In the ovine fetus, adrenalectomy at 90-120 days gestational age (dGA) results in a gradual increase in basal concentrations of fetal plasma ACTH beginning at -122 dGA. Bilateral adrenalectomy at 116-119 dGA also results in an increase in POMC mRNA in the fetal pituitary. It is not known whether both the paraventricular nuclei (PVN) of the hypothal-amus and the anterior pituitary of the ovine fetus are responsive in late gestation to the removal of cortisol negative feedback. The purpose of this study was to determine the subsequent effect of fetal adrenalectomy at 118-121 dGA on the CRH mRNA content in fetal PVN and on POMC mRNA in the fetal anterior pituitary at 134 dGA. Mature Rambouellet-Columbia cross-bred ewes (n = 10), bred on a single occasion only and carrying fetuses of known gestational ages, were used. Both fetal adrenal glands were exposed via a retroperitoneal approach and removed [ad- renalectomized (ADX); n = 5]. In control fetuses (CONT; n = 5) adrenal glands were exposed and isolated, but not removed. At 134 dGA, fetal plasma cortisol concentrations were significantly greater in CONT fetuses (7.2 ± 2.5 ng/ml) than in ADX fetuses (mean ± SD, 1.97 ± 0.9 ng/ml; P < 0.025). At 134 dGA the fetal PVN was removed by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was prepared by the guanidi- nium isothiocyanate-cesium chloride method and subjected to Northern analysis using specific cDNA probes to CRH and POMC. After autoradiography, quantification of mRNA was performed by scanning densitometry. Quantities of specific hy-bridization signal for POMC and CRH were normalized to the content of actin mRNA in each individual sample. RNA prepared from PVN exhibited a single specifically hybridizing band for CRH of -1300 nucleotides. RNA prepared from anterior pituitary exhibited a single specifically hybridizing band for POMC at -1300 nucleotides. Anterior pituitary POMC mRNA was significantly increased (P < 0.025) in ADX fetuses (236 ± 32% of CONT). CRH mRNA in PVN was greater in ADX fetuses than in CONT fetuses (P < 0.05; mean ± sem, 179 ± 21% of CONT). Adrenalectomy in fetal sheep significantly increased expression of CRH and POMC. We conclude that the increased levels of mRNA for CRH and POMC indicate that both the fetal PVN (CRH) and the anterior pituitary (POMC) are responsive to removal of the primary source of circulating glucocorticoid at this gestational age. This provides evidence that increased fetal plasma ACTH after fetal adrenalectomy may be mediated at both the hypothalamic and anterior pituitary levels through increased expression of POMC and CRH.
ASJC Scopus subject areas