Acrosomal Membrane Protein Research Articles

Sperm plasma membrane breakdown during Drosophila fertilization requires Sneaky, an acrosomal membrane protein

Fertilization typically involves membrane fusion between sperm and eggs. In Drosophila, however, sperm enter eggs with membranes intact. Consequently, sperm plasma membrane breakdown (PMBD) and subsequent events of sperm activation occur in the egg cytoplasm. We previously proposed that mutations in the sneaky (snky) gene result in male sterility due to failure in PMBD. Here we support this proposal by demonstrating persistence of a plasma membrane protein around the head of snky sperm after entry into the egg. We further show that snky is expressed in testes and encodes a predicted integral membrane protein with multiple transmembrane domains, a DC-STAMP-like domain, and a variant RING finger. Using a transgene that expresses an active Snky-Green fluorescent protein fusion (Snky-GFP), we show that the protein is localized to the acrosome, a membrane-bound vesicle located at the apical tip of sperm. Snky-GFP also allowed us to follow the fate of the protein and the acrosome during fertilization. In many animals, the acrosome is a secretory vesicle with exocytosis essential for sperm penetration through the egg coats. Surprisingly, we find that the Drosophila acrosome is a paternally inherited structure. We provide evidence that the acrosome induces changes in sperm plasma membrane, exclusive of exocytosis and through the action of the acrosomal membrane protein Snky. Existence of testis-expressed Snky-like genes in many animals, including humans, suggests conserved protein function. We relate the characteristics of Drosophila Snky, acrosome function and sperm PMBD to membrane fusion events that occur in other systems.

Effects of Sulfasalazine on Sperm Acrosome Reaction and Gene Expression in the Male Reproductive Organs of Rats

Sulfasalazine (SASP) has been reported to depress the fertility in men and experimental male animals, but the fundamental mechanisms of infertility caused by SASP are still unknown. This study was designed to investigate the mechanisms of infertility in rats treated with SASP at a dose of 600 mg/kg for 28 days, including monitoring of sperm motility using computer associated sperm analysis system and acrosome reaction by FITC-concanavalin A lectin staining. The sperm motility and acrosome reaction, which are important for fertilization, were significantly reduced by SASP. Furthermore, to investigate the molecular mechanisms of infertility induced by SASP, mRNA expression analysis in the testes was performed using cDNA microarray as a first screening. It was revealed that CD59, which is located on the acrosomal membrane and is known to be important for the reproductive function of sperm, was affected in the testes; this was also confirmed by real-time PCR analysis, but the spermatogenesis-related genes examined in this study were not affected. Therefore, we focused on CD59 and two other acrosome membrane related-genes: MCP and DAF. CD59, MCP, and DAF in the epididymides of SASP-treated rats were significantly decreased as assessed by real-time RT-PCR analysis and additionally, the expression of CD59 protein was found to be decreased by Western blotting. These results allowed us to hypothesize that the suppression of epididymal acrosomal membrane proteins synthesis with their consequent reduced incorporation to the sperm membrane leads to a depressed sperm motility and acrosome reaction, and thereby leads to infertility in SASP treated male rats.

A 70 kDa protein is transferred from the outer acrsomal to the plasma membrane during capacitation

Bull sperm plasma and outer acrosomal membranes were analyzed by SDS-PAGE. Analysis of the plasma membrane proteins revealed the presence of a 70 kDa band the prominence of which is enhanced after capacitation. This protein was found to bind to zona pellucida intact oocytes. PAGE analysis of outer acrosomal membrane proteins also reveals the presence of a 70 kDa band, but its prominence decreases after capacitation. This protein also binds to zona pellucida intact oocytes. Futhermore, the 70 kDa outer acrosomal membrane protein is recognized in Western blot analysis by antibodies to plasma membrane proteins and vice versa. The results indicate that the 70 kDa acrosomal and plasma membrane proteins are the same. This 70 kDa protein would thus be a zona pellucida binding protein which is initially stored in the outer acrosomal membrane and transferred to the plasma membrane during capacitation, enabling it to function in egg-sperm binding.

Changes in membrane proteins of cauda spermatozoa of goat bucks during in vitro capacitation

Sperm membrane (SM) proteins from in vitro capacitated and acrosome-reacted (AR) cauda spermatozoa of goat bucks were extracted with 1.0% Brij-35, deoxycholate and saponin. Spermatozoa showed capatitation after 3 h in BWW medium (pH 7.4, containing 3% BSA and 10 mM Ca 2+. The AR occurred by sequential shrinkage/decondensation of acrosomal contents, vesiculation and acrosome shedding, with concomitant release of SM proteins into the medium. Lesser amounts of SM proteins extracted from capacitated spermatozoa indicated their release during AR. Polyacrylamide gel electrophoresis pattern indicated that proteins/glycoproteins of low/high molecular weight were released and those with relatively higher molecular weight were preferentially retained in the membranes during AR. Glycoprotein nature of the released components indicated their membrane origin. Preferential retention of high molecular weight glycoproteins indicated that these were deeply embedded in membranes. The immunodiffusion with heteroantisera Ig against testicular cells and epididymal spermatozoal extracts demonstrated removal of 140–160 kDa surface antigens during AR. Membrane extracts of capacitated spermatozoa did not react with Ig from isoantisera raised against caput and cauda spermatozoa. The acrosome reaction in bucks involves sequential shrinkage/decondensation, vesiculation, shedding of acrosomal membranes and membrane proteins. Such proteins may be involved in fertilization.

Characterization of human sperm antigens and antisperm antibodies in infertile patients

To identify which sperm antigens may elicit the production of functionally important antisperm antibodies. Immunoblot analysis was performed on 69 serum and 9 seminal plasma samples from infertile patients, using detergent extracts of pooled donor sperm as the antigen source. Serum and seminal plasma had been previously tested by an indirect immunobead binding test (IBT); 61 IBT-positive and 17 IBT-negative samples were included in the study. Proteins recognized by IBT-positive but not IBT-negative samples were most likely to be cell surface antigens, whereas proteins recognized by both IBT-positive and IBT-negative samples were probably intracellular. Antibodies directed toward surface antigens would be most likely to affect fertilization. Characterization of sperm surface proteins on both acrosome-intact and -reacted sperm used labeling of cell surface proteins with an N-hydroxysuccinimide ester of biotin, fractionation of sperm heads and tails, and lectin binding to determine glycosylation. Specific immunoreactivity (with respect to IBT results) was observed to 35K, 40 to 45K, 57K, 66K, and 88 to 90K MW proteins. Characterization studies identified an 88K MW glycosylated plasma membrane protein, a 66K MW inner acrosomal membrane protein, a 34K MW inner acrosomal membrane protein, and a 35K MW prominent tail protein. Immunological infertility may involve several antigens characterized in this study. Further studies are necessary to determine if antibodies to these specific proteins interfere with sperm function.

Interactions of human sperm acrosomal protein SP-10 with the acrosomal membranes.

The interaction of the human acrosomal protein SP-10 with the acrosomal membranes was analyzed by the ability of Triton X-114 (TX-114) and other agents to release SP-10 from the acrosome. Treatment of human sperm with TX-114 revealed a pool of SP-10 that was released by TX-114 and a pool of SP-10 that was TX-114-resistant. TX-114-resistant SP-10 was associated with the equatorial segment and with TX-114-resistant portions of the acrosomal matrix and the inner acrosomal membrane. Phase partitioning of TX-114-released and TX-114-resistant SP-10 pools showed that both were hydrophilic, indicating that these pools consist of proteins that are peripherally associated with, rather than integral to, the acrosomal membranes. Sequential treatments of human sperm with various agents showed that repeated washes with TX-114 or 1.5 M NaCl had little or no effect on TX-114-resistant SP-10, whereas treatment with a chaotropic salt (150 mM sodium thiocyanate) and buffers at pH extremes (pH 2.0 and 10.0) completely released this pool of SP-10 from the acrosome. Together the results suggest that SP-10 is a hydrophilic peripheral acrosomal membrane protein that may be associated with a TX-114-resistant "anchor."

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 M r = 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.

Intra‐acrosomally located acrosin‐inhibitors: Evolution and developmental patterns in mammals

AbstractThe acrosome of the mammalian spermatozoon contains proacrosin that is autocatalytically activated to become the proteolytic enzyme acrosin during the process of fertilization. Tschesche et al [1982] isolated specific acrosin inhibitors and suggested that they block prematurely activated acrosin. Antibodies against acrosin inhibitors purified from boar spermatozoa were used to demonstrate the evolutionaary relationship and the developmental pattern of the inhibitors in mammals. Using immunofluorescent techniques the following results were obtained: (1) The spermatozoa of man, boar, bull, ram, rat, rabbit, beaver, and mole stained positive in the acrosomal portion. (2) The round‐headed spermatozoa of patients with globozoospermia and those in the ejaculates of fertile men lacked immunostaining for the inhibitors. (3) During spermatogenesis in all species, immunofluorescence for the acrosin inhibitors was first demonstrable in haploid spermatids and increased thereafter during spermatid differentiation. The stained area was found adjacent to the nuclear membrane, the position of the acrosome. (4) During teratogenesis of round‐headed spermatozoa, the immunofluorescent staining for the inhibitors becomes separated from the nuclear membrane of the spermatids and is lost in late spermatids. Since identical results have been described for acrosin and acrosomal membrane proteins both in spermatozoa and spermatids of mammalian species and during spermiogenesis of patients with globozoospermia, our results are consistent with the localisation of the inhibitors in the acrosome. Immunostaining of spermatozoa of species belonging to five different mammalian orders with the antibody against boar acrosin‐inhibitors is indicative of an evolutionary conservation of the inhibitors and their underlying genes.

The molecular transformation of rabbit testis proacrosin into acrosin

Partially purified rabbit testis proacrosin formed only one acrosin of 73,000 ± 3000 apparent molecular weight ( M r ) during the early phase of “autoactivation” at pH 8. Complete “autoactivation” then converted this acrosin to a 38,000 ± 3000 M r , acrosin. These results suggest the existence of a proacrosin dimer (73,000) or a dimer of proacrosin and an acrosomal membrane protein which were converted first to an acrosin dimer (73,000 M r ) or to an acrosin-membrane protein dimer and then to the acrosin monomer (38,000). The formation of a 73,000 ± 3000 M r acrosin from a 73,000 ± 3000 proacrosin is explainable by assuming that either a small activation pertide(s) is released or none at all.