Shanghai Key Laboratory for Reproductive Medicine, Department of Histology and Embryology, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China.
Reprod Biol Endocrinol. 2010 Feb 4;8:10. doi: 10.1186/1477-7827-8-10.
Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process.
Laser scanning confocal microscopy (LSCM) and Western blot analysis were employed to probe for ERp29 protein in BALB/c mouse epididymal and acrosome-reacted spermatozoa. We prepared rabbit polyclonal antibodies against mouse recombinant ERp29 (rERp29) to characterize: 1) fertilization rate (FR); 2) fertilization index (FI); 3) sperm motility and 4) acrosome reaction (AR).
Confocal microscopy indicated that ERp29 was partially localized at the sperm head of the epididymal caput as well as over the whole head and part of the principal piece of the tail region from the epididymal cauda. However, when the acrosome reacted, ERp29 remained in the equatorial and post-acrosomal regions of the sperm head, which is the initial site of sperm-oocyte membrane fusion. Such localization changes were confirmed based on the results of Western blot analysis. Furthermore, the antibodies against mouse rERp29 inhibited the spermatozoa from penetrating into the zona pellucida (ZP)-free oocytes. The functional blocking antibodies reduced both mouse sperm-oocyte FR and FI at concentrations of 100 and 200 micro g/ml compared with pre-immunized rabbit IgG or with anti-mouse recombinant bactericidal/permeability-increasing protein (BPI, a sperm surface protein unrelated to sperm-oocyte fusion) antibodies (100 micro g/ml), but they had no effect on sperm motility and AR.
This study demonstrates that ERp29 on mouse spermatozoa membrane changes during epididymal transit and AR. Accordingly, in mice this protein may be one of the important factors involved in sperm fertilization by facilitating sperm-oocyte membrane fusion.
精卵融合是受精过程中的一个关键步骤，需要来自精子和卵子的一系列蛋白质来介导膜黏附和随后的融合。大鼠精子膜蛋白内质网蛋白 29（ERp29）在精子表面以及精子从头部到尾部的附睾上皮细胞的细胞质中显著增加，因为精子经历附睾成熟。此外，ERp29 通过介导膜穿透促进病毒感染。我们确定除了促进精子成熟外，ERp29 是否也在受精过程中促进配子融合中发挥作用。
采用激光共聚焦显微镜（LSCM）和 Western blot 分析检测 BALB/c 小鼠附睾和顶体反应精子中的 ERp29 蛋白。我们制备了针对小鼠重组 ERp29（rERp29）的兔多克隆抗体，以表征：1）受精率（FR）；2）受精指数（FI）；3）精子运动和 4）顶体反应（AR）。
共聚焦显微镜显示，ERp29 部分定位于附睾头部的精子头部，以及尾部区域的整个头部和部分主段。然而，当顶体反应时，ERp29 仍然存在于精子头部的赤道和顶体后区，这是精子卵膜融合的初始部位。这种定位变化基于 Western blot 分析的结果得到了证实。此外，针对小鼠 rERp29 的抗体抑制了精子穿透透明带（ZP）自由卵的能力。功能阻断抗体将小鼠精子-卵 FR 和 FI 在 100 和 200μg/ml 浓度下降低与预免疫兔 IgG 或针对抗小鼠重组杀菌/通透性增加蛋白（BPI，一种与精子-卵融合无关的精子表面蛋白）抗体（100μg/ml）相比，但对精子运动和 AR 没有影响。
本研究表明，小鼠精子膜上的 ERp29 在附睾转运和顶体反应过程中发生变化。因此，在小鼠中，该蛋白可能是通过促进精卵膜融合而参与精子受精的重要因素之一。
