Uzumcu Mehmet, Westfall Suzanne D, Dirks Kristen A, Skinner Michael K
Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman 99164-4231, USA.
Biol Reprod. 2002 Dec;67(6):1927-35. doi: 10.1095/biolreprod.102.006254.
Mesonephric cell migration and seminiferous cord formation are critical processes in embryonic testis development at the time of male sex determination. Extracellular growth factors shown to influence seminiferous cord formation such as neurotropin-3 utilize in part the phosphotidylinositol 3-kinase (PI3K) signal transduction pathway. The current study investigates the hypothesis that the PI3K pathway is critical in seminiferous cord formation and testis development. The role of the PI3K signaling pathway in testicular cord formation was examined using an Embryonic Day 13 organ culture system and a PI3K-specific inhibitor LY294002. The actions of a mitogen-activated protein (MAP) kinase-specific inhibitor PD98059 was also examined. The PI3K inhibitor blocked cord formation or reduced the number of cords in a concentration-dependent manner. The actions of LY294002 were found to have a developmental stage specificity in that cord inhibition was observed in organs from embryos with 16-17 tail somites, while organs from embryos with 19 or more tail somites had no block in cord formation and only a small reduction in cord number. In contrast, the MAP kinase inhibitor PD98059 did not block cord formation and only caused a slight reduction in cord number. Neither PI3K or MAP kinase inhibitor altered apoptotic cell number, suggesting apoptosis was not the reason for the inhibition of cord formation. Embryonic testis cell migration assays showed that the PI3K inhibitor LY294002 blocked mesonephros cell migration into the testis, while the MAP kinase inhibitor had no effect. Observations suggest the interference of cell migration is the cause for the inhibition of cord formation. Western blot analysis confirmed that LY294002 and PD98509 inhibited phosphorylation of Akt and ERK1/ERK2, respectively. Combined observations demonstrate that the PI3K signaling pathway is involved in embryonic testis cord formation and mesonephros cell migration.
在雄性性别决定时期，中肾细胞迁移和生精索形成是胚胎睾丸发育过程中的关键步骤。已证实，诸如神经营养蛋白-3等影响生精索形成的细胞外生长因子部分通过磷脂酰肌醇3激酶（PI3K）信号转导途径发挥作用。本研究旨在探讨PI3K途径在生精索形成和睾丸发育中起关键作用这一假说。利用胚胎第13天的器官培养系统和PI3K特异性抑制剂LY294002，研究了PI3K信号通路在睾丸索形成中的作用。同时也检测了丝裂原活化蛋白（MAP）激酶特异性抑制剂PD98059的作用。PI3K抑制剂以浓度依赖的方式阻断索形成或减少索的数量。发现LY294002的作用具有发育阶段特异性，即16-17个尾节胚胎的器官中观察到索形成受到抑制，而19个或更多尾节胚胎的器官中索形成未受阻断，仅索数量略有减少。相比之下，MAP激酶抑制剂PD98059未阻断索形成，仅使索数量略有减少。PI3K或MAP激酶抑制剂均未改变凋亡细胞数量，表明凋亡不是索形成受抑制的原因。胚胎睾丸细胞迁移试验表明，PI3K抑制剂LY294002阻断了中肾细胞向睾丸的迁移，而MAP激酶抑制剂则无此作用。观察结果表明，细胞迁移的干扰是索形成受抑制的原因。蛋白质免疫印迹分析证实，LY294002和PD98509分别抑制了Akt和ERK1/ERK2的磷酸化。综合观察结果表明，PI3K信号通路参与胚胎睾丸索形成和中肾细胞迁移。
