Ohleth K M, Bagnell C A
Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08903, USA.
Biol Reprod. 1995 Dec;53(6):1286-92. doi: 10.1095/biolreprod53.6.1286.
Relaxin stimulates in vitro DNA synthesis and cell proliferation of porcine granulosa cells (GC) and theca cells. The objective of the study reported here was to determine whether components of the ovarian insulin-like growth factor (IGF) system mediate relaxin's growth-promoting effects on porcine GC in vitro. In small follicle GC, relaxin (1-100 ng/ml) significantly (p < 0.05) increased IGF-I secretion to 25-34% above control. Hormonal responsiveness of GC was shown by incubation with FSH (200 ng/ml), which resulted in 125% stimulation of IGF-I secretion relative to that in cells incubated alone. When IGF-I activity in the GC cultures was neutralized with a specific IGF-I antibody, relaxin (10 and 100 ng/ml)-induced [3H]thymidine incorporation was inhibited (p < 0.05). Coincubation with IGF-I antibody also suppressed basal and IGF-I (10 ng/ml)-induced [3H]thymidine incorporation into GC DNA, but had no effect on insulin (1 microgram/ml)-induced DNA synthesis, demonstrating the specificity and lack of toxicity of the IGF-I antibody. Ligand blot analysis showed no change in secretion of GC IGF binding protein (IGFBP) in response to relaxin (1, 10, and 100 ng/ml). In contrast, IGF-I (10 ng/ml) increased secretion of IGFBP-3 and -5, whereas FSH (200 ng/ml) decreased IGFBP-3 secretion and increased IGFBP-4 secretion (p < 0.05). In IGF-I receptor competition studies, IGF-I, but not relaxin, displaced [125I]IGF-I from the GC IGF-I receptor. These studies provide direct evidence for an interaction of relaxin and the ovarian IGF system. They are the first to show 1) a stimulatory effect of relaxin on IGF-I secretion; 2) the necessity of IGF-I activity for relaxin-induced GC DNA synthesis; and 3) the absence of an effect of relaxin on GC IGFBPs or IGF-I receptor. These findings support a paracrine role for relaxin in the porcine follicle and show that relaxin acts indirectly to promote follicle growth by stimulating GC IGF-I secretion.
松弛素可刺激猪颗粒细胞（GC）和卵泡膜细胞的体外DNA合成及细胞增殖。本文报道的这项研究的目的是确定卵巢胰岛素样生长因子（IGF）系统的成分是否介导松弛素对猪颗粒细胞的促生长作用。在小卵泡颗粒细胞中，松弛素（1 - 100 ng/ml）可使IGF - I分泌显著增加（p < 0.05），比对照组高出25% - 34%。通过与促卵泡激素（FSH，200 ng/ml）共同孵育显示颗粒细胞具有激素反应性，相对于单独孵育的细胞，其IGF - I分泌受到125%的刺激。当用特异性IGF - I抗体中和颗粒细胞培养物中的IGF - I活性时，松弛素（10和100 ng/ml）诱导的[3H]胸腺嘧啶核苷掺入受到抑制（p < 0.05）。与IGF - I抗体共同孵育也抑制了基础状态及IGF - I（10 ng/ml）诱导的[3H]胸腺嘧啶核苷掺入颗粒细胞DNA，但对胰岛素（1 μg/ml）诱导的DNA合成无影响，这证明了IGF - I抗体的特异性和无毒性。配体印迹分析显示，颗粒细胞IGF结合蛋白（IGFBP）的分泌对松弛素（1、10和1 ng/ml）无反应。相反，IGF - I（10 ng/ml）可增加IGFBP - 3和 - 5的分泌，而FSH（200 ng/ml）可降低IGFBP - 3的分泌并增加IGFBP - 4的分泌（p < 0.05）。在IGF - I受体竞争研究中，IGF - I而非松弛素能将[125I]IGF - I从颗粒细胞IGF - I受体上置换下来。这些研究为松弛素与卵巢IGF系统的相互作用提供了直接证据。它们首次表明：1）松弛素对IGF - I分泌具有刺激作用；2）IGF - I活性对于松弛素诱导的颗粒细胞DNA合成是必需的；3）松弛素对颗粒细胞IGFBP或IGF - I受体无影响。这些发现支持了松弛素在猪卵泡中的旁分泌作用，并表明松弛素通过刺激颗粒细胞IGF - I分泌间接促进卵泡生长。
