0031-3998/01/5002-0210 PEDIATRIC RESEARCH Vol. 50, No. 2, 2001 Copyright © 2001 International Pediatric Research Foundation, Inc. Printed in U.S.A. Role of Hypothalamic-Pituitary Axis in EGF Action on Maturation of Adrenal Gland in Fetal Rhesus Monkey In Vivo CATHERINE L. COULTER, LEANNA C. READ, SEAN J. BARRY, ALICE F. TARANTAL, AND DENNIS M. STYNE Department of Physiology, University of Adelaide, Adelaide, SA, Australia, 5005 [C.L.C.]; Child Health Research Institute, North Adelaide, SA, Australia 5006 [L.C.R.]; and Department of Pediatrics [S.J.B., A.F.T., D.M.S.] and California Regional Primate Research Center [A.F.T.], University of California, Davis, Davis, California 95616, U.S.A. ABSTRACT We determined the route of action of epidermal growth factor aldosynthase immunoreactivity was induced in the DZ. EGF (EGF) [intraperitoneal (IP) versus intraamniotic administration] intraamniotic administration had no significant effect on the width of on adrenal development and whether its effects are mediated via the DZ or cortical width of 3␤HSD staining compared with controls. the fetal hypothalamic-pituitary axis in the fetal rhesus monkey These data suggest that EGF acts via the hypothalamic-pituitary axis in vivo. EGF (40 ␮g) was administered IP (n ϭ 9) or intraam- to modulate adrenal cortical growth and functional maturation of the niotic (n ϭ 6) at 121, 123, 125, and 127 d gestation (term, transitional zone (the putative zona fasciculata), whereas EGF can approximately 165 Ϯ 10 d gestation). In addition, a competitive act independently of the hypothalamic-pituitary axis to stimulate 12 corticotropin-releasing factor antagonist ([D-phenylalanine , functional maturation of the DZ (the putative zona glomerulosa). Norleucine21,38] corticotropin-releasing factor12–41 to block fetal (Pediatr Res 50: 210–216, 2001) pituitary ACTH secretion; 400 ␮g IP) and metyrapone (11␤- hydroxylase inhibitor to block adrenal cortisol synthesis; 15 mg Abbreviations IP and 15 mg intraamniotic) were administered, in combination ACTH, adrenocorticotropic hormone with EGF (EGFϩBLOCK; 40 ␮g IP; n ϭ 4 fetuses). Control CYP11B2, aldosynthase (EC 1.14.99) fetuses (n ϭ 6) received saline injections in an equivalent 3␤HSD, 3␤-hydroxysteroid dehydrogenase (EC 1.1.) volume. On gestational d 128, a hysterotomy was performed, and CRF, corticotropin-releasing factor fetal adrenals were collected for morphometric analyses and CYP11B1, cytochrome P-450 11␤-hydroxylase (EC 1.14.15.4) immunocytochemical localization of 3␤-hydroxysteroid dehy- CYP11B, cytochrome P-450 11␤-hydroxylase/aldosynthase drogenase (3␤HSD) and cytochrome P-450 11␤-hydroxylase/ CYP11B-ir, cytochrome P-450 11␤-hydroxylase/aldosynthase aldosynthase. Definitive zone (DZ) width and cortical width of immunoreactivity 3␤HSD staining were significantly greater (p Ͻ 0.05) in the EGF DZ, definitive zone 12 21,38 12,41 IP-treated fetuses compared with controls and EGFϩBLOCK. BLOCK, [D-phenylalanine , Norleucine ] CRF plus With EGF IP, 3␤HSD was increased in the DZ and induced metyrapone extensively in the transitional zone of the fetal adrenal cortex, EGF, epidermal growth factor and cytochrome P-450 11␤-hydroxylase/aldosynthase immuno- FZ, fetal zone reactivity was induced to detectable levels in the DZ. The H&E, hematoxylin and eosin administration of EGFϩBLOCK inhibited the expression of IA, intraamniotic 3␤HSD in the transitional zone, but 3␤HSD expression was still TGF␣, transforming growth factor-␣ increased in the DZ and cytochrome P-450 11␤-hydroxylase/ TZ, transitional zone The development of the steroidogenic capacity of the fetal homeostasis, development of enzyme systems necessary for adrenal gland is necessary for the maintenance of intrauterine extrauterine life, and, at least in some species, the timing of Received January 20, 2000; accepted January 17, 2001. 990275 to C.L.C.), an NHMRC Block Grant to the Baker Medical Research Institute, and Correspondence and reprint requests: Catherine L. Coulter, Ph.D., Department of by NIH Grants HD17814, HD24959, and HD 08478. Physiology, University of Adelaide, North Terrace, Adelaide, South Australia, Australia, Presented in part, at the X International Congress on Hormonal Steroids, Quebec, 5005; e-mail: [email protected] Canada, June 1998. Supported by a Jean B. Reid Fellowship, the Faculty of Medicine, University of Current address (S.J.B.): The Rowe Program in Genetics, University of California, Adelaide (C.L.C.), the National Health and Medical Research Council of Australia (Grant Davis, Davis, CA 95616, U.S.A. 210 MATURATION OF THE PRIMATE FETAL ADRENAL 211 parturition. Secretion of cortisol from the fetal adrenal is EGF administration and the mode of action of EGF on adrenal necessary for the maturation of essential organ systems and growth variables using morphometric techniques and deter- plays a key role in the fetal response to intrauterine stress mined the pattern of localization of 3␤HSD and CYP11B in the (1–5). After birth, the secretion of aldosterone from the new- fetal rhesus adrenal using immunocytochemical techniques. born adrenal is a key component of the renin-angiotensin system and is essential for the maintenance of fluid balance and blood pressure during postnatal life. Previous studies in the METHODS human and nonhuman (rhesus monkey) primate fetus have indicated that the adrenal gland comprises three functional Animals and EGF administration. Twenty-five pregnant zones: 1) the large, inner FZ, which has the enzymes necessary rhesus monkeys (Macaca mulatta) were obtained from the for dehydroepiandrosterone sulfate production beginning early time-mated breeding colony at the California Regional Primate in gestation; 2) the TZ between the inner FZ and outer DZ, Research Center, University of California, Davis, CA, U.S.A. which possesses enzymes necessary for cortisol production Animals were maintained in accordance with the National beginning in late gestation; and 3) the outer, DZ, which appears Institutes of Health Guide for the Care and Use of Animals, to function as a reservoir of progenitor cells, which may and the protocols used in this study were approved by the populate the remainder of the gland, and does not acquire a Institutional Animal Use and Care Committee at the University steroidogenic phenotype with the capacity to produce miner- of California, Davis. Before IA or IP injections, the dams were alocorticoids until near term (1, 2, 6–13). Recently, we have administered ketamine hydrochloride (10 mg/kg), and EGF shown that timing of the induction of 3␤HSD in the TZ and administration was performed with ultrasound guidance as CYP11B-ir in the DZ are coincident with increased circulating described previously (18). Recombinant human EGF was pro- concentrations of cortisol and aldosterone in late gestation vided by Chiron (Emeryville, CA, U.S.A.). EGF (40 ␮gin1 (12). These data suggest that functional maturation of the fetal mL of saline) was administered via the IP route (n ϭ 9) or IA adrenal cortex is dependent on the specific zonal expression of (n ϭ 6) at 121, 123, 125, and 127 d gestation (term, 165 Ϯ 3␤HSD and CYP11B, which are pivotal enzymes in adrenal 10 d). In four fetuses, a competitive CRF antagonist ([D- steroidogenesis (1, 2). Thus, to understand the functional mat- phenylalanine12, Norleucine21,38] CRF12–41 to block fetal pi- uration of the primate fetal adrenal, it is important to determine tuitary ACTH secretion; 400 ␮g IP) and metyrapone the factors that regulate induction of 3␤HSD and CYP11B (CYP11B1 inhibitor to block adrenal cortisol synthesis; 15 mg expression. IP and 15 mg IA) were also administered, in combination with Recently, we have demonstrated that administration of EGF EGF IP (EGFϩBLOCK; 40 ␮g) at the same gestational times. into the fetal peritoneal and amniotic fluid cavities stimulated The CRF antagonist was provided by Dr. Jean Rivier [The Salk adrenal cortical growth and the expression of 3␤HSD in the Institute, San Diego, CA, U.S.A (19)], and the dose chosen was adrenal cortex of the fetal rhesus monkey in vivo (14). Studies previously shown to be effective in the fetal rhesus monkey at have shown that EGF can stimulate the secretion of CRF from modulating pituitary ACTH secretion by blocking the fetal the hypothalamus and ACTH from the pituitary (15). The hemorrhage-induced increase in fetal plasma ACTH concen- presence of specific receptors for EGF on both DZ and FZ cells trations (1, 20). Metyrapone treatment was included in the has been demonstrated, and EGF stimulates proliferation of experimental paradigm, to determine whether the stimulatory human fetal adrenal cells in vitro (16). It has also been shown effects of EGF treatment observed on fetal lung maturation (18, that EGF stimulates secretion of cortisol and aldosterone as 21–23) were direct or mediated via the fetal adrenals. The data well as angiotensin II–induced aldosterone secretion from the on the effects of EGFϩBLOCK, EGF IP, or EGF IA on lung adrenal gland (17). Thus, EGF may act both directly and development are the subject of a separate publication (in indirectly via the fetal hypothalamic-pituitary axis to stimulate preparation). In six control fetuses, saline was administered (1 adrenal growth and steroidogenesis. In the rhesus monkey, mL of saline IP and 1 mL of saline IA). At 128 d gestation amniotic fluid contains substantial amounts of a factor that (78% of gestation), hysterotomies were performed, and fetal binds to the EGF receptor in radioreceptor assays. This could tissues were collected for analysis (see below), as described reflect EGF or TGF␣, the latter having been identified in previously (18). Body and adrenal weights were obtained, and amniotic fluid in other studies. TGF␣ is a member of the EGF fetal adrenals were fixed in formalin (10%) for morphologic family and acts specifically via the EGF receptor (15). In these and immunocytochemical analyses as described (14). studies, we have used EGF, rather than TGF␣, because it was Immunocytochemistry.