Sperm Storage And Its Correlative Structures In Soft-Shelled Turtle, Trionyx Sinensis

Unlike mammals and birds, most of the studied reptilian species exhibit an unusualreproductive cycle whereby spermatogenesis and ovulation are out of phase with each other.Thus, storage by either males or females promotes the wide dissemination of gametesproduced in a narrow time window. However, the studies of sperm storage in reptiles arelimited and the only reports are available in turtles, lizard and snake. Soft-shelled turtle,Trionyx sinensis distribute widely in China and this species is famous for its importantvalue in economy and in pharmacy. This species exhibit an unusual reproductive cyclewhereby spermatogenesis and ovulation are out of phase with each other. But there are nostudies that have been reported the character of sperm storage in Trionyx sinensis. Inpresent study, we examined the ultrastructure characters of the sperm storage as well as therelationship between stored spermatozoa and the host cells that surround them in male andfemale turtles, and then give a further analysis of the cytology of the stored spermatozoa inthe sperm storage tubules.ExperimentⅠSperm storage in male soft-shelled turtles, trionyx sinensis Reproductiveducts of male soft-shelled turtles, Trionyx sinensis were examined throughout the year(March, May, September, December) using brightfield and electron microscopes(TEMand SEM), to determine the location and histomorphological characteristics of spermstorage structures as well as their changes at different phases of the seasonal reproductivecycle. Sperm stored in the epididymis was also examined. In male, spermatogenesisinitiates in spring (May), and then the mature sperm were released in autumn as an episodicevent. Spermatogenesis was inactive in winter. However, in this species, the epididymiscontains sperm throughout the entire year. The epithelial cell type of epididymal ductchanged in different seasons. The cells were fully developed with a highly secretoryactivity in September. Sperm observed in the epididymis were intact in structures and somestructures were unique differently from that of other of reptiles, which is characterized by35-40 concentric mitochondria with a dense core in the center. Lots of glycogen granuleswere observed in the cytoplasm of midpiece. Cells comprising the pseudostratified epithelium of the epididymis are principal cell, base cell and clear cell. The epithelial cellsare active in secrete and reabsorption, numerous secretory granules and vesicles were foundin the cytoplasm The rough endoplasmic reticulum (RER) is present in the wholecytoplasm and the cisternae of RER are inflated due to the filling of the secretory materials.The materials secreted from the epithelium might have the function as the noruishment forthe stored sperm.ExperimentⅡHeterogeneity of the spermatogenic cells in the epididymis ofsoft-shelled turtle, Trionyx sinensis The development of the spermatogenic cells in theepididymis of soft-shelled turtle, Trionyx sinensis was examined by electron and scanningmicroscopes. The mature sperm observed in the epididymis is vermifom in shape, whichconsists of a head, connecting piece and tail (middle piece,principal piece and end piece).However, portions of spermatogenic cells observed are presumed to be immature ones.Based on the development stage of spermatogenic cell, we divided these immature cellsinto types as follows:The first type is round sperm, of which the chromatins are observedas small, low electron density, homogeneous granules. Golgi complexes are accumulatednear the nuclear, which have functions in the forming of the pre-acrosomal vesicle.Mitochondria are sparse. Under scanning microscope, the surface of round sperm isconcavoconvex, and several holes could be found. The second type is developingspermatogenic cells. The acrosomal complex has been formed, and the chromatins are inthe condensing process, which become into big granules. The oblong nuclear then appearsas homogeneous, high electron density. Various mitochondria could be found in this stage,which are in the process of remolding, and then develop into concentric structure. Thecytoplasm of the sperm cells are reduced and discharged. The third type is those thatpremature sperm, which is characterized by the long head and the developing tail. Themicrotubules in the cytoplasm are observed which have the function in the forming of thetail. Under scanning microscope, cage-shaped structure is found. Finally, the cytoplasmlobes in premature sperm were discharged and the mature sperm come into being. Inconclusion, the spermatogenic cells in the epididymis of soft-shelled turtle are heteroideous,even all stages of the developed sperm were found in present study.ExperimentⅢUltrastructure of sperm storage in female soft-shelled turtle, Trionyxsinensis was examined under light and electron microscopes. Sperm storage tubules are restricted to the anterior of the uterus. These tubules developed either by folding or fusionof the oviductal mucosal folds and are lined by both ciliated and secretory cells. Ciliatedcells are characterized by a few microvilli and prominent cilia in the apical membranes. Aprominent feature of the secretory cell is the presence of secretory granules in thesupranuclear region. The size, shape, and electron density of these granules vary markedly.The secretory product is released mainly by exocytosis into the oviductal lumen, where itappears as flocculent material. The unique structure in the base of the epithelium, the basalborder of the cell—the basal lamina—and a blood vessel layer, is presumed to be aimportant barrier, by which the nourishment exchange and the microenvironmentmaintenance are assured. The gland cell is presented with numerous, round,membrane-bound secretory granules of moderate to high electron densities. We dividethese granules into 3 types according to their appearance: (ⅰ) membrane bounded granuleswith high-homogeneous electron density, (ⅱ) membrane bounded granules withmoderate-homogeneous electron density, (ⅲ) membrane bounded, electron dense granuleswith concentric structures. These granules are presented as different stage of the secretionsin the gland cell. The junction complexes are markedly distributed between cells, which areimportant in keeping stability and the microenvironment maintenance of the sperm storagetubules. Sperm stored in the tubules are heterogeneous in cytology. Besides the maturesperm in the lumen, sperm with large chromatic granules are found, which are presumed tobe immature sperm and are being in the process of nuclear condensation. A number ofspermatozoa in the tubules are exhibited with definitive indications of degeneration of thenuclei. The volume increase and the electron density decline of the central cores inmitochondria combined with the deterioration of concentric membrane structure areobserved, which are possibly due to long time storage period in SSTs, and the leakage ofreactive oxygen species is suggested to be a major cause. It is concludes that theultrastructure character of sperm storage in the oviduct of Trionyx sinensis is unique, inaddition to having a basal function in secretion and the cilia swing, the tubules also providean available microenvironment for the sperm to long time stored. The cell junctions and thecalled blood-epithelium barrier might have their co-functions to keep thismicroenvironment. The secretory granules both in secretory cell and gland cell arespecialized by their unique appearance, which have not been reported in other reptiles. Thedegenerative sperm in the tubules might be related to paternity-specific reproductiveadaptations, and the sperm competition might occur during long time storage. The degeneration of mitochondria plays an important role in the elimination of unavailablespermatozoa, which is presumed to be a time associated pattern.