College of Animal Science and Technology, Northwest A&F University, Shaanxi, 712100, China,
Mol Biol Rep. 2014 Jan;41(1):217-23. doi: 10.1007/s11033-013-2854-2. Epub 2013 Nov 12.
Our previous studies indicated that a purified rabbit antiserum against X-sperm contained sex-specific antibodies (SSAbs) which preferentially bound to sex-sorted X-sperm. The specificity of sex-specific antiserum was initially demonstrated using flow cytometry only, which resulted in uncertainty. In this study, the putative SSAbs against bovine X-sperm (XSSAb) were produced by a series of immunological approaches, and the effectiveness of separation of sperm using putative XSSAb was validated. Subsequently, the XSSAb was used to immunoprecipitate sex-specific proteins (SSPs) in bovine sperm, followed by two-dimensional gel electrophoresis. The results showed 7.6, 15.2 and 52.1 % of sex-sorted Y-sperm, sex-sorted X-sperm and unsorted sperm were recognized by the neutralized rabbit antisera against X-sperm, respectively. Also the purity of separation of sperm using putative XSSAb reached 74.3 % when the immunologically separated sperm were injected into oocytes. In addition, three candidate SSP sports about 30 kDa were captured by the XSSAb. Our results confirmed that the putative XSSAb contained SSAbs, and implied that these three protein sports might be SSPs in bovine X-sperm. This provides a potentially more efficient method for sorting sperm and lays a foundation for future search for SSPs.
我们之前的研究表明，一种针对 X 精子的纯化兔抗血清中含有性别特异性抗体（SSAbs），这些抗体优先与分选的 X 精子结合。性别特异性抗血清的特异性最初仅通过流式细胞术来证明，这导致了不确定性。在这项研究中，我们通过一系列免疫学方法制备了针对牛 X 精子的假定 SSAbs（XSSAb），并验证了使用假定 XSSAb 分离精子的有效性。随后，我们使用 XSSAb 免疫沉淀牛精子中的性别特异性蛋白（SSPs），然后进行二维凝胶电泳。结果表明，经中和的兔抗 X 精子抗血清分别识别 7.6%、15.2%和 52.1%的分选 Y 精子、分选 X 精子和未分选精子。此外，当用假定的 XSSAb 免疫分离的精子注入卵母细胞时，精子的分离纯度达到 74.3%。此外，XSSAb 还捕获了三个约 30 kDa 的候选 SSP 蛋白。我们的结果证实了假定的 XSSAb 含有 SSAbs，并暗示这三个蛋白可能是牛 X 精子中的 SSPs。这为精子分选提供了一种更有效的方法，并为未来寻找 SSPs 奠定了基础。
