Abstract：
BACKGROUND: Testicular hypoplasia can affect the sexual and reproductive ability in adulthood, and even increase the risk of cancer. Abnormal development of the gubernaculum is one of the important factors of testicular hypoplasia. Therefore, a study of the structure and function of the gubernaculum is an important but neglected new breakthrough point for investigating the normal/abnormal development of the testis. Previous findings showed that Insulin like factor 3 (INSL3) is a key factor regulating the growth of gubernaculum, however, the mechanism by which INSL3 acts on the gubernaculum remains unknown. Therefore, we probed the mechanism associated with INSL3-induced the proliferation, migration, and apoptosis of gubernacular cells in mice.
METHODS: A culture cell model of neonatal mice gubernaculum is established by INSL3 intervention. We blocked PLC/PKC signaling pathway with U73122 pretreat to investigate the role of the PLC/PKC signaling pathway. The changes of cell proliferation, migration, and apoptosis were detected by molecular biological methods. In addition, the levels of PCNA and F-action were detected by immunofluorescence and western blotting.
RESULTS: We found that INSL3 can promote the proliferation and migration of gubernacular cells and inhibit their apoptosis, meanwhile, INSL3 significantly up-regulated PLC/PKC protein phosphorylation. However, treatment with the PLC/PKC signaling pathway inhibitor U73122 significantly inhibited these effects of INSL3. Besides, we found that INSL3 could up-regulate the protein expression level of PCNA and F-actin, while the PCNA and F-actin expression was significantly weakened after U73122 pretreatment.
CONCLUSIONS: This research revealed that INSL3 binding to RXFP2 may up-regulate the expression levels of PCNA and F-actin by activating the PLC/PKC signaling pathway to promote the proliferation and migration of gubernacular cells. It suggests that the RXFP2-PLC/PKC axis may serve as a novel molecular mechanism by which INSL3 regulates growth of the gubernaculum.
METHODS: A culture cell model of neonatal mice gubernaculum is established by INSL3 intervention. We blocked PLC/PKC signaling pathway with U73122 pretreat to investigate the role of the PLC/PKC signaling pathway. The changes of cell proliferation, migration, and apoptosis were detected by molecular biological methods. In addition, the levels of PCNA and F-action were detected by immunofluorescence and western blotting.
RESULTS: We found that INSL3 can promote the proliferation and migration of gubernacular cells and inhibit their apoptosis, meanwhile, INSL3 significantly up-regulated PLC/PKC protein phosphorylation. However, treatment with the PLC/PKC signaling pathway inhibitor U73122 significantly inhibited these effects of INSL3. Besides, we found that INSL3 could up-regulate the protein expression level of PCNA and F-actin, while the PCNA and F-actin expression was significantly weakened after U73122 pretreatment.
CONCLUSIONS: This research revealed that INSL3 binding to RXFP2 may up-regulate the expression levels of PCNA and F-actin by activating the PLC/PKC signaling pathway to promote the proliferation and migration of gubernacular cells. It suggests that the RXFP2-PLC/PKC axis may serve as a novel molecular mechanism by which INSL3 regulates growth of the gubernaculum.
摘要：
背景：睾丸发育不全会影响成年期的性和生殖能力，甚至增加患癌症的风险。葡萄胎的异常发育是睾丸发育不全的重要因素之一。因此,对gubernaculum的结构和功能的研究是研究睾丸正常/异常发育的重要但被忽视的新突破口。先前的研究结果表明，胰岛素样因子3（INSL3）是调节葡萄膜生长的关键因素，然而,INSL3对Gubernaculum的作用机制尚不清楚.因此,我们探讨了与INSL3诱导的增殖相关的机制，迁移,和小鼠杯状细胞的凋亡。
方法：通过INSL3干预建立新生小鼠gubernaculum培养细胞模型。我们用U73122预处理阻断PLC/PKC信号通路，以研究PLC/PKC信号通路的作用。细胞增殖的变化，迁移,用分子生物学方法检测细胞凋亡。此外,通过免疫荧光和蛋白质印迹法检测PCNA和F-action的水平。
结果：我们发现INSL3能促进银耳细胞的增殖和迁移，抑制其凋亡，同时,INSL3显著上调PLC/PKC蛋白磷酸化。然而,用PLC/PKC信号通路抑制剂U73122处理显著抑制了INSL3的这些作用。此外,我们发现INSL3可以上调PCNA和F-actin的蛋白表达水平,U73122预处理后PCNA和F-actin表达明显减弱。
结论：本研究发现，INSL3与RXFP2的结合可能通过激活PLC/PKC信号通路上调PCNA和F-actin的表达水平，从而促进银耳细胞的增殖和迁移。这表明RXFP2-PLC/PKC轴可能是INSL3调节gebernaculum生长的新型分子机制。
方法：通过INSL3干预建立新生小鼠gubernaculum培养细胞模型。
结果：我们发现INSL3能促进银耳细胞的增殖和迁移，抑制其凋亡，
结论：本研究发现，INSL3与RXFP2的结合可能通过激活PLC/PKC信号通路上调PCNA和F-actin的表达水平，从而促进银耳细胞的增殖和迁移。
