| Background and objectives:Hyperprolactinemia（HPRL）is a class of endocrine diseases and could be caused by various reasons,characterized with high levels of serum prolactin（PRL）（>1.14nmol/L or 25 μg/L）.The main clinical features include galactorrhea,oligomenorrhea,amenorrhea and endocrine dysfunction.However,the pathway and mechanism of HPRL on the reproductive endocrine disorder are still unclear.It is known that PRL can inhibit the release of gonadotropin-releasing hormone（GnRH）by acting on GnRH neurons,resulting in the reduction of FSH and LH secretion,thereby the inhibition of estradiol and progesterone secretion.However,some studies showed that very few prolactin receptors（PRLR）were expressed in GnRH neurons,and most were expressed in kisspeptin neurons and some peripheral tissues such as ovaries and testes.Moreover,our previous studies showed that Kiss1/GPR54 system could be expressed in ovary luteal tissues and involved in the regulation of endocrine function of corpus luteum,suggesting that HPRL may exert its actions on endocrine function of corpus luteum by Kiss1/GPR54 system.Here,by constructing metoclopramide（MC）-induced HPRL model in vivo and luteal cells cultured in vitro,the aim of our study was to explore whether HPRL affected the endocrine function of rat corpus luteum through the peripheral ovarian Kiss1/GPR54 system and its mechanism.Methods:1.Indirect immunofluorescence was used to observe the localization and relationship with Kiss1 and PRLR protein in normal rat ovarian luteal tissues and cells.2.It was observed the effects of HPRL on the organizational structure and endocrine function of rat ovarian corpus luteum by HPRL model in vivo.3.Immunohistochemistry and real-time quantitative PCR were used to observe the effect of HPRL on the expression levels of the Kiss1 and GPR54 of rat luteal tissues.4.Radioimmunoassay（RIA）was used to observe the effects of kisspeptin and PRL on the hCG-stimulated estradiol and progesterone secretion in the rat luteal cells in vitro.5.Real-time quantitative PCR was used to explore the effects of kisspeptin and PRL on the hCG-stimulated expressions of three key steroidogenic enzymes mRNA in cultured rat luteal cells.6.Enzyme immunoassay（EIA）was used to detect the effects of HPRL on hCG-stimulated PGF2α and PGE2 secretion in the rat luteal cells.7.Western blot was used to observe the effects of kisspeptin and PRL treatment on hCG-stimulated phosphorylation of Erk1/2 in cultured rat luteal cells.Results:1.Using indirect immunofluorescence,we found that Kiss1 and PRLR protein were co-expressed in ovarian luteal cells of gonadotropin-primed immature rats.2.H&E staining showed that there were no significant difference in the weight of rat ovaries,the numbers and areas of rat corpora lutea between control group and HPRL group（P>0.05）.However,RIA analysis showed that the serum estradiol and progesterone levels in HPRL rats were decreased significantly compared to control group（P<0.01）.3.Compared with control group,we found that the expressions of Kiss1 and GPR54 protein in HPRL rat corpus luteum were decreased distinctly by immunohistochemistry method;likewise,Kiss1 and GPR54 mRNA expression levels in HPRL rat luteal cells were also decreased significantly（P<0.01）.4.In cultured luteal cells,high PRL treatment markedly inhibited hCGstimulated estradiol and progesterone production compared with control group（P<0.01）;compared with PRL group,PRL+kisspeptin-10 treatment caused a significant increase in progesterone level of luteal cells（P<0.01）,while estradiol level did not change significantly（P>0.05）.5.In cultured luteal cells,the expressions of three key steroidogenic enzymes（St AR,CYP11 A,3β-HSD）mRNA treated with high PRL were significantly inhibited compared with control group（P<0.05）;nevertheless,the expression levels of three key steroidogenic enzymes treated with PRL+kisspeptin-10 were significantly increased（P<0.05）.6.Furthermore,we found that treatment with high PRL significantly increased the levels of PGF2α secretion compared with control group in rat luteal cells（P<0.01）,and kisspeptin-10 could significantly inhibit the levels of PGF2α secretion（P<0.01）.On the other hand,PRL could dramaticlly inhibit the PGE2 secretion in rat luteal cells（P<0.01）,which could be improved by kisspeptin-10 treatment（P<0.05）.7.Compared with control group,high PRL treatment reduced the phosphorylation of Erk1/2 mitogen-activated protein kinase in cultured luteal cells.However,PRL+kisspeptin-10 treatment increased the phosphorylation of Erk1/2mitogen-activated protein kinase.Conclusion:HPRL can inhibit the endocrine function of rat luteal cells,at least in part,via the down-regulation of Kiss1/GPR54 expression in the corpus luteum of rat ovary. |
