Mara E. Lieberman

Prolactin synthesis in primary cultures of pituitary cells: regulation by estradiol.

Pituitary cells cultured with estradiol respond by a specific increase in prolactin synthesis. Extensive inhibition of DNA synthesis (61-78%) with hydroxyurea or cytosine arabinoside resulted in only 28-33% decrease in estrogen-induced prolactin synthesis. To assess the role of prolactin cell proliferation in the estrogen-induced response, mammotrophs were identified by immunocytochemistry. Cultures treated with estradiol for 1, 2 or 5 days contained 101 +/- 1, 113 +/- 2 and 132 +/- 1% of the number of mammotrophs in controls. Estradiol treatment for corresponding periods resulted in prolactin synthesis representing 94 +/- 5, 144 +/- 11 and 270 +/- 22% of controls and prolactin mRNA levels representing 115 +/- 7, 160 +/- 7 and 322 +/- 22% of controls. Thus estrogen caused a considerable increase in prolactin synthesis which paralleled the increase in prolactin mRNA levels and a much smaller relative increase in the number of mammotrophs. We conclude that regulation of prolactin synthesis by estrogen is mediated predominantly but not exclusively through stimulation of gene expression in existing pituitary cells.

Regulation of pituitary growth and prolactin gene expression by estrogen.

We presented evidence that primary cultures of rat pituitary cells respond to estradiol by increased incorporation of precursors into prolactin. The response is specific for estrogenic hormones and is maximal at physiological concentrations of estradiol. The time course and magnitude of the response in cultured cells is in agreement with that observed under in vivo conditions, suggesting that estrogen exerts its effect mainly through a direct action on the pituitary gland. The data presented indicate that estrogen stimulates prolactin synthesis predominantly through increased prolactin mRNA accumulation, and to a lesser extent, through mammotroph cell proliferation. Chronic treatment with DES caused sustained proliferation of pituitary cells leading to prolactin producing pituitary tumors in the Fischer 344 rat, but not in the Holtzman rat. The genetic basis for these differences are currently under investigation.

The inhibition of prolactin synthesis in GH3 rat pituitary tumor cells by monohydroxytamoxifen is associated with changes in the properties of the estrogen receptor

E2 (1 nM) stimulated the synthesis of PRL in GH3 cells. OH TAM (100 nM) did not affect basal PRL synthesis, but completely inhibited the increase produced by 1 nM E2. [3H]E2 and [3H]OH TAM both bound to the cytosolic 8S ER and these were split into 4S subunits on sucrose gradients containing 0.4 M KCl. By comparison, ER complexes extracted from nuclei of GH3 cells cultured in media containing [3H]E2 or [3H]OH TAM both sedimented at 5S on sucrose gradients containing 0.4 M KCl. Both 4S and 5S ER complexes were recognized by the monoclonal antibody D547 which increased their sedimentation coefficients to 8-9S. In contrast, a polyclonal antibody raised to calf uterine ER in the goat, interacted with the cytosolic ER so that the binding of [3H]E2 was inhibited but the binding of [3]OH TAM was only slightly reduced. A molecular model is proposed to describe the binding of E2 and OH TAM to the ER that might contribute to an understanding of estrogen and antiestrogen action.

THE ROLE OF ESTROGEN IN THE DIFFERENTIATION OF PROLACTIN PRODUCING CELLS

The capacity to synthesize PRL and GH was studied in normal mice and in Snell and Ames dwarfs. In normal mice GH synthesis turned on dramatically between day 16-17 of gestation whereas PRL was not detectable throughout gestation or the first week of life, was barely detectable in 8-day-old mice and was clearly demonstrable by 12 days of age. However, transplantation of pituitaries from newborn mice into adult female hosts resulted in substantial PRL synthesis. Treatment of mice with DES at various ages showed that neonates failed to respond to the hormone; in older pups, an age-dependent increase in the magnitude of PRL synthesis was observed. There was a direct correlation between nuclear estrogen receptor levels and PRL cell function. Snell and Ames dwarf mice failed to synthesize PRL or GH at any stage of development, nor could we detect immunoreactive peptides which might represent mutant forms of the hormones. Our findings suggest that: (1) GH gene expression precedes that of PRL by about 2 weeks; (2) the development of PRL cell function is dependent on estrogen; (3) the capacity to respond to estrogen is present before the endogenous initiation of PRL synthesis and is limited by the availability of estrogen receptor; and (4) in Snell and Ames mutants, both types of alleles result in failure of the pituitary to initiate PRL and GH synthesis.

Genomic organization of prolactin and growth hormone coding sequences in dwarf and normal mice

The genomic organization of DNA encoding growth hormone (GH) and prolactin (PRL) genes has been investigated in two types of homozygous dwarf mice and their normal counterparts. We have previously shown that dwarf mice of either strain fail to initiate pituitary synthesis of GH or PRL during perinatal development. Analysis by Southern transfer of restriction enzyme-digested DNA and hybridization to 32P-labeled cloned probes derived from bovine GH and rat PRL mRNAs revealed no evidence for deletions or rearrangements in or around the structural genes for GH or PRL in either dwarf genotype. In situ hybridization of the probes to pituitary and liver tissue slices failed to detect specifically hybridizing RNA species in dwarf pituitaries. These findings suggest that the pleiotropic effects of the dwarf mutations, which lead to abnormalities in transcription of the genes or in the processing of transcripts, may be due to blocks in the development of functional somatotrophs and mammotrophs.