Sperm acrosome antigen-1. Specific tissue distribution of immunoreactivity in a teleost fish,the swordtail ( Xiphophorus helleri)

The acrosome reaction is a fundamental requirement for mammalian fertilization. Its exact molecular mechanisms and cellular elements are still poorly understood. We have detected an acrosomal sperm antigen, SAA-1, by monoclonal antibodies directed against SAA-1, that appears to be critically involved in the regulation of the acrosome reaction. SAA-1 is conserved within a broad range of mammalian species, emphasizing its important role in mammalian reproduction. Here we demonstrate that SAA-1 is immunohistochemically detectable in a nonmammalian vertebrate whose sperm do not possess an acrosome. In the swordtail, a live-bearing teleost with special reproductive tactics, we were able to demonstrate immunoreactivity of sperm heads of spermatids and mature sperm in the testis using monoclonal antibodies against SAA-1. Due to the cystic spermatogenesis with synchronous sperm maturation, immunoreactive maturational stages could be clearly identified. Stored immunoreactive sperm were also identified in spermathecal tissue of the female genital tract. Interestingly, immunoreactivity was furthermore detected in defined cells of the compounded endocrine organs pituitary and endocrine pancreas. All these different cell systems are involved in paracrine regulation and exhibit exocytotic properties. The possible nature of SAA-1 is discussed. Additionally, some new aspects of the morphologic composition of the swordtail pituitary are described.     

Mouse sperm antigens that participate in fertilization. I. Inhibition of sperm fusion with the egg plasma membrane using monoclonal antibodies

Monoclonal antibodies (mAbs) have been generated to determine the sperm components responsible for interaction with an egg that results in fertilization. Here, we report upon a group of six different mAbs, all of which localize to a restricted region of the sperm head, the equatorial segment. Several of these mAbs demonstrated cross-reactivity with sperm from the other species tested (human, hamster, rabbit); when cross-reaction occurred, the mAb distribution was restricted to the equatorial segment despite the various configurations that this homologous region assumes in different species. When tested for an effect upon the fertilization process in vitro, ascites fluids containing two of the six mAbs, M29 and M37, displayed significant inhibition. The concentration dependency of this inhibition was observed using purified M29 immunoglobulin M, over a range of 0 to 0.2 mg/ml. The mAb inhibition of fertilization was independent of the presence of either the cellular (the cumulus) or acellular (the zona pellucida) layers surrounding the egg, indicating that the specific locus of inhibition for both of these antisperm mAbs was the egg plasma membrane. Immunologic detection of sperm components separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose sheets was used to identify the sperm components recognized by two of the mAbs in this group: M29, which inhibited fertilization, and M2, which did not inhibit fertilization. Using M29 mAb, a single sperm component with an apparent subunit molecular weight of approximately 40,000 was detected, whereas in the nitrocellulose strips incubated with M2 mAb two components displayed reactivity, a very prominent band at approximately 44,000 and a tight cluster of bands at approximately 36,000. Parallel nitrocellulose strips of mouse liver did not display these reactivities, consistent with indirect immunofluorescence data in which only testis and sperm, and not liver, kidney, ovary, and epididymal epithelium, demonstrated positive reactivity. These results indicate that the use of mAbs permits identification of sperm components that participate, putatively, in individual events of the fertilization process. Furthermore, using this strategy, we have identified a specific sperm component that appears to be a candidate for a role in sperm fusion with the egg plasma membrane.     

Mouse sperm antigens that participate in fertilization. IV. A monoclonal antibody prevents zona penetration by inhibition of the acrosome reaction

To investigate the molecular basis of gamete interaction in mammals, monoclonal antibodies (mAbs) have been generated by syngeneic immunization with mouse testis. Previous work has described two particular mAbs, M41 and M42, which localize indistinguishably to the plasma membrane overlying a restricted portion of the acrosome, but recognize different antigens. One of the mAbs, M42, inhibits mouse fertilization in vitro significantly, but only in the presence of the zona pellucida, whereas M41 has no apparent effect upon any assayable event in the fertilization process. The experiments described here were performed to identify the precise event of sperm-zona interaction (sperm-zona binding, induction of the acrosome reaction, or penetration through the zona) that is affected by M42 mAb. Capacitated mouse sperm binding to the zona pellucida was undiminished following pretreatment with M42 mAb, when compared to levels achieved using either no mAb- or to M41 mAb-treated control sperm. When the effect of mAbs on the zona-induced AR was examined, the percentage of acrosome reacted (AR) sperm at the zona surface increased with time, plateauing at approximately 90 min post-insemination, with 78% of the bound cells AR in the control and the M41 mAb-treated groups. M42-treated sperm never achieved greater than 23% AR cells over the 120-min interval assayed. To quantitate this effect, capacitated sperm were exposed to increasing concentrations of acid-solubilized zonae. Increased proportions of AR sperm were found in the control and M41 mAb-treated groups, up to a maximum of 70-76% AR cells with 8 or 12 zonae/microliter. In contrast, M42-treated sperm displayed only 21-28% AR cells over the entire range of zonae concentrations tested. An entirely different result emerged when acrosome reactions were induced with A23187: M42 was no longer able to prevent the AR. This ability of A23187 to override M42 mAb's inhibitory effect on the AR permitted specific examination of the possible effect of M42 mAb on sperm penetration through the zona pellucida. In the presence of A23187, zona penetration levels for M42 mAb-treated sperm were equivalent, both qualitatively and quantitatively, to control and to M41 mAb-treated sperm under the same conditions. It appears, therefore, that M42 mAb identifies a high molecular weight doublet (220-240 kDa) of mouse sperm that participates specifically in the induction of the sperm's acrosome reaction as it occurs under physiological conditions.     

Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion

Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.     

Mouse sperm antigens that participate in fertilization. II. Inhibition of sperm penetration through the zona pellucida using monoclonal antibodies

To dissect the process of mammalian sperm interaction with the egg at a molecular level, we have generated monoclonal antibodies (mAbs) to mature mouse sperm using syngeneic mouse testis as the immunogen. In this paper, we report upon three members of a mAb family, all of which displayed identical immunofluorescence patterns on cauda epididymal mouse sperm. Each of these mAbs, termed M42, M5, and M41, localized to a restricted region of plasma membrane overlying the acrosome. When tested for an effect on the fertilization process in vitro, two of the mAbs, M42 and M5, demonstrated significant inhibition. The inhibitory capacity was dependent upon the presence of the zona pellucida; neither M42 nor M5 was capable of blocking fertilization when zona pellucida-free mouse eggs were used. Identification of the antigens recognized by this group of mAbs was achieved by immunologic detection of sodium dodecyl sulfate-extracted sperm components separated via electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose. M42, which blocked fertilization, recognized a high molecular weight cluster of bands with Mr of approximately 220,000 to 240,000. M5, which also prevented fertilization, specifically recognized a sperm component with subunit molecular weight of approximately 54,000. M41, which did not interfere with fertilization, did not interact with any high molecular weight components, but recognized components with Mr of approximately 60,000, 35,000, and 21,000. Taken together with the work presented in a companion paper (Saling, Irons, and Waibel, this issue), we have demonstrated that it is possible to describe particular cellular regions of mammalian sperm with respect not only to location and function, but also to the molecules that are candidates for a role in that function.     

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.     

Anti-idiotype antibodies prevent antibody binding to mouse sperm and antibody-mediated inhibition of fertilization

Antibodies to components of sperm can interfere with sperm function and prevent fertilization by blocking specific steps of gamete interaction. It can be proposed that anti-idiotype antibodies (anti-Ids) that recognize determinants located close to or within the antigen-binding site of an anti-sperm antibody could block antibody binding to sperm antigen and antibody-mediated inhibition of fertilization. To test this hypothesis, rabbit polyclonal antibodies to idiotypic determinants of the monoclonal anti-sperm antibody M42.15 were developed and characterized. Previous studies demonstrated that M42.15 monoclonal antibody (mAb) inhibits fertilization in vitro and in vivo by inhibiting sperm-zona pellucida interaction. Anti-idiotype antibodies to M42.15 mAb (anti-Id M42) were isolated by affinity chromatography on immobilized M42.15 mAb. Binding specificity of anti-Id M42.15 was demonstrated in a solid-phase radioimmune binding assay and by specific immunoprecipitation of soluble M42.15 mAb. Anti-Id M42 competitively inhibited M42.15 mAb, but not P220.2 mAb, binding to mouse sperm, confirming that the anti-Id preparation contained antibodies directed against idiotopes within or adjacent to the antigen-binding site of the mAb. At equimolar concentrations, anti-Id M42 inhibited binding of 125I-labeled M42.15 mAb to sperm by greater than 80%. These results showed that anti-Id M42 efficiently blocked antibody binding to sperm and suggested that anti-Id M42 could be used to neutralize the anti-fertility activity of the M42.15 mAb. When tested in in vitro fertilization assays, anti-Id M42, but not rabbit immunoglobulin, prevented M42.15 mAb-induced inhibition of fertilization. Together, these results show that the inhibitory activity of anti-sperm antibodies capable of interfering with gamete interaction can be neutralized by anti-Id that recognize determinants close to the antigen-combining site of the anti-sperm antibody.     

Changing localizations of site-specific surface antigens during sea urchin spermiogenesis

Whole mount preparations of dissociated testicular cells from the sea urchin, Strongylocentrotus purpuratus, were exposed to monoclonal antibodies (mAbs) directed against sperm surface proteins. Indirect immunofluorescence microscopy and Western immunoblot analysis show that mAb J18/29 binds to the entire surface of the mature spermatozoon and membrane proteins ranging in relative molecular masses from 25 to 340 kDa. MAb J18/2 binds to the acrosomal and tail regions of the mature spermatozoon and mainly to a 210-kDa membrane protein. MAb J17/30 binds to the midpiece and tail regions and monospecifically to a 60-kDa membrane protein. MAb J16/33 binds specifically to the sperm midpiece but does not bind to Western immunoblots of sperm membrane proteins. With the exception of J16/33, which shows a punctate binding pattern, all of these mAbs show uniform binding over the entire surface of the early spermatid. This uniform and complete surface binding is observed through all stages of spermiogenesis for mAb J18/29. By the midspermatid stage, when tail formation first begins, but before the nucleus condenses and the cytoplasm decreases in volume, localized binding patterns of mAbs J17/30 and J16/33 become evident. Localized binding of mAb J18/2 is not observed until the late spermatid stage. These results show that the sea urchin sperm surface is composed of at least four different domains and provide the first insight into differentiation of the cell surface during sea urchin spermatogenesis.     

A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida

After the acrosome reaction, the PH-20 surface antigen of guinea pig sperm migrates from its original location on the posterior head surface to a new location on the inner acrosomal membrane (Myles, D.G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). We have isolated three monoclonal antibodies (MAbs) of the IgG1 subclass, PH-20, PH-21, and PH-22, that bind to the PH-20 antigen. The PH-20 MAb strongly inhibited (approximately 90%) sperm binding to the guinea pig egg zona pellucida at saturating antibody concentrations (greater than 20 micrograms/ml). Half-maximal inhibition of sperm binding to the zona was obtained with approximately 2 micrograms/ml PH-20 MAb. The PH-21 MAb at saturating concentration (50 micrograms/ml) partially inhibited (approximately 45%) sperm-zona binding, and the PH-22 MAb (50 micrograms/ml) did not inhibit (0%) sperm-zona binding. Essentially the same amounts of the three MAbs were bound to sperm under the conditions where inhibition (PH-20, PH-21) or no inhibition (PH-22) of sperm-zona binding was observed, which indicates that the different levels of inhibition did not arise from different levels of MAb binding. Competition binding assays with 125I-labeled MAbs showed that PH-21 binding to sperm was not affected by the binding of PH-20 or PH-22. However, that PH-20 and PH-22 blocked each other's binding to sperm suggests that their recognized determinants may be relatively close to one another. The results indicate that the migrating PH-20 antigen has a required function in sperm binding to the zona pellucida and that the PH-20 MAb affects is active site.     

Monoclonal antibodies to rabbit sperm autoantigens. I. Inhibition of in vitro fertilization and localization on the egg

Biochemical and antigenic similarities exist among members of what can be considered a family of low molecular weight rabbit sperm autoantigens. These autoantigens are intrinsic plasma membrane glycoproteins specific to spermatogenic cells and spermatozoa. The amino acid and carbohydrate compositions of rabbit sperm autoantigen-1 (RSA-1) and RSA-2 were compared and monoclonal antibodies (mAb) were raised in mice against rabbit sperm autoantigens. The epitopes recognized by the antibodies were present on RSA-1, 2 and 3. A monoclonal anti-RSA-1, 2 and 3 (designated A.F. 1) was used to localize the antigen on spermatozoa and testis cells and investigate the epitope's tissue specificity. This mAb inhibited in vitro fertilization but did not block the sperm from dispersing the cumulus cells surrounding the egg. The mAb also demonstrated the presence of RSA-1, 2 and 3 on the plasma membrane of the egg after fertilization. It is concluded that the RSA family plays a central role in zona penetration.     

SNAREs in mammalian sperm: possible implications for fertilization

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Monoclonal antibodies to a membrane glycoprotein induce the phosphorylation of histone H1 in sea urchin spermatozoa

Two groups of mAbs reacting with external domains of a major sea urchin sperm membrane glycoprotein of 210 kD were isolated. Previous studies have shown that group I mAbs inhibit the acrosome reaction induced by egg jelly and also cause large increases in intracellular Ca2+ [( Ca2+]i). Group II mAbs, at comparable levels of cell surface binding, neither inhibit the egg jelly-induced acrosome reaction nor cause increases in [Ca2+]i. In this paper, we investigate the ability of these mAbs to induce the cAMP-dependent phosphorylation of sperm histone H1. Group I mAbs induce H1 phosphorylation to the same level and on the same peptide, as occurs upon treatment of sperm with egg jelly. These mAbs also activate adenylate cyclase to the same extent as egg jelly. Group II mAbs do not induce H1 phosphorylation and are only poor activators of adenylate cyclase. Group I mAbs compete with each other, but not with group II mAbs, for binding to the cell surface. These data indicate that the activation of adenylate cyclase is an initial event in the pathway leading from the binding of mAbs to a specific domain of the 210-kD protein at the cell surface, to the discrete phosphorylation of histone H1 in highly condensed sperm chromatin. The domain on the 210-kD protein recognized by group I mAbs plays a critical role in signal transduction during the early events of fertilization.     

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.     

Analysis of a sperm surface molecule that binds to a vitelline envelope component of Xenopus laevis eggs

To analyze sperm surface molecules involved in sperm–egg envelope binding in Xenopus laevis, heat‐solubilized vitelline envelope (VE) dot blotted onto a polyvinylidene difluoride (PVDF) sheet was incubated with a detergent extract of sperm plasma membrane (SP‐ML). The membrane components bound to the VE were detected using an antibody library against sperm plasma membrane components, and a hybridoma clone producing a monoclonal antibody (mAb) 16A2A7 was identified. This mAb was used in a Far Western blotting experiment in which VE was separated by electrophoresis, and then transferred to a PVDF strip that was incubated with SP‐ML. It was found that SP‐ML binds to the VE component gp37 (Xenopus homolog of mammalian ZP1). The antigens reactive to mAb 16A2A7 showed apparent molecular weights of 65–130 and 20–30 kDa, and were distributed relatively evenly over the entire sperm surface. Periodate oxidation revealed that both the pertinent epitope on the sperm surface and the ligands of VE gp37 were sugar moieties. VE gp37 was exposed on the VE surface, and the mAb 16A2A7 dose‐dependently inhibited sperm binding to VE. The sperm membrane molecules reactive with mAb 16A2A7 also reacted with mAb 2A3D9, which is known to recognize the glycoprotein SGP in the sperm plasma membrane and is involved in interactions with the egg plasma membrane, indicating that the sperm membrane glycoprotein has a bifunctional role in Xenopus fertilization. Mol. Reprod. Dev. 77: 728–735, 2010. © 2010 Wiley‐Liss, Inc.     

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     

Protein dynamics in sperm membranes: implications for sperm function during gamete interaction.

A number of mammalian sperm plasma membrane antigens have been implicated as playing a functional role in sperm-egg interaction, by virtue of the fact that antibodies against these antigens interfere with fertilization. Two such mouse sperm plasma membrane antigens are M42, a 200/220 kD glycoprotein doublet, and M5, a 150-160 kD glycoprotein. We show that both of these antigens are concentrated on the posterior region of caudal epididymal and capacitated mouse sperm heads and are relatively diffusible, as determined by fluorescence recovery after photobleaching measurements (D = 3-8 x 10(-9) cm2/s with approximately 23% diffusing). Crosslinking of these antigens with bivalent antibodies causes them to redistribute into the anterior region (acrosomal crescent) of the sperm head. In contrast, we describe a third antigen, P220, which is also localized to the posterior region of the sperm head on caudal epididymal sperm but which exhibits very little diffusion and does not redistribute upon crosslinking. Bivalent anti-M42 blocks the ZP3-induced acrosome reaction. We have found that monovalent Fab fragments of anti-M42 do not block the ZP3-induced acrosome reaction, but that inhibition is restored by addition of a second antibody which crosslinks the Fabs. Thus, crosslinking is required for both inhibition of the acrosome reaction and redistribution. This suggests that redistribution of antigen away from the posterior region of the head may be part of the mechanism of inhibition of the ZP3-induced acrosome reaction.     

Fertilization in a chiton: Acrosome-mediated sperm-egg fusion

Contrary to the widely accepted view that chiton sperm lack acrosomes and that fertilization in this group occurs via a micropyle, we demonstrate here that fertilization in Tonicella lineata occurs by acrosome-mediated sperm-egg fusion. The acrosome is a small vesicle containing two granules located at the tip of the sperm. The eggs have an elaborate hull (=chorion), which is formed into cupules that remain covered by follicle cells until maturity. When dissected ripe eggs were exposed to sperm in vitro, the sperm were attracted only to open cupules, inside which they swam through one of seven channels to the base where they penetrated the hull. The acrosome fired on contact with, or in, the hull, and during passage through it the apical granule was exhausted while the basal granule was exposed. If sperm contacted follicle cells between the cupules the acrosome did not react. The vitelline layer beneath the hull contains pores arranged in a regular pattern. Embedded in the base of each pore is an egg microvillus. Having penetrated the hull the sperm anterior filament located a pore and fused with the tip of the egg microvillus projecting into it. This created a membranous tube, through which the sperm nucleus was injected into the egg. The egg membrane appeared to be raised up into a small fertilization cone around the penetrating sperm, the vitelline layer became slightly elevated, and some cortical granules were released by exocytosis.     

Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization

We have previously defined distinct localizations of antigens on the surface of the guinea pig sperm using monoclonal antibodies. In the present study we have demonstrated that these antigen localizations are dynamic and can be altered during changes in the functional state of the sperm. Before the sperm is capable of fertilizing the egg, it must undergo capacitation and an exocytic event, the acrosome reaction. Prior to capacitation, the antigen recognized by the monoclonal antibody, PT-1, was restricted to the posterior tail region (principle piece and end piece). After incubation in capacitating media at 37 degrees C for 1 h, 100% of the sperm population showed migration of the PT-1 antigen onto the anterior tail. This redistribution of surface antigen resulted from a migration of the surface molecules originally present on the posterior tail. It did not occur in the presence of metabolic poisons or when tail-beating was prevented. It was temperature-dependent, and did not require exogenous Ca2+. Since the PT-1 antigen is freely diffusing on the posterior tail before migration, the mechanism of redistribution could involve the alteration of a presumptive membrane barrier. In addition, we observed the redistribution of a second surface antigen after the acrosome reaction. The antigen recognized by the monoclonal antibody, PH-20, was localized exclusively in the posterior head region of acrosome-intact sperm. Within 7-10 min of induction of the acrosome reaction with Ca2+ and A23187, 90-100% of the acrosome-reacted sperm population no longer demonstrated binding of the PH-20 antibody on the posterior head, but showed binding instead on the inner acrosomal membrane. This redistribution of the PH-20 antigen also resulted from the migration of pre-existing surface molecules, but did not appear to require energy. The migration of PH-20 antigen was a selective process; other antigens localized to the posterior head region did not leave the posterior head after the acrosome reaction. These rearrangements of cell surface molecules may act to regulate cell surface function during fertilization.     

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer’s disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.