Shores E M, Picton H M, Hunter M G
Division of Animal Physiology, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK.
J Reprod Fertil. 2000 Mar;118(2):211-9.
The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.
通过建立一种生理性无血清培养系统来研究猪卵泡膜细胞类固醇生成的调节机制，随后该系统被扩展用于研究卵泡膜细胞与颗粒细胞之间潜在的相互作用。从直径6 - 9毫米的窦状卵泡中分离出卵泡膜细胞，并检测接种密度（每孔50 - 150×10³个活细胞）、促黄体生成素（LH，0.01 - 1.0纳克/毫升）、长效R3胰岛素样生长因子I（IGF - I，10、100纳克/毫升）和胰岛素（1、10纳克/毫升）对细胞数量和类固醇生成的影响。通过生化和组织学方法验证了卵泡膜细胞制备物的纯度。共培养时每孔含有50×10³个活细胞，颗粒细胞与卵泡膜细胞的比例为4:1。仅含有颗粒细胞的孔补充了“生理”剂量的雄烯二酮或100纳克/毫升。将共培养物产生的雌二醇产量与分别培养的颗粒细胞和卵泡膜细胞合成的雌二醇总量进行比较。在整个培养过程中持续产生雌二醇和雄烯二酮。高接种密度会降低类固醇生成（P < 0.01）。LH增加雄烯二酮（P < 0.001）和雌二醇（P < 0.05）的合成，并且随着培养时间的延长细胞的敏感性增加。10纳克/毫升的IGF - I可增加雌二醇生成（P < 0.001），但雄烯二酮需要100纳克/毫升（P < 0.001）。无论雄烯二酮剂量如何，共培养物产生的雌二醇都比分别培养的卵泡膜细胞和颗粒细胞合成的雌二醇总量更多（P < 0.001）。这种用于猪卵泡膜细胞的无血清培养系统维持了体内类固醇生成和促性腺激素反应性。卵泡膜细胞的雄烯二酮和雌二醇生成受到不同调节，分别主要由LH和IGF - I刺激。卵泡膜细胞与颗粒细胞之间的相互作用刺激了雌二醇合成，并且这种相互作用是由除了向颗粒细胞提供卵泡膜雄激素底物之外的其他因素介导的。
