The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo
The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo
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DOI：
10.1093/humrep/den028
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发表时间：
2008-05-01
影响因子：
6.1
通讯作者：
Calvo, J. C.
中科院分区：
文献类型：
--
作者：
Romanato, M.;Julianelli, V.;Calvo, J. C.
背景：我们实验室以前的研究结果表明，在体内人类精子解凝过程中，硫酸肝素(HS)可能是一种鱼精蛋白受体。这篇论文的目的是为了更好地支持这一观点，研究哺乳动物卵母细胞中是否存在糖胺聚糖。方法：采用两种实验方法：卵母细胞标记以确定HS的存在和分析新鲜卵母细胞在有或没有特定糖苷酶的情况下的精子解凝能力。结果：用荧光阳离子染料Rubipy在pH 1.5的条件下对小鼠卵母细胞进行染色，可在共聚焦显微镜下检测卵浆中的硫酸盐残留。免疫细胞化学方法检测卵浆中HS的表达。用肝素和谷胱甘肽成功地建立了精子解聚微量分析方法。用小鼠去透明带卵母细胞代替肝素可以达到同样的解凝效果。在卵母细胞/谷胱甘肽混合物中添加肝素酶可显著降低精子去凝集率(P=0.0159)，而添加软骨素酶ABC或透明质酸酶均无影响。结论：首次证实了HS存在于哺乳动物卵母细胞中，表明HS是新鲜卵母细胞在体外表达精子解聚能力所必需的。
BACKGROUND: Previous results from our laboratory have led us to propose heparan sulfate (HS) as a putative protamine acceptor during human sperm decondensation in vivo. The aim of this paper was to investigate the presence of glycosaminoglycans in the mammalian oocyte in an effort to better support this contention. METHODS: Two experimental approaches are used: oocyte labeling to identify the presence of HS and analysis of sperm decondensing ability of fresh oocytes in the presence or absence of specific glycosidases. RESULTS: Staining of mouse zona-intact oocytes with the fluorescent cationic dye, Rubipy, at pH 1.5 allowed for the detection of sulfate residues in the ooplasm by confocal microscopy. HS was detected in the ooplasm by immunocytochemistry. A sperm decondensation microassay using heparin and glutathione was successfully developed. The same level of sperm decondensation could be attained when heparin was replaced by mouse zona-free oocytes. Addition of heparinase to the oocyte/glutathione mixture significantly reduced sperm decondensation (P = 0.0159), while there was no effect following addition of either chondroitinase ABC or hyaluronidase. CONCLUSIONS: The results presented in this paper demonstrate for the first time that HS is present in the mammalian oocyte and show that HS is necessary for fresh oocytes to express their sperm decondensing ability in vitro.