Shakeeb Ullah

Identification and characterization of telocytes in the uterus of the oviduct in the Chinese soft-shelled turtle, Pelodiscus sinensis: TEM evidence

Telocytes (Tcs) are cells with telopodes (Tps), which are very long cellular extensions with alternating thin segments (podomers) and dilated bead‐like thick regions known as podoms. Tcs are a distinct category of interstitial cells and have been identified in many mammalian organs including heart, lung and kidney. The present study investigates the existence, ultrastructure, distribution and contacts of Tcs with surrounding cells in the uterus (shell gland) of the oviduct of the Chinese soft‐shelled turtle, Pelodiscus sinensis. Samples from the uterine segment of the oviduct were examined by transmission electron microscopy. Tcs were mainly located in the lamina propria beneath the simple columnar epithelium of the uterus and were situated close to nerve endings, capillaries, collagen fibres and secretory glands. The complete morphology of Tcs and Tps was clearly observed and our data confirmed the existence of Tcs in the uterus of the Chinese soft‐shelled turtle Pelodiscus sinensis. Our results suggest these cells contribute to the function of the secretory glands and contraction of the uterus.

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 extends the time window for fertilisation in several animal species, from a few days to several years. The underlying storage mechanisms, however, are largely unknown. In this study, spermatozoa from the epididymis and oviduct of Chinese soft-shelled turtles were investigated to identify potentially relevant morphological features and transformations at different stages of sperm storage. Large cytoplasmic droplets (CDs) containing lipid droplets (LDs) were attached to the midpiece of most spermatozoa in the epididymis, without migrating down the sperm tail. However, they were absent from the oviductal spermatozoa, suggesting that CDs with LDs may be a source of endogenous energy for epididymal spermatozoa. The onion-like mitochondria recovered their double-membrane morphology, with typical cristae, within the oviduct at a later stage of storage, thus implying that mitochondrial metabolism undergoes alterations during storage. Furthermore, a well developed fibrous sheath on the long principal piece was the integrating ultrastructure for glycolytic enzymes and substrates. These novel morphological characteristics may allow turtle spermatozoa to use diverse energy metabolism pathways at different stages of storage.

Telocytes: novel interstitial cells present in the testis parenchyma of the Chinese soft-shelled turtle Pelodiscus sinensis.

Telocytes (TCs) are novel interstitial cells that have been found in various organs, but the existence of TCs in the testes has not yet been reported. The present ultrastructural and immunohistochemical study revealed the existence of TCs and differentiate these cells from the peritubular cells (Pc) in contact with the surrounding structures in the testes. Firstly, our results confirmed the existence of two cell types surrounding seminiferous tubules; these were Pc (smooth muscle like characteristics) and TCs (as an outer layer around Pc). Telocytes and their long thin prolongations called telopodes (Tps) were detected as alternations of thin segments (podomers) and thick bead‐like portions (podoms), the latter of which accommodate the mitochondria and vesicles. The spindle and irregularly shaped cell bodies were observed with small amounts of cytoplasm around them. In contrast, the processes of Pc contained abundant actin filaments with focal densities, irregular spine‐like outgrowths and nuclei that exhibited irregularities similar to those of smooth muscle cells. The TCs connected with each other via homocellular and heterocellular junctions with Pc, Leydig cells and blood vessels. The Tps of the vascular TCs had bands and shed more vesicles than the other TCs. Immunohistochemistry (CD34) revealed strong positive expression within the TC cell bodies and Tps. Our data confirmed the existence and the contact of TCs with their surroundings in the testes of the Chinese soft‐shelled turtle Pelodiscus sinensis, which may offer new insights for understanding the function of the testes and preventing and treating testicular disorders.

Ultrastructure of epididymal epithelium and its interaction with the sperm in the soft-shelled turtle Pelodiscus sinensis

The epididymis of the soft-shelled turtle Pelodiscus sinensis was examined under light and transmission electron microscopes to determine the morphological characteristics, as well as their changes at different phases of the seasonal reproductive cycle. Three distinct regions, viz., cranial, middle and caudal were identified in the epididymis based on anatomical characteristics. The epididymal epithelium consists of five different cell types: principal, narrow, apical, clear and basal cells. Principal cells, which are the most abundant, together with basal cells are present along the entire length. Ultrastructural evidence suggests that all of the principal cells in each of the regions function in both absorption and secretion. Narrow cells and apical cells are rare and only confined to the cranial region. The clear cells, for the first time reported in the turtle epididymis, are confined to middle and caudal regions; these cells showed strong PAS-positive granulation in apical position, and secretory activity by a holocrine process, especially in the middle region. There was a significant difference in the epithelium height of all the regions between the reproductive season and the non-reproductive season. Sperm are stored in the epididymis throughout the year. Apart from the mature spermatozoa, immature spermatozoa with normal morphology are also observed. Under TEM, the immature spermatozoa showed a large amount of cytoplasm located eccentrically on the midpiece wrapped by plasma membrane, with some cytoplasm extended to the posterior of the head. Furthermore, the interactions of sperm with the epididymal epithelium were observed. Some sperm are associated with the secretory material in the lumen; other sperm are inserted into the intercellular space between the epithelial cells.

Sperm storage and spermatozoa interaction with epithelial cells 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 ovulation. 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 throughout 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 sparse 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 benefited spermatozoa survival for a long time as extraneous cells in the oviduct of this species. These findings would help to improve the 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.

Identification and structural composition of the blood–spleen barrier in chickens

To identify the existence and composition of the blood-spleen barrier (BSB) in chickens, the microanatomical features of the spleen were investigated by light and transmission electron microscopy, intravenous injection of ink, acid phosphatase reaction, and silver impregnation. The results showed that the white pulp in chicken spleen consists of lymphoid nodules, periarteriolar lymphatic sheaths (PALS) and periellipsoidal lymphatic sheaths (PELS). There was no evidence for the presence of a marginal zone. The splenic ellipsoid was a unique structure, which functioned as a barrier for filtering and phagocytosis. Uptake of carbon particles was limited to the ellipsoid and PELS, 60 min after injection of carbon particles. Reticular fibres were densely distributed in the ellipsoid and extended into the PELS. Ellipsoid-associated cells (EACs), reticular cells and macrophages were acid phosphatase positive. The sheathed capillaries, surrounded by the ellipsoid, were similar to high endothelial venules (HEVs). These findings suggest that the BSB of chickens is present in the ellipsoid and PELS, protecting the spleen from invasion from circulating pathogens. The BSB was a reticular framework, between the arterial and venous vessels, which included cuboidal-shaped endothelial cells, supporting cells, EACs, macrophages, reticular cells and fibres. Lymphocyte migration into the spleen parenchyma is most likely via the HEV-like vessels. These research findings contribute to better understanding of avian immunology and provide an insight into evolutionary differences in the immune system.

B-Cell Lymphoma-2 Localization in the Female Reproductive Tract of the Chinese Soft-Shelled Turtle, Pelodiscus Sinensis and Its Relationship With Sperm Storage.

The aim of the present study was to investigate the expression and localization of B‐cell lymphoma‐2 (Bcl‐2) in the oviduct of the Chinese soft‐shelled turtle, Pelodiscus sinensis, during the reproductive cycle to analyze the relationship between Bcl‐2 and sperm storage. Bcl‐2 expression was confirmed in the P. sinensis oviduct by western blot analysis. Hematoxylin‐eosin staining showed that female P. sinensis stored sperm from November to April of the following year. The oviduct showed positive immunostaining for Bcl‐2 of epithelial ciliated cells, gland ducts, and gland cells. Bcl‐2 expression in the oviduct was associated with sperm storage occurrence. This indicates that the survival factor Bcl‐2 may play a role in P. sinensis sperm storage. Anat Rec, 298:2011–2017, 2015.

Expression of TLR2/4 in the sperm‐storing oviduct of the Chinese soft‐shelled turtle Pelodiscus sinensis during hibernation season

Abstract The initiation of innate immunology system could play an important role in the aspect of protection for sperms long‐term storage when the sperms got into oviduct of turtles and come into contact with epithelium. The exploration of TLR2/4 distribution and expression in oviduct during hibernation could help make the storage mechanism understandable. The objective of this study was to examine the gene and protein expression profiles in Chinese soft‐shelled turtle during hibernation from November to April in the next year. The protein distribution of TLR2/4 was investigated in the magnum, isthmus, uterus, and vagina of the turtle oviduct using immunohistochemistry, and the gene expression of TLR2/4 was analyzed using quantitative real‐time PCR (qRT‐PCR). The results showed positive TLR2 protein expression primarily in the epithelium of the oviduct. TLR4 immunoreactivity was widely observed in almost every part of the oviduct, particularly in the epithelium and secretory gland membrane. Analysis of protein, mRNA expression revealed the decreased expression of TLR2/4 in the magnum compared with the isthmus, uterus, and vagina during hibernation. The protein and mRNA expression of TLR2 in the magnum, isthmus, uterus, and vagina was decreased in April compared with that in November. TLR4 protein and mRNA expression in the magnum, isthmus, uterus and vagina was decreased in November compared with that in April. These results indicated that TLR2/4 expression might protect the sperm from microbial infections. In contrast to the function of TLR2, which protects sperm during the early stages of hibernation, TLR4 might play a role in later stages of storage. The present study is the first to report the functions of TLR2/4 in reptiles.

Cellular Evidence of Telocytes as Novel Interstitial Cells within the Magnum of Chicken Oviduct

Telocytes are a novel type of interstitial cell that has been identified in many organs of mammals, but there is little information available on these cells in avian species. This study shows the latest findings associated with telocytes in the muscular layer and lamina propria of the magnum of chicken oviduct analyzed by transmission electron microscopy. Telocytes are characterized by telopodes, which are thin and long prolongations, and a small amount of cytoplasm rich with mitochondria. Spindle- or triangular-shaped telocytes were detected at various locations in the magnum. In the muscular layer, telocytes have direct connection with smooth muscle cells. The cell body of telocytes along with their long telopodes mainly exists in the interstitial space between the smooth muscle bundles, whereas large numbers of short telopodes are scattered in between the smooth muscle cells. In the lamina propria, extremely long telopodes are twisting around each other and are usually collagen embedded. Both in the lamina propria and muscular layer, telocytes have a close relationship with other cell types, such as immune cells and blood vessels. Telopodes appear with dichotomous branching alternating between the podom and podomer, forming a 3D network structure with complex homo- and heterocellular junctions. In addition, a distinctive size of the vesicles is visible around the telopodes and may be released from telopodes because of the close relation between the vesicle and telopode. All characteristics of telocytes in the magnum indicate that telocytes may play a potential, but important, role in the pathogenesis of oviduct diseases.

Novel cellular evidence of oviduct secretions in the Chinese soft-shelled turtle Pelodiscus sinensis

The 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 is lined with ciliated, secretory and other cells; the first two cell types span the entire epithelium, with secretory cells being predominant. The ciliated cells are 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 have wider proximal portion, abundant mitochondria, vacuoles, and narrow nuclei. The storage of spermatozoa is restricted to the isthmus, uterus, and vagina. In addition, the gland cells show 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 this study. Mitochondria appear to play an important role in the formation of dense granules, the rough endoplasmic reticulum and microfilaments may also play a role in the maturation of these dense granules. After completing the maturation process, these granules are released into the lumen of the gland cells.