Studies On Characteristics Of Reproductive Biology In The Male Soft-Shelled Turtle, Pelodiscus Sinensis

Breeding is the most important link in the animal life, whether they can normal and successful breed, is related to the continuation of species and species diversity. Because reptiles were in the special status during the evolution of animals, and was the first landing in vertebrate groups, whether they can successful breed in the land became the principal contradiction for further development. Therefore, a thorough study of this link is particularly important. However, they are few studies that have been reported the reproductive characteristics of soft-shelled turtle, Pelodiscus sinensis. Soft-shelled turtles, which are distributed widely in China, are one of the most representative classes of terrapin. This species is famous for its economical and pharmacological value, and hence they are subject to harvest pressure. Because the soft-shelled turtles have physiology characteristics which own typical hibernation and sperm storage in the reproductive periodicity, it is an ideal model for studying on reptile biology. In present study, we observed the characteristics of spermatozoon as well as spermiogenesis in the Pelodiscus sinensis by electron microscope; and examined the relationship between seasonal changes of testes histological structure, and the apoptosis and proliferation of germ cells in different season; then gave a further analysis of the distribution of the hormone receptor in the gonad tissues (Because the oviduct in the female soft-shelled turtle has a function for sperm storage, here we study this structure with the male reproductive organ). Those researches explain the reproductive characteristics of cell and molecule in the male Pelodiscus sinensis, and provide basic datum for the works of reproduction, protection and resource utilization of Chinese soft-shelled turtle.ExperimentⅠUltrastructure of spermatozoon in soft-shelled turtle, Pelodiscus sinensis Electron and scanning microscopies were undertaken to determine the ultrastructure of spermatozoon in soft-shelled turtle in present study. The mature spermatozoon is vermiform in shape, which consists of a head, connecting piece, middle piece, principal piece and end piece. In the head, acrosomal complex covers the anterior protrusion of the nucleus, where there is a narrow lucent space between the subacrosomal cone and the outer acrosomal cap. 3～5 intranuclear tubules go through the anterior tow-thirds of the nucleus and their central cores extend towards the perforatorial rods. The connecting piece is located in the concave implantation fossa in the caudal end of the nucleus. The mitochondrial sheath in the middle piece is composed of concentric mitochondria arranged in 7～38 circles each of which contains 5 mitochondrion. Concentric layers in the mitochondrion are double-membranes and they are cristae mitochondriales actually. The concentric cristae mitochondriales and lots of glycogens show the relationship to the vitality maintaining during long-time storage of the spermatozoon in the turtle. There is a wedge-shaped annulus around the end of the mitochondriall sheath. The structural modal of proximal centriole is 9×3+0, in which the typical nine triplet microtubules arrange in a pinwheel fashion and are surrounded by a dense material. By comparison, the structural modal of distal centriole is 9×3+2, and the central microtubules and outer triplet microtubules are embed in dense fibers respectively. The distal centriole stands vertically to the proximal one. The axonemal complex of the principal piece is surrounded by several layers of circumferential fibers, which obviously produces a fibrous sheath reducing backward gradually in thickness. In the axonemal complex that posses a structural model of 9x2+2, there are two thick fibers corresponding to No.3 and No.8 doublet microtubules respectively, which merge with the fibrous sheath. On the transection of principal piece, two dynein arms attached to a microtubule of the doublet which connected with the central microtubules through an obvious radial spoke. Two central microtubules are linked by a bridging substance. The end piece only contains an axonemal complex surrounded by cell membrane, the microtubules in which are dispersed gradually. The spermatozoon of Pelodiscus sinensis are unique in several structures and numbers to that of the turtles, Chrysemys picta and Chimemys reevesii. The structural characteristics above were different evidently from other class animals or other class reptile.ExperimentⅡSpermiogenesis in the soft-shelled turtle, Pelodiscus sinensis Spermiogenesis in the Pelodiscus sinensis, was examined by transmission electron microscopy. The process includes nuclear elongation, chromatin condensation, acrosomal and flagellar development, and elimination of excess cytoplasm. In stageⅠ, the proacrosomal vesicle occurs next to a shallow fossa of the nucleus, and a dense acrosomal granule forms beneath it. A smaller subacrosomal granule in the middle of the fibrous layer is related to the development of intranuclear tubules. The nucleus begins to move eccentrically. In stageⅡ, the round proacrosomal vesicle is flattened by protrusion of the nuclear fossa, and the dense acrosomal granule diffuses into the vesicle, as the fibrous layer forms the subacrosomal cone. Circular manchettes develop around the nucleus, and the chromatin coagulates into small granules. The movement of the nucleus causes rearrangement of the cytoplasm. In stageⅢ, the front of the elongating nucleus protrudes out of the spermatid and is covered by the flat acrosome; the spermatids come to lie within deep recesses of the Sertoli cell cytoplasm; the caudal end of the nucleus is wide and now lodged into a depression; coarse granules replace the small ones within the nucleus. At the posterior pole of the head, mitochondria move backwards. Numerous microtubules begin to assemble the axoneme of flagellum. In stageⅣ, the chromatin concentrates to dense homogeneous phase. The circular manchette is reorganized longitudinally. The Sertoli process covers the acrosome and the cytoplasm move to the posterior pole of the spermatid. The residual bodies are derived from cytoplasmic lobes of elongated spermatids, which are eliminated gradually. In stageⅤ, the sperm head matures. Following dissolution of the longitudinal manchette and the collar of the Sertoli cell process shrinks, the mitochondria arrange themselves around the proximal and distal centrioles. Caudal to the mitochondrial mass, a fibrous sheath surrounds the proximal portion of the flagellum.ExperimentⅢAnnual variation in testis and epididymis of male Pelodiscus sinensis The morphology and microstructure of testis, epididymis in male adult soft-shelled turtle, Pelodiscus sinensis were observed and analyzed relatively systemically using histological methods during the annual reproduction cycle, in an attempt to investigate its reproductive rule. The spermatogenetic cycle of soft-shelled turtle comprises seven stages with significant features: stageⅠin April, the testis was at the stage of spermatogonic proliferation; StageⅡfrom May to June, most spermatogonia proliferated and differentiated into the spermatocytes. StageⅢfrom July to August, spermatocytes were in meiotic division state and divided to form spermatides. StageⅣfrom September to October, spermiogenesis was well underway, and spermatids were transformed to spermatozoa. StageⅤin November, seminiferous tubules became filled with spermatogonia and lots of spermatozoa. StageⅥfrom December to January, the spermatogenesis was largely complete in this stage. Some residual spermatozoa scattered in the lumen. StageⅦin February, the testes were in the regression. The changes of testis index and epididymis index during the annual cycle were response the rule of the reproductive activity, and to accord with the results of histology. Spermatogenesis in the Pelodiscus sinensis was limited in some stages during one year, and spermatogenesis and ovulation were out if phase with each other. Therefore, the testicular cycle of soft-shelled turtle follows a disassociated pattern. This germ cell development strategy is temporal in the spermatogenesis. This strategy is different from bird and mammalia.ExperimentⅣSeasonal effects on apoptosis and proliferation of germ cells in the testes of the soft-shelled turtle, Pelodiscus sinensis To elucidate the proliferation and apoptosis of germ cells in the testes of the soft-shelled turtle during the spermatogenically active phase and nonspermatogenic phase, we used electron microscopy, the proliferating-cell nuclear antigen (PCNA), TdT-mediated dUTP nick end labeling (TUNEL) assay. The PCNA was expressed in nuclei of spermatogonia and primary spermatocytes during the spermatogenically active phase (July and September). During the regressive phase (December and January), PCNA-positive cells also included spermatogonia and spermatocytes, but the number of positive spermatocytes was less than that during the spermatogenically active phase. The TUNEL method detected few apoptotic cells in spermatogenic testis, with much larger numbers during the spermatogenically quiescent phase. Spermatocytes were the most common germ cell types labeled by the TUNEL assay and a few spermatides were also labeled. Using the electron microscope, apoptotic spermatocytes had membrane blebbing and chromatin condensation being mostly half-moon or horseshoe shaped during the resting phase, but not during active spermatogenesis. Apoptotic cells were absent from the spermatogenically active seminiferous epithelium, which exhibited intact membranes and intact organelle morphology. Sections from the spermatogenically quiescent phase that were stained with toluidine blue for light microscopic examination also had apoptotic characteristics. We inferred that accelerated apoptosis of spermatocytes and spermatides partly accounted for germ cell loss during the nonspermatogenic phase. We concluded that seasonal variations in spermatogenesis in the soft-shelled turtle spermatogenesis were stage- and process-specific.ExprementⅤDistribution of androgen receptor and estrogen receptor a in the genital organs of Pelodiscus sinensis The localization of androgen receptor, estrogen receptor a in the testis, epididymis and oviduct were studied by immunocytochemistry in order to discuss their effects on the gonad. The specific AR immunostaining was observed in Leydig cells, peritubular myoid cells, Sertoli cells, spermatogonia, spermatocytes, spermatids. As a result, androgen receptor localized in spermatogeneous cells in phasic changing, which binding with androgen can exerts regulation role directly on spermatogenous cells. Therefore, there may be close relationship between androgen receptor and spermatogonia proliferation, spermatocytes meiosis and spermiogenesis. Estrogen receptor a positive cells were distributed mainly in the Ledyig cells. Spermatogenic cells only showed weak positive reaction in the spermatogenic active. Those results indicated that estrogen might be regulated through somatic cell mainly, and the stages of distribution in the spermatogenic cells showed that it might regulate directly on the spermatogenic cells. Because the oviduct in the female soft-shelled turtle has a function for sperm storage, here we study this structure with the male reproductive organ. Two sex steroid hormone receptors were localized in the oviduct and they were localized mainly in the luminal epithelial cell, gland cell and stromal cell, which indicated that these receptors may be all associated with sperm storage and gland secretion.ExperimentⅥExpression of androgen receptor mRNA in testis cells of Pelodiscus sinensis This study was to detect the expression and localization of androgen receptor mRNA in testis by means of the in situ hybridization histochemiatry (ISHH). Frozen sections of testis in ten healthy Pelodiscus Sinensis were made. In situ hybridization was conducted on the tissue sections by oligonucleotide probe marked by cardiox. The distribution of androgen receptor mRNA were detected in the cells of testis tissue. The results of in situ hybridization indicate that the AR positive cells distributing in the Leydig cells. The labeling cells are round or oval-shap in shape, and the signals are distributed in nuclei of all positive cells. The Sertoli cells and germ cells in the seminiferous tubules were negative for the reaction. This finding show that the AR mRNA are transcribed in the testis, and would provide further evidence that androgen might be regulation the function of the testis in the soft-shelled turtle.