Mishra S R, Bharati Jaya, Rajesh G, Chauhan V S, Taru Sharma G, Bag S, Maurya V P, Singh G, Sarkar M
Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India.
Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India.
Anim Reprod Sci. 2017 Apr;179:88-97. doi: 10.1016/j.anireprosci.2017.02.006. Epub 2017 Feb 21.
The present study investigated the combined effect of fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGF-A) on estradiol (E) secretion and relative abundance of mRNA for aromatase enzyme (CYP19A1), proliferating cell nuclear antigen (PCNA) and BCL-2 associated X protein (BAX) in cultured buffalo granulosa cells (GCs). Follicles were isolated and classified into four groups based on size and E concentration in follicular fluid (FF): Small, 4-6mm diameter, E<0.5ng/ml; Medium, 7-9mm, E=0.5-5ng/ml; Large, 10-13mm, E=5-40ng/ml; Preovulatory (PFs), >14mm, E>180ng/ml. The GCs of PF were cultured in 24 well cell culture plates and allowed to become 75-80% confluent. Then cultured GCs were treated with FGF2 (200ng/ml) and VEGF-A (100ng/ml) separately and in combination for three incubation periods (24, 48 and 72h). Estradiol secretion was greater in GCs treated with FGF2+VEGF-A compared to FGF2 or VEGF-A at all incubation periods and was greatest (P<0.05) at 72h of incubation. The relative abundance of CYP19A1 and PCNA mRNA were relatively consistent with the amount E secretion. In contrast, the relative abundance of Bax mRNA was less in GCs treated with the combination of FGF2 and VEGF-A as compared to either FGF2 or VEGF-A alone and the least concentration (P<0.05) was at 72h of incubation. Findings with use of immunocytochemistry of cells treated with these factors were consistent to the relative abundance of mRNA transcript for the factor. The present findings indicate that FGF2 and VEGF-A may function in a synergistic manner to promote steroidogenesis and survival of cultured buffalo GCs.
本研究调查了成纤维细胞生长因子2（FGF2）和血管内皮生长因子A（VEGF-A）对培养的水牛颗粒细胞（GCs）中雌二醇（E）分泌以及芳香化酶（CYP19A1）、增殖细胞核抗原（PCNA）和BCL-2相关X蛋白（BAX）mRNA相对丰度的联合作用。根据卵泡大小和卵泡液（FF）中的E浓度，将卵泡分离并分为四组：小卵泡，直径4-6mm，E<0.5ng/ml；中卵泡，7-9mm，E=0.5-5ng/ml；大卵泡，10-13mm，E=5-40ng/ml；排卵前卵泡（PFs），>14mm，E>180ng/ml。将PF的GCs接种于24孔细胞培养板中，使其汇合度达到75-80%。然后，将培养的GCs分别用FGF2（200ng/ml）和VEGF-A（100ng/ml）单独处理以及联合处理，共孵育三个时间段（24、48和72小时）。在所有孵育时间段内，与单独使用FGF2或VEGF-A相比，联合使用FGF2+VEGF-A处理的GCs中雌二醇分泌量更高，且在孵育72小时时最高（P<0.05）。CYP19A1和PCNA mRNA的相对丰度与E分泌量相对一致。相反，与单独使用FGF2或VEGF-A相比，联合使用FGF2和VEGF-A处理的GCs中Bax mRNA的相对丰度更低，且在孵育72小时时浓度最低（P<0.05）。使用这些因子处理细胞的免疫细胞化学结果与该因子mRNA转录本的相对丰度一致。本研究结果表明，FGF2和VEGF-A可能以协同方式发挥作用，促进培养的水牛GCs的类固醇生成和存活。
