背景:PRAME构成了欧瑟利亚人最大的多拷贝基因家族之一,编码具有富含亮氨酸重复(LRR)结构域的癌症睾丸抗原(CTA),在癌细胞和配子生殖细胞中高度表达。这项研究旨在阐明两个成员之间的遗传相互作用,Pramex1和Pramel1,在小鼠Prame家族中使用基因敲除方法进行配子发生。
结果:Pramex1或Pramel1的单基因敲除(sKO)导致约7%的异常生精小管,以仅支持细胞(SCO)表型为特征,显著影响精子数量和繁殖力。值得注意的是,sKO雌性小鼠表现出正常的生殖功能。相比之下,Pramex1/Pramel1双敲除(dKO)小鼠在两种性别中均表现出降低的繁殖力。在dKO女性中,与sKO和WT小鼠相比,卵巢初级卵泡计数减少了50%,与繁殖力减少50%相关。这表明在Pramex1或Pramel1sKO雌性的卵子发生过程中具有代偿作用。相反,dKO男性表现出18%的SCO小管频率,凋亡生殖细胞增加,与sKO和WT睾丸相比,未分化的精原细胞减少。在sKO睾丸上使用PRAMEX1或PRAMEL1特异性抗体进行的蛋白质印迹分析显示,每种蛋白质(30-50%)的代偿性上调响应于另一个基因的缺失。双KO男性在精子数量和产仔数方面表现出更严重的缺陷,超越Pramex1和Pramel1sKO累积效应,表明精子发生过程中的协同增强相互作用。在sKO中施用反式维甲酸(RA)及其抑制剂(WIN18,446)的其他实验,dKO,和WT小鼠提示PRAMEX1和PRAMEL1在精子发生过程中协同抑制RA信号通路。
结论:来自sKO和dKO小鼠的数据揭示了在配子发生过程中通过RA信号通路在Pramex1和Pramel1基因之间的协同相互作用。这一发现为调查Prame家族其他成员之间的互动奠定了基础,促进我们对生殖细胞形成和功能涉及的多拷贝基因家族的理解。
BACKGROUND: PRAME constitutes one of the largest multi-copy gene families in Eutherians, encoding cancer-testis antigens (CTAs) with leucine-rich repeats (LRR) domains, highly expressed in cancer cells and gametogenic germ cells. This study aims to elucidate genetic interactions between two members, Pramex1 and Pramel1, in the mouse Prame family during gametogenesis using a gene knockout approach.
RESULTS: Single-gene knockout (sKO) of either Pramex1 or Pramel1 resulted in approximately 7% of abnormal seminiferous tubules, characterized by a Sertoli-cell only (SCO) phenotype, impacting sperm count and fecundity significantly. Remarkably, sKO female mice displayed normal reproductive functions. In contrast, Pramex1/Pramel1 double knockout (dKO) mice exhibited reduced fecundity in both sexes. In dKO females, ovarian primary follicle count decreased by 50% compared to sKO and WT mice, correlating with a 50% fecundity decrease. This suggested compensatory roles during oogenesis in Pramex1 or Pramel1 sKO females. Conversely, dKO males showed an 18% frequency of SCO tubules, increased apoptotic germ cells, and decreased undifferentiated spermatogonia compared to sKO and WT testes. Western blot analysis with PRAMEX1- or PRAMEL1-specific antibodies on sKO testes revealed compensatory upregulation of each protein (30-50%) in response to the other gene\'s deletion. Double KO males exhibited more severe defects in sperm count and litter size, surpassing Pramex1 and Pramel1 sKO accumulative effects, indicating a synergistic enhancement interaction during spermatogenesis. Additional experiments administering trans-retinoic acid (RA) and its inhibitor (WIN18,446) in sKO, dKO, and WT mice suggested that PRAMEX1 and PRAMEL1 synergistically repress the RA signaling pathway during spermatogenesis.
CONCLUSIONS: Data from sKO and dKO mice unveil a synergistic interaction via the RA signaling pathway between Pramex1 and Pramel1 genes during gametogenesis. This discovery sets the stage for investigating interactions among other members within the Prame family, advancing our understanding of multi-copy gene families involved in germ cell formation and function.