Biphasic Effects of Estrogen on Gonadotropin-Releasing Hormone-Induced Luteinizing Hormone Release in Monolayer Cultures of Rat and Monkey Pituitary Cells*

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Abstract
The negative feedback effect of estrogen on LH [luteinizing hormone] secretion has been difficult to demonstrate in monolayer cultures of rat pituitary cells. The experimental conditions required for manifestation of this response were established and, in the process, models for investigating the actions of steroids on pituitary cells were developed. Both dynamic and static incubation systems were used. For perfusion 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, 2 chambers were perifused concurrently: one with medium containing 1.8 .times. 10-10 M 17.beta.-estradiol and the other with medium alone. GnRH [gonadotropin-releasing hormone] (4.2 .times. 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 < 0.01) suppressed LH release in response to the first 5 GnRH pulses compared to the control value. This inhbiition was most pronounced early in the perifusion, but had disappeared by 6 h. 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, a static incubation system was modified 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 .times. 10-10 M) and various concentrations of GnRH (5 .times. 10-10 to 1 .times. 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 facilitatory 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. 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 [Macaca mulatta] pituitary cells yielded similar results. These findings indicate that cultured pituitary cells are responsive to the biphasic actions of estradiol and demonstrate the utility of 2 model systems for investigating these phenomena.