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P Primakoff

Publications and source records attributed to P Primakoff.

At least 37 records · Page 2Linked to original sources
PubMed1996

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.

Acrosome↗
PubMed1996

Initial evaluation of fertilin as an immunocontraceptive antigen and molecular cloning of the cynomolgus monkey fertilin beta subunit.

Fertilin (PH-30) is a sperm surface protein that functions in sperm adhesion and fusion with the egg plasma membrane. Because of its essential function in fertilization, fertilin is a potential target for novel contraceptive approaches. In a pilot fertility trial, immunization of male guinea pigs with purified guinea pig fertilin resulted in complete infertility. The contraceptive effect was partial (two out of six animals were infertile) when female guinea pigs were immunized with the antigen. These results suggest that fertilin or domains of fertilin may be effective as immunocontraceptive antigens. As a step toward achieving this goal, we communicate the cDNA and deduced amino acid sequence of the monkey fertilin beta subunit.

ADAM Proteins↗
PubMed1996

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.

Acrosome↗
PubMed1996

Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding.

In previous studies, we have found that the sperm membrane protein PH-20 acts during two different stages of fertilization. On acrosome-intact sperm, PH-20 has a hyaluronidase activity that is required for sperm penetration through the cumulus cell layer that surrounds the oocyte. On acrosome-reacted sperm, PH-20 has a required function in sperm-zona binding (secondary binding). Because hyaluronic acid (HA) has been detected in the zona pellucida, secondary sperm-zona adhesion could depend on repetitive binding and hydrolysis of HA by PH-20 acting as a hyaluronidase. Alternatively, PH-20 may be bifunctional and have a second, different activity required for secondary binding. To distinguish between these two possibilities, in this study we used reagents that inhibit either PH-20's function in sperm-zona binding or its hyaluronidase activity. We found that an anti-PH-20 monoclonal antibody that inhibited sperm-zona binding (approximately 90%) had no effect on hyaluronidase activity. Conversely, apigenin, a hyaluronidase inhibitor, blocked PH-20 hyaluronidase activity 93% without inhibiting sperm-zona binding. Similarly, another anti-PH-20 monoclonal antibody that inhibited hyaluronidase activity 95% only partially inhibited sperm-zona binding (approximately 45%). We also extensively pretreated oocytes with hyaluronidase to remove all accessible HA on or in the zona pellucida and found little or no effect on secondary sperm-zona binding. Our results suggest that PH-20 is bifunctional and has two activities: a hyaluronidase activity and a second, separate activity required for secondary sperm-zona binding.

Acrosome↗
PubMed1995

Mouse egg integrin alpha 6 beta 1 functions as a sperm receptor.

Binding between sperm and egg plasma membranes is an essential step in fertilization. Whereas fertilin, a mammalian sperm surface protein, is involved in this crucial interaction, sperm receptors on the egg plasma membrane have not been identified. Because fertilin contains a predicted integrin ligand domain, we investigated the expression and function of integrin subunits in unfertilized mouse eggs. Polymerase chain reactions detected mRNAs for alpha 5, alpha 6, alpha v, beta 1, beta 3, and beta 5. Immunofluorescence revealed alpha 6 beta 1 and alpha v beta 3 on the plasma membrane. GoH3, a function-blocking anti-alpha 6 monoclonal antibody, abolished sperm binding, but a nonfunction-blocking anti-alpha 6 monoclonal antibody, a function-blocking anti-alpha v beta 3 polyclonal antibody, and an RGD peptide had no effect. Somatic cells bound sperm avidly, but only if they expressed alpha 6 beta 1. A peptide analog of the fertilin integrin ligand domain inhibited sperm binding to eggs and alpha 6 beta 1+ cells and diminished GoH3 staining of eggs. Our results indicate a novel role for the integrin alpha 6 beta 1 as a cell-cell adhesion receptor that mediates sperm-egg binding.

ADAM Proteins↗
PubMed1995

ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain.

Fertilin alpha and beta, previously known as PH-30 alpha and beta, are two subunits of a guinea pig sperm integral membrane protein implicated in sperm-egg binding and fusion. They are derived from sequence-similar precursors which contain a metalloprotease-like and a disintegrin-like domain and which are related to a family of metalloprotease and disintegrin domain-containing snake venom proteins. We report here the cloning, sequencing, and characterization of mouse fertilin alpha and beta as well as five additional sequence-similar cDNAs from guinea pig and mouse testis. We name this gene family ADAM, for proteins containing A Disintegrin And Metalloprotease domain, and in honor of its dual origins in the fields of snakes and fertility. In situ hybridization demonstrated that, in testis, RNA encoding these ADAMs is expressed only in spermatogenic cells and that this expression is developmentally regulated. PCR analysis of mouse tissue cDNA showed that these ADAMs display different patterns of tissue distribution. Some ADAMs (e.g., fertilin alpha) have the consensus active-site sequence for a zinc-dependent metalloprotease in their metalloprotease-like domain. All have a disintegrin-like domain, which could bind integrins or other receptors. Some have sequences which may be active in membrane fusion. All encode potential membrane-spanning domains. Searches of sequence databases revealed that additional mammalian members of the ADAM gene family have been cloned from a variety of tissues. Thus, the ADAMs are a large, widely expressed, and developmentally regulated family of proteins with multiple potential functions in cell-cell and cell-matrix interactions.

ADAM Proteins↗
PubMed1995

Location of the PH-20 protein on acrosome-intact and acrosome-reacted spermatozoa of cynomolgus macaques.

Fluorescence microscopy and transmission electron microscopy (TEM) were used to determine the location of the membrane protein PH-20 on spermatozoa of cynomolgus macaques. Rabbit antiserum raised against recombinant cynomolgus macaque sperm PH-20 was used as the primary antibody, and the second antibody was goat anti-rabbit IgG conjugated with either fluorescein isothiocyanate or 15 nm gold particles. Spermatozoa were evaluated before capacitation and after capacitation and induction of acrosome reactions with calcium ionophore A23187. In sperm suspensions with a high percentage of intact acrosomes, fluorescence labeling was observed uniformly over most of the sperm head. The sperm midpiece and tail were not labeled. In sperm suspensions with a high percentage of acrosome reactions, most spermatozoa labeled intensely over the anterior sperm head, but labeling of the posterior sperm head was greatly reduced. TEM of acrosome-intact spermatozoa revealed gold particles distributed uniformly on the plasma membrane overlying the acrosome, the equatorial segment, and most of the post-acrosomal region. After the acrosome reaction, gold label was present on the inner acrosomal membrane and on the plasma membrane overlying the equatorial segment. Very little label was present on the plasma membrane in the post-acrosomal region of acrosome-reacted spermatozoa. The location of PH-20 on the surface of macaque spermatozoa suggests a function for this protein in primary and/or secondary binding to the zona pellucida. The apparent decrease in amount of PH-20 on the posterior head of macaque spermatozoa following the acrosome reaction is consistent with the migration of this protein to the inner acrosomal membrane, as demonstrated previously for the homologous PH-20 protein of guinea pig spermatozoa.

Acrosome↗
PubMed1994

Identification of a binding site in the disintegrin domain of fertilin required for sperm-egg fusion.

Fertilization and certain later stages in mammalian embryonic development require fusion between membranes of individual cells. The mechanism of eukaryotic cell-cell fusion is unknown, and no surface molecules required for this process have been unequivocally identified. The role of the sperm surface protein fertilin in sperm-egg fusion was tested by using peptide analogues of a potential integrin binding site in the fertilin beta subunit. Peptide analogues that include a TDE sequence from the disintegrin region of fertilin beta are able to bind to the egg plasma membrane and strongly inhibit sperm-egg fusion. These results show that the disintegrin domain of fertilin beta binds to the egg plasma membrane and that this binding is required for membrane fusion.

ADAM Proteins↗
PubMed1994

A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg.

A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well-supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model.

Animals↗
PubMed1993

The precursor region of a protein active in sperm-egg fusion contains a metalloprotease and a disintegrin domain: structural, functional, and evolutionary implications.

PH-30, a sperm surface protein involved in sperm-egg fusion, is composed of two subunits, alpha and beta, which are synthesized as precursors and processed, during sperm development, to yield the mature forms. The mature PH-30 alpha/beta complex resembles certain viral fusion proteins in membrane topology and predicted binding and fusion functions. Furthermore, the mature subunits are similar in sequence to each other and to a family of disintegrin domain-containing snake venom proteins. We report here the sequences of the PH-30 alpha and beta precursor regions. Their domain organizations are similar to each other and to precursors of snake venom metalloproteases and disintegrins. The alpha precursor region contains, from amino to carboxyl terminus, pro, metalloprotease, and disintegrin domains. The beta precursor region contains pro and metalloprotease domains. Residues diagnostic of a catalytically active metalloprotease are present in the alpha, but not the beta, precursor region. We propose that the active sites of the PH-30 alpha and snake venom metalloproteases are structurally similar to that of astacin. PH-30, acting through its metalloprotease and/or disintegrin domains, could be involved in sperm development as well as sperm-egg binding and fusion. Phylogenetic analysis indicates that PH-30 stems from a multidomain ancestral protein.

ADAM Proteins↗
PubMed1993

Molecular cloning of the human and monkey sperm surface protein PH-20.

The guinea pig sperm surface protein PH-20 has an essential function in sperm adhesion to the zona pellucida of guinea pig eggs. Fully effective contraception has been achieved by immunizing either male or female guinea pigs with purified guinea pig PH-20. Here we report the isolation of human and cynomolgus monkey PH-20 cDNAs as a key step toward testing the function of primate PH-20 and the contraceptive efficacy of PH-20 immunization in primates. The deduced amino acid sequence of human PH-20 has 509 residues and is 59% identical with guinea pig PH-20, suggesting they may have a conserved function and immunogenicity. Southern blots show that there is a single PH-20 gene in the human genome and Northern blots of human testis poly(A)+ RNA show a 2.4-kb message. Northern blots of tissues other than testis are negative for PH-20, indicating that human PH-20 is testis-specific.

Algorithms↗
PubMed1993

A role for the WH-30 protein in sperm-sperm adhesion during rouleaux formation in the guinea pig.

Mammalian spermatozoa participate in specific cell adhesion phenomena during their development and functional lifespan; this includes interaction with Sertoli cells, the zona pellucida, and the oolemma. In some species such as the guinea pig, an additional sperm-sperm adhesion occurs during epididymal maturation which results in the formation of rouleaux in which the sperm heads are stacked one upon the other and the periacrosomal plasma membranes of adjacent sperm are linked by periodic cross-bridges. In this study, we have used a monoclonal antibody to investigate the role of the WH-30 protein on the sperm surface in the formation of the junctional zones between adjacent guinea pig sperm in rouleaux. WH-30 monoclonal antibodies caused a dose- and time-dependent dissociation of rouleaux and an increase in the percentage of single, acrosome-intact sperm; there were no effects on sperm motility (maintained at 80-90%) or ultrastructure during the 120-min incubations. The maximal effect of about 80% single sperm was obtained with a 1:4 dilution of the WH-30 hybridoma supernatant or 5-50 micrograms/ml of purified WH-30 IgG. In contrast, incubation of sperm in AH-20 IgG, myeloma cell supernatants, or purified, nonspecific mouse IgG1 had no effect on rouleaux. Treatment of sperm with a WH-30 Fab fragment resulted in almost complete dissociation of rouleaux without any observed effect on sperm motility or acrosomal status. Surface labeling of sperm followed by immunoprecipitation and SDS-PAGE revealed that the WH-30 antibody recognizes a single polypeptide of 43-45 kDa. Using immunofluorescence, the WH-30 protein was localized over the entire surface of the sperm head (whole-head pattern), and immunogold labeling showed that WH-30 is localized in the glycocalyx on both the dorsal and ventral surfaces of the periacrosomal and postacrosomal plasma membranes. These results indicate that the WH-30 protein on the sperm surface is a cell adhesion protein which is involved in the molecular interactions that maintain guinea pig sperm in rouleaux.

Animals↗
PubMed1992

A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion.

The union of sperm and egg is a special membrane fusion event that gives a signal to begin development. We have hypothesized that proteins mediating cell-cell fusion events resemble viral fusion proteins and have shown that PH-30, a sperm surface protein involved in sperm-egg fusion, shares biochemical characteristics with viral fusion proteins. We report here the complementary DNA and deduced amino-acid sequences of the mature alpha and beta subunits of PH-30. Both are type-I integral membrane glycoproteins. The alpha subunit contains a putative fusion peptide typical of viral fusion proteins and the beta subunit contains a domain related to a family of soluble integrin ligands found in snake venoms. Thus, the PH-30 alpha/beta complex resembles many viral fusion proteins in both its membrane topology and its predicted binding and fusion functions.

ADAM Proteins↗
PubMed1991

Sperm proteins that serve as receptors for the zona pellucida and their post-testicular modification.

The search for molecules on the surface of mammalian sperm that are responsible for the binding of sperm to the zona pellucida has led to the identification of a number of different surface proteins. Different experimental approaches have been used to identify these proteins and the strength of the evidence for their putative role in zona binding is therefore quite variable. In this paper we have discussed some of the approaches that are used to identify cell adhesion molecules and criteria that might be applied in future research. Further research will be required to answer questions as to whether multiple surface antigens are involved in zona binding and if the sperm receptors for the zona are conserved among species. Both of these results would be expected based on what is known about cell adhesion in other systems. We have also discussed the modifications that occur to surface proteins after the sperm leave the testis and how these modifications can potentially activate or increase the efficiency of the function of a protein in zona binding.

Animals↗
PubMed1990

Proteolytic processing of a protein involved in sperm-egg fusion correlates with acquisition of fertilization competence.

A protein located on the surface of guinea pig sperm (PH-30) has been implicated in the process of sperm-egg fusion (Primakoff, P., H. Hyatt, and J. Tredick-Kline. 1987. J. Cell Biol. 104:141-149). In this paper we have assessed basic biochemical properties of PH-30 and have analyzed the molecular forms of PH-30 present at different stages of sperm maturation. We show the following: (a) PH-30 is an integral membrane glycoprotein; (b) it is composed of two tightly associated and immunologically distinct subunits; (c) both subunits are made as larger precursors; (d) processing of the two subunits occurs at different developmental stages; (e) the final processing step occurs in the region of the epididymis where sperm become fertilization competent; (f) processing can be mimicked in vitro; (g) processing exposes at least two new epitopes on PH-30-one of the newly exposed epitopes is recognized by a fusion-inhibitory monoclonal antibody. These results are discussed in terms of the possible role of PH-30 in mediating fusion with the egg plasma membrane.

Animals↗
PubMed1990

Evidence that proteolysis of the surface is an initial step in the mechanism of formation of sperm cell surface domains.

On terminally differentiated sperm cells, surface proteins are segregated into distinct surface domains that include the anterior and posterior head domains. We have analyzed the formation of the anterior and posterior head domains of guinea pig sperm in terms of both the timing of protein localization and the mechanism(s) responsible. On testicular sperm, the surface proteins PH-20, PH-30 and AH-50 were found to be present on the whole cell (PH-20) or whole head surface (PH-30, AH-50). On sperm that have completed differentiation (cauda epididymal sperm), PH-20 and PH-30 proteins were restricted to the posterior head domain and AH-50 was restricted to the anterior head domain. Thus these proteins become restricted in their distribution late in sperm differentiation, after sperm leave the testis. We discovered that the differentiation process that localizes these proteins can be mimicked in vitro by treating testicular sperm with trypsin. After testicular sperm were treated with 20 micrograms/ml trypsin for 5 min at room temperature, PH-20, PH-30, and AH-50 were found localized to the same domains to which they are restricted during in vivo differentiation. The in vitro trypsin-induced localization of PH-20 to the posterior head mimicked the in vivo differentiation process quantitatively as well as qualitatively. The quantitative analysis showed the process of PH-20 localization involves the migration of surface PH-20 from other regions to the posterior head domain. Immunoprecipitation experiments confirmed that there is protease action in vivo on the sperm surface during the late stages of sperm differentiation. Both the PH-20 and PH-30 proteins were shown to be proteolytically cleaved late in sperm differentiation. These findings strongly implicate proteolysis of surface molecules as an initial step in the mechanism of formation of sperm head surface domains.

Animals↗