Bhushan Vanya, Parashar Abhishek, Mahanandia Nimai Charan, Mohanty Tushar Kumar, Kaushik Jai Kumar, Kumar Sudarshan, Mohanty Ashok Kumar
Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, 132001, India.
Indian Agricultural Research Institute (IARI), PUSA, New Delhi, 110012, India.
Mol Biol Rep. 2025 Jun 21;52(1):619. doi: 10.1007/s11033-025-10657-2.
Semen sexing is a much sought-after technique in the livestock industry. Current research focuses on selecting DNA aptamers (Synthetic single-stranded DNA) against X-sorted sperm.
Four rounds of Cell-SELEX were performed, where the DNA aptamer library was incubated with X-sorted semen. Subsequently, affinity-depicting aptamers were sequenced. Fourteen out of thirty-four identified sequences were tested for specificity towards X-sorted sperm. The magnetic bead-based experiment revealed that the aptamers interacted preferentially with the acrosome region (sperm head) of sperm. We investigated the specificity of aptamers for X-spermatozoa by conducting real-time PCR assays targeting sex-specific genes. Aptamers X8, X19, X22 & X27 exhibited higher affinity for X-sorted sperm than Y in bovine semen. Western blot analysis showed an interaction between the aptamer and sperm protein at 35 kDa. The results underwent further analysis using in-gel mass spectrometry (MS) that predicted SPACA1 protein as the potential/putative aptamer binding protein on the sperm surface. Competitive western blot further confirmed aptamers' differential specificity for X-sperm.
The current work helps in creating new knowledge for the separation of Y-chromosome-bearing sperm from XY sperm populations that have the potential to produce good-quality cattle (cows). The work also generates proof of concept (POC) of using aptamers, which is cost-effective and could be used in sorting sex-specific spermatozoa without causing any ill effect on sperm.
精液性别分选是畜牧业中备受追捧的技术。当前研究聚焦于筛选针对X染色体分选精子的DNA适配体（合成单链DNA）。
进行了四轮细胞指数富集配体系统进化技术（Cell-SELEX），将DNA适配体文库与X染色体分选精液孵育。随后，对具有亲和性的适配体进行测序。在鉴定出的34个序列中，对14个进行了针对X染色体分选精子的特异性测试。基于磁珠的实验表明，适配体优先与精子的顶体区域（精子头部）相互作用。我们通过针对性别特异性基因进行实时聚合酶链反应（PCR）分析，研究了适配体对X精子的特异性。在牛精液中，适配体X8、X19、X22和X27对X染色体分选精子的亲和力高于Y染色体分选精子。蛋白质免疫印迹分析显示，适配体与35 kDa的精子蛋白之间存在相互作用。结果使用凝胶内质谱（MS）进一步分析，预测精子表面精子相关钙通道蛋白1（SPACA1）为潜在的适配体结合蛋白。竞争性蛋白质免疫印迹进一步证实了适配体对X精子的差异特异性。
当前工作有助于为从具有生产优质母牛潜力的XY精子群体中分离携带Y染色体的精子创造新知识。该工作还产生了使用适配体的概念验证（POC），其具有成本效益，可用于分选性别特异性精子且不会对精子造成任何不良影响。
