Frawley L S, Neill J D
Endocrinology. 1984 Feb;114(2):659-63. doi: 10.1210/endo-114-2-659.
The negative feedback effect of estrogen on LH secretion has been difficult to demonstrate in monolayer cultures of rat pituitary cells. The purpose of the present study was to establish the experimental conditions required for manifestation of this response and, in the process, to develop models for investigating the actions of steroids on pituitary cells. Both dynamic and static incubation systems were used. For perifusion experiments, trypsin-dispersed pituitary cells from rats at random stages of the estrous cycle were attached to glass coverslips with poly-L-lysine, incubated for 48 h, and then mounted in Sykes-Moore chambers. In each of these experiments, two chambers were perifused concurrently: one with medium containing 1.8 X 10(-10) M 17 beta-estradiol and the other with medium alone. GnRH (4.2 X 10(-9) M) was coinfused for 5 min out of every hour, and samples of perifusate were collected as 5-min fractions for assay of LH. Estrogen treatment significantly (P less than 0.01) suppressed LH release in response to the first five GnRH pulses compared to the control value. The inhibition was most pronounced early in the perifusion, but had disappeared by 6 h. These results demonstrate that estradiol exerts a potent but transient inhibition of GnRH-induced LH release in monolayer cultures of rat pituitary cells. In a subsequent set of experiments, we modified a static incubation system to assess sequentially the biphasic effects of estrogen on LH release by the same group of cells. Cultures of rat pituitary cells that had been established 42 h previously were treated simultaneously for 3 h with 17 beta-estradiol (3.7 X 10(-10) M) and various concentrations of GnRH (5 X 10(-10) to 1 X 10(-7) M) to measure the inhibitory effects of the steroid on LH secretion. This experiment was repeated on the same cells after 27 h of steroid exposure to estimate the facilitory actions of estrogen on LH release. The negative feedback of estrogen was demonstrable in static cultures of rat pituitaries provided that the period of estrogen exposure and duration of incubation were brief. Moreover, the results indicate that the same groups of cells can be used on consecutive days to investigate the inhibitory and stimulatory effects of estrogen on LH secretion. Experiments with cultures of monkey pituitary cells yielded similar results. Taken together, these findings indicate that cultured pituitary cells are responsive to the biphasic actions of estradiol and demonstrate the utility of two model systems for investigating these phenomena.
雌激素对促黄体生成素（LH）分泌的负反馈作用在大鼠垂体细胞单层培养中一直难以得到证实。本研究的目的是确定出现这种反应所需的实验条件，并在此过程中建立用于研究类固醇对垂体细胞作用的模型。研究中使用了动态和静态培养系统。对于灌流实验，将处于动情周期任意阶段的大鼠垂体细胞用胰蛋白酶分散后，通过聚-L-赖氨酸附着于玻璃盖玻片上，孵育48小时，然后安装在赛克斯-摩尔小室中。在每个此类实验中，同时对两个小室进行灌流：一个小室灌注含有1.8×10⁻¹⁰ M 17β-雌二醇的培养基，另一个小室仅灌注培养基。每小时共灌注GnRH（4.2×10⁻⁹ M）5分钟，收集灌流液样本，以5分钟的时间间隔进行LH测定。与对照值相比，雌激素处理显著（P＜0.01）抑制了对前五个GnRH脉冲的LH释放。这种抑制在灌流早期最为明显，但在6小时时消失。这些结果表明，在大鼠垂体细胞单层培养中，雌二醇对GnRH诱导的LH释放具有强大但短暂的抑制作用。在后续的一组实验中，我们改进了静态培养系统，以依次评估雌激素对同一组细胞LH释放的双相作用。对预先建立42小时的大鼠垂体细胞培养物同时用17β-雌二醇（3.7×10⁻¹⁰ M）和不同浓度的GnRH（5×10⁻¹⁰至1×10⁻⁷ M）处理3小时，以测量类固醇对LH分泌的抑制作用。在类固醇暴露27小时后，对同一组细胞重复该实验，以评估雌激素对LH释放的促进作用。如果雌激素暴露时间和孵育时间较短，雌激素的负反馈作用在大鼠垂体的静态培养中是可以得到证实的。此外，结果表明同一组细胞可在连续几天用于研究雌激素对LH分泌的抑制和刺激作用。对猴垂体细胞培养物的实验也得到了类似结果。综上所述，这些发现表明培养的垂体细胞对雌二醇的双相作用有反应，并证明了两种模型系统用于研究这些现象的实用性。
