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.
M. Hölpert and T. G. Cooper
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
T. G. Cooper and G. M. H. Waites
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.
T. G. Cooper and G. M. H. Waites
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.
C. H. Yeung and T. G. Cooper
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.
T. G. Cooper and D. E. Brooks
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.
C H Yeung and T G Cooper
AQP11 is one of the latest aquaporin (AQP) family members found, which differs from the other AQPs by its intracellular localisation and unusual water pore nucleotides with unclear function. Despite the highest mRNA expression among organs having been reported in the testis, the testicular molecule has not been studied in detail. Immunohistochemistry of rat adult testis localised AQP11 to the elongated spermatids (ES) and no other cell types except residual bodies inside Sertoli cells. It was absent from early ES at least until stage 13, and after a first diffuse appearance in the caudal cytoplasm became concentrated in intracellular organelles by stage 17, was strongest in vesicles in the anterior cytoplasm at the final ES stages and appeared in residual bodies. Staining was detected on the distal quarter of the sperm tail only immediately before spermiation. A similar localisation was found in the mouse and developmental profiles for both the open reading frame mRNA and protein expression in 8–50 dpp testis pinpointed its first appearance coinciding with late stage ES. Sequencing of PCR products of testicular Aqp11 containing the open reading frames confirmed a full match with GenBank databases for rat, mouse and human. Western blotting revealed two or more molecular forms with the 26/27 kDa species dominating in the rat/mouse testis and the 33/34 kDa form selectively allocated to the spermatozoa. In view of intracellular vacuolation leading to polycystic kidney in Aqp11-null mice, a possible role of testicular AQP11 in the recycling of surplus cytoplasmic components of the ES and sustaining Sertoli cell capacity in the support of spermatogenesis was discussed.
G. Oberländer, C. H. Yeung and T. G. Cooper
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−1 day−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−1 and 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.
T. G. Cooper, C. H. Yeung and G. F. Weinbauer
Summary. Unravelled tubules from the monkey caput and cauda epididymidis were perfused through the lumen in vitro during 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.
C. H. Yeung, G. Oberländer and T. G. Cooper
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