The Impact Of Autophagy Core Protein ATG5 Deficiency On Epididymal Function And Sperm Maturation

In the past 100 years,there has been a gradual and sharp global decline in sperm count and quality,and the problem of male infertility has gradually become a health issue and a major problem threatening human population.The epididymis is an important organ for sperm maturation and capacitation.Tapping into the mechanisms underlying sperm maturation and quality is essential to improve sperm quality and provide clinical solution to male infertility.Autophagy,an evolutionarily conserved lysosomal catabolism process that degrades and circulates damaged cellular components in the body as well as invading bacteria or viruses,is critical to mammalian reproductive development.The autophagy mechanism exists in the testis,epididymis,and sperm.It participates in the maintenance of the physiological functions of the testis and epididymis,the process of spermatogenesis,and the regulation of the vitality and fluidity of sperm cells.Abnormal levels of autophagy flux in the testis can cause defects in spermatogenesis and infertility,but thus far,there have been few studies on the function of autophagy in the epididymis.In this study,the Atg5 conditional knockout mouse model of principle cells in the 4-5th segments of the epididymal caput was created and used to study the effect of autophagy process in the epididymis on male reproductive system,especially on the maturation of sperm.This study aims to elucidate the regulatory mechanisms underlying sperm maturation in epididymis,and can potentially provide a new therapeutic target for the treatment of male infertility,and thus,bear important clinical significance.Objective:To investigate the role of autophagy core protein ATG5 in maintaining epididymal physiological functions and sperm maturation.Methods:（1）The Atg5 conditional knockout mouse model was constructed by Cre-lox P system in the principal cells of the caput epididymis segment 4-5.Western blot,PCR,q PCR,immunofluorescence and other experimental techniques were used to verify the expression level of ATG5 after Atg5 conditional knock out.（2）We evaluated the changes of autophagic flux in the 4-5th segments of the epididymal caput using Western Blot assays.（3）Proteomic sequencing was used to examine the changes of protein expression in the 4-5th segments of the epididymis caput under normal diet conditions.（4）H&E staining was conducted to observe the morphological changes of the epididymal caput.（5）Under the normal diet condition,the basic indexes of sperm quality were measured with computer-aided sperm analysis（CASA）.The fertility of mice was evaluated by cage mating experiments.（6）Under the condition of natural aging,the effect of Atg5 deficiency on sperm quality was evaluated with computer-aided sperm analysis（CASA）.（7）When the binding of androgen to androgen receptor（AR）was pharmacologically blocked,we used the Western Blot assays to examine the level of autophagy flux in the 4-5th segment of the caput epididymis,and various basic indicators of sperm quality by computer-aided sperm analysis（CASA）.Results:（1）Atg5 was successfully knocked out in the principal cells of the caput epididymis segment 4-5.And the mice were divided into three groups according to genotype:Atg5^f/f,Atg5^f/-and Atg5^-/-.（2）The autophagy marker proteins,LC3-Ⅰand p62,were accumulated,indicating that autophagy was successfully blocked.（3）Under normal diet,there was no significant difference in the morphology of epididymal tissues in the Atg5^f/f and Atg5^-/-mice.（4）Under normal diet,there was no statistically significant difference in sperm quality,litter size,and pregnancy rate.（5）Under normal diet,the loss of Atg5 in the 4-5th segment of the epididymis caput induced changes in the expression of a series of proteins,and some of the up-regulated proteins is functionally related to the Atg5-independent autophagy pathway.（6）Under the condition of natural aging,there has no effect on sperm quality,litter size and pregnancy rate.（7）When the binding of androgen to androgen receptor（AR）was pharmacologically blocked,certain indexes of sperm in Atg5^-/-mice changed significantly.However,no significant changes in autophagic flux were detected in the caput epididymis 4-5th segments,which may be due to the relatively small tissues of the epididymal head 4-5th segments and the total amount of protein expression of these segments was relatively low.Western Blot assays may not be able to accurately detect changes in autophagic flux.It might be possible to see changes in autophagic flux levels with flutamide or cimetidine treatment,or the time for the autophagy pathway to generate stress signals,and the length of action.In future research,it is necessary to further optimize the dose,time of drug administration,and time points of taking the material after drug administration.In addition,we can also combine different experimental techniques to see the changes in the level of autophagy flux in the epididymal head 4-5th segments under the condition of pharmacological block of testosterone receptors.Conclusion:Under normal diet without external challenge,Atg5 conditional knockout led to autophagy blocking of epididymal caput,but the physiological function and the process of sperm maturation are not affected.This phenotype might be due to the activation of some Atg5-independent autophagy pathway,thereby maintaining the normal functions of the epididymal head.More studies are required to delineate the functions of autophagy and ATG5protein in the epididymal caput and sperm maturation.