Cytological Study On Seasonal Changes Of Seminiferous Tubule Boundary Membrane In Pelodiscus Sinensis

Chinese soft shelled turtle,also known as Pelodiscus sinens,is one of the largest scale reptiles in China.Turtle meat has high nutritional value.The reproduction of male Chinese soft shelled turtle(Pelodiscus sinensis)is different from that of mammal,and its spermatogenesis has seasonal variation.In November,Pelodiscus sinens enters hibernation period,and its metabolism is slow until the end of next April.At this stage,spermatogenesis gradually stopped and only a small amount of spermatozoa remained in seminiferous tubules.In May,Pelodiscus sinensis entered the breeding period with vigorous metabolism until the end of October of that year.At this stage,spermatocytes divide into sperm cells and gradually metamorphose into mature sperm.The boundary membrane of seminiferous tubules in Pelodiscus sinensis consists of fibrocyte and extracellular matrix.The boundary membrane is an important part of the blood testis barrier,which can promote sperm transport to epididymis and nourish sertoli cells and spermatogonia in seminiferous tubules.The distribution of telocytes in the interstitial tissues of male reproductive system is still unknown.In this experiment,hematoxylin eosin staining,masson staining and toluidine blue staining were used to observe the changes of the limiting membrane and seminiferous tubules of Pelodiscus sinensis.Immunohistochemistry was used to determine the location of TCs in the testicular limiting membrane of Pelodiscus sinensis.Immunofluorescence double labeling was used to determine the immunophenotype of TCs.Transmission electron microscopy was used to verify the above results.The cytoplasmic components of TCs were determined by mitochondrial tracer staining.The purpose of this research is to study the peritesticular changes from the perspective of cell morphology,and provide morphological theoretical support for TCs to participate in the formation of blood testis barrier,maintain the homeostasis of internal environment,and participate in the seasonal changes of sperm.Experiment Ⅰ histological characteristics of testicular limiting membrane in Pelodiscus sinensis.In order to provide a theoretical basis for the further study of the limiting membrane of Pelodiscus sinensis,the testis of two reproductive stages were taken as the research object to explore the changes of cell components in the limiting membrane of testis.The testicles of healthy and mature wild Pelodiscus sinensis were harvested during hibernation(April)and breeding(September).HE staining,masson staining,toluidine blue staining and immunohistochemical staining were used to observe the changes of testicular tissue and cell composition in different stages of Pelodiscus sinensis.The expression of myoid fibroblasts was detected by immunohistochemistry.The changes of seminiferous tubules and testicular limiting membrane were statistically analyzed.The results showed that the area of seminiferous tubules in reproductive period was larger than that in hibernation period,and the thickness of spermatogenic epithelium was thicker than that in hibernation period;the area of testicular boundary membrane in hibernation period was larger than that in reproductive period,and the boundary membrane was thicker than that in reproductive period.The results showed that there were a lot of cells and collagen fibers in the intercellular matrix,but there were less cells and collagen fibers in the boundary membrane of testis during the reproductive period.The myofibroblasts in testis of Pelodiscus sinensis during the hibernation period had an obvious multilayer diffuse structure compared with those in the reproductive period,but the stratified structure of myofibroblasts in Pelodiscus sinensis testis during the reproductive period was not obvious.It is suggested that this structural change may be related to the physiological function of Pelodiscus sinensis at different reproductive stages,which also provides a reference for the study of the changes in the composition of the limiting membrane and spermatogenesis disorder.Experiment Ⅱ changes of TC in seminiferous tubules of Pelodiscus sinensis.This experiment focused on the peritubule of testis in Pelodiscus sinensis,and used immunohistochemistry,immunofluorescence,cell culture,TEM and other methods to study the seasonal changes of peritubule of testis in Pelodiscus sinensis,identify the characteristics of peritubule TCs of testis in Pelodiscus sinensis,and analyze the morphological changes of peritubule TCs of testis in Pelodiscus sinensis so as to explore its potential function.The results showed that CD34 could be regarded as a reliable TCs marker in different reproductive stages;Vimentin,as a common TCs marker,could not label the testicular TCs in hibernating Pelodiscus sinensis,but could only label partial telopodes of TCs in reproductive stage,but Vimentin could be used as a reliable marker of sertoli cells in different reproductive stages;androgen receptor could express a large number of leydig cells and label partial tp in hibernating stage.The results of cell culture showed that the growth of TCs was not obvious 24 hours after culture,but grew rapidly 48 hours after culture.By 96 hours,TCs could form junctions with the same or different cells.Cell immunofluorescence assay showed that the cultured TCs could be labeled with CD34.The results showed that there were a lot of mitochondria in the cytoplasm of TCs.The distribution of TCs in different seasons was confirmed by transmission electron microscope.During hibernation,TCs were widely distributed in the testicular stroma of Pelodiscus sinensis.During reproduction,TCs distributed close to the outside of myoid fibroblasts,and formed cell connections between TCs and myoid fibroblasts.The labyrinth like structure of TCs in the testis of Pelodiscus sinensis was observed,which proved that TCs in the testis of Pelodiscus sinensis had strong bioavailability TCs can also form a three-dimensional network structure with myoid fibroblasts,blood vessels and leydig cells.TCs plays the role of transmitting information through secretory vesicles.TCs,myoid fibroblasts and extracellular matrix form the blood testis barrier.TCs may play an important role in nourishing sperm and transporting energy,which is of great significance to the reproductive development of Pelodiscus sinensis.