Retinoic Acid Promotes The Differentiation Of Spermatogonia In Mouse Model Of Spermatogenesis Disorder Through MTOR Pathway

Background: Since the beginning of the 21st century,the incidence of infertility in couples of childbearing age in China has been increasing.With the rapid development of social science and technology,increased life pressure,environmental pollution,disease factors,etc.,the quality of male semen is abnormal,which manifests as dead sperm,weak sperm,and deformed sperm.Spermatogenesis requires precise spermatogonia differentiation.Abnormal proliferation and differentiation of spermatogonia can lead to spermatogenesis disorders.Studies have found that retinoic acid(RA)plays an important role in spermatogenesis.When mammals lack RA,they cannot produce sperm normally,and after a proper amount of RA,they can restore spermatogenesis.Recent studies have found that RA can promote the differentiation of spermatogonial stem cells into spermatocytes in in vitro experiments.The mTOR signaling pathway plays an important role in regulating the proliferation,differentiation and self-renewal of spermatogonia.However,whether RA promotes the differentiation of spermatogonia through the mTOR pathway in the spermatogenesis disorder model has not yet been reported.The purpose of this topic is to explore the role of mTOR pathway in the process of RA regulating the differentiation of spermatogonia,and to provide more experimental evidence for the clinical application of RA to improve the reproductive function of males with impaired spermatogenesis.Methods: 1)After replicating the mouse model of spermatogenesis disorder,the changes of testicular histomorphology and the number of sperm in epididymis were observed by HE staining,and testicular index,sperm density,sperm viability,forward sperm rate and sperm deformity rate were analyzed statistically.The expression of Stra8,C-kit and mTOR in mouse testicular tissue was detected by immunohistochemical(IHC)and Western Blot;2)The spermatogenic disturbance model mice were treated with RA(5mg/kg/d)and rapamycin(RAPA,2.5mg/kg/d).The morphology of testis and the number of sperm in epididymis were observed,and the expressions of Stra8,C-kit and mTOR in testis were detected by IHC and Western Blot;3)The mouse spermatogonial stem cells(C18-4spermatogonia)were cultured in vitro,and Western Blot and immunocytochemistry were used to detect the expression of Stra8,C-kit and mTOR in C18-4 spermatogonia treated with 40μM DES alone or in combination with 75 n M RAPA or 100 n M RA.The effect of RA on spermatogonia differentiation and the role of mTOR pathway in this process were analyzed comprehensively.Results: 1)Compared with the control group,the spermatogenic cell layers in the spermatogenic epithelium of the testis in the spermatogenic disorder model group were significantly reduced,a large number of deletions,disordered arrangement,loose interstitial cells,decreased sperm density in the epididymis,testicular index,sperm density and sperm motility,increased sperm deformity rate,and decreased the expression of Stra8 and C-kit in testicular tissue.It indicated that the spermatogenic disorder model was successfully established and the differentiation of spermatogonia was inhibited in the spermatogenic disorder model mice;2)After 2 weeks of RA treatment,the spermatogenesis function of spermatogenic disorder model mice was improved,and the expression of Stra8 and C-kit in testicular tissue was up-regulated,which suggested that RA can promote spermatogonia differentiation and repair spermatogenic function of spermatogenic disorder model mice.After the intervention of RAPA,the effect of RA on improving spermatogenesis in spermatogenic disorder model mice was inhibited,and the expression of Stra8,C-kit and mTOR in testicular tissue of mice was down-regulated.It is suggested that RA can promote spermatogonia differentiation and repair spermatogenic function in spermatogenic disorder model mice through mTOR pathway;3)The expression of Stra8 and C-kit in C18-4 spermatogonia treated with40μM DES was significantly lower than that in the control group,suggesting that DES can inhibit the differentiation of spermatogonia.Compared with 40μM DES alone,the expression of Stra8 and C-kit in C18-4 spermatogonia treated with 40μM DES combined with 100 n M RA was up-regulated,suggesting that RA can promote the differentiation of spermatogonia inhibited by DES;4)Compared with 40μM DES plus100 n M RA group,the down-regulated expression of Stra8 and C-kit in C18-4 spermatogonia treated with 75 n M RAPA(40μM DES+100n M RA+75n M RAPA group)suggested that RA promoted the differentiation of C18-4 spermatogonia inhibited by DES through mTOR pathway.Conclusion: 1)The combination of cyclophosphamide and diethylstilbestrol can effectively establish a stable mouse model of spermatogenic disorder.2)mTOR pathway is involved in the effect of RA on the differentiation of spermatogonia with spermatogenic disorders.