Metabolism of testosterone by tes epididymis and ventral prostate of rat and its inhibition by cyproterone acetate

The metabolism of ³H-testosterone by the epididymis and accessory organs of adult male rats exposed continuously to microdoses of cyproterone acetate from subcutaneous capsules were studied. The major metabolite of ³M-testosterone in the epididymis, vas deferens and ventral prostate of control rat was dihydrotestosterone while the formation of androstanediol by these tissues was low. The highest percentage of DHT was formed by the ventral prostate and cauda epididymis. In rats exposed to cyproterone acetate for four months, the conversion of testosterone to DHT was inhibited in all the tissues but maximally in the ventral prostate and cauda epididymis. In these rats, the secretory function of the ventral prostate was normal while that of the epididymis was markedly decreased. These data are discussed based on the differential thresholds of androgens required to regulate the functions of the accessory organs.     

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.     

Androgen-dependence of ornithine decarboxylase in the rat epididymis

Ornithine decarboxylase (ODC) activity was measured in epididymides of 45-day-old rats. Higher ODC activity was detected in the corpus and cauda than in the caput epididymidis. Bilateral castration for 7 days caused epididymal ODC to fall to undetectable values, whereas testosterone restored activity to normal values. The effect of the androgen was significantly inhibited by cyproterone acetate. The caput was more sensitive to the action of testosterone than were the corpus and caudal segments. Unilateral castration for 4 or 8 days did not affect ODC on the control or castrated side, but the activity fell in epididymides of both sides after removal of the remaining testis. These results show that epididymal ODC activity is androgen-dependent.     

Effects of sulphapyridine on sperm transport through the rat epididymis and contractility of the epididymal duct

This study was undertaken to investigate the effects of sulphapyridine on the transport of spermatozoa through different regions of the epididymis and on the contractility of the epididymal duct in the rat. Sperm transport was investigated by labelling testicular spermatozoa with [3H]thymidine and measuring intraluminal pressures of the epididymis by micropuncture, using a servo-nulling pressure transducer system. In control rats, the transit times of epididymal spermatozoa from the initial segment to the caput, from the caput to the proximal cauda, and from the proximal cauda to the distal cauda were 2, 6 and 3 days, respectively, giving a total transit time of 11 days. The total transit time was shortened to 8 days after treatment with sulphapyridine at a dosage of 450 mg kg-1 for 38-52 days. The rate of sperm transport was most affected in the caput epididymidis. Measurements of intraluminal pressures showed that sulphapyridine had no effect on spontaneous contractions in any regions of the epididymis. However, the frequency of contraction of the corpus and cauda epididymides in response to administration of 10 micrograms noradrenaline kg-1 in the sulphapyridine-treated rats was significantly higher (P < 0.05) than it was in the controls. Methacholine, at a dose of 20 micrograms kg-1, produced a smaller increase in basal pressure in the caput epididymidis of sulphapyridine-treated rats (P < 0.05) compared with controls. The results led to the conclusion that sulphapyridine increases the rate of sperm transport from the caput through the cauda epididymidis, in part, by changes in the responsiveness of the epididymis to the autonomic nervous system.     

Effect of antiandrogens on some key enzymes of glycolysis in epididymis and ventral prostate of rat

Effect of three antiandrogens: cyproterone acetate (5 mg/day, sc), flutamide (5 mg/day, sc) and STS-557 (5 mg/day, po) and an estrogen, estradiol dipropionate (5 micrograms/day, sc) on some key enzymes of carbohydrate metabolism was investigated in adult rat epididymis and ventral prostate. Antiandrogens were administered for 21 days and estrogen for 14 days. All of them caused a significant decrease in the weight of epididymis, seminal vesicles and ventral prostate. A significant decrease in the specific activities of enzymes (hexokinase, phosphofructokinase, aldolase, glyceraldehyde phosphate dehydrogenase, pyruvate kinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) occurred only in the organs of estrogen treated rats; activities of some of the enzymes were lowered also in the prostate of STS-557 treated rats. Flutamide and cyproterone acetate were ineffective in this regard. The possible factors responsible for the ineffectiveness of synthetic antiandrogens in influencing epididymal metabolism are discussed.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Effect of corticosterone on epididymal lipids in pubertal rat

Serum corticosterone excess was induced by the administration of corticosterone acetate to adrenal intact rats. Different lipid classes were studied in unwashed and washed (epididymal sperm and fluid free) caput and cauda epididymides. The unwashed caput epididymidis registered a significant decrease in total lipid, cholesterol and phospholipid while total glyceride glycerol and its fractions were not altered after corticosterone treatment. Among phospholipid fractions phosphatidyl inositol, choline and ethanolamine showed a significant decrease. Unlike the unwashed caput epididymidis, the washed caput region recorded a marked increase in total lipid, glyceride glycerol and its fractions. However, total lipid in the washed cauda region significantly increased and the increase was mainly due to triacyl glycerol. Though the phospholipid fractions phosphatidyl choline and ethanolamine showed an increase, the total phospholipid was not altered significantly. Serum testosterone and prolactin registered a significant decrease while gonadotropins were unaltered. On the withdrawal of corticosterone treatment, all the lipid classes turned to normalcy along with serum testosterone and prolactin. It is concluded that corticosterone excess favours lipid accumulation in the sperm free epididymal tissue and its influence on epididymis is region specific and reversible.     

Resorption versus secretion in the rat epididymis

Micropuncture samples were taken from the rete testis, caput epididymidis and cauda epididymidis of anaesthetized adult rats and assayed for total protein, sodium and potassium concentrations. Intraluminal sperm concentrations were determined and used to calculate the amount of fluid resorbed from the efferent duct and epididymal lumen. It was demonstrated that large amounts of protein (30.2 mg/ml cauda volume) and sodium (241.8 mequiv./l) and smaller amounts of potassium (19.4 mequiv./l) are resorbed from the rat epididymal lumen between the caput and corpus epididymidis. This occurs despite increases in intraluminal concentrations of protein (from 22 to 28 mg/ml) and potassium (from 16 to 50 mequiv./l). Resorption is an important aspect of epididymal control of the intraluminal environment.     

Changes in the composition of the excurrent duct system of the rat testis during postnatal development.

The gross composition of the testicular excurrent duct system of the rat was examined and compared along the length of the duct and with samples of testis, bladder and liver. Changes in composition with age were examined by analysing tissue from animals at postnatal ages of 19, 36, 48, 60, 90 and 120 days. In adult animals, testicular tissue was characterized by having the lowest dry weight, accompanied by low levels of total protein, lipid, RNA and glycogen; DNA, phospholipids and sialic acid were at levels similar to other tissues. A high proportion of the total protein was soluble. The ductuli efferentes plus initial segment of the epididymis were characterized by high levels of total lipid. The caput epididymidis contained a low level of total protein but a high level of acid-soluble phosphorus. The cauda epididymidis had a low dry weight and low levels of total protein, soluble protein, and lipid, but high levels of acid-soluble phosphorus, DNA and sialic acid. The ductus deferens contained small amounts of RNA and DNA but had a high dry weight, high total protein, soluble protein and glycogen. Several trends were apparent with increasing age. Dry weight increased in the ductuli efferentes plus initial segment, whilst total protein decreased in the caput and cauda epididymidis. Total lipid increased in the ductuli efferentes plus initial segment and acid-soluble phosphorus and sialic acid increased in all other segments of the excurrent duct system. In all segments the content of RNA and DNA decreased as the animals matured. The concentration of calcium and magnesium in the excurrent duct system was not significantly different from those levels found in the liver. High levels of spermine and spermidine were confirmed in the prostate, and were also detected in the testis, caput epididymidis and cauda epididymidis, but at a much lower concentration.     

The lipid content of epididymal spermatozoa of Rattus norvegicus.

1. Seven major lipids of rat spermatozoa from the caput, corpus and cauda epididymidis were separated and quantitated by TLC. 2. Spermatozoa from the caput epididymidis had a significantly greater (P less than 0.05) content of phospholipid, cholesterol, cholesterol ester and free fatty acid than those from the cauda epididymidis. 3. Spermatozoa from the corpus epididymidis had a significantly greater (P less than 0.05) content of monoglyceride than those from the caput epididymidis and a greater content of phospholipid, cholesterol, free fatty acids and monoglyceride than those from the cauda epididymidis.     

Distribution of carnitine and acylcarnitine in the hamster epididymis and in epididymal spermatozoa during maturation

The highest levels of carnitine and acylcarnitine were found in the cauda epididymidis, and spermatozoa from the cauda contained greater amounts of total carnitine (free carnitine plus acylcarnitine) than those removed from the corpus or caput epididymidis. Spermatozoa from the distal cauda contained significantly greater amounts of both free and total carnitine than those removed from the proximal cauda epididymidis. The acylcarnitine:carnitine ratio was 1.7 and 0.37 in caput and cauda spermatozoa, respectively and 1.7 and 1.3 in caput and cauda fluid, respectively. It is suggested that the accumulation of carnitine is involved in sperm maturation and that acylcarnitine serves as an energy substrate for epididymal spermatozoa.     

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.     

Ultrastructural and biochemical changes in epididymis and vas deferens of gossypol treated rats

Adult male Wister rats when administered with 15 mg/kg body weight/day of gossypol acetic acid proved to be sterile by 10 weeks of treatment. The weight of the whole epididymis did not deviate from the controls but when the caput, corpus and cauda epididymidis were considered separately, the cauda epididymidis weight was significantly reduced. The major changes were observed in the motor apparatus of the sperm. The most common defects in the sperm were the vacuolization and complete degeneration of the midpiece mitochondria and plasma membrane. The total LDH activity of caput and cauda epididymidis were within the range of control values. Sialic acid levels of the epididymis were not affected after the treatment. These results suggest a more proximal site of action of the drug than at the epididymal level.     

Identification of androgen-induced proteins in human epididymis

Androgenic stimulation (0.1 microM testosterone or 5 alpha-dihydrotestosterone in the medium) of cultured human epididymal tubules increased the synthesis of five proteins, identified by their mobility relative to albumin (Ra) in polyacrylamide gels as 0.31, 0.43, 0.67, 0.81 and 1.01. This effect was inhibited by the simultaneous presence of 10 microM cyproterone acetate in the medium. The caput epididymidis was the most active region in the production of these proteins and a gradient of decreasing activity was found in successive segments. The appearance of induced proteins in the culture medium suggests their secretory nature, while some data indicate that androgens may also affect the secretory process. Bands corresponding to Ra 0.31, 0.43, 0.68 and 1.01 were found in caput and cauda epididymidis fluids, while bands coincident with Ra 0.31 and 0.43 were consistently found in extracts (0.5 M NaCl) of caudal spermatozoa. Preliminary determinations of molecular weight and isoelectric point for the different bands yielded the following results: Ra 0.31, 38,000 and 5.8; Ra 0.43, 21,000 and 6.2; Ra 0.68, 69,000 and 5.1; Ra 0.81, 13,900 and 6.8; and Ra 1.01, 29,000 and 6.8.     

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.     

Comparison between epididymosomes collected in the intraluminal compartment of the bovine caput and cauda epididymidis

During their transit along the epididymidis, mammalian spermatozoa acquire new proteins involved in the acquisition of male gamete fertilizing ability. We previously described membranous vesicles called epididymosomes, which are secreted in an apocrine manner by the epididymal epithelium. Some selected proteins associated with epididymosomes are transferred to spermatozoa during epididymal transit. The present study compared epididymosomes collected from caput epididymal fluid with vesicles from the cauda epididymidis in the bull. Two-dimensional gel electrophoresis revealed major differences in protein composition of epididymosomes isolated from the caput and cauda epididymidis. LC-QToF analysis of major protein spots as well as Western blot analysis confirmed the differences in proteins associated with these two populations of epididymosomes. Biotinylated proteins associated with caput and cauda epididymosomes also revealed differences. When incubated with caput epididymal spermatozoa, epididymosomes prepared from these two segments transferred different protein patterns. By contrast, cauda epididymosomes transferred the same pattern of proteins to spermatozoa from the caput and cauda epididymidis. Transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa decreased in a dose-dependent manner when biotinylated epididymosomes were diluted with unbiotinylated vesicles. Caput epididymosomes added in excess were unable to inhibit transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa. Following transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa, addition of unbiotinylated cauda epididymosomes was unable to displace already transferred biotinylated proteins. These results established that epididymosomes from caput and cauda epididymidis have different protein composition and interact differently with maturing spermatozoa.     

Effect of diabetes mellitus on epididymal enzymes of adult rats.

Diabetes mellitus caused significant reduction in serum testosterone and accessory sex glands weight. The sperm content of epididymal regions also decreased. Among the epididymal regions, the cauda epididymidal tissue alone showed significant reduction in Na(+)-K+ ATPase activity. However, Mg2+ ATPase activity was lowered in caput epididymidis only. Specific activity of Ca2+ ATPase significantly decreased in caput and cauda epididymides. All three ATPases decreased significantly in caput epididymidal spermatozoa leaving cauda epididymidal spermatozoa unaffected. Specific activity of alkaline phosphatase was suppressed in caput epididymidis and in the spermatozoa collected from caput and cauda epididymides, while the acid phosphatase was unaffected. In general, the results are suggestive of definite influence of diabetes on epididymal phosphatases which is region specific. Diabetes induced decrease in phosphatases may have an impact on secretory and absorptive functions of epididymis and thus on sperm maturation.     

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.     

Ultrastructural localization of PGP 9.5 and ubiquitin immunoreactivities in rat ductus epididymidis epithelium

Summary The distribution of protein gene product 9.5 (PGP) and ubiquitin in the spermatozoa and epithelial cells in the different regions of the rat duetus epididymidis (proximal caput, distal caput, corpus and cauda) was studied by Western blotting analyses and electron microscopical immunogold labelling. Western blotting analyses showed that the PGP immunoreactive band was very intense in the caput and cauda epididymidis and almost irrelevant in the corpus, while the ubiquitin immunoreactive band was intense in the distal caput and cauda. No ubiquitin immunoreactive band was observed in the proximal caput and only a very weak band was seen in the corpus. The results of electron microscopical immunogold labelling varied from one epididymal region to another. The proximal caput epididymidis presented immunoreaction to PGP in the rough endoplasmic reticulum, cytosol, mitochondria and microvilli of most principal cells, and in the cytosol, rough endoplasmic reticulum and mitochondria of most basal cells. No ubiquitin immunoreaction was observed in this epididymal region. In the distal caput epididymidis, PGP immunoreactivity was detected in some principal and basal cells in the same intracellular locations as described in the proximal caput. In this region, ubiquitin immunoreactivity appears in the apical cytosol and mitochondria of principal cells. The corpus epididymidis showed no immunoreaction to PGP or ubiquitin. In the cauda epididymidis, immunostaining to PGP was observed in most clear cells and in isolated principal cells. The intracellular location of PGP in both cell types was the cytosol, mitochondria and microvilli. Ubiquitin immunoreactivity was detected in the perinuclear cytosol and mitochondria — but not in the digestive vacuoles — of some clear cells. Scanty ubiquitin immunolabelling was also found in the microvilli, cytosol and mitochondria of some principal cells. The head of the spermatozoa present in the ductal lumen in all epididymal regions immunoreacted intensely to PGP. Ubiquitin was detected in the intermediate piece and residual cytoplasm of intraluminal spermatozoa present in the corpus and cauda epididymidis. These findings suggest that a non-ubiquitinated PGP irnrnunoreactive protein is secreted by the principal cells in caput epididymidis and binds the spermatozoon heads. It is possible that the clear cells of the cauda epididymidis secrete the ubiquitin that binds to spermatozoon tail.