Study On The Role Of MTOR Signaling Pathway In The Epididymal Initial Segment Of Mice

According to statistics,about 5%of couples experience infertility in their reproductive lives,of which male infertility accounts for about half.Male infertility had transformed from a personal issue to a public health problem.Due to the complex structure of the male reproductive system,it is difficult to determine the mechanism leading to infertility.In the male reproductive system,the epididymal initial segment(IS)plays an important role in the transport and maturation of sperm by reabsorbing testicular fluid and maintaining the homeostasis of pH value and osmotic pressure.Its incomplete differentiation will lead to poorer quality sperm and infertility.In clinical practice,the sperm quality of patients treated with rapamycin,an inhibitor of the mTOR pathway,was found to decrease dramatically.Studies have also shown that the mTOR signaling pathway is involved in spermatogenesis,and is essential for spermatogonial differentiation.At present,the role of mTOR in the epididymal initial segment remains unclear.Therefore,the function of mTOR signaling pathway in the epididymal initial segment and its effect on sperm maturation were investigated in this study.For this purpose,we first generated a Lcn8-Cre mouse line using CRISPR/Cas9 technology,in which the Cre recombinase,driven by the endogenous Lcn8 promoter,is specifically expressed a in epididymal initial segment.From postnatal day 21(P21),Cre recombinase is specifically detected in the principal cells,with a significantly high level detected at postnatal 2 months.Although the insertion of Cre sequence disrupted the expression of endogenous Lcn8 protein,the development and reproduction of Lcn8-Cre/+mice were normal.In order to study the role of mTOR in the epididymal initial segment,an epididymis IS-specific Mtor knockout(Mtor cKO)mouse model was generated using the Cre-LoxP system.After mTOR deletion,hypertrophy of the epididymis and blockage of sperm transport in the epididymal duct were observed in Mtor cKO mice,which lead to the mice gradually losing their reproductive ability,and becoming infertile after 4 months old.Loss of Mtor decreased the level of p-ERK1/2,and impaired differentiation of epididymal epithelial cells,which progressively altered cell shape,size and the expression of connectin between cells.Impaired differentiation of principal cells led to decreased expression of some specific genes,which increased the pH value of epididymal luminal fluid.Moreover,the analysis of sperm in epididymal cauda showed a lower percentage of motility and a higher percentage of abnormal sperm compared with wild type,indicating compromised sperm maturation.In addition,the testis in Mtor cKO mice showed the phenomenon of enlargement and then atrophy,with a large number of spermatogenic cells apoptosis in the seminiferous tubules,resulting in reduced sperm count in the mice.mTOR signaling pathway regulates different physiological activities of cells mainly through mTORC1 and mTORC2 complex.To determine which mTOR complex play a role in epididymal initial segment,we then generated mouse models with conditional deletion of m TOR complex core protein Raptor or Rictor from the epididymal initial segment.Similar to the phenotype of Mtor cKO mice,the epididymal initial segment of Raptor cKO mice became hypertrophy at 6 months of age,with sperm obstructed in the epididymal duct.In the Raptor cKO mice,decreased activity of ERK1/2 signaling pathway impaired the differentiation of epithelial cells,and then altere the cell shape and size.As Raptor cKO mice aged,their sperm quality gradually declined and their fertility was impaired.Unlike in Raptor cKO mice,Rictor cKO mice had no obvious abnormalities in the structure of epididymis and testis,as well as sperm quality and fertility.Therefore,in epididymal initial segment,mTOR regulates the differentiation of epithelial cells mainly through its downstream mTORCl signaling pathway,to maintainstable epididymal luminal environment and ensure sperm maturation.However,the role of mTORC2 in epididymal initial segment was not distinct.This study elucidates the role of the mTOR signaling pathway in the development and differentiation of epididymal initial segment,and may provide new insights into the effect of rapamycin treatment on male reproduction.