睾丸特异性丝氨酸激酶 3 对于精子形态发生和男性生育力是必需的。
Testis-specific serine kinase 3 is required for sperm morphogenesis and male fertility.
机构信息
Center for Drug Discovery, Baylor College of Medicine, Houston, Texas, USA.
Department of Pathology and Immunology, Baylor College of Medicine, Houston, USA.
出版信息
Andrology. 2023 Jul;11(5):826-839. doi: 10.1111/andr.13314. Epub 2022 Nov 16.
BACKGROUND
The importance of phosphorylation in sperm during spermatogenesis has not been pursued extensively. Testis-specific serine kinase 3 (Tssk3) is a conserved gene, but TSSK3 kinase functions and phosphorylation substrates of TSSK3 are not known.
OBJECTIVE
The goals of our studies were to understand the mechanism of action of TSSK3.
MATERIALS AND METHODS
We analyzed the localization of TSSK3 in sperm, used CRISPR/Cas9 to generate Tssk3 knockout (KO) mice in which nearly all of the Tssk3 open reading frame was deleted (ensuring it is a null mutation), analyzed the fertility of Tssk3 KO mice by breeding mice for 4 months, and conducted phosphoproteomics analysis of male testicular germ cells.
RESULTS
TSSK3 is expressed in elongating sperm and localizes to the sperm tail. To define the essential roles of TSSK3 in vivo, heterozygous (HET) or homozygous KO male mice were mated with wild-type females, and fertility was assessed over 4 months; Tssk3 KO males are sterile, whereas HET males produced normal litter sizes. The absence of TSSK3 results in disorganization of all stages of testicular seminiferous epithelium and significantly increased vacuolization of germ cells, leading to dramatically reduced sperm counts and abnormal sperm morphology; despite these histologic changes, Tssk3 null mice have normal testis size. To elucidate the mechanisms causing the KO phenotype, we conducted phosphoproteomics using purified germ cells from Tssk3 HET and KO testes. We found that proteins implicated in male infertility, such as GAPDHS, ACTL7A, ACTL9, and REEP6, showed significantly reduced phosphorylation in KO testes compared to HET testes, despite unaltered total protein levels.
CONCLUSIONS
We demonstrated that TSSK3 is essential for male fertility and crucial for phosphorylation of multiple infertility-related proteins. These studies and the pathways in which TSSK3 functions have implications for human male infertility and nonhormonal contraception.
背景
精子发生过程中磷酸化在精子中的重要性尚未得到广泛研究。睾丸特异性丝氨酸激酶 3（Tssk3）是一个保守基因，但 TSSK3 激酶的功能和 TSSK3 的磷酸化底物尚不清楚。
目的
我们研究的目的是了解 TSSK3 的作用机制。
材料和方法
我们分析了 TSSK3 在精子中的定位，使用 CRISPR/Cas9 技术在 Tssk3 敲除（KO）小鼠中生成几乎所有 Tssk3 开放阅读框缺失（确保其为 null 突变），通过繁殖小鼠 4 个月来分析 Tssk3 KO 小鼠的生育能力，并对雄性睾丸生殖细胞进行磷酸蛋白质组学分析。
结果
TSSK3 在伸长精子中表达，并定位于精子尾部。为了在体内定义 TSSK3 的重要作用，杂合（HET）或纯合 KO 雄性小鼠与野生型雌性小鼠交配，并在 4 个月内评估生育能力；Tssk3 KO 雄性是不育的，而 HET 雄性产生正常的产仔数。TSSK3 的缺失导致睾丸生精小管上皮的所有阶段的组织紊乱和生精细胞的空泡化显著增加，导致精子计数显著减少和精子形态异常；尽管存在这些组织学变化，Tssk3 缺失小鼠的睾丸大小正常。为了阐明导致 KO 表型的机制，我们使用从 Tssk3 HET 和 KO 睾丸中纯化的生殖细胞进行了磷酸蛋白质组学分析。我们发现，与 HET 睾丸相比，许多与男性不育相关的蛋白质，如 GAPDHS、ACTL7A、ACTL9 和 REEP6，其磷酸化水平显著降低，尽管总蛋白水平没有改变。
结论
我们证明 TSSK3 对雄性生育力是必需的，并且对多个与不育相关的蛋白质的磷酸化至关重要。这些研究以及 TSSK3 发挥作用的途径对人类男性不育和非激素避孕具有重要意义。
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