Regulatory Mechanisms Underlying Spermatogenesis And Sperm Storage In Chinese Soft-shelled Turtle(Pelodiscus Sinensis)

Dissociated spermatogenesis and sperm storage are specific reproductive strategies evolved by some animals to adapt different living environment in evolution.Studies have shown that dissociated spermatogenesis and long-term sperm storage in vivo are used by a variety of seasonally breeding animal species,including birds,fish and reptiles.However,due to the lack of effective animal models as technical support and experimental platform,the cellular and molecular mechanisms underlying dissociated spermatogenesis and sperm storage have not been made substantial progresses.Elucidated the regulatory mechanism of dissociated spermatogenesis and sperm storage is a breakthrough to build new breeding technology on sperm liquid storage in vitro and also is a fruitful research field of animal reproductive biology.The Chinese soft-shelled turtle(Pelodiscus sinensis)belonging to ectothermic amniotic reptiles,has high economic and pharmaceutical value and is widely distributed in Asian countries such as China,Japan and Korea.Previous studies have shown that P.sinensis has a typical pattern of dissociated spermatogenesis and oviduct sperm storage.Thus,the Chinese soft-shelled turtle can be used as a desirable animal model to explore the molecular mechanisms of the dissociated spermatogenesis and sperm storage.In this study,the germ cells apoptosis of the P.sinensis during spermatogenesis were examined by transmission electron microscope(TEM)and TdT-mediated dUTP nick end labeling(TUNEL)assay,to uncover the close relationship of apoptosis and dissociated spermatogenesis at cellular level.RNA sequencing(RNA-Seq)and digital gene expression profiling(DGE)were performed from P.sinensis testis to identify apoptosis-related differentially expressed genes(DEG)and analysis their regulatory roles during spermatogenesis.Moreover,the identification of sperm storage-related critical DEG involved in immune,apoptosis and autophagy in the oviduct of P.sinensis were performed,which would clarify the regulatory mechanism underlying sperm storage.Detecting the dynamic level changes of androgen and androgen receptor(AR)would verify the regulatory roles of androgen in sperm storage in the oviduct of P.sinensis.The outcome of this study would provide a theoretical basis for the in-depth study of dissociated spermatogenesis and sperm storage at cellular and molecular levels.The main achievements obtained were as follows:Experiment I Apoptosis of germ cells during spermatogenesis in Chinese soft-shelled turtle The morphological characteristics of the P.sinensis seminiferous epithelium were examined by microscopy in different stages of spermatogenesis.The ultrastructural and structural characteristics of the seminiferous epithelium apoptosis were examined by TEM and TUNEL assay during normal spermatogenic cycle.The results showed that the microstructure of the P.sinensis seminiferous epithelium exhibited significant seasonal changes.In April,the spermatogenically quiescent phase,the seminiferous epithelium contained 2～3 layers of spermatogonia,the residual spermatids and spermatozoa from previous spermatogenesis cycle randomly distributed in the lumen of seminiferous tubules.In July,the active phase of spermatogenesis,the seminiferous epithelium formation sperm columns from the basal to the adluminal compartment with radial arrangement,and little scattered spermatozoa that located in the lumen.In October,the late phase of spermatogenesis,the majority of cells in the seminiferous epithelium were spermatids and many scattered spermatozoa that located in the lumen.A large number of apoptotic cells were observed in April by TUNEL staining.These TUNEL-positive cells were mainly spermatids and spermatozoa from previous spermatogenesis.In intermediate and later stages of spermatogenesis(July and October),the number of apoptotic cells decreased dramatically,and the apoptotic cells were mainly distributed among the spermatocytes and spermatids.The morphology of apoptotic germ cells under electron microscope includes chromatin condensation being mostly half-moon type or massive structures appear around the nucleus;Cell bodies became smaller and distinct separation with surrounding cells;Cytoplasm appeared vacuoles and small double-membrane-bound apoptotic bodies were occasionally detected.These results have implied that apoptosis may be employed as one of the key mechanisms to clear the seasonal degraded germ cells in P.sinensis.Experiment Ⅱ Transcriptomic analysis identifies apoptosis-related differentially expressed genes during spermatogenesis in Chinese soft-shelled turtle RNA-Seq and DGE tag profiling using the P.sinensis testis in different stages of spermatogenesis(April,July and October)were performed with the Illumina HiSeq 2500 platform.The results showed that a total of 4,622(2,514 up-and 2,108 down-regulated),4,572(2,555 up-and 2,017 down-regulated)and 380(163 up-and 217 down-regulated)DEG were obtained from three libraries,respectively.Kyoto Encyclopedia of Genes and Genomes(KEGG)-based analysis identified 42 DEG being involved in the Apoptosis pathway and p53 signalling pathway.Some critical anti-apoptosis genes(such as Bcl-2,BAG1 and BAG5)showed up-regulated patterns during intermediate and late spermatogenesis.Furthermore,the mitochondrial transmembrane potential and critical apoptosis protein were characterized and profiled by JC-1 fluorescent probe,immunohistochemistry(IHC)and Western blot analysis.The results showed that the mitochondrial transmembrane potential was significantly higher and the pro-apoptotic protein Cleaved caspase-3 and the release of CytC from the mitochondria were significantly lower in July and October.These results have indicated that the differential expression of apoptosis-related genes plays a major role in the regulation of dissociated spermatogenesis,and the CytC-Caspase model involved in the p53 signalling pathway might be responsible for the effects of germ cell apoptosis on seasonal spermatogenesis in P.sinensis.Experiment IH Immune,apoptosis and autophagy are involved in the molecular regulation of sperm storage in oviduct of Chinese soft shelled turtle The histological and ultrastructural characteristics of sperm storage in the oviduct of P.sinensis were examined by microscopy and TEM.The results showed that during the hibernation season(from November to the next April)spermatozoa were stored in the vagina,uterus and isthmus of the oviduct.These spermatozoa were interspersed among the epithelial cilia and in close contact with the epithelial cells.RNA-Seq and DGE tag profiling from the P.sinensis oviduct during reproductive(July,none sperm storage)and hibernation(January,sperm storage)seasons were performed using the Illumina HiSeq 2500 platform.The results showed that a total of 2,622 DEG including 1,224 up-and 1,438 down-regulated genes were obtained from two libraries.Functional annotation and enrichment analysis suggested that these DEG were predominately involved in the immune response,apoptosis signalling pathway and regulation of autophagy.Furthermore,the expression profiles of mRNA and protein in critical DEG related to sperm storage were examined by RT-qPCR,Enzyme linked immunosorbent assay(ELISA),IHC and Western blot analyses.During the phase of sperm storage,TUNEL analysis revealed the reduced number of apoptotic cells;IHC and TEM analyses found that autophagy occurred in the oviduct epithelial cells,where the spermatozoa were closely attached.These results provide fundamental insights into the immune response,apoptosis signalling pathway and regulation of autophagy involved in the seasonal regulation of sperm storage in oviduct of P.sinensis.Experiment Ⅳ Roles of androgen and androgen receptor regulate sperm storage in oviduct of Chinese Soft-Shelled Turtle The concentrations of circulating androgens in the serum of female P.sinensis were detected during hibernation(from November to the next April,sperm storage)and reproductive(July,none sperm storage)seasons using ELISA.The results showed that the concentrations of circulating testosterone and dihydrotestosterone were relatively higher during sperm storage.IHC analysis revealed that androgen receptor(AR)was distributed throughout the cytoplasm of gland cells and among the cilia of ciliated cells.Moreover,significantly increased in protein and mRNA levels of AR during sperm storage were validated through Western blot and RT-qPCR analyses.Furthermore,the roles of androgen regulate sperm storage was inversely analyzed after subcutaneous injection of flutamide(anti-androgen)to inhibitory androgens activity.The results showed that the concentrations of circulating androgens and the quantity of spermatozoa in the oviduct were significantly decreased,and the level of oviduct apoptosis was increased.These results implied that androgen regulating the long-term sperm storage and underlying mechanisms might be associated with oviduct apoptosis.