Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.     

Regional differences in composition of the perforatorium and outer periacrosomal layer of the rat spermatozoon as revealed by immunocytochemistry

The rat perforatorium is the part of the perinuclear theca that underlies the acrosomic system. It appears to be composed of several polypeptides. The main objective of this study was to determine the distribution of seven of these perforatorial polypeptides in the head of the rat spermatozoon. For this purpose, polyclonal antibodies were affinity purified from these polypeptides and tested 1) for their distribution on electron-microscope sections of late spermatids and spermatozoa by immunogold labeling and 2) for their specificity on Western blots of denatured perforatorial polypeptides by immunoblotting. Immunoblotting showed that all seven of the prominent perforatorial polypeptides had epitopes in common. Immunogold labeling of spermatozoa showed that antibodies against the 13, 13.4, and 16 kDa polypeptides were restricted in their localization to the thicker apical portion of the perforatorium and to the inner zone of the ventral spur. However, antibodies against the 34, 43, 57, and 63 kDa polypeptides reacted with the entire perforatorium but, in addition, reacted with the inner part of the ventral spur and with a portion of the "outer periacrosomal layer" lying between the plasma membrane and the outer acrosomal membrane. These results suggest 1) that there are regional differences in protein composition of the perforatorium, of the outer periacrosomal layer, and of the postacrosomal dense lamina; and 2) that perforatorial polypeptides may not necessarily be restricted to the subacrosomal region, but may also compose portions of the outer periacrosomal layer and postacrosomal dense lamina. Based on both immunoblotting and immunocytochemical results, using an antiactin monoclonal antibody that recognizes all known isoforms of actin, actin was not detected in the perforatorium of step 19 spermatids or spermatozoa. Actin, however, together with the seven perforatorial polypeptides tested, was present in the subacrosomal space of elongating spermatids before the process of condensation of the perforatorium takes place.     

Fine structure distribution of non-specific acid phosphatase in the head region of mouse spermatozoa from various regions of the male reproductive tract.

The fine structure distribution of non-specific acid phosphatase was determined in the head region of mouse spermatozoa from the testes, the caput, corpus and cauda epididymidis and the ductus deferens. Enzymatic localization was achieved by the Gomori technique. The postacrosomal dense lamina, the nuclear side of the inner acrosomal membrane and the space between the plasmalemma and the outer acrosomal membrane showed reaction product in spermatozoa from the testis and caput epididymidis. Spermatozoa from the cauda epididymidis exhibited reaction product only between the plasmalemma and the outer acrosomal membrane. Spermatozoa from the corpus epididymidis and from the ductus deferens showed no reaction product in the head region. The changes observed in the distribution of acid phosphatase in the sperm head during epididymal transport may reflect maturational events.     

Binding of zona pellucida proteins to a boar sperm polypeptide of Mr 53,000 and identification of zona moieties involved

Experiments have been carried out to identify proteins on boar spermatozoa that bind to components of the zona pellucida. Polypeptides in sodium deoxycholate extracts of boar spermatozoa and in whole seminal plasma have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose sheet by electroblotting and probed with 125I-labelled heat-solubilized zona pellucida from pig oocytes or ovulated eggs. Zona proteins bound avidly and consistently to a polypeptide of Mr 53,000 on blots of capacitated and noncapacitated sperm and weakly to polypeptides of Mr 67,000, 38,000 and 18,000. On blots of seminal plasma the 125I-labelled probes bound to two polypeptides of Mr 65,000 and 19-24,000. Identification of the zona proteins that were binding to the aforementioned proteins on blots showed that all the major zona pellucida glycoproteins were involved, including those acquired from oviduct secretions. Binding of 125I-ovulated zona pellucida to the polypeptide of Mr 53,000 also occurred in extracts of testicular and epididymal boar spermatozoa. The results are discussed in relation to sperm-egg recognition in the pig.     

The Acrosome Reaction in Ciona intestinalis (Ascidia, Tunicata)

An acrosome reaction occurs by fusion between the acrosomal outer membrane and the plasmalemma enclosing the acrosome in Ciona intestinalis spermatozoa. The fusion seems to proceed along the peripheral margin of the acrosome, which causes vesiculation. The membrane bound vesicle formed by this process is probably shed by the sperm. The acrosomal inner membrane is exposed and becomes a part of the plasmalemma enclosing the anterior region of the sperm head. During this process, any acrosomal substance might be released through the opening formed by membrane fusion. The acrosome reaction most likely occurs in C. intestinalis spermatozoa, via vesiculation, in fundamentally the same way as observed in mammalian spermatozoa.     

Characterization of an acrosomal matrix protein in hamster and bovine spermatids and spermatozoa.

Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.     

Silver-staining patterns of mammalian epididymal spermatozoa

Epididymal spermatozoa from 12 species of mammals were stained using silver nitrate and examined with the light microscope. Silver nitrate differentiates many of the gross morphological features of spermatozoa, including the acrosome, subacrosomal region, perforatorium, postacrosomal sheath, neck, dense outer fibers of the core of the midpiece, annulus, principal piece, and end piece. Silver-staining patterns of spermatozoa reveal both species-specific and strain-specific differences, particularly of the sperm head. The biochemical basis of silver staining may be due in part to the presence of sulfhydryl- and disulfide-rich proteins; however, it cannot be explained entirely by the presence of these moieties. The detail obtained using silver nitrate staining, coupled with the ease and rapidity of the procedure, should be useful to workers in many areas of biological and medical research.     

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.     

Protein composition of the ventral processes on the sperm head of Australian hydromyine rodents

The sperm head of the plains rat, an Australian hydromyine rodent, is highly complex in structure and contains, in addition to an apical hook, two large ventral processes (VPs) that extend from its upper concave surface and that are largely composed of a huge extension of the sperm head cytoskeleton surrounded by postacrosomal dense lamina. In this study we have attempted to determine their protein composition. For this, the VPs were isolated, the proteins within them separated by SDS-PAGE, and the resultant polypeptide bands Western blotted and probed with antibodies against laboratory rat perforatorial and bull perinuclear theca sperm proteins. Antibodies were also used to determine the perforatorial and perinuclear theca proteins by immunogold labeling of transmission electron microscopic sections. The results indicate that the material within the VPs is largely composed of perforatorial cross-reacting proteins together with F-actin with the dominant protein being PERF 15. The perinuclear theca proteins are, by contrast, restricted to a narrow region adjacent to the acrosomal and nuclear membranes. In conclusion, this study has shown that the VPs of the spermatozoa of Australian rodents are perforatorial-like appendages that contain similar proteins to the perforatorium of the apical hook together with F-actin; their functional significance remains unknown.     

Characterization of matrix domains of the hamster acrosome

In this study we describe the purification and the structural and biochemical properties of a detergent-stable complex of the hamster sperm acrosome. This complex consists of two distinct acrosomal matrix domains and a layer of electron-dense material, termed the acrosomal lamina, derived from the luminal surface of the outer acrosomal membrane. This complex has been isolated by centrifugation of detergent-extracted sperm suspensions on Percoll density gradients. The complex contains two major polypeptides of Mr 29,000 and Mr 22,000 and minor polypeptides of Mr 64,000-62,000, 56,000 and 35,000. Gelatin-containing sodium dodecyl sulfate-polyacrylamide gels demonstrate that bands of proteinase activity are not the major polypeptide components of the complex. These data demonstrate that the matrix of the acrosome is compartmentalized into domains of differing structural properties that occupy specific locations in the intact acrosome and that matrix components are physically associated with the outer acrosomal membrane. These data indicate that a structural framework is present within the acrosome and we speculate that it may be involved in sequestering hydrolases into specific spatial domains and could affect the temporal release of activity of selected hydrolases during the acrosome reaction.     

Ultrastructure of the testes and spermatogenesis in the mite Anystis baccarum

The epithelial lining of testes in Anystis baccarum is glandular and produces a secretory product necessary to form spermatophores. The main stages of spermatogenesis occur in the lumen of the testis in groups of synchronously developing sister cells. Spermatogonia and late spermatids are encircled by glandular cells. Reorganization of developing spermatids is typical of the trombidiform mites and includes formation of the acrosomal complex, cytoplasm elimination, disappearance of the nuclear envelope and formation of invaginations of plasmalemma. The chromatin material condensation is not followed by the entire chromatin body formation. In mature spermatoza, dense chromatin strands (80b nm in diameter) lie along the cell in the peripheral layer of the cytoplasm. Mature spermatozoa lack axonema or any protrusions. A layer of microtubules, visible underneath the outer membrane, may serve for sperm movement in the female genital duct. The acrosomal complex consists of acromal granule, acrosomal filament and subacrosomal substance. This, as well as two aggregates of typical mitochondria, looks plesiomorphic.     

Outer acrosomal membrane of guinea pig spermatozoa: Isolation and structural characterization

We describe a protocol to isolate a highly enriched fraction of outer acrosomal membrane from guinea pig spermatozoa and present new data on the ultrastructure of this membrane domain. Cauda epididymal spermatozoa were suspended into a low ionic strength buffer and subjected to brief homogenization; this stripped the plasma membrane from the spermatozoa and severed the acrosomal apical segment from the spermatozoon. The crescent-shaped apical segments retained the outer acrosomal membrane and specific components of the acrosomal matrix. Enriched fractions of apical segments were isolated on discontinuous sucrose gradients and the outer acrosomal membrane purified by subsequent centrifugation onto Percoll density gradients. The isolated outer acrosomal membrane did not form vesicles, but instead rolled up into spiral sheets. Both thin section and negatively stained specimens revealed a paracrystalline arrangement of filaments associated with the luminal surface of the membrane. The isolated outer acrosomal membrane revealed a limited number of polypeptides by SDS-PAGE, and the polypeptide pattern was distinct from the plasma membrane fraction. The isolated acrosomal membranes possessed no oubain sensitive Na+, K+-ATPase activity, whereas about 20% of the ATPase activity of the plasma membrane enriched fraction was inhibited by oubain. The potential function of the structural differentiations of the outer acrosomal membrane in the membrane fusion events of the acrosome reaction is discussed.     

Characterisation of an epitope shared by an acrosomal acrosin-like protein and the surface of tammar wallaby (Macropus eugenii) spermatozoa

Monoclonal antibodies (mAb) have been raised against marsupial sperm proteins to provide insights into the molecular nature of marsupial spermatozoa, and the proteins that mediate sperm maturation and interaction with the oocyte. This study reports the production of a mAb, designated WSA-1, which bound acrosomal and surface determinants on tammar wallaby spermatozoa. The acrosomal antigen was first detected in the wallaby testis; however, ejaculated spermatozoa demonstrated whole cell WSA-1 immunoreactivity as a result of binding an epididymal protein. Ultrastructural and agglutination analyses localised the WSA-1 epitope to the acrosomal matrix and the whole sperm plasmalemma. The WSA-1 mAb bound three polypeptides with relative molecular weights of 35, 31 and 15 kDa on western blots under reducing conditions. The N-terminal amino acid sequence obtained for the 35 kDa wallaby sperm polypeptide demonstrated identity with the eutherian acrosomal protein acrosin. The 31 kDa polypeptide was of epididymal origin and will be the subject of a separate study. Further studies of the WSA-1 antigens are likely to provide useful insights into the function and maturation of marsupial sperm since proacrosin has a number of putative roles in eutherian fertilisation, and epididymal proteins are thought to mediate sperm maturation and storage.     

Localization of intracellular calcium during the acrosome reaction in ram spermatozoa

Calcium was identified by a pyroantimonate-osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39°C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time. Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a “sawtooth” arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. © 1995 Wiley-Liss, Inc.     

Observations on the postacrosomal region and the neck membranes of rabbit spermatozoa stained en bloc with uranyl acetate

Summary The postacrosomal region (PAC) of the head and the neck membranes of rabbit spermatozoa have been reinvestigated with the use of en bloc uranyl acetate staining. The fine structure of the PAC and neck membranes is visualized with great clarity and departs from that seen in rabbit sperm prepared by other methods. In cells so treated, the PAC contains a heavily enfolded nuclear envelope, a dense lamina attached to the plasmalemma by periodic connecting links and scattered dense material between the lamina and nuclear envelope. Evidence is presented that the dense lamina is a discrete structure, separated from the plasmalemma by the connecting links. The latter may be of a different composition from both the lamina and the plasmalemma. The lamina is a homogenous structure which resists degeneration under conditions which affect other components of the PAC. The membranes of the neck are a complex labyrinth of nuclear envelope, individual membranes, and membranes coursing through a matrical gound substance.This investigation was supported by Ford Foundation Grant 67–650.     

Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000-74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane.     

Electron microscopical studies of membrane injuries in blue fox spermatozoa subjected to the process of freezing and thawing

Disintegration of blue fox sperm membranes is studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In unfrozen spermatozoa studied by SEM, the plasmalemma and the acrosome appeared to be intact, except for a few cases of disruption of the former structure at the anterior part of the head. In semen frozen in 0.5-ml plastic straws by use of N2 vapor after dilution with Tris-fructose-citric acid with 8 vol % glycerol and 20 vol % egg yolk and thawed at 70 degrees C for 8 sec, the spermatozoa displayed different degrees of membrane damage. These alterations could be classified into three main categories of which the first included only minor changes in the plasmalemma, but vesiculation and disintegration of the outer part of the acrosomal membrane. In the second category (also the most frequent one) the outer part of the acrosomal membrane was extensively vesiculated, and the plasmalemma was discharged proximal to the equatorial segment. Extensive loss of plasmalemma and complete absence of the outer part of the acrosomal membrane characterized the last category of membrane damage. The functional implications of the three categories of membrane alterations are discussed.     

Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit

Using a monoclonal antibody as a highly specific probe and a seminal particle-free fraction of rabbit ejaculated spermatozoa, actin has been localized in the postacrosomal region of mature rabbit spermatozoa. The sperm actin has been extracted and identified on two-dimensional PAGE immunoblots as a single spot of pI = 5.45 and Mr = 43,000. Rabbit sperm actin is present in a nonfilamentous form and is not removed by removing the plasma membrane. Unlike mature spermatozoa, however, filamentous actin is present in spermatogenic cells, as determined by rhodamine phalloidin staining. Starting as diffusely distributed in spermatocytes, actin accumulates in the subacrosomal space and appears as a band in conjunction with the developing acrosome. This band lengthens throughout the spermatid stage and becomes continuous with the postacrosomal region staining in testicular spermatozoa. Actin may therefore function during spermatogenesis to both shape the acrosome to the nucleus and to anchor inner acrosomal membrane proteins.     

Characterization of isolated acrosomal matrices from hamster spermatozoa

The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5.2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70-80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin-SDS-PAGE. The major activity corresponded to bands of relative molecular masses (Mr) of 56,000, 51,000 and 48,000 with two minor bands of Mr 30,000 and 28,000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC-PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of Mr 60,000 and 72,000.     

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.