Velásquez José Guillermo, Canovas Sebastián, Barajas Patricia, Marcos Julián, Jiménez-Movilla María, Gallego Ricardo Gutiérrez, Ballesta José, Avilés Manuel, Coy Pilar
Department of Physiology, Veterinary Faculty, University of Murcia, Murcia, Spain.
Mol Reprod Dev. 2007 May;74(5):617-28. doi: 10.1002/mrd.20619.
Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.
在透明带（ZP）糖蛋白中已检测到精子结合活性，并且人们普遍认为这种活性存在于碳水化合物部分。在本研究中，我们旨在鉴定一些参与牛精子与ZP相互作用的特定碳水化合物分子。我们进行了精子结合竞争试验、结合不同凝集素、抗体和神经氨酸酶消化的体外受精（IVF），以及对牛ZP的化学和细胞化学分析。识别α-2,3-连接唾液酸的MAA凝集素和具有针对α-2,3-连接唾液酸催化活性的鼠伤寒沙门氏菌神经氨酸酶，均对精子与ZP的结合及卵母细胞穿透表现出高度抑制作用。这些结果表明，牛精子与ZP的结合是由α-2,3-连接的唾液酸介导的。用三糖（唾液乳糖、3'-唾液乳糖胺和6'-唾液乳糖胺）和糖蛋白（胎球蛋白和去唾液酸胎球蛋白）进行的实验证实了这一点，并表明至少序列Neu5Ac(α2-3)Gal(β1-4)GlcNAc参与了精子与ZP的相互作用。此外，这些结果表明存在一种针对唾液酸的精子质膜特异性蛋白。化学分析表明，牛ZP糖蛋白主要含有Neu5Ac（84.5%）和Neu5GC（15.5%）。凝集素细胞化学分析表明，这两种类型的唾液酸残基可能分别通过α-2,3-和α-2,6-连接与Galβ1,4GlcNAc和GalNAc相连。使用神经氨酸酶抑制剂导致与ZP结合并穿透卵母细胞的精子数量增加。基于这一最后的结果，我们推测皮质颗粒中的神经氨酸酶可能通过从ZP上去除唾液酸而参与多精受精阻断。
