De novo biosynthesis and localization of inhibin in marmoset (Callithrix jacchus) and rat epididymis.

Using specific polyclonal antibodies generated against a 13 Kd human testicular inhibin, immunocytochemical localization was carried out in epididymis of intact and castrated marmoset monkey and rat epididymis. Inhibin was found to be present in the cytoplasm of epithelial cells of caput, corpus and cauda epididymis. The intensity of staining and pattern of distribution did not change following castration. Further, the in vitro biosynthesis of inhibin studied by incorporating 3H-leucine and precipitating it with specific antibody indicated maximum biosynthesis in the corpus epididymis in case of marmosets and cauda in case of rats. Following castration in rats, the epididymal tissue still retained the capacity to biosynthesize inhibin. These studies indicate that marmoset and rat epididymis are capable of biosynthesizing/absorbing inhibin and whose synthesis does not depend on androgens.     

Androgen receptors in the rat epididymis and their hormonal control.

The concentrations of cytoplasmic receptor sites for androgens in the caput, corpus and cauda epididymidis, and the effect of ligation of the efferent ducts and testosterone treatment after bilateral castration on the concentration of receptors in the caput have been measured. Androgen receptors in the ventral prostate have been measured in the same animals for comparison. The caput has the highest concentration of receptor sites, the corpus the lowest. The ligation of the efferent ducts has no effect on this concentration which is dependent on testicular androgens. The present data do not yet allow explanation of the differential response of the different regions of the epididymis and of the other accessory glands to the administration of androgens.     

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.     

Expression and regulation of lipocalin-type prostaglandin d synthase in rat testis and epididymis

Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.     

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.     

Effects of castration, 5 alpha-dihydrotestosterone and cyproterone acetate on enzyme activity in the mouse epididymis

The influence of castration, androgen replacement therapy and cyproterone acetate on the activity of beta-glucuronidase, acid and alkaline phosphatases and glucose-6-phosphate dehydrogenase was studied in the caput, corpus and cauda epididymidis of the mouse. The results add further evidence that the epididymis is not uniform but has regional differences in activity. Thus beta-glucuronidase was found to be androgen-dependent only in the cauda epididymidis, whereas glucose-6-phosphate dehydrogenase was under androgenic control in the caput epididymidis. The response of alkaline and acid phosphatases to castration and to androgen replacement was different in different segments.     

Short-term effect of androgen deprivation on intraluminal pressure and contractility of the rat epididymis

Short-term effects of bilateral castration, cyproterone acetate and unilateral efferent duct ligation on intraluminal pressures and spontaneous contractions in different regions of the epididymis were studied in the rat. Ligation of the efferent ducts for 5 days did not alter pressures or spontaneous contractions in any region of the epididymis. However, bilateral castration produced time-dependent changes in pressures and contractions in different segments. In the caput, the amplitude, but not the basal pressure or the frequency, of spontaneous contractions increased by Day 1 after operation. In the corpus, increments in the basal pressure and the amplitude of contractions occurred by Day 5 whilst the frequency of contractions was not changed. Similar effects were observed in the cauda by 3 days after castration. Changes in all regions of the epididymis were also mimicked by cyproterone acetate treatment (10 mg/rat per day, s.c. for 21 days). In addition, this drug increased the amplitude of contractions in the cauda. The effect of castration was abolished by testosterone propionate (0.2 mg/kg per day, i.m. for 5 days). The results support the suggestion that an enhancement of sperm transport through the rat epididymis occurs shortly after castration. The results also suggest that, in normal rats, androgens suppress the contractility of the epididymal tubule to ensure an optimal rate of sperm transport.     

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.     

Acetyl-coenzyme A carboxylase from avocado (Persea americana) plastids and spinach (Spinacia oleracea) chloroplasts.

1. Protein synthesis has been investigated in different regions of the rat epididymis by measuring incorporation of [35S]methionine in tissue minces incubated in vitro followed by analysis of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate. Rates of synthesis were highest in the proximal cauda > distal cauda > initial segment > ductuli efferentes > corpus > distal caput > proximal caput. One protein (mol.wt. 23 000) characterized the initial segment, three proteins (mol.wts. 18 500, 19 000 and 32 000) the caput and one protein (mol.wt. 47 000) the cauda. 2. After castration, [35S]methionine incorporation in all regions of the epididymis was reduced to < 10% of that in normal animals but could be restored to control levels within 5 days by testosterone treatment. Other steroids (corticosterone, oestrogen or progesterone) were ineffective. 3. The synthesis of the 18 500, 19 000, and 32 000 mol.wt. proteins in the caput and the 47 000 mol.wt. protein in the cauda were preferentially regulated by androgens, whilst the synthesis of 23 000 and approx. 80 000 mol.wt. proteins in the initial segment was dependent upon factors present in testicular fluid. 4. The androgen-dependent and testicular fluid-dependent proteins were major components of epididymal secretion. Purification and characterization of the 18 500, 19 000, 23 000 and 32 000 mol.wt. proteins showed them to be acidic glycoproteins with a carbohydrate content of 7.6-13.2%. The 47 000 mol.wt. protein, on the other hand, is highly basic. 5. A possible role for these proteins in the acquisition of motility, fertilizing capacity and storage of spermatozoa in the epididymis is discussed.     

Functional maturation of the epididymis in rabbit and rhesus monkey.

The changes in the weight, histology and biochemical composition of the epididymis (caput, corpus and cauda segments) in prepuberal rabbit and rhesus monkey in response to testosterone treatment were investigated. The increase in the weight of the organ was accompanied by increased levels of RNA and DNA. Androgen therapy caused an increase in the concentration of sialic acid, phospholipids and glycerylphosphorylcholine and activities of alkaline phosphatase, acid phosphatase and hyaluronidase. The cauda region of the epididymis exhibited relatively higher levels of sialic acid and glycerylphosphorylcholine. These findings are discussed in relation to the functional maturation of the organ and the role of androgen in this process.     

Proceedings: Effect of cyproterone acetate on the epididymis and accessory glands of the rat

Cyproterone acetate, a well-known potent anto-androgen, competitively inhibits the action of endogenously and expgenously administered androgens on target organs like the epididymis and ventral prostate (Prasad, Rajalakshmi & Reddy, 1972). Administration of cyproterone acetate to immature 30-day-old rats for 20 days or to adult, sexually mature rate for 15 days, resulted in a marked decrease in the content of sialic acid in the caput and cauda epididymides and a decrease in the secretory activity of the dorsolateral prostate and coagulating glands. The incorporation of (3H) uridine and (3H) phenylaline has been studied in adult rats treated with cyproterone acetate for 15 days. An increased incorporation of (3H) uridine and (3H) phenylalinine was observed in the caput and cauda epididymidis cauda epididymidis followed by the caput epididymidis. However, the increase in incorporation of (3H) phenylalanine was of the same order of magnitude in the caput and cauda epididymidis whereas it was almost insignificant in the ventral prostate.     

Effects of castration and androgen treatment on androgen-receptor levels in rat skeletal muscles.

The effects of castration and dihydrotestosterone (DHT) treatment on levels of skeletal muscle androgen receptor (AR) were examined in three groups of adult male rats: 1) intact normal rats, 2) rats castrated at 16 wk of age, and 3) rats castrated at 16 wk of age and given DHT for 1 wk starting at week 17. All animals were killed at 18 wk of age. Castration caused a decrease (P < 0.05) in the weights of the levator ani and bulbocavernosus muscles. The administration of DHT to the castrated rats increased (P < 0.05) the weights of the levator ani and bulbocavernosus muscles. Castration caused a significant downregulation of AR levels in the bulbocavernosus (P < 0.05) but had no significant effect on AR levels in the levator ani muscle. DHT administration to the castrated group upregulated AR levels in the bulbocavernosus and levator ani muscles. The plantaris muscle did not significantly (P > 0.05) change for any of the treatments. These findings suggest that the effects of castration and androgen replacement differentially affect skeletal muscle mass and AR levels.     

Activity and androgenic control of glycolytic enzymes in the epididymis and epididymal spermatozoa of the rat.

1. Procedures were developed for the extraction and assay of glycolytic enzymes from the epididymis and epididymal spermatozoa of the rat. 2. The epididymis was separated into four segments for analysis. When rendered free of spermatozoa by efferent duct ligation, regional differences in enzyme activity were apparent. Phosphofructokinase, glycerol phosphate dehydrogenase and glucose 6-phosphate dehydrogenase were more active in the proximal regions of the epididymis, whereas hexokinase, lactate dehydrogenase and phosphorylase were more active in the distal segment. These enzymes were less active in the epididymis of castrated animals and less difference was apparent between the proximal and distal segments. However, the corpus epididymidis from castrated rats had lower activities of almost all enzymes compared with other epididymal segments. 3. Spermatozoa required sonication to obtain satisfactory enzyme release. Glycolytic enzymes were more active in spermatozoa than in epididymal tissue, being more than 10 times as active in the case of hexokinase, phosphoglycerate kinase and phosphoglycerate mutase. 4. The specific activities of a number of enzymes in the epididymis were dependent on the androgen status of the animal. These included hexokinase, phosphofructokinase, aldolase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, glycerol phosphate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphorylase. 5. The caput and cauda epididymidis differed in the extent to which enzyme activities changed in response to an altered androgen status. The most notable examples were hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, 6-phosphogluconate dehydrogenase and phosphorylase.     

Segment- and cell-specific expression of D-type cyclins in the postnatal mouse epididymis

Sperm transport, maturation and storage are the essential functions of the epididymis. The epididymis in the mouse is structurally characterized by regional and segmental organization including caput, corpus and cauda epididymis that are comprised of 10 segments. Although several growth factor signaling pathways have been discovered in the epididymis, how these converge onto the cell cycle components is unknown. To begin to elucidate the growth factor control of cell cycle events in the epididymis, we analyzed the expression of D-type cyclins at different postnatal ages. At 7d, cyclin D1 was mainly expressed in the cauda epithelium, by 14d its expression occurred in the epithelium of caput, corpus and cauda that persisted up to 21d. By 42d, cyclin D1 was mostly detectable in the principal cells of the caput and corpus (segments 1–7) but not in the cauda epididymis. Expression of cyclin D2, unlike that of cyclin D1, was evident only at 42d but not earlier, and was mostly confined to corpus and cauda epithelium. In contrast to both cyclins D1 and D2, cyclin D3 was expressed primarily in the interstitium at 7d and by 21d its expression was localized to the epithelium of the corpus and cauda epididymis. By 42d, expression of cyclin D3 peaked in segments 6–10 and confined to basal and principal cells of the corpus and apical cells of the cauda epithelium. Ki67 immunoreactivity confirmed absence of cell proliferation despite continued expression of D-type cyclins in the adult epididymis. Collectively, on the basis of our immunophenotyping and protein expression data, we conclude that the D-type cyclins are expressed in a development-, segment-, and cell-specific manner in the postnatal mouse epididymis.     

Androgen binding to cytosol prepared from epididymides of sexually mature castrated rabbits: evidence for a cytoplasmic receptor.

The presence of androgen-binding activity in cytosol prepared from the major anatomical segments (caput, corpus, and cauda) of the epididymis of castrated sexually mature rabbits has been demonstrated. A portion of this binding activity is likely to be the epididymal androgen receptor. When epididymal cytosol from adult castrated rabbits is analyzed on low-ionic strength (0.01 MKCl) sucrose gradients, two peaks of macromolecular binding could be detected, one congruent to 4.6S and one congruent to 8S. On gradients containing 1.0 M KCl, only one sedimenting form congruent to 4.6S could be demonstrated, suggesting that the 8S component is composed of aggregates. If cytosol was preincubated with labeled androgen, followed by an incubation with unlabeled androgen, and subsequently analyzed for binding on low-ionic strength gradients, only the congruent to 8S peak could be detected, indicating that most of the binding in the congruent to 4.6S region was rapidly dissociable. This suggests that binding in this region was to moieties other than receptor. Since androgen binding proteins (ABP) of testicular origin would have been cleared from the epididymis at the timepoints that we concentrated on for most of these studies, the 4.6S binding probably represents the association of androgen with plasma testosterone binding globulin (TeBG). The binding of androgen to the receptor can be inhibited by cyproterone, while this antiandrogen does not inhibit binding to either ABP or TeBG at the concentration used.