Summary. Hamster spermatozoa collected from the caput and cauda epididymidis were washed, diluted in a medium containing Triton X-100 to dissolve the cell membrane and reactivated with various concentrations of MgSO4 and ATP. Stepwise increase in the concentrations of free ATP4− from 0·08 to 1·1 mm at constant concentrations of MgATP2− caused a dose-dependent delay of reactivation but the maximal percentage of motile spermatozoa was inhibited only at 1·1 mm. The inhibitory effect on caput spermatozoa was greater than that on cauda spermatozoa. When concentrations of ATP4− were fixed at 0·2 mm, 2·9 mm-MgATP2− suppressed the reactivation of cauda spermatozoa. When compared to 0·9 mm-MgATP2−, reactivation of caput spermatozoa was delayed at 1·9 mm and almost completely blocked by 2·9 mm-MgATP2−. Inhibition of cauda sperm reactivation by ATP4− and MgATP2− were both prevented by the presence of trypsin (50 ng/ml). Incubation of cauda spermatozoa in the reactivation medium for 1 and 2 min before the addition of ATP progressively reduced the inhibitory effect of ATP4−; inhibition by MgATP2− was reduced to a lesser extent. Addition of 100 μm-cyclic AMP to the medium abolished the delay of reactivation by ATP4− but not that by MgATP2−. Before reactivation occurred, inhibitory concentrations of ATP4− and MgATP2− both induced large-angle coiling of sperm tails but in opposite direction to each other with reference to the asymmetric sperm head. The results suggest that free-ATP4− and superoptimal concentrations of MgATP2− inhibit flagellar movement by different mechanisms.
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.
P. Y. D. Wong and C. H. Yeung
Daily injections of α-chlorohydrin (3-chloro-1,2-propanediol) at a dose of 7–8 mg/kg produce infertility in male rats (Ericsson, 1970; Ericsson & Baker, 1970). The mechanism of action of this compound is still unknown but previous studies have indicated that it has at least two sites of action. When given at high doses, α-chlorohydrin produces characteristic lesions in the caput epididymidis, leading to an occlusion of the epididymal duct and subsequent degeneration of the germinal epithelium (Ericsson, 1970; Hoffer, Hamilton & Fawcett, 1973; Cooper, Jones & Jackson, 1974). At lower doses, there is a functional interference with spermatozoa, causing a reversible phase of sterility (Coppola, 1969; Gunn, Gould & Anderson, 1969; Vickery, Erickson & Bennett, 1974), and the metabolism of testicular and epididymal spermatozoa is known to be affected (Mohri, Suter, Brown-Woodman, White & Ridley, 1975; Edwards, Dacheux & Waites, 1976).
P. Y. D. Wong and C. H. Yeung
Summary. A technique is described for measurement of rate of fluid reabsorption in a segment of the rat cauda epididymidis in vitro. The basal rate was 2·63 ± 0·22 μl/cm2/30 min and was dependent on temperature and inhibited by 2,4-dinitrophenol, suggesting that fluid reabsorption is an energy-dependent process. About 50% of the fluid reabsorption was dependent on intraluminal sodium ions. This Na+-dependent component was inhibited by addition of amiloride to the intraluminal fluid and ouabain to the peritubular fluid.
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.
T. G. Cooper, C. H. Yeung, R. Meyer and H. Schulze
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 situ but 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
C Callies, T G Cooper and C H Yeung
The nature of the membrane channels mediating water transport in murine spermatozoa adjusting to anisotonic conditions was investigated. The volume of spermatozoa subjected to physiologically relevant hypotonic conditions either simultaneously, or after isotonic pre-incubation, with putative water transport inhibitors was monitored. Experiments in which quinine prevented osmolyte efflux, and thus regulatory volume decrease (RVD), revealed whether water influx or efflux was being inhibited. There was no evidence that sodium-dependent solute transporters or facilitative glucose transporters were involved in water transport during RVD of murine spermatozoa since phloretin, cytochalasin B and phloridzin had no effect on volume regulation. However, there was evidence that Hg2 +- and Ag+-sensitive channels were involved in water transport and the possibility that they include aquaporin 8 is discussed. Toxic effects of these heavy metals were ruled out by evidence that mitochondrial poisons had no such effect on volume regulation.
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, J P Barfield and C H Yeung
The permeability of murine cauda epididymidal spermatozoa was determined from the swelling caused by penetrating agents at isotonicity, which lies between 422 and 530 mmol/kg. Spermatozoa were permeable to a range of solutes with size <200 Da. Relative entry rates of cryoprotective agents (CPAs) were ethylene glycol≈DMSO>propane-1,2-diol>glycerol>propane-1,3-diol. More polar compounds including major epididymal secretions were impermeant. None of the compounds entered spermatozoa through quinine-sensitive channels; rather, quinine increased the size of solute-swollen spermatozoa, suggesting that regulatory volume decrease and osmolyte loss occurred under these conditions. Volume responses to lowered osmolality revealed a greater volume-regulating ability of spermatozoa from the B6D2F1 strain than the C57BL6 strain. As the former strain displays better post-thaw fertility, their spermatozoa may have greater osmolyte loads enabling them to cope better with osmotic stress. Inadequate volume regulation, due to CPA-induced osmolyte loss, may affect post-thaw fertility. Knowing the permeability towards cryoprotectants will help to make a better choice of CPAs that are less damaging to sperm during cryopreservation.
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.