What is the core oscillator in the speract-activated pathway of the Strongylocentrotus purpuratus sperm flagellum?

Sperm chemotaxis has an important role in fertilization. Most of our knowledge regarding this phenomenon comes from studies in organisms whose fertilization occurs externally, like sea urchins. Sea urchin spermatozoa respond to sperm-activating peptides, which diffuse from the egg jelly coat and interact with their receptor in the flagellum, triggering several physiological responses: changes in membrane potential, intracellular pH, cyclic nucleotide levels, and intracellular Ca2+ concentration ([Ca2+]). In particular, flagellar [Ca2+] has been shown to oscillate. These [Ca2+] oscillations are correlated with changes in the flagellar shape and so with the regulation of the sperm swimming paths. In this study, we demonstrate, from a mathematical modeling perspective, that the reported speract-activated signaling pathway in Strongylocentrotus purpuratus (speract being a sperm-activating peptide specific to this species) has the necessary elements to replicate the reported [Ca2+] oscillations. We further investigate which elements of this signaling pathway constitute the core oscillator.

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 Ca2+ concentration ([Ca2+]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 Ca2+ 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 [Ca2+]i that correlate with changes in flagellar form and regulate sperm trajectory. The motility changes depend on [Ca2+]i influx through specific Ca2+ channels and not on the overall [Ca2+]i in the sperm flagellum. All cilia and flagella have a conserved axonemal structure and thus understanding how Ca2+ regulates cilia and flagella beating is a fundamental question.

Sperm channel diversity and functional multiplicity.

Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm-egg signaling and unveil novel contraception targets.

Phylogeny of the TMEM16 protein family: some members are overexpressed in cancer.

TMEM16 proteins are found in all eukaryotes and have eight putative transmembrane domains with NH2 and COOH termini located on the luminal side of the vesicle or plasma membrane. Nine homologues exist in humans and mice. Several of the human genes are overexpressed in cancer and could be valuable tumor markers, especially in profiling gene expression with microarrays. In Drosophila, homologues are involved in chromosome separation. In Baker's yeast, the one homologue is expressed exclusively in the bud and involved in osmotic regulation. In sea urchin embryos, the protein is associated with nuclei. In mammals, the functions of TMEM16 proteins are still unknown. We predict that the TMEM16 proteins will gain importance in the study of normal and malignant tissues. This report presents the first comprehensive phylogeny of TMEM16 proteins. The two phylograms show that these proteins fall into distinct families, which are differentiated from each other in all animal lineages. Here, all previous studies on these proteins are compiled in table form with the hope this compilation will facilitate future work on these genes and their protein products.

A new hyperpolarization-activated, cyclic nucleotide-gated channel from sea urchin sperm flagella.

A sea urchin sperm flagellar hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel is known (SpHCN1) that is modulated by cAMP. Here, we describe a second flagellar HCN channel (SpHCN2) cloned from the same sea urchin species. SpHCN2 is 638 amino acids compared to 767 for SpHCN1. SpHCN2 has all the domains of an HCN channel, including six transmembrane segments (S1-S6), the ion pore, and the cyclic nucleotide-binding domain. The two full-length proteins are 33% identical and 51% similar. The six transmembrane segments vary from 46-79% identity. S4, which is the voltage sensor, is 79% identical between the two proteins. The ion selectivity filter sequence is GYG in the ion pore of SpHCN1 and GFG in SpHCN2. By sequence, SpHCN2 is 73.5kDa, but it migrates on SDS-PAGE at 64kDa. Western immunoblots show localization to flagella, which is confirmed by immunofluorescence. A neighbor-joining tree shows that SpHCN2 is basal to all known HCN channels. SpHCN2 might be the simplest pacemaker channel yet discovered.

A third sea urchin sperm receptor for egg jelly module protein, suREJ2, concentrates in the plasma membrane over the sperm mitochondrion.

Sea urchin spermatozoa are model cells for studying signal transduction events underlying flagellar motility and the acrosome reaction. We previously described the sea urchin sperm receptor for egg jelly 1 (suREJ1) which consists of 1450 amino acids, has one transmembrane segment and binds to the fucose sulfate polymer of egg jelly to induce the sperm acrosome reaction. We also cloned suREJ3 which consists of 2681 amino acids and has 11 putative transmembrane segments. Both these proteins localize to the plasma membrane over the acrosomal vesicle. While cloning suREJ1, we found suREJ2, which consists of 1472 amino acids, has two transmembrane segments and is present in the entire sperm plasma membrane, but is concentrated over the sperm mitochondrion. Experimental evidence suggests that, unlike suREJ1 and suREJ3, suREJ2 does not project extracellularly from the plasma membrane, but is an intracellular plasma membrane protein. All three sea urchin sperm REJ proteins possess a protein module of > 900 amino acids, termed 'the REJ module', that is shared by the human autosomal dominant polycystic kidney disease protein, polycystin-1, and PKDREJ, a testis-specific protein in mammals whose function is unknown. In the present study, we describe the sequence, domain structure and localization of suREJ2 and speculate on its possible function.

Positive selection in the egg receptor for abalone sperm lysin.

The mechanism of speciation is a central problem in evolutionary biology. In free-spawning animals with no complex mating behavior, prezygotic reproductive isolation (speciation) could result from the rapid divergence of genes coding for sperm and egg proteins that bind each other during fertilization. In abalone, sperm lysin evolves rapidly by positive Darwinian selection. The egg vitelline envelope receptor for lysin had previously been shown to evolve neutrally and be subjected to concerted evolution. Several mathematical simulations predict that both male and female reproductive proteins should evolve rapidly by positive selection. Here we report that the sequence diversity of the amino-terminal end of the egg vitelline envelope receptor for lysin has been promoted by positive Darwinian selection. These data provide molecular support for theoretical models showing that the two sexes are locked in a "coevolutionary chase" that could be driven by processes such as sexual selection, sexual conflict, or microbial attack (pathogen avoidance). The result of this continuous coevolution of the gamete recognition system could be the splitting of one population into two that are reproductively isolated (speciation).

Full-length sequence of VERL, the egg vitelline envelope receptor for abalone sperm lysin.

Abalone sperm use 16 kDa lysin to create a hole in the egg vitelline envelope (VE) by a species-specific, nonenzymatic mechanism. To create the hole, lysin binds tightly to VERL (the VE receptor for lysin), a giant, unbranched glycoprotein comprising 30% of the VE. Binding of lysin to VERL causes the VERL molecules to lose cohesion and splay apart creating the hole. Lysin and VERL represent a cognate pair of gamete recognition proteins, one male the other female, which mediate fertilization. The coevolution of such cognate pairs may underlie the establishment of species-specific fertilization which could be a component of the mechanism to achieve reproductive isolation and hence new species. Here we present the full-length cDNA sequence (11,166 bp) of VERL from the red abalone (Haliotis rufescens). There are 42 amino acids from the start Met residue to the beginning of the first 'VERL repeat'. Most of VERL (9981 bp; 89.4%) consists of 22 tandem repeats of a approximately 153 amino acid sequence that is predicted to be beta-sheet. The last VERL repeat is followed by 353 non-repeat amino acid residues containing a furin cleavage site (RTRR), a ZP domain and a hydrophobic COOH-terminus with a 3' UTR of only 10 nucleotides. VERL repeats 3-22 have been subjected to concerted evolution and consequently have almost identical sequences. Curiously, comparisons of repeats from other species shows that repeats 1 and 2 of red abalone VERL have not been subjected to concerted evolution since the divergence of the red species from the other six California species.