Orna Therapeutics, Cambridge, MA 02139, USA.
Department of Animal and Veterinary Sciences, The University of Vermont, Burlington, VT 05405, USA.
J Anim Sci. 2022 Jul 1;100(7). doi: 10.1093/jas/skac105.
Intraovarian growth factors play a vital role in influencing the fate of ovarian follicles. They affect proliferation and apoptosis of granulosa cells (GC) and can influence whether small antral follicles continue their growth or undergo atresia. Transforming growth factor-alpha (TGFα), an oocyte-derived growth factor, is thought to regulate granulosa cell function; yet its investigation has been largely overshadowed by emerging interest in TGF-beta superfamily members, such as bone morphogenetic proteins (BMP) and anti-Mullerian hormone (AMH). Here, effects of TGFα on bovine GC proliferation, intracellular signaling, and cytokine-induced apoptosis were evaluated. Briefly, all small antral follicles (3-5 mm) from slaughterhouse specimens of bovine ovary pairs were aspirated and the cells were plated in T25 flasks containing DMEM/F12 medium, 10% FBS, and antibiotic-antimycotic, and incubated at 37 °C in 5% CO2 for 3 to 4 d. Once confluent, the cells were sub-cultured for experiments (in 96-, 12-, or 6-well plates) in serum-free conditions (DMEM/F12 medium with ITS). Exposure of the bGC to TGFα (10 or 100 ng/mL) for 24 h stimulated cell proliferation compared to control (P < 0.05; n = 7 ovary pairs). Proliferation was accompanied by a concomitant increase in mitogen-activated protein kinase (MAPK) signaling within 2 h of treatment, as evidenced by phosphorylated ERK1/2 expression (P < 0.05, n = 3 ovary pairs). These effects were entirely negated, however, by the MAPK inhibitor, U0126 (10uM, P < 0.05). Additionally, prior exposure of the bGC to TGFα (100 ng/mL) failed to prevent Fas Ligand (100 ng/mL)-induced apoptosis, as measured by caspase 3/7 activity (P < 0.05, n = 7 ovary pairs). Collectively, the results indicate TGFα stimulates proliferation of bGC from small antral follicles via a MAPK/ERK-mediated mechanism, but this action alone fails to prevent apoptosis, suggesting that TGFα may be incapable of promoting their persistence in follicles during the process of follicular selection/dominance.
卵巢内生长因子在影响卵泡命运方面起着至关重要的作用。它们影响颗粒细胞（GC）的增殖和凋亡，并影响小窦卵泡是继续生长还是发生闭锁。转化生长因子-α（TGFα）是一种卵母细胞衍生的生长因子，被认为调节颗粒细胞功能；然而，其研究在很大程度上被新兴的转化生长因子-β超家族成员（如骨形态发生蛋白（BMP）和抗苗勒管激素（AMH））的兴趣所掩盖。在这里，评估了 TGFα对牛 GC 增殖、细胞内信号传导和细胞因子诱导的凋亡的影响。简要地说，从屠宰场牛卵巢对的标本中抽吸所有小窦卵泡（3-5mm），并将细胞接种在含有 DMEM/F12 培养基、10% FBS 和抗生素抗真菌剂的 T25 培养瓶中，并在 37°C 下 5% CO2 中孵育 3 到 4 天。一旦汇合，将细胞在无血清条件下（含 ITS 的 DMEM/F12 培养基）进行亚培养用于实验（在 96 孔、12 孔或 6 孔板中）。与对照相比（P<0.05；n=7 对卵巢），TGFα（10 或 100ng/mL）孵育 24 小时刺激了 bGC 的细胞增殖。增殖伴随着处理后 2 小时内有丝分裂原激活的蛋白激酶（MAPK）信号的协同增加，表现为磷酸化 ERK1/2 表达（P<0.05，n=3 对卵巢）。然而，MAPK 抑制剂 U0126（10uM，P<0.05）完全消除了这些作用。此外，bGC 预先暴露于 TGFα（100ng/mL）并不能防止 Fas 配体（100ng/mL）诱导的凋亡，如 caspase 3/7 活性所测量的（P<0.05，n=7 对卵巢）。总之，这些结果表明，TGFα通过 MAPK/ERK 介导的机制刺激小窦卵泡 bGC 的增殖，但这种作用本身并不能防止凋亡，这表明 TGFα可能无法在卵泡选择/优势过程中促进它们在卵泡中的持续存在。
