Jesús García-Soto

High pH-induced acrosome reaction and Ca2+ uptake in sea urchin sperm suspended in Na+-free seawater

The egg jelly-induced acrosome reaction of sea urchin sperm requires the presence of Ca2+ and Na+ in seawater at its normal pH 8. Sperm suspended in seawater at pH 9 undergo the acrosome reaction in the absence of jelly. We have attempted to understand the role of external Na+ in this reaction. Sperm were suspended in Na+-free seawater and the percentage of acrosome reaction and the amount of Ca2+ uptake were determined as a function of external pH. High pH (9.0) in Na+-free medium without jelly triggered a high percentage (above 65%) of sperm acrosome reactions and a two to fourfold increase in Ca2+ uptake. Both the percentage of acrosome reactions and the amount of Ca2+ uptake were similar to those induced by either jelly or pH 9 in Na+-containing seawater. On the other hand, the absence of Na+ in seawater inhibits jelly from inducing Ca2+ uptake and acrosome reactions at pH 8.0 and even at pH 8.5. These results indicate that the Na+ requirement for the acrosome reaction induced by jelly is lost when triggering is by high pH. In contrast, Ca2+ was strictly required since sperm did not react in Ca2+-free seawater at pH 9. We also found that like the jelly-induced acrosome reaction the high-pH-induced acrosome reaction and Ca2+ uptake in complete and Na+-free seawater were inhibited by D600. This finding suggests that the same transport system for Ca2+ uptake associated with the acrosome reaction operates at both triggering conditions, i.e., jelly or pH 9. Although D600 is not now considered a specific blocker, its effect has suggested the involvement of Ca2+ channels in the acrosome reaction. This proposal is supported by our results with nisoldipine, a highly specific inhibitor of calcium channels. The drug inhibited both the sperm acrosome reaction and Ca2+ uptake induced by jelly or pH 9 in complete seawater.

Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.

A Rho-signaling pathway mediates cortical granule translocation in the sea urchin oocyte.

Cortical granules are secretory vesicles of the egg that play a fundamental role in preventing polyspermy at fertilization. In the sea urchin egg, they localize directly beneath the plasma membrane forming a compact monolayer and, upon fertilization, undergo a Ca(2+)-dependent exocytosis. Cortical granules form during early oogenesis and, during maturation, translocate from the cytosol to the oocyte cortex in a microfilament-mediated process. We tested the hypothesis that these cortical granule dynamics were regulated by Rho, a GTPase of the Ras superfamily. We observed that Rho is synthesized early in oogenesis, mainly in a soluble form. At the end of maturation, however, Rho associates with cortical granules. Inhibition of Rho with the C3 transferase from C. botulinum blocks cortical granule translocation and microfilaments undergo a significant disorganization. A similar effect is observed by GGTI-286, a geranylgeranyl transferase inhibitor, suggesting that the association of Rho with the cortical granules is indispensable for its function. In contrast, the anchorage of the cortical granules in the cortex, as well as their fusion at fertilization, are Rho-independent processes. We conclude that Rho association with the cortical granules is a critical regulatory step in their translocation to the egg cortex.

MULTIPLE GTP-BINDING PROTEINS IN SEA URCHIN SPERM: EVIDENCE FOR GS AND SMALL G-PROTEINS

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide‐binding regulatory protein) and small G‐proteins. Flagellar plasma membranes incubated with [32P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX‐catalyzed [32P]ADP‐ribosylation in conjunction with immunoprecipitation with anti‐Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP‐ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP‐ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP‐ribosylation by PTX was higher in flagellar than in head membranes. Small G‐proteins were investigated by [32P]GTP‐blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α‐32P]GTP‐binding in a concentration‐dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G‐proteins, including Ras. Gs, Gi and Ras are enriched in flagellar membranes while the other small G‐proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G‐proteins in sea urchin sperm is presently under investigation.

Glyceraldehyde-3-phosphate dehydrogenase is negatively regulated by ADP-ribosylation in the fungus Phycomyces blakesleeanus

Dormant spores of Phycomyces blakesleeanus contain a 37 kDa protein that is endogenously mono-ADP-ribosylated. This protein was purified and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal sequencing and homology analysis. GAPDH enzymic activity changed dramatically upon spore germination, being maximal at stages where ADP-ribosylation was nearly undetectable. The presence of glyceraldehyde 3-phosphate in this reaction affected the [(32)P]ADP-ribosylation of the GAPDH. ADP-ribosylation of the GAPDH occurred by transfer of the ADP-ribose moiety from NAD to an arginine residue. A model for the regulation of GAPDH activity and its role in spore germination in P. blakesleeanus is proposed.

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratus sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.

Increasing intracellular pH of sea urchin sperm with NH4Cl induces Ca2+ uptake and acrosome reaction in the absence of egg jelly

Intracellular alkalinization and Ca2+ uptake accompany the egg jelly‐induced acrosome reaction of sea urchin sperm. Here, sperm were incubated in NF4Cl‐containing seawater maintained at its physiological pH (8.0). Under these conditions, 20 mM NH4C1 induced intracellular alkalinization, Ca2+ uptake and acrosome reaction in the absence of egg jelly. The NH4Cl‐triggered acrosome reaction required external Ca2+. Our results indicate that internal alkalinization activates Ca2+ influx into sperm possibly through a pH sensitive Ca2+ channel. Thus egg jelly could first stimulate the sperm plasma membrane Na+/H+ exchanger inducing intracellular alkalinization which would then activate Ca2+ uptake and acrosome reactions.

The GTP-binding protein Gαs is present in dormant spores and expressed differentially during spore germination of the fungus Phycomyces blakesleeanus

A DNA sequence homologous to a G alpha s DNA probe, and the corresponding G alpha s protein (stimulatory alpha-subunit of GTP-binding protein) were detected in Phycomyces blakesleeanus. The protein was demonstrated in membrane fractions of dormant spores of this fungus using three different experimental approaches. Photoaffinity-labelling experiments with [alpha-32P]GTP of the membrane fraction revealed two bands, of 56 and 32 kDa. The 56 kDa GTP-binding protein was detected by this method in all the stages of early development and growth investigated. Also, a spore protein of 56 kDa was ADP-ribosylated by cholera toxin, and a 56 kDa protein was detected by Western blotting with a specific antibody against mammalian G alpha s. These results indicate that G alpha s (56 kDa) is present in dormant spores of P. blakesleeanus. Using the ADP-ribosylation and Western blotting assays, G alpha s was detected during all stages of spore germination before the hyphae became highly branched, but it was not detected in the branched hyphae that formed 18 h after the initiation of spore germination. Therefore, G alpha s is expressed differentially during Phycomyces development.

The small GTP-binding protein Rho is expressed differentially during spore germination of Phycomyces blakesleeanus.

Evidence has been obtained for the presence of the small 22 kDa GTP-binding Rho protein in dormant spores of Phycomyces blakesleeanus. Immunoblotting with a polyclonal antibody against RhoA revealed a soluble and membrane-associated 22 kDa protein. When [32P]ADP-ribosylated by Clostridium botulinum C3 exotoxin the protein had a pI of 5.7, a value similar to that reported for other RhoA proteins. The 22 kDa protein was expressed at all stages of growth investigated, but radiolabelling of the [32P]ADP-ribosylated protein increased when tube-formation occurred and decreased as the hyphae branched. Localization of RhoA during spore germination studied by immunofluorescence microscopy revealed the presence of RhoA in the cell membrane of the spore. When the spore started to swell, RhoA was observed as patches in the cell membrane which become concentrated in the neck region of the site of the protuberation tube, but this protein was never observed at the point of growth of the hyphal tip. The above results suggest that RhoA associated with one or more membrane proteins could participate in the molecular mechanism involved in maintaining cell integrity during the extrusion of the germ-tube of P. blakesleeanus.

Endogenous activity of cyclic nucleotide-dependent protein kinase in plasma membranes isolated from Strongylocentrotus purpuratus sea urchin sperm

Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [gamma-32P]ATP showed in the presence of 1 microM cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 microM of cAMP. In contrast, higher concentrations (100 microM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a detergent-solubilized fraction. Membranes pretreated with 200 microM cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit.