Distribution Of Microtubule-Associated Proteins Katanin,CAMSAP2 And EB3 During Testis Development And Spermatogenesis Of Hu Sheep

The testis,the male reproductive organ of mammals,has the primary responsibility of producing sperm and secreting androgens.The testis is one of the most abundant sources of microtubule networks.These networks include mitotic and meiotic spindles,the spermatid manchette and axoneme,and the Sertoli cell cytoskeleton.It is important to investigate the morphological structure and developmental patterns of microtubules in sheep testis development and spermatogenesis.In this study,the localization and relative expression of microtubuleassociated proteins Katanin,CAMSAP2 and EB3 in testis tissues of sheep at different months of age were firstly determined by western blotting and immunofluorescence.Subsequently,the morphological analysis of testicular tissue structure was performed to divide the cycle of the seminiferous epithelium of sheep,and then the distribution of microtubules in different stages of the cycle of the seminiferous epithelium of sheep was analyzed.Then the spermatozoa in the spermatogenesis of Hu sheep were divided by spermatids isolation and immunofluorescence technique,and then the changes of Katanin,CAMSAP2 and EB3 in the spermatogenesis were analyzed,in order to analyze the roles of these three microtubule-associated protein in spermatogenesis.Results of the study is shown as follow:(1)Morphological analysis of the testicular histology of Hu sheep showed that the area and the diameter of the varicose ducts and the number of germ cells increased gradually with the development of the testis at 1 month,4 months,6 months and 10 months of age.Western blotting and immunofluorescence analysis of microtubuleassociated proteins Katanin,CAMSAP2 and EB3 in testis of Hu sheep at different months of age showed no significant difference in the expression levels of CAMSAP2;the expression levels of Katanin and EB3 increased with the increase of Hu sheep’s age.In the testis of 4-and 6-month-old Hu sheep,Katanin,CAMSAP2 and EB3 signals were localized near the sperm cells in the seminiferous tubules.(2)The spermatids at each step during spermiogenesis were distinguished by labeling the developing acrosome with the Schiff’s periodate reagent,thus dividing the cycle of the seminiferous epithelium of Hu sheep testis into 12 stages and the spermiogenesis of Hu sheep into 14 steps.Among them,manchette appeared in stage VIII and disappeared in stage Ⅻ in the cycle of the seminiferous epithelium of Hu sheep.(3)By immunofluorescence on microtubule-associated proteins,Katanin was localized near the basal body of step 8 spermatids during the spermiogenesis in Hu sheep.EB3 was localized on the manchette of Hu sheep spermatids and mainly clustered near the marginal ring.CAMSAP2 was localized on the manchette of steps10-12 spermatids.In summary,The cycle of the seminiferous epithelium of Hu sheep can be divided into 12 stages and the spermiogenesis can be divided into 14 steps,in which the manchette starts at step 8 spermatids and begins to disappear at step 12 spermatids.The microtubule-associated proteins Katanin,EB3 and CAMSAP2 are expressed during spermatogenesis.