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Infertility is induced in male rats by oral administration of 400 mg ornidazole kg−1body mass day−1within 10 days, without drastic effect on the motility of epididymal spermatozoa obtained after 15 days of treatment. Spermatozoa were recovered from the female tract 9 h after mating with males treated with ornidazole for 10 days to identify the cause of infertility. The number and motility of spermatozoa in the uterus indicated normal seminal deposition. Spermatozoa could pass the utero–tubal junction but total numbers of spermatozoa and their velocities in the oviductal isthmus were significantly lower than those recovered from females mated to fertile, control males fed vehicle alone. However, the number of spermatozoa recovered from the ampulla was not different from control and the number of spermatozoa that had penetrated the cumulus mass was lower and none of the eggs was fertilized by spermatozoa from rats treated with ornidazole. The ability of cauda epididymal spermatozoa from ornidazole-fed rats to penetrate viscous media in vitrowas lower than that of the controls. When incubated in media containing different concentrations of substrates and ions, a reduction in movement of spermatozoa was observed when glucose was the only exogenous substrate or after preincubation in substrate-free medium. These results indicate that the antifertility action of orally administered ornidazole occurs via damage to spermatozoa in the upper regions of the female tract, possibly reflecting a failure in capacitation as a result of reduced energy production.

Summary.When [3H]testosterone was infused into the general circulation of the rat, perfusion of a length of the cauda epididymidis (17 ± 1·0 (s.e.m.) cm, n= 36) with perfusates of varied composition revealed a low entry of radioactivity (1–10% plasma levels; 10 exps) with protein-free perfusates, and a greater entry (15–48%; 10 exps) when the perfusate contained bovine serum albumin (38 mg/ml). When the perfusate contained ovine or rat testicular fluid, or rat epididymal fluid at protein concentrations of 3 mg/ml or less, the entry of radioactivity into the epididymis was greater than when the perfusate contained 3 mg BSA/ml. The addition of ovine rete testis fluid protein (3 mg/ml) to BSA (38 mg/ml) in the perfusate increased the uptake of radioactivity (58–106%; 6 exps).Radioactivity in blood was principally associated with testosterone (90, 95% total blood activity, 2 rats), whereas both [3H]testosterone (37, 41% total perfusate activity) and [3H]dihydrotestosterone (42, 63% total perfusate activity) were present in BSA-containing perfusates. The proportion of dihydrotestosterone appeared to increase when the perfusate contained protein of testicular origin.

Summary.Controlled perfusion through the lumen of the distal cauda epididymidis in the anaesthetized rat has been explored as a means of examining physiological exchanges from blood across the epididymal epithelium. The mean length of the perfused, sperm-free, tubule was 14·5 cm (±1·5 s.e.m., n= 9). No cholesterol, protein or sialic acid was detected in the perfusate at flow rates exceeding 10 μl/min, but at rates of 0·4–1·2 μl/min, protein appeared at concentrations of 0·21–0·55 mg/ml (i.e. secretion rates of 0·21–0·83 μg/min; 3 rats). Glucose was detected at all perfusion rates (3–27 μl/min) at concentrations of 0·06–0·58 mm(0·8–6·8% blood levels).During intravenous infusions of 3H2O, radioactivity in the perfusate rapidly attained 87% blood plasma concentrations; no radioactivity was detected when carboxy-[14C]dextran or methoxy-[3H]inulin were infused. Radioactivity appeared in the epididymal perfusate to 1–7% of blood levels during intravenous infusions of d-[U-14C]glucose or 3-O-methyl[1-3H]glucose.This evidence suggests that the preparation is physiological and could be used to explore the dynamics of exchanges between blood and epididymis.

Summary.Inositol was detected by gas—liquid chromatography in fluid perfused at 1 · 3 μl/min through the lumen of the distal cauda epididymidis of anaesthetized rats. The concentration (247 μm) exceeded that in blood (48 μm) and the secretion rate was constant for 5 h. d-[3H]Myo-inositol infused for 3 h into the general circulation of rats (1 μCi/min) also appeared in fluid perfusing the lumen, whether or not 50 mm-inositol was present in the perfusing solution. No plateau of radioactivity was reached during infusion, and by the end of 3 h perfusate activity was 26% of that in blood. Calculation of the specific activity of inositol in perfusates and blood plasma suggested that blood was not the immediate source of luminal inositol, and that any endogenous pool of cyclitol has a turnover time of greater than 3 h. [3H]Myo-inositol perfused through the lumen was not absorbed by the tissue. These data suggest that the high concentration of inositol in epididymal fluid (49 mm) is derived in part by epididymal secretion from a pool that is only slowly replenished from blood, and maintained by the impermeability of the epithelium. Glucose also appeared in fluid perfused through the epididymal lumen, but its concentration (461 μm) was much less than in blood (8·5 mm), so this sugar may diffuse down a concentration gradient.

Summary.A computer-aided sperm analysis system was optimized for objective assessment of the movement characteristics of mature and immature rat spermatozoa by testing different settings. Measurements of straight line velocity of individual motile cells were validated by manual tracking with a digitizer. Better agreement between the two methods and better performance in distinguishing between mature and immature spermatozoa was obtained by reducing the tracking rate to increase the time of analysis. However, numbers of motile and immotile cells could not be determined accurately. Manual counting of videotaped images revealed no significant differences in percentage motility of spermatozoa from five epididymal regions. Caput spermatozoa were characterized by low straight-line (VSL) and averaged-path (VAP) velocities and low path straightness (STR), whereas mature cells displayed high VSL, VAP and STR. An increase in curvilinear velocity on maturation was less obvious. Spermatozoa in the proximal corpus epididymidis were heterogeneous in their acquisition of motility maturation and the uniformity of movement pattern achieved in the distal corpus and proximal cauda regions tended to decrease again in the distal cauda epididymidis. Such objective measurements of motility patterns will facilitate studies on the regulation of motility development upon sperm maturation.Keywords:epididymal spermatozoa; motility maturation; sperm analysis; computer-aid; rat

The chlortetracycline fluorescence assay was used to study the status of capacitation and the extent of induced acrosome reactions in cauda epididymidal spermatozoa from fertile and infertile rats fed, respectively, with vehicle or ornidazole (400 mg kg−1day−1) for 10 days. Uniform bright fluorescence over the whole head was classified as the uncapacitated pattern, whereas a postacrosomal dark band, and a uniformly weaker fluorescence over the acrosome, reflected patterns intermediate between the uncapacitated and acrosome-reacted states. Acrosome-reacted spermatozoa displayed a dark head but always retained fluorescence at their tip. There was no difference between experimental and control groups of rats with regard to the development of the chlortetracycline fluorescence patterns during incubation. Under basal incubation conditions, the acrosome reaction was slightly delayed in spermatozoa from ornidazole-treated animals. In contrast, more spermatozoa were acrosome reacted in this group after incubation for 5 h when the concentration of BSA was increased from 4 to 20 mg ml−1. The Ca2+-ionophore A23187 induced a similar stimulation of capacitation and acrosome reactions in spermatozoa from control and ornidazole-fed animals, but in the latter group A23187 caused strong immobilization of spermatozoa. In the capacitation medium containing 5 mmol lactate l−1and 5 mmol glucose l−1, the straight-line velocity of spermatozoa from ornidazole-treated rats was reduced by 50% compared with controls, irrespective of the concentration of BSA. Two glycolytic enzymes, triose phosphate isomerase and glyceraldehyde 3-phosphate dehydrogenase, displayed reduced activity (48% and 68% of controls, respectively) in cauda epididymidal spermatozoa from ornidazole-fed rats, whereas the activities of hexokinase and lactate dehydrogenase remained unchanged. This finding suggests that the fertility-compromising action of ornidazole is due to a disturbed glycolytic pathway.

The activity of epididymal α-glucosidase in adult rats was rapidly suppressed to histochemically undetectable levels within 2 days by the continuous release of the enzyme inhibitor castanospermine via a peritoneal osmotic pump at a rate of 100–200 nmol h−1. It was established that mating activities overnight depleted 72% of the spermatozoa in the distal cauda, which was replenished in 2 days, and that fertility began to decline 3 weeks after efferent duct ligation. Male rats of proven mating proficiency and fertility were treated with castanospermine, or buffered saline as control, for up to 30 days and enzyme inhibition was confirmed at the end of treatment by histochemistry. Fertility was normal at the first mating test on day 7, significantly decreased at the second mating on day 9, but recovered in a stepwise manner at subsequent matings on days 12 and 14. Delaying the third mating until day 25 did not sustain the transient subfertility. However, prolonging sperm storage in the distal cauda epididymides and preventing replenishment with freshly matured spermatozoa, by efferent duct ligation for 14 days performed on day 15 during castanospermine administration, caused a decrease in fertility and a change in the kinematics of epididymal spermatozoa of the castanospermine-treated group. In control rats, binding of epididymal spermatozoa to Vicia faba, a lectin specific for glucose and glucosamine, and mannose and mannosamine residues, decreased from the proximal caput to the distal corpus coincident with the increase in α-glucosidase activity on the epithelial brush border. Lectin binding then increased in the cauda where enzyme activity was absent. However, castanospermine treatment did not significantly alter this binding profile. The findings suggest that epididymal α-glucosidase does not play a crucial role in the development of sperm fertilizing capacity, but may be involved in the preparation of spermatozoa for storage.

Ornidazole (400 mg kg−1day−1) given by oral gavage rendered male rats infertile by 6.6 ± 0.7 days (mean ± sem, n= 9, range 3–10) after beginning the treatment and fertility returned within 5–10 days after treatment with ornidazole for 6–7 days. At 200 mg ornidazole kg−1day−1, fertility was reduced but total infertility was not achieved. No differences were found in the percentage motility of spermatozoa recovered from any region of the epididymides of ornidazole-treated rats compared with controls. However, computer aided sperm analysis revealed significantly lower straight-line and average path velocities in ornidazole-treated animals (400 mg kg−1day−1) for spermatozoa from the distal regions of the tract than for controls. Curvilinear velocity was significantly lower than that of controls in the distal corpus and cauda regions. The motility characteristics of spermatozoa from animals receiving 200 mg ornidazole kg−1day−1were lower than, but not significantly different from, motility in controls. There were no differences between the total protein, l-carnitine, glycerophosphocholine or total α-glucosidase content in epididymal homogenates from fertile control and infertile ornidazole-treated animals. Spermatozoa released from the cauda epididymidis of untreated rats into ornidazole solutions displayed no changes in the percentage motility up to 20 mmol l−1and were only depressed at 50 mmol l−1. All velocities revealed a biphasic response with an initial increase in motility and then inhibition at higher concentrations, but a significant difference from velocities in the absence of ornidazole was evident only for straight line velocity (VSL) at 50 mmol l−1. The rapidity of action in inducing infertility is compatible with post-testicular action and an action on the epididymis is suggested by the decline in motility parameters of luminal spermatozoa. The lack of effect on epididymal secretions in vivoand on sperm motility in vitro, except at very high doses, suggests that there may be a direct action of an ornidazole metabolite on epididymal spermatozoa.

Summary.Cells liberated by enzyme treatment of tubules dissected out of human epididymides obtained at castration for prostatic carcinoma were cultured for up to 42 days on permeable supports. Outgrowth and monolayer formation was unrelated to the age of the patient or his treatment with anti-androgens. The cells comprising the monolayer were cuboidal and shorter than those in situbut their ultrastructure was characteristic of epithelial cells. There was no evidence of smooth muscle cells or fibroblast overgrowth. The cells manifested fluid-phase and adsorptive endocytosis from both apical and baso-lateral aspects and released into the medium alkaline and acid phosphatases and N-acetylglucosaminidase.Keywords:man, epididymis; cell culture; ultrastructure; endocytosis; secretion

Summary.Using an assay for α-lactalbumin in which galactosyltransferase activity was stabilized and a tissue phosphatase inhibitor was present, no evidence was found for α-lactalbumin-like activity in rat epididymal tissue, epididymal fluids or medium from cultured epididymal epithelial cells with either glucose or N-acetylglucosamine as acceptor. However, when assay conditions were suboptimal, apparent transfer of radioactivity to both acceptors could be demonstrated in the epididymis and other tissues. In these assays the amount of α-lactalbumin registered was linearly correlated to the extent of stimulation of α-lactalbumin added exogenously to tissue extracts as internal standards. When rete testis fluid from rats was used as source of galactosyltransferase under suboptimal conditions, no transfer to glucose was demonstrable in epididymal fluid and an apparent decreased transfer to N-acetylglucosamine could be explained by increases in (pyro)phosphatase activity. Putative α-lactalbumin activity in the epididymis may be an artefact of unoptimized assays.Keywords:epididymis; mammary gland; α-lactalbumin; optimized assay; non-specific stimulation; rat

Summary.Unravelled tubules from the monkey caput and cauda epididymidis were perfused through the lumen in vitroduring immersion in an organ bath kept at scrotal temperature and containing [3H]carnitine and [14C]inulin. The specific transport of carnitine from the bath to the lumen was constant for 4 h and reached a steady-state value of about 90 pmol/30 min per cm perfused length in the cauda and about 30 pmol/ 30 min/cm in the caput. These regional variations in carnitine transport differ from those found in the rat epididymis but may be relevant to human epididymal physiology.

Summary.A technique for perfusing the lumen of rat epididymal tubules maintained in vitroshowed that [3H]inulin was largely excluded from the lumen of unravelled tubules from the cauda and tubules from the corpus if the connective tissue capsule was removed. The preparation transported [3H]inositol from the bath fluid for 3 h against a concentration gradient in both regions with activity rising (16–29% of bath fluid values) in the cauda and reaching a plateau (18%) in the corpus epididymidis. HPLC showed that radioactivity was solely associated with inositol and its movement to the lumen was reduced by raising inositol in the bath fluid from 50 μm(plasma levels) to 10 mm, but not affected by reducing the glucose concentration in the bath fluid or introducing physiological concentrations of inositol (30 mm) into the lumen. Secretion into the caudal lumen of unlabelled inositol measured by g.l.c. was maintained for 3 h at concentrations (300 μm) greater than those in the bath fluid and was not reduced when glucose or inositol were removed from the bath. In contrast, glucose was only detectable in the lumen when it was present in the bathing medium, reaching 1% of this concentration. Radioactivity appeared in the epididymal lumen reaching a plateau (19% of bath fluid values) in the corpus and cauda when [3H]glucose was added to the bath fluid, but no radiolabelled inositol was found in the lumen. We conclude that epididymal tissue is a major source of secreted inositol.

Summary.The availability of glycerol in the epididymal lumen, and the extent of its utilization by spermatozoa, was studied by measurement of the substrate concentration in epididymal fluid and its oxidation in vitro. The source of luminal glycerol was sought by examining its penetration through the epididymal epithelium of a sperm-free tubule by the technique of luminal perfusion during intravenous infusion of glycerol. [3H]Glycerol was rapidly metabolized to a volatile and mobile molecule that quickly equilibrated between blood plasma and the epididymal lumen and so could not be used to monitor transfer. When blood levels of glycerol were raised by infusion of large amounts of the unlabelled compound, glycerol was detected in epididymal perfusates and a positive linear correlation existed between the rate of secretion into the epididymal lumen and the blood plasma concentration, suggesting that passive diffusion across the epithelium from blood could occur. In normal rats, however, the concentration of glycerol in sperm-free epididymal fluid (1·15 mm) exceeded that in blood plasma (0·35 mm). Luminal glycerol is therefore thought to arise from degradation of epididymal lipid.