A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation.

The sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm-egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca(2+) signaling and homeostasis modules present in the egg and zygote. The sea urchin "calcium toolkit" as predicted by the genome is described. Emphasis is on the Ca(2+) signaling modules operating during egg activation, but the Ca(2+) signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca(2+) at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca(2+) serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies.

Evidence for a secretory pathway Ca2+-ATPase in sea urchin spermatozoa.

Plasma membrane, sarco-endoplasmic reticulum and secretory pathway Ca2+-ATPases (designated PMCA, SERCA and SPCA) regulate intracellular Ca2+ in animal cells. The presence of PMCA, and the absence of SERCA, in sea urchin sperm is known. By using inhibitors of Ca2+-ATPases, we now show the presence of SPCA and Ca2+ store in sea urchin sperm, which refills by SPCA-type pumps. Immunofluorescence shows SPCA localizes to the mitochondrion. Ca2+ measurements reveal that approximately 75% of Ca2+ extrusion is by Ca2+ ATPases and 25% by Na+ dependent Ca2+ exchanger/s. Bisphenol, a Ca2+ ATPase inhibitor, completely blocks the acrosome reaction, indicating the importance of Ca2+-ATPases in fertilization.

A sodium bicarbonate transporter from sea urchin spermatozoa.

Bicarbonate (HCO3-) transporters play crucial roles in cell-signaling pathways and are essential for cell viability. Here we describe the first cloning and localization of a HCO3- transporter from sperm of the sea urchin, Strongylocentrotus purpuratus. The deduced protein is 1214 amino acids and has a calculated molecular mass of 135 kDa. The annotated protein coding region of the transporter gene consists of 24 exons. The most similar human protein is the Na+/HCO3- cotransporter-2 (NBC2), which has 53% identity and 68% similarity to the sea urchin protein. The sea urchin protein shares the major structural features of HCO3- transporters, including 13 transmembrane segments, a DIDS (4,4-diiodothiocyanatostilbene-2, 2-disulfonic acid) binding motif and N-linked glycosylation sites. It has longer N- and C-terminal cytoplasmic domains compared to human HCO3- transporters. The sea urchin protein possesses a relatively long 3rd extracellular loop with four conserved cysteine residues. This is characteristic for Na+/HCO3- cotransporters, but not for anion exchangers, suggesting that the sea urchin protein is a Na+/HCO3- cotransporter. It is therefore designated as Sp-NBC. A neighbor-joining tree shows that Sp-NBC branches closer to the electroneutral type of HCO3- transporters. Western immunoblots and immunoflourescence show that Sp-NBC is concentrated in the flagellar plasma membrane, suggesting a role in motility regulation.

Plasma membrane calcium ATPase is concentrated in the head of sea urchin spermatozoa.

Plasma membrane Ca2+ATPases (PMCAs) export Ca2+ from cells in a highly regulated manner, providing fine-tuning to the maintenance of intracellular Ca2+ concentrations. There are few studies of PMCAs in spermatozoa, which is surprising considering the importance of this enzyme in all cell types. Here we describe the primary structure and localization of the PMCA of sea urchin spermatozoa (suPMCA). The suPMCA is 1,154 amino acids and has 56% identity and 76% similarity to all 4 human PMCA isoforms. The suPMCA shares the features of a typical PMCA, including domains for calmodulin binding, ATP binding, ATPase phosphorylation, and 10 putative transmembrane segments with two large cytoplasmic loops. Southern blots show that suPMCA is a single copy gene. Treatment of live sea urchin sperm with the PMCA inhibitor, 5-(-6)-carboxyeosin, results in elevations of intracellular Ca2+ and loss of flagellar motility. Immunoblotting and immunoflorescence show that suPMCA is concentrated in the sperm head plasma membrane. In previous work, we showed that a plasma membrane K+ dependent Na+/Ca2+ exchanger (suNCKX), which also keeps Ca2+ low in these cells, is concentrated in the sperm flagellum. Thus, the sperm head and flagellum localize different gene products, both functioning to keep intracellular Ca2+ low, while the sperm swims in seawater containing 10 mM Ca2+.

Cloning of a sea urchin sarco/endoplasmic reticulum Ca2+ ATPase.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), a vesicular integral membrane protein, is the best-characterized member of the P-type ion translocating ATPase superfamily. Here we describe the cloning and structural analysis of a sea urchin SERCA (suSERCA) cloned from testis cDNA. The approximately 112 kDa suSERCA is 1022 amino acids with approximately 70% identity and 80% similarity to all known mammalian SERCA isoforms. suSERCA shares all the structural features of mammalian SERCAs, including domains: A, actuator; N, nucleotide-binding; and P, phosphorylation, and also 10 transmembrane helices. Like human SERCA2, the suSERCA has a possible 11th transmembrane segment in its extreme C-terminus. The alignment of three sequences (suSERCA, human SERCA2, and rabbit SERCA1a) shows that the Ca2+ binding residues and kinks (required to form the ion-binding pocket) are 100% conserved. The annotated suSERCA gene consists of 24 exons separated by 23 introns and is approximately 30 kb. Western blots show that suSERCA is present in sea urchin eggs and testis, but not in mature spermatozoa. Treatment of live sperm with SERCA inhibitors has no effect on intracellular calcium, suggesting the absence of SERCA in sea urchin spermatozoa.