雄鼠生殖细胞中FHAD1表达与功能的初步研究  

Expression and function of FHAD1 in male mouse germ cells:a pilot study

作　　者：张茜 刘辰辰 张舒雅 ZHANG Xi;LIU Chen-chen;ZHANG Shu-ya(The Affiliated Huai’an First People’s Hospital of Nanjing Medical University,Huai’an First People’s Hospital,Huai’an 223300;State Key Laboratory of Reproductive Medicine,Department of Histology&Embryology,Nanjing Medical University,Nanjing 210000;Department of Basic Medicine,Kangda College of Nanjing Medical University,Lianyungang 222000)

机构地区：[1]南京医科大学附属淮安第一医院,淮安市第一人民医院,淮安223300 [2]南京医科大学生殖医学国家重点实验室组织胚胎学系,南京210000 [3]南京医科大学康达学院基础医学部,连云港222000

出　　处：《生殖医学杂志》2021年第10期1365-1370,共6页Journal of Reproductive Medicine

基　　金：淮安市卫生健康科研立项项目(HAWJ202007)。

摘　　要：目的探索叉头相关的磷蛋白结合结构域1(Forkhead-associated phosphopeptide-binding domain 1,FHAD1)在雄性小鼠睾丸中的表达及功能。方法购买不同周龄C57BL/6J雄性小鼠(包括0.5 d雄性5只,1周、2周、3周、4周、5周龄雄性小鼠各3只)以及成年C57BL/6J雌、雄性小鼠各3只。收集成年小鼠的心、肝脏、脾、肺、肾脏、脑、肌肉、肠、脂肪、卵巢以及各周龄雄性小鼠睾丸等组织,提取组织mRNA和蛋白。RT-PCR检测fhad 1在成年小鼠不同脏器以及在不同周龄小鼠睾丸中mRNA表达情况;Western Blot法检测FHAD1在小鼠不同脏器以及不同周龄小鼠睾丸中的蛋白表达。密度梯度沉降法分选出成年雄鼠睾丸的支持细胞、间质细胞、精原干细胞、粗线期精母细胞和圆形精子细胞,RT-PCR检测fhad1在小鼠睾丸组织中各细胞的基因表达情况。运用RNA干扰技术,敲低GC-2细胞中fhad1的表达,Western Blot法检测FHAD1和DNA损伤标志物磷酸化的组蛋白(γH2AX)及磷酸化的DNA损伤修复关键蛋白(p-NBS1)的表达水平。结果RT-PCR及Western Blot结果显示,fhad1 mRNA及FHAD1蛋白在雄性小鼠的睾丸中特异表达,从2周龄开始表达并持续至成年;不同时期生精细胞表达结果显示,fhad1 mRNA主要在粗线期精母细胞和圆形精子细胞中表达。RNA干扰实验结果表明,与阴性对照组相比,敲低FHAD1后GC-2细胞中γH2AX水平显著上升[(1.99±0.14)vs.(0.99±0.10)]、p-NBS1水平显著下降[(0.24±0.07)vs.(1.01±0.10)](P<0.001)。结论FHAD1为雄性小鼠睾丸特异蛋白,主要表达在粗线期的精母细胞以及圆形精子细胞中;下调FHAD1使GC-2细胞的DNA损伤增加,机制上可能与NBS1的磷酸化相关。Objective:To explore the expression and function of forkhead-associated phosphopeptide-binding domain 1(FHAD1)in mice testis.Methods:Five C57BL/6J male mice at different ages(including 5 males at 0.5 day old,3 adult male mice at 1,2,3,4,and 5 weeks old respectively),and 3 adult female mice and 3 adult male mice were purchased.The tissues of heart,liver,spleen,lung,kidney,brain,muscle,intestine,fat,and ovary,as well as testis of mice at different ages were collected to extract tissue mRNA and protein.The mRNA expression of fhad1 in the different organs of adult mice and testis of mice at different ages was detected by RT-PCR.The protein expression of FHAD1 in different organs and testis of mice at different ages was detected by Western blot.Sertoli cells,Leydig cells,spermatogonial stem cells,pachytene spermatocytes and round spermatids of adult mice testis were separated by density gradient sedimentation method.The fhad1 expression in various cells of mouse testis was detected by RT-PCR.The RNA interference technique was used to knock-down the expression of fhad1 in GC-2 cells.Western blot was used to detect the expression levels of FHAD1 and DNA damage marker phosphorylated histone(γH2AX)and phosphorylated DNA damage repair key protein(p-NBS1).Results:The results of RT-PCR and Western blot showed that fhad1 mRNA and FHAD1 protein are specifically expressed in the testis of mice from 2 weeks old to adulthood.The results of spermatogenic cell expression in different stages showed that fhad1 mRNA was mainly expressed in pachytene spermatocytes and round spermatids.The results of RNA interference technique showed that the level of γH2AX increased significantly[(1.99±0.14)vs.(0.99±0.10)]and the level of p-NBS1 decreased significantly[(0.24±0.07)vs.(1.01±0.10)]after knock-down of FHAD1 in GC-2 cells,compared with the negative control group(P<0.001).Conclusions:FHAD1 is a specific protein of mice testis,which is mainly expressed in pachytene spermatocyte and round spermatid.Down-regulation of FHAD1 increases DNA

关 键 词：叉头相关的磷蛋白结构域 睾丸特异蛋白 生精细胞 DNA损伤修复 

分 类 号：Q952.4[生物学—动物学]