Cellular Characteristics Of Autophagy And Secretions In The Male Reproductive Systme Of The Chinese Soft-shelled Turtle,pelodiscus Sinensis

The male reproductive system mainly accountable for the production of spermatozoa in association with the Leydig cell steroidogenic activity and transition of spermatozoa through and storage in the excurrent duct for post-testicular maturation.Testosterone produced by the Leydig cell showed seasonal variations in the reptiles and responsible for the production of viable spermatozoa and male development.Though,it is also interconnected with the functional ability of excurrent duct and assist in spermatozoa survival during post-testicular maturation.Excurrent duct specifically efferentes ductuli and epididymis provide enormous rich secreted micro milieu in the lumen to the spermatozoa for gaining the functional and fertility ability.Autophagy is an evolutionary conserved cell programming machinery and has shown its active involvement in male reproductive system such as in spermatogenesis,testosterone production and sperm longevity in epididymis.Nevertheless,these events which accompanied for production of fertile and mature spermatozoa in male reproductive tract of reptile species is still largely unknown.The Chinese soft-shelled turtle is one of the representatives of the reptile species and possess an historic position in China.This turtle specie is widely raised and possessed economical and medicinal significance in China.Therefore,it is worthy to understand the events that accompanied for successfully viable and fertile spermatozoa in the male reproductive tract.Understanding of these cellular features will assist to build the knowledge that may be used to evaluate the reproductive tract in healthy and diseased conditions to produce more productive animals in the future.Our study is the first to demonstrate and characterized the steroidogenic cellular features of Leydig cells and the role of autophagy in consuming the lipid droplet for steroidogenesis during the turtle annual reproductive cycle.The Leydig cell revealed a dynamic steroidogenic structure with strong activity of 3?-HSD,vimentin as an intermediate filament,tubular endoplasmic reticulum,and abundant lipid droplets during hibernation.We also investigate the transit passage of spermatozoa "the efferentes ductuli"that involved in secretions of extracellular vesicles via apical blebbing,ciliary blebbing and from basolateral regions of epithelia.Interestingly at the ultrastructure level these secreted extracellular vesicles interacted with the transited spermatozoa.During sperm storage in the epididymis to gain the functional and morphological ability,the epididymal segments showed distinct interactions with stored spermatozoa by providing the nourishment through epithelial secretion either in the lumen or in the cytoplasm.Furthermore,the role of epididymis as a quality control organ demonstrated by epithelial phagocytosing weak or abnormal spermatozoa through lysosome and autophagy activity,which is a vital phenomenon to assure the quality of fertile spermatozoa.These accompanied events in the male reproductive system provide a deliberate phenomenon in turtle specie and to understand the diver's knowledge among different species.Experiment-1 Lipophagy:A novel way of steroidogenic activity within the Leydig cell during reproductive cycle in the turtle.Steroidogenesis is indispensable that indirectly associate with spermatogenesis in Leydig cell(LC)to utilize the lipid droplets(LDs)is critical to maintaining the normal testosterone synthesis.The regulation of LDs mobilization,known lipophagy,in LC is still largely unknown.In present study,LC of Chinese soft-shelled turtle was investigated to identify steroidogenic structure and lipophagy during the annual reproductive cycle by light microscopy,immunohistochemistry(IHC),immunofluorescence(IF),and transmission electron microscopy(TEM).The LC showed dynamic steroidogenic structure with strong activity of 3?-HSD,vimentin and tubular ER during hibernation by immunohistochemistry(IHC)and TEM.Tubulo-vesicular ER with weak immunopositivity of 3?-HSD in LC during spermatogenesis,suggesting the persistent activity of steroidogenic activity.The ORO staining and TEM demonstrated that the larger number of LDs had accumulated in LC during hibernation than spermatogenesis phase.These LDs were closely existed with mitochondria and lysosome by dynamic surrounding of intermediate filaments to facilitate the LDs utilization.Lysosome was found directly attached with large LDs by forming autophagic tube and showed LDs engulfing,suggesting the micro-lipophagy during hibernation.Furthermore,immunohistochemistry of ATG7 and immunofluorescence of LC3,P62 and LAMP1 results demonstrated the strong expressions and further confirmation by TEM showed the existence of autophagosome,autolysosome and their fusion during hibernation season.In conclusion,present study provides clear evidence of LD consumption in LC by lipophagy,lysosome and mitochondria during the hibernation period,which is a key aspect of steroidogenesis in Chinese soft-shelled turtle.Experiment-2 Characterization of Extracellular Vesicles from Cilia and Epithelial Cells of Ductuli Efferentes in a Turtle(Pelodiscus sinensis)The ductuli efferentes(DE)form a transit passage for the passage of spermatozoa from the rete testis to the epididymis.After spermiation,various epithelial secretory proteins are transferred via extracellular vesicle's(EVs)to the spermatozoa for their maturation and long-term viability.The aim of the present study was to investigate the distribution,classification,and source of multivesicular bodies(MVBs)and their EVs in the epithelia of the efferentes duct in a turtle species,the soft-shelled freshwater turtle Pelodiscus sinensis by using light and transmission electron microscopy.The results showed that CD63 as a classical exosome marker was strongly immunolocalized within the apical and lateral cytoplasm of the ciliated cells(CC)and moderate to weak in the nonciliated cells(NCC)of DE.The ultrastructure revealed that early endosome was present at the basement membrane and perinuclear cytoplasm of both CC and NCC,whereas MVBs were located over the nucleus in the cytoplasm of NCC and adjacent to the basal bodies of cilia within the CC.Many EVs,as sources of MVBs,were located within the blebs that were attached to the cilia of CC,within the apical blebs from NCC,and the lateral spaces of CC and NCC.There was ultrastructure evidence of EVs associated with spermatozoa in the lumens of DE.Collectively,the present study provides cytological evidence that the DE epithelium secreted EVs to the lumen by(1)apical blebs,(2)ciliary blebs,and(3)from the basolateral region.These EVs were associated with spermatozoa in the DE lumen of this turtle.Characterization and cellular distribution of these EVs in the DE of a turtle may provide a study model to further investigate the transferring of micromolecules via EVs to the spermatozoa.Experiment-3 Interaction of epididymal epithelia and their secretion with spermatozoa support functional and morphological changes during long-term storage in the Chinese soft-shelled turtle(Pelodiscus sinensis)Post-testicular maturation of spermatozoa is crucial for attaining the morphological and functional capabilities needed for successful fertilization.Epididymal epithelia offer a favorable milieu for sperm that are stored for long term in turtle epididymis,however sperm-epithelial interaction during storage which is enormously important for sperm functional and morphological maturation is still largely unknown in turtle.The present study examined the epididymis during the sperm-storage period(Nov-Apr)in Chinese softshelled turtle(Pelodiscus sinensis)by using light and transmission electron microscopy to determine the cellular features of each epididymis segments(caput,corpus and cauda)and their epithelial interaction with the spermatozoa.When viewed with light and ultrastructure,spermatozoa mainly located in the lumena of caput,corpus and cauda epididymis.Numerous spermatozoa were bound to apical surfaces of epithelia and several were even embedded in the epithelial cytoplasm of caput and corpus epididymis,while no embedded spermatozoa were found in the cauda epididymis.In all epididymis segments,principal and clear cells showed synthetic activity,evidenced by a well-developed endoplasmic reticulum network and,high and low electron-density secretory materials respectively.Principal and clear cells in the caput and corpus segments showed embedded spermatozoa in electrondense secretions and in the lipid droplets within the cytoplasm.No lysosomes were observed around the embedded spermatozoa.The lumena of the caput and corpus segments showed few apocrine and low electron density secretions.In the lumen of the cauda epididymis,we found and summarized different secretions such as holocrine with low and high electron density and their fragmentation,apocrine and dictyosome.Altogether,sperm physical interactions with secretions either in the cytoplasm of epithelium or in the lumen may provide the nourishment,morphological maintenance,and transfer of various proteins for the long-term sperm storage in the turtle.This interaction could help to understand the long-term storage and provide more insight to the reproductive strategies of turtle sperm preservation.Experiment-4 In vivo cellular evidence of autophagic associated spermiophagy within the principal cells during sperm storage in epididymis of the turtle.The epididymis plays a significant role as a quality control organ for long-term sperm storage,maturation,and fertilizing ability and perform filtration function to eliminate abnormal or residual spermatozoa by phagocytosis.However,the role of autophagy in spermiophagy during sperm storage in turtle epididymis still needs to be studied.In this study,we reported in vivo spermiophagy via the cellular evidence of lysosome engulfment and autophagy within the principal cells during sperm storage in the turtle epididymis.Using immunofluorescence,Lysosome associated membrane protein-1(LAMP1)and microtubule-associate protein light chain 3(LC3)showed strong immunosignals within the apical cytoplasm of epididymal epithelia during hibernation than non-hibernation.Co-immunolabeling of LAMP1 and LC3 was strong around the phagocytosed spermatozoa in the epididymal epithelia and protein signaling of LAMP1 and LC3 was confirmed by western blotting.During hibernation,ultrastructure showed epididymal principal cells was involved in spermiophagy and was characterized by the membrane's concentric layers around phagocytosed segments of spermatozoa,degenerative changes in the sperm head and lysosome direct attachment,and with the existence of cellular components related to autophagy(autophagosome,autolysosome).In conclusion,spermiophagy occurs by lysosomal engulfment and autophagic activity within the principal cells of the turtle epididymis during sperm storage.