Shimizu Takashi, Jayawardana Barana C, Tetsuka Masafumi, Miyamoto Akio
Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Japan.
J Reprod Dev. 2007 Feb;53(1):105-12. doi: 10.1262/jrd.18088. Epub 2006 Oct 14.
Vascular endothelial growth factor (VEGF) isoforms (VEGF 120 and VEGF 164) secreted by granulosa cells are involved in thecal angiogenesis during follicular development in the bovine ovary. However, whether the transcript of the VEGF120 and VEGF164 isoforms differs during follicular development in the ovary is still unknown. We first examined the gene expression of VEGF120, VEGF164, fms-like tyrosine kinase (Flt-1), and fetal liver kinase (Flk-1) in the granulosa cells (GCs) and theca cells (TCs) of pre-selection and post-selection follicles (PRF and POF respectively) from the bovine ovary. Then we examined the effects of FSH and estradiol (E2) on these factors in cultured bovine GCs. Messenger RNA (mRNA) expression was quantified using real-time PCR methods. The concentrations of E2 and P4 in the follicular fluid (FF) of the PRF and POF were estimated using an enzyme immunoassay (EIA). The concentrations of E2 and P4 in the FF were significantly higher in the POF than in the PRF. The ratio of E2/P4 in PRF and POF was 0.37 and 3.8, respectively. The expression levels of the VEGF120, VEGF164, and Flk-1 mRNAs in the GCs of POF with high E2 concentration were higher than those of PRF. The levels of the Flt-1 and Flk-1 mRNAs in the TCs were not different between PRF and POF. Since E2 in the FF of the POF used in the present study was high compared with the PRF, we examined the effects of E2 and FSH on the expression of the above genes using cultured GCs. Expression of VEGF120 mRNA was induced by a low concentration (1 ng/ml) of E2, whereas the levels of VEGF164 and Flk-1 mRNAs were not affected by E2. FSH stimulated the expression of the VEGF isoforms and Flk-1 genes. Moreover, the expression of those genes was enhanced when low E2 (1 ng/ml) was added to FSH. In conclusion, our data indicates that the VEGF isoforms have a follicle stage-dependent expression pattern. Thus, our results suggest that the expression of VEGF isoforms may be associated with characterization of the preovulatory phenotype during follicle development in the bovine ovary.
颗粒细胞分泌的血管内皮生长因子（VEGF）亚型（VEGF 120和VEGF 164）参与牛卵巢卵泡发育过程中的卵泡膜血管生成。然而，VEGF120和VEGF164亚型的转录本在卵巢卵泡发育过程中是否存在差异仍不清楚。我们首先检测了牛卵巢中预选卵泡（PRF）和排卵前卵泡（POF）的颗粒细胞（GCs）和卵泡膜细胞（TCs）中VEGF120、VEGF164、fms样酪氨酸激酶（Flt-1）和胎儿肝激酶（Flk-1）的基因表达。然后我们检测了促卵泡激素（FSH）和雌二醇（E2）对培养的牛颗粒细胞中这些因子的影响。使用实时PCR方法定量信使核糖核酸（mRNA）表达。使用酶免疫测定（EIA）估算PRF和POF卵泡液（FF）中E2和孕酮（P4）的浓度。POF卵泡液中E2和P4的浓度显著高于PRF。PRF和POF中E2/P4的比值分别为0.37和3.8。E2浓度高的POF颗粒细胞中VEGF120、VEGF164和Flk-1 mRNA的表达水平高于PRF。PRF和POF卵泡膜细胞中Flt-1和Flk-1 mRNA的水平没有差异。由于本研究中使用的POF卵泡液中的E2比PRF高，我们使用培养的颗粒细胞检测了E2和FSH对上述基因表达的影响。低浓度（1 ng/ml）的E2诱导VEGF120 mRNA的表达，而VEGF164和Flk-1 mRNA的水平不受E2影响。FSH刺激VEGF亚型和Flk-1基因的表达。此外，当向FSH中添加低浓度E2（1 ng/ml）时，这些基因的表达增强。总之，我们的数据表明VEGF亚型具有卵泡阶段依赖性表达模式。因此，我们的结果表明VEGF亚型的表达可能与牛卵巢卵泡发育过程中排卵前表型的特征有关。
