Sperm—oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa

Summary. Successful sperm-oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona-bound spermatozoa, and (3) the effect of human follicular fluid on the zona-binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 10⁷ cells ml⁻¹ after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h underwent the acrosome reaction (range 9–43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome-reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors' understanding of critical events occurring before fertilization.     

Physiological action of oestradiol on the acrosome reaction in human spermatozoa

The acrosome is a secretory vesicle located in the sperm head. The acrosome reaction consists in the fusion of the sperm plasma membrane with the external acrosomal membrane. It has been observed that this reaction does not take place in spermatozoa incubated in cervical mucus, hydrogel that contains high concentrations of oestradiol in the peri-ovulatory period. The objective of the present study was to analyse the influence of oestradiol on the acrosome reaction in human spermatozoa to evaluate the possible inhibitory effect of this hormone. Spermatozoa were incubated in progesterone (10.1 nmol l⁻¹); oestradiol plus progesterone (oestradiol at 840 pmol l⁻¹ and progesterone at 10.1 nmol l⁻¹), oestradiol (840 pmol l⁻¹) and control (without steroidal hormones) for 30 min, 60 min, 240 min and 24 h. The acrosome reaction was evaluated by stain with Hoechst 33258 and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin lectin. Progesterone-incubated spermatozoa showed the highest percentage of acrosome reaction ( P  <   0.05). Spermatozoa incubated with oestradiol and oestradiol plus progesterone showed the lowest percentage of acrosome reaction. The present study demonstrates the inhibitory role of oestradiol on the acrosome reaction, stimulated by progesterone in human spermatozoa under physiological conditions.     

Assessment of the acrosomal status of ram spermatozoa by RCA lectin-binding and partition in an aqueous two-phase system

The acrosome reaction is an important marker for sperm function. Because different laboratory techniques may be used to detect this exocytotic process, the objective of this study was to investigate the use of fluoresceinated lectins to assess the acrosomal status of nonpermeabilized ram spermatozoa. In addition, we used centrifugal countercurrent distribution (CCCD) in an aqueous 2-phase system to assess the sperm surface modifications associated with the acrosome reaction by observing changes in their partition behavior. We analyzed the binding of 5-fluorescein isothiocyanate (FITC)-conjugated lectins to ram sperm to select a lectin that bound preferentially to the acrosomal region, which would allow differentiation of acrosome-intact from acrosome-damaged ram spermatozoa. Ricinus communis agglutinin (RCA) bound intensely to the anterior and weakly to the equatorial acrosomal regions. Acrosomal labeling changed when spermatozoa were induced to acrosome-react with calcium ionophore A23187. RCA acrosomal labeling significantly increased (P < .0001) after incubation (84% versus 28% in control samples). To determine if RCA lectin labeling could be used to assess the acrosomal status of fresh ram spermatozoa in suspension, we compared the percentage of acrosome-reacted sperm detected by the carboxyfluorescein diacetate/propidium iodide (CFDA/PI) double-fluorescent staining with the percentage detected by FITC-RCA labeling. The incidence of acrosome-reacted spermatozoa detected by CFDA/PI was not significantly different (P = .704; 13 comparisons in 6 different experiments) from the incidence of spermatozoa detected by FITC-RCA staining. The evaluation of the spontaneous acrosome reaction by RCA labeling (5.83%) was not significantly different (P = .644) from that assessed by CFDA/PI (6.88%). The percentage of induced acrosome reactions detected by CFDA/PI staining (56%) significantly correlated (P < .0001; r = 0.876) with that detected by RCA labeling (56.67%). We simultaneously carried out a comparative CCCD in an aqueous 2-phase system to analyze sperm surface changes associated with the acrosome reaction. Results revealed that sperm surface hydrophobicity decreased in samples that had been incubated with ionophore compared with the untreated-control samples. Likewise, RCA binding after CCCD showed that all acrosome-reacted cells were stained, whereas only 42% of cells were lectin-labeled in the untreated semen sample. This change in lectin reactivity of acrosome-reacted spermatozoa signals the presence of some deep membrane or intracellular residues that would affect partitioning. Therefore, the FITC-RCA-labeling procedure can be used to accurately assess the acrosomal status of ram spermatozoa in suspension.     

Pentoxifylline acts synergistically with A23187 to increase the penetration of zona-free hamster oocytes by cryopreserved human spermatozoa

The number of cryopreserved human spermatozoa which penetrated zona-free hamster oocytes afier stimulation with 2μmol A23187 per litre was increased by the hrther addition of 0.6 or 3.6 mmol pentoxifjrlline per litre. With spermatozoa prepared by washing by repeated centrifugation, the median numbers of sperm headd/egg were 1.9, 7.9 and 10.8 in the presence of 0, 0.6 or 3.6 mmol pentoxifylline per litre, respectively. A similar effect was observed with spermatozoa prepared on a Percoll gradient. As A23187 inhibited sperm motility, and this was exacerbated by pentoxifylline, the increased penetration rate of hamster oocytes cannot be explained by improved sperm motility. The number of spermatozoa stimulated to acrosome react by 2 μmol A23187 per litre was increased 3-fold by 3.6 mmol pentoxifylline per litre and 4-fold by 5 mmol caffeine per litre. These data suggest that CAMP may act synergistically with Ca²⁺ to stimulate the acrosome reaction. Pentoxifjrlline may improve the fertility of poor-quality human spermatozoa by enhancing their ability to respond to the Ca²⁺ signal produced by binding to the zona pellucida.     

Remodeling of the plasma membrane in preparation for sperm–egg recognition: roles of acrosomal proteins

The interaction of sperm with the egg''s extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm–ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm–ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm–ZP binding step.     

Separation of human spermatozoa with hyaluronic acid induces, and Percoll® inhibits, the acrosome reaction

The effect on the acrosome reaction of human spermatozoa of two widely used sperm separation media, hyaluronic acid (Sperm Select®) and Percoll®, was studied. Viable and highly motile fractions of human spermatozoa were separated from seminal plasma using self-migration on a Percoll® gradient. After translocation of separated spermatozoa from the Percoll® solution to a culture medium, serum, Percoll® or hyaluronic acid (Sperm Select®) was added to aliquots of the spermatozoa containing culture medium. At increasing time intervals, the influx of ⁴⁵Ca²⁺ into spermatozoa was measured and the concentration of viable spermatozoa that had undergone the acrosome reaction was analysed using the triple stain technique. Serum was found to be necessary to support sperm motility and viability. Compared to culture medium with serum only, addition of hyaluronic acid induced influx of ⁴⁵Ca²⁺ and the acrosome reaction, whilst Percoll® inhibited both of these actions. Hyaluronic acid (Sperm Select®) added to spermatozoa separated by a 'swim-up' method induced, and the addition of Percoll® inhibited, influx of ⁴⁵Ca²⁺ when compared to the addition of culture medium with serum only. This study demonstrates that both hyaluronic acid (Sperm Select®) and Percoll® affect the acrosome reaction and the prerequisite for Ca²⁺ influx in human spermatozoa. These effects should be taken into consideration when using these media for preparation of spermatozoa for insemination or for fertilization in vitro.     

Scavenging effect of N-acetyl-L-cysteine against reactive oxygen species in human semen: a possible therapeutic modality for male factor infertility?

Summary. A new approach to reduce the level of reactive oxygen species (ROS) in human semen by using N-acetyl-L-cysteine (NAG) was evaluated. Semen samples were incubated with or without NAC (1.0 nig ml⁻¹) at room temperature. The chemiluminescent signal of the oxidation of luminol was detected by means of an MTP reader after 0, 20, 40, 60 and 120 min, respectively, using 200 μM luminol. In addition, the dose-dependent action of NAC (0.1, 1.0 and 5.0 mg ml⁻¹) and the influence of NAC on functional sperm parameters (motility and acrosome reaction) were studied.
ROS levels decreased significantly after 20 min incubation with NAG. This reduction was greater in the high ROS group (>30 000 counts/10⁷ viable sperm at t = 0) than in the low ROS group (<30 000). In addition, a marked dose-dependence of NAC was observed. Concerning sperm function, total sperm motility improved after incubation with NAC, but no significant change was observed with respect to the acrosome reaction.
NAC (at concentrations of 1.0 mg ml⁻¹) significantly reduced ROS in human semen and showed the possibility of improving impaired sperm function. After further testing NAC might be useful for the treatment of male infertility patients.     

Evaluation of the acrosome reaction and viability in buffalo spermatozoa using two staining methods: the effects of heparin and calcium ionophore A23187

In this study, we investigated the effect of heparin and calcium ionophore A23187 on in vitro induction of buffalo sperm acrosome reaction (AR). Two methods for detection of the AR and viability were employed. Fluorescein isothiocyanate-conjugated Arachis hypogea agglutinin (FITC-PNA) was used as a vital stain in combination with ethidium homodimer-1 (EthD-1) to determine the acrosome status of viable spermatozoa. In another experiment, trypan blue replaced EthD-1 to differentiate live and dead spermatozoa having undergone AR. The results from the two methods were significantly correlated (r > 0.9). Four different staining patterns were found in both methods. The FITC-PNA intensely labels the acrosome region of acrosome-intact spermatozoa. EthD-1 and trypan blue stained red and blue at the post-acrosomal region of dead spermatozoa, respectively. Spermatozoa incubated with heparin showed a significant increase ( p < 0.05) in the percentage of live acrosome-reacted sperm after 30 min incubation period. This trend continued and was significantly different over the entire incubation period when compared with the control group at the same interval. In the ionophore-treated group, the proportion of changes in live acrosome-intact and live acrosome-reacted spermatozoa was statistically significantly different ( p < 0.001) when compared with those treated with heparin at the same interval. The AR occurred sooner and to a greater extent when incubated with the ionophore but at 5 h of incubation the percentage of false acrosomal reaction was significantly higher than those in the control and heparin-treated groups. The results in this study indicated that the in vitro induction of AR by heparin and calcium ionophore evaluated by both methods could be used to assess sperm fertilizing capacity for in vitro fertilization of this species.     

Zona pellucida mediated acrosome reaction and sperm morphology

Summary. Sperm samples from 29 men randomly selected from the andrology laboratory, were used to evaluate acrosome reaction response to solubilized human zona pellucida. Capacitated sperm samples were exposed to a solution containing 2 zona pellucidae (ZP) per μl for 60 min, after which acrosomal status were recorded using a PSA-FITC technique. Controls included samples supplied by fertile sperm donors. After completion of acrosome reaction studies, patient samples were divided according to the percentage of morphologically normal spermatozoa. Three basic groups were identified, namely, fertile donors, teratozoo-spermic (normal sperm morphology 5–14%; n = 25) and severely teratozoospermic (normal sperm morphology <4%; n = 4) groups. The mean percent normal sperm were 15.8 ± 0.9, 10.4 ± 0.7 and 2.7 ± 0.7, respectively, for normozoospermic donors, teratozoospermic and severely teratozoospermic men. The mean percentage (± SE) ZP mediated acrosome reacted sperm among teratozoospermic and severely teratozoospermic cases was 25.8% ± 0.9 and 19.0% ± 0.9 (P = 0.001), compared to 36.8% ± 0.9 for the donor controls. Results were analysed and expressed as correlations between sperm morphology and acrosomal response to human solubilized zona pellucida, spontaneous and calcium ionophore induced acrosome reaction. Predictive values for acrosome responsiveness were depicted with ROC curve analyses. Sperm morphology evaluated by strict criteria correlated positively and highly significantly with the responsiveness of the acrosome reaction (r = 0.91, P = 0.0001). At a morphology cut-off value of 4%, the ROC curve analysis showed sperm morphology to be highly predictive of zona pellucida induced acrosome responsiveness with a sensitivity of 100% and negative predictive value of 100%. Spontaneous and calcium ionophore induced acrosome reactions revealed no correlation with sperm morphology. It was concluded that (i) morphological features of human spermatozoa are indicative of specific functional characteristics; (ii) zona pellucida induction of the acrosome reaction is superior, as a predictor of sperm morphology, compared to calcium ionophore induced and spontaneous acrosome reactions.     

The fate of acrosomal staining during the acrosome reaction of human spermatozoa as revealed by a monoclonal antibody and PNA-lectin

A monoclonal anti-human sperm antibody, raised against an acrosomal antigen and indicated to recognize in boar sperm the serine protease, acrosin, stained in human spermatozoa a 50 Kd antigen and several others in the region 24-34 Kd by immunoblotting. The 50 Kd band and the region of 30-34 Kd showed proteolytic activity by zymographic enzyme detection. The fate of the antigen was studied in the acrosome reaction induced by the calcium ionophore A23187. In control incubations 69.5 +/- 14.2% (mean +/- SD) of the spermatozoa had intact acrosomal staining according to indirect immunofluorescence using this antibody whereas in acrosome-reacted samples only 21.0 +/- 2.0% of the sperm were stained. Another marker for the acrosome, peanut agglutinin-lectin (PNA), was used to detect the acrosome with similar results. Acrosome reactions were verified by electron microscopy. The present results indicate that the corresponding antigen, evidently acrosin, and PNA-positive material are liberated during the acrosome reaction which suggests that they are not bound to the inner acrosomal membrane but are components of the acrosomal matrix.     

Pentoxifylline increases sperm penetration into zona-free hamster oocytes without increasing the acrosome reaction

Summary. Several drugs have been used to stimulate human sperm motility, including 3-deoxy-adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona-free hamster oocytes. Twenty-seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim-up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml⁻¹ of pentoxifylline at 37 °C, 5% CO₂ for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline-free medium, at a sperm concentration of 10 × 10⁶ cells ml⁻¹. One hundred microlitres of each sperm suspension was then deposited under oil and 30–40 zona-free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome-reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona-free hamster oocytes penetrated was increased after pentoxifylline-treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline-treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.     

Paramagnetic beads coated with Pisum sativum agglutinin bind to human spermatozoa undergoing the acrosome reaction

Summary. For the evaluation of sperm functions it is important to assess the acrosome reaction after induction with various stimuli. Acrosome reaction tests normally include the capacitation of spermatozoa, treatment with an inducer, and detection of acrosomal loss by dyes, lectins or antibodies. Since most of these methods are time-consuming or require expensive equipment, paramagnetic beads coated with Pisum sativum agglutinin (PSA) were investigated for their usefulness in facilitating the detection of human sperm acrosome reaction.
Binding of PSA beads to the acrosomal region increased significantly after incubation of capacitated spermatozoa with 10 μM A23187 (20.3±6.7% [mean±SD, absolute binding], n = 21), 1 mM dibutyryladenosine cyclic monophosphate (17.1±8.5%, n = 25) and 10 mM phorbol myristate acetate (21.1±12.5%, n = 10). Bead binding was significantly reduced by preincubation with a protein kinase inhibitor. Beads bound to Concanavalin A (ConA) were also attached to the acrosomal region after induction of the acrosome reaction by A23187 or dbcAMP, but a lower number of spermatozoa were bound to ConA-beads than to PSA beads. Pre-treatment of spermatozoa with α-methyl-D-mannoside before addition of the PSA beads markedly decreased bead binding, which indicates its mannose-specificity. Electron microscopic examinations demonstrated that PSA beads mainly bound to membrane structures of spermatozoa that were undergoing, but had not completed the acrosome reaction.     

Selective isolation of acrosome-reacted human spermatozoa with progressive motility by using cell affinity chromatography on Concanavalin A Sepharose

Summary. In order to ensure fertility, mammalian spermatozoa have to undergo acrosome reaction, the most obvious morphological change during this being the exposure of the inner acrosomal membrane. In the present study, the acrosome-reacted human spermatozoa were successfully separated without loss of viability by using cell affinity chromatography on Concanavalin A (Con A) Sepharose. Con A demonstrated affinity for both the intact and the acrosome-reacted spermatozoa regardless of their viability; the latter, however, gave higher affinity than the former against Con A. Prior to the column chromatography, the immotile spermatozoa and the seminal plasma were excluded by means of a modified swim-down procedure and the resulting spermatozoa were subsequently immobilized by slow rate cooling in ice-cold water. Cell affinity chromatography was performed at 4 °C. To prevent mechanical trapping of the spermatozoa among the packed gel beads, the column was interconnected with a reservoir, the vertical drive of which was allowed to lose the gel bed and thereby release the trapped spermatozoa. Stepwise competitive elution with 5.0 μM mannose and 25% heat-inactivated human serum was capable of separating the intact spermatozoa and the acrosome-reacted spermatozoa from each other. The acrosome reaction rate of sperm fraction which was adsorbed to Con A Sepharose and eluted with 25% serum was found to be 83±2.3%, and motility and viability of these fractions were measured to be 80±6.3% and 83±7.6%, respectively ( n = 8, mean±SD). The status of the acrosome in a final preparation (motility 92%, acrosome reaction rate 88%) was observed by scanning electron microscopy, and 81% spermatozoa lost their acrosome cap.
Acrosome-reacted spermatozoa—     

Influence of urogenital infections on sperm functions

Summary. Many studies have examined the impact of genital tract infections on male fertility; however, the effect of bacteriospermia on sperm quality is still controversial. Bacterial infections are more frequently found in semen samples from asymptomatic infertile patients than in those from fertile men. Bacteriospermia is also a common problem of male partners from couples undergoing IVF. Therefore, the effects of microorganisms on human sperm acrosome reaction of oocytes have been studied in vitro and in vivo.
Incubation of spermatozoa with Escherichia coli or Mycoplasma hominis in vitro resulted in reduced sperm motility and inducibility of acrosome reaction (ΔAR) after exposure to calcium ionophore A23187. To show possible effects of E. coli and mycoplasma species on sperm functions in vivo , data from 488 patients were evaluated, in whose ejaculates microbiological examinations and determinations of acrosome reaction after exposure to low temperature had been performed. U. urealyticum and E. coli were found in semen samples from 52 and 31 men, respectively. M. hominis was only present in a minor number of samples and was not included in this study. Semen concentrations of E. coli and U. urealyticum ranged between 500–100000 cfu x ml⁻¹ and 100–80000 cfu x ml⁻¹. No correlation was found between ΔAR and concentration of bacteria (Spearman rank correlation coefficient, E. coli : r-0.081, P = 0.6644; U. urealyticum : r = -0.081, P = 0.5698).
In 69% of cases with U. urealyticum infection and reduced inducibility of acrosome reaction, this sperm function was normal after antibiotic therapy. However, improvement of acrosomal function may only be due to intra-individual variations of acrosome reaction. While E. coli and mycoplasma species affect sperm functions in vitro , the present data and a review of the literature fail to demonstrate similar effects in vivo.     

Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa

Summary.  Bovine spermatozoa from frozenthawed semen are sensitive to lipid peroxidation. Vitamin E protects sperm membrane against oxidative damage. Sperm capacitation produces structural changes on the plasma membrane. Reactive oxygen species could be involved in the capacitation process. The aim of this work was to study the influence of natural antioxidants on the plasma membrane and the influence of reactive oxygen species during bovine sperm capacitation. Sperm samples were frozen in a standard diluent, with and without vitamin E (1 mg ml^-1). Heparin (60 μg ml^-1) was used as a sperm capacitation inductor. Sperm capacitation was evaluated by chlorotetracycline assay. Lipid peroxidation was determined by the 2-thiobarbituric acid assay. A diminution of thiobarbituric acid reactive substances was observed in sperm samples frozen with vitamin E ( P < 0.05). The addition of vitamin E to the freezing diluent had no effect on the capacitated pattern ( P > 0.05).
When vitamin E and vitamin E + vitamin C were added to the capacitation medium, a significant decrease in the percentage of capacitated spermatozoa ( P < 0.05) was observed in both cases. The addition of superoxide dismutase (0.1 mg ml^-1) or H₂O₂ (50 μM) in the incubation medium, decreased the percentage of capacitated spermatozoa ( P < 0.05). Vitamin E protects the plasma membrane against lipid peroxidation during sperm capacitation, and the presence of superoxide anion would be necessary for frozen-thawed bull sperm capacitation.     

Acrosome content release in streptolysin O permeabilized mouse spermatozoa

Summary. Sperm cell plasma membrane and the outer acrosomal membrane fuse profusely during the acrosome reaction. The process is triggered by extracellular signals that elicit several intracellular events leading ultimately to membrane fusion. We have developed a streptolysin O permeabilizing protocol that selectively affects the spermatozoon plasma membrane without causing a significant loss of the acrosomal content. Most of the acrosomal acid phosphatase remained sperm-associated even after a 20 min incubation at 37°C. However, the presence of 100 μM Ca²⁺ in the incubation buffer stimulates the release of the enzyme. The reaction was followed biochemically, measuring the acid phosphatase activity released to the medium and morphologically by the binding of fluorescein isothiocynate-conjugated peanut agglutinin and by electron microscopy. The results show that the streptolysin O permeabilized spermatozoon is a promising model for studying the complex set of events mediating and regulating the acrosome reaction.
Spermatozoa—     

Sperm-zona pellucida interaction in cynomolgus and rhesus macaques.

These experiments were carried out to establish and validate an in vitro system for studying macaque sperm-zona pellucida interaction. Sperm of rhesus and cynomolgus macaques were capacitated in vitro and incubated with cryopreserved zonae pellucidae. Homologous gamete incubations were tested, as well as cross-species combinations. Approximately 25% of macaque sperm bound to the zonae acrosome reacted within 1 minute of gamete coincubation, although the percentage of acrosome reactions in the sperm suspension was less than 1%. There was a small but consistent increase in the percent of acrosome reactions of zona sperm after an additional hour of incubation in sperm-free media. Similar results were obtained in the cross-species experiments, suggesting that zonae from the two macaque species can be used interchangeably in sperm-zona binding assays. Differences in the physiologic characteristics of the sperm of the macaque species were demonstrated. Cynomolgus sperm required activation with caffeine and dibutyryl cyclic adenosine monophosphate (dbcAMP) in order to bind to the zonae. Rhesus sperm were able to bind to the zonae and acrosome react in the absence of activators, although both sperm binding and percentage of acrosome reactions increased with the addition of activators. Large numbers of sperm from both macaque species bound to the zonae of hamster oocytes after treatment with activators, but the bound sperm did not acrosome react. These experiments demonstrate the importance of evaluating the acrosomal status of sperm when sperm-zona binding assays are performed with macaque gametes.     

Differential activities of malate and isocitrate NAD(P)-dependent dehydrogenases are involved in the induction of capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm catabolic processes produce energy for capacitation and acrosome reaction induction required for oocyte fertilization. The aim was to determine metabolic enzymes' activities and their participation in the supply of energy and generation of the redox state to acquire fertilizing capacity. Capacitation was induced with heparin and quercetin, and the acrosome reaction with progesterone. Enzymatic activities were determined spectrophotometrically. The chlortetracycline and differential-interferential contrast microscopy/tryptan blue techniques were used to evaluate capacitation and acrosome reaction, acrosomal integrity and sperm viability respectively. A 2 : 1 and 3 : 1 ratio were obtained for isocitrate dehydrogenase (IDH)-NADP/NAD and malate dehydrogenase (MDH)-NADP/NAD activities respectively. MDH-NADP activity remained constant with different treatments, unlike MDH-NAD activity, which diminished with both capacitation inducers and in acrosome-reacted spermatozoa previously treated with heparin (P < 0.05). IDH-NADP decreased its activity 50% in spermatozoa capacitated with heparin and acrosome reacted with progesterone (P < 0.05). Capacitation and acrosome reaction processes induced with heparin and progesterone, respectively, involve a differential oxidative metabolism, with the participation of MDH-NAD(P) and IDH-NAD(P) enzymes, whose activities would be linked to the malate-aspartate, lactate-pyruvate and isocitrate cytosolic-mitochondrial shuttles. These enzymes play a major role in supplying reduction equivalents and/or energy required for capacitation and acrosome reaction in cryopreserved bovine spermatozoa.     

Relationship between acrosome reaction and in vitro fertilization of zona-free hamster eggs by bonnet monkey (Macaca radiata) spermatozoa

The relationship between acrosome reaction, as studied by FITC-RCA staining technique, and the penetration of bonnet monkey spermatozoa into zona-free hamster eggs was investigated. The acrosomes of unreacted spermatozoa fluoresced, whereas those of acrosome-reacted sperm did not fluoresce owing to decreased binding of the lectin. The percentage of acrosome-reacted sperm increased following 3 h of incubation in BWW medium. The assessment of acrosome reaction by the FITC-RCA staining technique correlates well with the in vitro fertilization of zona-free hamster eggs.     

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μ m Fe²⁺/1 m m ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) ( p  < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI ( p  < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 ± 0.8% OXI vs. 17.4 ± 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.