Signal transduction in gamete recognition

在海胆受精过程中，精子在接触卵子表面之前必须穿过包裹着卵子的果冻层。果冻外壳含有两种高分子量糖缀合物和精子激活肽（SAPs）。一种聚焦硫酸盐糖缀合物（FSG）是果冻外壳中的糖缀合物之一，据报道是诱导顶体反应的主要物质。sap - 1 (gly - phep - asp - leu - asn - gly - gly - gly - gly - val - gly)，最初被认为是在海胆精子中诱导一系列生化事件的因子，如1)细胞内cAMP、cGMP和[Ca^<2+>]水平的短暂升高，以及鸟苷酸环化酶膜形式的短暂激活。它还诱导质子穿过精子质膜流出，导致细胞内ph值增加。除了上述，该肽已被证明作为FSG的特定辅助因子，促进了半圆心精子的顶体反应。利用多克隆抗体对海胆卵巢中FSG的免疫细胞化学定位进行了研究，结果表明FSG是由附属细胞产生的，最初沉积在卵黄细胞表面形成果冻。从黄鼠卵巢cDNA文库中分离到一个编码sap - 1的cDNA克隆，并对其核苷酸序列进行了测定。cDNA全长1282 bp，在开放阅读框中预测该蛋白含有334个氨基酸，包含5个sap - 1和7个sap - 1样十肽，每个十肽由一个赖氨酸残基分开。以1.3kb cDNA为模板合成的非放射性RNA探针原位杂交研究了sap - 1前体基因的表达，结果表明，在副细胞中表达了大量的sap - 1前体转录物，而在生长的卵母细胞中则没有表达。我们利用结合和交联技术对海胆H.PULCHERRIMUS精子中可能的sap - 1特异性受体进行了表征。从放射性捐赠的SAP- 1类似物与精子的平衡结合中获得的数据分析显示，精子中存在两类SAP-特异性受体（高亲和和低亲和）。SAP-I在活性方面有两类EC50值。一类EC_<50>值为亚纳摩尔水平，包括呼吸刺激活性和细胞内ph升高活性。另一类值范围为7-32 nM，见于细胞内Ca^<2+>-升高活性和细胞cgmp升高活性。因此，我们推测，在sap - 1的生物活性中，EC_<50>的两个不同范围可能反映了两类sap - 1受体的存在；高亲和受体可能具有细胞内ph升高活性和呼吸刺激活性，低亲和受体可能具有cGMP-和细胞内Ca^<2+>-升高活性。用放射性碘化的sapi类似物和化学交沉试剂二琥珀酰亚酸孵育完整的精子以及从H.pulcherrimus精子制备的精子尾部或精子膜，得到了71 kDa的放射性标记蛋白。该蛋白似乎与一个220 kDa的小麦胚芽凝集素（WGA）结合蛋白有关。纯化得到的71kDa蛋白末端氨基酸序列为EQNYREAVEGNIRLIHGRTENEGS。从睾丸H.pulcherrimus testis cDNA文库中分离到一个编码71kDa蛋白的cDNA克隆，并测定其核苷酸序列。cDNA全长2443 bp，在开放阅读框中预测该蛋白含有532个氨基酸，含有30个残基的氨基末端信号肽，其序列与71kDa蛋白的n端序列相同。该蛋白由一个大的n - tersap - 1组成，导致H.pulcherrimus精子鸟苷酸环化酶的电泳迁移率从131kDa转移到128kDa。131kda和128kda形式的鸟苷酸环化酶每mol蛋白质含有约24和4 mol磷酸盐。从睾丸H.pulcherrimus testis cDNA文库中分离到一个膜型鸟苷酸环化酶cDNA克隆，并测定其核苷酸序列。开放阅读框预测了1125个氨基酸的蛋白质，包括21个位点的表观信号肽；一个由25个氨基酸组成的单跨膜结构域将成熟蛋白分成由485个氨基酸组成的氨基端胞外结构域和由594个氨基酸组成的羧基结构域。用巯基琥珀酰亚胺- 6-（生物素胺）己酸盐（nhss - lc -生物素）孵育完整的H.pulceerrimus精子，导致几种精子膜蛋白的生物素化，如220kDa的wga结合蛋白、128kDa的膜结合鸟苷酸环化酶、71kDa的sap - i交联蛋白、63kDa蛋白和50kDa蛋白。在这些蛋白中，只有50kDa蛋白与H.pulcherrimus FSG表现出特异性相互作用。少

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 … More .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-terSAP-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