Weil C, Hansen P, Hyam D, Le Gac F, Breton B, Crim L W
Gen Comp Endocrinol. 1986 May;62(2):202-9. doi: 10.1016/0016-6480(86)90110-3.
To study the regulation of gonadotropin secretion in rainbow trout in vitro, a method for preparing primary cultures of dispersed pituitary cells is described. Cells were dispersed by collagenase 0.1% in Hank's saline solution for 20 hr at 12 degrees and a high yield of viable cells was obtained. Attempts to improve cell functioning were made by varying culture conditions (density of cells initially plated, age of the culture). Cell functioning was assessed by their ability to respond to increasing doses of mammalian and salmon GnRH. Pituitaries were collected from spermiating males whose pituitaries are known to be sensitive to mammalian GnRH in vivo. Using 96-well plates, optimal conditions for good biological activity, are initial plating with 6.2 X 10(4) cells, incubation with GnRH for 24 hr on the third day after plating. In these conditions mammalian analog and salmon GnRH induced an increase in GtH release for doses ranging from 10(-9) to 10(-6) M. The GtH released during the GnRH incubation period does not decrease the sensitivity of the system since addition of 20 ng of GtH at the beginning of incubation does not modify the response profile.
为了在体外研究虹鳟鱼促性腺激素分泌的调节机制，本文描述了一种制备分散垂体细胞原代培养物的方法。细胞在含有0.1%胶原酶的汉克斯盐溶液中于12℃分散20小时，获得了高产量的活细胞。通过改变培养条件（最初接种的细胞密度、培养时间）来尝试改善细胞功能。通过细胞对递增剂量的哺乳动物和鲑鱼促性腺激素释放激素（GnRH）的反应能力来评估细胞功能。垂体取自正在排精的雄性虹鳟鱼，已知其垂体在体内对哺乳动物GnRH敏感。使用96孔板，良好生物活性的最佳条件是最初接种6.2×10⁴个细胞，在接种后第三天用GnRH孵育24小时。在这些条件下，哺乳动物类似物和鲑鱼GnRH在10⁻⁹至10⁻⁶M的剂量范围内诱导促性腺激素（GtH）释放增加。在GnRH孵育期间释放的GtH不会降低系统的敏感性，因为在孵育开始时添加20 ng的GtH不会改变反应曲线。
