Fine structural observation on the oogenesis and vitellogenesis of the Chinese soft-shelled turtle ( Pelodiseus sinensis).

Fine structure observations were performed by means of electron microscopy on oogenesis and vitellogenesis and the special functions of follicular cells in the Chinese soft-shelled turtle (Pelodiseus sinensis). Histological examination of the ovary showed a well developed lacunae system containing fine granules, fibres or gelatiniform materials with one or two germinal beds dispersed on the dorsal surface of the ovarian cortex. The process of oogenesis comprised 10 consecutive phases according to the morphology of the yolk platelets, follicular cells and zona pellucida (ZP). Electron microscopy of vitellogenesis revealed some of the mitochondria gradually being transformed into yolk granules. In the advanced stage of vitellogenesis, large amounts of rough endoplasmic reticula, Golgiosomes and other cell organelles that are involved in synthesis and secretion were observed in follicular cells. The ZP was formed by microvilli, thus increasing the absorptive surface of the oocyte and facilitating transport of nutrients from the follicular epithelium to the ooplasm. This study demonstrated that the ovaries of members of the Testudinidae share more features with Archosaurs than with Squamates, indicating that these features were phylogenetically conserved in the Archosauria. The present observations suggest that the accumulation of yolk materials was controlled by the intrinsic and extrinsic pathways as well as by the activity of follicular cells. These results might also support a sibling relationship of the Testudinidae with the Archosauria and not with all extant reptiles.

Lymphocyte migration in the micro-channel of splenic sheathed capillaries in Chinese soft-shelled turtles, Pelodiscus sinensis.

The structural characteristics of the splenic sheathed capillary were investigated using light microscopy and transmission electron microscopy (TEM). This study mainly focused on lymphocyte migration to the splenic white pulp via micro-channels in Chinese soft-shelled turtles, Pelodiscus sinensis. The results showed that the sheathed capillaries in the turtle spleen were high endothelial venule (HEV)-like vessels. These capillaries consist of micro-channels that facilitate lymphocyte migration to the splenic white pulp. The micro-channel is a dynamic structure comprising processes of endothelial cells, supporting cells, and ellipsoid-associated cells (EACs), which provides a microenvironment for lymphocyte migration. The pattern of lymphocyte migration in the micro-channel of the turtle spleen includes the following steps: (i) lymphocyte first adheres to the endothelium of the sheathed capillary, passes through the endothelial cells, and traverses through the basement membrane of the sheathed capillary; (ii) it then enters into the ellipsoid combined with supporting cells and EACs; and (iii) lymphocyte migrates from the ellipsoid to the periellipsoidal lymphatic sheath (PELS) via the micro-channel. This study provides morphological evidence for lymphocyte migration in the micro-channels of turtle spleens and also an insight into the mechanism of lymphocyte homing to the splenic white pulp of reptiles.

Ultrastructure of the nephron in the soft-shelled turtle, Pelodiscus sinensis (Reptilia, Chelonia, Trionychidae).

The structure of the nephron in adult soft-shelled turtles (Pelodiscus sinensis) was studied by light microscopy, transmission and scanning electron microscopy. The kidney contained 5-6 renal lobes. Nephrons of P. sinensis are composed of a renal corpuscle (RC) and of a renal tubule that appears divided morphologically into five distinct segments: neck segment (NS) (This segment is only present in approximately 10% of the nephrons), proximal tubule (PT), intermediate segment (IS), distal tubule (DT), and collecting duct (CD). The RCs and most of the convoluted DTs lie in the central zone, while the PTs and the CDs lie in the peripheral zone of the renal lobe. The renal corpuscle is relatively large with especial processes in podocytes and a thick basement membrane. The podocyte processes covering a large capillary area can be observed by TEM, and the major podocyte processes formed a very specific pattern in SEM. The podocyte processes expand to form a flattened network over the whole capillary loops surface, and only may observe little filtration slits in glomerular area. The neck segment when presentis short and has a relatively narrow lumen, consisting of cuboidal or squamous cells. There is a well-developed endocytic-lysosomal apparatus in the apical cytoplasm of the PT. The proximal tubule and intermediate segment cells show some differences between male and female. It showed that proximal tubule cells of male soft-shelt turtle contain lateral intercellular spaces, into which extensions of the cell membrane protrude, and the basal cell membrane forms a conspicuous labyrinth. Whereas, the basal and lateral cell membranes of the female are smooth, and no later-basal intercellular spaces. The differences between male and female in the middle segment cells is similar to proximal tubule cells. Not previously reported in vertebrate kidneys. The IS is the narrowest nephron segment, formed by multiciliated as well as nonciliated cells. In DT cells, basolateral interdigitations and infoldings are particularly well-developed. The CD contains clear cells with numerous secretory granules and dark cells with dense mitochondria and an elaborate Golgi complex. This study was undertaken in order to disclose specific kidney features in P. sinensis that could be related to function. In addition, the ultrastructure of the nephrons in P. sinensis are discussed in relation to other turtles and vertebrates.

An immunohistochemical study of S-100 protein in the intestinal tract of Chinese soft-shelled turtle, Pelodiscus sinensis.

The present work describes the distribution of S-100 protein in the intestinal tract of a Chinese soft-shelled turtle specimen (Pelodiscus sinensis). S-100 protein positive cells were located in the intestinal tract, from the proximal small to distal large intestine. S-100 protein positive dendritic cells had irregular shape and were positive in both cytoplasm and nucleus. Most of them were located both lamina propria and submucosa in the small intestine, while few were found in the large intestine. S-100 protein, C-kit positive ICCs and Silver staining glial cells were predominantly observed in three locations: (1) in the interspace between the submucosa and circular muscle layer; (2) in the circular muscle layer; and (3) between the circular and longitudinal muscle layers of the intestine. Fewer were found in the large intestinal lamina propria and submucosa. Three types of positive cells (S-100 protein positive cells, C-kit positive ICCs and Silver staining glial cells) with 1-2 long or 2-3 short processes were distributed as lace-like or surrounding blood vessels in the different locations mentioned above. In the lamina propria, all the positive cells with irregular processes were connected with each other and formed a network. In the submucosa, all the positive cells were found surrounding the blood vessels.

Architecture of the blood-spleen barrier in the soft-shelled turtle, Pelodiseus sinensis.

We investigated the structure of the soft-shelled turtle, Pelodiseus sinensi, spleen and demonstrated that there were several microanatomical peculiarities by light and transmission electron microscopy. In the spleen, the white pulp of the spleen was composed of two compartments, the periarteriolar lymphatic sheath (PALS) and periellipsoidal lymphatic sheath (PELS). No lymph nodules and marginal zones were found. The spleen-blood barrier stood in the PELS and the ellipsoid. The high endothelial lining of penicilliform capillary contained small channels. These channels allowed circulating substances or lymphocytes to enter the ellipsoid. The distal portion of the penicilliform capillaries directly opened to pulp cords. The ellipsoid-associated cell (EAC) was located at the surface of the ellipsoid. Reticular fibers were mainly distributed in ellipsoid and the outer PELS. Both reticular cells and macrophages were distributed in the outer layers of PELS. S-100 protein positive dendritic cells were mainly distributed in out cells layer of the PELS and all over the PALS. Forty minutes after injection, carbon particles of Indian ink were mainly observed in the ellipsoid. Few carbon particles were observed in the outer PELS and fewer carbon particles in the red pulp. These findings suggested that a blood-spleen barrier indeed existed in the soft-turtle, P. sinensi, and it was a complex composed of an ellipsoid (including supporting cells, EAC, and reticular fibers) and the outer compartments of PELS (including dendritic cells, reticular fibers and cells, macrophages).

Ultrastructure of anterior uterus of the oviduct and the stored sperm in female soft-shelled turtle, Trionyx sinensis.

Ultrastructure of sperm storage in female soft-shelled turtle, Trionyx sinensis 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 fusion of the oviductal mucosal folds and are lined by both ciliated and secretory cells. Ciliated cells are characterized by a few microvilli and prominent cilia in the apical membranes. A prominent feature of the secretory cell is the presence of secretory granules in the supranuclear 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 it appears as flocculent material. The unique structure in the base of the epithelium, the basal border of the cell -- the basal lamina -- and a blood vessel layer, is presumed to be a important barrier, by which the nourishment exchange and the microenvironment maintenance are ensured. The gland cell is presented with numerous, round, membrane-bound secretory granules of moderate to high electron densities. We divide these granules into three types according to their appearance: (1) membrane bounded granules with high-homogeneous electron density, (2) membrane bounded granules with moderate-homogeneous electron density, (3) membrane bounded, electron dense granules with concentric structures. These granules are presented as different stages of the secretions in the gland cell. The junction complexes are markedly distributed between cells, which are important in keeping stability and the microenvironment maintenance of the sperm storage tubules. Sperm stored in the tubules are heterogeneous in cytology. In addition to the mature sperm in the lumen, sperm with large chromatic granules are found, which are presumed to be immature sperm and are being in the process of nuclear condensation. Several spermatozoa in the tubules are exhibited with definitive indications of degeneration of the nuclei. The nuclear volume increases. The electron density of the central cores in mitochondria declines, combined with the deterioration of concentric membrane structure. Those changes are possibly due to the long time storage of the sperm in sperm storage tubules, and the leakage of reactive oxygen species is suggested to be a major cause. We conclude that the ultrastructure character of sperm storage in the oviduct of Trionyx sinensis is unique, in addition to having a basal function in secretion and the cilia swing, the tubules also provide an available microenvironment for the sperm to long time stored. The degenerative sperm in the tubules might be related to paternity-specific reproductive adaptations, and the sperm competition might occur during long time storage.

Spermiogenesis in soft-shelled turtle, Pelodiscus sinensis.

Spermiogenesis in the soft-shelled turtle, 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 stage I, 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 stage II, 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 stage III, the front of the elongating nucleus protrudes out of the spermatid and is covered by the flat acrosome; coarse granules replace the small ones within the nucleus. At the posterior pole of the head, mitochondria move backward. Numerous microtubules begin to assemble the axoneme of flagellum. In stage IV, the chromatin concentrates to dense homogeneous phase. The circular manchette is reorganized longitudinally. The Sertoli process covers the acrosome and the residues of the cytoplasmic lobes are eliminated. In stage V, the sperm head matures. After dissolution of the longitudinal manchette, 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.

The sympathetic postganglionic and sensory innervation of oviducal magnum in hen: a choleratoxin subunit B-conjugated horseradish peroxidase study.

The anatomy of the extrinsic innervation of the avian magnum has not been accurately demonstrated previously. In the present study, choleratoxin subunit B-conjugated horseradish peroxidase (CB-HRP) was used as a retrograde tracer to determine the sympathetic postganglionic and sensory innervation of the magnum of hens. With regard to the sympathetic postganglionic innervation, following CB-HRP injections under the serosa of the magnum, CB-HRP-positive neurons were found bilaterally in the C12-LS13 ganglia of the sympathetic chain, splanchnic ganglia and adrenal ganglia. The number of labelled neurons in the left ganglia of the sympathetic chain and splanchnic ganglia was approximately 2.1 times that in the right ganglia. This suggests that the unilateral magnum is bilaterally innervated with sympathetic postganglionic nerves, the left nerves being predominant. With regard to the sensory innervation, following tracer injections, CB-HRP-positive neurons were found bilaterally in the spinal ganglia C13-LS12, jugular ganglia and nodose ganglia. The number of positive cells in the left ganglia was about 2.2 times that in the right ganglia. In the spinal ganglia, 85.6% of the labelled neurons were in the T5-LS2 and LS8-LS11 ganglia. These results suggest that the sensory nerve fibres of the magnum reach the central nervous system principally via two groups of spinal ganglia and vagus nerves, and that the innervation is bilateral although the left-hand route predominates. Moreover, 45.7% of all the CB-HRP-labelled neurons were found in the rectal region of the intestinal nerve of Remak (INR), which suggests that the INR plays a very important role in the functional regulation of the magnum.