Production of insulin-like growth factor binding proteins (IGFBPs) by porcine granulosa cells: identification of IGFBP-2 and -3 and regulation by hormones and growth factors.
Production of insulin-like growth factor binding proteins (IGFBPs) by porcine granulosa cells: identification of IGFBP-2 and -3 and regulation by hormones and growth factors.
复制标题
DOI：
10.1210/endo-127-5-2298
复制
发表时间：
1990-11
期刊：
影响因子：
4.8
通讯作者：
J. Mondschein;Sheila A. Smith;J. Hammond
中科院分区：
文献类型：
--
作者：
J. Mondschein;Sheila A. Smith;J. Hammond
使用配体印迹和免疫沉淀，我们对培养的猪颗粒细胞产生的胰岛素样生长因子结合蛋白（IGFBP）进行了表征。颗粒细胞条件培养基的配体印迹分析揭示了 5 个 IGF 结合活性条带，表观分子大小分别为 44、40、34、29 和 22 千道尔顿 (kDa)。通过用猪 IGFBP-3（150 kDa GH 依赖性血清 IGFBP 复合物的酸稳定亚基）抗体进行免疫沉淀来鉴定颗粒条件培养基的 40-44 kDa 带。 34 kDa 带通过大鼠 IGFBP-2 抗体进行免疫沉淀，IGFBP-2 是胎鼠血清和 BRL-3A 细胞培养物中发现的主要 IGFBP。迄今为止，我们还无法使用任何测试的抗体（包括一组针对人 IGFBP-1（羊水 IGFBP）的单克隆抗体）对 29 和 22 kDa 条带进行免疫沉淀。分泌模式随获得颗粒细胞的卵泡的大小和培养条件而变化。对于来自小（2-4 mm）卵泡的细胞，短期培养物主要分泌 IGFBP-3 和 IGFBP-2，而 IGFBP-2 以及 29 和 22 kDa 条带在长期培养物中明显。在短期培养中，来自中等大小（4-6毫米）猪卵泡的颗粒细胞产生的 IGFBP 数量明显高于来自小型（1-3毫米）卵泡的颗粒细胞，但表现出类似的条带模式。 IGFBP 的产生受到放线菌酮的抑制。培养物中颗粒细胞产生的 IGFBP 受激素和生长因子的调节。最显着的作用是通过转化生长因子β和FSH抑制IGFBP-3的分泌。相反，表皮生长因子可增强 IGFBP-3 水平。培养的猪颗粒细胞产生的 IGFBP 在大小和免疫反应性方面与之前在猪卵泡液中发现的 IGFBP 相同。因此，滤泡细胞可能是滤泡液IGFBP的来源。 IGFBP 可能是卵巢中 IGF 自分泌/旁分泌系统的重要调节剂。
Using ligand blotting and immunoprecipitation we have characterized the insulin like growth factor binding proteins (IGFBPs) produced by cultured porcine granulosa cells. Ligand blot analysis of granulosa cell conditioned medium revealed 5 bands of IGF binding activity with apparent molecular sizes of 44, 40, 34, 29, and 22 kilodaltons (kDa). The 40-44 kDa bands of granulosa- conditioned medium were identified by immunoprecipitation with an antibody to porcine IGFBP-3, the acid-stable subunit of the 150 kDa GH-dependent serum IGFBP complex. The 34 kDa band was immunoprecipitated by an antibody to the rat IGFBP-2, the major IGFBP found in fetal rat serum and in BRL-3A cell cultures. To date we have been unable to immunoprecipitate the 29 and 22 kDa bands with any of the antibodies tested including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGFBP. The pattern of secretion varied with size of the follicles from which granulosa cells were obtained and the culture conditions. With cells from small (2-4 mm) follicles, short term cultures secreted mainly IGFBP-3 and IGFBP-2, while IGFBP-2 and the 29 and 22 kDa bands were pronounced in longer term cultures. In short term culture, granulosa cells from medium sized (4-6 mm) porcine follicles produced IGFBPs in substantially greater amounts than did those from small (1-3 mm) follicles, but exhibited comparable band patterns. The production of IGFBPs was inhibited by cycloheximide. IGFBP production by granulosa cells in culture was regulated by hormones and growth factors. The most striking effects were the inhibition of IGFBP-3 secretion by transforming growth factor beta and FSH. In contrast, IGFBP-3 levels were enhanced by epidermal growth factor. The IGFBPs produced by cultured porcine granulosa cells are identical in size and immunoreactivity to those previously found in porcine follicular fluid. Thus, follicular cells may be the source of follicular fluid IGFBPs. The IGFBPs may be important modulators of the IGF autocrine/paracrine system in the ovary.