Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

Three-dimensional analysis of mesonephric tubules remodeling into efferent tubules in the male mouse embryo.

BACKGROUND: Spermatozoa are transported to the epididymal duct through efferent tubules. Although the origin of the efferent tubules is thought to be mesonephric tubules (MTs), their detailed developmental process, for example, where the rete testis and efferent tubules are connected, is unclear. We investigated the structural changes of the MTs in the male mouse embryo using a three-dimensional reconstruction method. RESULTS: Three to six MTs were connected to the Wolffian duct, and some of them branched, resulting in five to nine tips. Rete cells contacted the three to six tips. The MTs showed a folded shape when the gonadal fate was determined. After the testis development started, they became short and straight but emerged as long and twisting by birth. Before birth, the efferent duct was composed of MTs and a cranial portion of the folded Wolffian duct. CONCLUSIONS: The contact between the rete testis and efferent tubules is possibly established at the tip of each MT. The MTs regress after gonadal fate is determined but is remodeled to the twisting efferent tubules by birth. The efferent tubules are composed of the MTs but also a cranial portion of the folded Wolffian duct in the mouse.

Comparative anatomy on the development of sperm transporting pathway between the testis and mesonephros.

The male genital tract is diverse among vertebrates, but its development remains unclear, especially in the rete region. In this study, we investigated the testis-mesonephros complex of rabbit, chicken, and frog (Xenopus tropicalis) by immunohistochemistry for markers such as Ad4BP/Sf-1 (gonadal somatic and rete cells in mammals) and Pax2 (mesonephric tubules), and performed a three-dimensional reconstruction. In all investigated animals, testis cords were bundled at the mesonephros side. Rete cells positive for Ad4BP/Sf-1 (rabbit) or Pax2 (chicken and frog) were clustered at the border region between the testis and mesonephros. The cluster possessed two types of cords; one connected to the testis cords and the other to the mesonephric tubules. The latter rete cords were contiguous to Bowman's capsules in rabbit and chicken but to nephrostomes in frog. In conclusion, this study showed that mammals, avian species, and frogs commonly develop the bundle between the testis cords (testis canal) and the cluster of rete cells (lateral kidney canal), indicating that these animals share basic morphogenesis in the male genital tract. The connection site between the rete cells and mesonephric tubules is suggested to have changed from the nephrostome to the Bowman's capsule during vertebrate evolution from anamniote to amniote.

Dynamics of Spermatogenesis and Change in Testicular Morphology under 'Mating' and 'Non-Mating' Conditions in Medaka (Oryzias latipes).

Here, we report that the gross morphology of the testes changes under 'non-mating' or 'mating' conditions in medaka (Oryzias latipes). During these conditions, an efferent duct expands and a histological unit of spermatogenesis, the lobule, increases its number under 'non-mating' conditions. Based on BrdU labeling experiments, lower mitotic activity occurs in gonial cells under 'non-mating' conditions, which is consistent with the reduced number of germ cell cysts. Interestingly, the total number of type A spermatogonia was maintained, regardless of the mating conditions. In addition, the transition from mitosis to meiosis may have been retarded under the 'non-mating' conditions. The minimum time required for germ cells to become sperm, from the onset of commitment to spermatogenesis, was approximately 14 days in vivo. The time was not found to significantly differ between 'non-mating' and 'mating' conditions. The collective data suggest the presence of a mechanism wherein the homeostasis of spermatogenesis is altered in response to the mating conditions.

Connection between seminiferous tubules and epididymal duct is originally induced before sex differentiation in a sex-independent manner.

BACKGROUND: Spermatozoa in mammals develop in seminiferous tubules in a testis and are transported through the male reproductive tract. Their developmental origins are, however, different from each other; the seminiferous tubules are testicular (gonadal) structure but the subsequent ducts stem from the mesonephros. Although some mechanisms should function for the connection between these ducts, there are few reports on them. In the present study, basic information such as timing, localization, and cell types involved in the connection was obtained by sequential immunohistochemistry. RESULTS: At the time when the undifferentiated gonad differentiates into the testis or ovary, Adrenal-4 binding protein/steroidogenic factor-1 (Ad4BP/SF-1)-positive gonadal cells were noted in the mesonephric tubules (MT) in both sexes. At an earlier stage, although Ad4BP/SF-1-positive coelomic epithelial cells were adjacent to the MT, a basal membrane around them was not observed. CONCLUSIONS: The connection between the testis cords and MT is suggested to be induced between Ad4BP/SF-1-positive gonadal cells and the MT before sex differentiation in a sex-independent manner.

Androgens are essential for epithelial cell recovery after efferent duct ligation in the initial segment of the mouse epididymis†.

Efferent duct ligation (EDL) induces epithelial cell degeneration followed by regeneration in the epididymal initial segment. We tested here the role of androgens in the recovery phase. EDL was performed at post-natal weeks (PNW) 3, 4, 5, 6, and 7, and apoptotic and proliferating epithelial cells were quantified 24 h, and at days 2 and 2.5 post-EDL, respectively. A progressive increase in the number of apoptotic basal cells (BCs) and principal cells (PCs) was detected from PNW3 to 6, 24 h after EDL. Two days after EDL, no increase in proliferating BCs and PCs was observed at PNW3 and 4, despite the induction of apoptosis by EDL. A progressive increase in the number of proliferating BCs was then observed from PNW5 to 6, while the number of proliferating PCs remained low. 2.5 days after EDL, the number of proliferating BCs and PCs remained low at PNW3, 4, and 5, but a marked increase in the number of proliferating PCs was observed at PNW6. Flutamide pretreatment for 3 weeks followed by EDL at PNW7 dramatically decreased the number of proliferating BCs on EDL day 2, and the number of proliferating PCs on EDL day 2.5, compared to controls. We conclude that (1) BCs are the first to show recovery after EDL, followed by PCs; (2) androgens are essential for BC and PC repair after injury in the postpubertal epididymis; and (3) the prepubertal epididymis lacks repair ability following injury.

Mutation of cyp19a1b results in sterile males due to efferent duct obstruction in Nile tilapia.

The involvement of estrogen in male fertility has been well established in mammals. However, less is known about the role of estrogen in fish male reproduction. Our recent study revealed that Cyp19a1a deficiency had no effect on fertility in male fish. In this study, expression of Cyp19a1b, but not Cyp19a1a, was detected by immunohistochemistry in Leydig cells of tilapia testes. cyp19a1b mutation resulted in a significant decrease in the concentration of 17ß-estradiol in serum and sterility in XY fish, as no offspring were obtained when crossed with control XX fish at 240 days after hatching (dah). No sperm was obtained from the mature mutants by in vitro extrusion. Further examination of the mutant gonads revealed excessive semen accumulation and testicular hypertrophy. Semen collected from the mutant testes during autopsy contained sperm with a normal morphology that showed no significant differences in motility, VCL, BCF, STR, or fertility compared with control sperm. Efferent ducts from the mutant testes, which had low-convolution levels, fewer branches, and no blood vessels observed inside the walls, were significantly smaller in size. qRT-PCR analyses showed downregulated expression of ion exchange genes. There was increased apoptosis in the epithelial cells of the efferent ducts and other somatic cells of the testes as revealed by TUNEL staining, as well as upregulation of apoptosis gene expression in the mutants. At 360 dah, mutant fish showed testicular atrophy and efferent duct fibrosis. These results demonstrated that estrogen deficiency caused by Cyp19a1b mutation resulted in male sterility due to efferent duct obstruction.

MT-Feeding-Induced Impermanent Sex Reversal in the Orange-Spotted Grouper during Sex Differentiation.

In this study, we systematically investigated the process of sex reversal induced by 17-methyltestosterone (MT) feeding and MT-feeding withdrawal at the ovary differentiation stage in orange-spotted groupers, Epinephelus coioides. Gonadal histology showed that MT feeding induced a precocious sex reversal from immature ovaries to testes, bypassing the formation of an ovarian cavity, and MT-feeding withdrawal led to an ovarian fate. In both the MT feeding and MT-feeding withdrawal phases, cytochrome P450 family 11 subfamily B (cyp11b) gene expression and serum 11-KT levels were not significantly changed, suggesting that the MT-treated fish did not generate endogenous steroids, even though active spermatogenesis occurred. Finally, by tracing doublesex-expressing and Mab-3-related transcription factor 1 (dmrt1)-expressing cells and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) assays, we found that the efferent duct formed first, and then, the germ cells and somatic cells of the testicular tissue were generated around the efferent duct during MT-feeding-induced precocious sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex reversal induced by exogenous hormones during sex differentiation in the protogynous orange-spotted grouper.

Novel roles of Pkd2 in male reproductive system development.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited genetic diseases, caused by mutations in PKD1 and/ or PKD2. Infertility and reproductive tract abnormalities in male ADPKD patients are very common and have higher incidence than in the general population. In this work, we reveal novel roles of Pkd2 for male reproductive system development. Disruption of Pkd2 caused dilation of mesonephric tubules/efferent ducts, failure of epididymal coiling, and defective testicular development. Deletion of Pkd2 in the epithelia alone was sufficient to cause reproductive tract defects seen in Pkd2(-/-) mice, suggesting that epithelial Pkd2 plays a pivotal role for development and maintenance of the male reproductive tract. In the testis, Pkd2 also plays a role in interstitial tissue and testicular cord development. In-depth analysis of epithelial-specific knockout mice revealed that Pkd2 is critical to maintain cellular phenotype and developmental signaling in the male reproductive system. Taken together, our data for the first time reveal novel roles for Pkd2 in male reproductive system development and provide new insights in male reproductive system abnormality and infertility in ADPKD patients.

A Single Administration of Progesterone during the Neonatal Period Shows No Structural Changes in Male Reproductive Tracts in Mice.

The concentration of female-dominant steroid hormones, such as progesterone and estrogen, drops after birth in neonates. We have reported that neonatal estrogen treatment results in inflammation in the epididymis after puberty in male mice. Our recent study discovered that progesterone receptor was specifically expressed in efferent ducts just before birth in male mice. Therefore, this study aimed to reveal the impact of neonatal progesterone administration on the efferent ducts after puberty. Progesterone was subcutaneously administered to neonatal mice on their birthday in three groups: high-dose (200 mg/kg), low-dose (8 mg/kg), and control (cottonseed oil). Their testis and epididymis were collected at 12 weeks old. Semi-serial paraffin sections of these tissues were prepared and evaluated through PAS-hematoxylin staining. Efferent ducts were reconstructed into a three-dimensional structure, and their length and volume were analyzed. Spermatogenesis in the testis and epithelium of the tracts appeared normal, even in individuals administered with progesterone. There were no significant differences in the length and volume of the efferent ducts among the three groups. This study suggests that progesterone treatment in neonatal mice does not cause any structural changes in the male reproductive tracts at puberty, unlike the neonatal estrogen treatment.

Examination of testicular lumicrine regulation of activins and immunoregulatory genes in the epididymal caput.

BACKGROUND: Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3-dioxygenase-1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. OBJECTIVES: This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune-related genes in the caput epididymidis. MATERIALS AND METHODS: The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls. RESULTS: Pressure-induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage-specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected. CONCLUSION: These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput.

Androgen and estrogen receptors immunolocalization in the sand rat (Psammomys Obesus) cauda epididymis.

Both androgens and estrogens play key, albeit incompletely described, roles in the functioning of the epididymis. Because this tightly-coiled tubular structure is compartmented, precise mapping of the distribution of sex steroid's receptors is important. Such receptors have been located in the first segments (caput, corpus), but the last part (cauda) remains poorly explored. We used immunochemistry to localize androgen (AR) and estrogen (ESR1 and ESR2) receptors in the cauda in the fat sand rat (Psammomys obesus). We compared results obtained during the breeding versus resting seasons. We also used individuals castrated, or castrated then treated with testosterone, or subjected to the ligation of their efferent ducts. During the breeding season, in principal cells, we found strong staining both for AR and ESR1 in the apical cytoplasm, and strong staining for ESR2 in the nucleus. During the resting season, principal cells were positive for AR and ESR1, but negative for ESR2. In castrated animals, staining was null for ESR2 and AR, and weak for ESR1. In castrated then treated animals, immuno-expression was restored but only for AR and ESR1. Following efferent duct ligation, AR reactivity decreased while ESR1 and ESR2 provided strong staining. Broadly similar, but not fully identical patterns were observed in basal cells. They were positive for ESR2 and AR during the breeding season, but not for ESR1. During the resting season, staining was modest for ESR1 and AR and negative for ESR2. In all experimentally treated animals, we observed weak staining for AR and ESR1, and a lack of signal for ESR2. Overall, this study provides strong evidence that androgens and estrogens are involved in the seasonal regulation of the whole epididymis in the fat sand rat, with marked differences between caput and cauda (the corpus is highly reduced in rodent).

Development of Testis Cords and the Formation of Efferent Ducts in Xenopus laevis: Differences and Similarities with Other Vertebrates.

The knowledge of testis development in amphibians relative to amniotes remains limited. Here, we used Xenopus laevis to investigate the process of testis cord development. Morphological observations revealed the presence of segmental gonomeres consisting of medullary knots in male gonads at stages 52-53, with no distinct gonomeres in female gonads. Further observations showed that cell proliferation occurs at specific sites along the anterior-posterior axis of the future testis at stage 50, which contributes to the formation of medullary knots. At stage 53, adjacent gonomeres become close to each other, resulting in fusion; then (pre-)Sertoli cells aggregate and form primitive testis cords, which ultimately become testis cords when germ cells are present inside. The process of testis cord formation in X. laevis appears to be more complex than in amniotes. Strikingly, steroidogenic cells appear earlier than (pre-)Sertoli cells in differentiating testes of X. laevis, which differs from earlier differentiation of (pre-)Sertoli cells in amniotes. Importantly, we found that the mesonephros is connected to the testis gonomere at a specific site at early larval stages and that these connections become efferent ducts after metamorphosis, which challenges the previous concept that the mesonephric side and the gonadal side initially develop in isolation and then connect to each other in amphibians and amniotes.

Review of injection techniques for spermatogonial stem cell transplantation.

BACKGROUND: Although the prognosis of childhood cancer survivors has increased dramatically during recent years, chemotherapy and radiation treatments for cancer and other conditions may lead to permanent infertility in prepubertal boys. Recent developments have shown that spermatogonial stem cell (SSC) transplantation may be a hope for restoring fertility in adult survivors of childhood cancers. For this reason, several centres around the world are collecting and cryopreserving testicular tissue or cells anticipating that, in the near future, some patients will return for SSC transplantation. This review summarizes the current knowledge and utility of SSC transplantation techniques. OBJECTIVE AND RATIONALE: The aim of this narrative review is to provide an overview of the currently used experimental injection techniques for SSC transplantation in animal and human testes. This is crucial in understanding and determining the role of the different techniques necessary for successful transplantation. SEARCH METHODS: A comprehensive review of peer-reviewed publications on this topic was performed using the PubMed and Google Scholar databases. The search was limited to English language work and studies between 1994 (from the first study on SSC transplantation) and April 2019. Key search terms included mouse, rat, boar, ram, dog, sheep, goat, cattle, monkey, human, cadaver, testes, SSC transplantation, injection and technique. OUTCOMES: This review provides an extensive clinical overview of the current research in the field of human SSC transplantation. Rete testis injection with ultrasonography guidance currently seems the most promising injection technique thus far; however, the ability to draw clear conclusions is limited due to long ischemia time of cadaver testis, the relatively decreased volume of the testis, the diminishing size of seminiferous tubules, a lack of intratesticular pressure and leakage into the interstitium during the injection on human cadaver testis. Current evidence does not support improved outcomes from multiple infusions through the rete testes. Overall, further optimization is required to increase the efficiency and safety of the infusion method. WIDER IMPLICATIONS: Identifying a favourable injection method for SSC transplantation will provide insight into the mechanisms of successful assisted human reproduction. Future research could focus on reducing leakage and establishing the optimal infusion cell concentrations and pressure.

Structure of the testis and spermatogenesis of the viviparous teleost Poecilia mexicana (Poeciliidae) from an active sulfur spring cave in Southern Mexico.

We describe the histological characteristics of the testis and spermatogenesis of the cave molly Poecilia mexicana, a viviparous teleost inhabiting a sulfur spring cave, Cueva del Azufre, in Tabasco, Southern Mexico. P. mexicana has elongate spermatogonial restricted testes with spermatogonia arranged in the testicular periphery. Germ cell development occurs within spermatocysts. As spermatogenesis proceeds, the spermatocysts move longitudinally from the periphery of the testis to the efferent duct system, where mature spermatozoa are released. The efferent duct system consists of short efferent duct branches connected to a main efferent duct, opened into the genital pore. Spermatogenesis consisted of the following stages: spermatogonia (A and B), spermatocytes (primary and secondary), spermatids, and spermatozoa. The spermatozoa are situated within spermatocysts, with their heads oriented toward the periphery and flagella toward the center. Once in the efferent duct system, mature spermatozoa are packaged as unencapsulated sperm bundles, that is, spermatozeugmata. We suggest that the histological characteristics of the testis and spermatogenesis of P. mexicana from the Cueva del Azufre, and the viviparous condition where the spermatozoa enter in the female without been in the water, have allowed them to invade sulfurous and/or subterranean environments in Southern Mexico, without requiring complex morphofunctional changes in the testis or the spermatogenetic process.

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

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.

Pkd1 is required for male reproductive tract development.

Reproductive tract abnormalities and male infertility have higher incidence in ADPKD patients than in general populations. In this work, we reveal that Pkd1, whose mutations account for 85% of ADPKD cases, is essential for male reproductive tract development. Disruption of Pkd1 caused multiple organ defects in the murine male reproductive tract. The earliest visible defect in the Pkd1(-/-) reproductive tract was cystic dilation of the efferent ducts, which are derivatives of the mesonephric tubules. Epididymis development was delayed or arrested in the Pkd1(-/-) mice. No sign of epithelial coiling was seen in the null mutants. Disruption of Pkd1 in epithelium alone using the Pax2-cre mice was sufficient to cause efferent duct dilation and coiling defect in the epididymis, suggesting that Pkd1 is critical for epithelium development and maintenance in male reproductive tract. In-depth analysis showed that Pkd1 is required to maintain tubulin cytoskeleton and important for Tgf-ß/Bmp signal transduction in epithelium of male reproductive tract. Altogether, our results for the first time provide direct evidence for developmental roles of Pkd1 in the male reproductive tract and provide new insights in reproductive tract abnormalities and infertility in ADPKD patients.