Studies On Mechanism Underlying Sperm Long-term Storage In Epididymis Of The Chinese Soft-shelled Turtle

The transformation of reproductive system in vertebrate from external fertilization to internal fertilization has a great significance in biological evolution.The requirement for internal fertilization sometimes results in a delay between mating and fertilization,and a need for spermatozoa to remain alive long enough to fertilize eggs.In fact,spermatozoa of animals with internal fertilization survive and are stored for variable periods of time before mating inside the male or after mating inside the female.Sperm storage is an important reproductive strategy in the animal kingdom,which can affect spermatozoa longevity,mating system evolution.Because sperm storage extends the time window for fertilization from a few days to several years,it increases the opportunity of successful conception.Sperm storage is also an adaptive strategy in ecologically diverse habitats and can result from different selective forces acting on females and/or males,sometimes resulting in coevolution.Sperm storage is likely ubiquitous in most animals with internal fertilization.The duration of sperm storage in different species can range from hours to days,months and even years.However,many of the species that long-term store spermatozoa are threatened and therefore protected by law.Thus,the molecular mechanism underlying long-term sperm storage is largely unknown.The Chinese soft-shelled turtle,Pelodiscus sinensis,belonging to ectothermic amniotic reptiles that hibernate from December to April in Nanjing,is widely raised in freshwater lakes of central and southern China.The Chinese soft-shelled turtle has an adaptive reproductive strategy of sperm long-term storage during species evolution.Spermatogenesis in P.sinensis is a seasonal event,which starts in spring and continues through summer and early autumn.After spermiation in October(late autumn in Nanjing,China),immature spermatozoa are transferred into the epididymis and stored there over hibernation until the following mating season(June to August).Spermatozoa of the P.sinensis can be stored in the epididymis and oviduct for at least six months and one year,respectively.Thus,the Chinese soft-shelled turtle can be used as a potential model organism to explore the molecular mechanisms of the long-term sperm storage in vivo.In this study,the morphological changes of epididymal spermatozoa were observed by TEM at different periods of sperm storage to reveal the relationship between structure changes and long-term storage.RNA-Seq analysis was performed in testis of P.sinensis to identify critical differentially expressed genes(DEGs)involved in intracellular lipid droplets(LDs)development during spermiogenesis.The LDs of spermatozoa serves as an important source of energy,which contributes to long-term sperm storage in the epididymis of P.sinensis.Based on the above findings,the detection of lipophagy and lipase activity in spermatozoa would further elucidate the molecular mechanism of sperm long-term storage.Moreover,exosomes released from epididymal epithelium are key factors for nutrient and message supply during sperm storage.The epididymal spermatozoa from January were able to survive more than 40 days at 4?.The spermatozoa showed apoptotic-like changes during the time of cooled storage.All of results in present study would clarify the molecular regulation mechanism underlying sperm long-term storage from different perspectives in the Chinese soft-shelled turtle.The main findings are as follows:Experiment ? Morphological changes of spermatozoa during epididymal storage in the Chinese soft-shelled turtle Spermatogenesis in P.sinensis is seasonal,with spermiation occurring in October(late autumn in Nanjing,China).Next,immature spermatozoa are transferred into the epididymis,where they are stored over hibernation until the following mating season(from June to August).The present study analysed the dynamic changes and ultrastructural characteristics of epididymal spermatozoa at different stages of storage in P.sinensis.The results showed that numerous spermatozoa were observed within the lumen of epididymis during storage periods.Oil Red O staining found that a number of LDs were present on epididymal spermatoza.TEM results indicated that a large cytoplasmic droplet(CD)was attached along the entire midpiece and posterior head of most epididymal spermatozoa in this turtle.There are several LDs inside the CD,and each of the LD has a diameter of approximately 0.5 ?m.In the early stage of sperm storage(January),LDs were distributed within the CD.During the intermediate stage of storage,the number of LDs inside CD reduced significantly.After long-term storage in the epididymis,the volume of CD in spermatozoa had decreased,and the LDs had been consumed.Finally,the CD disappeared completely and the mature spermatozoa were vermiform in shape.These findings suggest that LDs of spermatozoa serves as important energy source,which can sustain long-term sperm storage in the epididymis of the P.sinensis.Experiment ? Autophagy enhances LDs development during spermiogenesis in the Chinese soft-shelled turtle Spermatogenesis of P.sinensis is a seasonal event,and can be divided into three major developmental stages.Stage 1,the spermatogenically quiescent phase(from December to the next April);Stage 2,the round spermatids phase(intermediate spermatogenesis,from May to July);Stage 3,the elongating spermatids phase(later spermatogenesis,from August to October).TEM and Oil Red O staining results found that the number and size of LDs within elongating spermatid increased considerably during the process of spermiogenesis.IHC and IF results showed that the positive reaction of LC3 was mainly present in the elongating spermatids of the testis.RNA-Seq analysis revealed that autophagy was highly activated via the PI3K pathway during spermatogenesis.Inhibiting autophagy with 3-MA significantly decreased testicular triglycerides(TGs)and fatty acids(FAs)content.In comparison with the control group,the number and size of LDs within the elongating spermatids was reduced significantly in the 3-MA group.Taken together,our results showed that FAs released through the autophagic degradation of germ-plasm was replenished LDs of spermatid,increasing LD number and size,during spermiogenesis in testis.These LDs would be served as an important energy source to sustain long-term sperm storage in the epididymis of the P.sinensis.Experiment ? Lipophagy contributes to long-term storage of spermatozoa in the epididymis of the Chinese soft-shelled turtle Ultrastructural changes of epididymal spermatozoa were observed by TEM in different stages of storage.TEM results indicated that LDs were scattered within the CD and each of the LD was surrounded by pro-autophagic vesicles(isolation membrane,IM).Occasionally,large LD were penetrated by IM that appeared to sequester them into smaller LDs in the early stage of storage(January).In the later stage of storage(July),the number of autophagosomes inside a CD increased markedly,conversely,the number of LDs decreased.The seqeustered cargo could be clearly seen within autophagosome.Immunohistochemistry and western blot demonstrated that autophagy activity in spermatozoa increased gradually with the extension of storage time.LC3,a reliable marker of autophagy,was also detectable in LDs isolated from the spermatozoa.In vitro studies found that spermatozoa obtained from January can survive more than 40 days at 4?.Furthermore,IF and TEM indicated that autophagy was involved in the degradation of LDs with the extension of sperm cooled storage.After inhibition autophagy with 3-MA,the breakdown of LDs within CD was suppressed significantly.Moreover,adipose triglyceride lipase(ATGL),a critical hydrolase,was also involved in the degradation of LDs.These results indicate that lipophagy and lipolysis were activated to maximise LDs breakdown,which can supply endogenous energy to maintain long-term sperm storage in the epididymis of this species.Experiment ? Exosome released by epididymal epithelium contribute to long-term sperm storage After spermiation in the testis,spermatozoa are functionally incompetent and are unable to fertilize an oocyte even though they are morphologically mature.Fertilization ability is acquired during spermatozoa transit through the epididymis,a process called sperm maturation.The epididymis is also a place where spermatoza be stored for a long time.Recent studies have shown that nano-scale exosomes released by epithelium play vital roles during sperm maturation and storage.IHC results showed that the exosome marker CD63 was primarily localized to the apices of principal cells throughout the epididymal epithelium.There were many CD63-positive particles scattered between spermatozoa within the lumen of epididymis.Some CD63-positive structures appeared to be shed from principal cells and the CD63 expression was similar at different stages of sperm storage.Identification of exosomes secretory process and their absorption pathway were further investigated via TEM.TEM results indicated that the epithelium secreted epididymal exosomes(50?300 nm in diameter)through apocrine secretion and the multivesicular bodies(MVB)pathways.Intraluminal spermatozoa can absorb exosomes through endocytosis and membrane fusion pathways.Taken together,our results suggest that the epididymal exosomes may act as messengers for epithelium-spermatozoa communication.Meanwhile,exosomes contact and absorption with spermatozoa in different regions of epididymis are important factors for long-term sperm storage in the epididymis of the P.sinensis.Experiment ? Apoptotic-like changes in epididymal spermatozoa of the Chinese soft-shelled turtle during cooled storage Spermatozoa of the P.sinensis can be stored in the epididymis for at least six months.In the present study,spermatozoa from the epididymis in January were investigated to identify the survival period and structural changes during cooled storage in vitro.We observed that the spermatozoa were able to survive more than 40 days at 4?.During the time of cooled storage,sperm kinematics was evaluated by CASA system.All sperm motility parameters decreased over the period of storage,while the velocity curvilinear(VCL)and velocity average path(VAP)had a further decrease after 20 days of storage.On day 30,the beat cross frequency(BCF)decreased gradually,and most spermatozoa remained in their original position.Flow cytometry analysis indicated that more than 70%spermatozoa was non-viable on day 40,and there were almost no viable spermatozoa on day 45 of cooled storage.TEM and TUNEL assay found that the spermatozoa showed apoptotic-like changes after 20 days of cooled storage.The nuclear fragmentation of spermatozoa was observed on day 30,and the percentage of apoptotic spermatozoa increased significantly.After 20 days of cooled storage,the mitochondria were to swell and their shape changed from onion-like to elliptic.Meanwhile,the concentric membranes of mitochondria were dispersed.Intracellular ROS concentration was measured using DCFH-DA,and found that the ROS production was rapidly induced after 20 days of cooled storage.The mitochondrial membrane potential(MMP)of spermatozoa decreased over the time of cooled storage,which indicates that there was a lose of the MMP.Moreover,the high levels of pro-apoptosis proteins,such as Cyt c,Cleaved Caspase-9/3,were detected by western blot after 30 days of spermatozoa cooled storage.These findings showed that epididymal spermatozoa of P.sinensis can be stored for a long time at 4?.During the cooled storage,the ROS generation was critical for mitochondria damage,which can trigger apoptotic-like changes of spermatozoa.