Epididymal compounds and their influence on the metabolism and survival of spermatozoa

Epididymal fluid, which is derived from testicular fluid, contains several unusual compounds. Little information is available on the composition of the testicular fluid of primates, but the fluid of the ram, bull, boar, and rat contains high concentrations of inositol and certain amino acids. Analyses have been made of epididymal fluid collected from the cauda epididymis of the Rhesus monkey and several nonprimate species (e.g., ram, bull, dog, stallion, rabbit, guinea pig, rat, and hamster), but similar information on the human is lacking. Cauda epididymal fluid appears to be similar in composition from one mammalian species to another. However, the epididymal plasma differs considerably from blood, lymph, and other extracellular fluids. The environment of spermatozoa in the epididymis is, therefore, highly specialized, and presumably in some way contributes to the prolonged survival of spermatozoa in this organ, and provides substrates for the metabolism of the spermatozoa. The chief characteristics of the cauda epididymal plasma are the low concentration of inorganic ions and the high levels of several unusual organic constituents namely, glycerylphosphorylcholine, carnitine, sialic acid, amino acids, glycosidases, and phosphatases. At least one antifertility compound, namely, orally administered α-chlorohydrin, appears to be concentrated in the epididymis. Studies on laboratory animals, domestic species, and man, suggest that it inhibits enzymes of the glycyolytic pathway in spermatozoa, and this may be the basis for its antifertility activity.     

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.     

α‐Lipoic acid inhibits testicular and epididymal oxidative damage and improves fertility efficacy in arsenic‐intoxicated rats

The present study evaluates the protective effect of α‐lipoic acid (LA) against arsenic‐induced testicular and epididymal oxidative damage in rats. Arsenic caused significant reduction in the reproductive organ weights, serum testosterone levels, testicular daily sperm count, epididymal sperm count, sperm motility, sperm viability, and sperm membrane integrity. Significant reduction in the activity levels of superoxide dismutase, catalase, and glutathione levels with a concomitant increase in the lipid peroxidation and protein carbonyl content in the testis and the cauda epididymis of arsenic‐exposed rats. Arsenic intoxication also enhanced the testicular caspase‐3 mRNA levels, disorganization of testicular and cauda epididymal architecture as well as increased arsenic content in the testis and the cauda epididymis of rats. Arsenic exposure also deteriorated fertility ability in male rats over controls. Conversely, α‐LA negated the testicular and cauda epididymal oxidative stress and restored the male reproductive health in arsenic‐exposed rats.     

Effects of intravasal nylon thread on the spermatozoa & reproductive organs in rats.

An investigation was undertaken in rats to study the effects of intr avasal thread (IVT) on the spermatozoa in the vas deferens and reproduct ive organs at various intervals after IVT insertion. The quantity of sperm was slightly reduced and motility was greatly reduced in the distal portion of the vas. The percentage of head and tail separation of sperm in the distal vas decreased with time. The quantity of sperm always remained the same in the cauda epididymis although the percentage of motile sperm decreased at 1 and 6 months, but not at 9 months, after IVT insertion. Following IVT insertion there was insignificant change in the weight of the testis, epididymis, ventral prostate, and seminal vesicles and alkaline phosphatase activity in the ventral prostate. Although cause and significance of these findings are unclear, the sialic level in the epididymis was significantly reduced in all groups bearing IVT. The presence of IVT apparently causes a change to occur in the epididymis, but it is unknown whether this affects sperm maturation.     

Epididymal response to exogenous testosterone in rats sterilized neonatally by estrogen.

Male rats treated with estradiol dipropionate (250 mug) by subcutaneous injection on Day 5 of postnatal life failed to show maturation changes in the testis and epididymis at puberty. Histological examination at 60 days revealed inhibition of spermatogenesis at primary spermatocyte stage and atrophic Leydig cells. Epididymis presented infantile features and there was a marked reduction in the secretory activity as revealed by lowered concentration of sialic acid and glycerylphosphorylcholine. Testosterone therapy (1 mg/day) for a period 45 days prior to autopsy at 60 days failed to induce completion of spermatogenesis but restored the structural and functional integrity of the epididymis.     

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.     

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.     

Effect of 17 alpha-cyanomethyl-17 beta-hydroxy-estra-4, 9-dien-3-one on reproductive organs of the male laboratory mouse

The effect of subcutaneous administration (10, 15 and 20 mg/kg body weight/day, for 21 days; and 20 mg/kg body weight/day, for 28 days) of 17 alpha-cyanomethyl-17 beta-hydroxy- estra-4, 9-dien-3-one (STS 557) on the male reproductive organs of the Parkes strain mouse was investigated. The effect of the treatment on the testis was not uniform; both regressed and normal seminiferous tubules were observed in the same section of the organ. Furthermore, the histological changes observed in the seminiferous tubules in testes of STS 557--treated mice were not different in different dosage groups. In general, in moderately affected seminiferous tubules, the germinal epithelium was thin and consisted of Sertoli cells, spermatogonia, spermatocytes and spermatids; such tubules showed presence of many vacuoles in the epithelium. In severe cases, the tubules had collapsed and were lined by mainly Sertoli cells, spermatogonia and spermatocytes. The treatment also caused marked depression in motility and concentration of spermatozoa in cauda epididymidis, weight of accessory sex glands and in the levels of sialic acid and fructose in the epididymis and seminal vesicle, respectively. By 56 days of drug withdrawal, the alterations induced in the reproductive organs returned to control levels, suggesting that STS 557 treatment induces reversible alterations in the male reproductive organs of Parkes strain mouse.     

The effects of alpha-chlorohydrin on the composition of rat and rabbit epididymal plasma: a possible explanation of species difference.

The relationship between the antifertility effect of alpha-chlorohydrin and changes in composition of luminal plasma from the cauda epididymidis of rats and rabbits has been investigated. At each dose regimen studied, the fertilizing capacity of rats treated with alpha-chlorohydrin was reduced to zero. The levels of sodium, potassium, glycerylphosphorylcholine (GPC), acid phosphatase and alkaline phosphatase in epididymal plasma were not markedly affected by drug treatment. The most noticeable change was a considerable increase in the concentration of lactic dehydrogenase (LDH) at all dose levels and of glutamic-oxaloacetic transaminase (GOT) after 7 days of treatment with 8 and 16 mg/kg. The effect of cold shock on the composition of epididymal plasma showed that LDH and GOT are, at least in part, derived from spermatozoa. In contrast, alpha-chlorohydrin did not have an antifertility action in the rabbit, and the only notable change in the compositon of epididymal plasma was an increase in the level of GPC. These results provide evidence that, in the rat, alpha-chlorohydrin or a metabolite primarily exerts its antifertility effect by a direct action on the spermatozoa, whilst in the rabbit a barrier may exist to the entrance of the drug into the lumen of the epididymal duct.     

Histochemical analysis of sialic acids in the epididymis of the rat

 A variety of sialic acids contained in the rat epididymis were histochemically examined by means of lectin and pre-lectin methods by light microscopy. Epididymides from adult male Sprague-Dawley rats were fixed in Bouin’s fluid and routinely embedded in paraffin wax. Hydrated sections were subjected either to the lectin methods using biotinylated Limax flavus, Sambucus nigra, Sambucus sieboldiana or Maackia amurensis lectins or to the selective periodate oxidation–phenylhydrazine–thiocarbohydrazide–silver protein–physical development technique with or without saponification. The present results revealed that principal cells in the initial segment and caput contain sialic acid linked to α2,6-galactose/N-acetylgalactosamine, whereas those in the corpus and cauda include the sialic acidα2,3-galactose sequence. Narrow and clear cells involve all the types of sialic acids examined. Basal and halo cells mainly contain sialic acidα2,3-galactose. 8- And/or 9-O-acetylated sialic acids were predominantly distributed in principal cells of the initial segment and proximal caput. These findings are taken to indicate that various sialic acids in the epididymis could participate in different physiological functions characteristic of the regions in this organ. Accepted: 10 November 1997     

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.     

Changes in the lipid content of boar sperm plasma membranes during epididymal maturation

Plasma membranes of boar sperm from caput, corpus and cauda of the epididymis were purified by differential- and sucrose-density equilibrium centrifugation and were found to yield a single band at a density of 1.13 g/cm3. This fraction was enriched in acid and alkaline phosphatase, 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas it contained minimal amounts of hyaluronidase and N-acetylglucosaminidase and no succinic acid dehydrogenase activities. The plasma membrane of caput, corpus and cauda sperm had the same phospholipid/protein and cholesterol/phospholipid ratios but yielded different amounts of protein and individual lipid classes. Several changes in the plasma membrane were observed during transit of sperm through the epididymis. Within the phospholipid class a decrease in the percentage of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol was detected accompanied by an increase in amount of phosphatidylcholine, sphingomyelin and polyphosphoinositides. In the other lipid classes there was a decrease in the amount of free fatty acid and the major glycolipid. The amount of cholesterol decreased, while the amount of desmosterol and cholesterol sulfate increased. There was an increase in the amount of diacylglycerol. In addition, the changes in the fatty acid composition of the total membrane lipid and each phospholipid were determined. The above changes in the lipid composition of the plasma membrane during epididymal maturation may help to explain the decreased resistance to cold shock and changes in membrane fluidity of sperm during transit in the epididymis.     

Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethanesulfonic acid-deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (approximately 44,000). Keratanase, an endo-beta-galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti-proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000-Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in surface ATPase of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis were found to have a lower activity of the surface ATPase than the spermatozoa from the caput region. The enzyme from spermatozoa of both regions had the same Michaelis constant (Km) for ATP of 5 X 10(-4) M. It was partly inhibited by ouabain and fluoride, but strongly inhibited by Cu2+, Zn2+,p-chloromercuribenzoate, 8-anilino-1-naphthalenesulphonate Triton X-100, Lubrol-PX, urea, guanidine hydrochloride, sodium dodecyl sulphate and glycerylphosphorylcholine. The enzyme of the spermatozoa from the cauda epididymidis was more sensitive to inhibition by ouabain and fluoride but less sensitive to inhibition by Cu2+ than that of the cells form the caput region. The Arrhenius plot of the temperature dependence of enzymatic activity varied for the cells from the caput and cauda epididymidis. The differences in the enzyme properties of spermatozoa from the two regions of the epididymis suggested that the decline in the activity during epididymal maturation may reflect changes in the lipids and sulphydryl groups of the sperm membrane.     

Effect of gossypol on pituitary reproductive axis: ultrastructural and biochemical studies

Oral administration of gossypol induced sterility in male rats by 10 weeks, at a dose of 15 mg/kg body weight/day. The pituitary FSH gonadotroph cells showed dilated endoplasmic reticulum and accumulation of secretory granules in the cytoplasm. LH cells were degranulated. The Leydig cells showed enhanced synthetic activity. There was no change in testis weight and testicular RNA, lipids and cholesterol in the treated group while significant increase was observed in DNA content. Testicular sialic acid content decreased significantly over controls. The Sertoli cells, spermatogonia, spermatocytes and early spermatids were not affected after the treatment. The weights of prostate, seminal vesicle were recorded normal and there were no ultrastructural variations. The levels of acid and alkaline phosphatase and RNA in prostatic tissue were insignificant as compared with controls. However, DNA content of prostate gland showed a significant increase. Sialic acid of seminal vesicle + coagulating gland were within the control range. A marked reduction in fructose values from the same organ was noted.     

Inhibition of alkaline phosphatase by sialic acid.

The interaction of human organ alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) with sugars was studied. Hexosamines, N-acetylneuraminic acid (NANA or sialic acid), N-acetylmuramic acid and N-acetylglycolylneuraminic acid inhibited human organ alkaline phosphatase activities. Of these, sialic acid was the most effective inhibitor. The pH profiles for the enzymes in the absence and presence of sialic acid were similar. The sialic acid - enzyme complex was more heat stable than the free enzyme between 20 and 45 degrees C. Lineweaver-Burk plots of 1/v versus 1/S at various concentrations of sialic acid showed intersecting straight lines indicating that the mechanism of inhibition was a mixed type. The Ki value obtained from the plots of 1/v versus the square of sialic acid concentration was 0.07 mM for the hepatic, sialidase-treated hepatic, and intestinal alkaline phosphatases. The respective Hill coefficients varied somewhat with the alkaline phosphatase isoenzyme. Hyperbolic curves were obtained when the percentage of remaining activity was plotted against the substrate concentration at different concentrations of sialic acid. The Hill coefficient was lowered in the presence of sialic acid. The sialidase-treated hepatic enzymes used gave the most effective conversion. Partial denaturation of the enzyme with urea, or pronase digestion had a little if any effect on the sialic acid inhibition with constant time.     

Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit

Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda.     

Tissue and cell specificity of immobilin biosynthesis

The mechanisms for the initiation of sperm motility have been poorly understood until recently. Immobilin is a novel mucin glycoprotein of high molecular weight found in the cauda epididymis of the rat that, at concentrations equivalent to those found in native cauda epididymal fluid, reversibly inhibits sperm motility. In this study, immobilin was purified from rat cauda epididymal fluid to apparent homogeneity and used to generate polyclonal antibody in rabbits. The antibody was characterized by immunoblotting, and immunofluorescence was used to localize immobilin in paraffin sections of components of the reproductive system of adult male rats. Immobilin was not detectable in the efferent duct and was first detectable in the apical portion of some epithelial cells of the initial segment of the caput epididymis. Immobilin was detectable intracellularly only in cells of the caput epididymis. In the corpus and cauda epididymis immobilin was detectable only in the lumen of the tubules. Immunoprecipitation of immobilin radiolabeled in vitro confirmed that immobilin biosynthesis in the adult rat is restricted to the caput epididymis. Principal cells in the caput epididymis synthesize immobilin and secrete it into the lumen of the tubules to travel with the sperm into the cauda.     

Effects of short-term treatment of solasodine on cauda epididymis in dogs

Oral administration (80 mg/kg body wt/day for 30 days) of solasodine (extracted and isolated from the berries of the Solanum xanthocarpum) to intact dogs significantly decreased the epithelial cell height of cauda epididymides. The cells became atrophic and the lumen was devoid of spermatozoa. Castration followed by the adminstration of solasodine further reduced the epithelial cell height in comparison to castrated controls. Concurrent treatment of solasodine along with testosterone propionate was unable to restore the normal epithelial lumen parameters. Total protein, sialic acid, glycogen and acid phosphatase activities were significantly reduced in solasodine treated cauda epididymides. These result suggest antiandrogenic potency of solasodine.