ACTIVATION OF THE BABOON FETAL PITUITARY-ADRENOCORTICAL AXIS AT MIDGESTATION BY ESTROGEN - ENHANCEMENT OF FETAL PITUITARY PROOPIOMELANOCORTIN MESSENGER-RIBONUCLEIC-ACID EXPRESSION

We have proposed that estrogen, via regulation of placental metabolism of maternal cortisol, regulates the baboon fetal hypothalamic-pituitary-adrenal axis and the timing of the onset of de novo cortisol production. In support of this hypothesis, we demonstrated that the ontogenesis of fetal adrenal steroidogenic enzymes near term could be induced at midgestation by maternal estrogen treatment. In the present study, we determined whether maturation of the fetal adrenal near term and at midgestation after estrogen treatment reflects enhanced expression of the messenger RNA (mRNA) for the ACTH precursor molecule POMC. Fetal pituitaries were obtained on day 100 (n = 7) and day 165 (n = 5) of gestation (term = day 184) from untreated baboons and on day 100 after maternal treatment with estradiol-benzoate (sc; days 70-100; n = 6). Sections were fixed in paraformal dehyde and hybridized with saturating concentrations of an antisense (or sense) oligodeoxynucleotide complementary to bases 297-326 of human POMC mRNA that was 3' end-labeled with [S-35]dATP. After stringent washes, sections were placed against Kodak X-Omat film (Eastman Kodak, Rochester, NY) and then dipped in Kodak NTB-2, developed, and counterstained. POMC mRNA (antisense minus sense) was quantified by densitometry and image analysis of silver grains. Specificity of labeling was documented by selective distribution of grains over a dispersed population of cells in sections of anterior pituitary hybridized with antisense, the relative absence of grains in sections incubated with sense, and the absence of grains in neurohypophyseal sections incubated with antisense. Moreover, silver grains were not visible when sections were pretreated with excess radioinert probe. The mean (+/- SE) maternal serum estradiol concentration in baboons treated with estradiol benzoate at midgestation (2.9 +/- 0.4 ng/ml) was greater (P < 0.05) than that in untreated baboons on day 100 (1.0 +/- 0.3) but not significantly different from that in late gestation (1.9 +/- 0.3). In umbilical serum, estradiol concentrations were greater (P < 0.05) at term (3.7 +/- 0.9) than at midgestation (0.7 +/- 0.2) but, unlike maternal values, were not significantly increased at midgestation after treatment of the mother with estradiol (1.1 +/- 0.2). Based on densitometric analysis, mean (+/- SE) pituitary POMC mRNA (absorbance units) was greater (P < 0.05) in baboon fetuses at term (0.57 +/- 0.05) than at midgestation (0.28 +/- 0.03) and increased (P < 0.05) on day 100 (0.43 +/- 0.04) in estrogen-treated animals. Similar results were obtained when data were analyzed as the number of silver grains/0.025 mm(2). Using a rabbit polyclonal antibody to human ACTH, immunohistochemical studies confirmed that cellular expression of ACTH peptide paralleled changes in POMC mRNA. Thus, the mean number of fetal pituitary cells expressing ACTH peptide was greater (P < 0.05) at term (424 +/- 21) than at midgestation (267 +/- 13) and increased (P < 0.05) at midgestation after maternal treatment with estradiol (355 +/- 8). We conclude that the onset of fetal adrenal steroidogenic maturation normally near term and prematurely at midgestation after estrogen administration reflects increased expression of POMC mRNA and consequently ACTH by the fetal pituitary.