Meroni S B, Riera M F, Pellizzari E H, Galardo M N, Cigorraga S B
Centro de Investigaciones Endocrinológicas (CEDIE), Hospital de Niños 'R. Gutiérrez', Gallo 1330, 1425 Buenos Aires, Argentina.
J Endocrinol. 2004 Feb;180(2):257-65. doi: 10.1677/joe.0.1800257.
The gonadotropin FSH plays a key role in the control of Sertoli cell function. The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway. However, other signaling events have also been demonstrated in Sertoli cells. We have recently presented evidence that FSH can stimulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathway in 20-day-old Sertoli cells. At the same time, it was proposed that in 8-day-old Sertoli cells the effects of FSH on phosphorylated PKB (P-PKB) levels can be explained by a combination of increased secretion of endogenous IGF-I, decreased IGF-binding protein-3 (IGFBP-3) production, and a synergistic action of FSH on IGF-I-dependent PI3K activation. The aim of the present study was to determine whether the effect of FSH on 20-day-old Sertoli cells is mediated by IGF-I secretion. Twenty-day-old rat Sertoli cell cultures were used. FSH stimulation produced a time-dependent increment in P-PKB levels reaching maximal values in 60-min incubations. IGF-I stimulation was also time-dependent reaching maximal values in 15-min incubations. On the other hand, stimulation of the cultures with FSH showed time-dependent inhibition in phosphorylated mitogen-activated protein kinase (P-MAPK) levels. In sharp contrast, stimulation of the cultures with IGF-I showed time-dependent increments in P-MAPK levels reaching maximal stimulus in 15-min incubations. In order to rule out an IGF-I action on FSH stimulation of P-PKB levels, the effect of a specific IGF-I antibody on the ability of both hormones to increase P-PKB levels was evaluated. As expected, the antibody inhibited IGF-I stimulation of P-PKB levels. However, simultaneous addition of an IGF-I antibody with FSH did not modify the ability of the hormone to increase P-PKB levels. The next set of experiments intended to analyze the relevance of a PI3K/PKB pathway to two biological responses of Sertoli cells to FSH and IGF-I. The PI3K inhibitor, wortmannin, dose-dependently decreased FSH-stimulated lactate and transferrin production. On the other hand, wortmannin was not able to modify the ability of IGF-I to stimulate these metabolic events. In addition, the analysis of the participation of a MAPK pathway in IGF-I regulation of Sertoli cell biological responses showed that the MAPK kinase inhibitors, PD98059 and U0126, decreased IGF-I-stimulated transferrin secretion while not modifying IGF-I-stimulated lactate levels. In summary, results obtained so far support the hypothesis that FSH action on P-PKB levels and Sertoli cell metabolism in 20-day-old animals is not mediated by autocrine regulation of an IGF-I/ IGFBP-3 axis as previously proposed in 8-day-old Sertoli cells.
促性腺激素卵泡刺激素（FSH）在调控支持细胞功能方面发挥着关键作用。FSH的分子作用机制最为人熟知的是其对腺苷酸环化酶/cAMP途径的刺激作用。然而，在支持细胞中也证实了其他信号转导事件。我们最近提供的证据表明，FSH可刺激20日龄支持细胞中的磷脂酰肌醇3激酶/蛋白激酶B（PI3K/PKB）途径。同时，有人提出，在8日龄支持细胞中，FSH对磷酸化蛋白激酶B（P-PKB）水平的影响可通过内源性胰岛素样生长因子-I（IGF-I）分泌增加、胰岛素样生长因子结合蛋白-3（IGFBP-3）产生减少以及FSH对IGF-I依赖性PI3K激活的协同作用来解释。本研究的目的是确定FSH对20日龄支持细胞的作用是否由IGF-I分泌介导。使用了20日龄大鼠的支持细胞培养物。FSH刺激使P-PKB水平呈时间依赖性增加，在孵育60分钟时达到最大值。IGF-I刺激也呈时间依赖性，在孵育15分钟时达到最大值。另一方面，用FSH刺激培养物显示磷酸化丝裂原活化蛋白激酶（P-MAPK）水平呈时间依赖性抑制。形成鲜明对比的是，用IGF-I刺激培养物显示P-MAPK水平呈时间依赖性增加，在孵育15分钟时达到最大刺激。为了排除IGF-I对FSH刺激P-PKB水平的作用，评估了特异性IGF-I抗体对两种激素增加P-PKB水平能力的影响。正如预期的那样，该抗体抑制了IGF-I对P-PKB水平的刺激。然而，将IGF-I抗体与FSH同时添加并不会改变该激素增加P-PKB水平的能力。接下来的一组实验旨在分析PI3K/PKB途径与支持细胞对FSH和IGF-I的两种生物学反应的相关性。PI3K抑制剂渥曼青霉素剂量依赖性地降低了FSH刺激的乳酸和转铁蛋白产生。另一方面，渥曼青霉素无法改变IGF-I刺激这些代谢事件的能力。此外，对MAPK途径参与IGF-I对支持细胞生物学反应调节的分析表明，MAPK激酶抑制剂PD98059和U0126降低了IGF-I刺激的转铁蛋白分泌，而未改变IGF-I刺激的乳酸水平。总之，目前获得的结果支持以下假设：在20日龄动物中，FSH对P-PKB水平和支持细胞代谢的作用并非如先前在8日龄支持细胞中所提出的那样，由IGF-I/IGFBP-3轴的自分泌调节介导。
