FSH和17β-雌二醇联合作用对仔猪睾丸支持细胞Skp2表达的调节

Effect of Combination of FSH and 17β-estradiol on the Expression of Skp2 in Cultured Immature Boar Sertoli Cell

【目的】确定FSH和雌激素联合作用是否可通过ERK1/2级联调节培养条件下未成熟仔猪睾丸支持细胞中Skp2的表达。【方法】以培养的仔猪睾丸支持细胞为试验材料,通过添加各种信号通路的抑制剂,应用Western blotting和实时荧光定量PCR检测Skp2蛋白、mRNA的表达。【结果】FSH(50 ng.mL-1)和17β-雌二醇(10-9 mol.L-1)联合作用时以时间依赖的方式促进了Skp2蛋白和mRNA的表达(P<0.05),这一作用在30 min时到达高峰;FSH(50 ng.mL-1)、17β-雌二醇(10-9 mol.L-1)和forskolin单独作用均促进了Skp2蛋白和mRNA的表达(P<0.05),FSH(50 ng.mL-1)和17β-雌二醇(10-9 mol.L-1)联合作用对Skp2表达的影响与FSH或17β-雌二醇(10-9 mol.L-1)单独作用无显著差异(P≥0.05),而环磷酸腺苷抑制剂(Rp-cAMP)、蛋白激酶A(PKA)抑制剂(H-89)、L-Ca2+离子通道抑制剂(verapamil)和ERK1/2抑制剂(U0126)单独作用时对Skp2蛋白和mRNA的表达与空白对照相比无显著影响(P>0.05),但都抑制了FSH(50 ng.mL-1)与17β-雌二醇(10-9 mol.L-1)联合作用对Skp2蛋白和mRNA表达的影响(P<0.05)。H-89、verapamil单独作用对ERK1/2(细胞外信号调节的蛋白激酶1/2)活性没有影响,但降低了FSH(50 ng.mL-1)和17β-雌二醇(10-9 mol.L-1)联合作用对ERK1/2活性的影响。【结论】FSH与17β-雌二醇联合作用激活了cAMP-PKA级联和Ca2+内流,而PKA和Ca2+内流又通过影响ERK1/2的活性进而影响Skp2的表达。

【Objective】 The objective of the study was to identify whether the combination of FSH and 17β-estradiol regulates the expression of S-Phase kinase-associated proteins2（Skp2） in cultured immature boar sertoli cell via extracellular signal-regulated kinase1/2（ERK1/2）.【Method】The cultured immature boar sertoli cells were treated with the combination of 17β-estradiol（10-9 mol·L-1） and FSH（50 ng·mL-1）,in addition to a variety of signaling pathway inhibitors and Western blotting,Real-time PCR was applied to detect the expression of Skp2 protein and mRNA,respectively.【Result】Treatment with combined FSH and 17β-estradiol increased the concentration and mRNA abundance of Skp2 from 15 mins to 90 mins,and these activities peaked at 30 mins in comparison to the controls（P0.05）.FSH（50 ng·mL-1）,17β-estradiol（10-9 mol·L-1） and forskolin alone increased the amount of Skp2 protein and the abundance of Skp2 mRNA（P≥0.05 for all）.Rp-cAMP（cAMP inhibitor）,H-89（protein kinase A inhibitor）,verapamil（L-type Ca2＋ ionic channel inhibitor） and U0126（ERK1/2 inhibitor） impaired the effect of combined FSH and 17β-estradiol on the expression of Skp2 in both protein and mRNA levels（P0.05）,however,no significant effects were found on the expression of Skp2 in both protein and mRNA levels when they were tested alone.Likewise,there was no significant effects on activity of ERK1/2 by using H-89 and verapamil alone,but the activity of ERK1/2 was impacted by combination of FSH 17β-estradiol,H-89 and verapamil（P0.05）.【Conclusion】cAMP-PKA pathway and Ca2＋influx are activated by the combination of FSH and 17β-estradiol,and both of them are associated with ERK1/2 activation,which regulates the expression of Skp2.