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.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们之前报道过DEFB126(以前的ESP13.2)覆盖在猕猴精子的整个表面，并一直保持到精子获得能力。在精子获能过程中，DEFB126的释放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潜在地掩盖了精子细胞表面的成分，这一观察促使我们研究了这种蛋白质作为一种保护免疫识别的手段的潜在作用。食蟹猴的精子被暴露在一系列已知可以改变精子表面涂层的治疗方法中，包括获能。我们利用了一种新的体内试验来确定免疫识别：将醛固定的完整精子注射到兔体内。在加强注射后，对以不同方式制备的整个精子进行了免疫印迹分析。在初次免疫后60天和80天，抗血清对单一的34-36 kDa蛋白表现出强烈的反应，该蛋白被证明是DEFB126。用Percoll梯度更严格地洗涤过的精子免疫兔子的血清显示，尽管对DEFB126的免疫反应占主导地位，但整个精子蛋白质印迹上识别的蛋白质数量和强度都增加了。当获得能力的精子(其中大部分DEFB126已被释放)被用作免疫原时，对各种蛋白条带的免疫识别能力显著增加。在固定前，用神经氨酸酶去除DEFB126上的唾液酸的精子显示仍具有DEFB126，但缺乏糖蛋白的唾液酸成分。这些精子与获能精子一样具有免疫原性，尽管所需的DEFB126仍然覆盖整个细胞表面。这些精子失去了高度负电荷(DEFB126等电点由等电点pI 3.0移至等电点pI 6.4)。使用不同的精子质膜蛋白特异性免疫球蛋白的实验表明，当有DEFB126存在时，这种识别不会发生，但在获能后，这些免疫球蛋白很容易识别暴露的精子膜。我们进一步研究了DEFB126的糖基与凝集素的结合。在荧光素标记的L-多赖氨酸或AlexA-488结合组蛋白作用下，精子在整个精子表面显示出与抗DEFB126Ig类似的非常均匀的荧光标记图案。用咖啡因/cAMP处理后释放的精子表面成分被印迹，并用三种已知识别末端唾液酸残基的不同凝集素进行探测，这三种凝集素都识别35 kDa的DEFB126区带。神经氨酸酶处理精子使DEFB126的分子量从34-36 kDa移动到约38-40 kDa，并去除或极大地抑制唾液酸特异性凝集素的识别。O-葡聚糖酶单独处理不能有效去除低聚糖，但预先用神经氨酸酶处理足以使O-葡聚糖酶有效地将分子量改变到10 kDa，即去除碳水化合物后的多肽质量，证实70%的分子质量与糖类部分有关。经神经氨酸酶处理的DEFB126的Western blotts显示出与许多识别β-半乳糖的凝集素以及一些识别N-乙酰氨基半乳糖-丝氨酸/苏氨酸的凝集素的强烈识别，这是建议的O-连接寡糖的连接。所有在蛋白质印迹上显示识别DEFB126的凝集素也被用来荧光探测精子。荧光图谱与L-多赖氨酸、唾液酸特异性凝集素、半乳糖特异性凝集素和抗DEFB126Ig的荧光图谱相同，显示出均匀分布在整个精子表面。