Morphology and immunolocalization of aquaporins 1 and 9 in the agouti (Dasyprocta azarae) testis excurrent ducts

This study investigated the morphology and immunoexpression of aquaporins (AQPs) 1 and 9 in the rete testis, efferent ducts, epididymis, and vas deferens in the Azara’s agouti (Dasyprocta azarae). For this purpose, ten adult sexually mature animals were used in histologic and immunohistochemical analyses. The Azara’s agouti rete testis was labyrinthine and lined with simple cubic epithelium. Ciliated and non-ciliated cells were observed in the epithelium of the efferent ducts. The epididymal cellular population was composed of principal, basal, apical, clear, narrow, and halo cells. The epithelium lining of vas deferens was composed of the principal and basal cells. AQPs 1 and 9 were not expressed in the rete testis. Positive reaction to AQP1 was observed at the luminal border of non-ciliated cells of the efferent ducts, and in the peritubular stroma and blood vessels in the epididymis, and vas deferens. AQP9 was immunolocalized in the epithelial cells in the efferent ducts, epididymis and vas deferens. The morphology of Azara’s agouti testis excurrent ducts is similar to that reported for other rodents such as Cuniculus paca. The immunolocalization results of the AQPs suggest that the expression of AQPs is species-specific due to differences in localization and expression when compared to studies in other mammals species. The knowledge about the expression of AQPs in Azara’s agouti testis excurrent ducts is essential to support future reproductive studies on this animal, since previous studies show that AQPs may be biomarkers of male fertility and infertility.     

Differential expression of estrogen receptors alpha and beta in the reproductive tracts of adult male dogs and cats

Expression of estrogen receptors (ERs) in the reproductive tracts of adult male dogs and cats has not been reported. In the present study, ERalpha and ERbeta were localized by immunohistochemistry using ER-specific antibodies. ERalpha was found in interstitial cells and peritubular myoid cells in the dog testis, but only in interstitial cells of the cat. In rete testis of the dog, epithelial cells were positive for ERalpha staining, but in the cat, rete testis epithelium was only weakly positive. In efferent ductules of the dog, both ciliated and nonciliated cells stained intensely positive. In the cat, ciliated epithelial cells were less stained than nonciliated epithelial cells. Epithelial cells in dog epididymis and vas deferens were negative for ERalpha. In the cat, except for the initial region of caput epididymis, ERalpha staining was positive in the epithelial cells of epididymis and vas deferens. Multiple cell types of dog and cat testes stained positive for ERbeta. In rete testis and efferent ductules, epithelial cells were weakly positive for ERbeta. Most epithelial cells of the epididymis and vas deferens exhibited a strong positive staining in both species. In addition, double staining was used to demonstrate colocalization of both ERalpha and ERbeta in efferent ductules of both species. The specificity of antibodies was demonstrated by Western blot analysis. This study reveals a differential localization of ERalpha and ERbeta in male dog and cat reproductive tracts, demonstrating more intensive expression of ERbeta than ERalpha. However, as in other species, the efferent ductules remained the region of highest concentration of ERalpha.     

Expression of constitutively active Notch1 in male genital tracts results in ectopic growth and blockage of efferent ducts, epididymal hyperplasia and sterility

The Notch signaling pathway is involved in a variety of developmental processes. Here, we characterize the phenotypes developing in the reproductive organs of male transgenic (Tg) mice constitutively expressing the activated mouse Notch1 intracellular domain (Notch1(intra)) under the regulatory control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Tg expression was detected in testis, vas deferens and epididymis by Northern blot analysis. In situ hybridization with a Notch1-specific probe lacked sensitivity to detect expression in normal-appearing cells, but demonstrated expression in hyperplastic epithelial cells of the vas deferens, epididymis and efferent ducts. Tg males from three independent founder lines were sterile. Histological analysis of reproductive organs of young Tg males (postnatal ages 8 and 21) showed no difference compared to those of non-Tg males. In contrast, in adult Tg mice from day 38 onwards, the efferent ducts, the vas deferens and most epididymal segments revealed bilateral epithelial cell hyperplasia with absence of fully differentiated epithelial cells. Electron microscopy confirmed the uniformly undifferentiated state of these cells. Immunohistochemistry with anti-PCNA antibody also revealed enhanced proliferation of Tg epididymis. In adult Tg testis, the different generations of germ cells of seminiferous tubules appeared normal, although some tubules were highly dilated and revealed an absence of early and/or late spermatids. The epithelial cells of the Tg tubuli recti and rete testis were not abnormal, but the rete testis was highly dilated and contained numerous spermatozoa, suggesting a downstream blockage. Consistent with a blockage of efferent ducts often seen at the rete testis/efferent duct interface, spermatozoa were absent in epididymis of all adult Tg mice and in all highly hyperplastic efferent duct tubules of these Tg mice. Such a blockage was visualized by injection of Evans blue dye into the rete testis lumen. Finally, the presence of ectopic hyperplastic efferent duct tubules was observed within the testicular parenchyma itself, outside their normal territory, suggesting that Notch1 signaling is involved in the establishment of these borders. This phenotype seems to represent a novel developmental defect in mammals. Together, these results show that constitutive Notch1 signaling significantly affects the development of male reproductive organs.     

Immunolocalization of Aquaporin-1, -5, and -7 in the Avian Testis and Vas Deferens

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been found in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs have been poorly investigated in the male reproductive system of birds. The localization of AQP subtypes (AQP1, 2, 3, 4, 5, 7, 8, 9, and 11) in the goose testis and vas deferens has been studied through immunohistochemistry and immunobloting. Interestingly, the testicular and deferential tissues were positive for AQP1, -5, and -7 but not the others. AQP1 immunoreactivity was detected in the capillary endothelial cells of testis and vas deferens. AQP5 was localized in the interstitial tissue of the testis, including Leydig cells, as well as in the basal cells of vas deferens. Double-labeling confocal microscopy revealed coexpression of AQP5 with capillary AQP1 in the testis. AQP7 was expressed in elongated spermatid and spermatozoa tails in the testis, as well as spermatozoa tails in the vas deferens. These results suggest that several subtypes of AQPs are involved in the regulation of water homeostasis in the goose male reproductive system. (J Histochem Cytochem 57:915–922, 2009)     

Co-expression and interaction of cubilin and megalin in the adult male rat reproductive system

Cubilin is a peripheral membrane protein that cooperates with the endocytic receptor megalin to mediate endocytosis of ligands in various polarized epithelia. Megalin is expressed in the male reproductive tract where it has been implicated in the process of sperm membrane remodeling. A potential role for cubilin in the male reproductive tract has not been explored. Using RT-PCR, we found that cubilin and megalin mRNAs are expressed in the efferent ducts, corpus and cauda epididymis, and proximal and distal vas deferens. Immunohistological analysis revealed that cubilin was expressed in nonciliated cells of the efferent ducts, principal cells of the corpus and cauda epididymis and vas deferens. Immunogold EM showed cubilin in endocytic pits, endocytic vesicles, and endosomes of these cells. The expression profile of cubilin in the male reproductive tract was coincident with that of megalin except in principal cells of the caput epididymis. Double immunogold labeling showed that cubilin and megalin co-localized within the endocytic apparatus and recycling vesicles of efferent duct cells. Neither protein was found in lysosomes. Injection of RAP, an antagonist of megalin interaction with cubilin, reduced the level of intracellular cubilin in cells of the efferent ducts and vas deferens. In conclusion, cubilin and megalin are co-expressed in cells of the epididymis and vas deferens and the endocytosis of cubilin in these tissues is dependent on megalin. Together, these findings highlight the potential for a joint endocytic role for cubilin and megalin in the male reproductive tract.     

Histochemical evaluation of glycoconjugates in the male reproductive tract with lectin-horseradish peroxidase conjugates: I. Staining of principal cells and spermatozoa in the mouse

Several glycoconjugates are thought to bind spermatozoa as they pass through reproductive ducts. Paraffin sections of testis, ductuli efferentes, epididymis, and vas deferens of male mice were stained with ten different lectin-horseradish peroxidase conjugates to localize possible sites of synthesis and secretion of such glycoconjugates, based on the carbohydrate moieties in their constituent oligosaccharide side chains. Principal (columnar) cells lining the efferent ducts, germinal epithelium, and developing and maturing spermatozoa were examined with light microscopy. Staining of the Golgi and apical zones of cells was interpreted as evidence for synthesis and secretion of glycoconjugates. Principal cells synthesized and secreted glycoconjugates with sugar moieties as follows: sialic acid, all regions of the efferent ducts examined; the terminal disaccharide D-galactose- (beta 1----3) -N-acetyl-D-galactosamine, all regions of ducts except epididymis I; terminal alpha-D-galactosamine, some cells in epididymis III-V; N-acetyl-D-galactosamine, ductuli efferentes, epididymis I, II, and some cells in epididymis III-V; alpha-L-fucose, ductuli efferentes, vas deferens, and all regions of the epididymis except IV; N-glycosidic side chains, ductuli efferentes, vas deferens, and epididymis I, IV, and V. All of these sugar residues as well as N-acetyl-D-glucosamine were associated with the acrosomes and tails of spermatozoa throughout the ducts except for alpha-N-acetyl-D-galactosamine in epididymis I, and all occurred during one or more stages of spermiogenesis. The synthesis and secretion of glycoconjugates that bind to spermatozoa appear to involve more regions of the primary reproductive structures than was believed previously.     

Segmental and cellular expression of aquaporins in the male excurrent duct

The male reproductive tract and accessory glands comprise a complex but interrelated system of tissues that are composed of many distinct cell types, all of which contribute to the ability of spermatozoa to carry out their ultimate function of fertilizing an oocyte. Spermatozoa undergo their final steps of maturation as they pass through the male excurrent duct, which includes efferent ducts, the epididymis and the vas deferens. The composition of the luminal environment in these organs is tightly regulated. Major fluid reabsorption occurs in efferent ducts and in the epididymis, and leads to a significant increase in sperm concentration. In the distal epididymis and vas deferens, fluid secretion controls the final fluidity of the luminal content. Therefore, the process of water movement in the excurrent duct is a crucial step for the establishment of male fertility. Aquaporins contribute to transepithelial water transport in many tissues, including the kidney, the brain, the eye and the respiratory tract. The present article reviews our current knowledge regarding the distribution and function of aquaporins in the male excurrent duct.     

Segmental and cellular expression of aquaporins in the male excurrent duct

The male reproductive tract and accessory glands comprise a complex but interrelated system of tissues that are composed of many distinct cell types, all of which contribute to the ability of spermatozoa to carry out their ultimate function of fertilizing an oocyte. Spermatozoa undergo their final steps of maturation as they pass through the male excurrent duct, which includes efferent ducts, the epididymis and the vas deferens. The composition of the luminal environment in these organs is tightly regulated. Major fluid reabsorption occurs in efferent ducts and in the epididymis, and leads to a significant increase in sperm concentration. In the distal epididymis and vas deferens, fluid secretion controls the final fluidity of the luminal content. Therefore, the process of water movement in the excurrent duct is a crucial step for the establishment of male fertility. Aquaporins contribute to transepithelial water transport in many tissues, including the kidney, the brain, the eye and the respiratory tract. The present article reviews our current knowledge regarding the distribution and function of aquaporins in the male excurrent duct.     

Aquaporin 9 expression along the male reproductive tract

Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3--4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogeneous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.     

Distribution of the vacuolar H+ atpase along the rat and human male reproductive tract

Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.     

Expression of villin in the mouse oviduct and the seminiferous ducts

Summary Villin, a 95-kD cytoskeletal protein selectively expressed in the microvilli of some absorptive cells was localized immunohistochemically in the oviduct and the seminiferous excretory ducts of the mouse. Villin was found in the proximal part of the oviduct, comprising the preampulla, ampulla, and part of the isthmus. Distal to the isthmus the oviductal cells lining the junctura and the intrauterine colliculus tubaris were devoid of villin. No villin could be detected in the uterine cells.Ductuli efferentes, connecting the rete testis with the epididymis were the only portion of the male seminiferous ductal system expressing villin. The cells lining the epididymis and the vas deferens were devoid of villin. These data show that villin is selectively expressed in male and female reproductive systems and that it is limited to anatomically defined proximal portions of the reproductive ducts.     

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

Background  

Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite.     

Members of the Toll-like receptor family of innate immunity pattern-recognition receptors are abundant in the male rat reproductive tract

Protecting developing and maturing spermatozoa and reproductive tissues from microbial damage is an emerging aspect of research in reproductive physiology. Bacterial, viral, and yeast infections of the testis and epididymis can hinder maturation and movement of spermatozoa, resulting in impaired fertility. Toll-like receptors (TLRs) are a broad family of innate immunity receptors that play critical roles in detecting and responding to invading pathogens. Objectives of this study were to determine if organs of the rat male reproductive tract express mRNAs for members of the TLR family, to characterize expression patterns for TLRs in different regions of the epididymis, and to determine if TLR adaptor and target proteins are present in the male reproductive tract. Messenger RNA for Tlr1-Tlr9 was abundantly expressed in testis, epididymis, and vas deferens, as determined by RT-PCR, while Tlr10 and Tlr11 were less abundantly expressed. Tlr mRNA expression showed no region-specific patterns in the epididymis. Immunoblot analysis revealed relatively equal levels of protein for TLRs 1, 2, 4, and 6 in testis, all regions of the epididymis and vas deferens, and lower levels of TLRs 3, 5, and 9-11. TLR7 was primarily detected in the testis. The TLR adapter proteins, myeloid differentiation primary response gene 88 and TLR adaptor molecule 1, as well as v-rel reticuloendotheliosis viral oncogene homolog and NFKBIA, were prominent in testis, epididymis, and vas deferens. The abundant expression of a majority of TLR family members together with expression of TLR adaptors and activation targets provides strong evidence that TLRs play important roles in innate immunity of the male reproductive tract.     

Expression of cysteine sulfinate decarboxylase (CSD) in male reproductive organs of mice

Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of taurine, but it is still controversial whether the male reproductive organs have the function to synthesize taurine through CSD pathway. The present study was thus undertaken to detect CSD expression in male mouse reproductive organs by RT-PCR, Western blot and immunohistochemistry. The results show that CSD is expressed both at the mRNA and protein levels in the testis, epididymis and ductus deferens. The relative levels of both CSD mRNA and protein increase from the testis to the epididymis and to the ductus deferens. Immunohistochemical results demonstrate that the main cell types containing CSD are Leydig cells of testis, epithelial cells and some stromal cells throughout the efferent ducts, epididymis and ductus deferens. These results suggest that male genital organs have the function to produce taurine through the CSD pathway, although quantifying the relation of CSD expression to taurine synthesis and the exact functions of taurine in male genital organs still need to be elucidated in future studies.     

Microanatomy of the testes and testicular ducts of the butterfly lizard,Leiolepis ocellata Peters, 1971 (Reptilia: Squamata: Agamidae) during the active reproductive period

The microanatomy of the testes and testicular ducts (rete testis, ductuli efferentes, ductus epididymis and ductus deferens) of Leiolepis ocellata (Agamidae) was investigated using light microscopy including histochemistry. Each testis contains seminiferous tubules and interstitial tissues. The former house spermatogenic cells (spermatogonia A & B, preleptotene, primary and secondary spermatocytes, spermatids (steps 1–8) and spermatozoa) and Sertoli cells, while the latter comprise peritubular and intersitial tissues. The rete testis is an anastomosing duct, having intratesticular and extratesticular portions. The proximal region of ductuli efferentes has wider outer ductal and luminal diameters than those of the distal region. The convoluted ductus epididymis is subdivided into four regions (initial segment, caput, corpus and cauda), based on the ductal diameter, epithelium characteristics and cell components. The ductus deferens has the greatest diameter and is divided into the ductal and ampulla ductus deferens. The ductal portion is subdivided into the proximal and distal regions, based on the epithelium types and ductal diameters. The ampulla ductus deferens is a fibromuscular tube, having numerous mucosal folds projecting into the lumen. Spermiophagy is detectable in the ductus epididymis and ductus deferens. The present results contribute to improved fundamental knowledge on the microanatomy of the reptilian reproductive system.