The Expression And Effect Of Kisspeptin/Kiss1r System In The Hypothalamic Arcuate Nucleus Of Adult Male Rats With Diet-induced Obesity

Ⅰ.Effect of high-fat-diet induced obesity on reproduction function in male ratsObjective:High-fat-diet (HFD) induced obesity model was established to detect the reproductive endocrine hormones, spermatogenic cycle, the morphology and function of sperm and testis in order to investigate the effect of high-fat-diet induced obesity on reproduction function in rats.Methods:Pregnant SD rats were obtained and their male pups were divided into the control or HFD groups randomly. HFD rats were fed with high-fat diets.After 14 weeks, obesity rats were selected according to lee’s index. Rats in control group were fed with normal feed. We detected the level of leptin, follicule-stimulating hormone (FSH), luteinizing hormone(LH) in serum and the concentration of testosterone (T). We observed the morphological changes of testis and sperm. We also observed the changes of spermatogenic cells cycle and the expression of androgen binding protein (ABP) in testis with obesity. Details are as follows:(1) Model identification:body weight, lee’s index, retroperitoneal fat and testis weight of rats were recorded.(2) Radioimmunoassay (RIA) was performed to analyze the concentration of leptin, T, LH and FSH in serum, in order to explore the effect of high-fat-diet induced obesity on endocrinal hormones in male rats(3) Morphological changes of testis in the control and HFD rats were observed via hematoxylin-eosin staining.(4) The changes of spermatogenic cells cycle were analyzed by flow cytometry.(5) WLJY-9000 color sperm morphology detection system was adopted to improve the sperm morphology analysis.(6) Immunohistochemistry was used for ABP protein expressions in testis tissues of rats in two groups. And according to area and intensity of staining, results were divided into positive and negative.Results:(1) Compared to the controls, body weight, lee’s index, retroperitoneal fat and testis weight in obesity rats all increased obviously, but the testis index in obesity rats decreased.(2) Compared to the control rats, the level of leptin in serum increased significantly in HFD rats, while the concentration of T, LH and FSH decreased in obesity group.(3) According to hematoxylin-eosin staining, the testis tissue in controls maintained their normal architecture morphologically. The seminiferous tubules in obesity group were with thinner wall and smaller lumen. More spermatogenic cells spin off the basement membrane to the lumen. The gap between sperm cells and spermatocyte was bigger or disappeared connection. The number of mature sperm decreased significantly in obesity group.(4) In the end of model establishing, spermatogenic cells in G0/G1 phase in obesity group increased and in S phase decreased, while cells in G2/M phase significantly increased.(5) The results of sperm morphology analyzed indicated that the number and activate rate in sperms decreased significantly in obesity group than controls, while the rate of terato sperm increased, especially the head burst swelling or separated from tails.(6) The expression of ABP in testis was brown nuclear staining in Sertoli cells. The expression of ABP in testis of obesity rats decreased obviously, compared with the controls.Conclusion:The neuroendocrine metabolic disorder induced by high-fat-diets, led to abnormity in morphology of seminiferous tubule, spermatogenic cells cycle and the function of Sertoli cells, leading to impediment of spermatogenesis, which might result in infertility.Ⅱ. Effect of kisspeptin/kisslr system on infertility induced by nutritional obesity in male ratsObjective:To explore the expression and interaction of leptin receptor, kisspeptin /kisslr system and GnRH in the hypothalamic arcuate nucleus (ARC) of rats with HFD induced obesity, via detecting the effect of exogenous kisspeptin on changes of body parameters, endocrinal hormones and reproductive related peptides in rats of all groups.Methods:On the basis of the first part, a part of obesity rats were perfused with kisspeptin, 0.5nmol, by lateral ventricles, as kisspeptin groups. Animals were sacrificed after 2 hours. Details are as follows:(1) Body weight, lee’s index and retroperitoneal fat in rats of the control, obesity and kisspeptin groups were recorded.(2) Radioimmunoassay (RIA) was performed to analyze levels of leptin, LH, T and E2 in serum of male rats in three groups.(3) Immunohistochemistry was used for leptin receptor, kisspeptin/kisslr system and GnRH protein expressions in the hypothalamic arcuate nucleus of three groups. The results were divided into positive and negative by stain area and intensity.(4) Expressions of leptin receptor, kisspeptin/kisslr and GnRH protein and mRNA contents were investigated by western blot and qRT-PCR.Results:(1) As shown in the first part of the results, body weight, lee’s index, retroperitoneal fat and testis weight in HFD rats all increased obviously. The level of leptin in serum increased significantly in obesity group, while the concentration of T and LH decreased in obesity group compared with the controls. The level of T and LH increased significantly in rats perfused with kisspeptin. There were no obvious changes in other parameters and hormones.(2) According to the immunohistochemistry, the expressions of leptin receptor, kisspeptin/kisslr system and GnRH were detected in the hypothalamic ARC of all groups. Lepr-IR was brown nuclear staining, while kisspeptin-IR, kiss1r-IR and GnRH-IR were all brown cytoplasmic staining. The expressions of Lepr, kisspeptin and GnRH decreased obviously in obesity group, compared to the controls. The GnRH protein increased significantly after kisspeptin perfusing. There were no obvious changes in other peptides.(3) Results from western blotting showed that there were successive positive expression straps of Lepr, kisspeptin/kisslr system and GnRH proteins in all groups. In obesity group, all proteins decreased obviously, while GnRH contents increased in rats with exogenous kisspeptin.(4) Significantly lower levels of kiss1-, Lepr-, and GnRH-mRNA were observed in the hypothalamic ARC of rats in obesity group than that in the controls. The GnRH-mRNA increased significantly after kisspeptin perfusing. No difference was observed in kiss1rmRNA expression among the three groups.Conclusion:Kisspeptin/kisslr system mediated the effect of HFD induced obesity on the expression of GnRH in the hypothalamic arcuate nucleus of male rats, affecting the function of HPG axis, insulting in infertility. Exogenous kisspeptin perfusing could restore reproductive disorder induced by nutritional obesity in some degree.