Evans J P, Kopf G S, Schultz R M
Center for Research on Reproduction and Women's Health, University of Pennsylvania, Philadelphia 19104, USA.
Dev Biol. 1997 Jul 1;187(1):79-93. doi: 10.1006/dbio.1997.8611.
The sperm protein fertilin (also known as PH-30) is a candidate for mediating the interactions between sperm and egg plasma membranes. Fertilin is a heterodimer. The beta subunit, which has a region with homology to the family of integrin ligands known as disintegrins, has been hypothesized to be involved in the binding of sperm to the egg surface. To investigate this hypothesis and determine what role fertilin beta plays in fertilization, we have expressed the putative extracellular domain of mouse fertilin beta in bacteria as a fusion protein with maltose-binding protein (hereafter referred to as recombinant fertilin beta-EC) and used two assays to characterize its binding to mouse eggs. Immunocytochemistry was used to examine the localization of recombinant fertilin beta-EC binding. A luminometric assay was also developed to quantify levels of binding of recombinant fertilin beta-EC to single eggs. We find that recombinant fertilin beta-EC binds to the region of the plasma membrane of the egg to which sperm bind, thus providing the first direct evidence that fertilin beta has adhesive properties. Peptides corresponding to the disintegrin domain of fertilin beta reduce its binding to eggs, suggesting that this domain is at least partially involved in the recognition of fertilin beta by binding sites on the egg. Treatment of zona pellucida-free eggs with chymotrypsin reduces the ability of the eggs to support the binding of recombinant fertilin beta-EC, implicating an egg surface protein as a binding site for recombinant fertilin beta-EC. Binding of recombinant fertilin beta-EC to eggs is also reduced in the absence of divalent cations and is supported by 2.0 mM Ca2+, Mg2+, or Mn2+. Furthermore, eggs incubated in recombinant fertilin beta-EC prior to in vitro fertilization show reduced levels of sperm binding. Finally, we have examined the possible role of integrins on eggs as receptors for fertilin beta, since an anti-alpha6 integrin subunit monoclonal antibody, GoH3, has been shown to inhibit sperm binding (E. A. C. Almeida et al. (1995) Cell 81, 1095-1104). We find that: (a) an increased amount of GoH3 epitope on the egg surface does not correlate with an increased ability of the eggs to bind sperm or recombinant fertilin beta-EC; (b) the GoH3 antibody has virtually no inhibitory effect on recombinant fertilin beta-EC binding; and (c) recombinant fertilin beta-EC binding is reduced in the presence of anti-beta1 integrin antibodies. These results suggest that a beta1-containing integrin participates in the binding of recombinant fertilin beta-EC to mouse eggs.
精子蛋白受精素（也称为PH - 30）是介导精子与卵细胞质膜之间相互作用的候选蛋白。受精素是一种异源二聚体。其β亚基有一个区域与整合素配体家族中的解整合素具有同源性，据推测该亚基参与精子与卵表面的结合。为了研究这一假说并确定受精素β在受精过程中所起的作用，我们已在细菌中表达了小鼠受精素β的假定细胞外结构域，使其与麦芽糖结合蛋白融合（以下简称重组受精素β - EC），并使用两种检测方法来表征其与小鼠卵的结合情况。免疫细胞化学用于检测重组受精素β - EC结合的定位。还开发了一种发光测定法来量化重组受精素β - EC与单个卵的结合水平。我们发现重组受精素β - EC与精子结合的卵细胞质膜区域结合，从而提供了首个直接证据，证明受精素β具有黏附特性。与受精素β解整合素结构域对应的肽段会降低其与卵的结合，这表明该结构域至少部分地通过卵上的结合位点参与受精素β的识别。用胰凝乳蛋白酶处理无透明带的卵会降低卵支持重组受精素β - EC结合的能力，这意味着一种卵表面蛋白是重组受精素β - EC的结合位点。在没有二价阳离子的情况下，重组受精素β - EC与卵的结合也会减少，而2.0 mM的Ca2 +、Mg2 +或Mn2 +可支持这种结合。此外，在体外受精前用重组受精素β - EC孵育的卵显示精子结合水平降低。最后，我们研究了卵上的整合素作为受精素β受体的可能作用，因为抗α6整合素亚基单克隆抗体GoH3已被证明可抑制精子结合（E. A. C. Almeida等人（1995年）《细胞》81卷，1095 - 1104页）。我们发现：（a）卵表面GoH3表位数量的增加与卵结合精子或重组受精素β - EC能力的增强并无关联；（b）GoH3抗体对重组受精素β - EC结合几乎没有抑制作用；（c）在抗β1整合素抗体存在的情况下，重组受精素β - EC结合减少。这些结果表明含β1的整合素参与重组受精素β - EC与小鼠卵的结合。
