The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation

It is generally accepted that incubation with heparin is required to induce capacitation of ejaculated bovine spermatozoa in vitro. The capacitation process implicates many biochemical events, and is correlated with modified sperm motility and the phosphorylation of specific proteins on tyrosine residues. To better understand the molecular basis of heparin-induced capacitation, bovine spermatozoa were incorporated with a radioactive substrate of protein kinases [gamma32P]-ATP, to observe protein phosphorylation dynamics over time. Sperm motion parameters including the percentage of motile spermatozoa, amplitude of lateral head displacement (ALH) and flagellar beat cross frequency (BCF) were assessed to determine whether the protein phosphorylation patterns induced by heparin also promote changes in motility. Capacitation was confirmed using the chlortetracycline fluorescence assay and the appearance of 'pattern B' stained spermatozoa. Evaluation of the different motility parameters during capacitation reveal that heparin has a marked negative effect, over time, on the percentage of motile spermatozoa, consistent with hyperactivation. Indeed, the presence of heparin greatly increases the BCF of bull spermatozoa and induces a significant increase in the ALH compared to spermatozoa incubated without heparin. We detected several sperm proteins that are phosphorylated over time. A 45 kDa protein is the most intensely phosphorylated of the sperm proteins. However, it is visible regardless of the presence of heparin. Interestingly, a second phosphorylated protein of approximately 50 kDa undergoes more intense phosphorylation with heparin than without. In summary, the present study demonstrated that heparin induces physiological changes in several sperm motility parameters including ALH, BCF and the percentage of motile spermatozoa. Heparin also increases the intensity of phosphorylation of a 50 kDa sperm protein. These results suggest that capacitation of bovine spermatozoa and capacitation-associated motility changes may be regulated by a mechanism that includes protein phosphorylation, and that a presently unknown protein kinase is involved.     

Intracellular pH of bovine sperm increases during capacitation

The effect of heparin-induced capacitation on the intracellular pH (pH_i) of individual bovine sperm was determined with image analysis. Sperm were loaded with the acetoxymethyl ester of the pH sensitive fluorescent indicator, 2′,7′-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). The pH_i of 5303 sperm was evaluated from a total of five bulls at .5, 2, 3, 4, and 5 h of incubation. The pH_i did not differ between the sperm head and mid-piece (P > 0.05). An increase in sperm head pH_i was seen in heparin-treated sperm at 3, 4, and 5 h of incubation relative to sperm incubated without heparin (control, P < 0.05). At 5 h of incubation, the pH_i in heparin-treated sperm was 6.92 ± 0.07, while control-treated sperm pH_i was 6.70 ± 0.03. Initially a normal frequency distribution was seen for sperm pH_i in both heparin- and control-treated sperm. As the incubation progressed, the frequency distribution began to skew towards higher pH_i in both samples but was more dispersed for the heparin-treated sperm. Following an NH₄Cl-induced alkaline load, the pH_i of both control- and heparin-treated sperm recovered toward the resting pH_i with a half-time of recovery of 1.5–1.7 min. The recovery of sperm pH_i was not due to leakage of NH⁴⁺ into sperm because recovery also occurred with trimethylamine. The instantaneous velocity of the pH_i recovery (v_i) was dependent on pH_i and decreased as pH_i decreased. Capacitation by heparin was associated with an 81% decrease in v_i at a pH_i of 7.00, but there was no effect of capacitation on the proton buffering power of the sperm, which was 87 ± 8 mM/pH unit. Results demonstrate that both the regulation of pH_i and resting pH_i were altered during capacitation of bovine sperm by heparin. © 1995 Wiley-Liss, Inc.     

Mammalian sperm chemotaxis and its association with capacitation

Much progress has been made in recent years in establishing mammalian sperm chemotaxis and understanding sperm capacitation. Thus far, chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime of 50 min to 4 h) and they occur only once in the sperm's lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in humans) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the time during which capacitated spermatozoa are available in the female reproductive tract. The sperm chemoattractants have not been identified, but they appear to be heat‐stable peptides. Although the molecular mechanism and the in vivo location of sperm chemotaxis are not known, a number of possible mechanisms and locations are discussed. Dev. Genet. 25:87–94, 1999. © 1999 Wiley‐Liss, Inc.     

Changes in lectin binding to bovine sperm during heparin-induced capacitation

Changes in the plasma membrane of bovine sperm during heparin-induced capacitation were detected by the binding of fluorescent labeled lectins to unfixed sperm. Of the seven lectins evaluated, only binding of wheat germ agglutinin (WGA) changed with capacitation. Sperm were classified into one of 5 patterns (p1–p5) based on staining with WGA, presence or absence of propidium iodide (PI) staining (dead or alive), and acrosomal integrity (acrosome intact or reacted). The major changes associated with capacitation occurred in p1 and p2. Sperm in p1 exhibited diffuse WGA binding over the anterior sperm head, were alive, and had intact acrosomes. In p2, sperm were also acrosome intact and alive, but lacked WGA binding. When sperm were incubated under capacitating conditions with heparin, there was a decrease over time in the percentage of sperm classified in p1 (p < 0.05) and an increase in the percentage of sperm in p2 (p < 0.05). When capacitation by heparin was delayed by the inclusion of glucose in the culture medium, the same heparin-dependent changes in the percentage of sperm in p1 and p2 were delayed (p < 0.05). When capacitation by heparin was inhibited by including protamine in the culture medium, the percentage of sperm in p1 or p2 was not different from sperm incubated without heparin. Heparin-induced capacitation was associated with a loss of WGA binding to the bovine sperm head. © 1996 Wiley-Liss, Inc.     

Capacitation of bovine sperm by heparin

Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (10 micrograms/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 100 micrograms/ml LC for 15 min. When sperm were incubated for 4 h with heparin, exposure to 100 micrograms/ml LC for 15 min had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 10% to over 70%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 min, and differed (p less than 0.001) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-10 micrograms/ml heparin. The correlation between the mean fertilization and LC-induced AR percentages was 0.997 (p less than 0.01). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC.     

Oviduct fluid and heparin induce similar surface changes in bovine sperm during capacitation: A flow cytometric study using lectins

Eight different lectins conjugated to fluorescein isothiocyanate (FITC) were used to screen for sperm plasma membrane changes during in vitro capacitation of bovine sperm. Analysis of lectin binding to sperm was done using flow cytometry. Of the eight lectins, only Triticum vulgaris (wheat germ agglutinin, WGA) binding to sperm was altered with capacitation. Capacitation of bovine sperm by heparin was found to decrease WGA binding to sperm by 78% (P < 0.05). The effect of capacitation by oviduct fluid was next compared with capacitation by heparin for changes in WGA binding to sperm. The effect of inhibiting capacitation with glucose on WGA binding was also determined. WGA-bound sperm were detected by flow cytometry as being present in two fluorescence peaks defined as low fluorescence (A) or high fluorescence (B) intensity. The percentage of sperm in peak A was greater for heparin and oviduct fluid-treated sperm compared to sperm incubated under noncapacitating conditions in only culture medium (P < 0.001). Capacitation with either heparin or oviduct fluid was inhibited by glucose as assessed by the ability of lysophosphatidylcholine (100 μg/ml) to induce acrosome reactions. Glucose also reduced the percentage of sperm in peak A for both heparin- and oviduct fluid-treated sperm (P < 0.01). We conclude that heparin or oviduct fluid induced changes on the sperm plasma membrane during capacitation. Binding sites for WGA on sperm were either structurally altered or lost during capacitation. © 1996 Wiley-Liss, Inc.     

Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation.

125I-labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 x 10(6) sperm/ml) were incubated up to 4 h in a protein-free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I-labeled ODF proteins. Solubilized sperm membrane proteins and incubation media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm, previously incubated with ODF, adsorbed five 125I-proteins: A doublet at 85-95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I-protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95-kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85-95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95-kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa, ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins to the membrane during in vitro capacitation.     

Energy requirement of bovine spermatozoa for in vitro capacitation

The oxidative energy requirements of bovine spermatozoa capacitated with dilauroil-phosphatidylcholine liposomes (PC 12) and the effect of these liposomes on acrosome reaction necessary for in vitro fertilization were studied. Mitochondrial respiration was measured using 3 different substrates (pyruvate-lactate-glucose) and endogenous substrates. The samples were either treated with PC 12 or were left untreated and used as the control. A 2.8-fold increase in the consumption of oxygen was observed in the PC 12 treated spermatozoa in the presence of the 3 combined substrates (pyruvate-lactate-glucose). Respiration changes were not observed when the spermatozoa were capacitated with only 2 of the 3 substrates or with glucose alone. When endogenous substrates were used, the consumption of oxygen increased 1.7 times, and mitochondrial uncoupling was observed in the treated samples. The hypermotility characteristic of the capacitation process was not observed when glucose or endogenous substrates were used. When the percentage of intact acrosomes was determined using differential-interferential contrast (DIC) microscopy, it was found that in the presence of oxidative substrates there was a 26% decrease compared with that of the control sample. The proportion of reacted acrosomes was in the range of 41.3 to 49.6%, as measured by the chlortetracycline epifluorescence method in the presence of calcium ionophore A23187. Only 4% of the spermatozoa showed acrosome reaction with endogenous substrates. A higher percentage of fertilized oocytes were observed when the spermatozoa were capacitated in the presence of the 3 substrates (pyruvate-lactate-glucose), confirming that the success of in vitro fertilization depends on the energy conditions associated with the capacitation process. The results of these experiments indicate that the presence of oxidative energy is necessary to produce capacitation and the hyperactivation characteristic in frozen-thawed bovine spermatozoa treated with liposomes.     

Na+/K+ATPase as a signaling molecule during bovine sperm capacitation

A heteromeric integral membrane protein, Na+/K+ATPase is composed of two polypeptides, alpha and beta, and is active in many cell types, including testis and spermatozoa. It is a well-known ion transporter, but binding of ouabain, a specific inhibitor of Na+/K+ATPase, to Na+/K+ATPase in somatic cells initiates responses that are similar to signaling events associated with bovine sperm capacitation. The objectives of the present study were to demonstrate the presence of Na+/K+ATPase in bovine sperm and to investigate its role in the regulation of bovine sperm capacitation. The presence of Na+/K+ATPase in sperm from mature Holstein bulls was demonstrated by immunoblotting and immunocytochemistry using a monoclonal antibody developed in mouse against the beta 1 polypeptide of Na+/K+ATPase. Binding of ouabain to Na+/K+ATPase inhibited motility (decreased progressive motility, average path velocity, and curvilinear velocity) and induced tyrosine phosphorylation and capacitation but did not increase intracellular calcium levels in spermatozoa. Furthermore, binding of ouabain to Na+/K+ATPase induced depolarization of sperm plasma membrane. Therefore, binding of ouabain to Na+/K+ATPase induced sperm capacitation through depolarization of sperm plasma membrane and signaling via the tyrosine phosphorylation pathway without an appreciable increase in intracellular calcium. To our knowledge, this is the first report concerning the signaling role of Na+/K+ATPase in mammalian sperm capacitation.     

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.     

Different signaling pathways in bovine sperm regulate capacitation and hyperactivation

Hyperactivated sperm motility is characterized by high-amplitude and asymmetrical flagellar beating that assists sperm in penetrating the oocyte zona pellucida. Other functional changes in sperm, such as activation of motility and capacitation, involve cross talk between the cAMP/PKA and tyrosine kinase/phosphatase signaling pathways. Our objective was to determine the role of the cAMP/protein kinase A (PKA) signaling pathway in hyperactivation. Western blot analyses of detergent extracts of whole sperm and flagella were performed using antiphosphotyrosine antibody. Bull sperm capacitated by 10 microg/ml heparin and/or 1 mM dibutyryl-cAMP plus 100 microM 3-isobutyl-1-methylxanthine exhibited increased protein tyrosine phosphorylation without becoming hyperactivated. Procaine (5 mM) or caffeine (10 mM) immediately induced hyperactivation in nearly 100% of motile sperm but did not increase protein tyrosine phosphorylation. After 4 h of incubation with caffeine, sperm expressed capacitation-associated protein tyrosine phosphorylation but hyperactivation was significantly reduced. Sperm initially hyperactivated by procaine or caffeine remained hyperactivated for at least 4 h in the presence of Rp-cAMPS (cAMP antagonist) or PKA inhibitors H-89 or H-8. Pretreatment with inhibitors also failed to block induction of hyperactivation; however, the inhibitors did block protein tyrosine phosphorylation when sperm were incubated with capacitating agents, thereby verifying inhibition of the cAMP/PKA pathway. While induction of hyperactivation did not depend on cAMP/PKA, it did require extracellular Ca(2+). These findings indicate that hyperactivation is mediated by a Ca(2+) signaling pathway that is separate or divergent from the pathway associated with acquisition of acrosomal responsiveness and does not involve protein tyrosine phosphorylation downstream of the actions of procaine or caffeine.     

Effect of progesterone on bovine sperm capacitation and acrosome reaction

Progesterone (P) appears to stimulate sperm capacitation and/or induce the acrosome reaction (AR) in some species. In bovine, it is now well established that the BSP-A1/-A2 proteins (the major proteins of bovine seminal plasma) promote sperm capacitation. In this study, we investigated the effect of P on bovine sperm cholesterol efflux, capacitation, and the AR. Labeled bovine epididymal sperm were incubated (0-6 h) with different concentrations of P (0.01-10 microg/ml) in the presence or absence of BSP-A1/-A2 proteins (capacitating conditions). At different time intervals, aliquots of sperm were taken to determine the sperm cholesterol efflux, sperm capacitation (AR induced by lysophosphatidylcholine, lyso-PC), and sperm AR. The results show that the presence of P in the media did not affect the membrane cholesterol efflux potential of the BSP-A1/-A2 proteins. P alone did not stimulate the AR with or without lyso-PC unless the epididymal sperm were incubated in capacitating conditions (in the presence of BSP-A1/-A2). When washed ejaculated sperm were continuously incubated with P, the P did not stimulate AR. However, when ejaculated sperm were preincubated (6 h) with heparin (capacitation medium) and then incubated 15 min with P (2 microg/ml), the percentage of AR obtained was similar to that obtained with lyso-PC. The effect of P on sperm AR was concentration dependent with a maximum 2.2-fold increase at 2 microg/ml of P. These results demonstrate a potential role of P in bovine sperm AR but not in capacitation.     

Kinases, phosphatases and proteases during sperm capacitation

Fertilization is the process by which male and female haploid gametes (sperm and egg) unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the zona pellucida, acrosome exocytosis, sperm penetration through the zona and fusion of the sperm and egg plasma membranes. However, freshly ejaculated sperm are not capable of fertilizing an oocyte. They must first undergo a series of biochemical and physiological changes, collectively known as capacitation, before acquiring fertilizing capabilities. Several molecules are required for successful capacitation and in vitro fertilization; these include bicarbonate, serum albumin (normally bovine serum albumin, BSA) and Ca(2+). Bicarbonate activates the sperm protein soluble adenylyl cyclase (SACY), which results in increased levels of cAMP and cAMP-dependent protein kinase (PKA) activation. The response to bicarbonate is fast and cAMP levels increase within 60?s followed by an increase in PKA activity. Several studies with an anti-phospho-PKA substrate antibody have demonstrated a rapid increase in protein phosphorylation in human, mouse and boar sperm. The target proteins of PKA are not known and the precise role of BSA during capacitation is unclear. Most of the studies provide support for the idea that BSA acts by removing cholesterol from the sperm. The loss of cholesterol has been suggested to affect the bilayer of the sperm plasma membrane making it more fusogenic. The relationship between cholesterol loss and the activation of the cAMP/PKA pathway is also unclear. During early stages of capacitation, Ca(2+) might be involved in the stimulation of SACY, although definitive proof is lacking. Protein tyrosine phosphorylation is another landmark of capacitation but occurs during the late stages of capacitation on a different time-scale from cAMP/PKA activation. Additionally, the tyrosine kinases present in sperm are not well characterized. Although protein phosphorylation depends upon the balanced action of protein kinases and protein phosphatase, we have even less information regarding the role of protein phosphatases during sperm capacitation. Over the last few years, several reports have pointed out that the ubiquitin-proteasome system might play a role during sperm capacitation, acrosome reaction and/or sperm-egg fusion. In the present review, we summarize the information regarding the role of protein kinases, phosphatases and the proteasome during sperm capacitation. Where appropriate, we give examples of the way that these molecules interact and regulate each other's activities.     

Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.     

获能期间精子蛋白的酪氨酸磷酸化

哺乳动物精了获能是精子与卵子成功受精的前提。蛋白酪氨酸磷酸化对精子获能十分重要。精了获能期蛋白酪氨酸磷酸化程度增高与sAC/cAMP/PKA途径、受体酪氨酸激酶途径和非受体蛋白酪氨酸激酶途径调节有关。获能过程中酪氨酸磷酸化蛋白分布于精子细胞的不同区域,蛋白的酪氨酸磷酸化与精子功能密切相关。     

Effects of heparin dosage and sperm capacitation time on in vitro fertilization and cleavage of bovine oocytes matured in vitro

The present study was conducted to clarify the effect of heparin dosage and sperm capacitation time on in vitro fertilization (Experiment 1) and cleavage (Experiment 2) rates of bovine oocytes matured in vitro. For in vitro fertilization, seven dosages of heparin (0, 5, 10, 25, 50, 100 and 200 mug/ml) and nine incubation periods (0, 5, 15, 30, 45, 60, 120, 180 and 240 min) in a capacitation medium were examined, using 6,634 oocytes. The mean proportions of fertilized oocytes in 25, 50 and 100 mug/ml of heparin were significantly (P<0.05) higher (53 to 59%) than in the other dosages (3 to 44%). Incubation with heparin for longer than 60 min lowered the frequencies of fertilization (20 to 36%) compared with the shorter incubation periods (38 to 49%). Higher proportions of fertilized oocytes were obtained by 5, 15, 30 or 45 min of incubation (42 to 49%) than by the other time periods (20 to 38%). Cleavage rates were found by using 2,098 oocytes in a factorial study (4 x 4 x 15: dosages -25, 50, 100 and 200 mug/ml; incubation periods -0, 15, 30 and 60 min; and replicates). The incubation periods and replicates resulted in highly significant differences (P<0.001) in development rates to eight-cell stage, but the four dosages of heparin showed no significant differences. The present results indicate that heparin dosage and sperm capacitation time are important factors influencing in vitro fertilization and cleavage rates. Optimal heparin dosages for the capacitation of bull spermatozoa ranged from 25 to 100 mug/ml; optimal incubation periods ranged from 5 to 60 min.     

Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction

Sperm capacitation is necessary for the fertilization of oocytes. During capacitation intracellular and membrane changes occur, that culminate with an exocytotic event called the acrosome reaction. The aim of this work was to study the participation of the superoxide anion (O2-.) and of hydrogen peroxide (H2O2) in the capacitation process and acrosome reaction in spermatozoa from cryopreserved bovine semen. Samples were capacitated with heparin or treated with the xanthine-xanthine oxidase-catalase system (X-XO-C) for the production of O2-. The percentage of capacitated spermatozoa was determined using the chlortetracycline (CTC) technique, by means of epifluorescence microscopy. Addition of X-XO-C to the incubation medium significantly induced capacitation (P < 0.05), but there were no differences with samples incubated with heparin. When the medium contained heparin or the X-XO-C, addition of superoxide dismutase (SOD, 0.5 mg/mL) significantly inhibited capacitation (P < 0.05). In samples treated with heparin and with diverse concentrations of H2O2 (10, 25, 50 and 250 microM) in the incubation medium, the percentage of capacitated spermatozoa was significantly reduced (P < 0.05); however, acrosome reaction was produced at concentrations of 10 and 25 microM H2O2. At concentrations greater than 25 microM H2O2 a deleterious effect was observed on sperm motility. From these results it may be inferred that O2-. is required in the capacitation process and that H2O2 may participate as an inductor of the acrosome reaction in spermatozoa from cryopreserved bovine semen.     

Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP(2(4,5)), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM WT but not by 10 nM WT, whereas PI3K activity and PIP(3(3,4,5)) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP(2(4,5)) and F-actin formation. The increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP(2(4,5)), PIP(3(3,4,5)), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP(3(3,4,5)) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.     

Albumin-mediated changes in sperm sterol content during capacitation

The role of albumin in mouse sperm capacitation was studied in relation to its activities as a lipid-solubilizing protein and a sterol acceptor. Two bovine serum albumins (BSA) which supported capacitation, Fraction V and fatty acid-free, both contained cholesterol and phospholipid but were without detectable levels of serum high-density lipoprotein (HDL). The lipid content of BSA could be reduced by trichloroacetic acid (TCA) precipitation; however, removal of all detectable lipids required precipitation with ethanolic acetone and diethyl ether extraction. In medium supplemented with Fraction V, fatty acid-free, or TCA-precipitated BSA, mouse sperm were capacitated as evidenced by their ability to fertilize eggs, concomitant with decreases in total cellular sterol and increases in phospholipid content. Delipidated BSA, fractionated on Sephadex G-100 in guanidine HCl also supported capacitation and mediated a 20% decrease in sperm sterol content, while cellular phospholipid levels remained unchanged. When BSA was modified by cholesterol augmentation, fertilization was inhibited in a cholesterol dose-dependent manner. These findings suggest that modulation of sperm lipid levels comprises an event of capacitation and that albumin mediates this process through its activity as a sterol acceptor.