Prasad S V, Wilkins B, Skinner S M, Dunbar B S
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mol Reprod Dev. 1996 Apr;43(4):519-29. doi: 10.1002/(SICI)1098-2795(199604)43:4<519::AID-MRD15>3.0.CO;2-U.
A cDNA encoding the rabbit 55 kDa ZP protein was expressed using a baculovirus expression system and was evaluated for its ability to elicit antibodies which may interfere with sperm-ZP interaction. The expressed glycosylated protein, BV55, was purified by wheat germ agglutinin lectin affinity chromatography. Antisera made in guinea pigs immunized with BV55 (GP-alpha-BV55) is specific for the 55 kDa rabbit ZP protein. Indirect immunofluorescence studies indicate that GP-alpha-BV55 localizes to a filamentous meshwork on the surface of the ZP of isolated rabbit eggs. Immunohistochemical analysis of rabbit ovaries demonstrated that this antigen is localized within the ZP of primary and more advanced stage ovarian follicles but is not detected in primordial follicles. In addition, the 55 kDa antigen was detected in the granulosa cells of secondary stage follicles but not in the oocyte. GP-alpha-BV55 effectively blocked the binding of rabbit sperm to rabbit eggs in vitro. However, Fab fragments generated from GP-alpha-BV55 failed to block sperm binding, suggesting that the inhibitory effect of GP-alpha-BV55 was due to stearic hindrance rather than specific blocking of a sperm receptor site. Although the Fab fragment did not inhibit sperm binding, additional studies demonstrated that biotinylated BV55 protein bound to rabbit sperm in the acrosomal region in a manner consistent with ligand activity in the sperm-ZP interaction, and that BV55 bound to rabbit sperm in a dose-dependent manner. These studies therefore demonstrate that antibodies against recombinant ZP proteins recognize the native intact ZP and inhibit sperm-ZP interaction. They also provide evidence that the rabbit 55 kDa ZP protein, which is the homolog of the pig ZP3 alpha sperm receptor protein, has sperm receptor activity.
利用杆状病毒表达系统表达了编码兔55 kDa透明带（ZP）蛋白的cDNA，并对其引发可能干扰精子与ZP相互作用的抗体的能力进行了评估。表达的糖基化蛋白BV55通过麦胚凝集素亲和层析进行纯化。用BV55免疫豚鼠制备的抗血清（GP-α-BV55）对55 kDa兔ZP蛋白具有特异性。间接免疫荧光研究表明，GP-α-BV55定位于分离的兔卵ZP表面的丝状网络。兔卵巢的免疫组织化学分析表明，该抗原定位于初级及更高级阶段卵巢卵泡的ZP内，但在原始卵泡中未检测到。此外，在次级卵泡的颗粒细胞中检测到55 kDa抗原，但在卵母细胞中未检测到。GP-α-BV55在体外有效地阻断了兔精子与兔卵的结合。然而，从GP-α-BV55产生的Fab片段未能阻断精子结合，这表明GP-α-BV55的抑制作用是由于空间位阻而非对精子受体位点的特异性阻断。尽管Fab片段不抑制精子结合，但进一步的研究表明，生物素化的BV55蛋白以与精子-ZP相互作用中的配体活性一致的方式结合到兔精子的顶体区域，并且BV55以剂量依赖的方式结合到兔精子上。因此，这些研究表明，针对重组ZP蛋白的抗体识别天然完整的ZP并抑制精子-ZP相互作用。它们还提供了证据，表明兔55 kDa ZP蛋白是猪ZP3α精子受体蛋白的同源物，具有精子受体活性。
