Suspensions of rat anterior pituitary cells were prepared using a trypsin dissociation at 23°C. This procedure produced a high yield of cells (4 x 10⁶ cells/gland) with a viability of 95% or greater (dye elusion). Using cells obtained from intact female rats (mixed cycle), a significant (P<0.01) dose-related response to LHRH was observed for both LH and FSH even when tested immediately after enzymatic dissociation; however, cells allowed to recover for an additional 6-18 h after dissociation exhibited a significantly greater response to LHRH. Following an 18 h culture period, suspensions of anterior pituitary cells derived from animals at different stages of the estrous cycle were incubated with varying concentrations of LHRH for 2 h. The LH responses to LHRH were significantly greater (P<0.01) with cells taken from diestrous-II and proestrous animals compared with those from estrous or diestrous-I rats. At all stages of the estrous cycle maximal responsiveness was obtained with 10^-7 M LHRH. FSH responses to LHRH were similar to those described for LH. Cells from pituitaries of short-term (72 h) ovariectomized rats given estradiol benzoate (EB; 5 µg/100 g BW) 26-27 h prior to tissue dissociation showed a significantly (P<0.001) greater response to LHRH (100%) compared with those given oil. Cell suspensions from ovariectomized animals given oil or EB failed to show this difference in hormone release when exposed to 60 mM K⁺ for 2 h.

These results demonstrate that gonadotrophs in short-term culture maintain their characteristic hormone-dependent responsiveness to LHRH similar to that reported in vivo, and suggest that estradiol is at least partly responsible for the amount of LH and FSH available for release from these cells. Furthermore, the increased LH and FSH available for release after estrogen treatment appears specific for the action of LHRH.