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.     

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.     

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.     

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.     

Gamma-glutamyl transpeptidase, glutathione, and L-glutamic acid in the rat epididymis during postnatal development

During postnatal development, gamma-glutamyl transpeptidase (gamma-GT), reduced glutathione (GSH), and L-glutamic acid (L-Glu) were assayed in the epididymides of rats at 5-day intervals between 10 and 60 days of age and compared to adult levels. gamma-GT activity (with gamma-glutamyl-p-nitroanilide as substrate) and L-Glu (nicotinamide adenine dinucleotide conversion-dependent assay) were measured photometrically, while GSH (o-phthalaldehyde reaction) was quantified with a fluorometric assay. In immature rats, the epididymal gamma-GT was very low but increased after 25 days of age in the caput and after 50 days of age in the cauda. The enzyme level in the epididymal caput was by far the highest in the adult rat reproductive tissues. The postnatal increase of gamma-GT in epididymal caput and cauda was associated with a decline of its substrate GSH and an accumulation of the product L-Glu. These observations provide evidence for the in vivo hydrolytic activity of gamma-GT and explain the high levels of L-Glu found in the epididymis of rats and other mammals.     

Enhancement of sperm transport through the rat epididymis after castration

Transport of spermatozoa through different regions of the epididymis has been followed by labelling testicular spermatozoa with [3H]thymidine in intact rats and in rats in which the efferent ducts were ligated or the testes were removed. In intact rats, the transit times of epididymal spermatozoa from the initial segment to the caput, from the caput to the corpus, and from the corpus to the cauda were 2, 4 and 2 days, respectively, giving a total transit time of 8 days. After bilateral castration, labelled spermatozoa were transferred from the initial segment into the proximal cauda by 2 days and appeared in the ductus deferens by 4 days. This effect was prevented by a daily subcutaneous injection of testosterone propionate (0.2 mg/kg). Bilateral efferent duct ligation had only a slight effect on the passage of epididymal spermatozoa. The results indicate that epididymal sperm transport is enhanced after androgen withdrawal.     

Alteration of free sulphydryl content of rat sperm heads by suppression of intratesticular testosterone

Subcutaneous injections of testosterone propionate to adult male rats at a dose of 2.5 or 10 mg/kg body weight, 3 times per week for 7 weeks, resulted in a 75% reduction in serum LH and more than 50% reduction in intratesticular testosterone concentration, but serum FSH levels remained unchanged. The free -SH content, measured as iodo[14C]acetamide binding, increased by 70-100% in testicular sperm heads after suppression of testicular testosterone, and by 25-30% in caput epididymal sperm heads but was decreased by 70-80% in cauda epididymal sperm heads. These results demonstrate an alteration in the oxidative state of sperm nuclear basic proteins, suggesting incomplete nuclear maturation. These changes may be specific for the suppression of intratesticular testosterone, thus illustrating the androgen dependency of sperm head maturation. The contrast effects noted between the iodo[14C]iodoacetamide binding by the caput and the cauda epididymal sperm heads indicate that testosterone propionate treatment may affect the mechanisms regulating the oxidation of the sulphydryl residues in sperm heads during epididymal transit. This alteration may not directly relate to the tissue androgen concentrations.     

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.     

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.     

Epididymal specificity and androgen regulation of rat EP2

In primates, expression of the EP2 gene is androgen-dependent and epididymis-specific. EP2 mRNA expression was investigated in caput, corpus, and cauda regions of rat epididymis and in 15 other rat tissues. Polymerase chain reaction and Northern analyses showed that rat EP2 is expressed predominantly in the proximal caput epididymidis. EP2 mRNA expression was determined in proximal epididymides from castrated, sham-operated, and efferent duct-ligated rats. In castrated rats, EP2 mRNA decreased to <10% of that in sham-operated rats between Days 3 and 4 postcastration, demonstrating the androgen dependence of EP2 expression. In epididymides ligated unilaterally at the efferent ducts, EP2 mRNA levels were approximately equal to those in the unligated contralateral epididymides or in sham-operated rats, indicating that EP2 expression does not depend on testicular factors. In bilaterally castrated rats, immediate and delayed testosterone replacement showed the dependence of EP2 expression on circulating androgens. Injection of testosterone propionate (TP) on Days 0, 1, 2, and 3 postcastration maintained EP2 mRNA levels approximately equal to those in sham-operated rats. Starting at Day 4 postcastration, daily injection of TP for 7 days restored EP2 mRNA to approximately normal levels. These data indicate for the rat that EP2 is expressed specifically in the proximal caput epididymidis and that its expression depends on circulating androgens but not on testicular factors.     

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

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

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.     

Thyroid epididymal relationship: II. Influence of hyperthyroidism on epididymal lipids

The influence of experimentally induced hyperthyroidism on the lipid composition of the caput and cauda epididymides has been studied in pubertal and adult rats. Thyroxine treatment did not alter the major lipid classes in the epididymis. However, regional and age-related fluctuations in the concentration of mono-, diand triacylglycerols have been observed. While the diacylglycerols increase, mono- and triacylglycerols were found to decrease, suggesting an inverse relationship between these fractions. Among the phospholipid fractions, phosphatidylcholine and phosphatidylethanolamine were reduced. The changes in epididymal lipid profiles give an indication that the epididymis may be yet another site responsible for fertility disturbances in hyperthyroid males. The withdrawal of thyroxine from hyperthyroid animals returned the epididymal lipid profiles to normal levels. This indicates that the effects of thyroxine in the epididymis are temporary and reversible following thyroxine withdrawal.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

The effect of ligation of cauda epididymidis and vasectomy on testicular function in the adult gerbil (Meriones hurrianae).

1. The effects of ligation of caput, cauda epididymides and vasectomy were studied in adult gerbils. 2. The operations were performed unilaterally, the testis and epididymides on the contralateral side serving as controls. 3. Ligation of cauda epididymides decrease testicular weight, whereas caput ligation did not change the testis weight. Accessory sex glands were reduced in size. 4. Ligation caused a drastic degeneration of the spermatogenic cells. There was a complete disruption of testicular function. Leydig cell hypertrophy was conspicuous. Caput and cauda ligation led to degenerative changes in the epididymides. Epididymal epithelium was regressed and the lumen was devoid of spermatozoa. 5. Caput and cauda ligation inhibited the synthesis of RNA, protein and sialic acid in testis and epididymides and depleted the fructose concentration in the seminal vesicle. 6. Vasectomy did not cause any alteration in sperm production during the 8 week period on the lighted side. The cytology and the biochemistry of the testis and epididymides appeared to be normal.     

The uptake of L-(methyl- 3 H) carnitine by the rat epididymis

The uptake of radioactivity by the epididymis and other tissues was measured following administration of L-[methyl-³H]carnitine to male rats. Rapid uptake occurred in both the caput and cauda epididymides. This radioactivity was shown to be present in carnitine and was located almost exclusively within the epididymal lumen.     

Impact of experimental diabetes and insulin replacement on epididymal secretory products and sperm maturation in albino rats

The present study is aimed to explore the impact of experimental diabetes and insulin replacement on epididymal secretory products, sperm count, motility, and fertilizing ability in albino rats. Prepubertal and adult male Wistar strain rats were made diabetic with a single intraperitoneal injection of streptozotocin (STZ), at 120 and 65 mg/kg body weight for prepubertal and adult rats, respectively. After 3 days of STZ administration, insulin was given to a group of diabetic rats at a dose of 3 U/100 g body weight, subcutaneously and killed after 20 days of treatment. STZ‐diabetes significantly reduced the epididymal tissue concentrations of testosterone, androgen‐binding protein, sialic acid, glycerylphosphoryl choline, and carnitine, suggesting its adverse effects on the secretory activity and concentrating capacity of epididymal epithelium. Impaired cauda epididymidal sperm motility and fertility (in vivo) of STZ‐diabetic rats imply the defective sperm maturation. Insulin replacement prevented these changes either partially or completely. From the above findings, it is evident that STZ‐diabetes has an adverse effect on sperm maturation, which may be due to the decrease in the bioavailability of testosterone and epididymal secretory products. J. Cell. Biochem. 108: 1094–1101, 2009. © 2009 Wiley‐Liss, Inc.     

Thyroid-epididymal relationship. I. Influence of hypothyroidism on epididymal lipids

The influence of thyroidectomy on the lipid composition of caput and cauda epididymides have been studied. The analysis was conducted in epididymal tissues free from fluids and sperm. A general tendency towards accumulation of epididymal lipids was observed in hypothyroid rats. Hypothyroidism also brought about a differential regional response, which may be age-related. The existence of a relationship between triacylglycerols, phospholipids and diacylglycerols has been suggested. Since immediate thyroxine replacement to thyroidectomised rats maintained epididymal lipids at control levels, it is concluded that hypothyroidism has a definite influence on the epididymal lipid composition.     

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.