Copulatory behavior of adult tamarins (Saguinus fuscicollis) castrated as neonates or juveniles: Effect of testosterone treatment

The sexual interactions of Saguinus fuscicollis males castrated as neonates, at 37 days of age, or prepubertally with adult intact females were studied. Prepubertally castrated males were observed while receiving testosterone, and while being treated with saline. Males castrated neonatally or at 37 days of age were observed while receiving testosterone. Neonatal castrates had previously been studied without hormone treatment and therefore no control condition was included for these animals. Prepubertally castrated males showed Mounts, Mounts with Thrusts, and Sexual Tongue Flicking when treated with saline only. In three of the four males, all measures of sexual behavior increased with testosterone treatment. Neonatally castrated males had failed to display any mounting or thrusting without testosterone treatment during a previous study. During the present study, three of the four males did not respond to testosterone treatment with sexual behavior. The fourth male and one male castrated at 37 days of age displayed some sexual behavior. These results suggest that most neonatally castrated males are not able to respond to testosterone with the activation of copulatory behavior. The findings are consistent with the hypothesis that in callitrichids the sensitive period for behavioral differentiation is shifted into neonatal life. However, some neonatally castrated males show a weak response to testosterone. This may reflect an extended and perhaps partially prenatal period of sensitivity.     

Ethylene dimethane sulfonate (EDS) ablation of Leydig cells in adult rat depletes testosterone resulting in epididymal sperm granuloma: Testosterone replacement prevents granuloma formation

Sperm granuloma may develop in the epididymis following vasectomy or chemical insults. Inflammation due to sperm granuloma causes abdominal and scrotal pain. Prolonged and persistent inflammation in the epididymis due to sperm granuloma may lead to infertility. Extravasation of germ cells into the interstitium of epididymis following damage of the epididymal epithelium is one of the primary reasons for sperm granuloma-associated pathology. Since testosterone is vital for the maintenance of epididymal epithelium, we investigated the pathology of sperm granuloma and its relationship with testosterone. Adult rats were treated with a Leydig cell-specific toxicant ethylene dimethane sulfonate (EDS) to eliminate testosterone. At 7 days post-EDS, disrupted epididymal epithelium and sperm granuloma were observed in the caput epididymis. Sperm granuloma and caput were collagen-filled indicating fibrosis. Numerous round apoptotic cells were localized inside the caput lumen and dispersed through the sperm granuloma. Tnp1 (round spermatid marker) was significantly higher in the epididymis of the EDS-treated group compared to controls suggesting the apoptotic cells were round spermatids. Increases in CD68⁺ macrophages and T cells (CD4 and CD8) support an inflammatory immune infiltration in post-EDS epididymis. However, testosterone replacement following EDS prevented the sperm granuloma-associated pathology. We suggest that the immune response in the sperm granuloma may be due to the increased numbers of apoptotic round spermatids or other testicular tissue components that may be released, in addition to the regression of epididymal epithelium due to testosterone loss. Thus, testosterone replacement prevents EDS-induced sperm granuloma and ameliorates sperm granuloma-associated pathology.     

Androgen metabolism by rat epididymis. 3. Effect of castration and anti-androgens.

The in vivo and in vitro metabolism of 3H-testosterone by rat epididymis and the changes in epididymal weight have been studied after castration and treatment with anti-androgens. The utilization of 3H-testosterone was greatly reduced after castration as was the formation of 5alpha-reduced 17 beta-hydroxy metabolites. The formation of the 17 -keto metabolites was unaffected. Castration had no effect on the ratio between water and ether soluble radioactivity. Administration of testosterone propionate, necessary for giving normal stimulated prostate weight (150 mug/day), restored the metabolism of testosterone to approximately normal values. Estradiol benzoate and progesterone inhibited metabolism of testosterone in vitro and greatly reduced the formation of DHT (17 beta-hydroxy-5alpha-androstan-3-one) and 3 alpha-diol(5 alpha-androstane-3 alpha-17 beta-diol) by experiments both in vivo and in vitro. No effect of cyproterone acetate could be demonstrated on either the in vitro or in vivo metabolism of testosterone. Castration for 14 days reduced the epididymal weight to about 30% of that found in intact animals. Administration of testosterone propionate restored the epididymal weight to about 80% of normal. Estradiol benzoate and cyproterone acetate given to intact rats led to a decrease in the epididymal weight. Progesterone had no such effect. In 14 days castrated rats receiving testosterone propionate all three anti-androgens reduced the weight of the epididymis. In conclusion, our results show that the metabolic conversion of testosterone in epididymis to DHT and 3 alpha-diol is dramatically dependent on the hormonal status of the animal; castration or treatment with anti-androgens causes a reduced formation of the "active" androgens whilst testosterone replacement treatment restores the metabolism of testosterone to normal.     

Ornithine decarboxylase activity as a marker of androgen and antiandrogen action in the rat epididymis

After castration, there was a marked decrease in serum androgen concentration at 6 h, and a dramatic inhibition of ornithine decarboxylase (ODC) at 12 h. Administration of testosterone propionate to castrated rats at a dose of 0.05 mg/animal restored ODC activity to the normal value. However, no change was observed when intact rats were treated with testosterone even at a 40-fold higher dose, indicating that endogenous androgens present in intact rats are far in excess for maintenance of maximal levels of activity. Administration of the antiandrogen flutamide to intact rats caused a moderate decrease in epididymal weight, whereas this effect was more pronounced in castrated, androgen-treated rats. In the latter, the effect of flutamide was significant at the lowest dose used (0.5 mg/day). ODC activity was significantly decreased by flutamide treatment of intact rats, but even at the highest dose used (10 mg/day) only a 39% inhibition was observed. In flutamide-treated rats, LH concentrations were markedly increased, as were serum and epididymal androgens. In androgen-treated castrated rats, flutamide caused epididymal ODC to fall to undetectable values. These results show that: (1) androgens are essential for the maintenance of ODC activity in the epididymis; (2) epididymal ODC activity is maximally stimulated by endogenous androgens, at least in the pubertal rat; (3) the apparent potency of flutamide is substantially lowered by an increase in epididymal androgens. We suggest that ODC is a sensitive marker of the action of androgens and antiandrogens in the epididymis.     

Developmental changes in and hormonal regulation of estrogen and androgen receptors present in the rabbit epididymis

Both androgen and estrogen receptors (AR and ER) are present in the rabbit epididymis. We have used the sucrose gradient method to examine receptor sedimentation properties, receptor concentration, and distribution of receptors among the caput, corpus, and cauda of the epididymis to determine changes that occur in these parameters as the animals age. The 9S form of the ER is present in all three epididymal segments of the immature rabbit, with the highest concentration occurring in the cauda. The 8.2S form of the AR is also present in all three segments of the immature epididymis, with the highest concentration occurring in the caput. Short-term castration (3 days) leads to an increase in the amount of both AR and ER detected. ER are present in all segments of the immature epididymis at higher concentrations than AR. The functional 9S form of the ER disappears as the animals mature, the result of a tissue-specific protease that our laboratory previously has shown proteolyzes ER to a non-DNA-binding 3.8S form. Long-term castration (3 mo) of adult rabbits results in the reappearance of the 9S form of the ER in all segments of the epididymis. The reappearance of the 9S form of the ER is also seen in animals castrated for 1 mo, but not in those castrated for 2 wk. Administration of testosterone once daily for 2 wk to adult animals castrated for 6 wk results in the disappearance of the 9S form of the ER and the reappearance of the 3.8S form, suggesting that the tissue-specific protease is androgen-dependent. In this way, circulating androgens may play a role in regulating the concentration and form of the ER in the rabbit epididymis. There is little change in the concentration or distribution of AR in the epididymis of adult rabbits castrated for 3 mo as compared to those castrated for 3 days. This implies that circulating androgens are not required for maintenance of AR in the epididymis. Our data demonstrate that there are temporal differences in the presence and concentration of ER and AR in the epididymis and suggest that there is a differential, age-dependent regulation of the development and function of the epididymis by androgens and estrogens.     

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.     

Androgen metabolism by rat epididymis 4. The formation of conjugates

The ability to form androgen conjugates and the hormone dependency of the conjugating enzymes have been studied in the rat epididymis.Following the in vitro incubation of ³H-testosterone with epididymal slices from intact and castrated rats, the radioactivity recovered was partitioned between water and ether. Examination of the water soluble radioactivity demonstrated the presence of glucuronides and sulfates. The total radioactivity in the conjugate fraction was the same for both intact and castrated animals. However, castrated rats showed a 3-fold increase in the glucuronide fraction with a corresponding decrease in the formation of sulfates. Characterization of the ether soluble radioactivity after solvolysis of the conjugate fraction from castrated animals, showed DHT (17β-hydroxy-5α-androstan-3-one) and 3α-diol (5α-androstane-3α,17β-diol) to be the main metabolites. After β-glucuronidase hydrolysis of the same, only 3α-diol could be demonstrated at a significant level, although traces of DHT and δ¹⁶ compounds were present.Corresponding hydrolysis of the water phase from incubation of epididymis from intact rats, demonstrated a marked quantitative difference. Here approximately 40% of the conjugated aglycones consisted of Δ¹⁶ compounds, whilst only abot 12% was comprised of 3α-diol. The preferential conjugation of DHT and 3α-diol to a sulfate radical was demonstrated in both intact and castrated rats. Since the conjugated Δ¹⁶ compounds were detected only in the epididymis from intact animals, it is possible that these are formed by the spermatozoa.     

Seasonal changes in epididymis and the effects of FSH and testosterone in the spiny-tailed lizard,Uromastix hardwicki

Seasonal changes in epididymal weight and histology were studied in relation to testicular function in the adult spiny-tailed lizard, Uromastix hardwicki, over a period of 1 year. The eipdidymal weights, tubular diameter, and epithelial height increased in March, reaching a peak in April. This peak coincided with sperm maturation, elevated plasma testosterone levels, and release of sperm into the epididymis. The epididymal weights decreased in May following a sudden regression of the testis early in the month. The epididymal weights decreased further during June and remained low until February. The diameter of the duct and the height of the epithelial cells also decreased in May and the epididymal epithelium maintained a low histological profile from June to February. The fall testicular recrudescence was not accompanied by a change either in the weight or the histological structure of the epididymis. Administration of oFSH (0.1 mg) daily for 7 days during the sexually quiescent period induced a significant increase in the weight of the epididymis and epithelial height of the duct. Administration of testosterone alone, (2.0 mg) daily for the same period and under identical conditions, did not induce a change in the weight of epididymis or its histology. A possible permissive role of gonadotrophin in the hormonal regulation of the lizard epididymis has been suggested.     

Cellular observations and hormonal correlates of feedback control of luteinizing hormone secretion by testosterone in long-term castrated male rhesus monkeys

Testosterone at physiological levels cannot exert negative feedback action on LH secretion in long-term castrated male monkeys. The cellular basis of this refractoriness is unknown. To study it, we compared two groups of male rhesus macaques: one group (group 1, n = 4) was castrated and immediately treated with testosterone for 30 days; the second group (group 2, n = 4) was castrated and treated with testosterone for 9 days beginning 21 days after castration. Feedback control of LH by testosterone in group 1 was normal, whereas insensitivity to its action was found in group 2. Using the endpoints of concentrations of aromatase activity (P450(AROM) messenger RNA [mRNA]) and androgen receptor mRNA in the medial preoptic anterior hypothalamus and in the medial basal hypothalamus, we found that aromatase activity in both of these tissues was significantly lower, P: < 0.01, in group 2 compared with group 1 males. P450(AROM) mRNA and androgen receptor mRNA did not differ, however. Our data suggest that the cellular basis of testosterone insensitivity after long-term castration may reside in the reduced capacity of specific brain areas to aromatize testosterone. Because P450(AROM) mRNA did not change in group 2 males, we hypothesize that an estrogen-dependent neural deficit, not involving the regulation of the P450(AROM) mRNA, occurs in long-term castrated monkeys.     

Effects of castration on thiol status in rat spermatozoa and epididymal fluid

Mammalian spermatozoa gain their fertilizing ability as they mature in the epididymis, a process which is accompanied by oxidation of sperm protein thiols. Since sperm maturation is dependent upon normal androgenic support to the epididymis, the present work was designed to study the effects of castration on thiol status. Spermatozoa and epididymal fluid were isolated from the epididymides of male rats 5 days after castration or after 11 daily injections of the antiandrogen, cyproterone acetate. Spermatozoa and epididymal fluid were labeled with the fluorescent thiol labeling agent monobromobimane. Intact spermatozoa were evaluated by fluorescence microscopy, protein thiols were analyzed by electrophoresis, and fertilizing ability was examined after insemination of sperm suspension into the uterine horns of immature superovulated female rats. We found that both treatments resulted in an increase in cauda sperm thiols as shown by increased fluorescence in the intact spermatozoa. Protamines and nonbasic proteins were found to have increased levels of reactive thiols. The protein profiles of epididymal fluid from castrated rats were different from those of the controls, and the fluorescence patterns corresponded to the protein profiles. Our results indicate that testosterone withdrawal leads to inhibition of sperm thiol oxidation. Mol. Reprod. Dev. 47:295–301, 1997. © 1997 Wiley-Liss, Inc.     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse

Summary Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuramainidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

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.     

Clusterin (SGP-2) in epididymal luminal fluid and its association with epididymal spermatozoa in androgen-deprived rats.

Clusterin is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells and epididymal epithelium. The goal of this study was to determine the presence of clusterin in the luminal fluid of the cauda epididymides and its association with the membranes of developing spermatozoa in the presence and absence of androgen. We have previously demonstrated by two-dimensional (2-D) Western blot probing for clusterin that in epididymal fluid the amounts of clusterin were: caput greater than corpus greater than cauda. Luminal fluid from cauda epididymides was collected from control and orchiectomized rats (6 and 12 days) and orchiectomized animals that received testosterone implants. Equal volumes of fluid were analyzed by 2-D Western blot probing for clusterin. Following orchiectomy, there was an increase in clusterin in the luminal fluid after 6 days and maximal amount after 12 days compared with control cauda fluid. Orchiectomized animals which received testosterone treatment showed levels of clusterin comparable to that of controls. Serum clusterin was detected in fluid of orchiectomized animals with and without testosterone. Western blots of cauda sperm membrane extracts of control animals and orchiectomized animals treated with testosterone had a very low level of epididymal clusterin, whereas extracts collected from orchiectomized animals revealed high levels of clusterin. We suggest that, in the normal animal, clusterin is secreted into the lumen of the proximal epididymis where it binds to the sperm membrane. In the distal epididymis, clusterin dissociates from sperm and is processed (proteolysis/endocytosis). We hypothesize that, in the absence of androgen, the processing and regulation of clusterin is disrupted.     

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.     

Effects of PNU157706, a dual 5alpha-reductase inhibitor, on rat epididymal sperm maturation and fertility

Sperm entering the epididymis gain progressive motility and fertilizing ability in a process termed maturation. The functional dependence of the epididymis on dihydrotestosterone (DHT) is well established, yet few studies have examined the consequences on the epididymis of inhibiting DHT formation. We have shown that inhibition of both isoforms of 5alpha-reductase (types 1 and 2), the enzyme that converts testosterone to DHT, has pronounced effects on epididymal gene expression. In the present study, we investigate whether inhibiting 5alpha-reductase has consequences on epididymal sperm maturation. Rats were treated with vehicle or 10 mg/kg/day PNU157706, a dual-type inhibitor, for 28 days. Fertility and several key facets of sperm maturation were analyzed. Changes in sperm motility were assessed by computer-assisted sperm analysis (CASA). Changes in sperm morphology were assessed by CASA and electron microscopy. The motility of spermatozoa from the cauda epididymidis of treated animals showed a significant decrease in both the percentage of motile and progressively motile sperm as well as altered motion parameters. The morphology of cauda epididymal spermatozoa was also adversely affected by the treatment; the most prominent effect was a markedly elevated proportion of sperm that retained their cytoplasmic droplet. Matings with treated males resulted in fewer successful pregnancies and a higher rate of preimplantation loss. Progeny outcome was unaffected. The compromised sperm motility and morphology likely contribute to the subfertility of inhibitor-treated rats. Our results indicate a role for dual 5alpha-reductase inhibitors in further studies of epididymal physiology and as a potential component of a male contraceptive.     

Analysis of epididymal proteins during sexual maturation in male albino mice.

Androgen dependent epididymal proteins act as antigen to produce autoantibodies and affect normal fertility. In the present study, epididymal proteins were analyzed during the time of sexual maturation and their androgen dependency was studied in male albino mice. Epididymis of 21 days (Pre-pubertal), 45 days (Pubertal), 60 days (Post-pubertal), orchidectomized (15 days after surgery) and orchidectomized with testosterone-treated (15 days after treatment) mice were dissected out and analyzed. Caput, corpus and cauda epididymidis were separated and the protein extract was prepared with 0.1 M PBS for 10% SDS-PAGE analysis. Testosterone assay was performed in the experimental groups except the testosterone treated group. The electrophoretic analysis of proteins in caput, corpus and cauda epididymidis of orchidectomized animals showed the disappearance of several proteins as compared to the adult. However, the disappeared proteins started to reappear in testosterone treated animals. The results suggest that removal of testis depletes the testosterone level and causes significant alteration in epididymal proteins. These proteins need further investigation for the purpose of immunocontraception by using them as antigens.     

The effects of various steroids on testosterone metabolism by the sexually mature rabbit epididymis

We examined the influences of steroids present in the epididymis on androgen metabolism by epididymal tissue and on the binding of androgen metabolites to the epididymal androgen receptor in castrated adult rabbit epididymides under in vitro conditions. The conversion of [3H]testosterone to [3H]17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT) and to [3H]5 alpha-androstane-3 alpha (beta), 17 beta-diol was inhibited by unlabeled steroids in the following manner progesterone greater than testosterone greater than estradiol. Unlabeled 5 alpha-DHT did not inhibit [3H]testosterone metabolism indicating that product inhibition is not an important regulatory event. The antiandrogen cyproterone acetate did not inhibit the formation of 5 alpha-reduced metabolites of [3H]testosterone. All of the compounds used inhibited androgen binding to the classically defined cytoplasmic and nuclear androgen receptor.