Ultrastructural And Secretory Characteristics Of The Reproductive Tract In Chinese Soft-shelled Turtle, Pelodiscus Sinensis

In most of the reptile species, the ability of sperm storage is obvious in both male and female reproductive tracts. In female reproductive tract the sperm storage occurs in different segments of oviduct while in male reproductive tract, the epididymis and efferent ducts regarded as site of sperm storage and conduit of sperm transportation. Reptilian oviduct is an important reproductive organ which regulates various physiological functions.It is responsible for the transportation and maturation of the eggs while it also performs the function of sperm storage. The secretions of oviduct have been already reported in birds,amphibians, human and other reptiles. There are potentially different mechanisms which are explaining the role of these secretions in egg production, egg transport, sperm capacitation and sperm storage. Chinese soft-shelled turtle is an ancient type of reptile found in China; as well as in many other countries especially in Asia. The Chinese soft-shelled turtle is of great importance in aquaculture and as a food item, and is, of significant economic and pharmacological value. It is of great importance to understand the normal functioning of the male and female reproductive system in this species because it will be very helpful to plan healthy and more productive animals in the future. In mammals, birds and amphibians a considerable work has been already done to analyze the integrated functional axis of the oviduct and epididymis from the ultrastructure to complex mechanism; however, in most of the reptile species these information are missing. In turtles these studies are scarce or only limited to simple microscopy. In this context, the knowledge on the advancement in the structure of sperm storage segments is fundamental part to achieving such goals. In current study we have demonstrated the ultrastructure and secretory cytology in the reproductive tract of soft- shelled turtle Pelodiscus sinensis. In the view of sperm storage,we have also drawn a comparative model between turtle and hen for better understanding the oviduct ultrastructure and secretory morphology. Thus, present study increases our understanding about the ultrastructure, secretory activity and reproductive functionality in Chinese soft- shell turtle Pelodiscus sinensis.(Experiment 1) Novel cellular evidence of oviduct secretions in the Chinese soft-shelled turtle Pelodiscus sinensisThe oviduct is the location of fertilization and sperm storage. We examined the ultrastructure of the oviduct epithelium and its glandular secretions in the isthmus, uterus and vagina of Chinese soft-shelled turtle Pelodiscus sinensis using light and transmission electron microscopy. The epithelium in these segments was lined with ciliated, secretory and other cells; the first two cell types span the entire epithelium, with secretory cells being predominant. The ciliated cells were characterized by the presence of a secretory vacuole that releases apocrine secretions into the lumen, whereas the secretory cells contain typical biphasic granules with both dark and light aspects. The third type of cells observed with wider proximal portion, abundant mitochondria, vacuoles and narrow nuclei. The storage of spermatozoa was restricted to the isthmus, uterus and vagina. In addition, the gland cells showed prominent features, including the presence of granules of different shapes, sizes and electron densities. The synthesis of these granules is described for the first time in current study. Mitochondria played an important role in the formation of dense granules,the rough endoplasmic reticulum and microfilaments also played a role in the maturation of these dense granules. After completing the maturation process, these granules were released into the lumen of the gland cells.(Experiment 2) Epithelial and glandular evidence of exosomes and secretions in the oviduct of Chinese soft- shelled turtle Pelodiscus sinensis The Oviduct is a location of egg production, fertilization and sperm storage while,its secretions have broadly attributes towards different physiology functions. We examined the ultrastructure of oviduct epithelium and glands in relation with secretions particularly with exosomes origin in Chinese soft shelled turtle Pelodiscus sinensis using,immunohistochemistry and transmission electron microscopy. The epithelial ciliated and gland cells were involved in the release of exosomes and secretions into the lumen throughout the year; the exosomes were either released directly from epithelium or in relation with multi vesicular body (MVB). The average size of the particles varies between(50-130nm). These exosomes were also widely distributed to the epithelial ciliated cells and in peri-cytoplasm of glands lumen. Intracellular MVB was characterized with the different size membrane bounded exosomes. Exosomes were also found in close contact with the cilia and sperm membrane in the lumen which is suggestive of their fusogenic properties. Strong to moderate positive localization of exosomes was noted in ciliated and gland cells during January, September and December as it is the time of sperm storage in this turtle while, it showed moderate to weak level of localization during breeding season(May). This is first study about the identification of the exosomes in the reptiles. Epithelial and glandular intracellular and extracellular exosomes, intracellular MVB, secretions and secretory vesicles while, the release of exosomes throughout the year gives this turtle specie a unique secretory morphology and a potential model for investigating the secretory nature of the oviduct.(Experiment 3) Comparative study of the ultrastructure and secretions of oviduct and SSTs between the turtle and henIn order to compare the ultrastructure and secretory cytology between turtle and hen, we used the uterovaginal junction (UVJ) of hen and isthmus segment of turtle oviduct. The sperm storage tubules (SSTs) in the turtle were mainly localized in the isthmus part of oviduct while, in hen the UVJ had abundance of these tubules. Ultrastructuraly, the epithelium of UVJ in hen and isthmus in turtle was lined with ciliated and secretory cells.In UVJ of hen epithelial ciliated cells were characterized with light and dense secretory granules in supernuclear cytoplasm. Dense secretory granules in ciliated cell of UVJ epithelium were larger in size, surrounded with transparent rim and concentric layers. The secretory cells of UVJ were associated with the release of secretions via apocrine blebs from microvilli. The dense secretory granules in these cells were smaller in size, absence of transparent rim or layers and were released in the lumen through secretory vesicles. In SSTs of UVJ, secretory and ciliated cells with the presence of both dense and light granules span the entire epithelium. The dense secretory granules in ciliated cells of SSTs were smaller in size in comparison to epithelium of UVJ and characterized with the absence of both circular layer and transparent rim. The cytological distribution of the cells in the hen were more or less similar to that seen in the oviduct of turtle as the both ciliated and secretory cells present the entire epithelium and these epithelial cells were involved in secretions. Ciliated cells of UVJ and isthmus were also involved in the shedding of exosomes and secretory vesicles in the lumen. The intracellular multivesicular body (MVB) was also observed in supernuclear cytoplasm of secretory cells in UVJ, which is related to the production of exosomes. In general the apical protrusion of epithelium in the form of apocrine secretions,the releasing of exosomes, the identification of intracellular MVB and releasing of dense granules gives the epithelium distinct nature and more resemblance to the turtle oviduct.(Experiment 4) Morphological and ultrastructural study of the efferent ductules in the Chinese soft- shelled turtle Pelodiscus sinensisComparative study of the turtle excurrent duct system increases our understanding the evolution of sperm motility and fertility maintenance in higher vertebrates. Therefore in this study we observed the histology and ultrastructure organization of efferent ductules in the Pelodiscus sinensis using light and transmission electron microscopy. The efferent ductules were extra- testicular and 22- 28 in number originated from rete testis. The epithelium was entirely composed of two types of cells, the predominant non- ciliated and ciliated cells.The ciliated cells have long cilia that protrude into the lumen to form a meshwork. These cells associated with clusters of mitochondria in the supranuclear cytoplasm and possess coated vesicles, vacuole, intracellular spaces and junction complexes. Ciliated cells in the proximal portion of the ductules contained endocytic apparatus with coated pits and tubules in the apical cytoplasm. Interdigitations and lipid droplets were predominantly present around the nuclei of these cells. The non- ciliated cells had clusters of mitochondria present in both the supranuclear and perinuclear cytoplasm whereas, the nuclei of these cells were lightly stained. Moreover, the contour of the epithelium towards lumen was irregular as it had a deep indentation. The apical cytoplasm goes deep into the lumen to form cytoplasmic processes. This is the first study to describe the detailed features of efferent ductules in Pelodiscus sinensis with, special focus on the morphology of ciliated cells, as these cells are involved in the mixing of luminal fluid and transport of spermatozoa towards the distal region.