Transmembrane adenylyl cyclase regulates amphibian sperm motility through protein kinase A activation.

Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation.

ß-hexosaminidase from Xenopus laevis eggs and oocytes: from gene to immunochemical characterization.

Glycosidases are present both in sperm and eggs in vertebrates and have been associated with different fertilization steps as gamete binding, egg coat penetration, and polyspermy prevention. In this manuscript, we have analyzed the activity of different glycosidases of Xenopus laevis eggs. The main activity corresponded to N-acetyl-ß-D-glucosaminidase (Hex), which was reported to participate both in gamete binding and polyspermy prevention among phylogenetically distant animals. We have raised homologous antibodies against a recombinant N-terminal fragment of a X. laevis Hex, and characterized egg's Hex both by Western blot and immunohistochemical assays. Noteworthy, Hex was mainly localized to the cortex of animal hemisphere of full-grown oocytes and oviposited eggs, and remained unaltered after fertilization. Hex is constituted by different pair arrangements of two subunits (α and ß), giving rise to three possible Hex isoforms: A (αß), B (ßß), and S (αα). However, no information was available regarding molecular identity of Hex in amphibians. We present for the first time the primary sequences of two isoforms of X. laevis Hex. Interestingly, our results suggest that α- and ß-like subunits that constitute Hex isoforms could be synthesized from a same gene in Xenopus, by alternative exon use. This finding denotes an evolutionary divergence with mammals, where α and ß Hex subunits are synthesized from different genes on different chromosomes.

The integrin-binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (Amphibia) oocytes.

Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and ß1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-ß1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. ß1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation.

Cellular origin of the Bufo arenarum sperm receptor gp75, a ZP2 family member: its proteolysis after fertilization.

BACKGROUND INFORMATION: The egg envelope is an extracellular matrix that surrounds oocytes. In frogs and mammals, a prominent feature of envelope modification following fertilization is the N-terminal proteolysis of the envelope glycoproteins, ZPA [ZP (zona pellucida) A]. It was proposed that ZPA N-terminal proteolysis leads to a conformational change in egg envelope glycoproteins, resulting in the prevention of polyspermy. Bufo arenarum VE (vitelline envelope) is made up of at least four glycoproteins: gp120 (glycoprotein 120), gp75, gp41 and gp38. The aim of the present study was to identify and characterize the baZPA (B. arenarum ZPA homologue). Also, our aim was to evaluate its integrity and functional significance during fertilization. RESULTS: VE components were labelled with FITC in order to study their sperm-binding capacity. The assay showed that gp75, gp41 and gp38 possess sperm-binding activity. We obtained a full-length cDNA of 2062 bp containing one ORF (open reading frame) with a sequence for 687 amino acids. The predicted amino acid sequence had close similarity to that of mammalian ZPA. This result indicates that gp75 is the baZPA. Antibodies raised against an N-terminal sequence recognized baZPA and inhibited sperm-baZPA extracted from VE binding. This protein does not induce the acrosome reaction in homologue sperm. Northern-blot studies indicated that the transcript is exclusively expressed in the ovary. In situ hybridization studies confirmed this and pointed to previtellogenic oocytes and follicle cells surrounding the oocyte as the source of the transcript. baZPA was cleaved during fertilization and the N-terminal peptide fragment remained disulfide bonded to the glycoprotein moiety following proteolysis. CONCLUSION: From the sequence analysis, it was possible to consider that gp75 is the baZPA. It is expressed by previtellogenic oocytes and follicle cells. Also, it can be considered as a sperm receptor that undergoes N-terminal proteolysis during fertilization. The N-terminal peptide could be necessary for sperm binding.

Sperm binding glycoprotein (SBG) produces calcium and bicarbonate dependent alteration of acrosome morphology and protein tyrosine phosphorylation on boar sperm.

The oviduct is a dynamic organ which modulates gamete physiology. Two subpopulations of sperm have been described in the oviduct of sows, a majority with normal appearance in the deep furrows and a minority, centrally located, and showing damaged membranes. Sperm-oviduct interaction provides the formation of a sperm storage and allows the selection of sperm with certain qualities. Pig (Sus scrofa) oviductal sperm binding glycoprotein (SBG) binds to sperm and exposes Gal beta1-3GalNAc. This disaccharide may be recognized by boar spermadhesin AQN1, which seems to be involved in sperm interaction with the oviduct. SBG is present at the apical surface of the epithelial cells that surround the lumen of the oviduct rather than at the bottom of the crypts. These characteristics imply it could be involved in sperm interaction with this organ. In this study, we evaluate the effect of SBG over boar sperm. We show that the presence of SBG produces alterations of the acrosome morphology of sperm only when they are incubated in capacitating conditions. SBG binds to the periacrosomal region of sperm undergoing capacitation. Its presence induces an increase on the tyrosine-phosphorylation of a polypeptide of apparent molecular mass 97 kDa, as occurs with a 95 kDa protein in other mammalian sperm upon acrosomic reaction. Altogether, these results suggest that SBG might be involved in sperm selection by alteration of the acrosome of sperm that have already begun the capacitation process when they arrive to the oviduct.

Sperm binding glycoprotein is differentially present surrounding the lumen of isthmus and ampulla of the pig's oviduct.

In several mammals a sperm reservoir is formed at the isthmus of the Fallopian tube, providing viable, potentially fertile sperm for an extensive period. In pig (Sus scrofa) the spermadhesin AQN-1 seems to be involved in the establishment of the sperm reservoir. The pig oviductal protein, sperm binding glycoprotein (SBG), binds to sperm and exposes carbohydrate groups that can be recognized by AQN-1. In this study we obtain anti-SBG polyclonal antibodies and use them to localize SBG in the oviduct. Immunohistochemical analysis shows that SBG is present at the apical surface of isthmic and ampullar epithelial cells. The presence of SBG is limited to the upper two-thirds of the crypts of the isthmus and to cells located near the oviductal lumen in the ampulla. The ratio of the amount of SBG detected by western blot is 1:3 (ampulla:isthmus). Sperm entering the Fallopian tube probably contact the epithelial cells at the lumen before they reach the cells at the bottom of the folds. In vitro sperm can bind to isthmus and, at less extent, to ampulla. Thus, the localization and the relative amount of SBG in the isthmus and ampulla of pig's oviduct are compatible with its possible function in sperm binding to oviductal epithelial cells.

SOME FEMALE COORDINATING FACTORS IN AMPHIBIAN FERTILIZATION.

The influence of volume and ionic composition of the hydrating media upon the fertility of Bufo arenarum oocyte strings, has been studied with the following results: a) when immersed in equal volumes of ionic and non-ionic media, oocyte strings have been found to become more hydrated in the latter ones; b) oocytes are no longer fertile when hydration is 300-400% of their original weight (critical hydration). Fertility is recovered by adding ions or egg water to the medium or by modifying its pH; c) when hydration is beyond 500% of the original string weight (extensive hydration) fertility can also be recovered by these treatments, but only after a partial jelly-coat liquefaction by thioglycolate. Egg water was also studied, and shown to have a buffering capacity between pH 5.5 and 7.0. It seems not to influence acrosomic proteinase release.

Effect of Phosphatidylcholine on Acrosome Breakdown and Fertilizing Capacity of Amphibian Spermatozoa: (phosphatidylcholine vesicles/amphibian fertilization/acrosome breakdown).

The effect of vesicles of purified egg yolk phosphatidylcholine on the fertilizing capacity and acrosome breakdown of amphibian spermatozoa was studied. When Bufo arenarum spermatozoa were incubated with either small unilamellar vesicles (prepared by sonication) or with large unilamellar vesicles (prepared by reverse-phase evaporation) a decrease in the fertilizing capacity of spermatozoa was found. At the same phosphatidylcholine concentration, large unilamellar vesicles were more inhibitory than small unilamellar vesicles. The inhibition was dependent upon the phospholipid concentration and the length of the incubation period. Small unilamellar vesicles did not modify the time course of acrosome breakdown in Leptodactylus chaquensis, while large unilamellar vesicles markedly accelerated the rate of acrosome breakdown. In both biossays, the charge of the vesicles (made either positive or negative by the addition of 5% stearylamine or 5% phosphatidic acid) did not influence their biological effect. Multilamellar vesicles did not alter the fertilizing capacity nor the acrosome breakdown. We conclude that the size and the structure of the vesicles are important parameters in determining the inhibitory capacity of phosphatidyl choline on amphibian fertilization.

ACROSOME BREAKDOWN IN LEPTODACTYLUS CHAQUENSIS (AMPHIBIA ANURA) SPERMATOZOA.

Acrosome breakdown in Leptodactylus chaquensis is described: during this process acrosome enlarges, becomes round-shaped and finally disrupts. Low tonicity media (0.025 M sucrose and 1/10 Holtfreter's solutions) favor acrosome breakdown and sperm fertility loosing. High tonicity media (0.250 M sucrose and Holtfreter's solutions) maintain acrosomes in an unreacted stage and sperm fertilizing capacity is preserved. Sperm motility does not seem to be a sufficient condition for the sperm to fertilize and also does not seem to be related with acrosome breakdown. The presence of lectins in the incubation media does not modify the time-course of acrosome breakdown.

The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum.

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.

Vitellogenesis in Bufo arenarum: identification, characterization and immunolocalization of high molecular mass lipovitellin during oogenesis.

Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100 kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190 kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late).

Dynamics of heparin-binding proteins on boar sperm.

The presence, topology and dynamics of heparin-binding proteins (HBP) on boar sperm were evaluated. HBP distribution was analyzed by subcellular parting, using biotinylated heparin followed by colorimetric detection. HBP were detected as peripherical and integral periacrosomal membrane proteins. Indirect fluorescence microscopy of sperm incubated with biotinylated heparin was used to evidence heparin binding on sperm at different physiological stages. Two different fluorescent patterns (A and B) were found, which probably correspond to non-capacitated and capacitated sperm as assessed by the ability to undergo acrosome reaction with calcium ionophore A23187 and by the increase of p32 phosphorylated protein. In A pattern, corresponding to untreated sperm, fluorescence located mostly on the post-acrosomal region; in B pattern, corresponding to incubated sperm, on the acrosomal region. Upon incubation under capacitating conditions (TALP), sperm having the B pattern was augmented compared with non-incubated sperm (p<0.001). Differences in the HBP patterns (p<0.0001) were observed in sperm incubated under non-capacitating conditions in relation to sperm incubated in TALP, indicating that the modification of HBP patterns is probably related to capacitation. No difference was observed when untreated sperm were permeabilized prior to staining, suggesting that HBP are present on the sperm surface. The effect of heparin on capacitation dependent protein tyrosine phosphorylation was also analyzed, finding a decrease in p32 phosphorylation in the presence of heparin. This suggests that the capacitation enhancement mediated by this glycosaminoglycan involves an alternative intracellular pathway. The finding that heparin binds to sperm differently according to its physiological state, is a new evidence of the remodelling of sperm membrane surface upon capacitation and may provide a useful and relatively simple method to evaluate in vitro modification of boar sperm physiological state.

Human tubal secretion can modify the affinity of human spermatozoa for the zona pellucida.

OBJECTIVE: To study the effect of the human tubal tissue conditioned medium (CM) on sperm parameters related to sperm-zona pellucida interaction. DESIGN: Controlled experimental laboratory study. SETTING: Research laboratory. SUBJECT(S): Semen samples from donors with normozoospermia. Human tubal tissue obtained from women undergoing hysterectomies. Human follicular fluids (hFF) and oocytes collected from patients undergoing IVF-ET. INTERVENTION(S): Incubation of spermatozoa with CM proteins obtained from human tubal tissue culture; sperm binding to the zona pellucida assessment. MAIN OUTCOME MEASURE(S): Explants' viability was assessed by tissue DNA analysis. Sperm ability to interact with zona was tested with use of the whole oocyte test. Expression of d-mannose binding sites was assessed with use of a fluorescent probe on mannose coupled to bovine serum albumin. Human FF-induced acrosome reaction was assessed by the Pisum sativum technique. RESULT(S): Although treatment with 0.8 microg/microL of CM allowed sperm binding to the zona and the expression of d-mannose binding sites comparable with sperm in control medium, with 3.2 microg/mL of CM resulted in a significant decrease of both parameters. No effect of CM on spontaneous or hFF-induced acrosome reaction or in sperm viability was observed. CONCLUSION(S): The results indicate that the incubation of spermatozoa in the presence of CM reduces sperm affinity for the zona pellucida. This effect can be partly explained by the decreased expression of d-mannose binding sites on the sperm surface.

Egg water from the amphibian Bufo arenarum modulates the ability of homologous sperm to undergo the acrosome reaction in the presence of the vitelline envelope.

Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed "egg water" (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca(2+) stores (induced by thapsigargin) promoted [Ca(2+)](i) rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca(2+) chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni(2+) and mibefradil prevented [Ca(2+)](i) rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca(2+) channel.

Effect of human oviductal in vitro secretion on human sperm DNA integrity.

PURPOSE: In the female genital tract spermatozoa interact with the oviductal secretion. The aim of the study was to evaluate the effect of conditioned media (CM) from cultures of human oviductal tissue, on sperm DNA integrity. The effect of H(2)O(2) on sperm DNA integrity, before and after incubation under capacitating conditions, was also investigated. METHODS: Motile sperm obtained from normozoospermic semen samples were incubated (4 h or 22 h) in the presence or absence of CM and further exposed to H(2)O(2). DNA damage was detected by the comet assay. RESULTS: The CM significantly reduced the DNA damage associated with sperm incubation, and also decreased the effect of H(2)O(2) after 4 h incubation, compared to controls. The H(2)O(2) caused a dose-dependent deleterious effect on sperm DNA integrity both before and following 22 h of capacitation. CONCLUSION: The oviductal tissue CM increased the stabilization of the sperm DNA structure under culture conditions.

Egg water from the amphibian Bufo arenarum induces capacitation-like changes in homologous spermatozoa.

Mammalian sperm acquire fertilizing capacity after residing in the female tract, where physiological changes named capacitation take place. In animals with external fertilization as amphibians, gamete interactions are first established between sperm and molecules of the egg jelly coat released into the medium. Since dejellied oocytes are not normally fertilized, the aim of this study was to determine if the jelly coat of the toad Bufo arenarum promotes a "capacitating" activity on homologous sperm. We found that sperm incubation in diffusible substances of the jelly coat (egg water) for 90-180 s is sufficient to render sperm transiently capable of fertilizing dejellied oocytes. The fertilizing state was correlated with an increase of protein tyrosine phosphorylation and a decrease of sperm cholesterol content. Inhibition of either the increase in tyrosine phosphorylation or cholesterol efflux affected the acquisition of fertilizing capacity. Phosphorylation and fertilization could be promoted with NaHCO(3) and also by addition of beta cyclodextrin. Moreover, sperm could gain the ability to fertilize dejellied oocytes in the presence of these compounds. These data indicate that sperm should undergo a series of molecular changes to gain fertilizing capacity; these changes are reminiscent of mammalian sperm capacitation and take place before the acrosome reaction.

Glycoproteins of the vitelline envelope of Amphibian oocyte: biological and molecular characterization of ZPC component (gp41) in Bufo arenarum.

The vitelline envelope (VE) participates in sperm-egg interactions during the first steps of fertilization. In Bufo arenarum, this envelope is composed of at least four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa and molar ratio of 1:1.3:7.4:4.8, respectively. These components were isolated and covalently coupled to silanized glass slides in order to study their sperm-binding capacity. When considering the molar ratio of the glycoproteins in the egg-envelope and assuming that each protein is monovalent for sperm, the assay showed that gp41 and gp38 possess 55 and 25% of total sperm-binding activity. We obtained a full-length cDNA of gp41 (ZPC), comprising a sequence for 486 amino acids, with 43.3% homology with Xenopus laevis ZPC. As in the case of mammalian ZP3 and Xenopus ZPC, Bufo ZPC presented a furin-like (convertase) and a C-terminal transmembrane domain (TMD) reflecting common biosynthetic and secretory pathways. As it was reported for some fishes, we obtained evidence that suggests the presence of more than one zpc gene in Bufo genome, based on different partial cDNA sequences of zpc, Southern blots and two-dimensional SDS-PAGE of deglycosylated egg-envelope components. As far as we are aware, this is the first observation of the presence of different zpc genes in an Amphibian species.

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm.

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.

Characterization and biological properties of L-HGP, a glycoprotein from the amphibian oviduct with acrosome-stabilizing effects.

BACKGROUND INFORMATION: The role of the jelly coat that surrounds the amphibian oocytes has been widely discussed, but is poorly understood. The presence of the jelly coat is essential for fertilization. However, the structure and function of the molecules that comprise the jelly coat have not been thoroughly documented. L-HGP (low-molecular-mass highly glycosylated protein) is a highly glycosylated protein that is present in the jelly coat of the toad, Bufo arenarum, oocytes and diffuses to the surrounding media. L-HGP, when purified from egg water, protects the sperm acrosome from breakdown induced by hypotonic solutions. RESULTS: L-HGP is an acidic glycoprotein, formed by two different subunits, linked by disulphide bonds. We raised polyclonal antibodies in rabbits against the deglycosylated protein. We determined that L-HGP is secreted along the oviduct, being hence present in all the jelly layers. The molecular mass of L-HGP is higher in the most cephalic region of the oviduct. The lower-M(r) L-HGP isoform, produced in the caudal regions of the oviduct, presents an acrosome-protecting property. L-HGP is produced by secretory cells in the oviduct and is deposited on the cilia at the oviduct lumen. CONCLUSIONS: Biochemical characterization of L-HGP has been carried out. It is synthesized by secretory cells in the oviduct and, when secreted, is deposited over the ciliated surface of the cells. The lower-M(r) isoform, secreted by the caudal region of the oviduct, protects acrosome integrity. This isoform diffuses into the medium. The role of the higher-M(r) L-HGP isoform in fertilization remains unknown.

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.