Immunolocalization of androgen receptor in the epididymis of rats with dihydrotestosterone deficiency

Dihydrotestosterone (DHT), 5alpha-reduced metabolite of testosterone, is the most potent androgen in the epididymis. The conversion of T into DHT is carried out by 5alpha-reductase. The activity of 5alpha-reductase type 2, preferentially expressed in the epididymis can be inhibited by a finasteride (a steroid-based specific inhibitor of 5alpha-reductase type 2) which results in DHT deficiency. The aim of the study was to examine the morphology of epididymis and the immunolocalization of an androgen receptor (AR) in the initial segment, caput and cauda epididymis of rats treated with finasteride for 56 days. There were no morphological changes in the morphology of epididymal epithelium in the experimental rats. Immunostainable AR was localized in nuclei of epithelial cells, smooth muscle cells and mainly in the cytoplasm of interstitial cells in the epididymis of control rats. In the epididymis of experimental rats, AR immunostaining was noticed mainly in the cytoplasm of epithelial cells and interstitial cells. The single cells of the initial segment epithelium, basal cells and smooth muscle cells of cauda epididymis showed nuclear AR staining. In conclusion, finasteride affected the expression of the AR in the rat epididymis without changing the morphology of epididymal epithelium. Altered AR expression reflected the hormonal status within the epididymis.     

The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.

We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.     

Epididymal maturation and the acrosome reaction in mouse sperm: response to zona pellucida develops coincident with modification of M42 antigen

Development of the sperm's capacity to interact with the zona pellucida was investigated at the stage when the acrosome reaction (AR) is induced. The response of epididymal sperm to agents that affect the occurrence of the AR was used to monitor maturational changes. Despite the finding that sperm from the three main epididymal regions were competent to undergo ARs induced by the divalent cation ionophore A23187 (56% AR, 74% AR, and 83% AR in caput, corpus, and cauda, respectively), the cells' responses to solubilized zonae pellucidae were different. When challenged with 5 zonae equivalents/microliter, both corpus and cauda sperm shed their acrosomes in high numbers (75% AR and 86% AR, respectively), whereas caput sperm did not (23% AR). Previous work has shown that the presence of M42 monoclonal antibody (mAb) during in vitro and in vivo fertilization inhibits sperm penetration through the zona pellucida by specific interference with zonae pellucidae-induced ARs. In this study, presence of the M42 mAb did not affect the incidence of A23187-induced ARs, whereas the zona-induced ARs that occurred in both corpus and cauda sperm were inhibited fully with M42 immunoglobulin (Ig) G. In addition, the antigen recognized by M42 mAb on sperm, termed M42 Ag, was examined during epididymal maturation. Although antigen localization appeared indistinguishable by immunofluorescence on sperm taken from the caput, corpus, and cauda regions of the epididymis, modification of this antigen during epididymal transit was detected. Equilibrium-binding studies using 125I-M42 IgG demonstrated a progressive increase during epididymal transit in the amount of M42 mAb that bound to fixed cells. Corpus and cauda sperm bound 185% and 240%, respectively, of the 125I-M42 IgG detected on caput sperm. These changes in expression of M42 Ag paralleled a structural change: the Mr of the antigen decreased from a 195,000/210,000 doublet in caput sperm to a 185,000/200,000 doublet in corpus and cauda sperm, as determined by immunoblot analysis of sodium dodecyl sulfate (SDS)-extracted sperm. Results presented here demonstrate that mouse sperm develop the capacity to undergo a zona-induced AR during epididymal maturation. The M42 antigen, which is involved in the zona-induced AR, is modified during epididymal transit coincident with development of the sperm's responsiveness to zonae. Our working hypothesis, based on these results, is that development of the sperm's capacity to undergo a physiological AR is related to modification of M42 Ag.     

Epididymal phenotype in luteinizing hormone receptor knockout animals and its response to testosterone replacement therapy

Previous studies reported that epididymis contains functional LH receptors. The LH receptor knockout mice, which have epididymal phenotypes, gave us an opportunity to test the hypothesis that testosterone replacement alone may not be sufficient to reverse phenotypes to wild-type epididymis. The morphological phenotype in knockout animals includes a decrease in luminal diameter of the proximal and distal caput and cauda epididymis, the absence of clear and halo cells in the epithelial lining, a decrease in the height of principal cells and the number of cells containing cilia, a decrease in cilia length, and a change from basal to central location of nuclei in the principal cells. The biochemical phenotype includes a decrease in periodic acid-Schiff reaction product, reflecting the glycogen and glycoprotein synthesis and secretion, a decrease in androgen receptor (AR) and estrogen receptor (ER)beta, and an increase in ERalpha levels. Twenty-one-day testosterone replacement therapy in 30-day-old knockout animals reversed some, but not all, morphological and biochemical phenotypes. Those that did not reverse include luminal diameters of proximal and distal caput and cauda epididymis, the percentage of ciliated principal cells in caput epididymis, and nuclear AR localization. In summary, while our results reaffirm that androgens are important for normal epididymal morphology and function, they indicate that LH could be required for certain facets of epididymal morphology and/or function.     

Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa.

We have attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals [this technique was thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) high efficiencies (more than 50%) of gene transmission were achieved in the mid-gestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and (iii) overt gene expression was not found in these fetuses. These findings suggest the possibility that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa. The purpose of this study was to elucidate the mechanism of TMGT by introducing trypan blue (TB) or Hoechst 33342 directly into testis. We found that TB is transported to the ducts of the caput epididymis via rete testis within 1 min after testis injection, and TB reached the corpus and cauda epididymis within 2-4 days after injection. Staining of spermatozoa isolated from any portion of epididymis was observed 4 days after injection of a solution containing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) expression vector/liposome complex into testis resulted in transfection of epithelial cells of epididymal ducts facing the lumen, although the transfection efficiency appeared to be low. In vivo electroporation toward the caput epididymis immediately after injection of EGFP expression vector into a testis greatly improved the uptake of foreign DNA by the epididymal epithelial cells. PCR analysis using spermatozoa isolated from corpus and cauda epididymis 4 days after injection of a DNA/liposome complex into testis revealed exogenous DNA in these spermatozoa even after treatment with DNase I. These findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermatozoa.     

Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.     

Immunolocalization of estrogen and androgen receptors and steroid concentrations in the stallion epididymis

The presence of steroids and their receptors throughout development, specifically androgen receptor (AR), estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta), in the epididymis of a high estrogen producing species like the stallion has not been determined. Epididymal and testicular samples were collected for analysis of testosterone and estradiol-17beta (E(2)) concentrations and for immunolocalization of AR, ERalpha and ERbeta. The concentration of testosterone in the testis and epididymis were not different among age groups (P>0.05). AR was localized in the principal cells of the caput, corpus and cauda in all four age groups. This lack of change in testosterone concentration and receptor localization suggests that testosterone is important for both development and maintenance of epididymal function. There was an age-related increase in E(2) concentrations in all regions of the epididymis (P<0.05), suggesting that E(2) is also important for adult function. ERbeta was localized in the principal cells of the caput, corpus and cauda in all four age groups, but the localization of ERalpha was regional and age dependent. In peri-pubertal animals, ERalpha immunostaining was most prominent and estradiol was similarly present in all three epididymal regions; this suggests that estradiol also plays a key role in the maturation of the stallion epididymis during the pubertal transition when sperm first arrive in the epididymis. In conclusion, these results suggest that the stallion epididymis is regulated by both androgens and estrogens throughout development and that estradiol is more important to epididymal function in the stallion than previously believed.     

Region-specific gene expression in the epididymis

The epididymis is responsible for post-testicular sperm maturation, which consists in the acquisition of forward motility and fertilizing ability. This organ is composed of three main anatomical regions - the caput, corpus and cauda epididymidis - which possess distinct gene expression profiles, ensuring different epididymal functions essential to the different steps of sperm maturation. Since many genes display spatially restricted expression in the epididymis, this organ constitutes a model of choice to study the mechanisms that govern region-specific gene expression. Factors such as steroid hormones, lumicrine factors and temperature affect the pattern of gene expression in the epididymis. Recently, the contribution of small RNAs in epididymal gene regulation has been investigated and constitutes a promising avenue for clinical application with regard to male fertility.     

Regional and seasonal differences in concentrations of androgen and estrogen receptors in ram epididymal tissue

Dihydrotestosterone (DHT) is essential for sperm maturation within the epididymis, but the roles of estradiol-17 beta (E2) and progesterone (P) in epididymal function are unknown. To identify sites of potential action of these hormones, and any effect of season on their concentrations, specific binding of steroids to receptors in extracts of ram epididymal tissue was quantified in two studies. Tissue was taken from three broad regions of the epididymis (caput, corpus, and cauda; Study 1) or from seven discrete regions of the epididymis (Study 2) in February to May (nonbreeding season; NBS) or late August to October (breeding season; BS). Specific binding of P was not detected. Saturable high-affinity binding sites specific for DHT (Ka = 2.6 x 10(8).M-1) and E2 (Ka = 5.4 x 10(8).M-1) were detected. Binding was not to androgen-binding protein, testosterone-estradiol-binding globulin, or sperm nuclei. There was no regional or seasonal difference in affinity of DHT or E2 binding. In both studies, concentration of DHT-binding sites (fmol/mg protein for low- plus high-salt extracts) was higher (p less than 0.05) in the BS than NBS. In Study 1, mean concentration of DHT-binding sites was higher (p less than 0.05) in the caput than in the corpus and cauda. The more definitive localization possible in Study 2 revealed that concentration of DHT-binding sites was highest in the distal caput, lowest in the proximal cauda (p less than 0.05), and intermediate in other regions. For E2, however, concentration of binding sites was higher (p less than 0.05) in the BS than NBS only in Study 1, and was higher (p less than 0.05) in the cauda or corpus than in the caput epididymidis. In Study 2, the season by region interaction was significant (p less than 0.05); concentration of E2-binding sites was higher in the distal cauda during the NBS. These data support the concept that the central caput through proximal corpus epididymidis are most dependent on androgenic stimulation, whereas distal regions may respond to estrogenic stimulation.     

Tissue and cell specificity of immobilin biosynthesis

The mechanisms for the initiation of sperm motility have been poorly understood until recently. Immobilin is a novel mucin glycoprotein of high molecular weight found in the cauda epididymis of the rat that, at concentrations equivalent to those found in native cauda epididymal fluid, reversibly inhibits sperm motility. In this study, immobilin was purified from rat cauda epididymal fluid to apparent homogeneity and used to generate polyclonal antibody in rabbits. The antibody was characterized by immunoblotting, and immunofluorescence was used to localize immobilin in paraffin sections of components of the reproductive system of adult male rats. Immobilin was not detectable in the efferent duct and was first detectable in the apical portion of some epithelial cells of the initial segment of the caput epididymis. Immobilin was detectable intracellularly only in cells of the caput epididymis. In the corpus and cauda epididymis immobilin was detectable only in the lumen of the tubules. Immunoprecipitation of immobilin radiolabeled in vitro confirmed that immobilin biosynthesis in the adult rat is restricted to the caput epididymis. Principal cells in the caput epididymis synthesize immobilin and secrete it into the lumen of the tubules to travel with the sperm into the cauda.     

Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.     

Region- and cell-specific differences in the distribution of carbonic anhydrases II, III, XII, and XIV in the adult rat epididymis.

We employed RT-PCR followed by light microscope immunocytochemistry on St. Marie's- and Bouin's-fixed tissues to define the distribution of carbonic anhydrase (CA) isoforms in the male reproductive tract. The data revealed that CA II, III, IV, XII, and XIV were expressed in rat epididymis. Whereas CA III was found in principal cells of all epididymal regions, CA II was localized in narrow cells of the initial segment and principal cells of all regions. CA XII expression was most intense in the corpus and proximal cauda regions, where it appeared over the basolateral plasma membranes of principal cells. Narrow cells of the initial segment also revealed intense reactions, as did basal cells of the corpus and proximal cauda regions. Principal cells of the initial segment and proximal caput regions showed diffuse apical cytosolic reactions and occasional basolateral staining for CA XIV, whereas principal cells of distal regions showed more diffuse cytosolic reactions highlighting both apical and basal regions of the cell, with basal cells also being reactive. These data suggest subtle differences in cell type and subcellular- and region-specific distributions for CAs in their role of fine-tuning pH in the lumen, cell cytosol, and intervening intercellular spaces of the epididymis.