缺氧诱导基因1C在牦牛隐睾中的表达及其调控机制研究

Expression of HIG1 Hypoxia Inducible Domain Family Member 1C in Cryptorchidism of Yak and Its Regulatory Mechanism

旨在探究缺氧诱导基因1C(HIG1 hypoxia inducible domain family member 1C,HIGD1C)对牦牛睾丸支持细胞凋亡的影响。本研究分别选择3头3~6岁龄健康状况良好的公牦牛和隐睾症公牦牛,以其睾丸组织作为研究材料。通过HE染色、牦牛睾丸支持细胞分离培养、间接免疫荧光、RT-qPCR、Western blot、HIGD1C过表达与干扰载体构建及体外细胞转染等技术,探究HIGD1C对牦牛睾丸支持细胞凋亡的调控机制。结果显示:正常睾丸基底膜及间质组织完整,曲细精管、管周肌细胞及支持细胞等排列紧密,隐睾中曲细精管断裂萎缩且HIGD1C在牦牛隐睾组织中转录及蛋白的表达水平极显著于高于正常睾丸组织(P<0.01);HIGD1C蛋白主要分布在睾丸间质细胞和支持细胞;成功分离培养出牦牛睾丸支持细胞且HIGD1C蛋白定位于原代睾丸支持细胞胞核;HIGD1C过表达支持细胞后,Bcl-2 mRNA表达水平显著上调(P<0.05),蛋白表达水平极显著上调(P<0.01),Bax和Caspase-3 mRNA和蛋白表达水平均极显著上调(P<0.01),流式细胞术结果显示过表达HIGD1C组支持细胞凋亡率极显著上升(P<0.01);反之,HIGD1C敲除后,Bax、Bcl-2和Caspase-3 mRNA和蛋白表达水平均极显著下调(P<0.01),流式细胞术结果显示HIGD1C敲除组支持细胞凋亡率极显著下调(P<0.01)。本研究结果表明:正常睾丸组织结构完整,隐睾间质疏松,且HIGD1C在牦牛隐睾组织中高表达。体外细胞试验表明上调HIGD1C,可以激活促凋亡蛋白,促进牦牛支持细胞凋亡;反之,下调HIGD1C表达水平会抑制支持细胞凋亡。提示HIGD1C参与调控牦牛睾丸支持细胞的凋亡,为揭示牦牛隐睾的发生机制提供了参考。

The study aimed to investigate the effect of HIG1 hypoxia inducible domain family member 1C(HIGD1C)on apoptosis of sertoli cells in yak testis.In this study,3 healthy male yaks aged 3-6 years and male yaks with cryptorchidism were selected,respectively as research materials,and their testis was collected.The regulation mechanism of HIGD1C on apoptosis of yak testicular sertoli cells was explored by HE staining,isolation and culture of yak testicular sertoli cells,indirect immunofluorescence,RT-qPCR,Western blot,HIGD1C overexpression and interference vector construction and in vitro cell transfection.The results showed that the basement membrane and interstitial tissue of normal testis were intact,the seminiferous tubules,peritubular muscle cells and sertoli cells were closely arranged,the seminiferous tubules in cryptorchidism were broken and atrophied,and the transcription and protein expression level of HIGD1C in yak cryptorchidism was significantly higher than that in normal testis(P<0.01);HIGD1C protein was mainly distributed in leydig cells and sertoli cells;yak sertoli cells were successfully isolated and cultured;and HIGD1C protein was located in the nucleus of primary sertoli cells;after overexpression of HIGD 1C,the mRNA expression level of Bcl-2 was up-regulated(P<0.05),the protein expression level was significantly up-regulated(P<0.01),and the mRNA expression levels and protein expression levels of Bax and Caspase-3 were significantly up-regulated(P<0.01),the results of flow cytometry showed that the apoptosis rate of sertoli cells in HIGD 1C overexpression group was significantly increased(P<0.01).On the contrary,after HIGD 1C knockout,the mRNA and protein expression levels of Bax,Bcl-2 and Caspase-3 were significantly down-regulated(P<0.01),the results of flow cytometry showed that the apoptosis rate of sertoli cells in HIGD 1C knockout group was significantly down-regulated(P<0.01).The results of this study show that the normal testicular tissue structure is complete,the cryptorchidism stroma is loose,and HIGD1C is highly expressed in the cryptorchidism of yak.In vitro cell experiments showe that up-regulation of HIGD1C could activate pro-apoptotic proteins and promote apoptosis of yak sertoli cells.On the contrary,down-regulation of HIGD1C expression level will inhibit the apoptosis of sertoli cells.It is suggested that HIGD1C is involved in the regulation of apoptosis of yak testicular sertoli cells,which provides a reference for revealing the mechanism of cryptorchidism in yak.