Monoclonal antibody ACR.4 recognizing specifically the 28-kDa intra-acrosomal protein was prepared by immunization of mice with acetic acid extract of boar spermatozoa, but cross-reacted also with bull intra-acrosomal protein. This monoclonal antibody was used for immunostaining analysis of bull spermatozoa before and during capacitation and ionophore-induced acrosome reaction. Immunostaining analysis showed changes of 28-kDa protein in the acrosome during capacitation and loss of this protein after induced acrosome reaction by ionophore A23187. Therefore, this monoclonal antibody can be used in the bull spermatozoa as an immunological test for detection of the acrosome state after manipulation with spermatozoa or after freezing/thawing. This test could be useful (apart from morphology and motility) for the selection of suitable spermatozoa for insemination or in vitro fertilization.

Changes of protein tyrosine phosphorylation in ejaculated boar sperm incubated in vitro were examined with the use of antiphosphotyrosine antibodies and immunoblotting. The intracellular levels of cAMP were modulated by treatment with various combinations of caffeine, 3-isobutyl-1-methylxanthine (IBMX), and dibutyryl cyclic AMP (dbcAMP), and acrosome reactions (ARs) were induced via treatment with divalent cation ionophore A23187. Proteins of M-r 34, 38, 40, and 44 (p34... p44) were strongly phosphorylated on tyrosine residues in freshly prepared sperm samples and at the same level during all subsequent treatments. Incubation of sperm in vitro for various periods of time induced an increase of tyrosine phosphorylation of p20, p93, and p175. The tyrosine phosphorylation of p93, p175, and several other sperm proteins was up-regulated in a concentration-dependent manner following treatment of the sperm with dbcAMP, caffeine, or IBMX alone, or with combinations of caffeine and IBMX, respectively, with dbcAMP; the tyrosine phosphorylation of p20 was not correlated with treatment of sperm with cAMP-elevating reagents. The percentage of sperm cells undergoing spontaneous ARs was not affected by the manipulation of cAMP levels and was not correlated with protein tyrosine phosphorylation. In contrast, the addition of calcium to the incubation media decreased protein tyrosine phosphorylation and elevated percentage of spontaneous ARs. The induction of ARs with A23187 caused a significant decrease of tyrosine phosphorylation of p93, p175, and p220/230, indicating that dephosphorylation on protein tyrosine residues might be associated with calcium influx during physiological ARs as well. Proteins p93 and p175 were effectively solubilized in greater than 9M urea/1% triton and in SDS sample buffer, but to only a small extent in triton, while p20 was virtually completely extractable with triton. In conjunction with the previously reported isolation of active tyrosine kinase sp42 from triton extracts of noncapacitated boar sperm cells (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149-154), our results suggest that a cAMP-dependent event is required for tyrosine phosphorylation of triton-insoluble proteins such as p93 and p175. On the other hand, the tyrosine phosphorylation of p20 (and potentially other triton-soluble substrates) might not strictly require such cAMP up-regulation. We discuss the differences in the regulation of cAMP-dependent tyrosine phosphorylation in mouse, human, and boar sperm, and suggest that sensitivity to calcium and distinct basal levels of cyclic nucleotide PDE might correspond to species-specific reproduction strategies in mammals. (C) 1998 Wiley-Liss, Inc.

The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP ave added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF. (C) 1998 Wiley-Liss, Inc.

Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm acrosome were prepared by immunization of mice with acetic acid extracts of dog spermatozoa. Electron microscopy and indirect immunofluorescence localized the site of Ds-1 and Ds-2 proteins inside the acrosomal vesicle. Ds-1 antibody detected 55, 76, 115, 120 and 190 kDa proteins under non-reducing conditions, and 73 kDa and 54 kDa proteins after reduction (p73/Ds-1 and p54/Ds-1). 92 kDa and 40 kDa proteins recognized by Ds-2 (p92/Ds-2 and p40/Ds-2) migrated at > 200 kDa in the absence of reducing agent. In vivo, p73/Ds-1 and p54/Ds-1 are therefore likely to be present both in free and complexed form, while all of p92/Ds-2 and p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of acrosomes stained with Ds-1 and Ds-2 was correlated with the progress of capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal antibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capacitation and A23187 treatment compared to Ds-1 and Ds-2, indicating that proteins recognized by Ds-1 and Ds-2 are localized in a specific compartment of acrosome, distinct from acrosin and possibly representing fraction of acrosomal matrix.

Summary Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8h of initial culture) or metaphase I (12h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.

Mouse eggs arrested in metaphase II display high levels of cdc2/cyclin B1 and MAP protein kinase activities. Following fertilization there is a time-dependent decrease in the activity of each of these protein kinases. The decline in cdc2/cyclin B1 protein kinase correlates with the resumption of meiosis and the emission of the second polar body and precedes the decline in MAP kinase activity, which correlates temporally with the formation of the male and female pronuclear envelopes. These results suggest that high levels of MAP kinase activity are incompatible with the presence of a pronuclear envelope. To test this possibility, we expressed in mouse eggs a constitutively active form of MAP kinase kinase (MEK) whose only known target is p42/p44 MAP kinase. We show that following fertilization cdc2/cyclin B1 kinase activity declines and a second polar body is emitted. The endogenous MAP kinase remains active, however, and no pronuclear envelopes form. Thus, high levels of MAP kinase activity by itself in mouse eggs appear incompatible with the presence of a pronuclear envelope.

Fertilization of metaphase II-arrested mouse eggs results in resumption of meiosis and a decrease in both cdc2/cyclin B kinase and MAP kinase activities; the decrease in cdc2/cyclin B kinase activity precedes the decrease in MAP kinase activity. Cycloheximide treatment of metaphase II-arrested mouse eggs also results in resumption of meiosis but bypasses the fertilization-induced Ca2+ transient. However, it is not known if cycloheximide treatment results in the same temporal changes in cdc2/cyclin B kinase and MAP kinase activities that are intimately associated with resumption of meiosis. We report that cycloheximide-treated mouse eggs manifest similar temporal changes in the decrease in both cdc2/cyclin B kinase and MAP kinase activities that occur following fertilization, although cortical granule exocytosis is not stimulated. The decrease in cdc2/cyclin B kinase activity, however, does not seem to be required for the decrease in MAP kinase activity, since the decrease in MAP kinase activity still occurs in cycloheximide-treated eggs that are also incubated in the presence of nocodazole, which inhibits cyclin B degradation and hence the decrease in cdc2/cyclin B kinase. Following removal of these drugs, cdc2/cyclin B kinase activity remains high, MAP kinase activity increases to levels similar to that in the metaphase II-arrested eggs, and a spindle(s) forms with the chromosomes aligned on a metaphase plate. Results of these experiments suggest that some other protein with a relatively short half-life, e.g. cmos, a known upstream activator of MAP kinase, may be responsible for events leading to the decrease in MAP kinase activity.

Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+ or in a medium containing the Ca2+ ionophore A23187. This Ca2+-induced dephosphorylation was calmodulin-dependent, suggesting that calcineurin was involved. In this regard, the calcineurin inhibitor deltamethrin inhibited the Ca2+ ionophore-induced dephosphorylation. A limited number of Mr 80,000-105,000 polypeptides were the most prominent phosphotyrosine-containing proteins present in human sperm. Unlike mouse sperm, which contains a tyrosine-phosphorylated isoform of hexokinase, a phosphotyrosine-containing hexokinase in human sperm was not detected. Most of the tyrosine-phosphorylated proteins were Triton X-100-insoluble and were localized to the principal piece of the flagellum, the region where the cytoskeletal fibrous sheath is found. Prominent phosphotyrosine-containing proteins of Mr 82,000 and 97,000 were identified as the human homologues of mouse sperm AKAP82, the major fibrous sheath protein, and pro-AKAP82, its precursor polypeptide, respectively. These proteins are A Kinase Anchor Proteins, polypeptides that sequester protein kinase A to subcellular locations. Taken together, these results suggest that protein tyrosine phosphorylation may be part of a signal transduction cascade(s) regulating events pertaining to capacitation and/or motility in mammalian sperm and that an interrelationship between tyrosine kinase and cAMP signaling pathways exists in these cells.

The composition of individual human zonae pellucidae and modifications to this extracellular coat both before and after fertilization were analysed using a rapid, sensitive, non-radioactive biotinylation- or lectin-based detection system; these assays use commercially available reagents and can be performed on fragments of individual zonae pellucidae. The zona pellucida from unfertilized eggs is composed of three glycoprotein species designated as huZP1, huZP2 and huZP3. Under non-reducing conditions, the molecular weights of these proteins are approximately 150 kDa, approximately 100 kDa, and approximately 55-65 kDa respectively. Following fertilization, huZP1 was not detected under either non-reducing or reducing conditions. In contrast, after fertilization huZP2 was detected under non-reducing conditions, but not under reducing conditions. The ability to detect pre- and post-fertilization changes in a single human zona pellucida is discussed in relation to its value in assessing deficiencies in clinical and laboratory protocols used for in-vitro fertilization.

Changes in the activities of the p34cdc2/cyclin B complex and mitogen-activated protein (MAP) kinase were analyzed after insemination of mouse eggs in vitro. Whereas histone H1 kinase activity (p34cdc2/cyclin B) fell to negligible levels by 90 min postinsemination, a decrease to negligible levels of myelin basic protein kinase activity (i.e., MAP kinase) was not observed until about 7 h postinsemination. The decrease in MAP kinase activity appeared to be linked to the prior decline in p34cdc2/cyclin B kinase activity, since inhibiting the fertilization-induced destruction of cyclin B by treating eggs with the microtubule inhibitor nocodazole prevented the decrease in each of these protein kinases; an intact spindle is required for cyclin destruction. Moreover, experimental elevation of MAP kinase activity by okadaic acid treatment under conditions that maintain negligible levels of p34cdc2/cyclin B kinase activity suggested that MAP kinase could be involved in pronuclear envelope dynamics. Specifically, preventing the fertilization-induced decrease in MAP kinase activity was correlated with inhibiting pronucleus formation, and elevating MAP kinase activity subsequent to pronucleus formation resulted in precocious pronuclear envelope breakdown prior to entry into M phase.

Three monoclonal antibodies that stained acrosin of 55, 53, 45 and 38 kDa (ACR.2), acrosomal protein of 28 kDa (ACR.4) and sperm protein of 17 kDa (ACR.3) were used for immunostaining and immunoblotting analysis of boar spermatozoa before and during capacitation and ionophore-induced acrosome reaction. Two proteins (acrosin and the 28 kDa protein) showed similar changes in the acrosome during capacitation and induced acrosome reaction. For detection of these proteins, immunoelectron microscopy was also used. The immunofluorescence test revealed that most spermatozoa lost acrosin and the 28 kDa protein after incubation with ionophore A 23187. The lost proteins were detected in the culture medium by immunoblotting. During capacitation and induced acrosome reaction, sperm protein of 17 kDa (ACR.3) showed a different pattern of labelling. The changes in relocation of specific proteins during capacitation and acrosome reaction support a concept of physiological preparation of spermatozoa for sperm-egg interaction.

A rapid, nonradioactive method to monitor the ZP2 to ZP2f conversion in the zona pellucida of single mouse eggs has been developed. This assay is based on the chemiluminescent detection of biotinylated ZP2 and ZP2f following electrophoresis under reducing conditions and electrophoretic transfer to Immobilon P. This method is about 10 times faster and detects similar extents of ZP2 to ZP2f conversion following A23187‐induced egg activation, when compared to the commonly used radioiodination procedures. © 1994 Wiley‐Liss, Inc.

The progesterone-induced Ca2+ influx and acrosomal exocytosis in human sperm are recently described examples of non-genomic steroid action on the cell surface. These progesterone effects are known to be inhibited by synthetic protease inhibitors. In this study we tested a hypothesis that a physiological activator of the sperm protease acrosin modulates the sperm response to progesterone. It was found that the activator augments the amplitude of the progesterone-induced Ca2+ transient and accelerates the progesterone-induced acrosomal exocytosis. These observations suggest a physiological significance of the modulation of nongenomic steroid effects by protease regulators.

In this study we used previously characterized monoclonal antibodies to acrosin (ACR.2) and to an acrosomal matrix antigen (ACR.4) to analyze the acrosin-binding activity of a 28-kDa putative acrosin-binding protein from the acrosomal matrix. The 28-kDa protein bound proacrosin and the 49-kDa form of acrosin (alpha-acrosin) but it did not bind the 36-kDa acrosin form (beta-acrosin). The acrosin-binding activity of the 28-kDa protein was stimulated by Ca2+, inhibited by Mg2+, and removed by disulphide bond reduction. Induction of the acrosome reaction by a calcium ionophore resulted in proteolytic cleavage of the 28-kDa protein, giving rise to a 12-kDa degradation product that was the only form of ACR.4 antigen released to incubation media; the release of the ACR.4 antigen was closely correlated with that of acrosin. The release of alpha-acrosin to incubation media was accelerated in the presence of ACR.4 antibody. In a cell-free system, a limited cleavage of the purified 28-kDa protein into immunoreactive degradation products was catalyzed by acrosin but not by trypsin or chymotrypsin. The data suggest that the 28-kDa acrosomal protein helps to maintain acrosomal matrix integrity and controls the acrosin release from acrosome-reacted cells.

In this study we used a previously characterized monoclonal antibody to analyze the molecular conversions of acrosin during the acrosomal exocytosis induced by ionophore A23187. Before sperm exposure to the ionophore, most of the sperm acrosin was in the form of proacrosin (55-kDa and 53-kDa forms). Upon exposure to the ionophore, the concentration of proacrosin in sperm samples decreased rapidly and was negatively correlated with the progression of exocytosis. After 1 h of ionophore treatment, proacrosin was quantitatively converted into the two active acrosin forms, alpha-acrosin (49 kDa) and beta-acrosin (36 kDa). However, products of further acrosin conversions were not found after this treatment. As compared with the speed of acrosin activation during sperm contact with the ionophore, the ionophore-induced release of acrosin from the sperm cells into the soluble fraction was apparently delayed, and only the active acrosin forms (49 kDa and 36 kDa) were found in sperm incubation media. External Ca2+ influenced the speed of proacrosin conversion in a concentration-dependent manner. The ionophore-induced activation of proacrosin and acrosome reaction were partially inhibited by trypsin inhibitors. The results suggest that proacrosin activation is an essential step in the mechanism of the acrosomal exocytosis.

Mature human sperm initiate a rapid Ca2+ influx and the acrosomal exocytosis in response to progesterone. Recent evidence indicates that both events can be induced by antibody-mediated cross-linking of a sperm surface progesterone receptor. In many other systems in which signal is generated by receptor cross-linking, protein phosphorylation on tyrosine residues is involved in the signal transduction across the plasma membrane. In this study we examined whether tyrosine phosphorylation is implicated in the function of the sperm surface progesterone receptor, too. The effect of progesterone on the phosphorylation of proteins from a sperm membrane lysate was evaluated by in vitro kinase assay and by phosphoamino acid analysis using [gamma-32P]ATP as precursor. These experiments revealed a selective increase in the tyrosine phosphorylation of a 94-kilodalton phosphoprotein in the presence of progesterone. To decide whether the progesterone-induced increase in protein tyrosine phosphorylation is actually due to the hormone action on the cell surface, living sperm were treated with a cell-impermeant progesterone receptor agonist, and the resulting changes in the cellular level of phosphotyrosine proteins were examined. These experiments showed a clear relationship between the agonist binding and an increase in the phosphotyrosine concentration in the respective cells. This relationship was lost in the presence of genistein, which also efficiently inhibited the phosphorylation of the 94-kilodalton protein and the progesterone-induced acrosomal exocytosis. These results lead to the hypothesis that protein tyrosine phosphorylation is involved in signal transduction through the sperm surface progesterone receptor and may be implicated in nongenomic steroid effects in other cell types.

OBJECTIVE:To evaluate the implication of fucose binding sites on the sperm surface in the sperm-zona pellucida (ZP) binding by examining the ability of a fucosylated neoglycoprotein to compete with solubilized zona proteins for sperm binding.DESIGN:The binding of a fucosylated neoglycoprotein to spermatozoa was analyzed in relation to sperm capacitation and the actual acrosomal status. The effect of solubilized ZP material on the binding was examined.SETTING:Private hospital, public research center, and university department.PATIENTS, PARTICIPANTS:Healthy volunteers with normal sperm parameters.INTERVENTIONS:None.MAIN OUTCOME MEASURES:Fluorescence signal detected by fluorescence-activated cell sorting or fluorescence microscopy on spermatozoa incubated with fluorescein isothiocyanate-labeled fucosylated neoglycoprotein.RESULTS:There was a small subpopulation of spermatozoa that showed a strong binding activity for the fucosylated neoglycoprotein, and the size of this subpopulation increased with the time of exposure to capacitating conditions. The binding sites were located on the head (mainly on the acrosomal region) of acrosome-intact spermatozoa. The neoglycoprotein binding was competitively inhibited by solubilized ZP.CONCLUSIONS:The data show that a commercially available fucosylated neoglycoprotein competes with solubilized ZP proteins for binding sites on the sperm surface. Further research into the relationship between the neoglycoprotein binding and zona binding is warranted to evaluate the potential diagnostic usefulness of this neoglycoprotein.