Badrhan Shiva, Karanwal Seema, Pal Ankit, Chera Jatinder Singh, Chauhan Vitika, Patel Aditya, Bhakat Mukesh, Datta Tirtha K, Kumar Rakesh
Animal Genomics Laboratory, Animal Biotechnology Division, National Dairy Research Institute, Karnal, India.
ICAR- Central Institute of Research on Goat, Mathura, Uttar Pradesh, India.
Front Cell Dev Biol. 2024 Jul 29;12:1400323. doi: 10.3389/fcell.2024.1400323. eCollection 2024.
Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7 nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200 nm, and concentrations varying from 2.84 to 6.82 × 10 and 3.57 to 7.74 × 10 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity.
水牛是印度乳业的支柱，而供精公牛的精液质量决定了奶牛场的整体生产效率。精浆中含有数以百万计的脂质双分子层纳米囊泡，即细胞外囊泡（EVs）。这些细胞外囊泡携带多种必需生物分子，包括与生育相关的蛋白质，这些蛋白质有助于精子的受精潜能。在本研究中，我们探索了来自两个不同生育力组的精浆细胞外囊泡的大小、浓度和完整蛋白质组图谱，以揭示影响公牛生育力的蛋白质。通过动态光散射（DLS）发现，在高生育力（HF）和低生育力（LF）公牛中，纯化的细胞外囊泡存在于7 - 14个尺寸排阻色谱（SEC）级分中，大小范围为146.5至258.7nm。纳米颗粒跟踪分析（NTA）证实了精浆细胞外囊泡的大小可达200nm，HF公牛和LF公牛中细胞外囊泡的浓度分别为2.84至6.82×10和3.57至7.74×10个颗粒/毫升。两组之间精浆细胞外囊泡的大小和浓度未观察到显著差异。我们使用高通量液相色谱 - 串联质谱（LC - MS/MS）方法，分别在HF和LF公牛的精浆细胞外囊泡中鉴定出总共1862种和1807种蛋白质。在这些总蛋白质中，两组共有1754种蛋白质，约87种蛋白质在HF组中高度丰富，而与LF公牛相比，有1292种蛋白质含量较低。基因本体（GO）分析表明，HF组中高度丰富的蛋白质主要是细胞核的一部分，参与核小体组装以及DNA结合。此外，发现HF组细胞外囊泡中高度丰富的蛋白质参与精子发生、运动、顶体反应、获能、配子融合和耐冻性。两种高度丰富的蛋白质，蛋白质二硫键异构酶A4和凝溶胶蛋白，分别与精子 - 卵母细胞融合和顶体反应相关，它们在精子上的免疫定位可能表明这些蛋白质是通过细胞外囊泡转移的。我们的证据支持细胞外囊泡中的蛋白质以及随后它们在精子上的存在与精子功能密切相关。总之，我们的研究表明精浆细胞外囊泡拥有对提高精子受精能力至关重要的关键蛋白质库。
