To determine the direct effect of estrogen on primate PRL production, cultures of dispersed monkey pituitary cells were established in serum-free medium on an extracellular matrix secreted by bovine corneal endothelial cells. Medium PRL levels were monitored by RIA every fourth day after plating. PRL secretion was maintained for 24-28 days in cultures from female monkeys and for 16-20 days in cultures from male monkeys using a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium H-16 (DME) and Ham’s F-12 medium (F12) supplemented with insulin (I), transferrin (T), PTH, T4, fibroblastic growth factor, putrescine, ethanolamine, lipids (oleic, lecithin, and cholesterol), selenium (S), and cadmium (C). The mean percent changes in medium PRL of seven cultures of female pituitaries and two cultures of male pituitaries compared to the first sample (day 4) were: +26% (day 8), +17% (day 12), +11% (day 16), -4% (day 20), -13% (day 24), and -24% (day 28). The elimination of supplements except ITSC did not appear to affect PRL secretion in a culture of cells from ovariectomized female monkeys, but secretion in two cultures of male pituitary cells declined after day 12. Addition of 10-8 M estrogen to three cultures of male pituitary cells in DME/F12 containing all supplements significantly elevated PRL over control levels and, in addition, prevented the decline observed in control wells of one culture for 28 days. In two cultures of male pituitary cells maintained in DME/F12 plus ITSC, estrogen significantly elevated medium PRL levels for the 28-day period, but did not totally prevent the decline observed in control wells after day 12. In two cultures of female pituitary cells maintained in DME/F12 plus all supplements and in three cultures of female pituitary cells maintained in DME/F12 plus ITSC, medium PRL levels were significantly higher when estrogen was present. However, estrogen addition had no effect on the pattern of PRL secretion in two cultures of female pituitary cells maintained in DME/F12 plus 10% charcoal-treated fetal calf serum. In five of seven cultures, the presence of estrogen for 28 days resulted in a significantly higher cellular content of PRL. These experiments suggest that estrogen can directly increase PRL production by primate mammotrophs and that this effect is best seen in serumfree medium. In summary, extracellular matrix and serum-free medium provide an adequate in vitro environment for studies of PRL processing by primate pituitary cells for periods up to 1 month. In this system, estrogen effectively elevated PRL secretion and cell content.
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