| Kisspeptin is a kind of protein hormone encoded by Kiss-1 gene, which is mainly secreted in hypothalamus to control gonadtrophin-releasing hormone (GnRH) secretion, then to regulate the onset of puberty in vertebrates.It's reported that kisspepin also exists in peripheral reproductive organs. Till now, although the positive signal for kisspeptin has been identified in ducks, the data about the function of kisspeptin in birds is really scarce. In birds, progesterone (P4), as a positive signal to induce LH surge before ovulation, is a vital factor for initiating egg production and maintain egg laying performance. In order to investigate the direct effect of kisspeptin on P4 secretion in ovary granulosa cells of hens, in vitro cultured method was established and identified by immunocytochemistry method. The objective of this study was to:1, whether kisspeptin was secreted in granulosa cells of chicken ovary, as reported in mammalian species.2, the effect of kisspeptin on P4 secretion of the ovary granulosa cells in vitro.3, the mechanism of kisspeptin regulating P4 secretion in granulosa cells.1 Effect of kisspeptin-10 on P4 secretion in granulosa cells of chicken ovary200-day-old ISA laying hens were sacrificed before the expected time to ovulation. The follicles 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 Kp-10 alone or in combination with Verapamil (the calcium channel blocker) and EGTA, respectively. The medium and the cells were collected for assay. Immunocytochemistry and immunofluorescence were used to identify the expression of FSHR and kisspeptin in granulosa cells cultured in vitro. The cell viability was measured by MTT. P4 and Ca2+ concentration were measured by radioimmunoassay (RIA) and flow cytometry (FCM), respectively. The results showed that there was positive signal for the receptor of follicle stimulating hormone (FSH) expression in cells. Combination with the morphologic and growth characteristics, these cells were identified as granulosa cells. With the specific antibody against Kp-10, these cells were also showed obvious signals for kisspeptin expression, which indicated that the granulosa cells of chicken have the ability to autocrine kisspeptin, as reported in other animals. After 24 h treatment, Kp-10 significantly increased the viability of granulosa cells as well as P4 secretion (P<0.05). Verapamil suppressed P4 secretion in a dose-dependent manner, reaching the statistical significance at 100μM dosage (P<0.05). Under low dosage of Verapamil (1μM) background,100 nM Kp-10 can still significantly increase P4 secretion (P<0.05). However, under higher dosage of Verapamil (10 or 100μM),100 nM 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 mM), while this effect was converted by adding 1.5 mM Ca2+in medium (P<0.05). These results suggested that the mechanism of kisspeptin-10 on promoting P4 secretion in granulosa cells of chicken might be associated with the changes of intracellular Ca2+concentration.2 The mechanism of kisspeptin-10 regulating P4 secretion in granulosa cells of chicken ovaryAs described in the experiment 1, the follicles (F1-F3) were collected and the follicular granulosa cells were isolated and cultured in serum-free medium. The granulosa cells were treated with Kp-10 alone or in combination with U73122 (PLC inhibitor). The media and the cells were collected for further assay. The cell viability was measured by MTT. P4 concentetion was measured by radioimmunoassay (RIA). Gene expression of StAR, P450scc and 3β-HSD was quantitated with relative quantitative real time RT-PCR, and western blot analysis was used to detect the protein content in cells. The results showed that, after 24 h treatment, Kp-10 significantly increased the viability of F1-F3 cells (P<0.05), and markedly increased P4 secretion in F1 and F2 cells (P<0.01). The expression of StAR, P450scc and 3β-HSD mRNA was significantly increased by 100 nM Kp-10 in F1 and F2 cells (P<0.05) as well as in F3 cells (P<0.01). However, western blot results showed that there was no effect of Kp-10 on these three enzyme proteins expression in cells (P>0.05). After the cells were treated with U73122 in the concentration of 0.5 or 2μM, there was no changes of U73122 on the viability of cells (P>0.05). Compared with the control group, P4 secretion significantly increased after treated with Kp-10 (P<0.05).0.5 and 2μM U73122 blocked the effect of Kp-10 on stimulating P4 secretion markedly (P<0.05), while 2μM U73122 alone treatment had no effect on P4 secretion. Compared with the control group, kp-10 and/or U73122 had no effect on the expression of StAR and P450scc mRNA (P>0.05), but significantly increased that of 3β-HSD (P<0.05). Compared with the group of 100 nM Kp-10, the level of 3p-HSD mRNA in treated groups (0.5 or 2μM U73122) was significantly lower (P<0.05). These results suggested that, the mechanism underlying the stimulation of Kp-10 on P4 secretion in ovary granulosa cells of hens might be associated with the up-regulating of several key enzymes transcription in P4 synthesis and celluar phospholipase C signal pathway. |
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