RFRP-3在湖羊雄性生殖器官中的表达及其受营养水平的影响
发布时间：2018-12-18 05:36
【摘要】：湖羊作为一个高繁殖力绵羊品种,目前的研究主要集中在雌性湖羊的繁殖生理,而对雄性湖羊的繁殖生理关注较少。2000年,日本科学家Tsutsui等从日本鹌鹑脑内成功分离提纯一种RF酰胺肽,进一步分析发现,其为C末端具有RF酰胺的十二肽,而且对鸟类垂体前叶GnRH的分泌有抑制作用,故命名为促性腺激素抑制激素(Gonadotropin-inhibitory hormone,GnIH)。RF 酰胺相关肽-3(RFamide-related peptide-3,RFRP-3)是GnIH在哺乳动物体内的同系物,它可以调节哺乳动物的繁殖、摄食、焦虑、应激和能量平衡等行为,是目前生殖生物学领域研究的热点之一,已在多种动物的不同组织均有表达,但其在雄性湖羊生殖系统中的表达情况还不清楚。本试验主要利用荧光定量PCR、免疫组化等技术,以10日龄、4月龄和8月龄的雄性湖羊的睾丸、附睾头、附睾体、附睾尾和输精管为研究对象,检测RFRP-3在其发育过程中各组织的定位及表达情况;再以6月龄湖羊雄性生殖器官为研究对象,分析不同营养水平饲喂下,RFRP-3、GnRHR、FSHR和LHR基因在雄性湖羊生殖器官的表达变化,旨在研究分析RFRP-3在雄性湖羊生殖器官生长发育过程中的作用,以及其参与繁殖营养调控的作用。主要的研究结果如下:1.RFRP-3蛋白在湖羊雄性生殖器官发育过程中的定位研究。RFRP-3蛋白主要分布于8月龄湖羊睾丸的生精小管内部、输精管及附睾;RFRP-3蛋白在8月龄湖羊附睾的假复层柱状上皮和输精管的粘膜层中强烈表达,而在4月龄和10日龄湖羊生殖器官中微弱表达。2.RFRP-3基因在湖羊雄性生殖器官发育过程中的定量研究。在10日龄湖羊组中,RFRP-3基因在附睾头、附睾体、附睾尾、睾丸和输精管组织中表达量差异不显著。在4月龄和8月龄湖羊组中,RFRP-3基因在附睾头、附睾体、附睾尾和睾丸的表达量差异不显著,而在输精管中的表达量显著高于其他组织(P0.05)。此外,在同一月龄不同组织中RFRP-3基因mRNA表达水平也存在一定的差异。3.不同营养水平下对湖羊雄性生殖器官中RFRP-3、GnRHR、FSHR和LHR基因mRNA表达的影响。在高能高蛋白水平饲喂下,RFRP-3基因在雄性湖羊生殖器官中表达量显著低于正常饲喂水平,而GnRHR、FS和LHR基因mRNA表达量显著高于正常饲喂水平。以上试验结果证明,RFRP-3在湖羊雄性生殖器官中广泛分布,其表达量在湖羊不同生殖器官、不同月龄和不同饲喂水平条件下差异显著。本研究为进一步探讨RFRP-3的生物功能以及其参与动物繁殖营养调控研究提供了理论依据,但其具体的调节机制以及与其它调节因子之间的相互作用还需要进一步的研究。
[Abstract]:As a high fecundity sheep breed, the current research focuses on the reproductive physiology of female Hu sheep, but less attention on the reproductive physiology of male Hu sheep. Japanese scientist Tsutsui et al successfully isolated and purified a RF amide peptide from the brain of Japanese quail. Further analysis showed that it was a dodeceptide with RF amide at the C-terminal and inhibited the secretion of GnRH in the anterior pituitary of birds. Therefore, gonadotropin inhibitory hormone (RFamide-related peptide-3,RFRP-3) is a homologue of GnIH in mammals, which can regulate mammalian reproduction, feeding and anxiety. The behaviors of stress and energy balance are one of the hotspots in the field of reproductive biology. They have been expressed in different tissues of many animals, but their expression in the reproductive system of male Hu sheep is still unclear. In this study, the testis, head of epididymis, body of epididymis, tail of epididymis and vas deferens of 10 days old, 4 months old and 8 months old male Hu sheep were studied by fluorescence quantitative PCR, immunohistochemical technique. The localization and expression of RFRP-3 in various tissues during its development were detected. The expression of RFRP-3,GnRHR,FSHR and LHR genes in male reproductive organs of six month old Hu sheep was analyzed under different nutrition levels. The purpose of this study was to investigate the role of RFRP-3 in the growth and development of reproductive organs of male Hu sheep and its role in reproductive and nutritional regulation. The main results are as follows: the localization of 1.RFRP-3 protein in the development of male reproductive organs in Hu sheep. The RFRP-3 protein was mainly distributed in the seminiferous tubules, vas deferens and epididymis of 8 months old Hu sheep testis. RFRP-3 protein was strongly expressed in the pseudostratified columnar epithelium of the epididymis and the mucosal layer of the vas deferens in the 8-month-old Hu sheep epididymis. The expression of 2.RFRP-3 gene in male reproductive organs of Hu sheep at the age of 4 months and 10 days was studied quantitatively. There was no significant difference in the expression of RFRP-3 gene in the head of epididymis, corpus epididymidis, cauda epididymidis, testis and vas deferens in 10-day-old Hu sheep group. The expression of RFRP-3 gene in the head of epididymis, body of epididymis, cauda epididymidis and testis was not significantly different in 4 and 8 months old Hu sheep group, but the expression level in vas deferens was significantly higher than that in other tissues (P0.05). In addition, the mRNA expression level of RFRP-3 gene was also different in different tissues of the same month. 3. 3. Effects of different nutritional levels on mRNA expression of RFRP-3,GnRHR,FSHR and LHR genes in male reproductive organs of Hu sheep. Under high energy and high protein levels, the expression of RFRP-3 gene in male Hu sheep reproductive organs was significantly lower than that in normal feeding, while the mRNA expression of GnRHR,FS and LHR genes was significantly higher than that of normal feeding. The results showed that RFRP-3 was widely distributed in male reproductive organs of Hu sheep, and the expression of RFRP-3 was significantly different in different reproductive organs, different months of age and different feeding levels of Hu sheep. This study provides a theoretical basis for further study on the biological function of RFRP-3 and its involvement in animal reproductive and nutritional regulation, but its specific regulation mechanism and interaction with other regulatory factors need to be further studied.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826
本文编号：2385429
[Abstract]:As a high fecundity sheep breed, the current research focuses on the reproductive physiology of female Hu sheep, but less attention on the reproductive physiology of male Hu sheep. Japanese scientist Tsutsui et al successfully isolated and purified a RF amide peptide from the brain of Japanese quail. Further analysis showed that it was a dodeceptide with RF amide at the C-terminal and inhibited the secretion of GnRH in the anterior pituitary of birds. Therefore, gonadotropin inhibitory hormone (RFamide-related peptide-3,RFRP-3) is a homologue of GnIH in mammals, which can regulate mammalian reproduction, feeding and anxiety. The behaviors of stress and energy balance are one of the hotspots in the field of reproductive biology. They have been expressed in different tissues of many animals, but their expression in the reproductive system of male Hu sheep is still unclear. In this study, the testis, head of epididymis, body of epididymis, tail of epididymis and vas deferens of 10 days old, 4 months old and 8 months old male Hu sheep were studied by fluorescence quantitative PCR, immunohistochemical technique. The localization and expression of RFRP-3 in various tissues during its development were detected. The expression of RFRP-3,GnRHR,FSHR and LHR genes in male reproductive organs of six month old Hu sheep was analyzed under different nutrition levels. The purpose of this study was to investigate the role of RFRP-3 in the growth and development of reproductive organs of male Hu sheep and its role in reproductive and nutritional regulation. The main results are as follows: the localization of 1.RFRP-3 protein in the development of male reproductive organs in Hu sheep. The RFRP-3 protein was mainly distributed in the seminiferous tubules, vas deferens and epididymis of 8 months old Hu sheep testis. RFRP-3 protein was strongly expressed in the pseudostratified columnar epithelium of the epididymis and the mucosal layer of the vas deferens in the 8-month-old Hu sheep epididymis. The expression of 2.RFRP-3 gene in male reproductive organs of Hu sheep at the age of 4 months and 10 days was studied quantitatively. There was no significant difference in the expression of RFRP-3 gene in the head of epididymis, corpus epididymidis, cauda epididymidis, testis and vas deferens in 10-day-old Hu sheep group. The expression of RFRP-3 gene in the head of epididymis, body of epididymis, cauda epididymidis and testis was not significantly different in 4 and 8 months old Hu sheep group, but the expression level in vas deferens was significantly higher than that in other tissues (P0.05). In addition, the mRNA expression level of RFRP-3 gene was also different in different tissues of the same month. 3. 3. Effects of different nutritional levels on mRNA expression of RFRP-3,GnRHR,FSHR and LHR genes in male reproductive organs of Hu sheep. Under high energy and high protein levels, the expression of RFRP-3 gene in male Hu sheep reproductive organs was significantly lower than that in normal feeding, while the mRNA expression of GnRHR,FS and LHR genes was significantly higher than that of normal feeding. The results showed that RFRP-3 was widely distributed in male reproductive organs of Hu sheep, and the expression of RFRP-3 was significantly different in different reproductive organs, different months of age and different feeding levels of Hu sheep. This study provides a theoretical basis for further study on the biological function of RFRP-3 and its involvement in animal reproductive and nutritional regulation, but its specific regulation mechanism and interaction with other regulatory factors need to be further studied.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826
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