Spermatozoa are produced in the testis but gain their fertilizing ability during epididymal migration. This necessary step in sperm maturation includes posttranslational modification of sperm membrane proteins that includes protein processing by proteases. However, the molecular mechanism underpinning this epididymal sperm maturation remains unknown. In this study, we focused on transmembrane serine protease 12 (Tmprss12). Based on multi-tissue expression analysis by PCR, Tmprss12 was specifically expressed in the testis, and its expression started on day 10 postpartum, corresponding to the stage of zygotene spermatocytes. TMPRSS12 was detected in the acrosomal region of spermatozoa by immunostaining. To reveal the physiological function of TMPRSS12, we generated two knockout (KO) mouse lines using the CRISPR/Cas9 system. Both indel and large deletion lines were male sterile showing that TMPRSS12 is essential for male fertility. Although KO males exhibited normal spermatogenesis and sperm morphology, ejaculated spermatozoa failed to migrate from the uterus to the oviduct. Further analysis revealed that a disintegrin and metalloprotease 3 (ADAM3), an essential protein on the sperm membrane surface that is required for sperm migration, was disrupted in KO spermatozoa. Moreover, we found that KO spermatozoa showed reduced sperm motility via computer-assisted sperm analysis, resulting in a low fertilization rate in vitro. Taken together, these data indicate that TMPRSS12 has dual functions in regulating sperm motility and ADAM3-related sperm migration to the oviduct. Because Tmprss12 is conserved among mammals, including humans, our results may explain some genetic cases of idiopathic male infertility, and TMPRSS12 and its downstream cascade may be novel targets for contraception.

中文翻译：

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Tmprss12 是小鼠精子活力和输卵管交界处迁移所必需的†。

精子在睾丸中产生，但在附睾迁移过程中获得受精能力。精子成熟的这一必要步骤包括精子膜蛋白的翻译后修饰，包括蛋白酶对蛋白质的加工。然而，支持这种附睾精子成熟的分子机制仍然未知。在这项研究中，我们专注于跨膜丝氨酸蛋白酶 12 ( Tmprss12 )。基于 PCR 多组织表达分析，Tmprss12在睾丸中特异性表达，其表达开始于产后第 10 天，对应于合子精母细胞的阶段。通过免疫染色在精子顶体区域检测到 TMPRSS12。为了揭示 TMPRSS12 的生理功能，我们使用 CRISPR/Cas9 系统生成了两个基因敲除 (KO) 小鼠品系。indel 和大缺失系都是雄性不育系，表明 TMPRSS12 对雄性生育力至关重要。尽管 KO 雄性表现出正常的精子发生和精子形态，但射出的精子未能从子宫迁移到输卵管。进一步的分析表明，解整合素和金属蛋白酶 3 (ADAM3) 是精子迁移所需的精子膜表面的一种必需蛋白质，在 KO 精子中被破坏。而且，我们发现 KO 精子通过计算机辅助精子分析显示出精子活力降低，导致体外受精率低。综上所述，这些数据表明 TMPRSS12 在调节精子活力和 ADAM3 相关的精子向输卵管迁移方面具有双重功能。因为Tmprss12在包括人类在内的哺乳动物中是保守的，我们的结果可以解释一些特发性男性不育的遗传病例，并且 TMPRSS12 及其下游级联可能是避孕的新靶点。