Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India.
Anim Reprod Sci. 2013 Mar;137(3-4):163-76. doi: 10.1016/j.anireprosci.2013.01.004. Epub 2013 Jan 17.
The objective of the present study was to characterize the temporal patterns of gene expression for vascular endothelial growth factors (VEGF) and VEGF receptors during ovarian follicular growth, development and maturation in buffalo (Bubalus bubalis). Follicles were classified into four groups according to size and the concentration of estradiol-17β (E2) in follicular fluid (FF): Group I (small), 4-6mm diameter, E2>0.5ng/ml of FF; Group II (medium), 7-9mm, E2=0.5-5ng/ml; Group III (large), 10-13mm, E2=5-40ng/ml; Group IV(pre-ovulatory), >13mm, E2>180ng/ml). The mRNAs for FSH receptor (FSHR), LH receptor (LHR) and aromatase (CYP19A1) in theca interna and granulosa layers were also determined, further defining the maturational state of each group. The relative expression of VEGF isoforms (120, 164, and 188 amino acid forms), as determined by quantitative real-time PCR (qRT-PCR), increased during follicular development in both the granulosa (P<0.05) and theca layers. Relative amounts of VEGF receptors (VEGFR-1 and VEGFR-2) were least in granulosa cell (GC) and theca interna cell (TI) layers of Gp-I follicles. The amount of VEGFR-2 transcripts increased in the granulosa layer throughout development, reaching a maximum in Gp-IV follicles (P<0.05). The relative amount of VEGF isoforms and receptors in follicle lysates, as determined by western blotting, increased throughout follicular maturation to maximum amounts in pre-ovulatory follicles. Immunohistochemistry revealed a clear localization of VEGF isoforms and receptors in both steroidogenic cell types (GC and TI) and of VEGF receptors in the vascular endothelial cells of the thecal blood vessels. The most intense immunofluorescence was evident in pre-ovulatory follicles compared to other smaller follicles. These data provide evidence that the VEGF may contribute to the extensive capillary proliferation associated with the increase in size, selection, and maturation of the pre-ovulatory follicle. This may facilitate follicle maturation by enhancing the supply of nutrients, hormones, and other essential blood-borne signals to the follicle. VEGF may also promote maturation of follicles through recently recognized, non-angiogenic mechanisms.
本研究的目的是描述水牛（Bubalus bubalis）卵巢卵泡生长、发育和成熟过程中血管内皮生长因子（VEGF）和 VEGF 受体的基因表达的时间模式。根据卵泡大小和卵泡液中雌二醇-17β（E2）的浓度，将卵泡分为四组：I 组（小），4-6mm 直径，E2>0.5ng/ml 的 FF；II 组（中），7-9mm，E2=0.5-5ng/ml；III 组（大），10-13mm，E2=5-40ng/ml；IV 组（排卵前），>13mm，E2>180ng/ml）。还测定了卵巢内膜和颗粒层中的促卵泡激素受体（FSHR）、促黄体激素受体（LHR）和细胞色素 P45019A1（CYP19A1）的 mRNA，进一步定义了每个组的成熟状态。通过定量实时 PCR（qRT-PCR）确定的 VEGF 同工型（120、164 和 188 个氨基酸形式）的相对表达在卵泡发育过程中在颗粒层（P<0.05）和膜层中均增加。VEGF 受体（VEGFR-1 和 VEGFR-2）的相对数量在 Gp-I 卵泡的颗粒细胞（GC）和卵巢内膜细胞（TI）层中最少。VEGFR-2 转录物的量在整个发育过程中在颗粒层中增加，在排卵前卵泡中达到最大值（P<0.05）。通过蛋白质印迹法测定的卵泡裂解物中 VEGF 同工型和受体的相对量在卵泡成熟过程中增加，在排卵前卵泡中达到最大值。免疫组织化学显示 VEGF 同工型和受体在两种甾体生成细胞类型（GC 和 TI）以及血管内皮细胞中的清晰定位在卵巢膜血管中的 VEGF 受体。与其他较小的卵泡相比，在排卵前卵泡中观察到最强烈的免疫荧光。这些数据表明，VEGF 可能有助于与卵母细胞大小增加、选择和成熟相关的广泛毛细血管增殖。这可能通过增强向卵泡供应营养物质、激素和其他必需的血液信号来促进卵泡成熟。VEGF 也可能通过最近认识到的非血管生成机制促进卵泡成熟。
