Sharma G Taru, Dubey P K, Kumar G Sai
Reproductive Physiology & ETT Laboratory, Division of Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Reprod Domest Anim. 2011 Feb;46(1):114-20. doi: 10.1111/j.1439-0531.2010.01604.x.
Follicle-stimulating hormone (FSH) stimulates antral follicles to grow, but its role in earlier stages (pre-antral) of follicle development, if any, is obscure. Aim of this study was to study the expression of follicle-stimulating hormone receptor (FSHR) gene in different sizes of pre-antral follicles (PFs) (<150, 200, 250, 300, 350, 400 μm) and to find out an optimum dose of FSH for better growth, development and steroidogenesis of PFs in vitro. Buffalo ovaries were collected from a local abattoir, and PFs were isolated by mechanical method. A semi-quantitative RT-PCR amplification strategy was used for mRNA expression, while FSHR protein was localized by immunohistochemistry. Isolated pre-antral follicles (80-85 μm) were cultured in TCM-199 supplemented with 10% foetal bovine serum, 1% ITS and 30 ng/ml EGF served as control medium. Addition of three different doses of FSH (0.5, 1.0, 2.0 μg/ml) in control medium was considered as treatment groups. A single 2.184-kb receptor mRNA transcript was present in all sizes (<150-400 μm) of follicles. Follicle-stimulating hormone receptor was also localized immunohistochemically in granulosa cells of all sizes of follicles. Survival and growth rate of follicles significantly (p<0.05) increased following supplementation of FSH at a concentration of 1.0 μg/ml and the culture medium also showed a significantly (p<0.05) greater accumulation of oestradiol and progesterone. In conclusion, FSHR is expressed in all sizes of PFs and in vitro survival, growth and steroidogenesis of follicles are optimally stimulated by 1.0 μg/ml FSH. These findings demonstrate that FSH has an important role during the recruitment, growth and development of buffalo ovarian PFs.
促卵泡激素（FSH）刺激窦卵泡生长，但其在卵泡发育早期（窦前）阶段的作用（若有）尚不清楚。本研究的目的是研究促卵泡激素受体（FSHR）基因在不同大小的窦前卵泡（PFs）（<150、200、250、300、350、400μm）中的表达，并找出能使PFs在体外更好地生长、发育和进行类固醇生成的FSH最佳剂量。从当地屠宰场收集水牛卵巢，通过机械方法分离PFs。采用半定量RT-PCR扩增策略检测mRNA表达，同时通过免疫组织化学对FSHR蛋白进行定位。将分离的窦前卵泡（80 - 85μm）培养于添加10%胎牛血清、1%ITS和30 ng/ml EGF的TCM-199中作为对照培养基。在对照培养基中添加三种不同剂量的FSH（0.5、1.0、2.0μg/ml）作为处理组。所有大小（<150 - 400μm）的卵泡中均存在单一的2.184kb受体mRNA转录本。FSH受体在所有大小卵泡的颗粒细胞中也通过免疫组织化学定位。添加浓度为1.0μg/ml的FSH后，卵泡的存活率和生长率显著（p<0.05）提高，培养基中雌二醇和孕酮的积累也显著（p<0.05）增加。总之，FSHR在所有大小的PFs中均有表达，1.0μg/ml的FSH能最佳地刺激卵泡在体外的存活、生长和类固醇生成。这些发现表明FSH在水牛卵巢PFs的募集、生长和发育过程中具有重要作用。
