Dispersed monkey pituitary cells were cultured on extracellular matrix and in serum-free medium for up to 1 month, with medium changes every other day. The cells were treated with ethanol vehicle or 10 nM 17β-estradiol (E2β), 17α-estradiol (E2α), progesterone (P4), pregnenalone (P5), 17α-hydroxyprogesterone (17OH P4), and E2β plus P4. Immunoreactive PRL (IR-PRL) levels in 48 h medium samples were first determined with a homologous human PRL RIA and then with a heterologous monkey PRL RIA. On the last day of culture, the cells were incubated with [35S]methionine for 4 h, and 35S-labeled PRL in the medium and cells was determined and compared to 4 h IR-PRL. Differences between control and treated groups in 48 h IRPRL were greater with the heterologous monkey RIA. E2β, E2α, and E2β plus P4 significantly elevated IR-PRL in 48 h medium samples an average of 73%, as determined with the monkey RIA. P4, P5, or 17OH P4 had no effect on IR-PRL in 48 h medium samples. Secreted and intracellular [35S]PRL were consistently elevated by E2β and E2β plus P4, but less consistently elevated by E2α. P4, P5, or 17OH P4 had little effect on [35S]PRL in medium or cells. IR-PRL in the 4 h medium sample and cells generally paralleled [35S]PRL, although E2α caused a more consistent elevation in IR-PRL than in [35S]PRL. In summary, E2β and E2α directly stimulate PRL synthesis by monkey pituitary cells on extracellular matrix in long term serum-free culture. Nanomolar levels of progestins have little direct effect on PRL synthesis, nor does 10 nM P4 block the stimulatory effect of E2β.
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