C1型尼曼–匹克病小鼠雄性不育的睾丸病理研究

The Testicular Pathology of Male Infertility in Niemann-Pick Disease Type C1 Mice

该文主要研究C1型尼曼–匹克病小鼠(Npc1–/–小鼠)雄性不育的睾丸病理变化,并进一步探讨Npc1基因对雄性不育的影响。随机选取P60的Npc1–/–和Npc1+/+雄鼠各12只,观察隐睾发生率;然后,随机选取P20、P40和P60的Npc1–/–和Npc1+/+雄鼠睾丸组织,统计睾丸重量并计算睾丸指数, HE染色观察生殖细胞层数并测量生精小管直径, PAS糖原染色观察生殖细胞周期并统计精母细胞在总细胞数中的相对百分比,油红O染色观察间质细胞的脂质存储;最后,随机选择P60的Npc1–/–和Npc1+/+雄鼠睾丸,通过TUNEL染色观察生殖细胞凋亡,并通过Western blot检测凋亡蛋白Cleaved-caspase-3、Cleaved-caspase-9、p53、Bax和Bcl-2的表达水平。结果显示,与Npc1+/+雄鼠相比, Npc1–/–雄鼠均有隐睾发生,睾丸重量和睾丸指数均显著降低(P<0.05, P<0.001),生殖细胞层数不明显,生精小管管径显著减小(P<0.001),生精周期不规律且精母细胞数量显著减少(P<0.001),间质细胞的脂质存储显著减少(P<0.001);生殖细胞凋亡大幅增加(P<0.001), Cleaved-caspase-3、Cleaved-caspase-9、p53、Bax表达量显著升高(P<0.001),Bax/Bcl-2比率显著升高(P<0.001)。该文结果提示, Npc1基因突变导致隐睾、睾丸结构异常、间质细胞脂质储存减少及生殖细胞凋亡,因此, Npc1基因可能成为研究男性不育的新靶点。

In this paper,we mainly studied the testicular pathological changes of male infertility in Niemann-Pick disease type C1 mice(Npc1–/–mice),and further explored the effect of Npc1 gene on male infertility.Twelve P60 Npc1–/–and Npc1+/+male mice were randomly selected to observe the incidence of cryptorchidism.The testicular tissues were collected from P20,P40 and P60 Npc1–/–and Npc1+/+male mice to calculate testicular weight and testicular index.HE staining was performed to observe the layers of germ cells and measure the diameter of seminiferous tubules.PAS staining was used to observe the cycle of germ cells and count relative percentage of spermatocyte in the total number of cells.Oil red O staining was performed to observe the lipid storage of leydig cells.Finally,testicular tissues of P60 Npc1–/–and Npc1+/+male mice were randomly selected to observe the apoptosis of germ cells by TUNEL staining,and the expression levels of apoptotic proteins Cleaved-caspase-3,Cleavedcaspase-9,p53,Bax and Bcl-2 were detected by Western blot.The results showed,compared with Npc1+/+male mice,cryptorchidism occurred in all Npc1–/–mice,testicular weight and index were decreased significantly(P<0.05,P<0.001),germ cell layers were not obvious,diameter of seminiferous tubules were also decreased significantly(P<0.001),spermatogenic cycle was irregular and the spermatogonial cells number were decreased significantly(P<0.001),and lipid storage in leydig cells were also decreased significantly(P<0.001).Apoptosis increased significantly(P<0.001),and the expression of Cleaved-caspase-3,Cleaved-caspase-9,p53 and Bax increased significantly(P<0.001)and the ratio of Bax/Bcl-2 increased significantly(P<0.001).The mutation of Npc1 gene leads to cryptorchidism,the decrease of lipid storage in leydig cells,and the apoptosis of germ cells.Therefore,Npc1 gene may become the new targets for male infertility.