Morphological analysis of interstitial cells in murine epididymis using light microscopy and transmission electron microscopy.

Smooth muscle contraction of the epididymis plays an important role in sperm transport. Although PDGFRα-positive interstitial cells (PDGFRα (+) ICs) are thought to be involved in controlling smooth muscle movement via intercellular signaling, they have not yet been reported to date in the epididymis. Therefore, we aimed to investigate the morphological characteristics of PDGFRα (+) ICs in the interstitial space of the murine epididymis. Immunohistochemistry showed that PDGFRα (+) ICs co-labeled with CD34 (PDGFRα (+) CD34 (+) ICs were distributed in the interstitial space of the murine epididymis from the initial segment (IS) to the cauda of the epididymis. PDGFRα (+) ICs that were not co-labeled with CD34 (PDGFRα (+) CD34 (-) ICs) were observed just beneath the epithelium from the corpus to the cauda but not in the IS. Both types of PDGFRα (+) ICs were in close proximity to each other as well as the surrounding nerves and macrophages. In addition, PDGFRα (+) CD34 (-) ICs beneath the epithelium were also in close proximity to the basal cells. Using transmission electron microscopy, we identified ICs that possessed elongated and woven cellular processes and were in close proximity to each other, surrounding the cells in the interstitial space. In the murine epididymis, it is suggested that there are two subtypes of ICs that show different distribution patterns depending on the segment, which may reflect segmental differences in mechanisms of sperm transport, forming a cellular network by physical interactions in the murine epididymis.

ATP8a1, an IFT27 binding partner, is dispensable for spermatogenesis and male fertility.

Intraflagellar transport 27 (IFT27) is a key regulator for spermiogenesis and male fertility in mice. ATP8a1, a protein involved in the translocation of phosphatidylserine and phosphatidylethanolamine across lipid bilayers, is the strongest binding partner of IFT27. To investigate the role of ATP8a1 in spermatogenesis and male fertility, the global Atp8a1 knockout mice were analyzed. All mutant mice were fertile, and sperm count and motility were comparable to the control mice. Examination of testis and epididymis by hematoxylin and eosin staining did not reveal major histologic defects. These observations demonstrate that ATP8a1 is not a major spermatogenesis regulator. Given that a tissue-specific paralogue of ATP8a1, ATP8a2, is present, further studies with double-knockout models are warranted to delineate any compensatory functions of the two proteins.

Modulation of adipocyte size and fat pad weight via resveratrol releasing scaffolds implanted into the epididymal adipose tissue.

Lipid overload of the adipose tissue, which can be caused by overnutrition, underlies metabolic disease. We hypothesized that increasing the energy demand of adipose tissue is a promising strategy to combat excessive lipid accumulation. Resveratrol, a natural polyphenol, activates lipid catabolism in fat tissue; however, its clinical success is hindered by poor bioavailability. Here, we implanted resveratrol releasing poly(lactide-co-glycolide) scaffolds into epididymal fat to overcome its poor bioavailability with the goal of enhancing local lipid catabolism. In lean mice, resveratrol scaffolds decreased adipocyte size relative to scaffolds with no drug, a response that correlated with AMP kinase activation. Immunohistochemistry indicated that macrophages and multinucleated giant cells within the scaffold expressed carnitine palmitoyltransferase 1 (CPT1) at higher levels than other cells in the adipose tissue. Furthermore, resveratrol increased CPT1 levels in cultured macrophages. Taken together, we propose that resveratrol scaffolds decrease adipocyte size because resveratrol increases lipid utilization in scaffold-infiltrating immune cells, possibly through elevating CPT1 levels or activity. In a follow-up study, mice that received resveratrol scaffolds 28-day prior to a high-fat diet exhibited decreased weight gain, adipose tissue expansion, and adipocyte hypertrophy compared to mice with control scaffolds. Notably, this scaffold-based strategy required a single resveratrol administration compared to the daily regiment generally needed for oral administration. These results indicate that localized delivery of metabolism modulating agents to the adipose tissue may overcome issues with bioavailability and that the role of biomaterials should be further investigated in this therapeutic strategy for metabolic disease.

Structural Investigation of Epididymal Microvasculature and Its Relation to Telocytes and Immune Cells in Camel.

The vascular and perivascular cells, including telocytes (TCs) and immune cells, play an important role in male fertility. The current study intended to describe in detail the microvascular structures harboring special regulatory devices in addition to the interstitial cellular components of the one-humped camel epididymis. The samples were collected from 10 clinically healthy mature camels (Camelus dromedarius). The distribution and characteristics of TCs, peripheral blood vessels of the epididymis, and immune cells were investigated using the light, immunohistochemistry, immunofluorescence, and transmission electron microscopy analyses. Frequent occlusive or throttle arterioles were demonstrated in the epididymal interstitium and their tunica media consisted of glomus cells. In addition, some vein walls consisted of one or two layers of glomus cells. TCs, fibroblasts, muscle cells, and tunica media of the blood vessels, that present in the loose connective tissue surrounding the intertubular interstitium of camel epididymis, showed a positive reaction with vimentin. The endothelium of blood vessels and veins showed positive immunoreactivity for CD34 and vascular endothelial growth factor (VEGF). Furthermore, VEGF, CD34, and S100 proteins were expressed in dendritic cells (DCs) as well as TCs. The current data suggest the involvement of DCs and TCs in angiogenesis and a possible role for the interstitial components in creating an appropriate milieu for the full maturation of sperms.

The essential role of intraflagellar transport protein IFT81 in male mice spermiogenesis and fertility.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism that is indispensable for the formation and maintenance of cilia and flagella; however, the implications and functions of IFT81 remain unknown. In this study, we disrupted IFT81 expression in male germ cells starting from the spermatocyte stage. As a result, homozygous mutant males were completely infertile and displayed abnormal sperm parameters. In addition to oligozoospermia, spermatozoa presented dysmorphic and nonfunctional flagella. Histological examination of testes from homozygous mutant mice revealed abnormal spermiogenesis associated with sloughing of germ cells and the presence of numerous multinucleated giant germ cells (symblasts) in the lumen of seminiferous tubules and epididymis. Moreover, only few elongated spermatids and spermatozoa were seen in analyzed cross sections. Transmission electron microscopy showed a complete disorganization of the axoneme and para-axonemal structures such as the mitochondrial sheath, fibrous sheath, and outer dense fibers. In addition, numerous vesicles that contain unassembled microtubules were observed within developing spermatids. Acrosome structure analysis showed normal appearance, thus excluding a crucial role of IFT81 in acrosome biogenesis. These observations showed that IFT81 is an important member of the IFT process during spermatogenesis and that its absence is associated with abnormal flagellum formation leading to male infertility. The expression levels of several IFT components in testes, including IFT20, IFT25, IFT27, IFT57, IFT74, and IFT88, but not IFT140, were significantly reduced in homozygous mutant mice. Overall, our study demonstrates that IFT81 plays an essential role during spermatogenesis by modulating the assembly and elongation of the sperm flagella.

Evidence that glycosaminoglycan storage and collagen deposition in the cauda epididymidis does not impair sperm viability in the Mucopolysaccharidosis type I mouse model.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by a deficiency of the lysosomal hydrolase, α-L-iduronidase (IDUA). IDUA degrades heparan and dermatan sulfates, two types of glycosaminoglycan (GAG), important signalling and structural molecules of the extracellular matrix. Because many cell types store GAGs, MPS I has been investigated in human and animal models. Enzyme replacement therapy is available for MPS I patients and has improved their life expectancy, allowing them to achieve reproductive age. The aim of this study was to evaluate epididymal and sperm morphology and function in a murine model of MPS I. We used C57BL Idua+/+ and Idua-/- adult male mice (6 months old) to investigate epididymal morphology, sperm ultrastructure, GAG characterisation and mating competence. Epithelial GAG storage, especially in the cauda epididymidis, was seen in Idua-/- mice. Regardless of the morphologic change and GAG storage found in the cauda epididymis, sperm morphology and motility were normal, similar to wild types. In the interstitium, vacuolated cells were found in addition to deposits of GAGs. Mating was not impaired in Idua-/- males and litter sizes were similar between groups. At the time point of the disease evaluated, the deficiency in IDUA affected the morphology of the epididymis in male Idua-/- mice, whereas sperm appearance and motility and the male's capacity to mate and impregnate females were preserved.

Epididymal ultrastructural changes associated with chronic cholestasis after bile duct ligation in adult rats.

Testicular atrophy and testesterone insufficiency have been commonly reported associated with chronic liver diseases. Though testosterone dependent, the epididymal changes induced by liver disease have never been studied before. Thus, this study aimed to assess the ultrastructural events in the epididymis of rats with chronic obstructive jaundice. Chronic cholestasis induced many epididymal structural alterations manifested by the reduced tubular diameters, thickening of the tubular basement membrane, and regression of the principal cells. This was accompanied with reduction of principal cell organelles, cytoplasmic vacuolations, nuclear alterations, and stereovilli loss. The results establish that chronic cholestasis causes epididymal structural changes due to androgen deficiency.

Absence of CFAP69 Causes Male Infertility due to Multiple Morphological Abnormalities of the Flagella in Human and Mouse.

The multiple morphological abnormalities of the flagella (MMAF) phenotype is among the most severe forms of sperm defects responsible for male infertility. The phenotype is characterized by the presence in the ejaculate of immotile spermatozoa with severe flagellar abnormalities including flagella being short, coiled, absent, and of irregular caliber. Recent studies have demonstrated that MMAF is genetically heterogeneous, and genes thus far associated with MMAF account for only one-third of cases. Here we report the identification of homozygous truncating mutations (one stop-gain and one splicing variant) in CFAP69 of two unrelated individuals by whole-exome sequencing of a cohort of 78 infertile men with MMAF. CFAP69 encodes an evolutionarily conserved protein found at high levels in the testis. Immunostaining experiments in sperm from fertile control individuals showed that CFAP69 localized to the midpiece of the flagellum, and the absence of CFAP69 was confirmed in both individuals carrying CFPA69 mutations. Additionally, we found that sperm from a Cfap69 knockout mouse model recapitulated the MMAF phenotype. Ultrastructural analysis of testicular sperm from the knockout mice showed severe disruption of flagellum structure, but histological analysis of testes from these mice revealed the presence of all stages of the seminiferous epithelium, indicating that the overall progression of spermatogenesis is preserved and that the sperm defects likely arise during spermiogenesis. Together, our data indicate that CFAP69 is necessary for flagellum assembly/stability and that in both humans and mice, biallelic truncating mutations in CFAP69 cause autosomal-recessive MMAF and primary male infertility.

Morphometric and ultrastructural analysis of tramadol effects on epididymis: an experimental study.

The effect of tramadol addiction on epididymal structure was not investigated before. Therefore, this experimental study was carried out to investigate the effect of chronic tramadol use on the epididymal structure using light and electron microscopies. Thirty adult Wister Albino male rats were divided into two groups: control group (five rats) and tramadol-treated group (25 rats), which was further subdivided into five subgroups that received tramadol orally at 4.5, 9, 45, 90, and 135 mg/kg/day, respectively, for 18 weeks. Epididymal tissues were dissected and processed for histopathological examination. Morphometric analysis showed significantly reduced mean values of epididymal ducts' diameters and epithelial height in the tramadol-treated group compared with the control group. Light microscopic examination revealed degeneration and necrosis of epididymal cells in the tramadol-treated group. Electron microscopic (EM) examination showed ultrastructure alterations in a dose-dependent manner. In conclusion, tramadol can adversely affect all epididymal cells, which subsequently deteriorate epididymal function and may affect sperm maturation, leading to subfertility.

Ultrastructure and morphometric features of epididymal epithelium in Desmodus rotundus.

The blood-feeding behavior of Desmodus rotundus made this bat a potential vector of rabies virus and a public health issue. Consequently, the better understanding of its reproductive biology becomes valuable for the development of methods to control its population. In this study, we described morphological aspects of epithelial cells in D. rotundus' epididymis using light and transmission electron microscopy methods. The duct compartment was the main component of initial segment (83%), caput (90%), corpus (88%) and cauda (80%) regions. The epithelium lining the duct presented a progressive decrease in its height from initial segment to cauda regions. Moreover, the morphology of each cell type was the same along the entire duct. Similarly to rodents, columnar-shaped principal cells were the most abundant cell type throughout the epididymis, followed by basal and clear cells. Differently in rat and mice, the frequency of clear cells did not increase in the epididymis cauda, whereas the proportion of principal and basal cells was greater in this region. Furthermore, D. rotundus presented goblet-shaped clear cells with the nucleus located in the apical portion of the epididymal epithelium. This cellular portion also presented electron-lucid vesicles of different sizes that may correspond to vesicles enriched with proteins related to proton secretion. In addition to the findings regarding clear cells' structural organization, basal cells presented scarce cytoplasm and no axiopodia. Taken these findings together, we suggest that the mechanism of luminal acidification may have other pathways in D. rotundus than those described in rodents.

Histological study on the effect of aroclor 1254 on the epididymis of adult rats and the role of L-NAME administration.

INTRODUCTION: Aroclor 1254, a commercial polychlorinated biphenyls (PCBs) mixture, was found to elicit various adverse effects on human health. OBJECTIVES: This study aimed to investigate the structural alterations in the epididymis induced by aroclor 1254, and to assess the possible protective role of L-NAME (NG-Nitro-L arginine methyl ester). MATERIALS AND METHODS: Thirty-five adult male albino rats were divided into three groups: control group (15 rats), equally subdivided into subgroup a; negative control group, subgroup b: received intraperitoneal corn oil (5 ml/kg/day), and subgroup c: received intraperitoneal L-NAME (10 mg/kg/day). Aroclor-treated group (10 rats): received aroclor 1254 (2 mg/kg/day, intraperitoneal), and aroclor + L-NAME-treated group (10 rats): received aroclor 1254 combined with L-NAME in the same previous regimen. After 30 days, blood samples were collected for hormonal assay. Specimens from the epididymis were prepared for histological study and assessment of sperm count. RESULTS: Rats in aroclor-treated group revealed a significant reduction in serum testosterone level and sperm count, in comparison with the control group. The epididymal caput showed stratification and detachment of the epithelium with vacuoles, mitotic figures, and electron-dense bodies together with increased collagen fibers in the interstitium. In addition, a strong reaction of androgen receptors (ARs) was seen in the cytoplasm of epithelial and stromal cells. These effects were attenuated by L-NAME administration. CONCLUSION: Aroclor 1254 provoked morphological and functional changes in the epididymis of adult rats, which were attenuated by L-NAME administration.

Influence of iodine in excess on seminiferous tubular structure and epididymal sperm character in male rats.

Excess iodine induced public health problems are now emerging in many iodine sufficient regions for indiscriminate intake of iodine through various iodized products. It has been reported that excess iodine can disrupt overall male reproductive physiology by generating oxidative stress in the testis. However, information on the possible effect of iodine in excess on spermatozoa found less. In the present investigation flow cytometric techniques and scanning electron microscopy (SEM) have been used to study the spermatozoal functional as well as structural status under the influence of excess iodine; generation of ROS in the spermatozoa as evident by DCFDA, altered acrosomal integrity as observed by fluorescence lectin staining method and depolarized mitochondrial membrane potential (ΔΨm ) noticed by JC-1 staining. Ultrastructure of seminiferous tubule after excess iodine exposure indicated severe deterioration of seminiferous tubular surface architecture. Significant increase in spermatozoal DNA fragmentation and apoptotic sperms were found by acridine orange and Annexin V, respectively, however the plasma membrane integrity/viability was decreased as evident by propidium iodide staining in various incremental doses and durations under iodine excess. The study reveals that excess iodine could cause apoptosis of spermatozoal cells by inducing ROS that ultimately affects male fertility potential.

Impaired spermatogenesis in the twitcher mouse: A morphological evaluation from the seminiferous tubules to epididymal transit.

Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa. In this developmental process the interactions between different cell types are finely regulated, hence any disruption in these relationships leads to male infertility. The twitcher mouse, the murine model of Krabbe disease, is characterized by deficiency of galactosylceramidase, an enzyme also involved in the metabolism of the galactosyl-alkyl-acyl-glycerol, the precursor of sulfogalactosyl-alkyl-acyl-glycerol, the most abundant glycolipid in spermatozoa. Twitcher mice are sterile due to alterations of spermatogenesis resulting in the production of spermatozoa with abnormally swollen acrosomes and bent flagella, mainly at the midpiece-principal piece junction. The current study employs light, fluorescence, and electron microscopy to examine the defective spermiogenesis leading to the morphological abnormalities of mature sperm. This study reveals that alterations in germ cell development can be initially detected at the stage VIII and IX of spermatogenesis. The disrupted spermatogenetic process leads to a reduced number of elongating spermatids and spermatozoa in these mutant animals. Electron microscopy analysis demonstrates major acrosomal and chromatin condensation defects in the mutants. In addition, in twitcher mice, the epididymal architecture is impaired, with stereocilia of caput and corpus broken, detached and completely spread out into the lumen. These findings indicate that seminolipid expression is crucial for proper development of spermatocytes and spermatids and for their normal differentiation into mature spermatozoa. ABBREVIATIONS: GALC: galactosylceramidase; GalAAG: galactosyl-alkyl-acyl-glycerol; SGalAAG: sulfogalactosylalkylacylglycerol; PND: postnatal day; PAS: periodic acid-Schiff stain; TEM: transmission electron microscopy; SEM: scanning electron microscopy; PFA: paraformaldheyde.

CABYR is essential for fibrous sheath integrity and progressive motility in mouse spermatozoa.

Ca2+-binding tyrosine-phosphorylation-regulated protein (CABYR) has been implicated in sperm physiological function in several in vitro studies. It has also been implicated as a potential cause of and diagnostic tool in asthenozoospermic human males. CABYR is known to be localized to the fibrous sheath, an accessory structure in the flagellar principal piece. Utilizing the CRISPR-Cas9 technology, we have knocked out this gene in mice to understand its role in male fertility. Cabyr-knockout male mice showed severe subfertility with a defect in sperm motility as well as a significant disorganization in the fibrous sheath. Further, abnormal configuration of doublet microtubules was observed in the Cabyr-knockout spermatozoa, suggesting that the fibrous sheath is important for the correct organization of the axoneme. Our results show that it is the role of CABYR in the formation of the fibrous sheath that is essential for male fertility.

Morphological Examination and Transcriptomic Profiling To Identify Prednisolone Treatment in Beef Cattle.

In livestock production corticosteroids are licensed only for therapy; nevertheless, they are often illegally used as growth promoters. The aim of this study was to identify morphological or biomolecular alterations induced by prednisolone (PDN) in experimentally treated beef cattle, because PDN and its metabolites are no longer detectable by LC-MS/MS methods in biological fluids. Moreover, PDN does not induce any histological alterations in the thymus, different from dexamethasone treatments. Therefore, a marker of illicit treatment for this growth promoter could be useful. Eight male Italian Friesian beef cattle were administered prednisolone acetate 30 mg day-1 per os for 35 days, and seven beef cattle represented the control group. Six days after drug withdrawal, the animals were slaughtered. Morphological and morphometric modifications were evaluated in the epididymis and testis, whereas transcriptomic changes induced by PDN administration were investigated in peripheral blood mononuclear cells (PBMCs) at different sampling times and in skeletal muscle and testis sampled at slaughtering. In the epididymis, spermatozoa number decreased in PDN-treated animals, and in some cases they were totally absent. Correspondingly, in the testis of treated animals, down-regulation for serine/threonine kinase 11 (STK11) gene expression was detected (p < 0.01). DNA microarray analysis revealed a total of 133 differentially expressed genes in skeletal muscle and testis, and 907 and 1416 in PBMCs after 33 days of treatment and at slaughtering, respectively. Histological investigations on epididymal content could represent a promising marker for PDN treatment in beef cattle and could be used as a screening method to identify animals worthy of further investigation with official methods. Moreover, the clear transcriptomic signature of PDN treatment evidenced in PBMCs supported the possibility of using this matrix to monitor the illicit treatment in vivo during ranching.

Expression of TLR2/4 on Epididymal Spermatozoa of the Chinese Soft-Shelled Turtle Pelodiscus sinensis During the Hibernation Season.

Spermatozoa are known to be stored in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis for long periods during hibernation, but the mechanism that underlies the sperm storage is poorly understood. This study was carried out to confirm the presence of TLR2/4 (Toll-like receptor 2/4) in epididymal spermatozoa during the hibernation season and to analyze whether TLRs play a role in sperm storage. The structure and ultrastructure of a spermatozoon during the hibernation stage were investigated using light- and transmission electron-microscopy. RT-PCR was used to analyze mRNA expression, while protein expression was determined via Western blot. TLR2/4 mRNA and proteins were detected in spermatozoa. Immunofluorescence staining was used to confirm TLR2/4 localization in the spermatozoon, and TLR2/4 were localized in the midpiece and the posterior segment of the head of the spermatozoon, which corresponded to the cytoplasmic droplets (CDs) of the turtle spermatozoon. As TLRs play critical roles in detecting and responding to invading pathogens, this study provided molecular evidence that TLR2/4 might contribute to sperm storage in the epididymides. Anat Rec, 299:1578-1584, 2016. © 2016 Wiley Periodicals, Inc.

Reversible Anti-Spermatogenic Effect of Piperine on Epididymis and Seminal Vesicles of Albino Rats.

BACKGROUND: We have recently proved the interactions of piperine with androgen receptor and androgen binding protein. The present study was aimed to evaluate the antifertility effect of piperine on male albino rats after the treatment period i. e., after 60 days and withdrawal period i. e., after 120 days. MATERIALS AND METHODS: Adult male rats were divided into 4 groups (n=12). Group I: CONTROL: Rats were given vehicle p.o i. e., 0.5% carboxy methyl cellulose (CMC) in normal saline daily for 60 days, Group II: Rats were treated with piperine suspended in 0.5% CMC at a dose of 10 mg/kg daily/60 days. Group III: Rats were treated with piperine suspended in 0.5% CMC at a dose of 10 mg/kg on every 4(th) day for 60 days. Group IV: Rats were treated with piperine suspended in 0.5% CMC at a dose of 10 mg/kg on every 7(th) day for 60 days. RESULTS: Piperine significantly altered the epididymal sperm count, motility, viability, weight of the epididymis, cauda, caput, corpus and seminal vesicles. It also exhibited negative impact on biochemical markers via decreasing epididymal sialic acid levels, seminal fructose content, epididymal anti-oxidant enzyme activities of super oxide dismutase (SOD), catalase (CAT) and by increasing the malondialdehyde content after the treatment period. Histopathological observations also supported the above findings. All the altered values were reinforced after the withdrawal period. CONCLUSION: From the results of this study, we can conclude that piperine has the potential to become a good lead for the reversible male oral contraceptive research.

Cellular evidence for nano-scale exosome secretion and interactions with spermatozoa in the epididymis of the Chinese soft-shelled turtle, Pelodiscus sinensis.

The epididymis is the location of sperm maturation and sperm storage. Recent studies have shown that nano-scale exosomes play a vital role during these complicated processes. Our aim was to analyze the secretory properties of epididymal exosomes and their ultrastructural interaction with maturing spermatozoa in the Chinese soft-shelled turtle. The exosome marker CD63 was primarily localized to the apices of principal cells throughout the epididymal epithelium. Identification of nano-scale exosomes and their secretory processes were further investigated via transmission electron microscopy. The epithelium secreted epididymal exosomes (50~300 nm in diameter) through apocrine secretion and the multivesicular body (MVB) pathway. Spermatozoa absorbed epididymal exosomes through endocytosis or membrane fusion pathways. This study shows, for the first time, that nano-scale exosomes use two secretion and two absorption pathways in the reptile, which may be contribute to long-term sperm storage.

Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging.

Epithelial cells are generally considered to be static relative to their neighbours. Basal cells in pseudostratified epithelia display a single long cytoplasmic process that can cross the tight junction barrier to reach the lumen. Using in vivo microscopy to visualize the epididymis, a model system for the study of pseudostratified epithelia, we report here the surprising discovery that these basal cell projections--which we call axiopodia--periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we refer to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2-ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment.

Morphological and ultrastructural study of the efferent ductules in the Chinese soft-shelled turtle Pelodiscus sinensis.

Comparative 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 are extra- testicular and 22-28 in number originate from rete testis. The epithelium is 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 contain an endocytic apparatus with coated pits and tubules in the apical cytoplasm. Interdigitations and lipid droplets are predominantly present around the nuclei of these cells. The non-ciliated cells have clusters of mitochondria present in both the supranuclear and perinuclear cytoplasm whereas, the nuclei of these cells are lightly stained. Moreover, the contour of the epithelium towards lumen is irregular as it has 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.