Blücher Rheannon O, Lim Rachel S, Ritchie Matthew E, Western Patrick S
Centre for Reproductive Health, Hudson Institute of Medical Research and Department of Molecular and Translational Science, Monash University, Clayton, VIC, 3168, Australia.
Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.
BMC Biol. 2024 Oct 1;22(1):222. doi: 10.1186/s12915-024-02003-y.
Abnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4-eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor.
RNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes, the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5. In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane.
Together, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis.
子宫内睾丸发育异常与生殖健康状况密切相关，包括男性不育和睾丸癌。在小鼠睾丸中，SOX9和FGF9支持支持细胞的发育，而VEGF信号通路对于血管形成至关重要。丝裂原活化蛋白激酶（MAPK）通路是一条主要的信号级联，对于细胞增殖、分化以及初级性别决定过程中Sry的激活至关重要，但关于其在胎儿睾丸形态发生过程中的功能却知之甚少。我们使用MEK1/2抑制剂培养胚胎第12.5天（E12.5）的Oct4-eGFP转基因小鼠睾丸，在睾丸索建立后立即探索MAPK信号通路的潜在功能。
在分离的性腺体细胞中进行RNA测序，结果显示在MEK1/2抑制24小时和72小时后，分别有116个和114个差异表达基因。通路分析揭示了MEK1/2信号通路与血管生成、血管发生和细胞迁移等生物学功能之间的关联。这包括未能上调血管发育的主要转录调节因子Sox7和Sox17、VEGF受体基因、细胞粘附因子基因Cd31以及一系列其他内皮细胞标志物，如Cdh5（编码VE-钙粘蛋白）和缝隙连接基因Gja4和Gja5。相比之下，只有少数支持细胞富集基因受到影响。对照睾丸的免疫荧光分析显示，MEK1/2的下游靶点ERK1/2在内皮细胞和支持细胞中被磷酸化。抑制MEK1/2可消除胎儿睾丸中的pERK1/2，并且CD31、VE-钙粘蛋白、SOX7和SOX17以及内皮细胞消失。与VEGF在驱动睾丸内皮细胞发育中的作用一致，抑制VEGFR也可消除pERK1/2以及表达SOX7和SOX17的内皮细胞。此外，虽然抑制MEK1/2会破坏支持细胞的增殖及其在睾丸索基底膜的定位，但VEGFR抑制对其没有影响。相反，抑制FGF信号通路会损害支持细胞的增殖及其在睾丸索基底膜的定位。
总之，我们的数据突出了VEGF依赖性MEK1/2信号通路在促进血管生成中的重要作用，并表明通过MEK1/2的FGF信号通路调节发育中小鼠睾丸中支持细胞的组织。
