A low molecular weight factor from sea urchin eggs elevates sperm cyclic nucleotide concentrations and respiration rates.

A factor associated with sea urchin eggs that increases sperm cyclic nucleotide concentrations and respiration rates was identified as having a low molecular weight. The factor was more potent at elevating cyclic GMP concentrations than cyclic AMP concentrations, and represents the first demonstration of a factor associated with eggs that is capable of causing elevations of sperm cyclic GMP. Concentration-response curves of the crude mixture of egg factors to increase sperm cyclic AMP and cyclic GMP concentrations and respiratory rates were very similar, and comparable losses of these three activities were observed after extensive dialysis and heat treatment of the crude egg factors. The factor was partly purified by ethanol precipitation of a large molecular weight egg jelly component, and by charcoal adsorption and LH-20 chromatography of the resultant ethanol-soluble material. The factor was not extracted into a variety of organic solvents and had an apparent molecular weight of between 1000 and 2000, as estimated by gel filtration.     

Effects of factors released from eggs and other agents on cyclic nucleotide concentrations of sea urchin spermatozoa.

Factors released from eggs (FRE) of the sea urchin, Strongylocentrotus purpuratus, caused up to 20-fold increases in sperm cyclic AMP levels after a 1-min incubation. Putative cyclic nucleotide phosphodiesterase inhibitors such as theophylline acted in a synergistic manner with FRE to cause even greater increases in sperm cyclic AMP levels. This effect appeared to be specific for egg factors since various hormones (triiodothyronine, norepinephrine, histamine), nucleosides (adenosine, guanosine), nucleophiles (axide), anaesthetics (procaine), ionophores (X537A, A23187), metals (Mn2+) and neurotransmitters (acetylcholine) did not increase sperm cyclic AMP levels. Various mammalian tissue extracts (serum, uterus, adrenal, ovary, lung) also had no effect. We suggest that the activity which elevates the cyclic AMP of sea urchin spermatozoa is specifically associated with sea urchin eggs.     

A novel group of sperm activating peptides from the sea urchin Glyptocidaris crenularis

1. Six new sperm activating peptides were purified from the egg jelly of the sea urchin Glyptocidaris crenularis and their amino acid sequences were determined as follows: Ser-Ala-Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Ser-Phe-Lys-Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val, Lys-Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val and Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val. 2. The peptides were specific for G. crenularis spermatozoa and caused significant increases of sperm respiration rates and sperm cyclic nucleotide concentrations at concentrations as low as 10(-10) M. 3. The addition of the peptides to intact spermatozoa resulted in the change of the apparent mol. wt of a sperm protein from 195,000 to 200,000.     

INDUCTION OF CHROMOSOME MOTION IN THE GLYCEROL-ISOLATED MITOTIC APPARATUS: NUCLEOTIDE SPECIFICITY AND EFFECTS OF ANTIDYNEIN AND MYOSIN SERA ON THE MOTION*

Chromosome motion in glycerol-isolated mitotic apparatus (MA) of sea urchin and starfish eggs was investigated with respect to nucleotide specificity and the effects of antisera against tryptic fragment (Fragment A) of flagellar dynein and starfish egg myosin. The motion was highly specific for ATP. GTP, ITP, CTP, UTP, and ADP caused no displacement of the chromosomes towards the poles. The anti-Fragment A serum completely inhibited chromosome motion in the MA of the sea urchin egg, while antiserum against starfish egg myosin as well as its γ-globulin fraction did not inhibit the motion in the isolated MA of the starfish egg, suggesting that chromosome motion depends upon dynein-microtubule but not upon myosin-actin interaction. In addition, colchicine completely suppressed the chromosome motion in vitro.     

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis I. Factors Participating in the Acrosome Reaction

In contrast with the case in sea urchin sperm, in starfish the acrosome reaction is not spontaneously induced by simply increasing the extracellular Ca²⁺ concentration or pH. At higher pHs, starfish sperm undergo morphological changes accompanied by exocytosis of the acrosomal vacuole, but they do not form acrosomal filaments. Nomarski-microscopic observation confirmed that spermatozoa undergo the acrosome reaction within the jelly coat. Acrosome reaction-inducing substance, a glycoprotein from the egg jelly, required a diffusible cofactor(s) present in the egg jelly for full activity. Several lines of evidence showed that this diffusible factor(s) is not merely Ca²⁺.     

Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possibly involved in determining the length of linker DNA.

Comparison has been made between sea urchin and starfish sperm chromatin. The only protein by which chromatins from these sources differ significantly is histone H2B. Sea urchin sperm H2B is known to contain an elongated N-terminal region enriched in Arg. Analysis of the micrococcal nuclease digests of sea urchin and starfish nuclei in one- and two-dimensional electrophoresis has shown that sperm chromatin of both animals consists of repeated units similar in general features to those of rat thymus or liver. However, DNA repeat length in chromatin of sea urchin sperm (237 bp) is higher than that of starfish sperm (224 bp), while the core DNA length does not differ and is the same as in the chromatin of rat liver or thymus. A suggestion has been made that the N-terminal region of histone H2B is associated with the linker DNA and is responsible for the increased length of sea urchin linker DNA.     

Induction of cross-fertilization between sea urchin eggs and starfish sperm by polyethylene glycol treatment

Cross-fertilization between sea urchin eggs (Strongylocentrotus nudus) and starfish sperm (Asterina pectinifera) was induced by treatment with polyethylene glycol (PEG). Without treatment with PEG, the denuded egg surface (jelly coat- and vitelline coat-free) engulfed the head of acrosome-reacted sperm; however, sperm penetration did not occur [Kyozuka and Osanai, 1988]. When these eggs were exposed briefly to PEG (molecular weight 3,000) in seawater, the sperm entered the egg by membrane fusion. Cortical granules were discharged, and embryogenesis began following sperm penetration. PEG did not induce parthenogenesis in Strongylocentrotus eggs. Egg activation is thus closely linked with gamete membrane fusion.     

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.     

Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility

Echinoderm sperm use cyclic nucleotides (CNs) as essential second messengers to locate and swim towards the egg. Sea urchin sperm constitute a rich source of membrane-bound guanylyl cyclase (mGC), which was first cloned from sea urchin testis by the group of David Garbers. His group also identified speract, the first sperm-activating peptide (SAP) to be isolated from the egg investment (or egg jelly). This decapeptide stimulates sperm mGC causing a fast transient increase in cGMP that triggers an orchestrated set of physiological responses including: changes in: membrane potential, intracellular pH (pHi), intracellular Ca(2+) concentration ([Ca(2+)]i) and cAMP levels. Evidence from several groups indicated that cGMP activation of a K(+) selective channel was the first ion permeability change in the signaling cascade induced by SAPs, and recently the candidate gene was finally identified. Each of the 4 repeated, 6 trans-membrane segments of this channel contains a cyclic nucleotide binding domain. Together they comprise a single polypeptide chain like voltage-gated Na(+) or Ca(2+) channels. This new type of channel, named tetraKCNG, appears to belong to the exclusive club of novel protein families expressed only in sperm and its progenitors. SAPs also induce fluctuations in flagellar [Ca(2+)]i that correlate with changes in flagellar form and regulate sperm trajectory. The motility changes depend on [Ca(2+)]i influx through specific Ca(2+) channels and not on the overall [Ca(2+)]i in the sperm flagellum. All cilia and flagella have a conserved axonemal structure and thus understanding how Ca(2+) regulates cilia and flagella beating is a fundamental question.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

Hemocytes/coelomocytes DNA content in five marine invertebrates: cell cycles and genome sizes

The hemocytes/coelomocytes DNA content in five selected marine invertebrates (sea mouse Aphrodita aculeata, spiny crab Maja crispata, sea star Echinaster sepositus, sea urchin Paracentrotus lividus, and tunicate Phallusia mammillata) was investigated by flow cytometry. The cell cycle analyses identified sea mouse coelomocytes as proliferating cells and revealed that spiny crab hemocytes and sea urchin coelomocytes complete their division in the hemolymph and coelom, respectively. The genome sizes of sea mouse and spiny crab are reported for the first time. The diploid DNA content (2C) in sea mouse A. aculeate was 1.24 pg, spiny crab M. crispata 7.76 pg, red starfish E. sepositus 1.52 pg and sea urchin P. lividus 1.08 pg. The mean diploid DNA content in tunicate P. mammillata was 0.11 pg with a high interindividual variability (45%). The presented results provide a useful database for future studies in the field of invertebrate physiology, ecotoxicology, biodiversity, species conservation and phylogeny.     

A soluble adenylyl cyclase from sea urchin spermatozoa

A previously identified, calmodulin-binding, sea urchin sperm flagellar adenylyl cyclase (AC) was cloned and sequenced and found to be a homologue of mammalian sperm soluble adenylyl cyclase (sAC). Compared to the mammalian sAC, the sea urchin sAC (susAC) has several long amino acid insertions, some of which contain protein kinase A phosphorylation sites. The enzymatic activity of susAC shows a steep pH dependency curve, the specific activity doubling when the pH is increased from 7.0 to 7.5. This suggests that like sperm dynein ATPase, the susAC is probably activated by increases in intracellular pH occurring upon spawning into seawater and also when sperm respond to contact with the egg jelly layer. The susAC is strongly activated by manganese, but has low activity in magnesium. Gene database searches identified sAC homologues in species known to have cyclic AMP-dependent sperm motility. This implies (as shown in mouse) that susAC has a role in sperm motility, most probably through axonemal protein phosphorylation or ion channel regulation.     

Subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm.

The subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 80% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucleotide phosphodiesterase in sperm flagella were also briefly described.     

High levels of a calcium-dependent modulator protein in spermatozoa and its similarity to brain modulator protein

Sperm from hamster, human, rooster, rabbit and sea urchin were found to contain relatively high levels of calcium-dependent modulator protein. Using rabbit sperm the modulator protein was found to be exclusively located in the sperm head fraction (nuclei + acrosomes) with no activity present in the midpiece or tail regions. The modulator protein represents approximately 12% of the total soluble protein found in the sperm head fraction and is similar to porcine and brain modulator proteins in its ability to activate brain cyclic nucleotide phosphodiesterase, its heat stability and electrophoretic migration. We have also observed modulator protein to be present in high levels in sea urchin eggs.     

Actin,more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig (1876), enormous progress and insights have been made on this topic. However, the precise molecular mechanisms underlying fertilization are largely unknown. The two most dramatic changes taking place in the zygote immediately after fertilization are: (i) a sharp increase of intracellular Ca²⁺ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave, and (ii) the concomitant dynamic rearrangement of the actin cytoskeleton. Traditionally, this has been studied most extensively in the sea urchin eggs, but another echinoderm, starfish, whose eggs are much bigger and transparent, has facilitated experimental approaches using microinjection and fluorescent imaging methodologies. Thus in starfish, it has been shown that the sperm-induced Ca²⁺ increase in the fertilized egg can be recapitulated by several Ca²⁺-evoking second messengers, namely inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP), which may play distinct roles in the generation and propagation of the Ca²⁺ waves. Interestingly, it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca²⁺ signaling. As it is well known that Ca²⁺ regulates the structure of the actin cytoskeleton, our finding that Ca²⁺ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca²⁺ signaling may form a ‘positive feedback loop’ that accelerates the downstream events of fertilization. Perturbation of the cortical actin networks also inhibits cortical granules exocytosis. Polymerizing actin bundles also compose the ‘acrosome process,’ a tubular structure protruding from the head of fertilizing sperm. Hence, actin, which is one of the most strictly conserved proteins in eukaryotes, modulates almost all major aspects of fertilization.     

Sperm-activating peptide type-V (SAP-V), a fifth member of the sperm-activating peptide family, purified from the egg-conditioned media of the heart urchin Brissus agassizii.

1. A novel type of sperm-activating peptide named sperm-activating peptide type-V (SAP-V) was isolated from the egg-conditioned media (egg jelly) of the heart urchin Brissus agassizii and the primary structure of the peptide was determined by fast atom bombardment mass spectrometry as follows: Gly-Cys-Glu-Gly-Leu-Phe-His-Gly-Met-Gly-Asn-Cys. 2. SAP-V and [Met(O)9]SAP-V stimulated the respiration of B. agassizii spermatozoa with half-maximal concentrations of 0.5 and 0.3 nM, respectively. However, half-maximal stimulation of the sperm respiration required 40 nM of S-carboxymethylated SAP-V. 3. SAP-V induced significant increases in the cyclic AMP and cyclic GMP levels in B. agassizii spermatozoa in a concentration-dependent manner. 4. The addition of SAP-V to B. agassizii spermatozoa resulted in a mobility shift of a major sperm protein (mol. wt from 133,000 to 129,000) on sodium dodecyl sulfate-polyacrylamide gels.     

Evidence for the presence of sexual pheromones in free-spawning starfish

Filtered natural seawater passed through a tank containing ripe female starfish of the forcipulate species Asterias forbesi (Desor 1848) and Orthasterias koehleri (de Loriol 1897) contains low concentrations of a substance which attracts the sperm of these species. Release of this substance is sex specific in at least one species, O. koehleri. Immature females apparently do not release the sperm attractant. Daily testing for sperm attractant in seawater in the Friday Harbor Laboratories' aquarium system, Washington, U.S.A., in the spring and summer of 1986 indicates that incursions of sperm attractant-bearing water occur frequently during the starfish reproductive season. Testing of seawater taken directly from the San Juan Channel in the summer of 1987 reveals that sperm attractant is also present at irregular intervals in natural seawater. There is a definite correlation between the sperm attractant incursions in the laboratory seawater and initiation of spawning of starfish in the laboratory. The periodic presence of a long-lived bio-active sex-specific substance in natural seawater during the reproductive season of starfish suggests that benthic invertebrates may communicate sexual identity and readiness to spawn by means of waterborne pheromones.     

Subcellualr localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm

The subcellular localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 980% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucelotide phosphodiesterase in sperm flagella were also briefly described.     

Positive selection in the carbohydrate recognition domains of sea urchin sperm receptor for egg jelly (suREJ) proteins

A wealth of evidence shows that protein-carbohydrate recognition mediates the steps of gamete interaction during fertilization. Carbohydrate-recognition domains (CRDs) comprise a large family of ancient protein modules of approximately 120 amino acids, having the same protein fold, that bind terminal sugar residues on glycoproteins and polysaccharides. Sea urchin sperm express three suREJ (sea urchin receptor for egg jelly) proteins on their plasma membranes. suREJ1 has two CRDs, whereas suREJ2 and suREJ3 both have one CRD. suREJ1 binds the fucose sulfate polymer (FSP) of egg jelly to induce the sperm acrosome reaction. The structure of FSP is species specific. Therefore, the suREJ1 CRDs could encode molecular recognition between sperm and egg underlying the species-specific induction of the acrosome reaction. The functions of suREJ2 and suREJ3 have not been explored, but suREJ3 is exclusively localized on the plasma membrane over the sperm acrosomal vesicle and is physically associated with sea urchin polycystin-2, a known cation channel. An evolutionary analysis of these four CRDs was performed for six sea urchin species. Phylogenetic analysis shows that these CRDs were already differentiated in the common ancestor of these six sea urchins. The CRD phylogeny agrees with previous work on these species based on one nuclear gene and several mitochondrial genes. Maximum likelihood shows that positive selection acts on these four CRDs. Threading the suREJ CRDs onto the prototypic CRD crystal structure shows that many of the sites under positive selection are on extended loops, which are involved in saccharide binding. This is the first demonstration of positive selection in CRDs and is another example of positive selection acting on the evolution of gamete-recognition proteins.