Retinoic Acid Improves Spermatogenesis In Mice Induced By Compound Drugs And Up Regulate The Expression Of CCND1 In Testis

Background: Spermatogenesis disorder is one of the main causes of male infertility.Spermatogonia proliferation and differentiation is the starting link of spermatogenesis.Its abnormal regulation can lead to spermatogenesis disorder and male infertility.Retinoic acid(RA)is the active metabolite of vitamin A in vivo,which can maintain the normal development of male reproductive system.RA is the key factor to start the differentiation of spermatogonia and enter into meiosis,and can promote the proliferation and differentiation of spermatogonia.Its mechanism may be related to the related protein CCND1 of spermatogonia proliferation and differentiation.In this study,two kinds of drugs,cyclophosphamide and diethylstilbestrol,were used to establish a stable spermatogenesis disorder mouse model.The effects of RA on spermatogenesis disorder and CCND1 expression in testis of the model mice were observed.The molecular mechanism of RA improving spermatogenesis disorder was discussed preliminarily,which provided experimental basis for clinical application of RA to improve thereproductive function of spermatogenesis disorder men.Methods: 136 male Kunming mice were randomly divided into normal control group and model group.The model group included cyclophosphamide(CP)group,diethylstilbestrol(DES)group and CP +DES group.The drug was injected intraperitoneally for 14 days.Sperm count,HE staining of testis and epididymis tissue sections were used to detect the success of spermatogenesis disorder model,and the most appropriate concentration and method of modeling drugs were selected.RA was dissolved in DMSO and diluted to target concentration with corn oil.The model mice were injected subcutaneously with different doses of RA(0,2.5,5,10 mg / kg / d),or treated with RA(5 mg / kg / d)and rapamycin(2.5 mg / kg / d).At the 2nd and 4th week after administration,the mice were killed,the epididymis and testis were separated and weighed,the morphology of spermatogenic epithelium was observed by HE staining,the sperm quality was observed by epididymal sperm smear;Western blotting,immunohistochemistry and immunofluorescence were used to detect the expression of CCND1,Bax and Bcl-2.Results: Compared with the normal control group,the sperm density,sperm activity rate and testis index of the model group decreased significantly,while the sperm deformity rate increased significantly,suggesting that CP combined with DES intraperitoneal injection can establish the model of spermatogenesis disorder;Compared with themodel control group,the model mice treated with medium and high dose RA were significantly improved in the fourth week after treatment.The testis quality,testis index and sperm density all tended to normal values,the sperm viability increased by about 50%(P < 0.05),the forward motile sperm ratio increased by more than 38%(P < 0.05),and the sperm deformity rate also decreased significantly(P < 0.05);At the same time,compared with the normal control group,the apoptosis of germ cells in the model group increased.The results of immunohistochemistry or Western blotting showed that the expression of CCND1,Bax and Bcl-2 in testis decreased significantly;Compared with the model control group,RA can significantly improve the apoptosis of germ cells in the model group.RA can up regulate the expression of CCND1 and Bcl-2,and down regulate the expression of Bax;Compared with the model group treated with RA alone,the spermatogenesis disorder of the group treated with RAPA + RA was more serious,and the expression of CCND1 protein was down regulated.Conclusion: 1.Cyclophosphamide combined with diethylstilbestrol was used to construct a stable spermatogenic disorder mouse model.2.RA can inhibit the apoptosis of spermatogonia and improve the spermatogenesis of mice induced by compound drugs.The mechanism may be related to the up regulation of CCND1 expression.