The sequence of the Arbacia punctulata bindin cDNA and implications for the structural basis of species-specific sperm adhesion and fertilization

Bindin is the major protein component of the acrosome granule of sea urchin sperm which mediates the species-specific adhesion of sperm to the egg surface during fertilization. Bindin isolated from both Arbacia punctulata and Strongylocentrotus purpuratus sperm demonstrate a distinct adhesive preference for eggs of the same species although a significant amount of cross-species reactivity is observed. Here we describe the isolation and sequence of A. punctulata bindin cDNA clones and a comparison of the predicted protein sequence with the sequence previously reported for S. purpuratus bindin (Gao et al., 1986, Proc. Natl. Acad. Sci., USA 83, 8634-8638). Bindins from these genera show substantial sequence similarity in both the mature bindin domain and the probindin precursor region. The most striking identity is a region of 42 conserved amino acids in the central part of the mature bindins. This conserved domain may be responsible for conserved functions of bindin. Regions flanking this conserved element on both the amino and carboxyl side are more highly divergent, suggesting that they are responsible for the species-specific properties of bindin. The mature A. punctulata sequence contains a putative transmembrane segment between residues 431 and 451 that is absent from S. purpuratus bindin. This structural element may account for the previous observation that isolated A. punctulata bindin uniquely forms multilamellar structures reminiscent of lipid bilayers and binds significant amounts of phospholipid and detergent. The structure of this hydrophobic segment also displays a number of similarities to viral fusion peptides.     

Sperm–egg binding: Identification of a species-specific sperm receptor from eggs of strongylocentrotus purpuratus

We have attempted to identify a surface component of echinoderm eggs that is involved in the species-specific binding of sperm. Cell surface membranes from eggs of the sea urchins Strongylocentrotus purpuratus or Arbacia punctulata were radioiodinated, detergent-treated, and subjected to density-gradient centrifugation. In the presence of bindin, the complementary binding protein isolated from sperm, one component of the membranes sedimented to a different density. This membrane component bound-species specifically to sperm that had undergone the acrosome reaction. This binding led to an inhibition of the ability of treated sperm to fertilize eggs. Exhaustive proteolytic digestion of this receptor fraction yields a high molecular weight glycopeptide that can also bind to bindin. It therefore appears that this egg surface membrane fraction contains a functionally intact, species-specific receptor for sperm.     

Species-specific inhibition of fertilization by a peptide derived from the sperm protein bindin.

The sperm protein bindin is responsible for the species-specific adhesion of the sperm to the egg. The regions of the bindin molecule responsible for forming the contact between the sperm and the egg were investigated by measuring the ability of peptides representing various regions of the bindin sequence to inhibit fertilization. Twenty-four peptides were studied: 7 based on the Strongylocentrotus purpuratus bindin sequence, 11 based on the S. franciscanus bindin sequence, and 6 control peptides. Values for the concentration of peptide required to inhibit 50% of the productive sperm contacts (IC50) were extracted from experimental measurements of the extent of fertilization in the presence of various concentrations. of these peptides. The IC50 value averaged 220 microM for the control peptides. Active peptides representing certain specific subregions of the bindin sequence displayed IC50 values < 10% of the average value for control peptides, and the IC50 for the most potent of the peptides tested was only approximately 1% of the control peptide value (IC50 = 2.2 microM). Furthermore, we found that a peptide representing a particular region of the S. franciscanus bindin sequence that differs from the S. purpuratus bindin sequence inhibits fertilization species specifically. For the reaction of S. purpuratus sperm and eggs, the IC50 of this peptide was approximately 120 microM, whereas for the reaction of S. franciscanus sperm and eggs it was only 8.6 microM. These results demonstrate that a few specific regions of the bindin molecule are involved in the sperm-egg contact and that certain of these regions mediate the species specificity of the interaction in a sequence-specific manner.     

Carbohydrate specificity of sea urchin sperm bindin: a cell surface lectin mediating sperm-egg adhesion

We have examined the carbohydrate specificity of bindin, a sperm protein responsible for the adhesion of sea urchin sperm to eggs, by investigating the interaction of a number of polysaccharides and glycoconjugates with isolated bindin. Several of these polysaccharides inhibit the agglutination of eggs by bindin particles. An egg surface polysaccharide was found to be the most potent inhibitor of bindin- mediated egg agglutination. Fucoidin, a sulfated fucose heteropolysaccharide, was the next most potent inhibitor, followed by the egg jelly fucan, a sulfated fucose homopolysaccharide, and xylan, a beta(1 leads to 4) linked xylose polysaccharide. A wide variety of other polysaccharides and glycoconjugates were found to have no effect on egg agglutination. We also report that isolated bindin has a soluble lectinlike activity which is assayed by agglutination of erythrocytes. The bindin lectin activity is inhibited by the same polysaccharides that inhibit egg agglutination by particulate bindin. This suggests that the egg adhesion activity of bindin is directly related to its lectin activity. We have established that fucoidin binds specifically to bindin particles with a high apparent affinity (Kd = 5.5 X 10(-8) M). The other polysaccharides that inhibit egg agglutination also inhibit the binding of 125I-fucoidin to bindin particles, suggesting that they compete for the same site on bindin. The observation that polysaccharides of different composition and linkage type interact with bindin suggests that the critical structural features required for binding may reside at a higher level of organization. Together, these findings strengthen the hypothesis that sperm-egg adhesion in sea urchins is mediated by a lectin-polysaccharide type of interaction.     

Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.     

Isolation and characterization of proteolytic fragments of the sea urchin sperm receptor that retain species specificity

The sea urchin sperm receptor isolated from the eggs of Strongylocentrotus purpuratus is a high molecular weight proteoglycan-like molecule. Previous studies in our laboratory suggested that the sperm receptor has two functional components, glycosaminoglycan chains that are responsible for sperm binding and polypeptide chains that control species specificity in the binding process. We have investigated this idea further by generating fragments of the receptor by limited proteolytic digestion of the egg cell surface. The results of experiments with these receptor preparations support the hypothesis that the species specificity of inhibition of fertilization observed in a competitive bioassay is conferred by the polypeptide portion of the receptor molecule. Studies with various receptor preparations reveal that the presence of at least 30% of the polypeptide by weight is required to inhibit fertilization species specifically. Receptor preparations containing less than 10% protein lack species specificity and inhibit fertilization in both S. purpuratus and Arbacia punctulata.     

Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata

The possibility that the surface of the egg of the sea urchin Arbacia punctulata contains a species-specific receptor for sperm has been investigated. The extent of fertilization of eggs of A. punctulata, which is proportional to the number of sperm, is unaffected by the presence of either eggs or membranes prepared from eggs of Strongylocentrotus purpuratus. In marked contrast, membranes prepared from eggs of A. punctulata quantitatively inhibit fertilization of A. punctulata eggs by A. punctulata sperm. Several lines of evidence indicate that this inhibition is due to the presence of a membrane-associated glycoprotein that binds to the sperm, thus preventing them from interacting with receptor on the surface of the eggs. First, eggs treated with trypsin are incapable of being fertilized, although they can be activated with the Ca2+ ionophore A23187. Moreover, membranes prepared from eggs pretreated with trypsin do not inhibit fertilization of eggs. Second, receptor isolated in soluble form from surface membranes binds to sperm and thus prevents them from fertilizing eggs; the inhibition by soluble receptor is species-specific. Third, the soluble receptor binds to concanavalin A-Sepharose. Fourth, eggs are incapable of being fertilized if they are pretreated with concanavalin A. The specificity of inhibition, and the affect of trypsin and concanavalin A on intact eggs, suggest that the receptor is a species-specific macromolecule located on the surface of the eggs. The sensitivity of the receptor to trypsin, and its ability to bind to concanavalin A, indicate that it is a glycoprotein.     

Immunocytochemical localization of the 35-kDa sea urchin egg trypsin-like protease and its effects upon the egg surface

Trypsin-like protease in sea urchin eggs is thought to reside in cortical granules since it is secreted at fertilization and has been isolated with cortical granule fractions from unfertilized eggs. A 35-kDa serine protease has been purified from Strongylocentrotus purpuratus eggs by soybean trypsin inhibitor-affinity chromatography. For this report the protease was localized by immunocytochemistry before and after fertilization, and its potential biological activity was examined by application of the isolated enzyme to the unfertilized egg surface. The protease was localized on sections by immunofluorescence and immunoelectron microscopy, and was found to reside in the spiral lamellae of S. purpuratus cortical granules and in the electron-dense stellate core of Arbacia punctulata granules. At fertilization the enzyme is secreted into the perivitelline space and accumulates only very briefly between the hyaline layer and the nascent fertilization envelope. Shortly thereafter the enzyme is lost from the perivitelline space and immunological reactivity is no longer associated with the egg surface. The 35-kDa cortical granule protease has vitelline delaminase activity but does not appear to destroy vitelline envelope sperm receptors as judged by the fertility of protease-treated eggs.     

Comparison of the bindin proteins of Strongylocentrotus franciscanus, S. purpuratus, and Lytechinus variegatus: sequences involved in the species specificity of fertilization.

Bindin is the sea urchin sperm acrosomal protein that is responsible for the species-specific adhesion of the sperm to the egg. Two new bindin cDNA sequences that contain the entire open reading frame for the binding precursor are reported: one for Strongylocentrotus franciscanus and one for Lytechinus variegatus. Both contain inverted repetitive sequences in their 3' untranslated regions, and the S. franciscanus cDNA contains an inverted repetitive sequence match between the 5' untranslated region and the coding region. The middle third of the mature bindin sequence is highly conserved in all three species, and the flanking sequences share short repeated sequences that vary in number between the species. Cross-fertilization data are reported for the species S. purpuratus, S. franciscanus, L. variegatus, and L. pictus. A barrier to cross-fertilization exists between the sympatric Strongylocentrotus species, but there is no barrier between the allopatric Lytechinus species.     

Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer

Resact, a peptide of known sequence isolated from the jelly layer of Arbacia punctulata eggs, is a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for millimolar external calcium. A. punctulata spermatozoa do not respond to speract, a peptide isolated from the jelly layer of Strongylocentrotus purpuratus eggs. This is the first report of animal sperm chemotaxis in response to a defined egg-derived molecule.     

Purification and characterization of an extracellular fragment of the sea urchin egg receptor for sperm

Fertilization in the sea urchin involves species-specific interaction between the ligand bindin on the surface of acrosome-reacted sperm and a receptor of high molecular weight on the surface of the egg. Efforts to understand this interaction and the resultant signal transduction events leading to egg activation have been limited because of the large size and extreme insolubility of the intact receptor on the egg surface. Earlier work suggested that an alternative strategy would be to isolate proteolytic fragments of the extracellular domain of this receptor. Consequently, we have treated S. purpuratus eggs with a specific protease, lysylendoproteinase C. This enzyme treatment abolished the ability of eggs to bind sperm and resulted in the release of proteolytic fragments that bound to sperm and showed inhibitory activity in a fertilization bioassay. One of these fragments, presumed to be a fragment of the extracellular domain of the receptor, was purified to homogeneity by gel filtration and anion exchange chromatography and shown to be a 70-kD glycosylated protein. Several lines of evidence support the contention that this fragment is derived from the receptor. First, the fragment inhibited fertilization species specifically. Second, species specific binding of the 70-kD glycoprotein to acrosome-reacted sperm was directly demonstrated by using 125I-labeled receptor fragment. Third, the fragment exhibited the same species specificity in binding to isolated bindin particles. Species specificity was abolished by Pronase digestion of the fragment. This observation supports the hypothesis that although binding is mediated by the carbohydrate moieties, species specificity is dependent on the polypeptide backbone. The availability of a structurally defined fragment of the receptor will facilitate further studies of the molecular basis of gamete interaction.     

Polysaccharide structural features that are critical for the binding of sulfated fucans to bindin, the adhesive protein from sea urchin sperm

We have investigated the structural features of sulfated fucose-containing polysaccharides which are responsible for their selective binding to Strongylocentrotus purpuratus bindin. The data presented demonstrate that the sulfate esters and a molecular weight in excess of approximately 15,000 are required for high affinity binding of the fucans to bindin. Desulfation destroys the binding activity of the fucans, which can be fully restored by chemical resulfation. Fucan fragments of an average molecular weight of 15,000 were nearly as active as the starting material (Mr 10(6)). The observed IC50 value for fragments of Mr congruent to 10,000 and Mr congruent to 5,000 were 1 and 2 orders of magnitude higher, respectively. The binding of fucoidan to bindin is stable in high salt (50% at 1.2 M NaCl) whereas the binding of fucoidan to DEAE-cellulose or polylysine is inhibited by the concentrations of salt normally found in sea water (50% at 0.2 and 0.5 M NaCl, respectively). This result suggests that the binding mechanism is not a simple ionic interaction and that hydrogen bonding and cooperativity may also be important determinants of the binding mechanism. We also found that polyvinyl sulfate binds to bindin with high affinity and inhibits the bindin-mediated agglutination of sea urchin eggs. The results of these investigations suggest that the spatial orientation of the sulfate esters plays a critical role in determining the selectivity of sulfated polysaccharide binding and that the polysaccharide backbone does not play a direct role in the binding mechanism.     

Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar

An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.     

Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization

Egg activation at fertilization requires the release of Ca(2+) from the egg's endoplasmic reticulum, and recent evidence has indicated that a Src family kinase (SFK) may function in initiating this signaling pathway in echinoderm eggs. Here, we identify and characterize a SFK from the sea urchin Strongylocentrotus purpuratus, SpSFK1. SpSFK1 RNA is present in eggs, and an antibody made against a SpSFK1 peptide recognizes an approximately 58-kDa egg membrane-associated protein in eggs of S. purpuratus as well as another sea urchin Lytechinus variegatus. Injection of both species of sea urchin eggs with dominant-interfering Src homology 2 domains of SpSFK1 delays and reduces the release of Ca(2+) at fertilization. Injection of an antibody against SpSFK1 into S. purpuratus eggs also causes a small increase in the delay between sperm-egg fusion and Ca(2+) release. In contrast, when injected into eggs of L. variegatus, this same antibody has a dramatic stimulatory effect: it causes PLCgamma-dependent Ca(2+) release like that occurring at fertilization. Correspondingly, in lysates of L. variegatus eggs, but not S. purpuratus eggs, the antibody stimulates SFK activity. Injection of L. variegatus eggs with another antibody that recognizes the L. variegatus egg SFK also causes PLCgamma-dependent Ca(2+) release like that at fertilization. These results indicate that activation of a Src family kinase present in sea urchin eggs is necessary to cause Ca(2+) release at fertilization and is capable of stimulating Ca(2+) release in the unfertilized egg via PLCgamma, as at fertilization.     

Egg jelly induces the phosphorylation of histone H3 in spermatozoa of the sea urchin Arbacia punctulata

When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.     

Specific recognition of sulfate esters by bindin, a sperm adhesion protein from sea urchins

Bindin specifically binds to egg surface sulfated fucan polysaccharides and mediates the attachment of sperm to the egg during fertilization. Sulfate esters are critical for this interaction. We have examined the effect of different anionic groups on the relative binding affinities of a series of homologous anionic polymers for bindin to determine the extent to which other charged moieties can substitute for sulfate. We found that bindin displays a remarkable specificity for sulfate- or sulfonic acid-containing polymers. The relative affinities of poly(vinyl sulfate) and poly(styrenesulfonic acid) are four orders of magnitude higher than polymers containing phosphate esters or carboxyl groups. The bindin-mediated aggregation of sea urchin eggs was inhibited by the sulfated polymers but not the other anionic polymers. This high degree of selectivity for sulfated polymers is not observed for the binding of the polyanions to most other proteins and basic polypeptides. These results suggest that the binding is not due to the formation of simple salt bridges, and that all three non-ester oxygen atoms of the sulfate groups are involved in multiple bonding interactions with a complementary 'docking site' on the bindin polypeptide. The orientation of the polysaccharide sulfate oxygen atoms relative to the protein binding site may be an important determinant of the specificity of polysaccharide binding.     

Molecular Reproduction & Development Volume 78, Issue 8 cover image

Transmission electron micrograph of a sea urchin (Strongylocentrotus purpuratus) spermatozoon (purple) bound to the tip of an egg microvillus (yellow) by the bindin protein (diffuse purple), which was released during exocytosis of the sperm acrosomal vesicle. Egg microvilli are approximately 0.25 µm in diameter. More on observing these events on the cellular level in a classroom laboratory setting is presented by VD Vacquier (this issue).     

Low rates of bindin codon evolution in lecithotrophic Heliocidaris sea urchins

Life-history variables including egg size affect the evolutionary response to sexual selection in broadcast-spawning sea urchins and other marine animals. Such responses include high or low rates of codon evolution at gamete recognition loci that encode sperm- and egg-surface peptides. Strong positive selection on such loci affects intraspecific mating success and interspecific reproductive divergence (and may play a role in speciation). Here, we analyze adaptive codon evolution in the sperm acrosomal protein bindin from a brooding sea urchin (Heliocidaris bajulus, with large eggs and nonfeeding or lecithotrophic larval development) and compare our results to previously published data for two closely related congeners. Purifying selection and low relative rates of bindin nonsynonymous substitution in H. bajulus were significantly different from selectively neutral bindin evolution in H. erythrogramma despite similar large egg size in those two species, but were similar to the background rate of nonsynonymous bindin substitution for other closely related sea urchins (including H. tuberculata, all with small egg size and feeding planktonic larval development). Bindin evolution is not driven by egg size variation among Heliocidaris species, but may be more consistent with an alternative mechanism based on the effects of high or low spatial density of conspecific mates.     

Structure of a major yolk glycoprotein and its processing pathway by limited proteolysis are conserved in echinoids

To study the fate of the yolk glycoproteins found in eggs and embryos of the sea urchin, Strongylocentrotus purpuratus, a polyclonal antibody to a 90-kDa polymannose glycoprotein found in the embryo was prepared. Immunoblot analysis of total proteins over the course of development showed that this antibody recognized a family of glycoproteins. Concomitant with the disappearance of the major 160-kDa yolk glycoprotein of the egg during embryogenesis, glycoproteins with a lower molecular mass appeared. These glycoproteins (115, 108, 90, 83, and 68 kDa) were purified from S. purpuratus and analyzed by limited proteolysis and peptide mapping. This analysis revealed that these glycoproteins were cleavage products derived from the major yolk glycoprotein. The antibody to the 90-kDa glycoprotein in S. purpuratus embryos was used to identify a homologous set of yolk glycoproteins with similar molecular masses in the embryos of three other species in the class Echinoidea: Arbacia punctulata, Lytechinus pictus, and Dendraster excentricus. However, eggs from other echinoderm classes and from Xenopus laevis, Drosophila melanogaster, and the chicken did not contain any cross-reactive molecules. Cross-reactivity within the class Echinoidea was not due to a common carbohydrate epitope, because the antibody recognized the glycoproteins even after the N-linked carbohydrate side chains were enzymatically removed. The major yolk glycoprotein (160-170 kDa) from each of the three sea urchin species was purified and analyzed. Comparison of the physical and chemical properties of these glycoproteins revealed striking similarities in pI and in amino acid and monosaccharide composition. The results of peptide mapping also supported the conclusion that the 160- to 170-kDa glycoproteins from the four echinoids are structurally homologous glycoproteins containing N-linked polymannose chains. Immunolocalization by electron microscopy in S. purpuratus showed that the yolk glycoproteins remained within the yolk platelet throughout development, and that externalization of the 160-kDa glycoprotein or its cleavage products was not detectable.     

A peptide associated with eggs causes a mobility shift in a major plasma membrane protein of spermatozoa

A peptide (resact) associated with the eggs of the sea urchin, Arbacia punctulata, which stimulates sperm respiration rates by 5-10-fold, was purified and its amino acid sequence was determined. The sequence was found to be Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2. The peptide was subsequently synthesized by solid phase methods, amidated at the carboxyl-terminal Leu, and shown to be identical to the isolated, native material. The peptide half-maximally stimulated A. punctulata spermatozoan respiration at 0.5 nM and half-maximally elevated cyclic GMP concentrations at 25 nM at an extracellular pH of 6.6. The increase in oxygen consumption was coupled with a stimulation of motility. However, at elevated extracellular pH (pH 8.0), resact failed to appreciably stimulate respiration while the elevations of cyclic GMP continued to occur. Resact did not cross-react with sperm cells obtained from Lytechinus pictus or Strongylocentrotus purpuratus; a peptide (speract) obtained from S. purpuratus eggs (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) which activates S. purpuratus sperm respiration did not stimulate A. punctulata spermatozoa. Resact caused a shift in the apparent molecular weight (160,000-150,000) of a major sperm plasma membrane protein; as with cyclic GMP elevations, this response was evident at extracellular pH values of both 6.6 and 8.0. The protein exists in the cell as a phosphoprotein and 32P is released coincident with the molecular weight change. Approximately 115 nM resact caused one-half-maximal conversion of the 160,000-dalton protein after 1 min of incubation. Resact caused the apparent molecular weight conversion of the protein within 5 s and appeared to do so in an irreversible manner. The molecular weight change of the protein was also observed after the addition of monensin A (25 microM) and NH4Cl (40 mM), two agents known to elevate intracellular pH and to increase sperm respiration rates. The membrane protein appears to be the enzyme guanylate cyclase, but since concentrations of resact causing one-half-maximal conversion of the Mr = 160,000 form of the enzyme are about 250 times higher than those causing one-half-maximal stimulation of respiration, the relationship of the apparent molecular weight conversion to a subsequent physiological event remains unclear.