Efferent Ducts Research Articles

G protein-coupled oestrogen receptor 1, oestrogen receptors and androgen receptor in the sand rat (Psammomys obesus) efferent ducts.

The efferent ducts are mainly involved in the reabsorption of the seminiferous tubular fluid. Testosterone and oestrogens regulate efferent ducts functions via their receptors. This paper presents an experimental investigation on the location of the P450 aromatase, the 17-b oestradiol (E2), the androgen receptor (AR), the oestrogen receptor 1 (ESR1), the oestrogen receptor 2 (ESR2) and the G protein-coupled oestrogen receptor 1 (GPER1) in the efferent ducts using Psammomys obesus as an animal model to highlight the effect of the season on the histology and the distribution of these receptors. We observed a proliferation of the connective tissue, decreasing in the height of the epithelium during the resting season compared to the breeding season. Ciliated cells expressed P450 aromatase, AR, E2, ESR1, ESR2 and GPER1 during both seasons. Basal cells showed a positive staining for the ESR1 and the GPER1 during both season, the AR and E2 during the breeding season and ESR2 during the resting season. Our result shows that the expression of androgen receptor and oestrogen receptors in the efferent ducts vary by season witch suggest that they are largely involved in the regulation of the efferent ducts functions.

Function and therapeutic potential of G protein-coupled receptors in epididymis.

Infertility rates for both females and males have increased continuously in recent years. Currently, effective treatments for male infertility with defined mechanisms or targets are still lacking. G protein-coupled receptors (GPCRs) are the largest class of drug targets, but their functions and the implications for the therapeutic development for male infertility largely remain elusive. Nevertheless, recent studies have shown that several members of the GPCR superfamily play crucial roles in the maintenance of ion-water homeostasis of the epididymis, development of the efferent ductules, formation of the blood-epididymal barrier and maturation of sperm. Knowledge of the functions, genetic variations and working mechanisms of such GPCRs, along with the drugs and ligands relevant to their specific functions, provide future directions and a great arsenal for new developments in the treatment of male infertility.

Mouse defensin beta 20 (Defb20) is expressed specifically in the caput region of the epididymis and regulated by androgen and testicular factors

Sperm cells undergo maturation during their transit throughout the epididymis. This process takes place in region-specific manner in which sperm are battered by proteins secreted by epithelium lining the epididymal duct. Most of the genes that encode for the proteins involved in the sperm maturation remain uncharacterized. Previous studies showed that family of β-defensins preferentially eaxpressed in male reproductive tracts and play an important role in both innate immunity and sperm fertility. In this study we characterized Defb20 to gain insight on its role in sperm maturation. Bioinformatic tools were used to analyzed functional domains and signal peptide. qRT-PCR analyses were used to analyzed tissue distribution, dependency on androgen and testicular factors and developmental-regulated expression analysis. Defb20 sequence contains important domains such as N-myristoilation and kinase binding sites which are putatively involved in the protein activation and protein-plasma membrane interaction. Moreover, DEFB20 contains a signal peptide indicating characteristic of secretory proteins. Defb20 was expressed exclusively in the epididymis with the highest expression in the caput region and was down-regulated by gonadectomy. Defb20 was also regulated by testicular factors in which the expression was down-regulated after efferent duct ligation (EDL). The dependency on the androgen was further confirmed by postnatal expression analysis in which Defb20 began to express at day-20 postnatal indicating specific stage of expression after initial development of the testis. In conclusion, Defb20 have a potential to be involved in the epididymal sperm maturation process.

Immunolocalization of androgen and vitamin D receptors in the epididymis of mature ram (Ovis aries).

This study illustrated the immunohistochemical distribution of androgen and vitamin D receptors of epididymis in 20 sexually mature ram (Rahmani breed) with average age ranged from (2_4) years and average weight ranged from (50_65kg). Androgen receptor was localized in the cytoplasm of both ciliated and non ciliated cells of efferent ductules, besides the principal cells via the entire epididymal duct. The principal cells of both corpus and proximal cauda epididymis showed the highest immunoreactivity to androgen receptors. Furthermore, vitamin D receptor was localized in the cytoplasm of all epithelium of the efferent ductules besides principal cells of all epididymal regions, however the immunoreaction was significantly higher in the efferent ductules, distal caput and distal cauda epididymis. In conclusion, these results suggest that the function of ram epididymis is regulated by both androgen and Vitamin D.

Neutrophil Extracellular Traps Promote the Development and Growth of Human Salivary Stones.

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. The mechanism behind the formation of sialoliths has been elusive. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland, and often results in sialadenitis with the need for surgical intervention. Here, we show by the use of immunohistochemistry, immunofluorescence, computed tomography (CT) scans and reconstructions, special dye techniques, bacterial genotyping, and enzyme activity analyses that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths in humans. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in the dense aggregation of the latter, and the subsequent mineralization creates alternating layers of dense mineral, which are predominantly calcium salt deposits and DNA. The further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis, in which an immune system-mediated response essentially participates in the physicochemical process of concrement formation and growth.

Formation of the rete testis during mouse embryonic development.

The rete testis connects seminiferous tubules of the testis with efferent ducts having a mesonephric origin. The development of the rete testis is insufficiently studied, but there is evidence suggesting that it originates from gonadal cells. Here, the formation of the rete testis was investigated from E11.5 to E16.5 using immunofluorescent staining and 3D-modeling. The rete testis became visible by SOX9 and PAX8 staining starting from E12.5. It was located in the mesonephros but connected with testis cords formed by Sertoli cells expressing SOX9, AMH, DMRT1. Between E13.5 and E14.5, AMH+ network of testis cords at the mesonephric side began to disintegrate in a gradient-dependent manner along the anterior-posterior axis of the gonad and connections between testis cords gradually lost AMH becoming a part of the rete. Cells combining features of Sertoli and rete cells (PAX8+ AMH+ and DMRT1+ AMH- cells) were detected starting from E14.5, suggesting that some rete cells originated from Sertoli cells. The rete ovarii, a female counterpart of the rete testis, developed in a similar way as the rete testis until E13.5. A part of the rete testis originates from connections between testis cords. Evidence that Sertoli cells contribute to rete cells is provided.

A NEW METHOD FOR ACCURATE PREDICTION OF SPERMATOGENESIS: FSH/T AND T IN SEMINAL PLASMA

A NEW METHOD FOR ACCURATE PREDICTION OF SPERMATOGENESIS: FSH/T AND T IN SEMINAL PLASMA

An atlas of human proximal epididymis reveals cell-specific functions and distinct roles for CFTR.

Spermatozoa released from the testis are unable to fertilize an egg without a coordinated process of maturation in the lumen of the epididymis. Relatively little is known about the molecular events that integrate this critical progression along the male genital ducts in man. Here, we use single cell RNA-sequencing to construct an atlas of the human proximal epididymis. We find that the CFTR, which is pivotal in normal epididymis fluid transport, is most abundant in surface epithelial cells in the efferent ducts and in rare clear cells in the caput epididymis, suggesting region-specific functional properties. We reveal transcriptional signatures for multiple cell clusters, which identify the individual roles of principal, apical, narrow, basal, clear, halo, and stromal cells in the epididymis. A marked cell type-specific distribution of function is seen along the duct with local specialization of individual cell types integrating processes of sperm maturation.

Anatomy and histology of the male reproductive tract in giant water bugs of the genus Belostoma Latreille, 1807 (Heteroptera, Belostomatidae)

Giant water bugs (Belostomatidae) are widespread in the tropical and subtropical regions and have important ecosystem functions. This study describes the structure of the male reproductive tract in three species of Belostoma Latreille, 1807. The reproductive systems of sexually mature males of B. plebejum, B. stollii and B. testaceopallidum have a pair of globular testes, deferent ducts, seminal vesicles, an ejaculatory duct and an aedeagus. Each testis has five folded follicles, and each follicle is continuous with an efferent duct (0.72 cm in length on average), which opens together in the deferent duct (0.85 cm in length on average). Inside the testis follicles, germ cells develop synchronously in cysts with seven mitotic and two meiotic cycles. The deferent ducts have a dilated portion for the storage of spermatozoa, the seminal vesicles. Accessory glands were absent. The morphological characteristics of the male reproductive tract in these three species of Belostoma and their similarities with other aquatic Heteroptera are discussed.

Ruavermis mikebargeri gen. et sp. n. (Digenea: Schistosomatoidea) infecting the yellow-headed temple turtle, Heosemys annandalii (Cryptodira: Geoemydidae), in Vietnam, including an updated phylogeny for the turtle blood flukes

Ruavermis mikebargeri gen. et sp. n. infects the yellow-headed temple turtles Heosemys annandalii (Boulenger et Robinson) in the Mekong River Basin. It resembles Platt Roberts et Bullard, 2018 and Coeuritrema Mehra, 1933 by having the anterior to posterior anatomical sequence of a ventral sucker, external seminal vesicle, cirrus sac, anterior testis, ovary, transverse vitelline duct, and posterior testis. These genera are further similar by having the combination of an elongate/ovoid aspinous body, a ventral sucker at the level of the body constriction, an oesophagus that terminates in the anterior 1/5 of the body and that is ventral to the anterior nerve commissure, intestinal caeca that bifurcate in the anterior 1/3 of the body (not immediately anterior to the ventral sucker), a sinistral caecum that bends toward the midline at level of the cirrus and common genital pore, an external seminal vesicle that abuts the anterodextral margin of the cirrus sac, an oviduct that emerges from the dextral margin of the ovary, and an oviducal seminal receptacle that comprises the middle portion of the oviduct. These genera lack lateral oesophageal diverticulae and a median oesophageal diverticulum. The new genus is unique by having a papillate ventral body surface, an external seminal vesicle lateral to the cirrus sac, vasa efferentia that are ventral to the gonads, an oviduct that is convoluted, a Laurer's canal pore that is preovarian, a Laurer's canal that extends anterolaterad, and an excretory vesicle that is Y-shaped. The 28S rDNA phylogenetic analysis recovered the new species sister to Coeuritrema platti Roberts et Bullard, 2016, with that clade sister to Hapalorhynchus spp. and Platt spp. The new turtle blood fluke is the fourth from Vietnam, second from a Vietnam geomydid, and first from Heosemys Stejneger as well as the first endohelminth from the yellow-headed temple turtle.

Seasonal Timing of Spermatogenesis and Mating in Squamates: A Reinterpretation

The squamates occur in a variety of climates from tropical to Arctic regions. Being poikilotherms, snakes and lizards in temperate regions, and high elevation tropical environments, must adjust their reproductive biology to reproduce at a time that optimizes offspring survival. The two major components of the reproductive cycle in both males and females are gametogenesis and mating. The reproductive cycle of males is the focus of this study. In snakes in temperate climates, sperm production (spermatogenesis) may occur immediately prior to mating (prenuptial spermatogenesis) or following mating (postnuptial spermatogenesis). In postnuptial spermatogenesis, sperm are produced following the mating season and stored in the efferent testicular ducts (primarily the ductus deferens) until the following spring mating season. Given that most recent phylogenetic reconstructions resolve snakes as a monophyletic group of highly specialized lizards, it is generally assumed that lizards have spermatogenic cycles similar to snakes. Lizard spermatogenic cycles are often described as prenuptial or postnuptial. We propose that the major difference between snake and lizard spermatogenic cycles is the presence of postnuptial spermatogenesis in snakes and the absence of true postnuptial spermatogenesis in lizards. Our interpretation of lizard spermatogenic cycles suggests that all lizards have prenuptial spermatogenesis (i.e., sperm are produced immediately prior to mating). If fertilization occurs months after mating, the female, and not the male, stores the sperm until spring ovulation and fertilization. Using a variety of analytical tools, we analyzed the reproductive strategies of snakes and lizards, and we have concluded that they differ in fundamental ways. Most notably, prenuptial spermatogenesis is the ancestral condition for Squamata with continuous spermatogenesis evolving multiple times independently within lizards and snakes. We also found that postnuptial spermatogenesis evolved early in the evolutionary history of snakes but, we argue, has never evolved in lizards. We suggest that the evolutionary origin of snakes may account for the differences observed in snake versus lizard reproductive cycles, and we present a scenario for the evolution of snake reproductive cycles.

Axisymmetric mixed convective propulsion of a non-Newtonian fluid through a ciliated tubule

Thousands of chemical reactions occur in the human body when certain biological fluids, such as blood, semen, mucus, and synovial joint materials, move in various organs. These reactions play a vital role in regulating the life sustaining metabolic processes in the body. Analysis of thermal effects on these chemical reactions is relatively a new area in modern clinical medications. The present study investigates a simulation of the combined response due to heat and mass transport mechanisms taking place in the human body during the flow of physiological fluids. In particular, we focus our attention on the human male reproductive system, wherein the semen transports through the ductus efferentes due to metachronal waves of cilia. The constitutive relations of the robust Jeffrey viscoelastic fluid are used to model the human semen. The mathematical model of the present problem constitutes the axisymmetric flow of a Jeffrey fluid inside a vertical tubule under the influence of mixed convective heat and mass transfers. The inner side of the tubule is covered with ciliated structures. The influence of thermal behaviors of various metabolic processes in the human body due to an external heat source or sink is also taken into account. The mathematical formulation consists of using the approach of lubrication theory approximation; the nonlinear momentum, energy, and concentration equations are simplified to get analytical solutions. Explicit expressions for temperature, concentration, velocity, pressure gradient, and volume flow rate of the proposed bodily fluid (i.e., human semen) are formulated. The expression for the volume flow rate is used to estimate the volume flux of the semen under the influence of various parameters. A comparison between the theoretical and experimentally obtained values of the flow rate of the human semen is also made. It is noted that our calculated values are very close to the estimated values. Industrial applications of the present results are obvious in the fabrication of artificial cilia pumping systems for microfluidic flow systems.

A novel hemizygous loss-of-function mutation in ADGRG2 causes male infertility with congenital bilateral absence of the vas deferens.

Cystic fibrosis transmembrane conductance regulator (CFTR) and adhesion G protein-coupled receptor G2 (ADGRG2) have been identified as the main pathogenic genes in congenital bilateral absence of the vas deferens (CBAVD), which is an important cause of obstructive azoospermia. This study aimed to identify the disease-causing gene in two brothers with CBAVD from a Chinese consanguineous family and reveal the intracytoplasmic sperm injection (ICSI) outcomes in these patients. Whole-exome sequencing and Sanger sequencing were used to identify the candidate pathogenic genes. Real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to assess the expression of the mutant gene. Moreover, the ICSI results from both patients were retrospectively reviewed. A novel hemizygous loss-of-function mutation (c.G118T: p.Glu40*) in ADGRG2 was identified in both patients with CBAVD. This mutation is absent from the human genome databases and causes an early translational termination in the third exon of ADGRG2. Expression analyses showed that both the ADGRG2 mRNA and the corresponding protein were undetectable in the proximal epididymal tissue of ADGRG2-mutated patients. ADGRG2 expression was restricted to the apical membranes of non-ciliated epithelia in human efferent ducts, which was consistent with a previous report in mice. Both ADGRG2-mutated patients had normal spermatogenesis and had successful clinical outcomes following ICSI. Our study verifies the pathogenic role of ADGRG2 in X-linked CBAVD and broadens the spectrum of ADGRG2 mutations. In addition, we found positive ICSI outcomes in the two ADGRG2-mutated CBAVD patients.

Disorders of the Lacrimal Apparatus, Part 2: Basics of Therapy

The tearing eye (epiphora) is deemed to be the leading symptome of efferent tear duct stenosis. Nevertheless, epiphora might be caused by ocular surface pathologies or even intraocular diseases. A distinguished anamnesis and sufficient clinical examination is most meaningful for the differential diagnostic distinction. Therapy is based on pathologic changes. In case of ocular surface disease, a suitable tear substitution and antiinflammatory approach is mandatory. Multifarious triggers have to be considered as well. Addressing tear film distribution, suitable oculoplastic surgery is required. Efferent tear duct stenosis necessitates surgical treatment as the level of suffering is often very high and acute exacerbations may develop. Besides recanalization, anastomosing techniques represent the available therapeutic principles. Possible surgical approaches offer a great variety and many influencing factors navigate therapy decisions. This part of the review comments on the most important therapeutic approaches for lacrimal apparatus diseases.

Heat transfer analysis of biological nanofluid flow through ductus efferentes

A theoretical investigation on heat transfer of nanofluid transport in a ciliated channel is presented. A mathematical model for cilia oriented nanofluid transport through the human male reproductive tract is exhibited. Considering the structure of tubules, the Reynolds number is considered very small. The concept of a long wavelength approximation is incorporated to model the nanofluid flow problem. The exact analytical expressions for the x-component of velocity, temperature, nanoparticle concentration, stream function, volume flow rate, and pressure gradient are calculated. The solution for the transverse component of velocity, pressure rise per wavelength, and friction force on the wall of the channel is explored numerically, while variations in crucial flow parameters are examined graphically.

Loss of Cyp11c1 causes delayed spermatogenesis due to the absence of 11-ketotestosterone.

The impacts of androgens and glucocorticoids on spermatogenesis have intrigued scientists for decades. 11β-hydroxylase, encoded by cyp11c1, is the key enzyme involved in the synthesis of 11-ketotestosterone and cortisol, the major androgen and glucocorticoid in fish, respectively. In the present study, a Cyp11c1 antibody was produced. Western blot and immunohistochemistry showed that Cyp11c1 was predominantly expressed in the testicular Leydig cells and head kidney interrenal cells. A mutant line of cyp11c1 was established by CRISPR/Cas9. Homozygous mutation of cyp11c1 caused a sharp decrease of serum cortisol and 11-ketotestosterone, and a delay in spermatogenesis which could be rescued by exogenous 11-ketotestosterone or testosterone, but not cortisol treatment. Intriguingly, this spermatogenesis restored spontaneously, indicating compensatory effects of other androgenic steroids. In addition, loss of Cyp11c1 led to undersized testes with a smaller efferent duct and disordered spermatogenic cysts in adult males. However, a small amount of viable sperm was produced. Taken together, our results demonstrate that cyp11c1 is important for testicular development, especially for the initiation and proper progression of spermatogenesis. 11-ketotestosterone is the most efficient androgen in tilapia.

Disorders of the Lacrimal Apparatus, Part 1: Anatomy, Physiology,Diagnostics

The lacrimal apparatus is a part of the ocular adnexa preserving ocular surface homoeostasis and therefore enabling sufficiant visual functioning. Tear producting tissues, eyelids and efferent tear ducts operate as one functional unit. Pathologic changes consist of inflammatory diseases, congenital disorders, degenerations and neoplasia. Common to all these conditions is a potential impairment of the ocular integrity as well as a substantial degree of suffering for the affected patients. This review reflects basics of the anatomy, physiology, and diagnostics of the lacrimal apparatus.

Differential gene expression profiles of human efferent ducts and proximal epididymis.

Gene expression patterns along the epididymis are established by specific transcription factor networks that coordinate region-specific functions. In rodents, the epididymis can be divided in up to 19 segments. Based on gross anatomy, the human epididymis is divided into caput, corpus, and cauda segments together with efferent ducts that occupy the proximal part of this organ. To determine to which extent gene expression pattern is segmented along the efferent ducts and the proximal region of the epididymis in men of reproductive age. Epididymal transcriptome profiling was performed on eight distinct regions from three donors. Microarray analysis was performed on a gene-chip microarray. Differentially expressed genes (DEGs)>2-fold change (P<.05) were clustered in relation to their intensity profiles. Overrepresented biological functions from gene ontology were searched using DAVID 6.8. Expression profiles were validated by qRT-PCR quantification of selected genes. There were no DEGs among segments 1-3 of efferent ducts region neither among segments 4-6 of the caput epididymis. 1058 DEGs were identified between efferent ducts and the epididymis, whereas 444 and 846 DEGs distinguished the caput from the corpus (section 7) and cauda (section 8) epididymis, respectively. A total of 131 DEGs were detected between corpus (7) and cauda (8) transcriptomes. Up-regulated DEGs in the efferent ducts were predominantly related to cilium assembly/movement and cell differentiation. Fertilization, defense, and immune responses were associated with caput epididymis (4-6), while spermatogenesis and protein binding were found all along the epididymis (4-8). The proximal human epididymis is exclusively occupied by efferent ducts with a distinct DEG profile compared with the downstream epididymal segments. Moreover, gene expression profiling revealed two regions in the human epididymis: the caput and the distal corpus/cauda region. Human epididymal transcriptome reveals limited DEGs, and efferent ducts have a distinct DEGs profile.

Seasonal histology and ultrastructure of the urogenital system in two sympatric lizards

AbstractThe urogenital system of male squamates consists of the testis, testicular ducts, and sexual segment of the kidney. These structures undergo seasonal variation, and their morphology is variable across taxa. Despite multiple studies examining the morphology of the various components of the reproductive system, few studies have examined squamates in sympatry. The purpose of this study was to investigate the morphology and seasonal variation of the reproductive system in two taxa that are sympatric with one another, Anolis carolinensis and Sceloporus undulatus, from southeastern Louisiana. Specimens were collected monthly for an entire year and processed for histological and ultrastructural analysis. Both species have a single rete testis leading from the testis to the ductuli efferentes, which leads into the proximal portion of the epididymis. The rete testis consists of a simple squamous epithelium with irregularly shaped nuclei, the ductuli efferentes consists of a ciliated simple cuboidal epithelium, and the epididymis a pseudostratified columnar epithelium. No differences were observed in the morphology of the urogenital systems between taxa but phenological variation was observed in terms of the tissue development for reproduction. Anolis carolinensis has an extended active period and two separated periods of maximal activity evidenced by two peaks in the ductuli efferentes epithelial height and tubular diameter (March and July) whereas Sceloporus undulatus only had a single peak occurring in June. These results suggest that reproductive morphology may be conserved at taxonomic levels above family level, and reproductive activity may be constrained phylogenetically and is not solely dependent on environmental factors, at least in terms of geographic range, though it is acknowledged that microhabitats may play a major role in cyclicity of reproductive events. Future studies examining species at various taxonomic levels, as well as species in sympatry, will provide additional insight into hypotheses concerning abiotic factors and reproductive biology.

Characterization of Extracellular Vesicles from Cilia and Epithelial Cells of Ductuli Efferentes in a Turtle (Pelodiscus sinensis).

Simple SummaryDuctuli efferent is a conduit passage of spermatozoa from rete testis to the epididymis. After spermiation, various extracellular vesicles are released by epithelial cells to assist and transfer micro-molecules for spermatozoa maturation. To date, there is a lack of data in ductuli efferent. In this study, we investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their extracellular vesicles (EVs) in the epithelia of efferent ducts in a turtle by using light and transmission electron microscopy. Immunohistochemistry of CD63 (Cluster of differentiation 63) and ultrastructure demonstrates that ciliated and non-ciliated cells of ductuli efferent possess well developed exocytic systems. Ciliated cells and non-ciliated cells secrete extracellular vesicles via ciliary blebs and apical blebs, respectively. Results also showed numerous extracellular vesicles between these cells and indicate basolateral secretion. Moreover, these extracellular vesicles are associated with spermatozoa in the lumens of ductuli efferentes. Collectively, the present study provides cytological evidence that the ductuli efferentes (DE) epithelium secretes EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. Characterization and cellular distribution of these extracellular vesicles in the ductuli efferent of turtles may provide a study model to further investigate the transferring of micro-molecules via extracellular vesicles to the spermatozoa.The ductuli efferentes (DE) form a transit passage for the passage of spermatozoa from the rete testis to the epididymis. After spermiation, various epithelial secretory proteins are transferred via extracellular vesicles (EVs) to the spermatozoa for their maturation and long-term viability. The aim of the present study was to investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their EVs in the epithelia of the efferentes duct in a turtle species, the soft-shelled freshwater turtle Pelodiscus sinensis by using light and transmission electron microscopy. The results showed that CD63 as a classical exosome marker was strongly immunolocalized within the apical and lateral cytoplasm of the ciliated cells (CC) and moderate to weak in the non-ciliated cells (NCC) of DE. The ultrastructure revealed that early endosome was present at the basement membrane and perinuclear cytoplasm of both CC and NCC, whereas MVBs were located over the nucleus in the cytoplasm of NCC and adjacent to the basal bodies of cilia within the CC. Many EVs, as sources of MVBs, were located within the blebs that were attached to the cilia of CC, within the apical blebs from NCC, and the lateral spaces of CC and NCC. There was ultrastructure evidence of EVs associated with spermatozoa in the lumens of DE. Collectively, the present study provides cytological evidence that the DE epithelium secreted EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. These EVs were associated with spermatozoa in the DE lumen of this turtle. Characterization and cellular distribution of these EVs in the DE of a turtle may provide a study model to further investigate the transferring of micromolecules via EVs to the spermatozoa.