Cross-linking a maturation-dependent ram sperm plasma membrane antigen induces the acrosome reaction

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.     

Lateral regionalization and diffusion of a maturation-dependent antigen in the ram sperm plasma membrane

We have used a monoclonal antibody ESA 152 in fluorescence recovery after photobleaching (FPR) studies of a maturation-dependent surface antigen of ram sperm. The antibody is an immunoglobulin G secreted by a hybridoma derived from NS1 mouse myeloma cells. The ESA 152 antigen is not detectable in testicular sperm. It is localized on the surface of ejaculated sperm where it is present on all regions of the surface, but tends to be concentrated on the posterior region of the head. The ESA 152 antigen can be extracted by detergents or chloroform-methanol. The extracted antigen is sensitive to proteases and migrates with an apparent Mr approximately 30,000 in SDS-containing 10-20% polyacrylamide gradient gels. FPR measurements of ESA 152 lateral mobility in the membrane yield diffusion coefficients in the range 10(-9)-10(-8) cm2/s, values typical of lipids but observed for proteins only at the fluid dynamic limit where diffusion is controlled by lipid fluidity. Immobile fractions, typical of membrane proteins, are observed on all regions. When the antigen is stained by a fluoresceinated Fab fragment of the ESA 152 antibody, the diffusibility is highly regionalized, with particularly low, but rapid, recovery on the midpiece. Cross-linking of the antigen with the intact ESA 152 antibody induces a redistribution in which the antigen is excluded from the posterior head region. This cross-linking is accompanied by increases in ESA 152 diffusibility on both the anterior head and the midpiece.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent M_rS of 38–26 kD, whereas the memrane-associated form of the molecule has an M_r of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley-Liss, Inc.     

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.     

Identification of membrane antigens of goat epididymal spermatozoa

Purified goat sperm plasma membrane was used as antigen to raise the antibody in rabbit. Using this antisera four groups of antigenic membrane polypeptides are determined in caput and cauda epididymal sperm. The immunoresponsiveness of the polypeptides in caput and cauda sperm differs significantly. In case of cauda epididymal sperm, the polypeptides of region A (96KDa, 82KDa, 78KDa, 68KDa) and region D (24KDa, 20KDa, 18KDa) are highly immunoresponsive whereas in case of caput epididymal sperm the same antisera recognized the polypeptides of region B, C and D. By surface labelling with lactoperoxidase iodination and subsequent immunoprecipitation in the iodinated cell extract we demonstrate eight of these above polypeptides (96KDa, 82KDa, 68KDa, 50KDa, 29KDa, 24KDa, 20KDa and 18KDa) as surface antigen. The 96KDa, 82KDa and 68KDa surface polypeptides are highly immunoresponsive than the other lower molecular weight surface antigens in cauda epididymal goat spermatozoa.     

Identification of bovine CD52-like molecule by monoclonal antibody IVA-543: distribution of CD52-like molecule in the bull genital tract

The bovine maturation-associated sperm membrane antigen CD52-like molecule has been analysed using a mouse anti-sperm monoclonal antibody developed against bull spermatozoa. The antigen recognised by monoclonal antibody IVA-543 was detected on blood mononuclear cells (including lymphocytes and monocytes) and on a minor population of polymorphonuclear leukocytes. The bovine CD52-like molecule is secreted by the epididymal epithelium and then it is inserted into the sperm membrane during the epididymal transport in the distal part of epididymis. The CD52-like molecule was absent from spermatozoa derived from testes, and the highest proportion of IVA-543-reactive sperm was observed in the cauda epididymis (91.6%).This study has shown that the new molecule identified on bovine cells has properties analogous to those previously described for CD52 molecules in man, mouse, rat, monkey, and dog.     

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.     

Localization and expression of a 70 kDa protein in goat spermatozoa having Na+,K+-ATPase inhibitory and arylsulphatase A activities

We have previously isolated and purified a goat sperm protein of 70 kDa molecular weight designated as P70 and characterized it as an inhibitor of Na(+),K(+)-ATPase. Our study reveals that the first 10 amino acid residues from the N-terminal end of P70 has high degree of homology with arylsulphatase A from mice, pig and human. Indirect immunofluorescence study shows the presence of the protein on goat sperm surface. Furthermore, live goat sperm and the extract of peripheral sperm plasma membrane proteins exhibit arylsulphatase A's desulphation activity. The P70 remains on the head surface of in vitro capacitated cauda epididymal sperm as shown by positive immunofluorescence staining of cauda sperm. Immunoblot and flow cytometric studies corroborate the above findings. The presence of P70 on capacitated cauda sperm surface suggest a possible role of this protein in sperm zona pellucida binding. In the present report we demonstrate arylsulphatase A like activity in P70 and describe its localization and expression in goat sperm.     

Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface.

The determinant of a mouse sperm maturation antigen was examined morphologically and biochemically with monoclonal antibody T21 as a probe. The plasma membrane components of cauda epididymal spermatozoa were extracted with nonionic detergent Nonidet P-40 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and by immunoblotting. Wheat germ agglutinin-lectin staining and immunoblotting indicated that the antigen recognized by T21 is a sialoglycoprotein of about 54,000 daltons (54 kDa). The antigenic determinant was more distinctly exposed after treatment with neuraminidase, as evaluated by immunohistochemistry, immunocytochemistry, and immunoblotting. The cryptic nature of the determinant was further confirmed by immunostaining nitrocellulose strips, subsequently digesting the strips with neuraminidase, and then reimmunostaining them. Results obtained by periodate oxidation treatment suggested that the epitope is a carbohydrate. Immunoperoxidase electron microscopy confirmed that the antigen is distributed on the flagellar plasma membrane of the sperm. This was demonstrated clearly when sperm were desialylated with neuraminidase. These results indicate that the 54 kDa sialoglycoprotein sperm maturation antigen has a cryptodeterminant which can be masked by a sialic acid residue, that is recognized by monoclonal antibody T21.     

Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa

The surface membrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of approximately 200 kDa in immature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of approximately 160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.     

Germinal angiotensin I-converting enzyme is totally shed from the rodent sperm membrane during epididymal maturation

Acquisition of sperm fertilizing ability is due, in part, to the reorganization of plasma membrane proteins that occurs during epididymal sperm transit. Using polyclonal antibodies against angiotensin I-converting enzyme (ACE), we showed that this enzyme is immunolocalized mainly on the middle piece of rat and mouse testicular sperm and with less intensity along the initial part of the principal piece of the flagellum. In both species, only some sperm from the caput epididymis were still reactive, whereas no labeling was observed on cauda epididymal sperm. The 105- to 110-kDa germinal ACE was absent from the rat testicular fluid but appeared in the fluid of the anterior epididymis. Thereafter, its molecular weight shifted to 94 kDa in the corpus epididymal fluid and remained at this weight in the caudal region. The 105- to 110-kDa immunoreactive protein was present in testicular rat sperm extract but was completely absent from epididymal sperm extracts. Western blot analysis of testicular and epididymal tissue extracts from the rat and mouse also confirmed that the germinal enzyme was absent from the epididymal sperm cell. Our results demonstrated that the rodent germinal ACE is released from the testicular sperm membrane when sperm enter the epididymis, a process similar to that observed in domestic mammals. This result is discussed in view of the suggested role for this enzyme in sperm fertility.     

Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein-glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27-kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27-kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed the loss of a 27-kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the ¹²⁵I-distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.     

Different glycoforms of the human GPI-anchored antigen CD52 associate differently with lipid microdomains in leukocytes and sperm membranes

CD52 is a human GPI-anchored antigen, expressed exclusively in the immune system and part of the reproductive system (epididymal cells). Sperm cells acquire the antigen from the epididymal secretions when transiting in the epididymal corpus and cauda. The peptide backbone of CD52, consisting of only 12 aminoacids, is generally considered no more than a scaffold for post-translational modifications, such as GPI-anchor and especially N-glycosylation which occur at the third asparagine. The latter modification is highly heterogeneous, especially in the reproductive system, giving rise to many different glycoforms, some of which are tissue specific. A peculiar O-glycan-containing glycoform is also found in reproductive and immune systems. We determined to locate CD52 in microdomains of leukocytes and sperm membranes using two antibodies: (1) CAMPATH-1G, the epitope of which includes the last three aminoacids and part of the GPI-anchor of glycoforms present in leukocytes and sperm cells; (2) anti-gp20, the epitope of which belongs to the unique O-glycan-bearing glycoform also present in both cell types. Using a Brij 98 solubilization protocol and sucrose gradient partition we demonstrated that the CD52 glycoforms recognized by both antibodies are markers of typical raft microdomains in leukocytes, whereas in capacitated sperm the O-glycoform is included in GM3-rich microdomains different from the cholesterol and GM1-rich lipid rafts with which CAMPATH antigen is stably associated. The importance of the association between GM3 and O-glycans for formation of specialized microdomains was confirmed by heterologous CD52 insertion experiments. When prostasomes from human seminal fluid were incubated with rat sperm from different epididymal regions, the CD52 glycoform recognized by anti-gp20 decorated rat epididymal corpus and cauda sperm, associated with the same low-cholesterol GM3-rich sperm membrane fractions as in human sperm. The glycoforms recognized by CAMPATH-1G were not found in rat sperm. The relationship between this differential insertion and differences in glycosylation of rat and human CD52 is discussed.     

Features of a seminal proteinase inhibitor- zona pellucida-binding component on murine spermatozoa

Murine cauda epididymal sperm contain sites on the plasma membrane over the apical portion of the acrosome that recognize proteinase inhibitors and the homologous zona pellucida. Ten times more of the component can be extracted from cauda and ductus sperm than from equal numbers of caput and corpus sperm. Likewise, few sperm from the upper epididymal regions are able to bind seminal inhibitor, while the majority of sperm from the cauda and ductus do bind. Cauda epididymal and ductus sperm lose little of their ability to bind inhibitor after a 4-hour in vitro incubation in either a capacitating or a noncapacitating medium. The percentage of naturally inseminated sperm with the seminal inhibitor bound to their surface decreases to about 10 after 4 hours in utero. Approximately 80% of these sperm show positive fluorescence when given the opportunity to rebind the inhibitor, and these sperm do have an intact plasma membrane over the apical portion of the acrosome. Furthermore, after 4 hours in utero, the inhibitor bound in the same region of the sperm head as it did on freshly ejaculated sperm. The seminal inhibitor inhibits the binding of sperm to the zona if added during the first 15 minutes of incubation but has no effect on attachment. The data indicate that sperm gain the ability to bind the seminal inhibitor during the epididymal sojourn. Furthermore, this binding capacity is not lost during in vitro or in utero incubation. The site is not involved in sperm-zona attachment but does participate in the binding of sperm to the zona.     

Maturational changes of the CD52-like epididymal glycoprotein on cynomolgus monkey sperm and their apparent reversal in capacitation conditions

A major epididymal secretory protein in men has a colinear cDNA sequence with lymphocyte CD52, a sialylated glycoprotein. Immunostaining and flow cytometric detection of cynomolgus monkey sperm CD52 during epididymal maturation showed increases from 20 to 85% stained sperm from the caput to the corpus with staining intensities doubled. Freshly prepared cauda sperm showed only 10% staining while they markedly increased in percentage and intensity of staining upon incubation at 37 degrees C under capacitating conditions, but not at 4 degrees C. Western blotting of proteins from fresh cauda sperm revealed no less antigen than corpus sperm. Staining of ejaculated sperm exhibited similar increases during incubation. Further washing with a high salt medium before staining to remove any electrostatically-bound molecules masking the antigen showed no effect. Incubation-induced increases in antigen binding were accelerated by the addition of neuraminidase (0.25 and 0.5 U/ml), but not affected by the sialyl residue-rich fetuin (5 mg/ml) competing for any endogenous neuraminidase. There were no concomitant decreases in the staining of sialic acid residues during capacitation-incubation. These findings suggest a cryptic antigen epitope site as a consequence of sperm maturation and subsequent re-exposure under capacitation conditions, but not due to the removal of sialic acid residues by endogenous neuraminidase. Involvement of endogenous proteases was also ruled out, as incubation in the presence of protease inhibitors did not hinder the increases but resulted in a dose-dependent enhancement in staining, suggesting some protease-sensitive unmasking process. In conclusion, the monkey epididymal secreted CD52 on sperm underwent changes in antigenic characteristics during sperm maturation which were reversed under capacitation conditions.     

Lectin binding sites on the plasma membrane of epididymal and ejaculated chimpanzee sperm

Lectins have been used to analyze variations in the distribution and density of exposed saccharides of the sperm plasma membrane during physiologic maturation and after ejaculation. Studies have been conducted in a number of nonprimate species but have been conducted to only a limited extent in nonhuman primates. In this study, pure suspensions of chimpanzee sperm from the caput and cauda epididymis and from the ejaculate were labeled with lectins conjugated to fluorescein isothiocyanate in order to visualize changes in the distribution of exposed membrane glycocomponents. The lectins used were Con A, DBA, RCA-I, and WGA. Con A binding showed minimal change during epididymal transit, with an increased binding to the flagellum after ejaculation. DBA binding was relatively constant in all specimens. RCA-I showed distinct changes in binding pattern between epididymal and ejaculated sperm. On ejaculated sperm strong fluorescence was limited to the posterior head and to the midpiece. WGA binding increased during epididymal passage and decreased after ejaculation. There appears to be a wide variety of saccharide groups available for lectin binding on the surface of epididymal and ejaculated chimpanzee sperm. The general similarity in binding patterns of caput and cauda epididymal chimpanzee sperm exposed to Con A and DBA might reflect the fact that sperm morphology does not change during epididymal transit in this species, thus implying a more stable membrane structure than is present in other primates so far studied.     

OBSERVATIONS ON SPERM PENETRATION IN THE RAT

The structural aspects of sperm penetration in the rat egg were investigated by electron microscopy. Eggs were recovered at intervals between 8 and 10:30 A.M. from females which had mated during the previous night. The oviducts were flushed with hyaluronidase and the eggs transferred into a 2 per cent osmium tetroxide solution, buffered at pH 7.8. After fixation, the eggs were mounted individually in agar, dehydrated in ethyl alcohol, and embedded in butyl-methyl methacrylate (3:1). The sperm penetrating the egg is covered by a plasma membrane which is present only on the side facing toward the zona pellucida; no membrane is visible on the side facing toward the vitellus. The sperm plasma membrane becomes continuous with the egg plasma membrane and forms a deep fold around the entering sperm. Cross-sections through the sperm midpiece in the perivitelline space show an intact plasma membrane. At the place of entrance, the plasma membrane of the sperm appears to fuse with the egg plasma membrane. After the sperm has penetrated the vitellus, it has no plasma membrane at all. The nuclear membrane is also absent. These observations suggest a new hypothesis for sperm penetration. After the sperm has come to lie on the plasma membrane of the egg, the egg and sperm plasma membranes rupture and then fuse with one another to form a continuous cell membrane over the egg and the outer surface of the sperm. As a result the sperm comes to lie inside the vitellus, leaving its own plasma membrane incorporated into the egg membrane at the surface of the egg.     

Molecular analysis of a carbohydrate antigen involved in the structure and function of zona pellucida glycoproteins

A lactosaminoglycan-associated antigen is associated with a carbohydrate moiety of all three zona pellucida (ZP) glycoproteins of pig and rabbit but is absent in the mouse and rat. A monoclonal antibody (PS1) recognizing this determinant was obtained by immunizing mice with a porcine ZP glycoprotein isoform purified by two-dimensional polyacrylamide gel electrophoresis. Conditions known to remove O-linked or sialic acid carbohydrate moieties (alkaline reduction; O-glycanase or neuraminidase enzymatic cleavage) did not remove the carbohydrate epitope. However, treatment with endo-beta-glycosidase, endoglycosidase F, or combinations of neuraminidase plus beta-galactosidase, totally removed the determinant, indicating that it is associated with a poly-N-acetyllactosaminoglycan structure present on an N-linked oligosaccharide. Molecular morphology studies using immunofluorescence and confocal microscopy techniques demonstrate that the PS1 antigen is localized at the surface of the ZP. Confirmation of this localization was obtained through studies that show that this antibody will inhibit homologous sperm binding to the pig ZP. Additional analyses using modular contrast microscopy and immunocytochemistry demonstrate that this carbohydrate-associated antigen is localized in discrete layers throughout the ZP matrix. These studies are the first to demonstrate the presence of a lactosaminoglycan type carbohydrate moiety in all three ZP proteins using a monoclonal antibody that appears to be involved in sperm recognition and structural organization.     

Effects of in vivo and in vitro fertilization environments on the expression of a surface antigen of the mouse sperm tail

Sperm maturation antigen 4 (SMA 4) is a glycoprotein secreted by the mouse epididymis that binds specifically to the sperm tail. Its fate has been examined on cauda epididymidal sperm in vivo and in vitro. SMA 4 was detected by indirect immunofluorescence (IIF) on sperm flushed from uteri of mice 5.5 h after natural or artificial insemination, but not on sperm attached to cumulus cells or zonae pellucidae of eggs recovered at that time. Detectable SMA 4 declines with time in vitro, as assayed by IIF on intact sperm or by enzyme immunoassay (EIA) of detergent extracts. After 3 h in vitro, 90% or more of sperm are not positive for SMA 4 by IIF. EIA of medium in which sperm have been incubated suggests that SMA 4 is being released from the cell surface. This time-dependent loss of SMA 4 is inhibited by mouse or rat cauda epididymidal fluid, low incubation temperature, or lack of protein in the incubation medium. However, the loss does not seem to be affected by the presence of eggs, cumulus cells, or oviduct fluids. SMA 4 is not removed from the sperm by selected treatments, suggesting that it is bound to the plasma membrane by strong, noncovalent interactions.