in vitro cultured granulosa cells. Flow cytometric analysis showed that the level of intracellular Ca24 was consistent with progesterone secretion. P4 secretion induced by Kp-10 was decreased significantly in the presence of EGTA (1 and 5 mmol-L-1), while this effect was converted by adding 1.5 mmol-L-1 Ca2+(P<0.05). [ Conclusion ] Kisspeptin-10 increase P4 secretion in granulosa cells of ISA hens' follicles cultrured in serum-free medium, and the mechanism might be associated with intracellular Ca2* concentration.关键词
kisspeptin-10/钙离子/孕酮/颗粒细胞/伊莎褐蛋鸡Key words
kisspeptin-10/ Ca2+/ progesterone/ granulosa cells/ ISA Brown hens引用本文复制引用
肖蕴祺,黄炎冰,吴晶,倪迎冬,赵茹茜..Kisspeptin-10对无血清培养鸡F1级卵泡颗粒细胞孕酮分泌的影响及机制研究[J].中国农业科学,2012,45(2):391-398,8.基金项目
国家自然科学基金-青年基金项目 (30800809) （30800809）
