Effects of temperature upon capacitation of guinea pig spermatozoa

Spermatogenesis in many mammalian species requires a temperature a few degrees below body core temperature. Upon ascent through the male tract and deposition in the female tract, the temperature of spermatozoa is increased to body core temperature. This report investigates the effects of temperatures above or below normal body core temperature, which is also the usual temperature of in vitro gamete incubations and fertilization, upon sperm acrosome reacting ability and fertility. Epididymal guinea pig spermatozoa were preincubated in a Ca2+-free medium at temperatures of 15 degrees C, 25 degrees C, 37 degrees C, or 44 degrees C for increasing periods of time. At 15 degrees C or 25 degrees C, no or very few spermatozoa acquired the ability to acrosome react upon exposure to Ca2+ even after 18 hr of culture or warming up to 37 degrees C. A known stimulator of acrosome-reacting ability, lysophosphatidylcholine, was ineffective in promoting acrosome-reacting ability in spermatozoa incubated at 15 degrees C or 25 degrees C. At 37 degrees C the percentage of acrosome reaction increased steadily over time, reaching about 65% after 18 hr. At 44 degrees C the time course of acquisition of acrosome-reacting ability was greatly accelerated with a percentage at 2 hr comparable to that achieved at 37 degrees C only after 18 hr of preincubation. This effect of incubation at 44 degrees C could be reversed by cooling the spermatozoa to 37 degrees C before they were exposed to Ca2+. Spermatozoa induced to undergo the acrosome reaction after preincubation at 44 degrees C were fully capable of fertilizing intact guinea pig eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic changes of red cell membrane thiol groups followed by bimane fluorescent labeling

Monobromobimane labels red cell membrane protein thiol groups; bands exhibit fluorescence after sodium dodecyl sulfate acrylamide gel electrophoresis and correspond to almost all of those staining with Coomassie blue. The response of membrane protein thiol groups to oxidative challenge and the dynamics of recovery of the thiol groups may be followed. Diminished labeling is found after oxidation with diamide, with both intrachain and interchain disulfide bond formation demonstrated by sodium dodecyl sulfate acrylamide gel electrophoresis. Regeneration of thiol groups under physiological conditions (incubation with glucose) after a moderate degree of diamide oxidation is shown to be complete (with respect to thiol group content and degree and distribution of bimane label) in normal human red blood cell membranes. Even after oxidation of almost half of the membrane protein thiol groups (maximum degree of oxidation achieved), regeneration of thiol groups is almost complete; a minor fraction resides in the form of disulfide-linked high molecular weight proteins (demonstrated by the electrophoretic profile) which may be reduced completely with dithiothreitol.Bimane fluorescent labeling provides a convenient and sensitive method for following membrane thiol group status under physiological conditions.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Immunocytochemical alterations in the intra-acrosomal antigen MN7 during epididymal maturation of guinea pig spermatozoa

We have previously shown that a 90-kDa intra-acrosomal antigen, MN7, is restricted to the anterior acrosomal region of mouse, rat, and hamster spermatozoa. The present study has examined the localization and the behavior of MN7 during sperm maturation in the epididymis of the guinea pig by immunoelectron microscopy. MN7 showed not only a specific localization in the apical segment of the guinea pig sperm acrosome, but also a distinct alteration during maturation, as follows. MN7 was exclusively found both at the dorsal matrix and on the outer acrosome membrane (OAM)/matrix-associated materials in the apical segment. MN7 was initially distributed throughout the electron-lucent dorsal matrix in immature sperm but, during maturation, became more restricted to the spherical bodies within the electron-lucent area. MN7 on OAM/matrix-associated materials was first distributed along the ventral margin and the small area posterior to the dorsal matrix but, during maturation, disappeared from the ventral margin and became restricted to the dorsal region. These results indicate that MN7 is a good tool for studying the stepwise maturation of epididymal spermatozoa.     

Glucose effect on respiration: possible mechanism for capacitation in guinea pig spermatozoa.

Guinea pig sperm respiration was determined in minimal capacitation medium (MCM) with different energy sources. The ZO2 observed for spermatozoa suspended in media containing pyruvate and lactate was 35.7 +/- 5.9, pyruvate alone, 27.9 +/- 3.8 and D-glucose alone 3.4 +/- 1.1. When D-glucose was added to spermatozoa rapidly respiring in media containing pyruvate as the only exogenous energy source, an immediate suppression in respiration was observed. Further reduction was caused by continued addition of D-glucose. Fructose and mannose also produced a suppression in respiratory rate. However, lactose, fucose, sucrose, L-glucose, and galactose did not alter the respiratory rate. The suppression of respiration by metabolizable sugars is paralleled by a suppression of acrosome reaction in guinea pig spermatozoa. The possibility that suppression of respiration is the mechanism for retardation of capacitation and the subsequent acrosome reaction by D-glucose and other metabolizable sugars is suggested.     

Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane

Mammalian spermatozoa undergo maturation as they pass through the epididymis. Maturation is accompanied by the oxidation of thiols to disulfides. Disulfides are probably involved in sperm chromatin condensation and tail structure stabilization. In this work, we used the fluorescent thiol-labeling agent monobromobimane to determine the changes occurring in thiols and disulfides in rat sperm heads and tails during maturation. Spermatozoa were obtained from testis, epididymis (caput, corpus, cauda, and vas deferens), and ejaculate. Intact spermatozoa were labeled with monobromobimane, with or without pretreatment with dithiothreitol. Labeling was evaluated microscopically, and quantitative analysis was carried out spectrofluorimetrically with labeled globin used as a standard. Samples were also analyzed by gel electrophoresis. The total amount of thiols and disulfides remained the same during the entire period of sperm maturation (26 +/- 0.5 nmoles thiols + disulfides/10(6) spermatozoa). However, the reactive thiols decreased markedly between the corpus and the cauda (from greater than 90% of total in testis and 75% in corpus to about 25% in cauda), with little or no further change in vas deferens and ejaculated sperm. Trypsin treatment followed by sucrose gradient was used to separate the heads from the tails. Thiols comprised 84% of the total SH + SS in the heads and 74% in the tails of caput spermatozoa, decreasing to 14% and 45%, respectively, in cauda sperm. Thus, the decrease in reactive thiols involved both heads and tails-oxidation to disulfides being very marked in the head. Electrophoresis revealed that oxidation of thiols to disulfides occurred in many protein fractions during maturation in the epididymis.     

Reversible inactivation of tyrosine aminotransferase from guinea pig liver by thiol and disulfide compounds.

Tyrosine aminotransferase from guinea pig liver is strongly inactivated by a variety of natural thiols and disulfides. L-cysteine was used as a model compound in the study of inactivation. Inactivation is due to the disulfide produced by spontaneous oxidation of thiol during incubation. Binding studies with [³⁵S]-cysteine revealed simultaneous incorporation of [³⁵S] into tyrosine aminotransferase and loss of enzyme activity. The reversibility demonstrates that the inactivation is the result of the formation of mixed disulfide between the disulfide and the sulfhydryl group of tyrosine aminotransferase. Some features of the enzyme active site are showed by the inactivation reaction.     

Gossypol-induced inhibition of guinea pig sperm capacitation in vitro

The effect of gossypol acetate at various concentrations (10(-6) to 10(-4) M) on guinea pig sperm forward progressive movement, capacitation, and the acrosome reaction was explored in vitro. We found that 10(-4) M gossypol completely abolished the forward progressive motility of the sperm, and that this inhibition of motility was proportional to the concentration of gossypol used. Also, a dose-dependent decrease in acrosome reactions occurred with concentrations of the agent as low as 5.0 X 10(-6) M. However, we observed that such prevention of the acrosome reaction apparently happens at the capacitation stage rather than during the acrosome reaction itself. Inhibition of capacitation by gossypol was reversible--once the spermatozoa were capacitated in gossypol-free medium, the compound did not block the reaction.     

Redox activity associated with the maturation and capacitation of mammalian spermatozoa

As rat spermatozoa undergo epididymal maturation, they acquire the ability to exhibit a spontaneous burst of luminol-peroxidase-dependent chemiluminescence when released into a simple, defined culture medium. This activity was suppressed by inhibitors of plasma membrane redox systems such as diphenylene iodonium, p-chloromercuribenzenesulfonic acid, and capsaicin, but was resistant to inhibition by resiniferatoxin and rotenone. The luminol-peroxidase signal was dependent on the presence of bicarbonate, enhanced by the substitution of fructose for glucose, and severely suppressed by desferoxamine, superoxide dimutase, and catalase. Both L- and D-arginine were stimulatory, suggesting the involvement of *NO in this spontaneous chemiluminescence activity. The L-arginine-dependent, but not the D-arginine-dependent, activity was significantly suppressed by an inhibitor of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester). L- and D-arginine could also stimulate redox activity observed in immature caput epididymal cells, but only after prolonged incubation. The inhibitory effects of uric acid and ascorbate suggested the chemiluminescence signal might be induced by peroxynitrite. This conclusion was supported by confocal imaging of the cells following treatment with 4-amino-5-methylamino-2',7'-difluorofluorescein. Stimulation or suppression of the redox activity detected by luminol-peroxidase led to corresponding changes in the ability of the spermatozoa to exhibit acrosomal exocytosis, indicating that this pathway is of fundamental biological significance.     

An investigation of capacitation and the acrosome reaction in dog spermatozoa using a dual fluorescent staining technique

The processes of capacitation and acrosomal exocytosis of dog spermatozoa in vitro have yet to be fully investigated. Firstly, we investigated the effectiveness of a technique for staining dog spermatozoa with the fluorescent labels Hoechst 33258 and chlortetracycline. A modified fluorescence microscopy staining method was shown to be effective for the assessment of both viability and functional status in this species. Secondly, the presence of the ionophore A23187 in culture medium was shown to promote capacitation and the acrosome reaction of dog spermatozoa. We have therefore established that this dual fluorescent staining method can be used for monitoring these events in the dog, and it may be useful in future studies of optimal in vitro culture conditions.     

Surface modification of guinea pig sperm during in vitro capacitation: an assessment using lectin-induced agglutination of living sperm

Plant lectins have been used to advantage to study carbohydrate-containing cell surface receptors in numerous systems. In this study, a simple, reliable assay was developed to quantitate lectin-induced agglutinability of sperm. This assay was used successfully to compare some of the surface properties of uncapacitated and capacitated guinea pig sperm. Capacitation was induced by incubating sperm in minimum capacitation medium (MCM) or modified Tyrodes solution (T-PL). Control incubations were done in Ham's F-10 or Hank's balanced salt solution which do not support capacitation. At timed intervals during incubation, sperm samples were assessed for pattern and degree of lectin-induced agglutination. Results establish that: (1) soybean agglutinin (SBA) and to a lesser extent concanavalin A (Con A) induced agglutinability of guinea pig sperm increase during in vitro capacitation in MCM; (2) a similar increase in SBA induced agglutinability occurs during capacitation in T-PL, but not in the non-capacitating media; and (3) for sperm incubated in MCM or T-PL, there is a significant increase in tail to tail agglutination after capacitation. The results with SBA demonstrate that D-galactose and/or N-acetyl-D-galactosamine containing receptor sites or the guinea pig sperm surface are affected by capacitation, and this effect occurs, at least in part, in the sperm tail. Possible explanations for the observed increase in agglutinability are discussed. The agglutination assay may prove useful as a direct test for the occurrence of capacitation and may be especially valuable for species having a small acrosome or limited number of eggs.     

Effects of polyamines upon capacitation and fertilization in the guinea pig

The effects of polyamines upon the ability of guinea pig spermatozoa to undergo the acrosome reaction and upon sperm-egg fusion were investigated. All of the polyamines (polyarginines and compound 48/80) stimulated the occurrence of the acrosome reaction, although differences in the duration of the preincubation period required were noted. The stimulatory effect of the polyarginines could not be reversed by washing, while that of 48/80 was abolished by its removal prior to induction of the acrosome reaction. Polyarginine-treated spermatozoa were fully capable of fertilizing intact guinea pig eggs. In contrast, 48/80 completely blocked sperm-egg fusion at only 5-10 micrograms/ml. This inhibition was reversible by washing either treated gamete. The ability of polyamines which may also be present at the time and site of fertilization, in vivo, to inhibit or stimulate gamete functions or interactions is discussed.     

Alteration of sperm thiol-disulfide status and capacitation in the guinea pig

Epididymal spermatozoa of the guinea pig were incubated under conditions known to promote a rapid synchronous capacitation in a large proportion of the spermatozoa (Ca²⁺-free medium with lysophosphatidylcholine, LC) or in Ca ²⁺-free medium without LC. To study the effects of altered thiol-disulfide status and content, incubations were conducted with reagents that maintain and increase thiol groups (DTT, GSH), maintain and increase disulfide groups (diamide, GSSG), or which irreversibly block thiol groups by alkylation (NEM). The permeable DTT inhibited LC-induced capacitation and at high concentrations diminished the percentage of acrosome reactions in capacitated spermatozoa. The permeable diamide exhibited a stimulatory effect upon capacitation. The largely impermeable GSH and GSSG exhibited effects similar to their respective permeable counterparts but their effects were moderate and required extremely high concentrations. The DTT inhibition of LC-induced capacitation was reversible by washing and a further 1 hr incubation. In this final incubation after removal of DTT by washing, LC was absent too so its stimulatory effect must have been accomplished prior to washing and in the presence of DTT. NEM-alkylation of the existing thiol population did not affect LC-induced capacitation but alkylation of the increased thiol population after prior DTT treatment was inhibitory of capacitation. These results suggest that the maintenance and/or formation of disulfide groups on enzymes or structural proteins may be a component of the capacitation process. In contrast, the formation and maintenance by alkylation of increased thiol groups but not the maintenance of existing thiol groups, is inhibitory of capacitation. The relevance of these findings to a role for a thiol-sensitive proteinase in capacitation is discussed.     

Surface protein changes in goat spermatozoa during capacitation

Polypeptides of goat sperm surface before and after capacitation were examined by radiolabelling and immunologically using polyclonal antisera. Radioiodination revealed five protein bands having mol wt of 14.8, 72.4, 81, 100 and 128 kDa in uncapacitated ejaculated spermatozoa and only three bands of 23.4, 27 and 72.4 KDa in capacitated spermatozoa. The protein band with mol wt 72.4 kDa was only feebly iodinated in uncapacitated sperm surface but in capacitated spermatozoa it was heavily labelled. Western blot analysis of detergent-extracted proteins using gamma-globulin fraction of antisera raised against purified goat sperm plasma membrane revealed six antigens (17.8, 29.1, 33.4, 45.6, 85.1, 123.2 kDa) in uncapacitated spermatozoa, four (26, 32.1, 40.1, 45.6 kDa) in capacitated spermatozoa and only one (45.6 kDa) in acrosome-reacted spermatozoa. High mol wt proteins were more numerous on the surface of uncapacitated spermatozoa while the capacitated spermatozoa had relatively low mol wt proteins. An apparent effect of capacitation is the metabolism and reorganisation of proteins on goat sperm surface. Polypeptides on capacitated sperm surface revealed through radiolabelling and polyclonal antisera may have a likely receptor(s) role in the recognition and binding to homologous zona pellucida during fertilization.     

Changes in the acrosome of guinea pig spermatozoa during passage through the epididymis

Summary Electron microscopic observations have been made on the structure of guinea-pig spermatozoa in successive segments of the epididymal duct. Marked changes are observed in the shape and internal structure of the acrosome as the sperm move through the epididymis. The morphological basis for this continuing differentiation of the sperm is traced back to certain unusual features of acrosome formation in the early spermatids of this species. The possible relation of the progressive development of the acrosome to the known increase in fertilizing capacity of the spermatozoa during their passage through the epididymis is discussed.Supported in part by grant RG-6729 and by GM-10182-8 from the Division of General Medical Sciences, National Institutes of Health, United States Public Health Service.It is a pleasure to acknowledge the technical assistance of Arthur Mitchell who made some of the initial observations that stimulated this investigation.     

Adhesion between plasma membrane and mitochondria with linking filaments in relation to migration of cytoplasmic droplet during epididymal maturation in guinea pig spermatozoa

High-resolution microscopy has been used to investigate the mechanism of the migration of cytoplasmic droplets during epididymal maturation of guinea pig spermatozoa. On testicular spermatozoa, droplets are located at the neck and, after passage through the middle cauda epididymidis, migrate only as far as the center of the midpiece. Initially, the space between the plasma membrane and outer mitochondrial membranes outside the droplet is 30.8±11.0 nm, whereas on mature spermatozoa, it significantly (P<0.01) narrows to a more consistent 15.9±1.3 nm. This is accompanied by the appearance of thin filaments cross-linking the two membranes above and below the droplet. Changes also occur in the arrangement of intramembranous particles (IMPs) in the plasma membrane overlying the midpiece. At the spermatid stage, linear arrays of IMPs are absent but appear on immature spermatozoa, where they are short with an irregular orientation, in the epididymis. On mature spermatozoa, numerous parallel linear arrays are present at the region where the plasma membrane adheres to the mitochondria. The membrane adhesion process can thus be observed two-dimensionally. The initial migration of the droplet from the neck is probably attributable to diffusion, with the formation of cross-linking filaments between the two membranes in the proximal midpiece preventing any backward flow and squeezing the droplet distally until it is arrested at the central midpiece by the filaments formed in the distal midpiece. The filaments might also stabilize the flagellum against hypo-osmotic stress encountered during ejaculation and within the female tract.     

An investigation using lectins of glycocomponents of mouse spermatozoa during capacitation and sperm-zona binding

Ten different lectins conjugated to fluorescein isothiocyanate (FITC) were used to study the distribution of surface carbohydrates on mouse spermatozoa, and to monitor the possible changes of their distribution during capacitation in vitro and sperm-egg interaction. Most of the lectins gave a restricted pattern of binding to fixed or unfixed epididymal spermatozoa. Binding was highly specific because no staining occurred in the presence of appropriate monosaccharides. Binding of UEA I, DBA and Con A was unaffected by the type of fixative used, but it was influenced by mild centrifugation. While unwashed spermatozoa showed binding mainly over the acrosomal cap and equatorial or postacrosomal regions, spermatozoa washed by mild centrifugation showed a change in the staining of the equatorial segment. Binding of 5 different lectins to spermatozoa did not change during capacitation in vitro. In contrast, capacitated spermatozoa bound to the zona pellucida exhibited a UEA I binding pattern which was strikingly different from that of the capacitated but unbound spermatozoa. We conclude that glycocomponents of specific regions of mouse spermatozoa do not change dramatically during capacitation, but do alter significantly during binding to the zona pellucida.     

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl₂ and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl₂ revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl₂ lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg²⁺, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca²⁺ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg²⁺, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.     

Changes in guinea pig sperm intracellular sodium and potassium content during capacitation and treatment with monovalent ionophores

Guinea pig spermatozoa were collected from the caudae epididymides in various isotonic solutions and the intracellular sodium and potassium content was determined by atomic absorption spectroscopy. The sperm intracellular Na and K content was found to be influenced by large variations in the extracellular concentrations of these ions. Treatment of spermatozoa suspended in a saline-based solution with the monovalent ionophores monensin or nigericin caused an approximate 2-fold increase in the intracellular Na content and a 3–6 fold decrease in the intracellular K content. Incubation of the spermatozoa in a K⁺-free minimal culture medium (MCM-PL) at a pH of 7.6 or 8.3 for 2 hr caused an approximate 2-fold increase in the sperm intracellular Na content and a 5-fold decrease in the intracellular K content. The motile spermatozoa incubated for 2 hr at pH 7.6 showed less than 5% acrosome reactions, compared with 30–40% acrosome reactions after incubation at pH 8.3, in response to the addition of 5 mM Ca²⁺. Changes in the sperm intracellular elemental composition during culture in vitro, which may lead to an acrosome reaction, are discussed.