背景:Cornichon是一个功能保守的跨膜蛋白家族,通常充当货物分选受体,并在ER和高尔基体之间循环。已经确定了四个Cornichon家族成员(CNIH1-4)。负责CNIH1-3结合AMPA受体的关键残基在CNIH4中不保守。此外,CNIH1-3在GPCR信号传导中的功能不太确定,而在CNIH4蛋白与GPCR相互作用并控制其出口的情况下更为确立。许多GPCRs因其在男性和女性性腺发育中的重要作用而闻名。但是CNIH4是否在配子发生中起作用仍然未知。
方法:携带Cnih4敲除等位基因(Cnih4tm1a-/-)的小鼠通过在外显子1后插入LacZ报道分子和聚腺苷酸化位点来产生。蛋白质印迹,免疫荧光,计算机辅助精子分析等方法用于功能分析。
结果:我们确定Cnih4tm1a-/-雄性和雌性小鼠均具有正常的生育力。不过,精子数,形态学,与野生型小鼠相比,Cnih4tm1a-/-小鼠的运动性略有受损,睾丸体重比和睾丸组织学与对照小鼠相似。Cnih4tm1a-/-卵巢的组织学检查从原始到窦阶段检测到卵泡,每个阶段的卵泡数量也与稀薄的野生型对照相当。经过六个月的生育力测试后发现正常的生育力。这可能是由于Chin3的代偿作用,在Cnih4tm1a-/-小鼠中显着上调以保持生育作用。
结论:尽管CNIH4在小鼠生殖细胞中显示出富集表达,我们的基因敲除研究表明,CNIH4对于小鼠的配子发生和生育能力不是必需的,尽管数量略有减少,雄性小鼠精子的运动和形态。
Cornichon is a functionally conserved transmembrane protein family that generally acts as a cargo-sorting receptor and cycles between the ER and the Golgi. Four
Cornichon family members (CNIH1-4) have been identified. The key residues responsible for CNIH1-3 to bind to AMPA receptors are not conserved in CNIH4. Additionally, the function of CNIH1-3 in GPCR signaling is less established, while more established in case of CNIH4 protein that interact with GPCR and control their exportation. Many GPCRs are known for their essential roles in male and female gonad development. But whether CNIH4 plays a role in gametogenesis remains unknown.
Mice carrying the Cnih4 knockout allele (Cnih4tm1a-/-) were generated by insertion of a LacZ reporter and a polyadenylation site after exon 1. Western blot, Immunofluorescence, computer-aided sperm analysis and other methods were used in the functional analysis.
We identified that both Cnih4tm1a-/- male and female mice have normal fertility. Though, the sperm count, morphology, and motility of Cnih4tm1a-/- mice were slightly impaired compared to those of wild-type mice, the testes to body weight ratio and testicular histology were similar to those in control mice. Histological examination of Cnih4tm1a-/- ovaries detected follicles from primordial to antral stages and the numbers of follicles at each stage were also comparable to wild-type controls. Normal fertility was noticed after six-month fertility tests. That was likely due to the compensatory role of Chin3, which significantly upregulated in the Cnih4tm1a-/- mice to preserve the fertility role.
Despite CNIH4 showing enriched expression in mouse germ cells, our genetic knockout studies demonstrated that CNIH4 is not essential for gametogenesis and fertility in mice although with a slight reduction in count, motility and morphology of sperm in male mice.