The Cytological Mechanism Of Spermatogenesis And Sperm Maturion In The Chinese Soft-shelled Turtle And The Zebrafish

The process of spermatogenesis and sperm maturation is related to the survival of a species.The abnormal process often leads to reproductive disorder and male sterility.Studies on reproductive biology of different male animals will helpful not only to understand the animal reproductive regularity,and is also closely related to animal production.The pattern of spermatogenesis altered during evolution of vertebrate from anamniote to amniote.Reptile has a transitional and evolutionary significance in amniote.The Chinese soft shelled turtle(Chelonia)is a traditional aquaculture animal in China,which has high nutritional value.The knowledge of the reproductive biology of Chinese soft shelled turtle,not only has theoretical significance and potential application value for turtle breeding,but can also provide richer information for the studies of reptile animal reproduction.Zebrafish is one of the important model animal,which is representative of aquatic experimental animal.Although there were some reports on the spermatogenesis,but the data is not exhaustive.In this paper we investigated the ultrastructural characteristics of spermatogenesis in both the zebrafish and the Chinese soft-shelled turtle.The feature of zebrafish sperm organization and the modification of the sperm ultrastructure during sperm storage in the Chinese soft-shelled turtle were detected.In addition,the expression of TLR2 and TLR4 were examined respectively in the testis of Chinese soft shelled turtle during breeding period and the zebrafish under the different temperature.We further analyzed the relationship between the organization and function.The characteristic of reproductive biology in this two kinds of male aquatic animal were uncovered at the cellular and molecular level,which would provide a theoretical basis for the study of the evolution of the spermatogenesis pattern,and is the basis for the aquatic animal breeding and resource utilization.Experiment?The spermatogenesis in the Chinese soft-shelled turtle,Pelodiscus sinensis The structural and ultrastructural characteristic of the germ cells were examined by microscopy and electron microscopy during spermatogenesis of Chinese soft shelled turtle.Spermatogenesis underwent type A spermatogonia,type B spermatogonia,primary spermatocyte,secondary spermatocyte,spermatid and sperm.Spermatogonia have apparent nucleolus and heterochromatin,and many organelles in the cytoplasm,including crista-scarce mitochondria,endoplasmic reticulum,Golgi complex and 3-5 small Nuages.Primary spermatocyte increased in volume,with clearly visible synaptonemal complex in nucleus,and developed Golgi complex,dense long mitochondria and Nuages in cytoplasm.Nuages were connected with mitochondria and vesicles.The spermatids experienced elongation and concentration of nuclei,acrosome formation and flagella formation and cytoplasmic exclusion.In addition,there were some special structural changes in the Chinese soft shelled turtle spermiogenesis.(1)During the chromatin condensation,spermatid nucleus was extruded by circular manchette and the redundancy nuclear membrane was discharged from nuclei by forming vacuolate at the end of acrosomal cap.(2)The tubulobulbar complexes appeared on the nuclei of spermatids.(3)The large and dense Nuages existed in spermatids,even in the last stage.(4)A unique endoplasmic reticulum developed in cytoplasm to form chrysanthemum-like structure,which emerged in the spermatids at the first stage,and became vacuolization vacuolization in the last stage.A large number of tubule originated from the chrysanthemum-like endoplasmic reticulum is closely related to the nuclear membrane,lipid droplets,mitochondria,Golgi complex and annulate lamella.(5)During spermiogenesis,the lipid droplets wee accumulated,which were retained in the spermatozoa,becoming the main component of sperm cytoplasmic droplets,and were located in the midpiece of spermatozoa and the posterior of sperm head.These characteristics could provide an important reference for the research on the phylogeny and further relative functional studies.Experiment?Modification of sperm morphology during long-term sperm storage in the reproductive tract of the Chinese soft-shelled turtle,Pelodiscus sinensis Sperm storage in vivo can extend the duration of fertilisation in many animal species for several years,but the storage mechanisms remain unknown.In this study,spermatozoa from the epididymis and oviduct of Chinese soft-shelled turtles were investigated using light and electron microscopy.Distinctive morphological features and transformations of the spermatozoa were found.Large cytoplasmic droplets(CDs)containing lipid droplets were attached to the midpiece and posterior head,without backward movement,of most spermatozoa in the epididymis,whereas they were absent from the spermatozoa in the oviduct,which suggested that CDs with lipid droplets may be endogenous pools of energy for the epididymal spermatozoa.The onion-like mitochondria recovered the double membrane morphology with typical cristae within the oviduct in a later stage of storage,which implied a change in the mitochondrial metabolism in turtle spermatozoa.Furthermore,a developed fibrous sheath on the long principal piece was the integrating ultrastructure for glycolytic enzymes and substrates.These novel morphological characteristics may allow the turtle spermatozoa to use diverse pathways of energy metabolism at different stages of storage.Additionally,Chinese soft-shelled turtles might be a potential model for uncovering the mechanisms responsible for the storage of spermatozoa.Experiment ? Sperm storage and spermatozoa interaction with epithelial cell in oviduct of Chinese soft-shelled turtle,Pelodiscus sinensis Spermatozoa are known to be stored within the female genital tract after mating in various species to optimize timing of reproductive events such as copulation,fertilization and egg laying.The mechanism supporting long-term sperm storage is still unclear in turtles.The aim of this study was to investigate the interaction between the spermatozoa and oviduct in Chinese soft-shelled turtle by light and electron microscopy to reveal the potential cytological mechanism of long-term sperm storage.Spermatozoa were stored in isthmus,uterine and vagina of the oviduct through the year,indicating long-term sperm storage in vivo.Sperm heads were always embedded among the cilia,and even intercalated into the apical hollowness of the ciliated cells in the oviduct mucosal epithelium.The stored spermatozoa could also gather in the gland conduit.There was no lysosome distribution around the hollowness of the ciliated cell suggesting that the ciliated cells of the oviduct can support the spermatozoa instead of phagocytosing them in the oviduct.Immune cells were sparsely distributed in the epithelium and lamina propria of oviduct,although few were found inside the blood vessel of mucosa,which may be an indication of immune tolerance during sperm storage in the oviduct of the soft-shelled turtle.These characteristics developed in the turtle would benefit spermatozoa survival for a long time as extraneous cells in the oviduct of this species.This findings would help to improve understanding of reproductive regularity and develop strategies of species conservation in the turtle.The Chinese soft-shelled turtle may be a potential model for uncovering the mechanism behind the sperm storage phenomenon.Experiment IV The expression of TLR2 and TLR4 in the testis of Chinese soft-shelled turtle,Pelodiscus sinensis Immunohistochemical(IHC)method and Western blot were used to examine the expression and distribution of TLR2/4 in the testis of Chinese soft-shelled turtle(Pelodiscus sinensis)in different months of breeding season.The results showed that TLR2 was expressed in spermatogonia at the basal of the seminiferous epithelium in April,July and November.In April,the spermatogonia showed weak TLR4 immunoreactivity;In July,the spermatogonia,spermatocytes and spermatids have strong TLR4 immunoreactivity.And it was stronger in spermatogonia;In November,TLR4 immunoreactivity became stronger even in the early spermatogenic cells which have been released into the lumen,but it was weak on spermatozoa.The expression of TLR2 protein in the testis had no significant difference between April,July and November.While the expression of TLR4 protein have increased gradually and increased significantly in November.These results suggest that TLR4 have closely related to the spermatogenesis and spermiation in the Chinese soft shelled turtle seminiferous epithelium and emissions.Experiment V The spermatogenesis in zebrafish,Danio rerio The microstructural and ultrastructural changes of germ cells during spermatogenesis of zebrafish(Danio rerio)were examined using light microscopy(LM)and transmission electron microscopy(TEM).Generally the process of spermatogenesis in zebrafish is similar to that of other teleosts,however,here we describe some peculiar features of zebrafish spermatogenic cells which have a limited report in this species.(1)The basic events of spermiogenesis are asynchronous,location of flagellum finished in initial stage,while chromatin condensation sharply occurred in intermediate stage and elimination of excess cytoplasm mainly taken place in final stages.(2)Surprisingly,the cilia or initial flagellae are created in spermatocytes,approach towards the nucleus of early stage spermatids,and then the centrioles depress into nuclear fossa and change their orientation to each other from right angle to obtuse angle about 125°.(3)During spermatogenesis,the chromatin compaction performs in a distinctive pattern,condensed heterogeneously from granular into chromatin clumps with central electron-lucent areas,round or long,which diminished to small nuclear vacuoles in spermatozoa;This finding demonstrates the origin of nuclear vacuoles in zebrafish spermatozoa for the first time.(4)Nuages are observed in both spermatogonia and spermatocytes;They are connected with the mitochondria and nuclear membrane,and are even located in the perinuclear spaces of spermatogonia nuclei.(5)Mitochondrial morphology and distribution shows diversity in different germ cells;The condensed mitochondria appear in pachytene spermatocytes,and mitochondria including membrane conglomerate exist in both spermatocytes and spermatids.This study was undertaken in order to disclose specific spermatogenic cells features in zebrafish that could be helpful for understanding the correlative function in this model species.Experiment VI Fine structure of spermatozoa in the zebrafish(Danio rerio)The microstructural and ultrastructural examination of zebrafish(Danio rerio)spermatozoa was performed to detect the structure,geometrical feature and morphological parameters.Milt samples from 30 zebrafish were examined by use of phase contrast microscopy(PCM)and transmission electron microscopy(TEM).The zebrafish spermatozoa measured 32.799±1.97?m in total length and had a non-acrosomal spherical head,a short cone-shaped midpiece and a flagellum.Heads were lopsided and almost occupied by nucleus with homogeneous dense chromatin,except for a few small electro-lucent vacuoles.The shallow nuclear fossa was mid-laterally located on the nuclear surface and contained the proximal centriole,which was tilted to one side of nucleus and placed at a 125° angle against distal centriole.In the midpiece,the cytoplasmic sheath showed asymmetric organization with round or bended mitochondria gathered at one side,while at the thinnest place,was only little cytoplasm.The flagellum,29.32±1.98?m in length and 242.56±19.27nm in width,was eccentrically positioned in relation to the nucleus.It had a "9×2+2" pattern axoneme,however,there was lack of central microtubules at transition between flagellum and distal centriole.In addition,a few vesicles appeared between the axoneme and the cytoplasmic membrane.Based on the observations,the schematic organization of zebrafish spermatozoon was drawn.The zebrafish spermatozoa possessed some structural features that were specific to cyprinid family,however,they exhibited interspecific differences.Experiment ? Identification of immunohistochemical characterization of TLR2 and TLR4 in the testis of zebrafish The expression and distribution of TLR2 and TLR4 in zebrafish testis under different temperature conditions was examined by immunohistochemistry.The zebrafishes were divided into low temperature group(10-15?)and common temperature group(25-30?).The testis of zebrafish from room temperature group showed weak TLR2 and TLR4 immunoreactivity.While in the low temperature group,the interstitial cells and Sertoli cells appeared TLR2 immunoreactivity,and the spermatogonia and Sertoli cells expressed TLR4 immunoreactivity.These results indicated that low temperature could increase the expression of TLR2 and TLR4 in zebrafish testis,and enhance the ability of the innate immunity.