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P Primakoff

Publications and source records attributed to P Primakoff.

At least 55 records · Page 3Linked to original sources
PubMed1990

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 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.

Amino Acid Sequence↗
PubMed1988

Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20.

Immunization of male and female animals with extracts of whole sperm cells is known to cause infertility. Also, men and women who spontaneously produce antisperm antibodies are infertile but otherwise healthy. Although the critical sperm antigens are unknown, these observations have led to the proposal that sperm proteins might be useful in the development of a contraceptive vaccine. The guinea pig sperm surface protein PH-20 is essential in sperm adhesion to the extracellular coat (zona pellucida) of the egg, a necessary initial step in fertilization. Here, we report that 100% effective contraception was obtained in male and female guinea pigs immunized with PH-20. Antisera from immunized females had high titres, specifically recognized PH-20 in sperm extracts, and blocked sperm adhesion to the egg zona pellucida in vitro. The contraceptive effect was long-lasting and reversible: immunized females, mated at intervals of six to fifteen months after immunization, progressively regained fertility.

Animals↗
PubMed1988

Restricted lateral diffusion of PH-20, a PI-anchored sperm membrane protein.

The rate of lateral diffusion of integral membrane proteins is constrained in cells, but the constraining factors for most membrane proteins have not been defined. PH-20, a sperm surface protein involved in sperm-egg adhesion, was shown to be anchored in the plasma membrane by attachment to the lipid phosphatidylinositol and to have a diffusion rate that is highly restricted on testicular sperm, being more than a thousand times slower than lipid diffusion. These results support the hypothesis that lateral mobility of a membrane protein can be regulated exclusively by interactions of its ectodomain.

Animals↗
PubMed1988

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.

Acrosome↗
PubMed1987

Binding of both acrosome-intact and acrosome-reacted guinea pig sperm to the zona pellucida during in vitro fertilization.

Mammalian sperm-egg adhesion occurs when sperm bind to the zona pellucida of the egg. In this study with guinea pig gametes, we have asked if sperm can initiate binding to the zona before and after the acrosome reaction and if the sperm surface protein PH-20 is involved in the binding at these two stages. Sperm binding to the zona was examined under a variety of conditions. Sperm were suspended in 0.9% NaCl or capacitated by two different methods. Eggs were immobilized on lectin-coated coverslips, compressed between a coverslip and a glass slide, or free in tissue culture dishes. The sperm-egg interaction was recorded on videotape or assessed after fixation of the eggs with bound sperm. Under all conditions studied, both acrosome-intact and acrosome-reacted sperm initiated binding to the zona. The binding was persistent and not transitory. In particular, acrosome-intact sperm that bound the zona were observed to remain bound for up to 80 min. One acrosome-intact sperm, bound to the zona, was videotaped while it acrosome-reacted. When mixed sperm populations (on the average 24% acrosome-intact and 76% acrosome-reacted) were incubated with eggs for 30 min, an average of 10% of the bound sperm were acrosome-intact. The PH-20 monoclonal antibody has previously been shown to inhibit zona binding by guinea pig sperm of undetermined acrosomal status (P. Primakoff, H. Hyatt, and D. G. Myles (1985), J. Cell Biol. 101, 2239-2244). In this study, when the two populations of sperm were counted separately, PH-20 inhibited the binding of acrosome-reacted but not acrosome-intact sperm. Our results show that both acrosome-intact and acrosome-reacted guinea pig sperm can initiate binding to the zona; however, the binding in the two cases may not occur by the same mechanism.

Acrosome↗
PubMed1987

A cholera toxin-sensitive G-protein stimulates exocytosis in sea urchin eggs.

To identify guanine nucleotide binding proteins (G-proteins) in sea urchin eggs and to investigate their role in signal transduction at fertilization, we used cholera toxin (CTX) and pertussis toxin (PTX), which catalyze the specific ADP-ribosylation of G-proteins. Cell surface complex, consisting of plasma membranes and adhering cortical vesicles, was prepared from eggs of Lytechinus variegatus and incubated with 32P-labeled NAD in the presence of CTX or PTX. CTX catalyzed the ADP-ribosylation of a 47-kDa polypeptide, whereas PTX catalyzed the ADP-ribosylation of a 40-kDa polypeptide. Microinjection of approximately 30 micrograms/ml whole CTX or approximately 20 micrograms/ml CTX subunit A into intact eggs caused exocytosis of cortical vesicles. However, if the eggs were first injected with EGTA (0.6-1.4 mM), injection of CTX did not cause exocytosis. Eggs injected with 0.8-2.8 mM cAMP or 1.0-4.0 mM adenosine 3':5'-monophosphotioate cyclic Sp-isomer (cAMP-S), a hydrolysis-resistant analog of cAMP, did not undergo exocytosis. These results suggest that a CTX-sensitive G-protein is involved in regulating Ca2+ release and exocytosis of cortical vesicles in sea urchin eggs.

Animals↗
PubMed1987

Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.

Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.

Animals↗
PubMed1987

Actin filaments, localized to the region of the developing acrosome during early stages, are lost during later stages of guinea pig spermiogenesis.

The presence and localization of actin was investigated in guinea pig spermatogenic cells and cauda epididymal sperm (CauE). Staining with rhodamine-phalloidin demonstrated the presence of actin filaments in the region of the developing acrosome in guinea pig spermatids. The actin filaments were visualized predominantly in the region of the inner acrosomal membrane in both round and elongating spermatids. As development progressed, the intensity of the staining diminished. No rhodamine-phalloidin staining was found in testicular sperm lacking a residual body or in CauE sperm. Analysis of actin levels by immunoblotting with an anti-actin monoclonal antibody showed that the disappearance of actin filaments is accompanied by a decrease in the level of actin per cell. By using immunoblotting techniques, actin was readily detected in preparations of purified spermatogenic cells, but not in preparations of purified CauE sperm. Actin was also not detected in cauda sperm by indirect immunofluorescence (IIF) with anti-actin antibodies or examination of whole cell extracts by two-dimensional gel electrophoresis.

Acrosome↗
PubMed1987

Rearrangement of sperm surface antigens prior to fertilization.

During spermiogenesis and epididymal transit, proteins on the sperm surface become localized to specific domains. In at least one case (PH-20), the protein is initially inserted throughout the membrane and subsequently becomes restricted to a domain by some mechanism that has not yet been determined. Other proteins could become localized through localized insertion. The sperm surface is a dynamic structure that is altered even after the spermatozoon leaves the male. In the female reproductive tract the spermatozoa undergo capacitation and the acrosome reaction that enables them to fertilize the egg. Both of these processes are accompanied by alterations in protein localization: the PT-1 protein migrates during capacitation, and the PH-20 protein migrates after the acrosome reaction. In addition, an upregulation of the surface expression of PH-20 occurs during the acrosome reaction. This additional PH-20 is incorporated into the plasma membrane by the irreversible fusion of the acrosomal membrane with the plasma membrane. The acrosomal membrane contains PH-20 protein that has been stored there since the formation of the acrosome at the spermatid stage of spermiogenesis. Proteins that are freely diffusing must be maintained in a domain by a mechanism that does not involve immobilization or slowing of protein diffusion. We have suggested that barriers to membrane protein diffusion exist at the equatorial region, the posterior ring, and the annulus and that they are responsible for maintaining a localized distribution of at least some of the surface proteins. The migration of surface proteins could result from an alteration of these barriers, a change in the protein structure so that it can pass through the barrier, or active transport across the barrier. These observed changes in surface expression (localization and the level of expression) may be acting to control surface function post-testicularly.

Acrosome↗
PubMed1986

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.

Acrosome↗
PubMed1986

An antisperm monoclonal antibody inhibits sperm fusion with zona-free hamster eggs but not homologous eggs.

The zona-free hamster egg penetration assay (HEPA) was evaluated as a test for identifying fertilization-blocking antibodies. A monoclonal antibody, AH-20, that binds to the surface of guinea pig sperm was used to test antibody inhibition of sperm-egg fusion. AH-20 strongly inhibited guinea pig sperm fusion with zona-free hamster eggs but had no effect on guinea pig sperm fusion with zona-free guinea pig eggs. No inhibition by AH-20 was found in the homologous fusion assay over a wide range of sperm concentration, fertilization rate, and fertilization index. The results suggest that although guinea pig sperm can fuse with both hamster and guinea pig eggs, some aspect of the fusion mechanism is different in the two cases. The findings also indicate that HEPA, which is frequently used to assess the fertility potential of human sperm, can identify as blockers of sperm-egg fusion antibodies that have no effect on homologous sperm-egg fusion.

Acrosome↗
PubMed1985

A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida.

After the acrosome reaction, the PH-20 surface antigen of guinea pig sperm migrates from its original location on the posterior head surface to a new location on the inner acrosomal membrane (Myles, D.G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). We have isolated three monoclonal antibodies (MAbs) of the IgG1 subclass, PH-20, PH-21, and PH-22, that bind to the PH-20 antigen. The PH-20 MAb strongly inhibited (approximately 90%) sperm binding to the guinea pig egg zona pellucida at saturating antibody concentrations (greater than 20 micrograms/ml). Half-maximal inhibition of sperm binding to the zona was obtained with approximately 2 micrograms/ml PH-20 MAb. The PH-21 MAb at saturating concentration (50 micrograms/ml) partially inhibited (approximately 45%) sperm-zona binding, and the PH-22 MAb (50 micrograms/ml) did not inhibit (0%) sperm-zona binding. Essentially the same amounts of the three MAbs were bound to sperm under the conditions where inhibition (PH-20, PH-21) or no inhibition (PH-22) of sperm-zona binding was observed, which indicates that the different levels of inhibition did not arise from different levels of MAb binding. Competition binding assays with 125I-labeled MAbs showed that PH-21 binding to sperm was not affected by the binding of PH-20 or PH-22. However, that PH-20 and PH-22 blocked each other's binding to sperm suggests that their recognized determinants may be relatively close to one another. The results indicate that the migrating PH-20 antigen has a required function in sperm binding to the zona pellucida and that the PH-20 MAb affects is active site.

Acrosome↗
PubMed1984

A localized surface protein of guinea pig sperm exhibits free diffusion in its domain.

Using the technique of fluorescence redistribution after photobleaching, we are studying the cellular mechanisms involved in localizing surface molecules to particular domains. A number of antigens localized to discrete surface regions have been identified with monoclonal antibodies on guinea pig sperm cells ( Primakoff , P., and D. G. Myles , 1983, Dev. Biol., 98:417-428). One of these monoclonal antibodies, PT-1, binds exclusively to the posterior tail region of the sperm cell surface. PT-1 recognizes an integral membrane protein that in complex with n-octyl-beta-D-glucopyranoside has a sedimentation coefficient of 6.8S in sucrose density gradients. Fluorescence redistribution after photobleaching measurements reveal that within its surface domain the PT-1 antigen diffuses rapidly (D = 2.5 X 10(-9) cm2/s) and completely (greater than 90% recovery after bleaching). These results rule out for this membrane protein all models that invoke immobilization as a mechanism for maintaining localization. We propose that the mechanism for localization of the PT-1 antigen may be a barrier to diffusion at the domain boundary.

Animals↗
PubMed1984

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.

2,4-Dinitrophenol↗
PubMed1983

A map of the guinea pig sperm surface constructed with monoclonal antibodies.

The surface of mammalian sperm is known to exhibit regional heterogeneity. Using monoclonal antibodies, we have analyzed the nature of this regional variation at the molecular level. A map of the surface of guinea pig sperm has been constructed that defines a number of regions in which surface antigens are localized and analyzes the diversity of antigens found in each region. In an initial screening of 117 hybridoma antibodies to a sperm membrane preparation, a remarkable result was obtained: all the antibodies bound to a localized region of the cell. From the initial hybrid lines, we established a collection of 56 stable hybridomas producing antibodies to surface antigens. These antibodies detect antigens localized in five surface regions: anterior head (AH), posterior head (PH), whole head (WH), posterior tail (PT), and whole tail (WT). At least 12 distinct surface antigens are recognized that bind antibodies in one of the localized regions (five AH antigens, three PH, two WH, one PT, and one WT). Some of the recognized antigens have been identified as proteins, comprised of either one or several 125I-labeled polypeptides. The identified AH antigens have labeled polypeptides of molecular weights (Mr) 52,000 (52K); 70K, 62K, 46K, 25K, and 18K; 62K, 52K, and 38K; 16K; and 38K. Identified PH antigens have polypeptides of Mr 60K; 66K, 48K, and 41K; and 58K and 48K. Identified WH antigens have polypeptides of Mr 89K and 45K; and 42K. We conclude that the sperm cell can maintain contiguous membrane domains which have quite different compositions. Its surface is a mosaic consisting of multiple regions and each region can contain several localized antigens.

Animals↗
PubMed1981

Surface domains of the guinea pig sperm defined with monoclonal antibodies.

Four monoclonal antibodies have been produced that recognize surface molecules of the guinea pig sperm. Each antibody binds to a unique, localized region of the sperm surface. The four antibody-binding patterns are restricted to the anterior head, of the sperm, the posterior head, the whole head and the posterior tail. Based on absorption experiments, the surface molecules detected by the antibodies are differentiation antigens. Three of the antibodies immunoprecipitate 125I-labeled protein antigens. The molecular weights of the polypeptides observed after SDS-PAGE are 52,000 (anterior head), 42,000 (whole head), and 60,000 (posterior head). The observed monoclonal antibody-binding patterns suggest that the guinea pig sperm surface is divided into a minimum of four domains.

Animals↗
PubMed1981

In vivo role of the relA+ gene in regulation of the lac operon.

Under conditions of amino acid limitation, beta-galactosidase was produced at a 70-fold higher rate in a relA+ strain than in an isogenic relA strain of Escherichia coli K-12. Under identical conditions with the relA+ and relA strains carrying various lac promoter mutations, rates of beta-galactosidase synthesis in relA+ (relative to relA) ranged from 26-fold higher (promoter mutant Pr 13) to only 5-fold higher (promoter mutant PrL8uv5). This promoter specificity was independent of strain background and the means of eliciting amino acid limitation. Addition of cyclic AMP to the growth medium altered the relA+/relA difference for beta-galactosidase synthesis from the wild-type lac promoter. The experiments suggest that the relA+/relA difference in lac expression arises primarily at the point of transcription initiation. The results are discussed in relation to recent in vitro data showing a promoter-specific guanosine 5'-diphosphate 3'-diphosphate stimulation of lac transcription (P. Primakoff and S. W. Artz, Proc. Natl. Acad. Sci. U.S.A. 76:1726-1730).

Cyclic AMP↗