Department of Animal and Food Sciences, Oklahoma State University, Stillwater, OK, 74078.
J Anim Sci. 2019 Jul 2;97(7):3034-3045. doi: 10.1093/jas/skz164.
Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis and is associated with increased vascularity in ovarian follicles of cattle. The objectives of this study were to investigate the developmental and hormonal regulation of VEGFA expression in ovarian granulosa and theca cells (TC) of cattle. Bovine ovaries were collected from a local slaughterhouse and granulosa cells (GC) and TC were collected from small (SM; 1 to 5 mm) and large (LG; 8 to 20 mm) follicles. Cells were collected fresh or cultured in serum-free medium and treated with various factors that regulate angiogenesis and follicular development. RNA was collected for analysis of VEGFA mRNA abundance via quantitative PCR. In SM-follicle GC (SMGC), prostaglandin E2 (PGE2) and FSH decreased (P < 0.05) VEGFA mRNA abundance by 30 to 46%, whereas in LG-follicle GC (LGGC), PGE2 and FSH were without effect (P > 0.10). In SMGC, dihydrotestosterone (DHT), sonic hedgehog (SHH), and growth differentiation factor-9 (GDF9) decreased (P < 0.05) VEGFA expression by 30 to 40%. Fibroblast growth factor-9 (FGF9) and estradiol (E2) were without effect (P > 0.10) on VEGFA mRNA in both SMGC and LGGC, whereas progesterone increased (P < 0.05) VEGFA mRNA in LGGC but had no effect in LGTC. Bone morphogenetic protein-4 (BMP4), LH, and FGF9 increased (P < 0.05) abundance of VEGFA mRNA by 1.5- to 1.9-fold in LGTC. Insulin-like growth factor-1 (IGF1) was without effect (P > 0.10) on VEGFA mRNA in both TC and GC. An E2F transcription factor inhibitor, HLM0064741 (E2Fi), dramatically (i.e., 8- to 13-fold) stimulated (P < 0.01) the expression of VEGFA mRNA expression in both SMGC and LGTC. Abundance of VEGFA mRNA was greater (P < 0.05) in LGGC and SMGC than in LGTC. Also, SMTC had greater (P < 0.05) abundance of VEGFA mRNA than LGTC. In conclusion, VEGFA mRNA abundance was greater in GC than TC, and VEGFA expression decreased in TC during follicle development. Some treatments either suppressed, stimulated, or had no effect on VEGFA expression depending on the cell type. The inhibition of E2F transcription factors had the greatest stimulatory effect of all treatments evaluated, and thus, E2Fs may play an important role in regulating angiogenesis during follicle growth in cattle.
血管内皮生长因子 A（VEGFA）刺激血管生成，并与牛卵巢卵泡中的血管生成增加有关。本研究的目的是研究 VEGFA 在牛卵巢颗粒细胞和卵泡膜细胞（TC）中的发育和激素调节。从当地屠宰场采集牛卵巢，从小（SM；1 至 5 毫米）和大（LG；8 至 20 毫米）卵泡中采集颗粒细胞（GC）和 TC。细胞新鲜采集或在无血清培养基中培养，并接受各种调节血管生成和卵泡发育的因子处理。通过定量 PCR 收集 RNA 以分析 VEGFA mRNA 丰度。在 SM 卵泡 GC（SMGC）中，前列腺素 E2（PGE2）和 FSH 降低（P <0.05）VEGFA mRNA 丰度 30%至 46%，而在 LG 卵泡 GC（LGGC）中，PGE2 和 FSH 无影响（P >0.10）。在 SMGC 中，二氢睾酮（DHT）、声 hedgehog（SHH）和生长分化因子-9（GDF9）降低（P <0.05）VEGFA 表达 30%至 40%。成纤维细胞生长因子-9（FGF9）和雌二醇（E2）对 SMGC 和 LGGC 中的 VEGFA mRNA 均无影响（P >0.10），而孕激素增加（P <0.05）LGGC 中的 VEGFA mRNA，但对 LGTC 无影响。骨形态发生蛋白-4（BMP4）、LH 和 FGF9 使 LGTC 中的 VEGFA mRNA 丰度增加 1.5 至 1.9 倍（P <0.05）。胰岛素样生长因子-1（IGF1）对 TC 和 GC 中的 VEGFA mRNA 均无影响（P >0.10）。E2F 转录因子抑制剂 HLM0064741（E2Fi）显著（即 8 至 13 倍）刺激（P <0.01）SMGC 和 LGTC 中 VEGFA mRNA 的表达。VEGFA mRNA 的丰度在 LGGC 和 SMGC 中均高于 LGTC（P <0.05）。此外，SMTC 中的 VEGFA mRNA 丰度高于 LGTC（P <0.05）。总之，GC 中的 VEGFA mRNA 丰度高于 TC，并且在卵泡发育过程中 TC 中的 VEGFA 表达降低。某些处理方法根据细胞类型抑制、刺激或对 VEGFA 表达没有影响。所有评估的处理方法中，E2F 转录因子的抑制作用具有最大的刺激作用，因此，E2F 可能在牛卵泡生长过程中的血管生成调节中发挥重要作用。
