Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (2): 391-398.doi: 10.3864/j.issn.0578-1752.2012.02.023

• VETERINARY SCIENCE • Previous Articles     Next Articles

Effect and Mechanism of Kisspeptin-10 on Progesterone Secretion in Granulosa Cells of Chicken F1 Follicles Cultured in Serum-Free Medium

 XIAO  Yun-Qi, HUANG  Yan-Bing, WU  Jing, NI  Ying-Dong, ZHAO  Ru-Qian   

  1. 1.南京农业大学动物医学院，南京 210095
  • Received:2011-03-09 Online:2012-01-15 Published:2011-12-07

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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

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