Antibodies to rat sperm tail polypeptides recognize Sertoli cell secretory proteins

We have previously reported that (a) polyclonal antisera raised against rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein recognize outer dense fiber polypeptides from rat sperm tail, and (b) protein S70 is antigenically related to polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein. We now report that polyclonal antisera generated against three different outer dense fiber polypeptides recognize (a) the putative antigen of the sperm tail and (b) Sertoli cell secretory protein S70 and its antigenically-related polypeptides. Immunogold electron microscopy shows that outer dense fibers of epididymal sperm crossreact with anti-S70 serum as well as with an antiserum raised against the polypeptide D complex of extracted outer dense fibers. Electron microscopy demonstrates that outer dense fibers consist of filamentous, coil-coiled units aligned side-by-side with each other. Results of this study strengthen the antigenic homology between Sertoli cell secretory proteins and outer dense fiber polypeptides of the sperm tail.     

Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Using commercial monoclonal antibodies against actin and tubulin (alpha and beta), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and - most intensely - in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail. Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

A sulfated glycoprotein synthesized by Sertoli cells and by epididymal cells is a component of the sperm membrane

We report here the purification, partial characterization and immunofluorescent localization of a dimeric acidic glycoprotein (DAG-protein) secreted by cultures of Sertoli cells of rat testes. Partially purified protein was obtained after chromatography over Sepharose 4B under conditions which favored a soluble, nonaggregated form of the protein. Rechromatography over the same column under reducing conditions yielded very pure monomers of 41,000 daltons and 29,000 daltons. Antibodies were prepared against the mixed monomers and used to immunoprecipitate proteins in spent medium from cultures incubated with [35S] methionine, 35SO4 = or tunicamycin. DAG-protein and another protein (Band 4, 70,000 daltons) were coprecipitated by the antiserum and all contained 35SO4 = in their structures. It was shown by Western blotting that the antiserum cross-reacted very weakly with Band 4 protein. The DAG-protein polypeptides secreted in the presence of tunicamycin were assumed to lack N-glycosylation and exhibited apparent molecular weights of 27,000 and 21,000 daltons. Immunoprecipitations of media from organ cultures of testis and epididymis yielded DAG-protein of slightly lower molecular weight than the protein secreted in Sertoli cell cultures. Indirect immunofluorescence of DAG-protein in paraffin sections of testis and epididymis revealed that the protein was concentrated in the cytoplasm of Sertoli cells, on the stereocilia of epididymal principal cells, in the cytoplasm of epididymal halo cells, and was associated with late spermatids and mature sperm. Sperm were specifically labeled on the acrosome, at the neck, and on the endpiece of the tail. No enzymatic or structural function has been ascribed to DAG-protein as yet, but the protein must play a pivotal role in spermatogenesis because it is secreted by both the testis and epididymis and becomes an integral component of sperm.     

Processing of two protein precursors yields four mature guinea pig seminal vesicle secretory proteins

The translation of the two most abundant guinea pig seminal vesicle epithelium mRNAs (1800 nucleotides and 950 nucleotides) and the subsequent processing of their protein products were studied in an effort to elucidate the mechanism by which the four mature guinea pig seminal vesicle epithelium (GPSVE) secretory proteins are produced. The primary translation products of the 1800 nt and 950 nt mRNAs are two secretory protein precursors of 45 kDa and 20 kDa, respectively. Removal of signal peptides from these two precursors produces proteins of 43 kDa and 18.5 kDa, which are recognized by polyclonal antisera directed against the four mature secretory proteins. The existence of further processing intermediates in the production of the secretory proteins is suggested by the appearance of other immunoreactive polypeptides following incubation of GPSVE in nutrient medium containing [3H] leucine. Immunological and pulse-chase analysis strongly suggests that the 43-kDa protein gives rise to SVP-1, -3, and -4 and that SVP-2 is derived from the 18.5-kDa protein.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Summary Using commercial monoclonal antibodies against actin and tubulin ( and ), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and — most intensely — in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail.Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

Temporal appearance and cyclic behavior of Sertoli cell-specific secretory proteins during the development of the rat seminiferous tubule

Electrophoretic and morphologic methods have been used to study the time course of [35S] methionine-labeled proteins accumulated in the incubation medium of rat fetal testes and seminiferous cords/tubules during their development. We have found that Sertoli cell-specific secretory proteins S70, S45 and S35 became progressively prominent as premeiotic, meiotic and postmeiotic spermatogenic events were established in the seminiferous tubules. In the sexually mature rat, S70, S45 and S35 were expressed in a spermatogenic stage-dependent manner. While S70, S45 and S35 were present in Stage VII-VIII, S45 and S35 were observed in Stages X and XIV. Neither S70, S45 nor S35 were detected in Stage IV. A relevant group of high molecular weight proteins, previously reported as characteristic products of cultured peritubular cells, accumulated in the incubation medium of seminiferous cords from postnatal Day 0-15 rats. This group of high molecular weight proteins appears when peritubular cells are proliferative and are engaged in the organization of the seminiferous tubular wall. A low molecular weight protein, designated T35, was also detected. T35 was prominent in the medium of incubated fetal testes and seminiferous cords of postnatal rats 0- to 5-days-old and disappeared gradually thereafter. A set of proteins (designated SP1 and SP2) previously ascribed to both cultured Sertoli and peritubular cells, were recognized during the early postnatal stages of seminiferous tubular development. SP1 and SP2 displayed age-dependent fluctuations in their [35S] methionine labeling. The timing of appearance of S70, S45, and S35 indicates both age- and spermatogenic stage-related activity that, in the future, may prove to be functionally significant in the spermatogenic process.     

Polyclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II

Polyclonal antibodies against rabbit skeletal muscle phosphatases C-I and C-II were raised in goats and in mice. The goat polyclonal antibodies to phosphatases C-I and C-II were examined for their ability to immunoblot the purified enzymes and crude rabbit muscle extracts. In preparations of phosphatases C-I and C-II that were apparently homogeneous, the expected ca. 35- to 38-kDa polypeptides were immunoblotted, but, in addition, immunoblotting of a 67-kDa polypeptide was observed. Both the antisera blotted only the 67-kDa polypeptide in crude rabbit muscle extracts and not the expected 35- to 38-kDa polypeptides. These findings are qualitatively similar to those reported previously (D.L. Brautigan et al. (1985) J. Biol. Chem. 260, 4295-4305) where immunoblotting experiments with a sheep antisera to phosphatase C-I indicated that the ca. 35-kDa polypeptide originates from a 70-kDa precursor. On further investigation, it was found that our antisera were strongly immunoreactive to rabbit serum albumin. The antisera blotted purified rabbit albumin, but not bovine serum albumin. After passage through a rabbit albumin-Sepharose column, the antisera lost immunoreactivity to rabbit albumin, and no longer blotted the ca. 70-kDa band in muscle extracts or in purified enzyme preparations. These findings show that the phosphatase preparations contained traces of albumin which produced a strong antigenic reaction. Production of antisera in BALB/c mice produced similar results; i.e., an antibody to the low-molecular-weight phosphatases was produced that was also a strong antibody to rabbit albumin. This antibody could be removed by affinity adsoption on rabbit albumin-Sepharose columns. In addition, the antibodies to phosphatase C-I displayed no cross-reactivity to phosphatase C-II, while antibodies to C-II showed no cross-reactivity to phosphatase C-I by immunoblotting methods.     

The identification and partial characterization of merosin--a new specific protein of the basement membranes of certain tissues

A tissue-specific basement membrane-associated protein has been identified by the use of monoclonal antibodies prepared against a protein fraction of human placenta. In frozen sections of human tissues the monoclonal antibodies decorated basement membranes of Schwann cells, striated muscle, and trophoblast. In antibody-affinity chromatography of limited pepsin digests of human placenta, a 65-kDa polypeptide was bound by the monoclonal antibodies. Polyclonal antisera and new monoclonal antibodies were raised against the isolated 65-kDa polypeptide, and they stained human tissues identically to the original monoclonal antibodies. An 80-kDa polypeptide was detected by these antibodies in placental extracts prepared without proteolysis. The 65-kDa and 80-kDa polypeptides were immunologically distinct from laminin, type IV collagen, fibronectin and major serum proteins. These polypeptides are presumably derived from a novel basement membrane-associated protein which we named merosin. Several cDNA clones were isolated which code for a protein specifically recognized by polyclonal antibodies to the 65-kDa fragment. In developing mouse tissues, merosin was first detected at the newborn stage. The restricted tissue distribution and the late development appearance of merosin suggest that the protein has a tissue-specific function in highly differentiated cells.     

Structural features of the 26S proteasome complex isolated from rat testis and sperm tail

We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.     

Molecular and functional characterization of the rabbit epididymal secretory protein 52, REP52

As part of a systematic study of rabbit epididymal proteins involved in sperm maturation, we have identified and characterized a novel glycoprotein (rabbit epididymal secretory protein 52 [REP52]) of 52 kDa. REP52 is synthesized and secreted in a tissue-specific manner by the mid (region 6) and distal (region 7) corpus epididymidis and associates weakly with the sperm surface overlying the principal piece of the tail. Sequencing of cloned REP52 cDNA demonstrated that this protein represents a novel member of the highly conserved fibronectin type II (FN2) module protein family. The protein appears related but not homologous to ungulate seminal plasma proteins and is the first known example to be identified as a rabbit epididymal secretory protein. Consistent with other members of this protein family, REP52 possessed a high level of sequence identity within the FN2 module-encoding domains, but a highly variable N-terminal sequence that failed to show significant homology with published sequences. By analogy with evidence from studies of the ungulate seminal plasma proteins it is hypothesized that the tandemly arranged FN2 modules could facilitate the association of REP52 with sperm phosphatidylcholine residues on the outer leaflet of the sperm tail. It is also considered likely that these domains represent key elements for the function of this novel protein, a conclusion supported by the fact that antisera raised against the REP52 protein blocked in vitro fertilization in a concentration-dependent fashion.     

Heat shock response of Pseudomonas aeruginosa.

The general properties of the heat shock response in Pseudomonas aeruginosa were characterized. The transfer of cells from 30 to 45 degrees C repressed the synthesis of many cellular proteins and led to the enhanced production of 17 proteins. With antibodies raised against the Escherichia coli proteins, two polypeptides of P. aeruginosa with apparent molecular weights of 76,000 and 61,000 (76K and 61K proteins) were shown to be analogous to the DnaK and GroEL heat shock proteins of E. coli due to their immunologic cross-reactivity. The major sigma factor (sigma 87) of P. aeruginosa was shown to be a heat shock protein that was immunologically related to the sigma 70 of E. coli by using polyclonal antisera. A hybridoma was produced, and the monoclonal antibody MP-S-1 was specific for the sigma 87 and did not cross-react with sigma 70 of E. coli. A smaller 40K protein was immunoprecipitated with RNA polymerase antisera from cells that had been heat shocked. The 40K protein was also associated with RNA polymerase which had been purified from heat-shocked cells and may be the heat shock sigma factor of P. aeruginosa. Exposure to ethanol resulted in the production of seven new proteins, three of which appeared to be heat shock proteins.     

Two major antigens of heart sarcolemma are Ca2(+)-binding glycoproteins that copurify with the dihydropyridine receptor.

Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.     

How probable are antibody cross-reactions?

Antibodies to polypeptides are increasingly being used in research. Their specificity and tight but reversible binding make them ideal for applications such as identification of proteins, immunological quantification or purification, and peptide mapping. Antibodies are also used in medicine to deliver loads to specific sites in tissues, and in electron microscopy as heavy metal conjugates to locate antigens in thin sections. While these techniques depend on specificity of antibody binding, it is occasionally observed that cross-reactions occur. These cross-reactions are attributed to the existence of one or more antibody binding sites common to both polypeptides. It is important to know whether these cross-reactions are expected due to chance alone, or if they are improbable and likely due to some causative agent. Examples of such causative agents might include gene duplication events or convergence due to functional constraints. At the present time, good methods for predicting the probability and therefore the frequency of cross-reactions are unavailable. In this paper we apply some recently reported mathematical results to address the following questions: (1) What is the probability that polyclonal or monoclonal antibodies raised against a given polypeptide will cross-react with another polypeptide due to chance alone? (2) What is the probability that polyclonal or monoclonal antibodies raised against a given polypeptide will cross-react with one or more polypeptides in a pool of polypeptides? Approximate answers to these questions are presented for cases where amino acid compositions of linear polypeptides are known or unknown, but the amino acid sequence of one or more of the polypeptides is not known. Implications of the results for antibody use in protein research are discussed.     

Inhibition of sea urchin sperm acrosome reaction by antibodies directed against two sperm membrane proteins : Characterization and mechanism of action

The egg jelly-induced acrosome reaction is inhibited by polyclonal antibodies raised against either of two S. purpuratus sperm-membrane proteins, of M_r 80 and 210 kD. Although the two antigens used have dissimilar CNBr peptide maps, antisera produced against each of them cross-react with both proteins. Inhibition of the egg jelly-induced acrosome reaction by the antisera is bypassed by a combination of the ionophores monensin and A23187. This result, along with data showing that the antisera inhibit egg jelly-induced uptake of ⁴⁵Ca²⁺, suggests that the antisera may block both Ca²⁺ uptake and Na⁺/H⁺ exchange in the sperm. The acrosome reaction blockage appears to be caused by the same component of the polyclonal sera responsible for cross-reaction; consequently, these antisera cannot be used to determine whether one or both of the crossreacting proteins modulate a critical step in the acrosome reaction.     

Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.     

Molecular cloning of Odf3 encoding a novel coiled-coil protein of sperm tail outer dense fibers.

The outer dense fibers (ODF) are the main cytoskeletal structures of the sperm tail found in animals with internal fecundation. They consist of at least 14 polypeptides from which only a few are identified due to difficulties in isolation of the protein components. Here we report the isolation and molecular characterization of Odf3, encoding a novel protein of rat sperm ODF. Odf3 is transcribed in testes and more specifically in spermatids but it is also expressed in epididymides and brain suggesting a possible involvement in building of the cellular cytoskeleton. Odf3 encodes a putative protein of approximately 110 kDa. Secondary structure predictions indicated that ODF3 is a coiled-coil protein. The identification of coiled-coil proteins as constituents of outer dense fibers reveals a model for ODF formation.     

Synthesis and application of peptide immunogens related to the sperm tail protein tpx-1, a member of the CRISP superfamily of proteins.

The synthesis of peptides containing 0, 1 and 2 cysteine residues related to the human sperm tail protein, tpx-1, is described. These synthetic peptides, following conjugation to keyhole limpet hemocyanin modified with maleimidobenzoic acid N-hydroxysuccinimide ester, were used as immunogens to generate polyclonal antibodies in female New Zealand white rabbits. The binding characteristics of the derived antipeptide sera were evaluated using indirect and competitive ELISA procedures. Western immunoblot experiments also confirmed that these synthetic peptide immunogens are able to generate high-titer polyclonal antibodies capable of cross-reacting with the mature tpx-1 protein present in crude rat sperm tail/testis preparations as well as in outer dense fiber preparations. Consequently, these synthetic peptides represent promising candidates for investigations into the role of tpx-1 in the immunoregulation of sperm function in the rat and other mammalian models, with the derived antisera also providing an avenue to explore possible sites of expression of tpx-1 proteins in other tissues.     

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.     

Surface protein changes in goat spermatozoa during capacitation

Polypeptides of goat sperm surface before and after capacitation were examined by radiolabelling and immunologically using polyclonal antisera. Radioiodination revealed five protein bands having mol wt of 14.8, 72.4, 81, 100 and 128 kDa in uncapacitated ejaculated spermatozoa and only three bands of 23.4, 27 and 72.4 KDa in capacitated spermatozoa. The protein band with mol wt 72.4 kDa was only feebly iodinated in uncapacitated sperm surface but in capacitated spermatozoa it was heavily labelled. Western blot analysis of detergent-extracted proteins using gamma-globulin fraction of antisera raised against purified goat sperm plasma membrane revealed six antigens (17.8, 29.1, 33.4, 45.6, 85.1, 123.2 kDa) in uncapacitated spermatozoa, four (26, 32.1, 40.1, 45.6 kDa) in capacitated spermatozoa and only one (45.6 kDa) in acrosome-reacted spermatozoa. High mol wt proteins were more numerous on the surface of uncapacitated spermatozoa while the capacitated spermatozoa had relatively low mol wt proteins. An apparent effect of capacitation is the metabolism and reorganisation of proteins on goat sperm surface. Polypeptides on capacitated sperm surface revealed through radiolabelling and polyclonal antisera may have a likely receptor(s) role in the recognition and binding to homologous zona pellucida during fertilization.