Kunkel Sarah, Johnston Stephen D, Nouwens Amanda, Pini Taylor
School of the Environment, The University of Queensland, Gatton, Australia.
School of Veterinary Science, The University of Queensland, Gatton, Australia.
Biol Reprod. 2025 Sep 15;113(3):581-591. doi: 10.1093/biolre/ioaf160.
While assisted reproductive technologies (ARTs) are widely used in domestic animals, successful implementation of ARTs to conserve wildlife species remains challenging. In macropods, crucial aspects of fundamental reproductive biology, including changes induced by epididymal maturation, remain unknown, limiting the development of ARTs. In this context, we performed a proteomic analysis of spermatozoa from the caput, corpus, and cauda epididymis of Eastern Grey Kangaroos (n = 6) to profile changes over epididymal maturation. Samples prepared by filter-aided sample preparation digestion were analyzed by liquid chromatography tandem mass spectrometry with sequential window acquisition of all theoretical acquisition. A total of 4304 proteins were identified, with significant overlap across epididymal regions. Highly abundant proteins in common across caput, corpus, and cauda spermatozoa had strong enrichment for tubulins and included four histone proteins. The most significant proteomic remodeling was observed in the corpus to cauda transition, late in epididymal transit (728 differentially abundant proteins). Overall proteomic changes across epididymal maturation (1131 differentially abundant proteins) suggested a loss of sperm glycosidases and an increase in flagellar proteins, including tubulins and dyneins. These findings serve to highlight both consistencies with eutherian sperm epididymal maturation (e.g. bias toward protein loss over transit, transfer of proteins via extracellular vesicles) and elements which are likely unique to marsupials (e.g. reduced chromatin stability, potential use of β-oxidation as a major metabolic pathway). This critical information can now be leveraged to further develop ARTs in marsupials.
虽然辅助生殖技术（ARTs）在家畜中被广泛应用，但将其成功应用于野生动物保护仍具有挑战性。在有袋类动物中，基本生殖生物学的关键方面，包括附睾成熟所引起的变化，仍然未知，这限制了辅助生殖技术的发展。在此背景下，我们对东部灰袋鼠（n = 6）附睾头、附睾体和附睾尾的精子进行了蛋白质组学分析，以描绘附睾成熟过程中的变化。通过滤膜辅助样品制备消化法制备的样品，采用液相色谱串联质谱结合全理论质量数的同步窗口采集法进行分析。共鉴定出4304种蛋白质，在附睾各区域有显著重叠。附睾头、附睾体和附睾尾精子中共同存在的高丰度蛋白质在微管蛋白方面有很强的富集，并且包括四种组蛋白。在附睾体至附睾尾的过渡阶段，即在附睾运输后期，观察到最显著的蛋白质组重塑（728种差异丰度蛋白质）。整个附睾成熟过程中的总体蛋白质组变化（1131种差异丰度蛋白质）表明精子糖苷酶减少，鞭毛蛋白增加，包括微管蛋白和动力蛋白。这些发现既突出了与真兽类精子附睾成熟的一致性（例如在运输过程中倾向于蛋白质丢失、通过细胞外囊泡转移蛋白质），也突出了有袋类动物可能独有的元素（例如染色质稳定性降低、可能将β-氧化作为主要代谢途径）。现在可以利用这些关键信息进一步开发有袋类动物的辅助生殖技术。
