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

doi:10.3864/j.issn.0578-1752.2012.02.023

摘要/Abstract

摘要： 【目的】研究kisspeptin-10对无血清培养的鸡卵泡颗粒细胞孕酮分泌的作用及可能机制。【方法】选取200日龄处于产蛋高峰期的伊莎蛋鸡，于排卵前剖腹收集F1卵泡，分离纯化颗粒细胞，于含胎牛血清培养基中培养24 h，换成无血清培养基稳定培养24 h后，用不同浓度的Kp-10、U73122、EGTA等单独或共同处理细胞，收集细胞上清，放射免疫学方法检测培养基中孕酮含量。【结果】（1）通过免疫细胞化学方法，显示颗粒细胞上有Kp-10免疫活性样物质的阳性表达；（2）Kp-10处理可显著增加无血清培养的颗粒细胞的活力，100 nmol•L-1时可显著促进孕酮的分泌（P＜0.05）；（3）与对照组相比，2 μmol•L-1 U73122（磷脂酶C抑制剂）对孕酮的分泌无显著影响（P＞0.05），而0.5、2 μmol•L-1 U73122能显著降低Kp-10对孕酮分泌的促进作用（P＜0.05）；（4）钙离子阻断剂Verapamil（1—100 μmol•L-1 ）呈剂量依赖性降低孕酮的分泌，于100 μmol•L-1时达显著降低水平（P＜0.05）；与1 μmol•L-1 Verapamil单独处理组相比，100 nmol•L-1 Kp-10与1 μmol•L-1 Verapamil同时处理可显著增加孕酮的分泌，而与10、100 μmol•L-1 Verapamil共同处理不能逆转其对孕酮分泌的抑制作用（P＞0.05），且胞内钙离子含量与上清液中孕酮分泌水平相一致；（5）与100 nmol•L-1 Kp-10处理组相比，1和5 mmol•L-1 EGTA共同处理时孕酮分泌显著降低，当再加入1.5 mmol•L-1 Ca2+时孕酮分泌量显著增加。【结论】Kp-10促进体外培养的鸡卵泡颗粒细胞孕酮的分泌，其机制可能与胞内Ca2+信号通路有关。

关键词: kisspeptin-10, 钙离子, 孕酮, 颗粒细胞, 伊莎褐蛋鸡

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 (F1) 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 μmol•L-1 and 2 μmol•L-1 blocked the effect of Kp-10 on stimulating P4 secretion markedly (P＜0.05), while 2 μmol•L-1 U73122 had no effect on P4 secretion. Verapamil (the calcium channel blocker) suppressed P4 secretion in a dose-dependent manner, reaching the statistical significance at 100 μmol•L-1 dosage (P＜0.05). Under low dosage of Verapamil (1 μmol•L-1) 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 μmol•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 Ca2+ 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.

Key words: kisspeptin-10, Ca2+, progesterone, granulosa cells, ISA Brown hens

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.

XIAO Yun-Qi, HUANG Yan-Bing, WU Jing, NI Ying-Dong, ZHAO Ru-Qian. Effect and Mechanism of Kisspeptin-10 on Progesterone Secretion in Granulosa Cells of Chicken F1 Follicles Cultured in Serum-Free Medium[J]. Scientia Agricultura Sinica, 2012, 45(2): 391-398.

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