| Budget Amount *help |
¥31,800,000 (Direct Cost: ¥31,800,000)
Fiscal Year 1993: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1992: ¥7,700,000 (Direct Cost: ¥7,700,000)
Fiscal Year 1991: ¥9,700,000 (Direct Cost: ¥9,700,000)
Fiscal Year 1990: ¥11,100,000 (Direct Cost: ¥11,100,000)
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| Research Abstract |
In sea urchin fertilization, before contacting an egg surface a spermatozoon must pss through the jelly coat which surrounds the egg. The jelly coat contains two high mlecular weight glycoconjugates and sperm-activating peptides (SAPs). A fucose sulfate glycoconjugate (FSG), one of the glycoconjugates in the jelly coat has been reported to be a major substance responsible for nduction of the acrosome reaction. SAP-I (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), which was originally identified as factors which induces a number of biochemical events in sea urchin spermatozoa such as 1) the transient elevation of intracellular levels of cAMP, cGMP and [Ca^<2+>], and transient activation of the membrane form fo guanylate cyclase. It also induces a proton efflux across the sperm plasma membrane, resulting in an increase in intracellular pH.In addition to the adove, the peptide has been shown to promote an acrosome reaction in Hemicentrotus pulcherrimus spermatozoa as a specific cofactor of FSG
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The immunocytochemical localization of FSG was invesitigated in ovaries of the sea urchin H.pulchherrimus by use of a polyclonal antibody, showing that FSG is produced by the accessory cells and is deposited initially on the surface of vitellogenic oocytes for te formation of jelly. A cDNA clone encoding SAP-I was isolated from a H.pulcherrimus ovary cDNA library and its nucleotide sequence was determined. The cDNA was 1282 bp long and an open reading frame predicted a protein of 334 amino acids containing five SAP-I and seven SAP-I-like decapeptides, each separated by a single lysine residue. An expression study of the SAP-I precursor gene by in situ hybridization with a non-radioact5ive RNA probe synthesized using the 1.3kb cDNA as template demonstrated that abundant SAP-I precursor transcripts were were expressed in the accessory cells, but not in the grwing oocytes.
We characterized putative receptors specific for SAP-I in spermatozoa of the sea urchin H.PULCHERRIMUS, USING BOTH BINDING AND CROSSLINKING TECHNIGUES.Anslysis of the data obtained from the equilibrium binding of a radioiodonated SAP-I analogue to the spermatozoa showed the presence of two classes of receptors specific (high-affinity and low-affnity) for SAP- in the spermatozoa. SAP-I has two classes of EC50 values with regard to activity. One class of EC_<50> values is at subnanomolar levels, which includes respirationstimulating activity and intracellular pH-increasing activity. The other class of vaues ranges from 7-32 nM and is found in intracellular Ca^<2+>-incresing activity and cellular cGMP-elevating activity. Therefore, we presume that the two different ranges of EC_<50> values fund in the biological activity of SAP-I may reflect the preseence of two classes of SAP-I receptors ; the high-affinity receptor may be for intracellular pH-increasing activity as well as wel as respirationstimulating activity and the low affinity receptor may be for cGMP- and intracellular Ca^<2+>-elevating activity. The incubation of intact spermatozoa as well as sperm tails or sperm-membranes prepared from H.pulcherrimus spermatozoa with the radioiodinated SAP-I analogue and a chemical crossinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)-dinding protein. The purified 71kDa protein had the Nterminal amino acid sequence EQNYREAVEGNIRLIHGRTENEGS.A cDNA clone encoding the 71kDa protein was isolated from a H.pulcherrimus testis cDNA library and its nulceotide sequence was determined. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30-residue amino terminal signal peptide, followed by the same sequence as the N-terminal sequence of the 71kDa protein. The protein consists of a large N-ter
SAP-I caused an electrophoretic mobility shift of H.pulcherrimus sperm guanylate cyclase from 131kDa to 128kDa. The 131kda and 128kda forms of guanylate cyclase contained about 24 and 4 moles of phosphate per mol protein. A cDNA clone for the membrane form of guanylate cyclase was isolated from a H.pulcherrimus testis cDNA library and its nucleotide sequence was determined. An open reading frame predicted a protein of 1125 amino acids including an apparent signal peptide of 21 resides ; a sisngle transmembrane domain of 25 amino acids divided the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl domain of 594 intracellular amino acids.
Incubation of intact H.pulceerrimus spermatozoa with sulfosuccinimidyl- 6-(biotinamido)hexanoate (NHS-LC-biotin) resulted in biotinylation of several sperm membrane proteins such as a 220kDa WGA-binding protein, a 128kDa membranebound guanylate cyclase, a 71kDa SAP-I-croslinked protein, a 63kDa protein and 50kDa protein. In these proteins, only the 50kDa protein showed specific interaction with H.pulcherrimus FSG. Less
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