Ovarian paracrine mediation by components of the wingless-type mouse mammary tumor virus integration site ligands (WNT1 to 11) and their receptors, frizzled family members (FZD1 to 10), has been proposed. Secreted truncated forms of FZD proteins (e.g., secreted frizzled-related protein 4 [SFRP4]) block the action of WNT ligands. Dickkopf-1 (DKK1) is another WNT antagonist, and R-spondin-1 (RSPO1) is one of a group of four secreted proteins that enhance WNT/β-catenin signaling. Our hypothesis was that granulosa cells signal theca cells (TCs) via SFRP4, DKK1, RSPO1, and WNT secretion to regulate TC differentiation and proliferation. Therefore, in vitro experiments were conducted to study the effects of WNT family member 3A (WNT3A), WNT5A, RSPO1, DKK1, insulin-like growth factor 1 (IGF1), bone morphogenetic protein 7 (BMP7), Indian hedgehog (IHH), and fibroblast growth factor 9 (FGF9) on bovine TC proliferation and steroidogenesis. TCs of large (8 to 20 mm) and small (3 to 6 mm) follicles were collected from bovine ovaries; TC monolayers were established in vitro and treated with various doses of recombinant human WNT3A, WNT5A, RSPO1, DKK1, IGF1, FGF9, BMP7, IHH, and/or ovine luteinizing hormone (LH) in serum-free medium for 48 h. In experiment 1, using LH-treated TC, IGF1, IHH, and WNT3A increased (P < 0.05) cell numbers and androstenedione production, whereas WNT3A and BMP7 inhibited (P < 0.05) progesterone production. In experiment 2, FGF9 blocked (P < 0.05) the WNT3A-induced increase in androstenedione production in LH plus IGF1-treated TC. In experiment 3, RSPO1 further increased (P < 0.05) LH plus IGF1-induced progesterone and androstenedione production. In experiment 4, SFRP4 and DKK1 alone had no significant effect on TC proliferation or progesterone production of large-follicle TC but both blocked the inhibitory effect of WNT5A on androstenedione production. In contrast, DKK1 alone inhibited (P < 0.05) small-follicle TC androstenedione production whereas SFRP4 was without effect. We conclude that the ovarian TC WNT system is functional in cattle, with WNT3A increasing proliferation and androstenedione production of TC.

中文翻译：

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无翼型小鼠乳腺肿瘤病毒整合位点对牛膜细胞的调控

已经提出通过无翼型小鼠乳腺肿瘤病毒整合位点配体 (WNT1 至 11) 及其受体、卷曲家族成员 (FZD1 至 10) 的组分介导卵巢旁分泌。分泌的截短形式的 FZD 蛋白（例如分泌的卷曲相关蛋白 4 [SFRP4]）阻断 WNT 配体的作用。Dickkopf-1 (DKK1) 是另一种 WNT 拮抗剂，而 R-spondin-1 (RSPO1) 是增强 WNT/β-catenin 信号传导的四种分泌蛋白之一。我们的假设是颗粒细胞通过 SFRP4、DKK1、RSPO1 和 WNT 分泌向膜细胞 (TC) 发出信号，以调节 TC 的分化和增殖。因此，进行了体外实验研究WNT家族成员3A（WNT3A）、WNT5A、RSPO1、DKK1、胰岛素样生长因子1（IGF1）、骨形态发生蛋白7（BMP7）、印度刺猬（IHH）、和成纤维细胞生长因子 9 (FGF9) 对牛 TC 增殖和类固醇生成的影响。从牛卵巢中收集大（8 至 20 毫米）和小（3 至 6 毫米）卵泡的 TC；TC 单层在体外建立，并在无血清培养基中用不同剂量的重组人 WNT3A、WNT5A、RSPO1、DKK1、IGF1、FGF9、BMP7、IHH 和/或羊黄体生成素 (LH) 处理 48 小时。在实验 1 中，使用 LH 处理的 TC，IGF1、IHH 和 WNT3A 增加（P < 0.05）细胞数量和雄烯二酮的产生，而 WNT3A 和 BMP7 抑制（P < 0.05）孕酮的产生。在实验 2 中，FGF9 阻断了 (P < 0.05) WNT3A 诱导的 LH 加 IGF1 处理的 TC 中雄烯二酮产生的增加。在实验 3 中，RSPO1 进一步增加（P < 0.05）LH 加 IGF1 诱导的孕酮和雄烯二酮的产生。在实验4中，SFRP4和DKK1单独对TC增殖或大卵泡TC的孕酮产生没有显着影响，但都阻断了WNT5A对雄烯二酮产生的抑制作用。相比之下，单独的 DKK1 抑制 (P < 0.05) 小卵泡 TC 雄烯二酮的产生，而 SFRP4 没有效果。我们得出结论，卵巢 TC WNT 系统在牛中是有功能的，WNT3A 增加了 TC 的增殖和雄烯二酮的产生。