As yet, there is little knowledge as to the possible side effects of vasectomy on the function of the epididymis. In rats and bulls, it has been reported that vasectomy causes a spermatocoele to develop at the site of ligation on the vas deferens or in the epididymis (Amann & Almquist, 1962; Igboeli & Rakha, 1970; Hooker & Gilmore, 1972); in other species, notably the rabbit, this does not occur (Macmillan, Desjardins, Kirton & Hafs, 1968; Paufler & Foote, 1969). Some reports have claimed that vasectomy causes atrophy of the testis and disruption of spermatogenesis (Laumas & Uniyal, 1967; Sacher & Schilling, 1973) but most workers agree that, at least in the early stages, vasectomy only affects the terminal parts of the epididymis. It is not known what influence this might have
Summary. Monoclonal antibodies to antigens located on surface and intracellular membranes of spermatozoa from the rat cauda epididymidis have been used as probes to assess the purity of putative plasma membrane fractions. Spermatozoa were demembranated by shearing forces generated on a vortex-mixer. Immunofluorescence and ultrastructural analysis of vortex-mixed spermatozoa showed that they were denuded of approximately 90% of surface membrane. Areas of acrosomal membranes were also removed. Crude plasma membranes were recovered in low-speed wash fluids and fractionated on a 13–23% Nycodenz density gradient. Three bands containing membrane vesicles were resolved. Absorption curves and direct binding assays using monoclonal antibodies specific for acrosomal membranes, mitochondrial membranes and fibrous sheath showed relatively strong binding to bands 1 and 2 but weak binding to band 3. Conversely a monoclonal antibody specific for a surface membrane antigen bound strongly to band 3 and weakly to bands 1 and 2. Identification on immunoblots of the antigens recognized by the monoclonal antibodies revealed that band 3 was positive for surface membrane antigens but gave no reaction for intracellular antigens. However, bands 1 and 2 were strongly positive for intracellular components. The results suggest that vortex-mixing is a simple and efficient means of removing the plasma membrane from spermatozoa and that a membrane fraction can be recovered from a Nycodenz density gradient that is enriched 40- to 50-fold in surface antigens.
R. C. Jones
Summary. Micropuncture samples of luminal fluid were collected from the rete testis and along the epididymis. Quantitative analyses showed that the ductuli efferentes reabsorb about half the protein leaving the testis. Considerable protein is secreted by the caput epididymidis (initial segment) and there is a net loss of protein from the corpus and cauda epididymidis.
Denatured, polyacrylamide gel electrophoresis showed that there are 5 proteins in rete testis fluid which are not present in blood (M r of 14 700, 22 800, 24 100, 43 200 and 44 800). One of these proteins (M r 14 700) is lost from plasma in the ductuli efferentes and 2 (M r 43 200 and 44 800) are lost in the corpus epididymidis. Twelve proteins appear in the epididymal plasma and are not present in rete testis fluid or blood: 6 appear in the caput epididymidis (M r 30 000, 31 000, 32 300, 17 400, 18 700 and 21 400), 3 in the corpus epididymidis (M r 12 800, 39 800 and 90 600) and 3 in the cauda epididymidis (M r 10 900, 56 300 and 63 000). A protein with the same molecular weight as a blood protein (149 500) accumulates in the corpus and cauda epididymidis.
None of the samples of luminal fluid contained particulate matter other than spermatozoa, indicating that the tammar is a useful animal for micropuncture studies.
R. C. Jones
Summary. The mean spermatocrit and sodium, potassium and protein concentrations of fluid collected from the rete testis of the elephant were similar to values described for the ram. Sperm maturation, as assessed by the location of the cytoplasmic droplet in the middle piece, occurred in the distal head and the isthmus of the epididymis (middle segment). Spermatocrit determinations indicated that 96% of the fluid leaving the testis was reabsorbed by the efferent ducts and proximal initial segment, and 53% of the remainder was reabsorbed in the more distal parts of the head of the epididymis (initial and proximal middle segments). Sodium was reabsorbed in the same concentration as luminal fluid and not in amounts equimolar with potassium. The potassium concentration increased from 12·1 mequiv./1 in rete testis plasma to 64·8 mequiv./1 in the proximal head of the epididymis. About two thirds of the protein in rete testis fluid (3·8 mg/ml) was reabsorbed by the efferent ducts and more was absorbed by the head of the epididymis.
R. C. JONES
The sequence of structural changes in the head of mammalian spermatozoa which have been associated with ageing or death (see review by Hancock, 1966) were determined in bull spermatozoa by Saacke & Marshall (1968). Bedford (1970) considered this acrosomal disintegration to be a `nonspecific degeneration' or `false' acrosomal reaction, quite different from the `true' acrosomal reaction (Barros, Bedford, Franklin & Austin, 1967) which involves vesiculation of the opposing acrosomal and plasma membranes. These reactions do not, however, account for all morphological modifications of the acrosome and the subject has therefore been examined further. This report summarizes the results of a number of studies, and records for boar spermatozoa the degenerative changes already described in bull spermatozoa by Saacke & Marshall (1968), which involved vacuolation of the acrosome. Another sequence of changes, not previously reported and which involves vesiculation of the acrosome, is also described.
Experiment 1 involved further examination of
R. Jones and T. Mann
Summary. We examined the damaging effects on spermatozoa of endogenous phospholipid peroxidation brought about by aerobic incubation at 37°C in the presence of 0·5 mM-ascorbic acid and 0·5 mM-FeSO4. As well as becoming immotile, such peroxidized spermatozoa also lost, through leakage, certain intracellular enzymes into the surrounding medium, on a scale resembling that produced by cold shocking nonperoxidized spermatozoa. Morphological observations revealed that peroxidation damaged the plasma membrane, particularly in the region of the acrosome. Further experiments showed that lipid peroxidation irreversibly abolished the fructolytic and respiratory activity of spermatozoa. The susceptibility of spermatozoa to peroxidation was greater when the cells were damaged before incubation with ascorbic acid and FeSO4. To some extent, peroxidation could be prevented, but not reversed, by the addition to sperm suspensions of dialysed egg yolk or dialysed bull seminal plasma. However, dialysed seminal plasma from ram, stallion or man had no protective effect.
R. Jones and T. Mann
Summary. The toxicity of unsaturated fatty acids towards spermatozoa was shown to be directly related to their degree of peroxidation. The toxicity was manifested by an immediate arrest of motility and irreversible loss of respiratory and fructolytic activity. Repeated washing of spermatozoa, or the addition of fructose, lactate, or ATP, failed to restore these functions. The structural damage incurred as a result of the fatty acid peroxides was particularly evident in the acrosomal region. Partial protection from the adverse effects of these peroxides was provided by prior treatment of spermatozoa with dialysed egg yolk or milk, but tocopherol, albumin and mercaptoethanol were ineffective. It is suggested that lipid peroxides or their degradation products, whether introduced exogenously or derived from the peroxidation of endogenous phospholipids in semen, constitute a potential hazard to the functional integrity of spermatozoa.
R. C. JONES
Spermatozoa of domestic animals may vary considerably in response to handling in the laboratory (Mann, 1964) and during preparation for artificial insemination (Jones, 1971a), but it is not known why such structurally similar cells should behave so differently. The plasma membranes of spermatozoa from the various species may differ in physical properties, but because spermatozoa are structurally unsuitable for tests which are performed on other cells, such as erythrocytes, this is not an easy hypothesis to test.
Jones (1971b) found that when boar semen was fixed in solutions of glutaraldehyde containing from 175 mm- to 100 mm-sodium cacodylate, the decrease in buffer concentration caused the plasma membranes of the spermatozoa to swell and break; around the head, where the response was greatest, the amount of swelling and incidence of breakage was inversely related to the buffer concentration of the fixative. As these methods may provide a measure
R. C. JONES
As a sample of semen contains spermatozoa of various types depending on their normality, stage of maturity and degree of cytolysis, a quantitative study of the structure of the sample must consider the frequency of the different morphological types in a representative sub-sample. The effects of treatments may be determined by comparing different sub-samples. Since the methods that have been described for preparing spermatozoa for electron microscopy (Birch-Andersen & Blom, 1963; Saacke & Almquist, 1964; Healey, 1969; Quinn, White & Cleland, 1969) have disadvantages which make them unsuitable for use in such a study, a number of different methods have been examined. The procedures described below were developed for speed and convenience. They have given consistent results when used to prepare samples of spermatozoa from various sources (e.g. epididymis, semen, diluted and stored semen) and from a variety of mammals including the domestic ungulates, carnivores and rodents. For electron microscopy,
D. Stevenson and A. R. Jones
Summary. The (S)-isomer of the male antifertility agent α-chlorohydrin was metabolized by mature boar spermatozoa in vitro to (S)-3-chlorolactaldehyde. This oxidative process, which did not occur when (R)-α-chlorohydrin was offered as a substrate, was catalysed by an NADP+-dependent dehydrogenase that converts glycerol to glyceraldehyde. (S)-3-chlorolactaldehyde, produced by this metabolic reaction or when added to suspensions of boar spermatozoa, was a specific inhibitor of glyceraldehyde 3-phosphate dehydrogenase as assessed by the accumulation of fructose 1,6-bisphosphate and the triosephosphates. When glycerol and (S)-α-chlorohydrin were added concomitantly to boar spermatozoa in vitro, the presence of glycerol decreased the degree of inhibition of glyceraldehyde 3-phosphate dehydrogenase. Extracts of glyceraldehyde 3-phosphate dehydrogenase that were obtained from boar spermatozoa incubated with (S)-α-chlorohydrin or (R,S)-3-chlorolactaldehyde showed significant reductions in their enzymic activity.