Nandi S, Girish Kumar V
National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India.
Acta Physiol (Oxf). 2008 Aug;193(4):341-55. doi: 10.1111/j.1748-1716.2008.01849.x. Epub 2008 Feb 28.
Regulation of folliculogenesis and oocyte-somatic cell interactions in the ovarian follicles is under the control of gonadotrophins and various local factors. In the present study, an attempt was made to isolate and examine the biological activities of ovarian follicular fluid protein(s) in sheep in vitro.
Follicular fluids aspirated from ovarian follicles of slaughterhouse-derived ovaries were made cell free by centrifugation (5000 g for 30 min) and steroid free by charcoal treatment. The follicular fluid was then subjected to ammonium sulphate precipitation and gel filtration chromatography using G-75 Sephadex. Protein detection was performed using a UV spectrophotometer at 280 nm. The 35-50% fraction yielded a detectable peak and a protein of 30.1 kDa as examined by SDS-PAGE. The effect of increasing doses of the 30.1 kDa ovine follicular fluid protein (oFFP) was tested at different doses on pre-antral and antral follicle growth; cumulus cell expansion; oocyte maturation; changes in protein, calcium and phosphorus levels of oocytes after culture in media containing different levels of isolated protein; mural granulosa cell, polar granulosa cell (cumulus cell), oviductal epithelial cell monolayer formation and granulosa cell proliferation in vitro.
The oFFP significantly inhibited antral follicle growth, cumulus expansion, oocyte maturation and somatic cell growth in vitro in a dose-dependent manner. The oFFP did not have a significant effect on the pre-antral follicle growth in vitro. The protein, calcium and phosphorus contents of oocytes were found to decrease in oocytes cultured in maturation medium containing the oFFP.
The present study demonstrates a follicular fluid factor regulating folliculogenesis and oocyte maturation in sheep.
卵巢卵泡中卵泡发生及卵母细胞-体细胞相互作用受促性腺激素及多种局部因子调控。本研究旨在体外分离并检测绵羊卵巢卵泡液蛋白的生物活性。
从屠宰场获取的卵巢卵泡中吸出的卵泡液，经离心（5000g，30分钟）去除细胞，并用活性炭处理去除类固醇。然后对卵泡液进行硫酸铵沉淀及使用G-75葡聚糖凝胶的凝胶过滤层析。使用紫外分光光度计在280nm处进行蛋白质检测。经SDS-PAGE检测，35%-50%的组分产生一个可检测峰及一种30.1kDa的蛋白质。检测不同剂量的30.1kDa绵羊卵泡液蛋白（oFFP）对窦前卵泡和窦卵泡生长、卵丘细胞扩展、卵母细胞成熟、在含有不同水平分离蛋白的培养基中培养后卵母细胞蛋白质、钙和磷水平的变化、壁颗粒细胞、极颗粒细胞（卵丘细胞）、输卵管上皮细胞单层形成及颗粒细胞体外增殖的影响。
oFFP在体外以剂量依赖方式显著抑制窦卵泡生长、卵丘扩展、卵母细胞成熟及体细胞生长。oFFP对体外窦前卵泡生长无显著影响。发现在含有oFFP的成熟培养基中培养的卵母细胞，其蛋白质、钙和磷含量降低。
本研究证明了一种调节绵羊卵泡发生和卵母细胞成熟的卵泡液因子。
