Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling.

The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membrane-permeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of ram sperm mitochondrial function by quantitative determination of sperm rhodamine 123 accumulation.

A simple procedure is described for determining the functional state of ram sperm mitochondria by quantitative measurement of sperm rhodamine 123 (R 123) accumulation. Sperm were incubated with 1 microgram/ml R 123, and the accumulated R 123 was measured fluorimetrically after release from washed sperm by detergent lysis. Ram sperm R 123 uptake was maximal after 30 min of incubation and responded to changes in both sperm (P < 0.01) and R 123 (P < 0.01) concentration. There was a linear relationship (r = 0.98) between R 123 uptake and the proportion of cold-shocked sperm present in a sperm sample. R 123 uptake was unaffected by 20 mM 2-deoxyglucose or by 10 mM malonate (the latter being sufficient to reduce O2 uptake; P < 0.01). R 123 accumulation in ram sperm was reduced by 6 mg/ml sodium pentobarbitone (P < 0.05), by 1 microM 2,4-dinitrophenol (P < 0.01), and by 0.05% Triton X-100 (P < 0.01). It is concluded that quantitative estimation of R 123 uptake complements oxygen uptake in detecting mitochondrial dysfunction in ram sperm. While it is largely unaffected by inhibition of glycolysis, and is less sensitive than oxygen uptake to trichloroacetic acid cycle inhibition, R 123 uptake is sensitive to factors directly reducing the mitochondrial membrane potential of ram sperm. It may therefore by useful in the evaluation of the effects of such membrane-mediated injuries as cold shock and freezing damage on ram sperm mitochondria.

Effects of the lipid peroxidation product (E)-4-hydroxy-2-nonenal on ram sperm function.

(E)-4-hydroxy-2-nonenal (HNE) is a lipid peroxide end-product which exerts powerful biological effects in a variety of cell and tissue systems. The effects of exogenous HNE on ram spermatozoa were examined in vitro. HNE inhibited the motility of diluted ram spermatozoa in a dose-dependent (100-400 mumol l-1) manner (P < 0.05). The extent of motility loss varied with sperm concentration as well as with HNE concentration, and was manifested as a progressive decrease in mean sperm velocity. The suppressive effect of 250-500 mmol HNE l-1 on the motility of reactivated ram sperm models (P < 0.05) was prevented by the addition of 1 mmol reduced glutathione l-1 to the reactivation medium, suggesting that HNE inhibits ram sperm motility via oxidation of sulfhydryl groups in the axoneme. Oxygen uptake by ram spermatozoa was inhibited (P < 0.05) by the addition of 100 or 200 mumol HNE l-1. Glucose utilization was maintained in the presence of 200 mumol HNE l-1, suggesting that fructolysis was unaffected by HNE. As was the case with motility, the inhibition of oxidative metabolism by HNE was not reversed by washing the spermatozoa. The activity of ram sperm acrosomal enzymes released by cold shock, as measured by hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), was reduced in the presence of 100 mumol HNE l-1 (P < 0.05). No evidence was found of disruption of the acrosomal outer membrane or the sperm plasma membrane as a result of exposure to HNE.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review.

When sperm of the ram, bull, boar and stallion are cold-shocked by rapid cooling to near freezing point, motility and metabolic activity are irreversibly depressed and the acrosome and plasma membrane disrupted. Ram sperm become susceptible to cold shock in the proximal corpus region of the epididymis when the cytoplasmic droplet has moved backwards to the distal portion of the sperm midpiece. The membrane constituents phospholipids and cholesterol are important in cold shock which causes loss of lipid from sperm. The susceptibility of sperm to cold shock is linked with a high ratio of unsaturated:saturated fatty acids in the phospholipids and a low cholesterol content. The high unsaturated fatty acid content of sperm also makes them susceptible to damage from peroxidation which adversely affects motility, metabolism, ultrastructure and fertility. Hydroxynonenal, a product of fatty acid peroxidation, depresses the motility and oxygen uptake of ram sperm in vitro and may react with the -SH groups of the axonemal microtubules. High calcium concentrations in the external medium may decrease the motility and metabolism of sperm and 'calcium intoxication' may be a factor in cold shock. Lowering the environmental temperature increases calcium uptake by sperm and the effect is aggravated if the rate of cooling is rapid. Phospholipids, particularly those in egg yolk, protect sperm to some extent from cold shock and also prevent increased calcium flux into the sperm. Suggestions are made for increasing the life span of sperm during preservation and microencapsulation by adding agents that may stabilize membranes, counter peroxidation and decrease calcium uptake.

Frequent semen collection and sperm reserves of the male Angora goat (Capra hircus).

Semen was collected from six mature and sexually rested Angora bucks at one-hour intervals five times a day on each of 5 consecutive days in the breeding season. There was a marked decline in semen volume (P less than 0.001), sperm concentration (P less than 0.05) and number of spermatozoa (P less than 0.001) on consecutive days. Successive ejaculates within days differed only in number of spermatozoa (P less than 0.001). The following year at the beginning of the breeding season, the weights of testes and epididymides and the reserves of spermatozoa in these parts were examined after slaughter of the six bucks. The mean number of spermatozoa in the paired testes, capita, corpora and caudae of the epididymides were (22.8 +/- 1.24) x 10(9), (9.4 +/- 1.19) x 10(9), (3.4 +/- 0.22) x 10(9) and (35.0 +/- 2.21) x 10(9), respectively. Epididymal reserves of spermatozoa were correlated with testicular weight (r = 0.50, P = 0.01) and number of spermatozoa in the testes (r = 0.42, P = 0.07), but not with epididymal weight. The daily production of spermatozoa per animal in the breeding season was estimated to be 4.0-6.4 x 10(9).

Biochemical changes in bull spermatozoa during capacitation in vitro.

To evaluate the metabolic changes of bull spermatozoa (SPZ) during capacitation in vitro, SPZ were incubated for 0, 5 or 10 hours in the presence (co-culture) and absence (control) of monolayers of bovine oviduct epithelial cells, which promote capacitation-like changes in vitro. There was little change in the oxygen uptake of the SPZ after 5 hours, but after 10 hours there was a decrease, particularly in the co-cultured sample. After 5 hours there was little change in the cyclic adenosine monophosphate (cAMP) concentration of the co-culture or control SPZ, but by 10 hours the levels of cAMP decreased in both the co-cultured and control SPZ (P=0.06). The concentration of adenosine triphosphate (ATP) was somewhat decreased after 5 hours in both the co-cultured and control SPZ and the percentage of decline was much higher after 10 hours. Overall, there was no significant change in oxygen uptake or cAMP and ATP levels specifically associated with capacitation of bull SPZ.

Electron microscopic observations on the effect of gossypol on rat cauda epididymis.

Gossypol administered orally to male rats at a daily dose of 20 mg/kg body weight for 63 days caused hypertrophy of the cauda epididymal epithelium, with more than fourfold increase in height of the cells. The principal cells lost most of their microvilli and formed apical blebs which appeared to produce the dense secretory material which was found in the lumen. Less dramatic but similar changes also occurred after 9 days on the same regimen, with the height of the epithelium doubling. However after 19 days on this regimen, with the height of the epithelium doubling. However after 19 days on this regimen, the epithelium looked fairly normal apart from a maintained hypertrophy. As reported in other studies, the cauda epididymal sperm were severely damaged and immotile; many were decapitated and the oxygen uptake was low. Ultrastructural defects were abnormal or absent mitochondria, absence of plasma membranes and axonemal components and accessory fibres.

Studies of chemical components of Angora goat seminal plasma.

Ejaculates were collected by artificial vagina from 11 Angora goats, once or twice weekly, between April and July in two successive years. The mean +/- SEM ejaculate volumes each year were 0.8 +/- 0.30 and 0.98 +/- 0.52 ml; the sperm concentrations were 3.33 +/- 0.49 and 2.94 +/- 0.45 x 10(9)/ml, and the pH values were 7.01 +/- 0.34 and 7.20 +/- 0.17. The concentrations (mg/100ml) of fructose (875 +/- 97) and lactic acid (73 +/- 17) in goat seminal plasma were sufficiently high to be important substrates for maintenance of sperm motility. Only trace amounts of glucose were present in seminal plasma. The glycerylphosphorylcholine (GPC) concentration of seminal plasma (809 +/- 154 mg 100 ml ) was correlated with whole semen sperm concentration (P < 0.001), indicating that GPC is of epididymal origin. Goat sperm are not likely to utilize GPC as a substrate and its metabolizable derivatives, glycerophosphate (3.3 +/- 1.1 mg 100 ml ) and glycerol (1.8 +/- 1.0 mg 100 ml ), were not present in sufficiently high concentrations to be significant as energy sources for the sperm. The mean concentration of citric acid was 331 mg 100 ml seminal plasma. Colored semen was consistently produced by eight bucks, and in yellow, light yellow and white ejaculates, the seminal plasma riboflavin (mug/ml) concentrations were 5.38 +/- 2.89, 3.09 +/- 0.85 and 1.73 +/- 0.88, respectively. This suggests that the color is due to riboflavin, which is probably produced by the vesicular glands since the concentration of riboflavin in the seminal plasma was correlated with fructose and citric acid levels.

Heavy metals and human spermatozoa. III. The toxicity of copper ions for spermatozoa.

The dissolution of copper ions from copper metal into a saline medium in vitro was quantified using a colourimetric assay. The presence of spermatozoa enhanced this dissolution and increasing the protein content of the medium further increased the rate of dissolution. Approximately 17% of the copper released was either tightly bound to the spermatozoa or was within the cell and could not be removed by repeated washing. Once spermatozoa were immobilized, they could not be revived by washing and repeated changes of medium, by addition of copper specific-chelating agent or by extensive dialysis. When the toxicity to spermatozoa of cuprous and cupric ions was compared with copper metal, it could be shown that the quantity of cupric ions required (0.2-0.4 mg/ml) was in excess of the total quantity of copper released into solution. The quantity of cuprous ion required (0.08-0.16 mg/ml) to exert similar toxic effects to copper, was within the range of copper released from the metal. Under the conditions of this study, it is possible that cuprous ion would be oxidised to the cupric form generating free radicals in the process. It is not known whether the toxic effect is due to the cuprous ion, per se, or to radicals generated in its oxidation. Increasing the protein content of the medium to levels similar to low (8 mg/ml) and high (64 mg/ml) values reported in human uterine fluid increased the dissolution rate of copper but also offered some protection against the toxic effects of copper metal and cuprous and cupric ions.

Studies of the mechanism of action of gossypol as a male antifertility agent.

Gossypol administered orally to male rats at a daily dose of 20 mg/kg body weight for 62 days caused infertility. There were changes in the epididymal epithelium and the sperm were severely damaged and immotile. The sperm head was often detached; other defects were abnormal mitochondria, absence of plasma membranes and axonemal and accessory fibres and a lower oxygen uptake. To study the effect of gossypol on the motor apparatus of sperm, ram sperm were demembranated with the detergent, Triton-X-100. Such sperm models can normally be reactivated with ATP but gossypol (2.5-12.5 microM) decreased reactivation and must have a direct effect on the axoneme. Gossypol also inhibited ram sperm adenyl cyclase which is essential for maintaining high levels of cAMP in sperm and, in turn, motility. Ram sperm adenyl cyclase required Mn2+ for activity and high Mn2+ concentrations protected the enzyme from gossypol inhibition. Electron spin resonance studies proved that gossypol chelated Mn2+ with the formation of a 2:1 complex.

Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2.

The highly selective fluorescent Ca2+ indicator 'quin 2' has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, 'quin 2/AM', which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean +/- S.E.M.) indicated a normal cytoplasmic free-calcium concentration, [Ca2+]i, of 193 nM +/- 0.2 (n = 10) for ejaculated ram sperm, 175 nM +/- 3.9 (n = 10) for cauda epididymal boar sperm and 105 nM +/- 10 (n = 10) for the caput sperm. After cold shock ejaculated ram and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm. Sodium azide, propranolol and caffeine did not affect the [Ca2+]i of ram and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rotenone and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of [Ca2+]i, most likely resulting from release of mitochondrial calcium. The increased [Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in [Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4-DNP and CCCP had no effect on [Ca2+ )i. Addition of filipin to ram and boar sperm resulted in a large increase in [Ca2+]i but addition of filipin to ionophore-treated sperm caused [Ca2+]i to fall well below control levels.

Control of ram sperm adenylate cyclase by divalent cations.

The adenylate cyclase activity of ram sperm increased on freeze-thawing and the enzyme was stable at 0 degrees C. Its activity was stimulated by Mn2+, Zn2+, Co2+, Mg2+ and Ca2+ in descending order of activity. The enzyme was insensitive to fluoride when Mn2+ concentration was in excess. Mn2+-stimulated enzyme activity was decreased by the simultaneous addition of Co2+, or Cd2+, or Ni2+, and particularly Cu2+. Sulfhydryl compounds (viz. dithiothreitol, glutathione, dithiocarbamate, 2-mercaptoethanol, ergothioneine and cysteine) and chelating agents (viz. D-penicillamine and 8-hydroxyquinoline) were effective, to varying degrees, in overcoming the inhibition by Cu2+. Ca2+ augmented the stimulatory effect of Mg2+, Co2+, Zn2+ and Mn2+ on enzyme activity.

Occurrence of unusual molecular species of sphingomyelin containing 28-34-carbon polyenoic fatty acids in ram spermatozoa.

The high levels of very long chain fatty acids found in ram spermatozoa are located almost exclusively in one of two separable species of sphingomyelin. Mass spectral analysis, including fast atom bombardment of the purified sphingomyelin, has shown the fatty acids to have a carbon chain length of between 28 and 34, with between four and six double bonds, and to belong predominantly to the n-3 series.

Susceptibility of epididymal boar sperm to cold shock and protective action of phosphatidylcholine.

Rapidly cooling (cold shocking) washed cauda boar sperm irreversibly reduced motility and respiration and greatly increased the uptake of 45Ca2+; the plasma membranes were removed and the acrosomes detached from nuclei. The motility, respiration, and calcium uptake of the less mature caput sperm were largely unaffected; and there was little damage to the ultrastructure. This indicates that boar sperm becomes less resistant to cold shock as they mature in the epididymis. The oxygen uptake, glucose breakdown, and lactic acid production of control caput sperm was less than that of cauda sperm. This suggest that the maturation of sperm in the epididymis of the boar involves an increase in both the glycolytic and oxidative phases of glucose metabolism. The presence of 2.0 mg/ml phosphatidylcholine (lecithin) in the medium prevented ultrastructural damage to cauda sperm on cold shock, and motility and respiration were maintained at levels similar to those of control sperm. Although the presence of phospholipid reduced the large calcium influx following cold shock, it was still greater that that of control sperm. The "protective" effect against cold shock was not maintained after rewashing the sperm free of phosphatidylcholine prior to cold shock, indicating a fairly "loose" interaction of the phospholipid with boar sperm membranes that was easily disrupted.

Calcium uptake, respiration, and ultrastructure of sperm exposed to ionophore A23187.

Calcium accumulation by ram sperm in the presence of 3.0 microM ionophore A23187 increased greatly when the pH of the medium was raised from 7.5 to 8.5. The increase in calcium uptake was remarkable between pH 7.5 and 8.0 and was paralleled by increased oxygen consumption. There was extensive vesiculation of the plasma membrane and membranes of the acrosome and postnuclear cap of ram sperm incubated with the ionophore and calcium at pH 8.0. The mitochondria of the midpiece contained pale and expanded cristae similar to ATP-deprived mitochondria in the "condensed" configuration. Such changes were not observed in ram sperm incubated under similar conditions at pH 7.0. The ionophore-induced calcium and oxygen uptake of cauda and caput epididymal boar sperm also increased with increased pH, but the effect commenced at a lower pH. Control cauda epididymal boar sperm (i.e., without ionophore) had higher oxygen uptake, glucose breakdown, and lactic acid production than caput sperm. The influence of pH on the operation of the membrane calcium pumps between ram boar sperm indicates species differences.

Effect of male reproductive tract fluids and proteins on the metabolism and motility of ram spermatozoa.

Cauda epididymal fluid (CEF) greatly stimulated the oxygen uptake of washed ejaculated ram spermatozoa; the effect was evident within 1 h and persisted over the 8 h of the experiment. The stimulus was comparable to that produced by 10 mM glucose and the effects were not additive, that is, CEF and CEF plus glucose elicited about the same oxygen uptake. This suggested that CEF suppressed the oxidation of added glucose and this was confirmed by measuring the amount of glucose oxidized in the presence and absence of CEF. Cauda epididymal fluid improved the motility of washed ram spermatozoa but it was somewhat less than that produced by glucose and usually only became evident after about 6 h or incubation. Electrophoretic analysis of cauda epididymal spermatozoa incubated in radioiodinated CEF showed that these cells absorb fluid components in the zone 82 to 56 kD. However, a molecular weight fraction less than 5 kD obtained by passing CEF through a Sephadex G-25 column, was not effective in stimulating the oxygen uptake of ram spermatozoa. The effects of CEF on the metabolism of ram spermatozoa could be mimicked by 2.5-4.0 mg/ml bovine serum albumin (BSA). Stimulation of oxygen uptake was apparent within 1 h and persisted over the 8 h of the experiment. As with CEF, stimulation of oxygen uptake by BAS was less than with 10 mM glucose but the effects were not additive. Like CEF, BSA reduced the amount of glucose oxidized. Bovine serum albumin also improved the motility of ram spermatozoa over 8 h. After passage through Sephadex G-25 to remove any low molecular weight contaminants (less than 5 kD), BSA was still effective in stimulating the oxygen uptake of spermatozoa over 4 h. Ram blood plasma and especially ram seminal plasma were also effective after passage through the Sephadex. Human serum albumin (HSA) was as effective as BSA in stimulating the oxygen uptake of ram spermatozoa but defatting decreases its effectiveness. The motility score of the spermatozoa was also adversely affected by this treatment. It is concluded that the stimulating effects of CEF and of the other fluids and proteins are due to substrates, at least some of which are present as or associated with macromolecules.

Mechanism of inhibition of fructolysis in ram spermatozoa by chlorinated antifertility agents.

When ejaculated ram spermatozoa were incubated with (S)-alpha-chlorohydrin (up to 0.25 mM) the oxidative metabolism of fructose to carbon dioxide was inhibited in a concentration-dependent manner. This appears to be due to inhibition of glyceraldehyde-3-phosphate dehydrogenase which leads to the accumulation of fructose-1,6-bisphosphate, dihydroxyacetone phosphate and, to a lesser extent, glyceraldehyde-3-phosphate. (R)-alpha-Chlorohydrin (10 mM) had no significant effect on the oxidative metabolism of fructose. The inhibition of the oxidative metabolism of fructose by (S)-alpha-chlorohydrin (0.1 mM) was not immediate but was detected after incubation for 15 min. By contrast, (R,S)-3-chlorolactaldehyde (5 mM) caused an immediate inhibition of this metabolic pathway. 1-Chloro-3-hydroxyacetone (0.5 mM) immediately decreased the oxidative metabolism of fructose which resulted in the accumulation of key fructolytic intermediates in a manner comparable to that produced by (S)-alpha-chlorohydrin. At a concentration of 20 mM, 6-chloro-6-deoxyglucose had no significant effect on the metabolic activity of ram spermatozoa. We suggest that the anti-fructolytic actions of (S)-alpha-chlorohydrin and 1-chloro-3-hydroxyacetone are mediated via a common metabolite, (S)-3-chlorolactaldehyde, and that the inactivity of 6-chloro-6-deoxyglucose is due to the inability of ram spermatozoa to metabolise this chlorinated sugar to (S)-3-chlorolactaldehyde.