FUNCTION OF MACAQUE SPERM PROTEINS IN FERTILIZATION

猕猴精子蛋白在受精中的功能

• 批准号: 7349619
• 负责人: PAUL PRIMAKOFF
• 金额: $ 4.97万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7349619
• 关键词: FUNCTION; MACAQUE; SPERM; PROTEINS; FERTILIZATION

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We previously reported that DEFB126 (formerly ESP13.2) coats the entire surface of macaque sperm and remains until sperm become capacitated. The release of DEFB126 from sperm during capacitation is required for sperm recognition and binding of the zona pellucida, suggesting that DEFB126 masks zona pellucida ligands on the sperm surface. The observation that ESP13.2 potentially masks sperm cell surface components prompted us to examine the potential role of this protein as a means of protection from immune recognition. Cynomolgus macaque sperm were exposed to a number of treatments that are known to alter sperm surface coats, including capacitation. We utilized a novel in vivo assay to determine immune recognition: aldehyde-fixed whole sperm injections into rabbits. Following booster injections, immunoblot analyses of whole sperm prepared in various manners was conducted. On days 60 and 80 post initial immunization, the antisera showed a remarkably strong reaction to a single 34-36 kDa protein, which was shown to be DEFB126. Sera from rabbits that were immunized with sperm washed more rigorously using Percoll gradients showed an increase in the number and intensity of proteins recognized on whole sperm western blots, although the immune response to DEFB126 was dominant. When capacitated sperm, from which most of the DEFB126 had been released, were used as the immunogen, there was a dramatic increase in the immune recognition to a variety of protein bands. Sperm treated with neuraminidase to remove sialic acid on DEFB126 prior to fixation were shown to still possess DEFB126, but lacked the sialic acid component of the glycoprotein. These sperm were as immunogenic as capacitated sperm even though the desialylated DEFB126 still covered the entire cell surface. These sperm lost their highly negative charge (isoelectric point of DEFB126 shifted from pI 3.0 to pI 6.4). Experiments using different sperm plasma membrane protein-specific Igs showed that recognition did not occur when DEFB126 was present, but following capacitation these Igs readily recognized the exposed sperm membrane. We further characterized the carbohydrate groups of DEFB126 with lectin binding. Sperm exposed to fluorescein conjugated L-poly-lysine or alexa-488 conjugated histone showed a very uniform fluorescent labeling pattern over the entire sperm surface similar to that seen with anti-DEFB126 Ig. Sperm surface components that were released following treatment with caffeine/cAMP were blotted and probed with three different lectins which are known to recognize terminal sialic acid residues, and all three recognized the 35kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight of DEFB126 from 34-36kDa to approximately 38-40kDa and removed or greatly inhibited sialic acid specific lectin recognition. O-glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was sufficient to enable the o-glycanase to effectively change the molecular weight to 10kDa, the polypeptide weight after removal of the carbohydrates, confirming that 70% of the molecule mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated DEFB126 showed strong recognition with a number of lectins that recognize beta-galactose and also some lectins that recognize the n-acetyl galactosamine-serine/threonine, which is the proposed linkage of o-linked oligosaccharides. All of the lectins that showed recognition of DEFB126 on a western blot were also used to fluourescently probe sperm. The fluorescent patterns were identical to those seen with L-poly-lysine, sialic acid specific and galactose specific lectins and anti- DEFB126 Ig, which showed an even distribution along the entire sperm surface.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。我们之前报道过 DEFB126（以前称为 ESP13.2）覆盖猕猴精子的整个表面，并一直保留到精子获能为止。获能过程中精子释放 DEFB126 是精子识别和结合透明带所必需的，这表明 DEFB126 掩盖了精子表面的透明带配体。 ESP13.2 可能掩盖精子细胞表面成分的观察结果促使我们研究这种蛋白质作为免受免疫识别的保护手段的潜在作用。食蟹猴精子接受了多种已知会改变精子表面涂层的处理，包括获能。我们利用一种新颖的体内测定来确定免疫识别：将醛固定的全精子注射到兔子体内。加强注射后，对以各种方式制备的完整精子进行免疫印迹分析。初次免疫后第 60 和 80 天，抗血清对单一 34-36 kDa 蛋白质（即 DEFB126）表现出非常强烈的反应。尽管对 DEFB126 的免疫反应占主导地位，但用 Percoll 梯度更严格洗涤的精子免疫的兔子血清显示，全精子蛋白质印迹识别的蛋白质数量和强度有所增加。当获能精子（大部分 DEFB126 已从其中释放出来）用作免疫原时，对多种蛋白质条带的免疫识别显着增加。在固定前用神经氨酸酶处理以去除 DEFB126 上的唾液酸的精子仍具有 DEFB126，但缺乏糖蛋白的唾液酸成分。尽管去唾液酸化的 DEFB126 仍然覆盖整个细胞表面，这些精子与获能精子一样具有免疫原性。这些精子失去了高度负电荷（DEFB126 的等电点从 pI 3.0 变为 pI 6.4）。使用不同精子质膜蛋白特异性 Igs 的实验表明，当 DEFB126 存在时，不会发生识别，但在获能后，这些 Igs 很容易识别暴露的精子膜。 我们进一步表征了具有凝集素结合的 DEFB126 的碳水化合物基团。暴露于荧光素结合的 L-聚赖氨酸或 alexa-488 结合的组蛋白的精子在整个精子表面显示出非常均匀的荧光标记图案，类似于抗 DEFB126 Ig 所观察到的荧光标记图案。用咖啡因/cAMP 处理后释放的精子表面成分被印迹并用三种不同的凝集素进行探测，这三种凝集素已知识别末端唾液酸残基，并且所有三种凝集素均识别35kDa DEFB126 带。精子的神经氨酸酶处理将DEFB126的分子量从34-36kDa改变至大约38-40kDa，并去除或极大地抑制唾液酸特异性凝集素识别。单独的 O-聚糖酶处理对于去除寡糖是无效的，但是用神经氨酸酶进行预先处理足以使 O-聚糖酶有效地将分子量改变至 10kDa，即去除碳水化合物后的多肽重量，证实了 70%分子质量与碳水化合物部分相关。神经氨酸酶处理的 DEFB126 的蛋白质印迹显示出对许多识别 β-半乳糖的凝集素以及一些识别 N-乙酰半乳糖胺-丝氨酸/苏氨酸的凝集素的强烈识别，这是 O-连接寡糖的拟议连接。所有在蛋白质印迹中显示出对 DEFB126 的识别的凝集素也被用于荧光探测精子。荧光模式与 L-聚赖氨酸、唾液酸特异性和半乳糖特异性凝集素以及抗 DEFB126 Ig 所观察到的荧光模式相同，显示出沿整个精子表面的均匀分布。