R-Spondin 1/Dickkopf-1/Beta-Catenin Machinery Is Involved in Testicular Embryonic Angiogenesis

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today’s most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors’ observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis.

睾丸血管生成是调控雄性性腺形态发生的关键过程之一。阐明该现象背后的分子调控机制，是当前领域内极具挑战性的研究课题，同时也可为深入理解睾丸形态发生异常的发病机制提供重要理论支撑。R-spondin 1（R-spondin 1）已被明确认定为哺乳动物卵巢发育决定的候选因子。与之相对，目前关于R-spondin 1在睾丸形态发生过程中的表达模式与生物学功能的研究信息仍较为匮乏。本研究旨在明确R-spondin 1及其调控通路的其他相关因子在睾丸形态发生全周期中的分布特征，并阐明该信号系统在睾丸发育中的作用。我们的整体免疫荧光染色结果显示，R-spondin 1始终在睾丸体腔分区——即睾丸血管组织构建的区域——中可被检测到，而Dickkopf-1（Dickkopf-1）在该区域中无表达。此外，胚胎雄性泌尿生殖嵴的器官培养实验证实，Dickkopf-1可作为睾丸血管生成的抑制剂。与之相符的是，实时荧光定量PCR分析结果显示，DKK1能够小幅但显著地下调内皮标志物Pecam1的表达水平。后续实验进一步发现，DKK1处理还会干扰血小板衍生生长因子B链（Pdgf-b chain）的表达水平，这与已有研究中该因子与产前睾丸模式形成及血管生成相关的结论一致。值得注意的是，联合施用R-spondin 1可挽救DKK1介导的睾丸血管生成抑制效应；同时，单独施用R-spondin 1即可在培养体系中增强睾丸血管生成。