Characterization of low Mr zona pellucida binding proteins from boar spermatozoa and seminal plasma.

A group of low Mr (16 kDa-23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N-terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin-ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract.     

Calcium-dependent binding of mouse epididymal spermatozoa to the zona pellucida.

The minimal requirements and characteristics of epididymal sperm binding to the zona pellucida of the mouse egg were investigated using a new stop-fix centrifugation technique. This assay provided a precise physical definition of the association between the spermatozoon and the zona and permitted quantitation of the binding reaction at short time intervals. The results demonstrated that Ca²⁺ is an essential physiological component required for binding to occur. Sperm preincubated for 60 min in a simplified medium lacking Ca²⁺ did not acquire the ability to bind to eggs. In contrast, if sperm preincubation occurred in this medium supplemented with 1.7 mM Ca²⁺, binding was identical to that observed following sperm preincubation in the complete culture medium which supports both capacitation and fertilization in vitro. The Ca²⁺-dependent binding reaction was rapid, reversed by EGTA, specific for Ca²⁺, and did not require the transport of Ca²⁺ into the cell. Sperm bound to the zona surface following preincubation with Ca²⁺ were capable of fertilization in vitro when the eggs were subsequently transferred to the culture medium. It is proposed that this binding reaction represents a part of capacitation and not the acrosome reaction.     

Identification of 17-18 kDa zona pellucida binding proteins from boar spermatozoa

Zona pellucida (ZP) binding proteins from boar spermatozoa were compared with antigens recognized by ACR.2 and ACR.3 monoclonal antibodies. The ZP binding proteins of 55, 53, 45 and 38 kDa are identical with various forms of boar acrosin immunologically detected by ACR.2 antibody. The ZP binding proteins of 17 and 18 kDa are recognized by ACR.3 antibody. The N-terminal amino acid sequence of the 17 kDa protein revealed that it is not derived from an acrosin molecule.     

Interaction of boar spermatozoa with porcine oocytes: Increase in proteins with high affinity for the zona pellucida during epididymal transit

Methods for the investigation of cell-associated calcium and intracellular calcium were studied in washed ejaculated human spermatozoa. Experiments using ⁴⁵Ca²⁺ indicated that human spermatozoa were permeant to calcium and that a significant proportion of the cellassociated calcium (approximately 50%) was accumulated in the mitochondrion. This necessitated the use of alternative procedures to measure cytoplasmic free calcium. The ability of human spermatozoa to accumulate and de-esterify the intracellular fluorescent calcium indicator Quin-2 was established. Using this technique, the resting level of free intracellular calcium in human spermatozoa was found to be 146.0 ± 19.9 nM, and was significantly elevated upon addition of the divalent cation ionophore ionomycin. In further experiments designed to illustrate the applications of the Quin technique, data was obtained suggesting that the mechanisms controlling intracellular calcium in human spermatozoa are temperature dependent but do not involve voltage-sensitive calcium channels.     

Capacity of boar spermatozoa to bind zona pellucida proteins in vitro in relation to fertilization rates in vivo

The purpose of this study was to determine variation among boars in the percentage of sperm in an ejaculate that express enhanced binding of zona pellucida proteins during treatment for capacitation in vitro, and to determine whether this relates to fertilizing ability in vivo. Ejaculates (n=35) were collected from 12 boars. A sample of each ejaculate was treated for capacitation in vitro. During incubation, the zona binding ability of spermatozoa was assessed at regular intervals with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP) and propidium iodide, using a flow cytometer. After incubation, a percentage of the sperm had enhanced FITC-sZP binding. The percentage of viable sperm with enhanced FITC-sZP binding, expressed as a percentage of the total sperm population, increased rapidly over the first 60 min and thereafter reached a plateau after 120-180 min. Averaged over all ejaculates, the percentage at 180 min was 46% (range 27-61%); this percentage was significantly different among boars. However, the variation between ejaculates within a boar was relatively small. There was no significant boar effect on the rate at which the percentage of viable cells with enhanced FITC-sZP binding reached the maximum. In ejaculates (n=14) from four boars (selected from the group of 12), we investigated the increase in the percentage of viable sperm with enhanced sZP binding during treatment for capacitation in vitro in relation to the ability to fertilize in vivo. Sows (n=44) were inseminated 4 h after ovulation with a suboptimal insemination dose (0.5x10(9) spermatozoa). Time of ovulation was determined using transrectal ultrasonography and sows were killed at 120 h after ovulation. The percentage of fertilized oocytes, embryo development, and numbers of accessory spermatozoa were determined. The percentage of spermatozoa that were viable and showed enhanced sZP binding after 180 min of incubation was 48 +/- 12% (range 28-56%). The percentage of fertilized oocytes was 85 +/- 27% and 64% of the sows had 100% fertilized oocytes. The percentage of sows with 100% fertilized oocytes correlated well (P< or =0.05, R2=0.98) with the percentage of viable spermatozoa with enhanced FITC-sZP binding after capacitation in vitro.     

Effect of recombinant boar beta-acrosin on sperm binding to intact zona pellucida during in vitro fertilization

In a previous paper we demonstrated that boar beta-acrosin recombinant proteins were able to bind non-enzymatically to solubilized pig zona pellucida (ZP) glycoproteins. Here we report the participation of boar beta-acrosin in the secondary binding of sperm to intact pig ZP. This was achieved by using two boar recombinant proteins: beta-acrosin and a mutant of the catalytic site, beta-acrosin Ser/Ala(222). Assays of binding between the iodinated recombinant beta-acrosin and whole ZP showed that this binding could be saturated, was specific, and was stable over time. Using autoradiography, we determined that recombinant beta-acrosin bound on the entire surface of the ZP but initially was distributed heterogeneously. This suggests that the ligands for beta-acrosin may not be homogeneously distributed on the ZP. To study the contribution of acrosin in sperm secondary binding to the ZP, we preincubated in vitro-matured oocytes with these recombinant proteins and then performed in vitro fertilization assays. Under the experimental conditions used, binding of beta-acrosin recombinant proteins did not block sperm penetration. These results suggest that there may be other proteins that participate in the secondary binding, and that these proteins may recognize ligands that are different from those blocked by beta-acrosin recombinant proteins.     

Adsorption of seminal plasma proteins by boar spermatozoa

Seminal plasma protein adsorption by boar spermatozoa was examined using ejaculated sperm from vesiculectomized boars and seminal plasma from vasectomized boars. Sperm adsorbed 14 pg protein/sperm in 10 min. When seminal plasma proteins were radiolabeled, most of the adsorbed radiolabel was present in low M(r) proteins, particularly a 12700 M(r) protein. A 349300 M(r) seminal plasma protein was also readily adsorbed. Three radiolabeled seminal plasma proteins (307600, 165400 and 7400 M(r)) were not detected on the sperm; either they are not adsorbed by the sperm or the sperm were previously exposed to these proteins in other accessory sex gland fluids and had already adsorbed them. A 29100 M(r) sperm protein was also radiolabeled (4.9% of the adsorbed radiolabel), although there was no corresponding seminal plasma protein. Large quantities of seminal plasma protein (particularly low M(r) proteins) are adsorbed by sperm not previously exposed to seminal vesicle secretion. The functions of these proteins are yet to be determined.     

Microfilaments appear in boar spermatozoa during capacitation in vitro

Boar spermatozoa were incubated in a capacitation medium and examined for the presence of filamentous actin by using the fluorescent probe NBD-phallacidin. F-actin was not observed in uncapacitated sperm, but developed in most regions of the cell during the capacitation period. Fluorescent staining was most intense in the flagellum. When fresh seminal plasma was added to capacitated sperm and the sperm was further incubated, F-actin was no longer observed. In view of previous experiments which indicated that plasma membrane proteins (PMPs), including a major integral PMP, move out of the sperm head into the flagellum during capacitation and that this movement is inhibited by the microfilament poison cytochalasin D (Peterson, Saxena, Saxena, and Russell: Biol. Reprod., in press, '86), we suggest that actin-PMP interactions play a major role in capacitating boar spermatozoa.     

Electron microscopic study of the in vivo zona pellucida binding ability of epididymal ram spermatozoa

In the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit.     

Stabilizing rôle of epididymal plasma in relation to the capacitation time of boar spermatozoa

Functional aspects of the maturation of epididymal spermatozoa have been examined by means of surgical insemination of two types of sperm suspension directly into the oviducts. Suspensions were prepared by macerating tissue from the upper corpus region of the epididymis, and cell-free plasma was prepared from the contents of the cauda epididymidis. Each comparison of the fertilizing ability of the two sperm suspensions was made within the same animal, known numbers of upper corpus spermatozoa in either medium TCM 199 or caudal plasma being instilled into separate oviducts close to the time of ovulation.Activated eggs were recovered from 11 of 12 inseminated animals some 4–6 h later, but within the intervals examined there was a distinct difference in the fertilizing ability of the two types of sperm suspension; 87% of the eggs were activated by upper corpus spermatozoa in TCM 199 compared with 9% of the eggs exposed to similar spermatozoa suspended in caudal plasma. Furthermore, the fertilization process was invariably more advanced when eggs had been activated by the upper corpus spermatozoa suspended in TCM 199, and the number of spermatozoa on or in the zona pellucida was likewise consistently higher with such sperm suspensions. The rôle of the factor(s) in cauda epididymal plasma contributing to the observed delay in fertilizing ability is discussed in the context of sperm transport and capacitation after natural mating.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding

Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.     

Influence of seminal plasma on the kinematics of boar spermatozoa during freezing

Sperm motility is, for its relation to cell viability and fertility, a central component of the spermiogram, where consideration of motion patterns allows discrimination of sub-populations among boar spermatozoa. Extension and cryo-preservation imposes changes in these patterns in connection to handling, additives, temperature changes and the removal of boar seminal plasma (BSP) which apparently makes spermatozoa susceptible to oxidative stress, thus affecting survival and motility post-thaw. Detailed kinematic analyses during sperm cooling are sparse, particularly when considering the instrumentation and settings used for analyses, the effect of extenders, and of the BSP the processed spermatozoa are exposed to. Spermatozoa present in the first collectable 10mL of the sperm-rich fraction of the ejaculate (portion 1, P1-BSP), have shown an increased ability to sustain motility during and after cryo-preservation than spermatozoa immersed in the rest of the ejaculate (portion 2, P2). When P2-spermatozoa were cleansed from their BSP and exposed for 60min to pooled P1-BSP, their motility post-thaw increased to similar levels as P1-spermatozoa. This BSP-influence is sire-dependent, presumably related to the protein concentration in the different ejaculate portions, and apparently unrelated to changes in membrane integrity or membrane stability through conventional, controlled cooling.     

Isolation, characterization, and localization of the zona pellucida binding proteins of boar sperm

In order to isolate the putative zona pellucida-binding proteins (ZPBPs) from boar spermatozoa, a new, simple method has been developed. The new isolation strategy made the most of the highly specific interactions between the components of the gametes. Detergent-extracted boar sperm proteins were submitted to affinity chromatography on a ZP-Sepharose column. SDS-PAGE analyses of the retained fraction under reducing conditions revealed that in addition to a component of Mr 38,000, the predominant ZPBPs contained at least three low molecular weight proteins (Mr less than 20 kDa). The isolated ZPBPs were effective in blocking sperm-oocyte binding in vitro. Using immunofluorescence microscopy, the ZPBPs were shown to be localized primarily in the sperm head, especially in the acrosomal cap region.     

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

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

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Identification of anti-agglutinin for spermatozoa in epididymal boar plasma

The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs–Ringer bicarbonate at 37°C (5% CO₂ in air). In the samples washed three or five times and then incubated for 3–5 h, higher rates (72–79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2–5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. © 1994 Wiley-Liss, Inc.     

A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa

Background

Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0.

Result

We identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction.

Conclusion

The results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.     

Interaction of human spermatozoa with the human zona pellucida and zona-free hamster oocyte following capacitation by exposure to human cervical mucus

The functional capacity for sperm interaction with the human zona pellucida and zona-free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed-capacitated sperm suspensions were more successful than mucus-capacitated sperm in attaching to the zona-free hamster oocyte and in fusing with its oolemma. However, the ability of mucus-capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.