Epididymal Epithelium Research Articles

Excision of Trpv6 Gene Leads to Severe Defects in Epididymal Ca2+ Absorption and Male Fertility Much Like Single D541A Pore Mutation

Replacement of aspartate residue 541 by alanine (D541A) in the pore of TRPV6 channels in mice disrupts Ca(2+) absorption by the epididymal epithelium, resulting in abnormally high Ca(2+) concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilization capacity, raising the possibility of residual activity of channels formed by TRPV6(D541A) proteins (Weissgerber, P., Kriebs, U., Tsvilovskyy, V., Olausson, J., Kretz, O., Stoerger, C., Vennekens, R., Wissenbach, U., Middendorff, R., Flockerzi, V., and Freichel, M. (2011) Sci. Signal. 4, ra27). It is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared with the deletion of the corresponding protein. Therefore, we generated TRPV6-deficient mice (Trpv6(-/-)) by deleting exons encoding transmembrane domains with the pore-forming region and the complete cytosolic C terminus harboring binding sites for TRPV6-associated proteins that regulate its activity and plasma membrane anchoring. Using this strategy, we aimed to determine whether the TRPV6(D541A) pore mutant still contributes to residual channel activity and/or channel-independent functions in vivo. Trpv6(-/-) males reveal severe defects in fertility and motility and viability of sperm and a significant increase in epididymal luminal Ca(2+) concentration that is mirrored by a lack of Ca(2+) uptake by the epididymal epithelium. Therewith, Trpv6 excision affects epididymal Ca(2+) handling and male fertility to the same extent as the introduction of the D541A pore mutation, arguing against residual functions of the TRPV6(D541A) pore mutant in epididymal epithelial cells.

Fibroblast Growth Factor 8b Causes Progressive Stromal and Epithelial Changes in the Epididymis and Degeneration of the Seminiferous Epithelium in the Testis of Transgenic Mice1

Transgenic (Tg) mice expressing human fibroblast growth factor 8b (FGF8-b) under the probasin promoter (Tg [Pbsn-FGF8] L2-L5Elo; hereafter referred to as FGF8-b-Tg) were shown to produce FGF8-b at high levels in the prostate and epididymis and at lower levels in the testis. The present study examined the effects of FGF8-b expression on the epididymis and testis. In old (age, >6 mo) FGF8-b-Tg mice, epididymides were frequently enlarged, with epithelial and stromal hypercellularity progressing upon aging to epithelial dysplasia and malignant transformation of stroma. In addition, oligospermia, dilatation of the duct, and inflammation were frequently observed in the epididymides. In association with the epididymal changes, some FGF8-b-Tg mice presented a degenerative seminiferous epithelium of the testis. Consistent with this observation, infertile males were found in two FGF8-b-Tg mouse lines. Masson trichrome staining and immunohistochemical analysis of smooth muscle actin, laminin, and androgen receptor revealed that changes in the epididymal stroma closely resembled those previously found in the prostates of the FGF8-b-Tg mice. Genes previously found to be upregulated in the prostate of FGF8-b-Tg mice, such as osteopontin (Spp1) connective tissue growth factor (Ctgf), apolipoprotein D (Apod), and FGF receptor 1c (Fgfr1-c), were also upregulated in the epididymides, suggesting that similar molecular mechanisms were active in both tissues. However, unlike in the prostate, the changes in the epididymal epithelium of the FGF8-b-Tg mice did not progress into invasive carcinoma. The results suggest that prolonged and enhanced FGF signaling induces dramatic changes in the epididymis and testis that lead to infertility in a portion of the FGF8-b-Tg males.

Molecular cloning and identification of mouse epididymis-specific gene mHong1, the homologue of rat HongrES1

Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHong1. The mHong1 gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHong1 mRNA shows similarity with HongrES1 in the expression patterns: (i) specific expression in epididymal tissue, especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHong1 with high specificity and sensitivity was raised. Like HongrES1, the mHong1 protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHong1 protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHong1 is localized to the equatorial segment, which is different from that of HongrES1. The mHong1 protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHong1 is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHong1.

Immunolocalisation of 11β‐HSD‐1 and ‐2, glucocorticoid receptor, mineralocorticoid receptor and Na+K+‐ATPase during the postnatal development of the rat epididymis

Glucocorticoids have been implicated in male reproductive function and 11β-HSD-1 and -2, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), all of which are known to modulate glucocorticoid action, have been localised in the adult rat epididymis, but their developmental expression has not been investigated. Na(+)K(+)-ATPase activity, responsible for sodium transport, is induced by both mineralocorticoids and glucocorticoids in the kidney and colon, and has been localised in epididymal epithelium. This study examined the immunolocalisation of 11β-HSD-1 and -2, GR, MR and Na(+)K(+)-ATPase in rat epididymal epithelium (n = 5) at postnatal days (pnd) 1, 7, 15, 28, 40, 60, 75 and 104, and relative mRNA expression of 11β-HSD-1 and -2, and GR at pre-puberty (pnd 28) and post-puberty (pnd 75). 11β-HSD-1, GR and MR were localised in the epididymal epithelium from pnd 1, and 11β-HSD-2 and Na(+)K(+)-ATPase reactivity from pnd 15. At pnd 28 there was maximal immunoreactivity for both the GR and MR and 11β-HSD-1 and -2. 11β-HSD-1 mRNA expression in the caput increased from pre- to post-puberty, whereas 11β-HSD-2 mRNA expression fell over the same period (P < 0.01). GR mRNA expression was similar at pre- and post-puberty in both caput and cauda. Developmental changes in expression of 11β-HSD-1 and -2 suggest that overall exposure of the epididymis to glucocorticoids increases post-puberty, but cell-specific expression of the 11β-HSD enzymes still provides a capacity for intricate local control of glucocorticoid exposure.

Morphological studies on the seasonal changes in the epididymal duct of the one-humped camel (camelus dromedarius)

The present work was carried out on 20 testes and epididymis of sexually mature camels to elucidate the gross anatomical, morphometerical, light microscopical and scanning electron microscopical features of the epididymis in different seasons. Anatomically, the epididymal duct of a camel consists of three parts head, body and tail. Histomorphologically, the epididymal duct is subdivided into initial, middle and terminal segments, of which the middle segment is further subdivided into proximal, intermediate and distal parts. There is a gradual decrease in the epithelial height of the epididymal duct from the initial to the terminal segments. This mechanically facilities passage of the sperms toward the terminal segment. High epithelium in the initial segment may indicate a more absorptive power of the epithelium in this segment. The seasonal reproductivety of the epididymal duct in the camel expressed by variations in the weight and volume of the epididymis, total diameter of the epididymal duct, epithelial height, length of the stereocilia, thickness of the muscular coat and cellular distributions in different segments. The spring months offer ideal circumstances for maximal reproductive activity in this species. The cellular components of the epididymal duct epithelium of the camel displays important morphological changes from season to another showing signs of increasing activity during spring in comparison to decreasing activity in other seasons. PAS positive granules are demonstrated in different segments of the epididymal duct and intraepithelial glands in different seasons. These granules are relatively more numerous in spring. The lamina propria surrounding the epididymal duct contains a layer of the elastic fibers which is very thick in winter, thick in spring and thin in other seasons. This increase in thickness of the elastic fibers predisposes for the increase in the total diameter of the epididymal duct in spring. It was conclude that the muscular coat of the middle and terminal segments is the thickest in spring that may be helpful for powerful ejaculation.

Changes in lectin-binding sites on epididymal cells during postnatal development of the mouse

Using lectin histochemistry combined with immunohistochemistry, we recently demonstrated that the principal, clear, and basal cells in the adult mouse epididymis specifically react with UEA-I, MAL-I, and GS-IB lectins, respectively. The present study examined the distribution of the lectin-binding sites for some lectins on the epididymal epithelium during postnatal development. Galactose staining with GS-IB was first detected in some of the undifferentiated epithelial cells in mice aged 1 week, and this characteristic became specific to basal cells in mice aged 2 weeks and above. Fucose staining with UEA-I was first detected in the principal cells in mice aged 3 weeks. Staining of sialic acid with MAL-I was first detected in all undifferentiated epithelial cells in mice aged 1 week, and this characteristic became specific to the narrow and clear cells in all regions and to the principal cells in regions IV and V in mice aged 3 weeks. The results indicate that epididymal differentiation is characterized by the expression of cell- and region-specific sugar chains that appear early during postnatal development before the sperm arrives in the epididymis.

Cell- and region-specific expression of sugar chains in the mouse epididymal epithelium using lectin histochemistry combined with immunohistochemistry

To understand the cytochemical properties of epididymal epithelial cells, the characteristics of glycoconjugates in the mouse epididymis were examined using the technique of lectin histochemistry combined with immunohistochemistry. Characteristic staining patterns depending on the type of lectins were observed in the epididymal epithelium. Principal cells expressed N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), and Fucose in the proximal region of the epididymis and Mannose, Glucose, and Galactose in the distal region of the epididymis. Basal cells expressed Mannose, Glucose, Galactose, and GlcNAc in the proximal region and Galactose in the distal region. On the other hand, clear cells expressed various sugar residues and differences among regions were not observed. Interestingly, principal cells, clear cells, and basal cells specifically reacted with Ulex Europaeus-Agglutinin I (UEA-I lectin), Maackia Amurensis-Lectin I (MAL-I lectin), and Griffonia simplicifolia Lectin I-B4 (GS-I B lectin), respectively. These findings indicate that the selectivity in lectin reactivity for distinct cell types and segment-dependent staining in the epididymis may be related to cellular and regional differences in function. Furthermore, because some lectins stain particular cells or cellular compartments selectively, these lectins could be useful markers for histopathological evaluation of diseases or diagnosis of male infertility.

Effects of Tripterygium wilfordii multiglycosides on sex hormones and receptors in mandarin vole Leydig cells

Multiglycosides ofTripterygium wilfordii(GTW) have been found to exhibit reversible antifertility effects through complex mechanisms. The positive expression of sex hormones and receptors in Leydig cells were investigated following GTW treatment to determine the impact of GTW on male mandarin vole (Microtus mandarinus) fertility. Compared to the control group, body weight, testis and epididymis weight were not affected by GTW, but the number of sperm sharply declined. There were significant decreases in the thickness of seminifeous epithelium and epididymal epithelium, and the diameter of seminiferous tubules. The expression of estrogen receptor alpha (ERα) in Leydig cells significantly decreased and estradiol (E) expression significantly increased. Testosterone (T) and androgen receptor (AR) expression were not affected. These results show that GTW may impair fertility in male mandarin voles via interference with spermatogenesis. This may be associated with an alteration in estrogen activation rather than suppression of T and AR in Leydig cells and suggests the estrogen system of Leydig cells is of major importance for the GTW’s antifertility effects.

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.

Sodium Coupled Bicarbonate Influx Regulates Intracellular and Apical pH in Cultured Rat Caput Epididymal Epithelium

BackgroundThe epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na+/HCO3 − cotransporter in the pH regulation in rat epididymis.Method/Principal FindingsImmunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit(KH) solution, the intracellular pH (pHi) recovery from NH4Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na+/H+ exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO3 − buffered would cause another pHi recovery. The pHi recovery in HCO3 − buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS), the inhibitor of HCO3 − transporter or by removal of extracellular Na+. The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH.ConclusionsThe present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;ARflox/Y mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.

The effects of prepubertal epididymal ligation upon androgen receptor distribution in the rat caput epididymis

The aim of the present study was to investigate the androgen receptor (AR) distribution in epididymal cells of developing rats and the effects of prepubertal epididymal obstruction upon AR distribution in the rat caput epididymis. At 15 days of age, the young rats were divided at random into groups for epididymal ligation or sham operation. In the ligation group the corpus epididymides were ligated bilaterally; in the sham group only laparatomy was performed. Both groups were sacrificed at 21, 56, 90, 120 days. The epididymes were removed, fixed in Bouin's fixative and embedded in paraffin wax. The tissues were sectioned at 5 &amp;mu;m and stained using the microwave stimulated antigen retrieval technique for immunohistochemistry. The features of the immunohistochemical staining of caput epididymal cells for the AR were similar across both groups. The operation did not affect AR distribution in caput epididymis. Positive immunohistochemical staining for the AR appeared in nuclei but not in the cytoplasm of caput epididymal cells at all ages beginning from 21 to 120 days old. The staining intensity of AR-positive cells did not change depending on age. In the caput epididymis, immunostainable AR were found in tubular epithelial cells (principal cells, basal cells and apical cells) and interstitial stromal cells (peritubular smooth muscle cells). There were no significant histological alterations in epididymal epithelium.

Histological study of the possible protective effect of pomegranate juice on bisphenol-A induced changes of the caput epididymal epithelium and sperms of adult albino rats

ObjectivesThe aim of our study was to throw light on the possible protective effect of pomegranate juice (PJ) after experimental oral administration of the widely used plasticizer Bisphenol A (BPA) on the epididymis by using light and electron microscopes, as well as cauda eididymis sperm count. MethodsForty adult male albino rats were divided into two main groups; (control and treated groups) Control group which was further subdivided into two equal subgroups group (Ia) was given corn oil (5mg/kg) and the other group (Ib) given oral PJ 1ml daily for 8weeks. The other group was subdivided into two subgroups, one group (IIa) was given BPA dissolved in corn oil in a dose of 20μg/kg, while the other group (IIb) given BPA in the same dose combined with 1ml of PJ daily both for 8weeks. All administrations were given by oral gavage. The rats were sacrificed. The right caput epididymis was subjected to histological study. The left cauda epididymis was used for caudal sperm count. ResultsThe caput epididymis of group IIa revealed structural alterations in the epididymal epithelium. Sperms showed marked affection and alteration of the mitochondrial sheath. All these changes were attributed to oxidative stress. These changes were found to be prevented after PJ administration. Caudal sperm count was significantly lower in the group given BPA as compared with the control groups. ConclusionBPA causes structural changes in the epididymis that would interfere with its function and contribute to infertility. On the other hand PJ had the ability to prevent these changes, increasing the number of caudal sperm and decreasing sperm abnormalities. Thus, it could have a role in improving male fertility.

Postnatal Changes in the Expression of Claudin‐11 in the Testes and Excurrent Ducts of the Domestic Rabbit (Oryctolagus cuniculus domesticus)

We examined the expression of claudin-11 (CLDN11) in the testes and male reproductive tracts of rabbits. The rabbit CLDN11 cDNA sequences were nearly identical with human, mouse, and bovine CLDN11. The levels of CLDN11 mRNA and protein (22 kDa) were markedly increased in the testis during adult development. On postnatal day (PND) 10, CLDN11 was colocalized with ZO-1 at the lateral contacts between adjacent Sertoli cells and was perpendicular to the basal lamina. In adult testis on PND 180, CLDN11 was codistributed with ZO1, and the pattern of immunoreactivity consisted of wavy linear tracts parallel to the basal lamina, which was different according to the spermatogenic stage. These results suggest that CLDN11 participates in inter-Sertoli cell tight junctions (TJs) at the blood-testis barrier in adult rabbits. CLDN11 was also found in the basal regions of Sertoli cells adjacent to the basal lamina in adult testis, suggesting that CLDN11 also participates in the adhesion between Sertoli cells and the basal lamina. CLDN11 mRNA and protein expressions were decreased in the adult epididymis compared with those in immature animals. In adults, CLDN11 mRNA levels were relatively high in the efferent duct, followed by those in the vas deferens, proximal corpus, and distal cauda, although low levels were observed in the initial segment and caput. On PND 10, CLDN11 immunoreactivity was identified at the apicolateral contacts between adjacent epithelial cells in the epididymis and vas deferens. In adults, CLDN11 was found in the nonciliated cells in the efferent duct and at the lateral contacts in the epithelial cells in the epididymal segments. In the caput, CLDN11 was found at the apicolateral contacts between adjacent epithelial cells, but expression was weak to negligible in the corpus of the vas deferens. CLDN11 may play an important role in TJs and cell adhesion in immature rabbit excurrent duct epithelia. In adult rabbits, CLDN11 in efferent duct epithelium and epididymal epithelium may contribute to the specific environment for sperm maturation.

Mice Lacking the USP2 Deubiquitinating Enzyme Have Severe Male Subfertility Associated with Defects in Fertilization and Sperm Motility

The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

Male Fertility Depends on Ca 2+ Absorption by TRPV6 in Epididymal Epithelia

TRPV6 [transient receptor potential vanilloid 6] is a calcium ion (Ca²+)-selective channel originally identified in the duodenal epithelium and in placenta; replacement of a negatively charged aspartate in the pore-forming region with an uncharged alanine (D541A) renders heterologously expressed TRPV6 channels nonfunctional. We found that male, but not female, mice homozygous for this mutation (Trpv6(D541A/D541A)) showed severely impaired fertility. The motility and fertilization capacity of sperm were markedly reduced, despite intact spermatogenesis. Trpv6 was expressed in epididymal epithelium where the protein was detected in the apical membrane, whereas it was not expressed in spermatozoa or the germinal epithelium. The Ca²+ concentration of the fluid in the cauda epididymis of Trpv6(D541A/D541A) males was 10 times higher than that of wild-type mice, which was accompanied by a seven- to eightfold decrease in Ca²+ absorption through the epididymal epithelium and was associated with reduced sperm viability. Thus, appropriate Ca²+ absorption and a consequent TRPV6-mediated decrease in the extracellular Ca²+ concentration toward the distal segments of the epididymal duct are essential for the acquisition of basic functions and the survival of spermatozoa.

Estrogen and Its Receptors in Efferent Ductules and Epididymis

Estrogens play key roles in the development and maintenance of male reproductive function and fertility. In this review, we briefly describe the localization and function of estrogen receptors ESR1 and ESR2 (also known as ERα and ERβ, respectively) and the expression of G protein-coupled estrogen receptor-1 (GPER, formerly known as GPR30) in efferent ductules and epididymis. The efferent ductules present the highest levels of ESR1 and ESR2 in the male reproductive system, and represent a major target of estrogen action. In efferent ductules, ESR1 has a crucial role in the regulation of fluid reabsorption, and in the epididymis the receptor helps to maintain fluid osmolality and pH. ESR1 expression in the epididymal epithelium shows considerable variation among species, but differences in laboratory techniques may also contribute to this variation. Here we report that Esr1 mRNA and protein are higher in corpus than in other regions of the rat epididymis. The mRNA level for Gper was also higher in corpus. Although ESR1 is expressed constitutively in efferent ductules and down-regulated by estrogen, in the epididymis, both testosterone (T) and estradiol (E2) may regulate its expression. T and E2 are, respectively, higher and lower in the corpus than in the initial segment/caput and cauda regions. It is important to determine the expression of GPER, ESR1, androgen receptor, and their respective cofactors in specific cell types of this tissue, as well as the intracellular signaling pathways involved in efferent ductules and epididymis. These studies will help to explain the consequences of exposures to environmental endocrine disruptors and provide potential targets for the development of a male contraceptive.

A dense network of dendritic cells populates the murine epididymis

One of the most intriguing aspects of male reproductive physiology is the ability to generate spermatogenic cells - which are 'foreign' to the host - without triggering immune activation. After leaving the testis, spermatozoa enter the epididymis where they mature and are stored. In this study, we report a previously unrecognized dense network of dendritic cells (DCs) located at the base of the epididymal epithelium. This network was detected in transgenic mice expressing CD11c-EYFP and CX3CR1-GFP reporters. Epididymal DCs (eDCs) establish intimate interactions with the epithelium and project long dendrites between epithelial cells toward the lumen. We show that isolated eDCs express numerous leukocyte markers described previously in other organs that are in contact with the external environment, and present and cross-present ovalbumin to T cells in vitro. eDCs are, therefore, strategically positioned to regulate the complex interplay between immune tolerance and activation, a balance that is fundamental to male fertility.

Essential Roles of Androgen Signaling in Wolffian Duct Stabilization and Epididymal Cell Differentiation

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.

Androgen Receptor Expression in the Caput Epididymal Epithelium Is Essential for Development of the Initial Segment and Epididymal Spermatozoa Transit

The epididymis plays an essential role in male fertility, and disruption of epididymal function can lead to obstructive azoospermia. Formation and function of the epididymis is androgen-dependent. The androgen receptor (AR) is expressed in both the stromal and epithelial compartments of the epididymis, and androgen action mediated via stromal cells is vital for its normal development and function. However the impact of epithelial specific AR-dependent signaling in the epididymis remains underexplored. To address this, we used conditional gene-targeting in mice to selectively ablate AR from the caput epididymal epithelium, and characterized the resulting phenotype at multiple postnatal ages. Caput epithelium androgen receptor knock-out (CEARKO) mice have normal serum testosterone concentrations at day (d) 21 and d100, but do not develop an epididymal initial segment. The remaining caput epithelium displays a significant decrease in epithelial cell height from d11 and lumen diameter from d21 and disruption of the smooth muscle layer of the caput epididymis at d100. From d21, CEARKO mice accumulate cell debris, proteinaceous material, and, at later ages, spermatozoa in their efferent ducts, which prevents normal passage of spermatozoa from the testis into the cauda epididymis resulting in infertility when tested at d100. This efferent duct obstruction leads to fluid back-pressure and disruption of the seminiferous epithelium of the adult testis. We conclude that epithelial AR signaling is essential for postnatal development and function of the epididymal epithelium and that disruption of this signaling can contribute to obstructive azoospermia.