Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada;; Department of Pathology and Laboratory Medicine.
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada;; Lunenfeld-Tanenbaum Research Institute.
Mol Cell Proteomics. 2019 Feb;18(2):338-351. doi: 10.1074/mcp.RA118.001170. Epub 2018 Nov 14.
TEX101 is a germ-cell-specific protein and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm-oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied due to the lack of human germ cell lines. Here, we genotyped 386 men for a common missense variant rs35033974 of and identified 52 heterozygous and 4 homozygous men. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with the near-complete degradation (>97%) of the corresponding G99V TEX101 form and suggested that spermatozoa of homozygous men could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified eight cell-surface and nine secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm-oocyte interaction (including LY6K and ADAM29) were confirmed by targeted proteomics and Western blotting assays. Because recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a monogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found fivefold lower ( = 0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction.
TEX101 是一种生殖细胞特异性蛋白，也是男性不育的一种已验证的生物标志物。研究发现，小鼠 TEX101 对雄性生育力至关重要，它可能作为一种细胞表面伴侣蛋白，参与精子迁移和精子-卵子相互作用所需蛋白的成熟。然而，由于缺乏人类生殖细胞系，人类 TEX101 的精确功能尚不清楚，也无法进行研究。在这里，我们对 386 名男性进行了常见错义变体 rs35033974 的基因分型，发现 52 名杂合子和 4 名纯合子男性。然后，我们通过靶向蛋白质组学发现，变体等位基因 rs35033974 与相应 G99V TEX101 形式的近乎完全降解（>97%）相关，并表明纯合子男性的精子可作为敲低模型，用于研究人类 TEX101 的功能。采用无标记定量的差异蛋白质组学分析，在 8 名男性的精子中测量了 8046 种蛋白质，在 4 名 rs35033974 纯合子患者的精子中发现了 8 种细胞表面蛋白和 9 种分泌型睾丸特异性蛋白显著下调。通过靶向蛋白质组学和 Western blot 分析，进一步证实了潜在参与精子迁移和精子-卵子相互作用的大量下调的睾丸特异性细胞表面蛋白（包括 LY6K 和 ADAM29）。由于最近的全基因组规模的遗传数据显示，纯合子父亲具有生物学后代，因此 rs35033974 不是人类男性不育的单基因因素。然而，我们发现欧洲血统的杂合子男性的精液中 TEX101 水平比野生型男性低五倍（=0.0005）。我们得出结论，rs35033974 纯合子男性的精子中 TEX101 水平显著降低，可作为阐明 TEX101 确切功能的模型，这将推动人类生殖生物学的发展。
