Chondroitin sulfate facilitates an acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in vitro fertilization

Bovine epididymal spermatozoa were incubated for 22 h in a modified Tyrode's medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. The addition of chondroitin sulfate A (CS-A) significantly increased the incidence of acrosome reaction. When observed by electron microscopy, acrosome-reacted sperm had undergone vesiculation of the plasma and outer acrosomal membranes. Sperm incubated in the presence of CS-A demonstrated a significantly higher incidence of vesiculation when compared to the controls. Additionally, rates of in vitro fertilization of bovine oocytes were significantly elevated when sperm and ova were exposed to CS-A. These results suggest that glycosaminoglycans in the female reproductive tract may be responsible for some of the biochemical changes associated with fertilization, and a light microscope procedure can be used to assess occurrence of the acrosome reaction.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

Interactions of seminal plasma and glycosaminoglycans on acrosome reactions in bovine spermatozoa in vitro

Previous reports indicate that glycosaminoglycans (GAGs) would enhance the occurrence of acrosome reactions in sperm in vitro, but continuous exposure of those sperm to seminal plasma prevented a significant incidence of acrosome reactions. This study was designed to evaluate the interaction of GAGs and seminal plasma to promote acrosome reactions in bull sperm in vitro. Epididymal sperm required 22 hr to exhibit acrosome reactions in response to GAGs whereas only 9 hr were needed to achieve the same effect with washed ejaculated sperm. Exposure of epididymal sperm to seminal plasma for 20 min shortened the time for induction of the acrosome reaction to 9 hr. Scatchard analyses of displacement data suggested an alteration in the binding affinity of ³H-heparin to epididymal sperm membrane following the short-term exposure to seminal plasma. High doses (250 and 500 μg/ml) of heparin, heparan sulfate, and chondroitin-4-sulfate were without effect, but doses <100 μg/ml were stimulatory in terms of enhancing acrosome reactions. Compositional studies with seminal plasma revealed a total GAG content of 1.6 mg/ml, proportioned as 61.6% chondroitin sulfates, 17.6% heparin-like material, 0.3% hyaluronic acid, and 20.5% undetermined GAG. It is proposed that seminal plasma can alter the ability of sperm to respond to GAGs, and the high concentrations of GAGs endogenous to seminal plasma may prevent premature initiation of the membrane perturbations necessary for the acrosome reaction.     

Identification of chondroitin sulfate E in human lung mast cells

Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans.     

Penetration by bull spermatozoa into the zona pellucida of dead bovine oocytes recovered from frozen-thawed ovaries

The present study was carried out to determine if the zona pellucida of dead bovine oocytes obtained from ovaries stored at -196 degrees C could be used to assess penetrability of capacitated bull spermatozoa. Follicular oocytes were recovered from bovine ovaries which were frozen slowly in a box containing dry ice, plunged into liquid nitrogen, and thawed at 37 degrees C. The dead oocytes were inseminated with various concentrations of spermatozoa preincubated for 0 to 4 h. Sperm penetration rates of the dead oocytes were significantly altered by sperm concentration and preincubation time. Dead and living oocytes matured in vitro (control) gave similar patterns of penetrability based on sperm preincubation time. When sperm concentration was increased, the rate of multiple sperm penetration into the dead oocytes also increased significantly, but the rate of penetration into living oocytes did not alter significantly. All dead oocytes from ovaries stored at -196 degrees C for 1 d to 3 mo were penetrated at similar rates by spermatozoa preincubated for 1-h. Thus, we conclude that dead follicular oocytes recovered from frozen ovaries are useful for the assessment of sperm capacitation and/or the acrosome reaction in cattle.     

Effects of metalloendoprotease substrates on the human sperm acrosome reaction

The substrates carbobenzyloxyserylleucylamide, carbobenzyloxyglycylleucylamide and carbobenzyloxyglycylphenylalanylamide were used as potential competitive inhibitors of endogenous metalloendoprotease activity. When the acrosome reaction was elicited by a potential physiological stimulus, human follicular fluid, each of the substrates (1-1.5 mM) inhibited exocytosis. Carbobenzyloxyserylleucylamide also inhibited the acrosome reaction when exocytosis was stimulated using the calcium ionophore ionomycin, but carbobenzyloxyglycylleucylamide was not inhibitory and carbobenzyloxyglycylphenylalanylamide actually enhanced exocytosis under these conditions. Experiments using the fluorescent indicator fura-2 revealed that the increase in intracellular, free calcium stimulated by follicular fluid in human spermatozoa was depressed by carbobenzyloxyglycylphenylalanylamide but not by carbobenzyloxyserylleucylamide. The peptide carbobenzyloxyglycylglycylamide, which is not a substrate for metalloendoproteases, had no effect on the acrosome reaction, whether stimulated by follicular fluid or ionomycin. While the results with carbobenzyloxyserylleucylamide suggest a possible involvement of a metalloendoprotease in the human sperm acrosome reaction, our other results demonstrate that these carbobenzyloxy peptides have complex effects on the process of exocytosis in human spermatozoa, and suggest caution in interpretation of data obtained using such peptides on intact cells.     

Acrosin activation follows its surface exposure and precedes membrane fusion in human sperm acrosome reaction

Evidence has accumulated suggesting multiple roles of acrosin in fertilization, including its participation in early steps of gamete recognition and binding. However, the implication of acrosin in many of these processes is not compatible with its presumptive sequestration within the sperm acrosome until a late phase of the acrosome reaction. In an earlier study (J. Tesarik, J. Drahorad, J. Peknicova, 1988, Fertil. Steril. 50, 133-141), we reported the binding of an anti-acrosin monoclonal antibody (MO-AKR.1) to the plasma membrane overlying the acrosome of human spermatozoa starting the acrosome reaction. In this study, we characterized further this antibody with regard to its reactivity with different forms of acrosin and found that it recognizes specifically an active form of this enzyme and does not react with its proenzyme form. MO-AKR.1 was thus used as a probe for in situ analysis of acrosin activation during the acrosome reaction. When suspensions of living spermatozoa were incubated with MO-AKR.1 and with another monoclonal antibody (T6) directed to an intra-acrosomal cytoskeletal protein, significantly more spermatozoa reacted with the former antibody than with the latter; this indicated that some of the spermatozoa showing acrosin immunoreactivity carried activated acrosin on the cell surface, while their acrosome was still impermeable to intra-acrosomal-directed probes. The size of this particular sperm subpopulation was increased by the action of follicular fluid (a natural acrosome reaction inducer), but not ionophore A23187 (an artificial acrosome reaction inducer); it corresponded to the proportion of spermatozoa showing acrosin immunoreactivity on the plasma membrane but neither intra-acrosomal staining nor perceptible membrane perturbations when examined by immunoelectron microscopy. When spermatozoa were pre-incubated with protease inhibitors before the addition of acrosome reaction-inducing agents, the percentage of cells binding MO-AKR.1 was markedly reduced. These data suggest that limited acrosin activation on the sperm plasma membrane is an early event in the physiological acrosome reaction.     

Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro

Significant differences in the frequency of spontaneous and induced acrosome reactions (identified by electron microscopy) were detected between 5 individual sperm samples. Maximal stimulation of the acrosome reaction was achieved with ionophore A23187, no differences being detected between the two preincubation times used (5 and 15 h). The cumulus oophorus and follicular fluid caused a similar increase in the proportion of acrosome-reacted spermatozoa after 5 h of preincubation. It is concluded that specific products of the cumulus and/or granulosa cells may contribute to the acrosome reaction-inducing activity of human follicular fluid.     

Evidences for the presence of chymotrypsin-like activity in human spermatozoa with a role in the acrosome reaction

The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 10⁷ cells/ml, 37°C, and 5% CO₂, After 4.5 hr, the chymotrypsin inhibitor TPCK (N-Tosyl-L-Phenylalanine-Chloromethyl Ketone) or the substrate ATEE (N-Acetyl-L-Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome-reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida-induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester), Succinyl-Ala-Ala-Phe-7-Amido-4-Methyl-Coumarin (Suc-Ala-Ala-Phe-AMC), and Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methyl-Coumarin (Suc-Leu-Leu-Val-Tyr-AMC) inhibited the human follicular fluid-induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca²⁺, and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin-like activity with a possible role in the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Chemotactic response of frozen-thawed bovine spermatozoa towards follicular fluid

The aim of this study was to verify whether cattle spermatozoa respond by chemotaxis to follicular fluid (FF). The experimental conditions were defined to maintain a frozen-thawed sperm population with great motility and capacitation, and lesser sperm agglutination. Several sperm preparation conditions were studied: sperm separation from the seminal plasma by Sephadex column or migration-sedimentation, incubation under capacitating conditions in the presence or absence of a superficial layer of mineral oil, and different pH of the culture medium. The percentage of motile and agglutinated spermatozoa was determined in plate dishes under inverted phase contrast microscope. The percentage of capacitated spermatozoa was calculated as the difference between the percentages of acrosome reacted spermatozoa with and without lysophosphatidylcholine stimulation. The most ideal experimental conditions to evaluate chemotaxis in frozen-thawed cattle spermatozoa were: to separate the cells from the seminal plasma by migration-sedimentation and to incubate them under oil, in culture medium at pH 7.2, for less than 2h. The chemotaxis assays were conducted with spermatozoa treated as mentioned above which were confronted to several dilutions of FF (1:10(3), 1:10(4), 1:10(5), 1:10(6)) in a chemotaxis chamber by videomicroscopy and computer image analysis. A subpopulation of capacitated spermatozoa ( approximately 10%) that responded chemotactically to a concentration gradient generated by FF (1:10(4) to 1:10(5)) was observed. Since cryopreserved spermatozoa are regularly used to artificially inseminate the cows, the sperm chemotactic response towards FF would be potentially used to diagnose the bull sperm sample or to select the spermatozoa in the most functional state.     

The effect of semen extension, cAMP and caffeine on in vitro fertilization of bovine oocytes

A previously reported in vitro system that used epididymal spermatozoa for fertilizing bovine follicular oocytes (1) has been expanded to include ejaculated semen as the sperm source. Frequency of fertilization was higher when semen was extended 1:1 prior to transport to the laboratory rather than transport as neat semen. Pretreatment of spermatozoa with cAMP, caffeine or both prior to insemination of oocytes did not increase frequency of either acrosome reactions or fertilization after sperm/oocyte incubation.     

猪精子体外获能与顶体反应的超微结构研究

用４种方法,检测了猪精子体外获得的效果。结果证明：高离子浓度的前培养液和猪镦泡液,具有促进获能过程的作用,实验还获得了获能后顶体反尖的一些重要的形态学变化资料,包括质膜的膨胀、断裂、顶体膨胀、顶体外膜内陷或原位局部囊泡化,质膜再全部丢失。顶体内膜直到与卵母细胞质膜融合,才发生可见的变化。受精过程无论体内或体外,都容易发生多精入卵,体外受精则更甚。在精子穿过卵丘细胞之间时,一方面开始进行顶体反应,另     

The site of the acrosome reaction during in vivo penetration of the sheep oocyte

In vivo fertilization of sheep eggs has been studied by electron microscopy. Remnants of the acrosome reaction were present at the zona surface of every penetrated egg, indicating that the acrosome reaction in sheep occurs at the surface of the zona pellucida. To determine whether follicular oocytes could specifically bind spermatozoa, oocytes isolated from different size classes of antral follicles were transferred into the oviducts of mated ewes, recovered 4 hr 30 min later, and analyzed by electron microscopy. Oocytes from follicles up to 1 mm in diameter failed to bind spermatozoa and were not penetrated. In contrast, the zona of oocytes from follicles ? 2 mm in diameter induced the acrosome reaction. These oocytes were penetrated but failed to achieve cortical granule exocytosis and so to mount a block to polyspermy. Moreover, sperm nuclei incorporated into the ooplasm did not decondense although the sperm nuclear envelope was dispersed.     

Effects of platelet activating factor on the motility and acrosome reaction of bovine spermatozoa

The effects of platelet activating factor (PAF) on motility and the acrosome reaction of ejaculated bull spermatozoa were evaluated. Washed spermatozoa (30 x 10(6)/ml) were incubated (39 degrees C) for up to 2 h with 10 to 200 muM PAF in a modified Tyrode's solution (pH 7.4) containing 3 mg/ml bovine serum albumin. Sperm motility was evaluated subjectively and by computer-assisted semen analysis. Percent acrosome-reacted spermatozoa was quantified microscopically from fixed smears following Giemsa staining. Percent fertilization by PAF-treated spermatozoa was determined using in vitro-matured bovine ova. Percent sperm motility decreased with >/= 50 muM PAF, while the rate of motility loss increased with PAF concentration (P<0.001). Percent acrosome reactions increased with PAF concentration during incubation (P<0.001). Acrosomal loss was rapid and complete with 200 muM PAF. At concentrations between 80 to 120 muM PAF, bull spermatozoa underwent acrosome reactions without a rapid loss of motility and penetrated in vitro-matured bovine ova at a rate comparable to that of heparin-capacitated spermatozoa (68 versus 54%, respectively). Incubation of bull spermatozoa with 10 to 50 muM PAF for 45 min had no effect on percent progressive motility, sperm velocity or other motility parameters. These results indicate that PAF can be used to induce acrosome reactions in bull spermatozoa and to promote in vitro fertilization of bovine ova. Under the conditions used in this study, PAF did not stimulate bovine sperm motility.     

Evidence for nonmuscle myosin in bovine ejaculated spermatozoa

Mammalian spermatozoa contain nonmuscle actin and many of the components of regulatory systems thought to be involved in nonmuscle actin-myosin function. An actin-stimulated adenosine triphosphate hydrolase (ATPase) activity has been fractionated from bovine ejaculated spermatozoa by immobilized-actin affinity chromatography. The actinstimulated ATPase activity has a specific activity (0.04 ± 0.02 mM phosphate released/min/mg protein) similar to nonmuscle myosins from other mammalian cells and tissues, but it does not have appreciable K⁺-EDTA ATPase activity. The sperm actin-myosin may function in sperm morphogenesis in the seminiferous tubule, in capacitated spermatozoa undergoing an acrosome reaction, or in decondensation of the sperm nucleus after fertilization.     

Isolation and characterization of glycosaminoglycans from bovine follicular fluid and their effect on sperm capacitation

The majority of published studies have reported the use of commercial heparin to capacitate bovine sperm. However, heparin is not present in the female genital tract fluids. In this study, we purified large amounts of glycosaminoglycans (GAGs) from bovine follicular fluid (FF), characterized them and determined their potential to capacitate sperm. FF-GAGs were isolated by protease digestion, lipid extraction, and by different precipitation conditions and then purified by ion exchange chromatography. Two GAGs, heparan sulfate and chondroitin sulfate B, were present in FF. To determine the capacitation potential of FF-GAGs, bovine ejaculated sperm were incubated 5 hr with or without 12 or 24 microg/ml of each of the FF-GAG fractions or with heparin (12 microg/ml). The purified FF-GAGs and heparin did not stimulate sperm acrosome reaction (AR), but stimulated sperm capacitation. Fractions 1 and 2 (heparan sulfate) were more active to promote capacitation (stimulated up to 3.2-fold) than fractions 3 and 4 (mostly chondroitin sulfate B). Fractions 3 and 4 stimulated capacitation two times more than the control (without FF-GAGs or heparin). When the heparan sulfate impurity was removed from fractions 3 and 4 by acid hydrolysis, the capacitation-promoting activity associated with these fractions did not change significantly. When 24 microg/ml of fraction 1 or 2 were used, the percentage of sperm capacitation observed was similar to the capacitation with 12 microg/ml of heparin. Our results also indicated that the FF-GAGs interact strongly with the BSP proteins. Therefore, it is concluded that heparan sulfate is the GAG that is the most potent capacitating factor present in bovine FF.     

Effects of bovine oviductal proteins on bull spermatozoal function

The effects of bovine oviductal proteins on bull sperm viability, acrosome reaction and motility were studied. Motile frozen/thawed spermatozoa from Percoll gradients were incubated with 1.0 mg/mL oviductal proteins (>8 kDa) extracted by ammonium sulphate precipitation from oviductal extract (OE) or serum-free oviductal epithelial cell-conditioned media (CM), treated in the presence (CM+) or absence (CM-) of 1 microg/mL 17beta-estradiol. Inclusion of oviductal proteins had a significant beneficial effect on sperm viability (76.3 to 80.6%+/-5.3) compared with the control (without oviductal proteins; 57.8%+/-5.3) immediately after the commencement of incubation. After 5 h, viability was significantly higher for CM- and OE treatments than for the control, although no differences were observed at 24 h. Acrosomal status only differed among treatments after 24 h, when higher percentages of acrosome- reacted spermatozoa were found in the control (46.0%+/-2.5) than in the oviductal protein treatments (33.1 to 38.2%+/-2.5). No differences in percentages of motile spermatozoa occurred within the first hour of incubation, although inclusion of CM proteins decreased sperm velocities, beat cross frequency, linearity, and straightness but increased values for mean angular displacement. These findings suggest that proteins secreted by oviductal epithelium promote viability, delay the acrosome reaction and suppress the motion of spermatozoa.     

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.