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Histochemical demonstration of zinc in rat epididymis using a sulphide-silver method.

We studied the histochemical distribution of zinc in rat epididymis using a sulphide-silver method. In the supranuclear cytoplasm of the principal cells that line the epididymis of rats, varying amounts of sulphide-silver-reactive zinc were visualized. In adult mating rats, significant amounts of zinc were found in the proximal portion of the epididymis, whereas in non-mating, mature and immature young rats, this heavy metal was most prominent in the distal portion of this organ. In all of the rats studied, zinc was sparsely distributed in the intermediate portion of the epididymis. From these results, it can be assumed that the zinc present in the epithelial lining of rat epididymis plays an important role in the maturation of spermatozoa. The present results represent a useful contribution to our understanding of the functional morphology of rat epididymis.

Animals↗

Vitamin D3 metabolites in rat epididymis: high 24,25-dihydroxy vitamin D3 levels in the cauda region.

Normal male rats received six subcutaneous injections of 8.0 pmoles of tritiated 25-hydroxy vitamin D3 ([3H]25(OH)D3) or one intrajugular injection of 8.0 pmoles of high specific radioactivity [3H]-25(OH)D3. Lipid extracts of several tissues including the reproductive organs were subjected to sephadex LH-20 chromatography to determine the tissue distribution of the injected material and of the in vivo produced dihydroxylated cholecalciferol metabolites. The nature of the putative 25(OH)D3 and the 24,25-dihydroxy vitamin D3 (24,25(OH)2D3) from epididymis tissue was confirmed by high performance liquid chromatography (HPLC). The epididymis levels of 24,25(OH)2D3 were considerably higher in the cauda epididymis compared to kidney and caput epididymis levels. The other metabolites levels in this tissue were similar to those determined in the kidneys. The amounts of the three metabolites found in all other tissues were well below the cauda epididymis or kidney levels. The findings suggest a possible physiological role for 24,25(OH)2D3 in the epididymis, and are also consistent with data of others which indicated a possible action of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) in rat reproductive tissues.

24,25-Dihydroxyvitamin D 3↗

Immunohistochemical localization of galectin-3 in boar testis and epididymis.

The presence and distribution of galectin-3, a beta-galactoside-binding protein, in boar testis and epididymis was studied. Western blot analysis detected galectin-3 in boar testis and epididymis. In particular, intense galectin-3 immunoreaction was seen in the tail of the epididymis, while it was moderate in the head and body. Galectin-3 immunolabelling was detected in the connective tissues of the testis. In the testis, galectin-3 was detected in some cells (presumably peritubular myoid cells), but not in Leydig cells or the cells of the seminiferous tubules. In the epididymis, the galectin-3 immunoreactivity in the connective tissues was the same as in the testis. Intense galectin-3 immunolabelling was seen in the covering epithelium of the epididymis tail, but in very few cells in the head and body. We postulate that galectin-3, immunodetected here in the connective tissues in the male reproductive organs, serves as an extracellular matrix. Furthermore, we postulate that intracellular galectin-3 in the epithelium of the epididymis tail plays a role in either the maintenance of the epithelium or as a source of galectin-3 in the seminal fluid; here it may play a role in sperm activation in the boar reproductive system.

Animals↗

Estrogen and androgen receptor expression in relation to steroid concentrations in the adult boar epididymis.

The steroid hormone regulation of the epididymis in a high estrogen producing animal like the boar is not currently understood. To test the hypothesis that the boar epididymis is an estrogen and androgen responsive tissue, the presence of estrogen and androgen receptors, in conjunction with steroid hormone concentrations were investigated in the boar epididymis. Epididymal (caput, corpus, cauda) and testicular samples of boars (1-2.5 years; n=5) were collected for immunolocalization of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and androgen receptor (AR). Concentrations of testosterone, estradiol and estrogen conjugates (EC) in the tissue were also determined. AR and ERbeta were localized in the principal and basal cells of all three epididymal regions. ERalpha was localized in the principal cells of the caput, some cells of the corpus and was not present in the cauda. Testosterone (p<0.0001), estradiol (p<0.0001) and EC (p<0.005) were significantly lower in the epididymis compared with the testis. The epididymal regions were not significantly different from each other for testosterone (p>0.15) or estradiol (p>0.09). EC were significantly higher in the corpus than either the caput (p=0.003) or cauda (p=0.002). These results suggest that the boar epididymis is responsive to both estrogens and androgens and that both steroid hormones are important for proper epididymal function. Since testosterone and estradiol concentrations are similar throughout the epididymis, regional differences in steroid hormone regulation are likely due to differences in receptor expression.

Animals↗

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.

Animals↗

Focused ultrasound ablation of the epididymis with use of thermal measurements in a canine model.

OBJECTIVE: To explore the epididymis as an alternative anatomical target to the vas deferens for noninvasive male sterilization using therapeutic focused ultrasound. DESIGN: Controlled preclinical study. SETTING: Canine animal model in an academic research environment. PATIENT(S): Four healthy male mongrel dogs (30-35 kg). INTERVENTION(S): A transducer mounted on a plastic clip delivered ultrasound energy to the canine epididymis. Thermocouples placed transcutaneously into the epididymis, intradermally, and on the skin surface recorded temperatures during ablation with a wide range of acoustic powers and sonication times (control, 3 W/120 s, 5 W/90 s, 7 W/60 s). MAIN OUTCOME MEASURE(S): Thermocouple temperature measurements determined the optimal range of ablation parameters that produced successful thermal occlusion of the epididymis without adverse effects (e.g., skin burns, testicular injury). RESULT(S): A large "therapeutic window" was determined (power = 3-7 W, time = 20-120 seconds) over which noninvasive thermal occlusion of the epididymis can be achieved. Thermal occlusion rates were higher, and complications lower, than found previously with vas deferens ablation. CONCLUSION(S): The epididymis represents a larger and easier target than the vas deferens for performing noninvasive male sterilization using focused ultrasound. Long-term azoospermia studies will be necessary to confirm permanent sterilization with this technique.

Animals↗

Endothelin-1 is synthesized and biologically active in human epididymis via a paracrine mode of action.

In a previous study, we reported the presence of endothelin-1 and endothelin receptors in the human testis. We have now extended our investigations to the human epididymis. Since sperm appear to be immotile during their transit through the epididymis, it is conceivable that specific local factors promote smooth muscle contraction, enabling sperm transport. In this paper, we show that endothelin-1 mRNA and protein are readily detectable in the epithelial compartment of the human epididymis, and that endothelin converting enzyme- 1, which converts the precursor pro-endothelin-1 into active endothelin-1, is expressed in the epididymis, consistent with active processing of the prohormone. In addition, two classes of endothelin receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the previously characterized endothelinA and endothelinB receptor. These receptors appear to be biologically active and mediate the contractile activity of the epididymis in vitro. Our data suggest that endothelin-1, via a paracrine mode of action, may be responsible for sperm progression through this organ.

Endothelin-1↗

Sequential histology of the adult chimpanzee epididymis.

The chimpanzee epididymis consists of three gross anatomical regions: caput (Cp), corpus (Cr), and cauda (Cd). During epididymal transit, chimpanzee sperm undergo maturational changes (affecting motility, capacitation, and the acrosome reaction), which enable fertilization. Elucidation of the sequential histology of the chimpanzee epididymis is preliminary to determining the role of each region of epididymal duct in the process of sperm maturation. We report the histology, in the adult male chimpanzee, of sequential segments of epididymis from the proximal efferent ductules through the Cp, Cr, and Cd regions of the epididymis proper to the proximal ductus deferens. These data illustrate the gross architecture of the tissue and reveal the histology characteristic of the diverse epithelia composing the epididymal ducts at each level. The tubules composing the epididymis of the adult male chimpanzee present at least 16 histologically-distinct epithelia and their transitional forms. Such diversity of epithelia suggests a corresponding diversity of function. Although there are some clear differences, the histological appearance of the ducts, and of the component epithelia of the chimpanzee epididymis, is remarkably similar to that reported for the human.

Animals↗

Angiotensinogen expression by rat epididymis: evidence for an intrinsic, angiotensin-generating system.

Previous studies have suggested the presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis with functions in epididymal activity and sperm maturation. In the present study, the localization and expression of angiotensinogen, the component of the RAS which is indispensable for intracellular angiotensin generation, were investigated by immunochemistry, hybridization histochemistry and by reverse-transcriptase polymerase chain reaction (RT-PCR). Western blot analysis of protein from the epididymis confirmed the presence of angiotensinogen with the expected molecular mass of about 60 kDa, in agreement with results from other tissues. Immunocytochemistry showed the regional localization of immunoreactivity for angiotensinogen in the rat epididymis. In situ hybridization histochemistry further demonstrated the expression of angiotensinogen mRNA by the epididymal epithelium in a region-specific manner along the length of the rat epididymis. RT-PCR confirmed that the rat epididymis expresses angiotensinogen mRNA. On the other hand, mRNA of renin was not detected in the rat epididymis using Northern blot and RT-PCR analyses. The present study strongly supports the existence of an intrinsic, angiotensin-generating system based on locally formed angiotensinogen as a precursor for angiotensin production. This epididymal RAS may have paracrine or autocrine roles in mediating the epididymal and sperm functions.

Angiotensinogen↗

Molecular cloning and characterization of the epididymis-specific glutathione peroxidase-like protein secreted in the porcine epididymal fluid.

The epididymis-specific glutathione peroxidase was purified from the porcine cauda epididymal fluid in order to analyze its enzymatic activity and roles in the epididymis. The purified protein was found to consist of four identical 23 kDa subunits. The complementary DNA encoding the 23 kDa subunit was cloned from the cDNA library of the porcine proximal caput epididymis, only where the 23 kDa subunit is expressed. Although the selenocysteine codon (TGA) is contained in the cDNA of the other cytosolic type of glutathione peroxidases, it is replaced by cysteine codon (TGT) in the 23 kDa subunit cDNA, similarly to the results previously obtained for cDNAs encoding the epididymis-specific form of the secreted glutathione peroxidases of mouse, rat and monkey. By the direct analysis of the selenium, the purified protein was proved to contain no selenium atom in the molecule. The activities of the purified epididymis-specific glutathione peroxidase toward hydrogen peroxide or organic hydroperoxides were by far lower than the activity of cytosolic selenium-dependent glutathione peroxidase (less than 0.1%). In addition, the concentration of glutathione in the porcine epididymal fluids was about 20 microM, which is much lower than the optimal concentration for the glutathione peroxidase activity of the purified protein. These results strongly suggest that this protein is enzymatically quiescent at least in the porcine epididymal fluid. An immunocytochemical study showed that this protein was found to bind to the acrosomal region of the epididymal sperm and to disappear during the acrosome reaction. Furthermore, this protein significantly retarded the acrosome reaction induced in vitro. The possibilities have been discussed that it protects sperm from the premature acrosome reaction and maintains sperm fertilizing ability in the epididymis.

Amino Acid Sequence↗

On the intrinsic innervation of the epididymis of the camel (Camelus dromedarius).

The innervation of the camel epididymis was studied in 26 apparently healthy, sexually mature animals aged between 4 and 12 years. The material was collected during the different seasons of the year. Generally, five samples were taken from each epididymis. To demonstrate the general innervation pattern, immunohistochemical reactions to protein gene product-9.5, neurofilaments and neuron-specific enolase were used, in addition to acetylcholinesterase histochemistry. The nerve supply of the epididymis comes from two sources: (1) The majority of fibers come from the N. spermaticus inferior and accompany the deferent duct. (2) Another contribution stems from the N. spermaticus superior and enters the head region of the epididymis. From the exterior, the nerves penetrate the capsule of the organ to reach the interductular connective tissue. The terminal ramifications are observed directly within the wall of the duct and the wall of the epididymal arteries. The veins of the camel epididymis are not innervated. In the wall of the ductus epididymidis, the nerve fibers form plexuses at the subepithelial level and in the muscular coat. The amount of nerve fibers increases from the head to the tail, paralleling an increase in the intrinsic musculature. The intramural and interductular innervation of epididymal body and tail shows clear seasonal variations: More fibers and stronger reactions are observed during the winter season; the lowest density and the weakest reactions occur during the summer season. All epididymal nerves of the camel are unmyelinated. The majority of the intramural fibers and all in the arterial wall represent postjunctional sympathetic axons, but in the intramural plexuses of the duct a considerable number of cholinergic fibers are also present. Neuropeptide Y is the most frequent peptidergic transmitter and generally co-localized with dopamine-beta-hydroxylase in the sympathetic axons. Vasoactive intestinal polypeptide has a distribution similar to that of the cholinergic fibers. Calcitonin gene-related peptide-positive axons occur in moderate numbers, but never in the arterial innervation. Together with the relatively rare substance P-containing fibers, the calcitonin gene-related peptide-positive axons seem to represent the only sensory nerves in the camel epididymis.

Acetylcholinesterase↗

Proteomic profiling of regionalized proteins in rat epididymis indicates consistency between specialized distribution and protein functions.

The epididymis is a key structure of the male reproductive system; its function is to mature, transport, and store sperm. Most of the research examining the epididymis to date has been limited to the study of the secreted proteins involved in the maturation of spermatozoa. However, it is also very important to understand the protein components, regulation and function of the tissue itself since these are the basis for all of its physiological processes. We investigated the differential expression of proteins among the caput, corpus, and cauda regions of rat epididymis and considered the possible links between the localization of these proteins and the different functions of these epididymal regions. High-resolution 2-D gel electrophoresis followed by mass spectrometry (MS) revealed 28 distinct proteins whose expression levels varied from the caput to the cauda epididymis. Sixteen of them were reported for first time to be expressed in the epididymis. Expression patterns of some proteins were validated by Northern blot or Western blot. Immunohistochemistry revealed that inducible carbonyl reductase (iCR), an important enzyme in the anti-oxidative system, exhibits primary and cell-type specific distribution in the distal cauda region. Moreover, analysis of iCR transcription in castrated animals showed that its expression is androgen-dependent. Together with its known functions, iCR may also be involved in androgen metabolism and maintaining a steady microenvironment in the duct of epididymis.

Alcohol Oxidoreductases↗

Detection and localization of antigens on the surface of mouse spermatozoa. Antigen synthesis in the epididymis.

Spermatozoa achieve functional maturity during their transit through the epididymis and this maturation process is accompanied by changes in the composition and proteins of their surface. The addition of secretory products from the epididymis to the plasma membranes of the spermatozoa is considered to be a prerequisite for the acquisition by the spermatozoa of the capacities for forward motility and ovum recognition. An antibody was purified from an antiserum raised in the rabbit against fluid from the cauda epididymis of the mouse. This antibody, in combination with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody, was used to demonstrate a progressive increase in the synthesis and secretion of antigens along the length of the epididymis. Immunoaffinity chromatography of [35S]methionine-labelled proteins, synthesized by segments of the epididymis maintained in vitro, showed that the predominant protein synthesized by the cauda, but not by the caput, epididymis, migrated on electrophoresis with an apparent Mr of 26,000. This same protein was the major antigen found on the plasma membrane of cauda spermatozoa that had been radioactively labelled with the non-penetrating probe isethionyl [1-14C]acetimidate.

Animals↗

Identification and characterization of antioxidant enzyme mRNAs in the rat epididymis.

Spermatozoa are highly sensitive to oxidative stress. The epididymis, a natural sperm reservoir, has maturational and storage functions. The epididymis may also protect spermatozoa from oxidative injury by elaborating scavengers of reactive oxygen species (ROS). Therefore, we have evaluated the mRNA expression of antioxidant enzymes in the normal rat epididymis and the effects of efferent duct ligation no the expression of these enzymes. Adult rat epididymides were harvested, divided into caput, corpus and cauda and processed for RNA extraction. Additional adult rats were subjected to unilateral efferent duct ligation and the epididymides harvested at 1, 4, 8, 16 or 28 days after the procedure. Antioxidant enzyme mRNA expression was assessed by Northern blot analysis using 32P-labelled DNA probes derived from known cDNA sequences for classical cellular glutathione peroxidase (GSHPx), phospholipid hydroperoxide glutathione peroxidase (PHGPX), secretory epididymal glutathione peroxidase (E-GPX), copper-zinc superoxide dismutase (SOD), secretory epididymal superoxide dismutase (E-SOD) and catalase. Specific mRNA levels were measured, with gene expression evaluated relative to total RNA, not per organ. Variations in lane loading were controlled by measuring the levels of 28S ribosomal RNA. GSHPx, PHGPX, SOD and catalase mRNA were detected in the caput, corpus and cauda epididymis. E-GPX mRNA was only detected in the caput, whereas E-SOD mRNA was primarily detected in the corpus. At 28 days after efferent duct ligation, epididymal weight decreased by 34% relative to controls (p < 0.05). With the exception of PHGPX, the relative mRNA levels of the antioxidant enzymes studied did not change after efferent duct ligation. This study demonstrates that mRNAs for multiple antioxidant enzymes are expressed in the epididymis and that the relative expression of these enzymes remains largely unchanged in response to efferent duct ligation. Taken together, these results suggest that antioxidant enzymes may play an important, region-specific role in epididymal function. Expression of the secretory antioxidant enzymes E-SOD and E-GPX is region-specific, indicating that the need for antioxidant enzymes may vary along the length of the epididymis.

Animals↗

Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated 'scoop'-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the 'scoop'. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation.

Acrosome↗

Nerve growth factor mRNA and protein in the testis and epididymis of mouse and rat.

In situ hybridization using beta-nerve growth factor (NGF) DNA probes was used to demonstrate NGF mRNA in spermatocytes and early spermatids of adult mouse. NGF mRNA-containing cells were also identified in the epithelium of convoluted ducts in mouse corpus epididymidis. Blot-hybridization analysis of RNA prepared from mouse testis and epididymis as well as from rat epididymis confirmed the presence of a 1.3-kilobase (kb) NGF mRNA in these tissues. In the rat testis, however, only a 1.5-kb NGF mRNA was found, corresponding in size to a minor NGF mRNA detected in the rat brain, heart, and epididymis. By using affinity-purified anti-NGF antibodies, NGF-like immunoreactivity was observed in germ cells of rat and mouse testis and in the lumen of epididymis. Extracts of both mouse epididymis and testis stimulated fiber outgrowth in cultured sympathetic ganglia, and the effect was blocked by antibodies to mouse NGF. A two-site enzyme immunoassay showed the presence of 10 and 70 ng of NGF per g of tissue in the mouse testis and epididymis, respectively. Furthermore, RNA blot analysis showed the presence of mRNA for the NGF receptor in mouse testis. These results suggest a nonneurotrophic role for NGF in the male reproductive system, possibly in survival maturation and/or motility of spermatozoa.

Animals↗

Pem homeobox gene regulatory sequences that direct androgen-dependent developmentally regulated gene expression in different subregions of the epididymis.

The epididymis is a useful model system to understand the mechanisms that govern region-specific gene expression, as many gene products display spatially restricted expression within this organ. However, surprisingly little is known about how this regulation is achieved. Here, we report regulatory sequences from the Pem homeobox gene that drive expression in different subregions of the mouse epididymis in vivo. We found that the 0.3-kb 5'-flanking sequence (region I) from the Pem proximal promoter (Pem Pp) was sufficient to confer androgen-dependent and developmentally regulated expression in the caput region of the epididymis. Expression was restricted to the normal regions of expression of Pem in the caput (segments 2-4), but there was also aberrant expression in the corpus region. This corpus misexpression was extinguished when 0.6 kb of Pem Pp 5'-flanking sequence was included in the transgene, indicating that one or more negative regulatory elements exist between 0.6 and 0.3 kb upstream of the Pem Pp start site (region II). When heterologous sequences were introduced upstream of the Pem Pp, expression was further restricted, mainly to caput segment 3, implying that the Pem Pp has segment-specific regulatory elements. To our knowledge, the regulatory regions we have identified are the shortest so far defined that dictate regionally localized expression in the epididymis in vivo. They may be useful for identifying the factors that regulate region-specific expression in the epididymis, for expressing and conditionally knocking out genes in different subregions of the epididymis, for treating male infertility, and for generating novel methods of male contraception.

Androgens↗

Effect of vasectomy on gene expression in the epididymis of cynomolgus monkey.

Vasectomy has been shown to affect the pattern of mRNA expression of P34H, a human sperm protein added to the acrosomal cap during epididymal transit. It has been reported that vasectomy alters the histology of the reproductive tract in various species as a result of the increased pressure in the epididymis. The aim of this study was to evaluate if other epididymis-specific mRNAs, which are expressed in different patterns along the duct, are altered by vasectomy as well. We analyzed the expression of P31m (a monkey homologue of human P34H) and three different HE-like (HE-l) mRNAs along the epididymis in the cynomolgus monkey (Macaca fascicularis). Sexually mature cynomolgus monkeys were vasectomized unilaterally; then the epididymides were surgically removed at different time points. The ipsilateral normal epididymis was used as a control. Histomorphometric measurements showed that the height of the epididymal epithelial cells started to be affected only at 14 wk postsurgery. However, Northern blot and in situ hybridization analysis showed that the expression pattern of P31m, HE1, and HE5-like mRNA along the epididymis was not affected by vasectomy. Only the HE2-like mRNA predominantly expressed in the normal corpus epididymidis was significantly lowered 14 wk after vasectomy. Thus, ductal obstruction differentially alters mRNA expression along the epididymis of the cynomolgus monkey.

Animals↗