膜联蛋白Al在小鼠睾丸中的表达、定位及其与受精的关系

发布时间：2018-08-30 15:32

【摘要】： 膜联蛋白是一类钙依赖性磷脂结合蛋白,形成进化上保守的多基因家族,在动、植物中广泛表达。在细胞各种生理功能中起重要作用。膜联蛋白A1(Annexin A1,简称AnxA1)是哺乳动物膜联蛋白家族中的一个重要成员,它与细胞增殖、分化、信号传递、炎症等密切相关,前期研究已发现它在精子中亦有表达,并呈区域性分布,推测与精子的一系列钙依赖事件如运动、顶体反应过程中的膜融合、受精过程中的精卵相互作用等有关。但目前它在生殖系统中的作用尚不明确,在睾丸中的表达定位尚未见报道,对其在精子中的定位研究还相当有限,且还未见关于它在精子中功能的较为直接的研究。本文以小鼠为实验对象,研究AnxA1在出生后不同发育时期睾丸中的表达与定位,以期了解它与睾丸出生后发育及精子发生的关系;同时,对处于不同成熟阶段及不同生理功能状态的精子进行AnxA1定位。此外,制备了AnxA1多克隆抗体并探讨AnxA1与体外受精的关系。本研究旨在为阐明AnxA1在生殖系统中的作用奠定一定的基础,可望为哺乳动物及人类受精机制的研究提供一定的理论依据,从而为辅助生殖技术、不育症诊疗与计划生育提供一定的理论指导。本实验分三部分。1膜联蛋白A1在小鼠睾丸不同发育阶段的表达与定位对1~8周龄小鼠睾丸进行AnxA1间接免疫组化染色及半定量RT-PCR以研究AnxA1的表达水平及其定位。结果显示:AnxA1在各级生精细胞中均有表达。其中精原细胞染色最强,主要分布在细胞核内;初级精母细胞多为弱阳性或阴性;圆形精子细胞AnxA1分布在细胞一侧顶体泡、顶体帽中,精子的AnxA1分布在顶体中。精子形成后留下的残余体亦呈强阳性染色。支持细胞未见明显阳性染色。对1、2、3、4、5、6、8周龄小鼠睾丸AnxA1免疫组化染色阳性强度即平均积分光密度进行图像分析结果分别是:2.94±0.36, 8.53±0.90, 7.32±1.22, 6.87±0.51, 5.59±0.53, 4.53±0.38, 4.64±0.40,单因素方差分析结果显示1、2、5周龄组的AnxA1免疫组化染色强度与其余各组均存在显著性差异(P0.01),3周龄与4周龄间(P=0.26, 0.05)、6周龄与8周龄间(P =0.78, 0.05)无显著性差
[Abstract]:Annexin is a kind of calcium dependent phospholipid binding protein, which forms an evolutionarily conserved polygene family and is widely expressed in animals and plants. It plays an important role in various physiological functions of cells. Annexin A1 (AnxA1) is an important member of mammalian cell binding protein family. It is closely related to cell proliferation, differentiation, signal transduction, inflammation and so on. It is supposed to be related to a series of calcium dependent events, such as movement, membrane fusion during acrosome reaction, sperm-egg interaction during fertilization and so on. However, its role in the reproductive system is still unclear, the expression localization in the testis has not been reported, and the study of its localization in spermatozoa is very limited, and there is no direct study on its function in spermatozoa. In this paper, we studied the expression and localization of AnxA1 in testis of different postnatal development stages in mice, in order to understand its relationship with postnatal development and spermatogenesis. The spermatozoa in different maturation stage and different physiological function were located by AnxA1. In addition, AnxA1 polyclonal antibodies were prepared and the relationship between AnxA1 and in vitro fertilization was investigated. The purpose of this study is to lay a foundation for elucidating the role of AnxA1 in the reproductive system, and to provide a theoretical basis for the study of fertilization mechanism in mammals and human beings. Diagnosis and treatment of infertility and family planning provide certain theoretical guidance. The expression and localization of integrin A1 in mouse testis at different stages of development were divided into three parts. AnxA1 indirect immunohistochemical staining of testis of 1-week-old mice was carried out. And semi-quantitative RT-PCR to study the expression level and localization of AnxA1. The results showed that: AnxA1 was expressed in spermatogenic cells at all levels. The primary spermatocytes were weakly positive or negative. The AnxA1 of round spermatocytes was distributed in one side of the cell acrosome vesicles, acrosome cap and sperm AnxA1 in acrosome. The residual bodies left after spermatogenesis also showed strong positive staining. Sertoli cells showed no positive staining. The results of image analysis on the positive intensity of AnxA1 immunohistochemical staining in testis of mice at 8 weeks of age were: 1: 2.94 卤0.36, 8.53 卤0.90, 7.32 卤1.22,6.87 卤0.51,5.59 卤0.53, 4.53 卤0.38, 4.64 卤0.40. The results of univariate variance analysis showed that the intensity of AnxA1 immunohistochemical staining was 12 weeks old, 8.53 卤0.90, 7.32 卤1.22, 4.53 卤0.38, 4.64 卤0.40, respectively. There was no significant difference (P0.01) between 3 and 4 weeks of age (P0. 26, 0. 05) and between 6 and 8 weeks of age (P = 0. 78, 0. 05).
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329

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