CHARACTERIZATION OF A RAT ANTERIOR-PITUITARY CELL BIOASSAY

We have described the protocols and characterization of a pituicyte cu lture, which became established as a reliable and reproducible bioassa y for the secretion of follicle-stimulating hormone (FSH) and luteiniz ing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protoc ol that was used involved the culling of female Wistar rats (200 to 25 0 g weight), at random stages of their cycle, and dispersal of their p ituicytes in a concentration of 0.4 X 10(6) cells . ml(-1). well(-1) i n serum-fr ee medium (Dulbecco's modified Eagle's medium/Ham's F12 mix ture, supplemented with insulin and transferrin) in Falcon 3047 24-wel l culture plates, After 24 h of pre-culture, the medium was changed an d the cells cultured for a further 48 h. The supernatant was removed a nd assayed for basal secretion of FSH and LH. The cells were then stim ulated with 10(-8) M GnRH for 4 h and the supernatant assayed for gona dotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion, Al l samples were assayed as pairs of duplicates (i.e. quadruplicate samp les) which were randomly added to the plates to minimize plate effects . Random number tables were used to achieve this randomization.