Lipid Regulation of Acrosome Exocytosis.

Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

Haprin, a novel haploid germ cell-specific RING finger protein involved in the acrosome reaction.

The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr approximately 82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.

Mammalian sperm acrosome: formation, contents, and function.

Sperm-egg interaction is a carbohydrate-mediated species-specific event which initiates a signal transduction cascade resulting in the exocytosis of sperm acrosomal contents (i.e., the acrosome reaction). This step is believed to be a prerequisite which enables the acrosome-reacted spermatozoa to penetrate the zona pellucida (ZP) and fertilize the egg. Successful fertilization in the mouse and several other species, including man, involves several sequential steps. These are (1) sperm capacitation in the female genital tract; (2) binding of capacitated spermatozoa to the egg's extracellular coat, the ZP; (3) induction of acrosome reaction (i.e., sperm activation); (4) penetration of the ZP; and (5) fusion of spermatozoon with the egg vitelline membrane. This minireview focuses on the most important aspects of the sperm acrosome, from its formation during sperm development in the testis (spermatogenesis) to its modification in the epididymis and function following sperm-egg interaction. Special emphasis has been given to spermatogenesis, a complex process involving multiple molecular events during mitotic cell division, meiosis, and the process of spermiogenesis. The last event is the final phase when a nondividing round spermatid is transformed into the complex structure of the spermatozoon containing a well-developed acrosome. Our intention is also to briefly discuss the functional significance of the contents of the sperm acrosome during fertilization. It is important to mention that only the carbohydrate-recognizing receptor molecules (glycohydrolases, glycosyltransferases, and/or lectin-like molecules) present on the surface of capacitated spermatozoa are capable of binding to their complementary glycan chains on the ZP. The species-specific binding event starts a calcium-dependent signal transduction pathway resulting in sperm activation. The hydrolytic and proteolytic enzymes released at the site of sperm-zona interaction along with the enhanced thrust of the hyperactivated beat pattern of the bound spermatozoon, are important factors in regulating the penetration of the zona-intact egg.

Structural relationship of sperm soluble hyaluronidase to the sperm membrane protein PH-20.

The sperm plasma membrane protein PH-20 has a hyaluronidase activity that enables acrosome-intact sperm to pass through the cumulus cell layer of the egg. In this study we analyzed the relationship of guinea pig PH-20 and the "classical" soluble hyaluronidase released at the time of the acrosome reaction of guinea pig sperm. PH-20 is a membrane protein, anchored in the plasma and inner acrosomal membranes by a glycosyl phosphatidyl inositol anchor. Several types of experiments indicate a structural relationship of PH-20 and the soluble hyaluronidase released during the acrosome reaction. First, an antiserum raised against purified PH-20 is positive in an immunoblot of the soluble protein fraction released during the acrosome reaction. In the released, soluble protein fraction, the anti-PH-20 antiserum recognizes a protein of approximately 64 kDa, i.e., identical in molecular mass to PH-20 (approximately 64 kDa). Second, the enzymatic activity of the released hyaluronidase is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the released protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that the released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20 undergoes endoproteolytic cleavage into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endoprotease on PH-20. We searched for a cDNA encoding sPH-20 but none was found. This result suggests that sPH-20 may arise from the enzymatic release of PH-20 from its membrane anchor, possibly at the time of acrosome reaction.

The PH-20 protein in cynomolgus macaque spermatozoa: identification of two different forms exhibiting hyaluronidase activity.

In these experiments, we have characterized the bifunctional sperm protein PH-20 in macaque sperm and studied its hyaluronidase activity. Intact sperm were evaluated before the acrosome reaction (AR), and a soluble form of PH-20 released during acrosomal exocytosis was also investigated. Western blots of SDS-PAGE of acrosome-intact sperm extracts revealed a 64-kDa form of PH-20 was recognized by a polyclonal antibody (R-10) raised in rabbits against purified, recombinant cynomolgus macaque sperm PH-20. The soluble components released during the AR which were recognized by the R-10 antibody included both the 64-kDa form and a 53-kDa form of PH-20. An ELISA-like procedure for determining PH-20 hyaluronidase activity indicated that acrosome-intact sperm exhibited two peaks of hyaluronidase activity near pH 4 and > or = pH 7. The majority of enzyme activity in acrosome-intact sperm extracts occurred at neutral pH, while the soluble hyaluronidase activity released at the AR was predominantly acid-active. Hyaluronidase activity of PH-20 at different pH optima was investigated using hyaluronic acid substrate gel electrophoresis, and results indicated that the 64-kDa polypeptide had a broad range, with the majority of activity at neutral pH (pH 7). The 53-kDa polypeptide in sperm extracts only exhibited activity at acid pH (pH 4). The hyaluronidase activities of both enzymes could be inhibited by apigenin. The soluble PH-20 hyaluronidase activity released during the AR was primarily of the acid-active 53-kDa form. Fine structural localization of PH-20 using Fab fragments of R-10 IgG demonstrated that PH-20 was associated not only with sperm membranes, but also with the dispersing acrosomal contents. These data suggest that the more neutral-active form of PH-20 (64 kDa) is present on the plasma and inner acrosomal membranes and gives rise to the soluble acid-active form at the time of the AR. The generation of the soluble form of PH-20 may result from the action of acrosomal enzymes, which could include proteases, glycosidases, and phospholipases.

The value of subcellular elemental analysis in the assessment of human spermatozoa.

The conventional sperm parameters of concentration, motility and morphology are descriptive and have proved of little value in predicting the fertilization potential of semen. New complementary tests of sperm function are required. Previous studies evaluating the subcellular elemental composition of spermatozoa using X-ray microanalysis with electron microscopy suggested that this technique may have the potential to confirm spermatozoal intracellular normality. This study compares the semen from 18 men of subfertile relationships and from 10 men of proven fertility with reference to concentration, motility, morphology and the elemental composition of spermatozoa. The elements phosphorus, sodium, potassium, chlorine and sulphur were detected and there were large intra- and intersample variations in the concentrations of these elements. There was no statistically significant difference in the elemental composition between the two groups. This study suggests that the spermatozoa of fertile and subfertile men are similar in subcellular elemental composition and in the maintenance of an ionic gradient. This test is therefore not a useful predictor of sperm function.

The sperm acrosomal matrix contains a novel member of the pentaxin family of calcium-dependent binding proteins.

The sperm acrosome is a regulated secretory granule that undergoes exocytosis during fertilization. To elucidate the structural organization of the contents within the acrosome, guinea pig sperm acrosomal apical segments were isolated and mapped by two-dimensional polyacrylamide gel electrophoresis (PAGE). Although complex, the two-dimensional PAGE map was dominated by two M(r) 50,000 polypeptides (p50 and proacrosin), a M(r) 67,000 polypeptide (p67), and a M(r) 32,000 polypeptide (sp32). Proacrosin (pI > 8.0), p67, and sp32 were extracted from apical segments by 1 M NaCl. Protein p50, a relatively acidic polypeptide, was not extracted in 1 M NaCl and/or 1% Triton X-100 at 4 degrees C, but was solubilized with 6 M urea. Protein p50 was purified from the urea extract by elution from DEAE-Sephacel with 100 mM guanidine HCl and appeared homogeneous by SDS-PAGE. Antibodies to p50 were monospecific as judged by Western blot analysis. Indirect immunofluorescence indicated that p50 was restricted to the acrosomal apical segment. Incubation of apical segments at pH 7.5 in the presence of 1 mM EDTA at 37 degrees C resulted in the release of p50 into the 200,000 x g supernatant fluid, a process that was reversed by a subsequent incubation with 1.5 mM CaCl2, but not with MgCl2. The Ca(2+)-dependent reassociation of p50 with the acrosomal apical segments was reversed by the addition of 2.0 mM EGTA, indicating that p50 binding is dependent on free Ca2+ concentrations. When acrosomal matrices were purified following Triton X-100 extraction, p50 was the major component, with p67, proacrosin, and sp32 as less prominent constituents. Molecular cloning demonstrated that p50 is a unique, testis-specific member of the pentaxin family of calcium-dependent binding proteins.