Biochemical characterization of the PH-20 protein on the plasma membrane and inner acrosomal membrane of cynomolgus macaque spermatozoa

Preparations of sperm membranes (plasma membranes and outer acrosomal membranes) and denuded sperm heads were isolated from macaque sperm, and the PH-20 proteins present were characterized by Western blotting, hyaluronic acid substrate gel analysis, and a microplate assay for hyaluronidase activity. Because we have shown previously that PH-20 is located on the plasma membrane and not on the outer acrosomal membrane, the PH-20 in the membrane preparations was presumed to be plasma membrane PH-20 (PM-PH-20). PM-PH-20 had an apparent molecular weight of 64 kDa and the optimum pH for its hyaluronidase activity was 6.5. The PH-20 associated with denuded sperm heads was localized by immunogold label to the persistent inner acrosomal membrane (IAM) and was presumed to be IAM-PH-20, which included a major 64 kDa form and a minor 53 kDa form. The 53 kDa form was not detected in extracts of denuded sperm heads from acrosome intact sperm that were boiled in nonreducing sample buffer, but was present in extracts of sperm heads from acrosome reacted sperm and in the soluble material released during the acrosome reaction, whether or not the samples were boiled. Substrate gel analysis showed that the hyaluronidase activity of the 53 kDa form of PH-20 was greatest at acid pH, and this activity was probably responsible for the broader and lower optimum pH of IAM hyaluronidase activity. When hypotonic treatment was used to disrupt the sperm acrosome and release the acrosomal contents, less than 0.05% of the total hyaluronidase activity was released. The PH-20 protein released by hypotonic treatment was the 64 kDa form and not the 53 kDa form, suggesting that its source might be the disrupted plasma membranes. Our experiments suggest that the soluble form of hyaluronidase, which is released at the time of the acrosome reaction, is derived from the IAM. This soluble hyaluronidase is composed of both the 64 kDa form and 53 kDa form of PH-20. The 53 kDa form appears to be processed from the 64 kDa form at the time of the acrosome reaction. Mol. Reprod. Dev. 48:356–366, 1997. © 1997 Wiley-Liss, Inc.     

Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm

PH-20 protein on the plasma membrane (PH-20PM) is restricted to the posterior head of acrosome-intact guinea pig sperm. During the exocytotic acrosome reaction the inner acrosomal membrane (IAM) becomes continuous with the posterior head plasma membrane, and PH-20PM migrates to the IAM. There it joins a second population of PH-20 protein localized to this region of the acrosomal membrane (PH-20AM) (Cowan, A.E., P. Primakoff, and D.G. Myles, 1986, J. Cell Biol. 103:1289-1297). To investigate how the localized distributions of PH-20 protein are maintained, the lateral mobility of PH-20 protein on these different membrane domains was determined using fluorescence redistribution after photobleaching. PH-20PM on the posterior head of acrosome-intact sperm was found to be mobile, with a diffusion coefficient and percent recovery typical of integral membrane proteins (D = 1.8 X 10(-10) cm2/s; %R = 73). This value of D was some 50-fold lower than that found for the lipid probe 1,1-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (C14diI) in the same region (D = 8.9 X 10(-9) cm2/s). After migration to the IAM of acrosome-reacted sperm, this same population of molecules (PH-20PM) exhibited a 30-fold increase in diffusion rate (D = 4.9 X 10(-9) cm2/s; %R = 78). This rate was similar to diffusion of the lipid probe C14diI in the IAM (D = 5.4 X 10(-9) cm2/s). The finding of free diffusion of PH-20PM in the IAM of acrosome-reacted sperm supports the proposal that PH-20 is maintained within the IAM by a barrier to diffusion at the domain boundary. The slower diffusion of PH-20PM on the posterior head of acrosome-intact sperm is also consistent with localization by barriers to diffusion, but does not rule out alternative mechanisms.     

Sperm exocytosis increases the amount of PH-20 antigen on the surface of guinea pig sperm

Evidence has been presented that the PH-20 protein functions in sperm adhesion to the egg zona pellucida (Primakoff, P., H. Hyatt, and D. G. Myles, 1985, J. Cell Biol., 101:2239-2244). The PH-20 protein migrates from its original surface domain to a new surface domain after the acrosome reaction (Myles, D. G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). The acrosome reaction is an exocytotic event that results in insertion of a region of the secretory granule membrane, the inner acrosomal membrane (IAM), into the plasma membrane. After the acrosome reaction, PH-20 protein migrates to the IAM from its initial domain on the posterior head surface. We have now found a new dynamic feature of the regulation of PH-20 protein on the sperm surface; exocytosis increases the surface expression of PH-20 protein. After the acrosome reaction there is an approximately threefold increase in the number of PH-20 antigenic sites on the sperm surface. These new antigenic sites are revealed on the surface by insertion of the IAM into the plasma membrane. Our evidence indicates that before the acrosome reaction an intracellular population of PH-20 antigen is localized to the IAM. When migration of the surface population of the PH-20 protein is prevented, PH-20 protein can still be detected on the IAM of acrosome-reacted sperm. Also, PH-20 protein can be detected on the IAM of permeabilized acrosome-intact sperm by indirect immunofluorescence. Thus, the sperm cell regulates the amount of PH-20 protein on its surface by sequestering about two-thirds of the protein on an intracellular membrane and subsequently exposing this population on the cell surface by an exocytotic event. This may be a general mechanism for regulating cell surface composition where a rapid increase in the amount of a cell surface protein is required.     

The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling

The ovulated mammalian oocyte is surrounded by the "cumulus ECM", composed of cells embedded in an extracellular matrix that is rich in hyaluronic acid (HA). The cumulus ECM is a viscoelastic gel that sperm must traverse prior to fertilization. Mammalian sperm have a GPI-anchored hyaluronidase which is known as PH-20 and also as SPAM 1. PH-20 is located on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH-20 appears to be a multifunctional protein; it is a hyaluronidase, a receptor for HA-induced cell signaling, and a receptor for the zona pellucida surrounding the oocyte. The zona pellucida recognition function of PH-20 was discovered first. This function is ascribed to the inner acrosomal membrane PH-20, which appears to differ biochemically from the PH-20 on the sperm surface. Later, when bee venom hyaluronidase was cloned, a marked cDNA sequence homology with PH-20 was recognized, and it is now apparent that PH-20 is the hyaluronidase of mammalian sperm. PH-20 is unique among the hyaluronidases in that it has enzyme activity at both acid and neutral pH, and these activities appear to involve two different domains in the protein. The neutral enzyme activity of plasma membrane PH-20 is responsible for local degradation of the cumulus ECM during sperm penetration. Plasma membrane PH-20 mediates HA-induced sperm signaling via a HA binding domain that is separate from the hyaluronidase domains. This signaling is associated with an increase in intracellular calcium and as a consequence, the responsiveness of sperm to induction of the acrosome reaction by the zona pellucida is increased. There is extensive evidence that GPI-anchored proteins are involved in signal transduction initiated by a diverse group of cell surface receptors. GPI-anchored proteins involved in signaling are often associated with signaling proteins bound to the cytoplasmic leaflet of the plasma membrane, typically Src family, non-receptor protein tyrosine kinases. PH-20 appears to initiate intracellular signaling by aggregating in the plasma membrane, and a 92-kDa protein may be the cell signaling molecule linked to PH-20.     

Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis.

The localization of proacrosin was determined by using colloidal gold labeling and electron microscopy of boar germ cells during spermiogenesis to post-ejaculation. Proacrosin was first localized in round spermatids during the Golgi phase of spermiogenesis; it was associated with the electron-dense granule, or acrosomal granule that was conspicuous within the acrosome. It remained within the acrosomal granule during the cap and acrosome phases of spermiogenesis. At these stages, there was no apparent association of the proacrosin molecule with the acrosomal membranes. During the maturation phase of spermiogenesis, proacrosin was seen to become dispersed into all regions of the acrosome except the equatorial segment. When sperm from different segments of the epididymis and ejaculated sperm were examined, localization was observed throughout the acrosome except for the equatorial segment. Here proacrosin appeared to be localized on both the inner and outer acrosomal membranes as well as with the acrosomal matrix, although further studies are required to verify the membrane localization. No labeling was seen on the plasma membrane. These data suggest that the synthesis and movement of proacrosin to sites in the acrosome are controlled by an as yet unknown process. The absence of proacrosin on the plasma membrane of mature ejaculated sperm makes it unlikely that this enzyme plays a role in sperm-zona adhesion prior to capacitation.     

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.     

大熊猫精子获能和顶体反应过程中钙分布变化规律的研究

应用焦锑酸钾原位定位法对大熊猫精子获能和顶体反应过程中进行钙定位研究,发现未获能精子的 Ｃａ２＋主要结合于顶体前区和赤道段质膜外侧和顶体内膜内侧（核膜侧）;随着获能的进行,Ｃａ２＋进入精子内部并主要结合于顶体区质膜内侧和顶体外膜外侧;顶体反应的精子,Ｃａ２＋结合于顶体内膜外侧、顶体后区质膜外侧和分散存在于释放的顶体内容物中,有些顶体反应精子的顶体内膜外侧结合的Ｃａ２＋特别丰富。精子尾部的Ｃａ２＋主要分布于中段线粒体内,且其内所含Ｃａ２＋含量随着获能和顶体反应而增加。另外尾部致密纤维和轴丝处也有少量Ｃａ２＋分布。     

Zonadhesin assembly into the hamster sperm acrosomal matrix occurs by distinct targeting strategies during spermiogenesis and maturation in the epididymis

Zonadhesin is the only sperm protein known to bind in a species-specific manner to the zona pellucida. The zonadhesin precursor is a mosaic protein with a predicted transmembrane segment and large extracellular region composed of cell adhesion, mucin, and tandem von Willebrand D domains. Because the precursor possesses a predicted transmembrane segment and localizes to the anterior head, the mature protein was presumed to be a sperm surface zona pellucida-binding protein. In this study of hamster spermatozoa, we demonstrate that zonadhesin does not localize to the sperm surface but is instead a constituent of the acrosomal matrix. Immunoelectron microscopy revealed that distinct targeting pathways during spermiogenesis and sperm maturation in the epididymis result in trafficking of zonadhesin to the acrosomal matrix. In round spermatids, zonadhesin localized specifically to the acrosomal membrane, where it appeared to be evenly distributed between the outer and inner membrane domains. Subsequent redistribution of zonadhesin resulted in its elimination from the inner acrosomal membrane and restriction to the outer acrosomal membrane of the apical and principal segments and the contents of the posterior acrosome. During sperm maturation in the epididymis, zonadhesin dissociated from the outer acrosomal membrane and became incorporated into the forming acrosomal matrix. These data suggest an important structural role for zonadhesin in assembly of the acrosomal matrix and further support the view that the species specificity of zona pellucida adhesion is mediated by egg-binding proteins contained within the acrosome rather than on the periacrosomal plasma membrane.     

SAMP14, a novel,acrosomal membrane-associated,glycosylphosphatidylinositol-anchored member of the Ly-6/urokinase-type plasminogen activator receptor superfamily with a role in sperm-egg interaction

We report a new member of the Ly-6/urokinase-type plasminogen activator receptor (uPAR) superfamily of receptors, SAMP14, which is retained on the inner acrosomal membrane of the human spermatozoan following the acrosome reaction and may play a role in fertilization. The SAMP14 sequence predicted a glycosylphosphatidylinositol (GPI)-anchored protein with a signal peptide, a transmembrane domain near the carboxyl terminus, and a putative transamidase cleavage site in the proprotein. Attachment of SAMP14 to the membrane by a lipid anchor was confirmed by its sensitivity to phosphatidylinositol phospholipase C. SAMP14 has a single functional domain similar to the Ly-6 and urokinase plasminogen activator receptor superfamily of proteins, and the gene mapped to 19q13.33, near the PLAUR locus for uPAR at 19q13.2. Northern and dot blotting showed that SAMP14 expression was testis-specific. Indirect immunofluorescence and immunoelectron microscopy with antisera to purified recombinant SAMP14 localized the protein to outer and inner acrosomal membranes as well as the acrosomal matrix of ejaculated human sperm. Acrosome-reacted sperm demonstrated SAMP14 immunofluorescence, indicating its retention on the inner acrosomal membrane following the acrosome reaction. However, SAMP14 localized to the entire sperm when unwashed swim-up sperm from the ejaculate were stained, indicating that some SAMP14 is loosely associated with the plasma membrane. Antibodies against recombinant SAMP14 inhibited both the binding and the fusion of human sperm to zona free hamster eggs, suggesting that SAMP14 may have a role in sperm-egg interaction. SAMP14 represents a GPI-anchored putative receptor in the Ly-6/uPAR family that is exposed on the inner acrosomal membrane after the acrosome reaction.     

Purification of the guinea pig sperm PH-20 antigen and detection of a site-specific endoproteolytic activity in sperm preparations that cleaves PH-20 into two disulfide-linked fragments

Previous work has indicated that the guinea pig sperm membrane protein, PH-20, functions in sperm-egg adhesion and that its surface expression is regulated by the acrosome reaction. The PH-20 protein was purified by monoclonal antibody affinity chromatography. Sixty-seven to one hundred percent of the PH-20 antigenic activity present in an octylglucoside (OG) extract of sperm was recovered in the purified protein. From 10(10) sperm, approximately 0.4 mg of PH-20 protein was obtained, which was about 0.24% of the total protein in the OG extract. The purified protein retained the ability to bind the three anti-PH-20 monoclonal antibodies we have isolated. Silver staining of purified PH-20 on overloaded sodium dodecyl sulfate (SDS) gels allowed the estimate that silver-stainable contaminants were present at a level of one part in 2000. The purified PH-20 protein exists in three forms separable on SDS-polyacrylamide gel electrophoresis: a major form with a molecular mass of 64 kDa, a minor form of 56 kDa, and an endoproteolytically cleaved form composed of two disulfide-linked fragments of 41-48 kDa and 27 kDa. Cleveland digests of the 64 kDa and 56 kDa polypeptides indicated that they were structurally related. A proportion of the 64 kDa polypeptide in each purified preparation had undergone endoproteolysis at a specific site, so that it was cleaved into the two disulfide-linked fragments, 41-48 kDa and 27 kDa. It is speculated that the site-specific endoproteolysis of PH-20 may occur during the acrosome reaction and have biological significance.     

Equine sperm-oocyte interaction: the role of sperm surface hyaluronidase.

The plasma membrane over the sperm head of several mammalian species has been shown to express a glycerolphosphatidylinositol-linked hyaluronidase known as PH-20. This protein has been associated with the sperm's interaction with the oocyte cumulus matrix and zona pellucida. The characteristics of PH-20 in equine sperm have not been clearly defined. In this study, ejaculated gel-free semen from five stallions and epididymal sperm from isolated epididymis from 10 stallions was used to characterize the PH-20 activity in equine sperm. Affinity purified anti-equine PH-20 polyclonal antibody was used to immunodetect sperm surface-associated PH-20 and immunolabel whole sperm. The intracellular calcium indicator, Fluo-3, was used to assess sperm intracellular calcium. Stallion sperm express a surface-associated hyaluronidase localized to the posterior sperm head region in ejaculated sperm. Following in vitro capacitation and acrosomal exocytosis, the inner acrosomal membrane (IAM) displays intense hyaluronidase fluorescence suggesting that the IAM and hyaluronidase plays a significant role in zona penetration by sperm. Sperm incubated in hyaluronan (HA)-containing capacitation medium display an elevated intracellular calcium concentration (P<0.01) that is associated with translocation of PH-20 antigenic sites on the sperm surface in addition to increases in protein tyrosine phosphorylation. Caput- and cauda-derived sperm display developmentally unique PH-20 immunofluorescence expression patterns. These data suggest that the differential expression of PH-20 in ejaculated and epididymal sperm could be involved in cumulus penetration, sperm-egg recognition, and oolemmal fusion in this species.     

Changes in distribution and molecular weight of the acrosomal protein acrin2 (MC41) during guinea pig spermiogenesis and epididymal maturation

In this study, we examined the localization and characteristics of an intra-acrosomal protein, acrin2 (MC41), during guinea pig spermiogenesis and post-testicular sperm maturation in the epididymis, using the monoclonal antibody MC41. Immunoelectron microscopy demonstrated not only a specific domain localization of acrin2 in the apical segment of the guinea pig sperm acrosome, but also its dynamic behavior according to the spermatid differentiation and passage through the epididymis, as follows: acrin2 was exclusively localized in the membrane of the endoplasmic reticulum of early-stage spermatids but was not detectable in the developing acrosome until spermatids reached the maturation phase. In the final stage of spermiogenesis, acrin2 became localized in the outer acrosomal membrane (OAM)/matrix-associated materials both in the small region posterior to the dorsal matrix and along the ventral margin of the acrosomal apical segment. The acrosomal location of acrin2 in caput epididymidal sperm was almost identical to that observed in the final step spermatids, but during maturation it became progressively more restricted in area until on distal cauda epididymidal sperm it remained only in the dorsal region. In Western blot analysis, the MC41 antibody recognized a 165-kDa protein in the mature sperm extract. Furthermore, it was demonstrated that molecular weight reduction of the protein occurred during sperm passage through the epididymis. These findings indicate that acrin2 changes progressively in both distribution and size during development and maturation of the acrosome.     

Acrosome differentiation in the ascidians Clavelina lepadiformis and Ciona intestinalis

The spermatozoa of both Clavelina lepadiformis and Ciona intestinalis have architectural features characteristic of ascidian spermatozoa that have been previously described. They have an elongated head (6 microm and 3 microm long, respectively) and a single mitochondrion that is closely applied laterally to the nucleus; they lack a midpiece. The acrosome of Clavelina lepadiformis spermatozoa is a moderately electron-dense, pear-shaped flattened vesicle, approx. 300 nm x 200 nm x 40 nm in length, width, and height, respectively. The acrosome of Ciona intestinalis spermatozoa is a moderately electron-dense, round flattened vesicle with an electron-dense plate in its central region. It is approx. 200 nm x 200 nm x 50 nm in length, width, and height, respectively. During spermiogenesis in both ascidians, several proacrosomal vesicles (50-70 nm in diameter) appear in a blister at the future apex of the spermatids. These vesicles appear to be associated with the inner surface of the plasma membrane enclosing the blister. They come into contact with each other along the inner surface of the plasma membrane and fuse to form a horseshoe-shaped acrosomal vesicle, which becomes a round, flattened vesicle during further differentiation. Some speculations about the mechanism of acrosome differentiation, the possible role of the acrosome during fertilization, and in the speciation of ascidians are presented.     

Studies on the acrosome. X. Differentiation of the starfish acrosome

The course of acrosomal differentiation observed during spermiogenesis in two starfishes shows that the central components of the mature acrosome are produced by Golgi activity. In the early spermatid, small Golgi-derived vesicles enter the hydrated acrosomal mass and appear to contribute their membrane constituents to the acrosomal-membrane precursor elements. A single lamella of smooth endoplasmic reticulum and fine-fibrillar material associated with it surround the membraneprecursor complex. In a drastic reorganization by which the spermatid acquires antero-posterior symmetry, the acrosome becomes embedded in the anterior part of the nucleus directly beneath the plasma membrane. All the other organelles congregate in the posterior cytoplasm; a thin layer of cytoplasm persists around the sides of the nucleus. During late spermiogenesis two additional acrosomal components become increasingly conspicuous. One is the layer of fine-fibrillar material associated with the smooth endoplasmic reticular vesicles surrounding the Golgi-derived elements. This material is finally pushed towards the center of the sperm head by a late accretion of fibrous product which appears to be synthesized throughout spermiogenesis by the ribosomes, and accumulates around the anterior part of the acrosome as the cytoplasmic matrix diminishes.     

Afaf, a novel vesicle membrane protein, is related to acrosome formation in murine testis

As a cell-specific organelle, acrosome (Acr) and its formation are an important event for spermiogenesis. However, the Acr formation is far more complicated than has been proposed. In this study, we have cloned a novel membrane protein Afaf (Acr formation associated factor) that was expressed abundantly in the round spermatids, localized in the inner and outer membrane of forming Acrs, and declined in the maturing Acrs. In the transfected Hela cells, Afaf protein was localized in the plasma membrane, EEA1-positive early endosomes (EEs) and occasionally in the nuclei. Therefore, we propose that EEs and plasma membrane may be also directly involved in the Acr biogenesis.     

Fusion of membranes during the acrosome reaction: a tale of two SNAREs

During spermiogenesis, hydrolytic enzymes are sorted from the Golgi apparatus to the acrosome, a supranuclear megavesicle. At fertilization, the enzymatic content of the acrosome is released by exocytosis when a portion of the plasma membrane enveloping the sperm head fuses with the outer membrane of the acrosome. Membrane fusion involves the interaction of a specific pair of proteins, called SNAREs (for soluble N-ethylmaleimide sensitive factor attachment protein receptor). v-SNARE is presumably associated with the membrane of the acrosomal vesicle. Target t-SNARE is associated with the plasma membrane. The interaction of v-SNARE and t-SNARE requires two additional proteins: Rab proteins, members of a family of small GTPases related to the Ras proteins, and a complex of two proteins, NSF-SNAP, recruited by the interacting v-SNARE-tSNARE pair. Syntaxin 2, a v-SNARE member, and Rab3A, a member of the Rab GTPases, have been localized in the acrosome of rodent sperm.     

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

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

cDNA cloning reveals the molecular structure of a sperm surface protein,PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals

Sperm binding to the egg zona pellucida in mammals is a cell-cell adhesion process that is generally species specific. The guinea pig sperm protein PH-20 has a required function in sperm adhesion to the zona pellucida of guinea pig eggs. PH-20 is located on both the sperm plasma membrane and acrosomal membrane. We report here the isolation and sequence of a full-length cDNA for PH-20 (available from EMBL/GenBank/DDBJ under accession number {"type":"entrez-nucleotide","attrs":{"text":"X56332","term_id":"49444","term_text":"X56332"}}X56332). The derived amino acid sequence shows a mature protein of 468 amino acids containing six N-linked glycosylation sites and twelve cysteines, eight of which are tightly clustered near the COOH terminus. The sequence indicates PH-20 is a novel protein with no relationship to the mouse sperm adhesion protein galactosyl transferase and no significant homology with other known proteins. The two PH-20 populations, plasma membrane and acrosomal membrane, could arise because one form of PH-20 is encoded and differentially targeted at different spermatogenic stages. Alternatively, two different forms of PH-20 could be encoded. Our evidence thus far reveals only one sequence coding for PH-20: Southern blots of guinea pig genomic DNA indicated there is a single PH-20 gene, Northern blots showed a single size PH-20 message (approximately 2.2 kb), and no sequence variants were found among the sequenced cDNA clones. Cross-species Southern blots reveal the presence of a homologue of the PH-20 gene in mouse, rat, hamster, rabbit, bovine, monkey, and human genomic DNA, showing the PH-20 gene is conserved among mammals. Since genes for zona glycoproteins are also conserved among mammals, the general features of sperm and zona proteins involved in mammalian sperm-egg adhesion may have been evolutionarily maintained. Species specificity may result from limited changes in these molecules, either in their binding domains or in other regions that affect the ability of the binding domains to interact.     

Biochemical and morphological characterization of the intra-acrosomal antigen SP-10 from human sperm

The human sperm protein SP-10 was previously defined as a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. By one- and two-dimensional immunoblots, we show that SP-10, extracted from ejaculated human sperm, demonstrated a polymorphism of immunogenic peptides from 18 to 34 kDa, a pattern that was conserved from individual to individual and was not altered by reducing agents. The majority of the antigenic peptides possessed isoelectric points of approximately 4.9. Immunocytochemistry on testis sections indicated that SP-10 was localized to round spermatids and spermatozoa within the adluminal compartment of the seminiferous epithelium. Immunofluorescence showed that SP-10 was not associated with the surface of acrosome-intact, ejaculated sperm. Light and electron microscopic immunocytochemistry localized SP-10 throughout the acrosome, and electron microscopic evidence demonstrated a bilaminar array in association with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After induction of the acrosome reaction with the ionophore A23187, SP-10 remained displayed on the sperm head in association with the inner acrosomal membrane and equatorial segment. The results indicate that the MHS-10 monoclonal antibody may be used as a marker of acrosome development in the human and as a probe to evaluate acrosome status. The results also support the hypothesis that inhibition of sperm-egg interaction by anti-SP-10 monoclonal antibody may occur as a result of antigen exposure following the acrosome reaction.     

The role of a Sertoli cell actin-myosin system in sperm bundle formation in the ratfish, Hydrolagus colliei (Chondrichthyes, Holocephali)

Sertoli cells in the ratfish entirely surround a clone of spermatids to form a spermatocyst. As spermiogenesis proceeds within the cyst cavity, the acrosome areas become apposed to the Sertoli cell plasma membrane lining the spermatocyst. The spermatids elongate and are gathered into an increasingly compact bundle oriented with acrosomal tips directed toward the Sertoli cell base. As all acrosome areas move closer together, Sertoli cell microfilaments oriented parallel to the long spermatid axis appear and increase in concentration. Actin and myosin were demonstrated in the microfilament area with fluorescent antibodies and NBD-Phallacidin. Simultaneously, endocytosis of Sertoli cell membrane between spermatid attachment sites removes the intervening membrane and allows the latter sites to approach each other. Sertoli cell endocytosis is spatially and temporally related to a unique projection at the basal rim of each acrosome. During midspermiogenesis, structured intercellular material appears between the Sertoli cell and the acrosomal region of each spermatid. Its periodicity is closely related to periodic arrangement of Sertoli cell actin and material within the spermatids. These attachment sites move together upon endocytosis, gathering a clone of spermatids into a closely packed bundle.