Campbell B K, Baird D T, Webb R
Department of Obstetrics and Gynaecology, University of Edinburgh, UK.
J Reprod Fertil. 1998 Jan;112(1):69-77. doi: 10.1530/jrf.0.1120069.
The study reports the development of a serum-free culture system for sheep thecal cells that overcomes the problem of spontaneous luteinization and the use of this system to study the control of proliferation and differentiation. Theca cells were isolated by enzymatic dispersion from small follicles (< 3.5 mm) and the effect of plating densities (25-100 x 10(3) cells per well), LH (0.001-100 micrograms l-1), insulin (1-5000 micrograms l-1), insulin-like growth factor I (IGF-I) analogue (1-100 micrograms LR3-IGF-I l-1) and epidermal growth factor (EGF) (0.005-50 micrograms l-1) on the number of cells and androstenedione and progesterone production were determined. Plating density had a marked effect on the pattern of hormone secretion with densities between 50 and 75 x 10(3) cells per well resulting in a high androstenedione: progesterone ratio at optimum doses of LH (0.1 micrograms l-1: P < 0.001). In the first 48 h, the production of both androstenedione and progesterone was stimulated in a dose-dependent manner by LH (P < 0.001). However, the production of androstenedione was ten times higher than that of progesterone and was more sensitive to LH (ED50 value 0.08 micrograms l-1 for androstenedione and 1 microgram l-1 for progesterone). From 48-144 h of culture higher doses of LH (> 1 ng ml-1) inhibited androstenedione (P < 0.001) and stimulated progesterone (P < 0.001) and resulted in a marked change in cell morphology, thus reflecting both functional and morphological luteinization. At optimum doses of LH, both insulin and IGF stimulated cell proliferation (P < 0.001) and androstenedione production (P < 0.001) in a dose responsive manner and there was a significant (P < 0.001) interaction between them. In contrast, both insulin and IGF-I inhibited (P < 0.001) progesterone production in a dose responsive manner. EGF stimulated cell proliferation (P < 0.001) and progesterone production (P < 0.001), but inhibited androstenedione production (P < 0.001), in a dose responsive manner. In conclusion, this culture system exhibits physiologically relevant responses to known in vivo modulators of follicle development. The biphasic nature of the theca cell response to LH emphasises the exquisite sensitivity of theca cells to LH stimulation and highlights the importance of dose-response relationships in the gonadotrophic control of ovarian function.
该研究报告了一种用于绵羊卵泡膜细胞的无血清培养系统的开发，该系统克服了自发黄体化问题，并利用此系统研究增殖和分化的控制。通过酶分散从小卵泡（<3.5毫米）中分离出卵泡膜细胞，并测定接种密度（每孔25 - 100×10³个细胞）、促黄体生成素（LH）（0.001 - 100微克/升）、胰岛素（1 - 5000微克/升）、胰岛素样生长因子I（IGF - I）类似物（1 - 100微克LR3 - IGF - I/升）和表皮生长因子（EGF）（0.005 - 50微克/升）对细胞数量以及雄烯二酮和孕酮产生的影响。接种密度对激素分泌模式有显著影响，每孔50至75×10³个细胞的密度在LH最佳剂量（0.1微克/升：P < 0.001）下导致高雄烯二酮：孕酮比率。在最初48小时内，LH以剂量依赖性方式刺激雄烯二酮和孕酮的产生（P < 0.001）。然而，雄烯二酮的产生比孕酮高十倍，并且对LH更敏感（雄烯二酮的ED50值为0.08微克/升，孕酮为1微克/升）。在培养48 - 144小时期间，较高剂量的LH（>1纳克/毫升）抑制雄烯二酮（P < 0.001）并刺激孕酮（P < 0.001），并导致细胞形态发生显著变化，从而反映了功能性和形态学上的黄体化。在LH的最佳剂量下，胰岛素和IGF均以剂量反应方式刺激细胞增殖（P < 0.001）和雄烯二酮产生（P < 0.001），并且它们之间存在显著（P < 0.001）相互作用。相比之下，胰岛素和IGF - I均以剂量反应方式抑制孕酮产生（P < 0.001）。EGF以剂量反应方式刺激细胞增殖（P < 0.001）和孕酮产生（P < 0.001），但抑制雄烯二酮产生（P < 0.001）。总之，该培养系统对已知的体内卵泡发育调节剂表现出生理相关反应。卵泡膜细胞对LH反应的双相性质强调了卵泡膜细胞对LH刺激的高度敏感性，并突出了剂量 - 反应关系在卵巢功能促性腺激素控制中的重要性。
