成年树鼩睾丸维生素D代谢酶CYP24A1表达与生育力相关研究

Expression of CYP24A1,a vitamin D metabolic enzyme,in the testes of adult tree shrews and its effect on fertility

目的研究成年树鼩睾丸中维生素D代谢酶和维生素D受体的转录水平,检测CYP24A1的蛋白表达,探索雄性实验树鼩生殖障碍的原因。方法选择2~3岁雄性树鼩,根据个体繁殖档案分为具有成功生育史的正常组,和无生育史的生殖障碍组,每组各5只。手术摘取一侧睾丸,通过病理学观察其组织病理变化;采用实时荧光定量PCR检测睾丸组织中维生素D受体、维生素D代谢酶mRNA水平的变化,以蛋白印迹法检测CYP24A1的蛋白表达,同时用免疫荧光观察精子CYP24A1表达情况。结果正常组与生殖障碍组相比曲细精管排列更紧密,生精细胞更多。与正常组相比,生殖障碍组树鼩睾丸组织VDR、CYP2R1、CYP27A1 mRNA水平无明显差异(P>0.05),而CYP24A1在mRNA表达水平和蛋白水平都明显低于正常组(P<0.05)。同时免疫荧光结果显示正常组睾丸来源的精子在其头部、颈部和中段的CYP24A1蛋白高表达,而生殖障碍组表现为低表达或不表达。结论CYP24A1与雄性树鼩成功生育有直接联系,提示CYP24A1可以作为判断雄性树鼩生育力的重要指标。

Objective To examine transcription levels of vitamin D metabolic enzymes and the vitamin D receptor(VDR)in the testes of adult tree shrews.Specifically,to detect protein expression of cytochrome P450 family 24 subfamily A member 1(CYP24A1)and investigate causes of reproductive disorders in male tree shrews.Methods Male tree shrews(aged 2 to 3 years)were selected.On the basis of individual records,shrews were divided into a normal group with a successful reproductive history and a reproductive disorder group without a reproductive history;there were five shrews in each group.One side of the testis was surgically removed and histopathological changes were observed by pathology.Realtime fluorescent quantitative PCR was used to detect changes in m RNA levels of VDR and vitamin D metabolizing enzymes in the testis.Protein expression of CYP24A1 was detected by western blotting.Expression of sperm CYP24A1 was detected by immunofluorescence.Results In the normal group,the seminiferous tubules were tightly arranged and there were more spermatogenic cells than in the reproductive disorder group.Compared with the normal group,there was no significant difference in m RNA levels of VDR,CYP2 R1,or CYP27 A1 in the testes of tree shrews(P>0.05).However,m RNA expression levels and protein levels of CYP24A1 were significantly lower than in the normal group(P<0.05).At the same time,immunofluorescence showed that sperm from the testis of the normal group had high expression of CYP24A1 in the head,neck,and midpiece,while the reproductive disorder group had low or no expression of CYP24A1.Conclusions CYP24A1 is directly related to successful fecundity of male tree shrews,suggesting that CYP24A1 can be used as an important index to evaluate the fecundity of male tree shrews.