Table_4_Proteome Informatics in Tibetan Sheep (Ovis aries) Testes Suggest the Crucial Proteins Related to Development and Functionality.XLSX

Testis has an indispensable function in male reproduction of domestic animals. Tibetan sheep (Ovis aries) is a locally adapted breed of sheep raised in the Qinghai-Tibet Plateau, with outsized roles in providing the livelihood for millions of residents. Nevertheless, less is known on how protein expression and their functional roles in developmental testes of such breed limit their use in breeding efforts. In this study, we obtained comprehensive protein profiles from testes of Tibetan sheep at three developmental stages (including pre-puberty, post-puberty, and adulthood) using data-independent acquisition-based proteomic strategy to quantitatively identify the differentially abundant proteins (DAPs) associated with testicular development and function and to unravel the molecular basis of spermatogenesis. A total of 6,221 proteins were differentially expressed in an age-dependent manner. The reliability of the gene expression abundance was corroborated by quantitative PCR and targeted parallel reaction monitoring. These DAPs were significantly enriched to biological processes concerning spermatid development and sperm deformation, mitosis, glycolytic process, cell-cell/extracellular matrix (ECM) junctions, cell proliferation, apoptosis, and migration and to the pathways including, developmental process and sexual reproduction-related (such as VEGF, estrogen, insulin, GnRH, Hippo, PI3K-Akt, mTOR, MAPK, and AMPK), and testicular cell events-related pathways (such as tight/gap/adherens junctions, ECM-receptor interaction, regulation of actin cytoskeleton, glycolysis, cell cycle, and meiosis). Based on these bioinformatics analysis, we constructed four protein–protein interaction network, among which the proteins are involved in mitosis, meiosis, spermiogenesis, and testicular microenvironment, respectively. Altogether, these bioinformatics-based sequencing results suggest that many protein-coding genes were expressed in a development-dependent manner in Tibetan sheep testes to contribute to the testicular cell development and their surrounding microenvironment remodeling at various stages of spermatogenesis. These findings have important implications for further understanding of the mechanisms underlying spermatogenesis in sheep and even other plateau-adapted animals.

睾丸在家畜雄性生殖过程中具有不可或缺的作用。藏羊（Ovis aries）是饲养于青藏高原的本土适配绵羊品种，为数百万居民的生计提供了重要支撑。然而，目前对于该品种发育中睾丸的蛋白质表达及其功能如何制约育种应用的相关研究仍较为匮乏。本研究采用基于数据非依赖采集（data-independent acquisition, DIA）的蛋白质组学策略，对处于青春期前、青春期后及成年期三个发育阶段的藏羊睾丸组织进行了全面的蛋白质谱分析，以定量鉴定与睾丸发育及功能相关的差异丰度蛋白（differentially abundant proteins, DAPs），并解析精子发生的分子机制。共计6221种蛋白质呈现出年龄依赖性的差异表达模式。本研究通过实时定量聚合酶链式反应（quantitative PCR, qPCR）与靶向平行反应监测（parallel reaction monitoring, PRM）技术，验证了蛋白质表达丰度定量结果的可靠性。上述差异丰度蛋白显著富集于精子细胞发育、精子变形、有丝分裂、糖酵解过程、细胞-细胞/细胞外基质（extracellular matrix, ECM）连接、细胞增殖、凋亡及迁移等生物学过程；同时显著富集于与发育过程及有性生殖相关的信号通路，包括血管内皮生长因子（VEGF）、雌激素、胰岛素、促性腺激素释放激素（GnRH）、Hippo、PI3K-Akt、mTOR、MAPK及AMPK等通路，以及与睾丸细胞事件相关的通路，如紧密连接/缝隙连接/黏着连接、ECM-受体相互作用、肌动蛋白细胞骨架调控、糖酵解、细胞周期及减数分裂等通路。基于上述生物信息学分析，本研究构建了4个蛋白质相互作用网络，各网络分别涉及有丝分裂、减数分裂、精子形成及睾丸微环境相关的蛋白质。综上，本研究的生物信息学分析结果表明，藏羊睾丸中众多蛋白质编码基因的表达呈发育依赖性特征，可在精子发生的不同阶段参与睾丸细胞发育及其周围微环境的重塑。本研究结果为深入解析绵羊乃至其他高原适配动物的精子发生机制提供了重要的理论参考。