0163-769X/90/1101-0151$02.00/0 Endocrine Reviews Copyright © 1990 by The Endocrine Society
Vol. 11, No. 1 Printed in U.S.A.
Regulation of the Primate Fetal Adrenal Cortex* GERALD J. PEPE AND EUGENE D. ALBRECHT Department of Physiology (G.J.P.), Eastern Virginia Medical School, Norfolk, Virginia 23501; and Department of Obstetrics and Gynecobgy and Department of Physiology (E.D.A), University of Maryland School of Medicine, Baltimore, Maryland 21201
I. Physiological Significance of the Fetal Adrenal II. Fetal Adrenal Development III. Regulation of Fetal Adrenal Growth A. Role of fetal pituitary B. POMC-derived peptides C. ACTH D. Placental factors E. Fetal growth factors IV. Regulation of Fetal Adrenal Steroidogenesis A. Substrate for fetal adrenal steroid production 1. LDL pathway 2. De novo biosynthesis B. Enzyme regulation of fetal adrenal steroidogenesis C. Multifactorial regulation of fetal adrenal steroidogenesis 1. Role/effect of ACTH and other peptides 2. Protein kinase A and protein kinase C pathways 3. Role of calcium 4. Peptide growth factors 5. Modulating effect of estrogen V. Placental Metabolism of Cortisol: Relationship to Maturation of the Fetal Adrenal Gland VI. Summary
enzymes in the fetal brain, retina, pancreas, and gastrointestinal tract (12-17) that normally are associated with late intrauterine life. It is well known, from the elegant work of Liggins and colleagues (7), that ablation of the fetal adrenal in sheep prevents parturition, whereas infusion of cortisol or ACTH1 to the fetus induces premature delivery. Although evidence that the fetal adrenal of the human or nonhuman primate participates in the initiation of labor appears less convincing (5, 8, 18-20), maturation of fetal adrenal steroidogenic enzyme systems that permit de novo synthesis of cortisol (5, 21, 22) must occur, as in sheep (23, 24), to ensure neonatal adrenocortical self-sufficiency in the perinatal period. In addition to these purported roles for fetal adrenal cortisol, it is well established that the fetal adrenal in primates, including the human, is important to the synthesis and secretion of androgen precursors essential to the production of estrogen by the placenta (25-32). It is apparent, therefore, that the fetal adrenal is important to several physiological homeostatic mechanisms and maturational events operative during fetal development. Thus, aberrations in pituitary-adrenal function in fetal life could have important consequences on fetal development and the onset of delivery. An understanding of the factors regulating growth, maturation, and hormonogenesis of the fetal adrenal, therefore, appears to be a prerequisite to formulating methods for reducing the incidence of abnormal human pregnancy
I. Physiological Significance of the Fetal Adrenal
I
N MOST mammalian species, products of the fetal adrenal gland appear to play an important role in regulating maturation of various organ systems in the fetus (1-5), providing the fetus homeostatic mechanisms to respond to stress (6), and initiating and/or participating in the cascade of events culminating in the birth of a newborn (7-9). Thus, cortisol, presumably of fetal adrenal origin, is one of the chemical messengers involved in the stimuli to lung maturation (3,4), deposition of glycogen in the liver (10, 11), and induction of several
1 The following abbreviations have been used in this article: ACTH, adrenocorticotropic hormone; CLIP, corticotropin-like intermediate lobe peptide; CRF, corticotropin releasing hormone; DHA, dehydroepiandrosterone; DHAS, dehydroepiandrosterone sulfate; MSH, melanocyte stimulating hormone; POMC, proopiomelanocortin; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor I; FGF, fibroblast growth factor; IGF-I, insulin-like growth factor I; IGF-II, insulinlike growth factor II; PDGF, platelet-derived-growth factor; TGF«, transforming growth factor a; TGF/3, transforming growth factor /?; 11/3-HSD, 11/3-hydroxysteroiddehydrogenase; 3/3-HSD, 3/?-hydroxysteroid dehydrogenase; HMG CoA, 3-hydroxy-3-methyl-glutaryl coenzyme A; HST, hydroxysteroid-sulfotransferase; P45011/?, 11/3-hydroxylase; P450i7a, 17a-hydroxylase; P450C2i, 21-hydroxylase; P4508CC, P450-cholesterol side chain cleavage; HDL, high density lipoprotein; LDL, low density lipoprotein; TPA, 12-O-tetracanoyl phorbol 13-acetate.
Address request for reprints to: Dr. Gerald J. Pepe, Department of Physiology, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, Virginia 23501-1980. * This work was supported by National Institutes of Health Grant HD-13294. 151
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
PEPE AND ALBRECHT
152
and thus neonatal morbidity and mortality. This review will attempt to summarize the present status of our understanding of the regulation of the fetal adrenal in humans and the more extensively studied nonhuman primate models of human pregnancy, namely the baboon and rhesus monkey. Attention will be focused on developing the perspective that fetal adrenal development is influenced by factors of uteroplacental origin and perhaps integrated with and/or directed by metabolic systems in the trophoblast and/or decidua. II. Fetal Adrenal Development During the course of gestation in humans (33, 34) and nonhuman primates (21, 35-38), the fetal adrenal gland not only undergoes extensive anatomical and biochemical changes but also exhibits a remarkable rate of growth, primarily during the final third of intrauterine development (Fig. 1). In the baboon and rhesus monkey, and presumably the human, increased weight is associated with marked growth of the inner zone (fetal cortical zone) which comprises between 80 and 90% of the gland during the majority of gestation. According to McClellan and Brenner (39), fetal cortical cells reach maximal size late in gestation with a marked increase in the number and size of the mitochondria and an expansion of smooth endoplasmic reticulum accounting for the majority of the increase in cytoplasmic volume. In addition, there is no apparent evidence of lipid droplet depletion or imminent cell death or necrosis with advancing gestation. Of particular interest, vascularization within the fetal zone is extensive and clearly exceeds that in the outer, adulttype cortical zone throughout gestation (39).
Vol. 11, No. 1
While there appears to be minimal maturation of the outer cortex during the majority of intrauterine development, by late gestation there is the initiation of substantial development of the adult-type definitive zone or neocortex in humans (40-42), baboons (21), and rhesus monkeys (39). Thus, by day 150 of gestation in the rhesus monkey (term = day 165) a presumptive zona glomerulosa and differentiation of cells into a zona fasciculata are evident (39). It is well established that the activity of the enzyme A5-3/3-hydroxysteroid dehydrogenase-isomerase (3/3HSD) is minimal during most of intrauterine development in the fetal adrenal of humans (43-47), baboons (21), and rhesus monkeys (43, 48, 49), as has been demonstrated in the fetal adrenal of the sheep (23, 50). Moreover, based on studies of human fetal adrenals obtained on weeks 18-24 of gestation and cultured in the absence of serum additives, the activity of 3/3-HSD is only 3-fold higher in the outer adult-type definitive tissue as compared with that in fetal cortex (51, 52). Consequently, throughout most of intrauterine development, the primate fetal adrenal has a limited capacity to synthesize A4-ketosteroids, such as cortisol, from precursors such as pregnenolone (53). In contrast, the activities of the enzymes catalyzing hydroxylations at steroid carbons 17, 21, and 11 (21, 54-56), as well as messenger RNAs (mRNAs) for the 17- and 21-hydroxylases (57-59), appear to be present earlier in gestation. These findings suggest that the baboon and monkey fetal adrenal have the capacity to synthesize cortisol from placental progesterone, as has been suggested in humans (60-62). However, based on in vivo studies in the baboon (63) and rhesus monkey (64), less than 5% of the cortisol meas1 900
FlG. 1. Changes in the DHAS concentrations (mean ± SE) in fetal plasma and in weight of fetal adrenals in the fetus of the rhesus monkey from midgestation to term. [Reproduced with permission from M. Seron-Ferre et al.\ J Clin Endocrinol Metab 57:1173, 1983 (37). © The Endocrine Society.]
E
2000
£
1500
O
GESTATIONAL AQE DAYS
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
February, 1990
FETAL ADRENAL DEVELOPMENT
ured in fetal plasma during gestation is derived from circulating progesterone. Thus, at least during early to midgestation, the fetus must obtain the majority of its cortisol from the maternal circulation. Indeed, in humans (65, 66), baboons (67), and rhesus monkeys (68, 69) there is extensive transplacental passage of maternal cortisol into the fetal compartment throughout gestation. Very late in gestation in the baboon (21), rhesus monkey (49, 70), and presumably the human (43-45, 71), there is a marked increase in fetal adrenal 3/3-HSD activity. Increased enzyme activity was associated with a rise in the serum concentrations of cortisol in the baboon fetus (67), and in umbilical artery obtained at select times during human gestation (72). A rise in fetal serum cortisol was also demonstrable in chronically catheterized rhesus monkeys studied longitudinally over the course of gestation (48). The increase in circulating cortisol in the baboon (67) and human (72) fetus late in gestation reflects the ontogenesis of fetal adrenal production and secretion and is not the result of placental transfer of maternal cortisol (65, 67) or alterations in the MCR of cortisol (73) due to changes in the concentrations of plasma binding proteins (74, 75) or patterns of fetal tissue metabolism (76). Moreover, studies in humans at midgestation (77), rhesus monkeys at term (69), and baboons at midgestation (78) and term (73) indicate that the ability of the primate fetus to utilize circulating cortisone as a precursor for the production of cortisol is also negligible. Thus, near term the primate fetal adrenal develops the capacity to synthesize cortisol, and there is an apparent increase in fetal cortisol concentrations due primarily to increased production by the fetal adrenal. It is apparent, based on the enzymatic profile of the fetal adrenal throughout most of intrauterine development, that quantitatively the major steroid products of the human (79-81) and nonhuman primate fetal adrenal (37, 48, 82-84) are the A5-hydroxysteroid androgen dehydroepiandrosterone (DHA) and its sulfurylated derivate DHA sulfate (DHAS). In the fetal rhesus monkey (Fig. 1), the serum levels and daily production rates of DHAS, which is formed primarily by the fetal cortical tissue, increase steadily during gestation and parallel fetal cortical growth (37). III. Regulation of Fetal Adrenal Growth A. Role of fetal pituitary Although the growth and differentiation of the definitive and fetal cortical zones of the human and nonhuman primate fetal adrenal are striking features of intrauterine development, our understanding of the factors regulating these processes is incomplete. It is well established that at mid to late gestation fetal adrenal size and weight in anencephalic human (41) and rhesus monkey (31) fetuses
153
are considerably reduced when compared with normal. Moreover, fetal adrenal atrophy and reduced steroidogenesis occur after treatment with glucocorticoids late in gestation in humans (81), rhesus monkeys (85, 86), and baboons (82, 87, 88). Collectively, these findings suggest that factors of fetal pituitary origin are important to fetal adrenal growth at this time in gestation. Indeed, fetal adrenal weight (13 ± 3 mg/100 g BW) in rhesus monkeys in which the fetal pituitary was removed on day 115 of gestation (term = day 165) was 85% lower than that (88 ± 16 mg/100 g BW) of sham-operated intact fetuses (89). Similar observations have been made in the fetal lamb (7) in which the maturation of immature fetal fascicular cells was prevented by removal of the fetal pituitary (90). Because treatment of hypophysectomized fetal lambs with ACTH reversed most of the effects of pituitary ablation, it appears that in this species fetal pituitary ACTH is critical to fetal adrenal development during the second half of gestation. While this may also be true in the human and nonhuman primate, it has not been unequivocally established, due in part to the absence of in vivo studies in nonhuman primate models and the inherent limitations with in vivo studies that have been performed in humans. For example, in studies of human anencephalics administered ACTH either during the second or third trimester (40, 91, 92) or in the immediate postnatal period (93-95) the number of patients was small, and for ethical reasons appropriate controls were limited. B. POMC-derived peptides Studies of Silman and colleagues (96, 97) indicate that there are several POMC-derived peptides in addition to ACTH present in the human fetal pituitary gland. Similar observations have been made in the fetal rhesus monkey in which desacetyl as well as mono and diacetyl a-MSH are specifically localized in the neurointermediate lobe by day 65 of gestation (98). Moreover, there appears to be a relative increase in the mono and diacetyl forms of a-MSH during the second half of gestation in the fetal rhesus monkey (98). A peptide consisting of XMSH with a carboxy terminal extension of 15 additional residues has also been isolated from the pituitary of the adult rat (99). This material, denoted X3-MSH, has the capacity to modulate adrenal function in the rat by stimulating pathways different from those responsive to ACTH!_24. In addition, compounds resembling a-MSH and corticotropin-like intermediate lobe peptide (CLIP) are quantitatively the dominant hormones in the human fetus after week 20 of gestation and may only be replaced by ACTH just before parturition (96). Based on these observations, Silman et al. (96) have suggested that several POMC-derived pituitary peptides, other than
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
154
PEPE AND ALBRECHT
ACTH, may serve to drive the fetal cortical zone of the adrenal during the course of pregnancy. In contrast, it was suggested that ACTH may be important only to the definitive zone. Thus, Silman et al. (96) have proposed that the switch in pituitary peptide synthesis to increased ACTH just before parturition may be a central mechanism in the chain of events leading to adrenal maturation and neonatal adrenocortical self-sufficiency. This hypothesis is interesting, because MSH and CLIP are thought to be synthesized primarily in the pars intermedia (100), a structure that is present in rudimentary form in normal human fetuses (101), and which disappears after birth. Indeed, a-MSH is not present in the anterior lobe of either the fetal or adult rhesus monkey pituitary gland (98). Moreover, a pars intermedia is only found occasionally in anencephalic fetuses (102), and there is evidence that when present, it is structurally (103) and functionally (104) abnormal. More recently, the studies of Facchinetti et al. (105) suggest that by the end of the second trimester, the processing of pituitary POMC in normal human fetuses is similar to that in the adult, and the functional activity of the anterior lobe appears to prevail over that of the "fetus-related" neurointermediate lobe around week 25 of gestation. C. ACTH Several investigators have examined the effects of ACTH on fetal adrenal maturation in vitro. After culture of collagenase-dispersed human fetal adrenal cells obtained early in gestation, there is marked proliferation of the adult-type definitive cells and an apparent involution and subsequent disappearance of the large fetal cortical cells (106-108). Under the culture conditions employed by Kahri and co-workers (106, 107), addition of ACTH increased the rate of growth and the cytoplasmic size (i.e. hypertrophy) of the definitive cells over an 8-day period but had no apparent effect on fetal cortical cells. Roos (109) demonstrated that ACTH enhanced growth and protein concentration of cultured fetal adrenal cells maintained for more than 10 days in 10%, but not in 20% fetal calf serum (FCS), while ACTH enhanced corticosteroid secretion in 10% and 20% FCS. These findings were interpreted to indicate that factors in fetal serum may modulate the effect of ACTH on growth of adrenal cells. In contrast to the stimulatory effects of ACTH, it has been well established that ACTH inhibits mitosis of adult human and bovine adrenocortical cells in culture (110-112), and ACTH has been reported to have inhibitory effects on human fetal definitive and cortical cells cultured for 6 days (113, 114). Clearly, therefore, the role of ACTH per se on growth (hyperplasia) and differentiation of fetal adrenal cortical cells appears tenuous. Rather, ACTH or other POMC-
Vol. 11, No. 1
derived peptides of fetal pituitary origin may interact with additional factors either produced in the adrenal itself or in other fetal and/or perhaps placental tissues to modulate adrenal growth during most of intrauterine development. In human anencephalic fetuses, the fetal adrenal gland develops normally up to 14-17 weeks of gestation and involutes sometime before week 20 (115-117), supporting the suggestion that extrapituitary factors perhaps of maternal and/or placental origin are important to adrenal maturation at least early in gestation. However, because the involution and/or remodeling of fetal cortical cells only ensues after delivery in neonates with normal pituitary function, it appears that placental and/or maternal factors may actually modulate fetal cortical development throughout the course of pregnancy. D. Placental factors
Several growth factors or oncogene-related peptides that may be important to fetal adrenal growth have been isolated from the human placenta, including transforming growth factors [TGFs a (118) and 0 (119)], fibroblast growth factor, [FGF (120)], platelet-derived-growth factor [PDGF (121)], placental GH (122), epidermal growth factor [EGF (121)], insulin-like growth factor I [IGF-I (123)], IGF-II (124), and nerve growth factor (125). In addition, a 34-kilodalton (KDa) growth factor exhibiting receptor binding specificity and antigenic structure quite different from placental FGF, TGFa, TGF/3, and PDGF, has recently been isolated from brush-border membrane preparations of human term placenta (126, 127). Simonian and Gill (113) and Crickard et al. (128) demonstrated that EGF and FGF have the capacity to stimulate growth of the human fetal adrenal in culture, although the mitogenic effect of these growth factors appeared greater on fetal definitive than on fetal cortical cells. The concentration of EGF required to elicit half-maximal stimulation of cell proliferation of both zones was considerably lower than the concentration of EGF required to saturate adrenal EGF receptors, suggesting that only a small percentage of occupied EGF binding sites is required to effect a maximal proliferative response. Of particular interest, definitive but not fetal cortical cells responded to EGF and FGF when the concentration of FCS in the medium was reduced from 10% to 1% (Fig. 2). This implies that EGF and FGF may interact with other factors present in serum to promote mitogenesis, particularly in the fetal cortex. Apparently ACTH is not one of the factors, because it attenuated the mitogenic effects of FGF and EGF on both definitive and cortical cells (113, 128). The mitogenic effects of EGF observed in vitro may also occur in vivo. Thus, in a preliminary report, Addiego et al. (129) demonstrated that fetal ad-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
February, 1990 2.5
FETAL ADRENAL DEVELOPMENT
factors involved, their regulation, secretion into the fetus, and potential interactions with the POMC-derived fetal pituitary peptides, particularly ACTH in vivo, remain to be elucidated.
- DEFINITIVE ZONE
2.0 (~"]i0% SERUM
m o
•
E. Fetal growth factors
i% SERUM
5 1-5
1.0 FETAL ZONE
0.5
ll fl CONTROL EGF
155
FGF CONTROL EGF
FGF
FIG. 2. Relationship between FGF, EGF, and serum concentration on culture growth. Separated human fetal adrenal cells were plated at 6000 cells per 3.5-cm dish in the presence of 1% or 10% fetal calf serum. EGF (20 ng/ml) or FGF (100 ng/ml) were added every other day. On day 9, the cells were trypsinized and counted. The results are expressed as the mean of duplicate plates; SD did not exceed 10% of the mean. [Reproduced with permission from K. Crickard et al.: J Clin Endocrinol Metab 53:790, 1981 (128). © The Endocrine Society.]
renal weight and maturation in rhesus monkeys were significantly increased after intraamniotic injection of EGF over a 7-day period at midgestation. Whether other factors similarly administered would be equally effective remains to be determined. Simonian et al. (130) have recently shown that an aqueous extract of media from cultures of human placenta stimulated proliferation of human definitive zone cells in culture. The effects of this extract of placentalderived mitogenic factor(s), as with purified EGF and FGF, were inhibited by concomitant treatment with ACTH. Branchaud and colleagues (131) have also demonstrated that conditioned media from cultures of human placenta (PM) stimulated fetal adrenal cell proliferation. According to these workers, PM was 2 to 5 times more potent than EGF or FGF, effective in the absence of serum, and not inhibited by ACTH. Based on these observations, plus the fact that conditioned PM is partially heat resistant, Branchaud et al. (131) have proposed that the active components in PM are not EGF, FGF, or TGFa. Moreover, because adrenal cortical growth in the presence and absence of exogenous EGF was inhibited by TGF/3, apparently TGF/3 is not one of the active components of PM. It is apparent, therefore, that the placenta may be a source of a variety of growth factors capable of modulating fetal adrenal mitogenesis. However, the requisite
Although the placenta has the capacity to provide the fetus with growth factors, it has recently become apparent that several fetal tissues, including the adrenal, have the biochemical machinery to produce peptide growth factors. Using in situ hybridization histochemistry, Han et al. (132) have shown that mRNAs for IGF-I and IGFII were measurable in 14 organs of the human fetus including the kidney, liver, and adrenal. These workers more recently demonstrated (133) that mRNA for IGFII in the fetal adrenal was present at concentrations 200fold greater than the mRNA for IGF-I. Of particular interest, all of the hybridizing regions appeared to be comprised predominantly of fibroblasts or other cells of mesenchymal origin. As pointed out by Han et al. (132), because these cells are widely distributed and anatomically integrated they are ideally located for the production of growth factors that may exert autocrine and/or paracrine effects on nearby target tissues, as originally suggested by D'Ercole and colleagues (134). Thus, Branchaud et al. (131) have shown that conditioned media of human fetal liver, lung, and kidney have the capacity to stimulate fetal adrenal mitogenesis, although at a rate lower than that of placental media. Voutilainen and Miller (135) demonstrated that the expression of the mRNA for IGF-II in the human fetal adrenal can be stimulated by concomitant treatment with ACTH. DiBlasio et al. (136) have also shown that the human fetal adrenal expresses message for basic FGF (bFGF); expression is increased by ACTH. These observations are of particular interest in light of previous studies (113, 128) demonstrating mitogenic effects of FGF on human fetal adrenal definitive and cortical cells in culture. What remains to be explained is the apparent inhibition of adrenal mitogenesis clearly exhibited by ACTH in vitro (113). Recently, McAllister and Hornsby (137) demonstrated that the phorbol ester 12-O-tetradecanoyl phorbol 13acetate (TPA), which activates protein kinase C by substituting for the physiological activator diacylglycerol (138), is also a mitogen for fetal human adrenal definitive zone cells in culture. Because IGF-I has the capacity to activate protein kinase C via increased hydrolysis of glycosyl-phosphatidyl inositols (139), factors capable of stimulating IGF-I or protein kinase C directly in the fetal adrenal may have profound effects on growth and differentiation. Rainey et al. (140) have demonstrated the presence of protein kinase C in the human fetal adrenal
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
and indicate that both the activity and content of this molecule are more abundant in the fetal than the definitive zone (Fig. 3). Interestingly, protein kinase C activity and content are very low in fetal adrenal tissues of anencephalics. The potential impact of protein kinase C as a promoter of fetal adrenal growth takes on additional import in light of recent observations by McAllister and Hornsby (141, 142), indicating that protein kinase C activation profoundly influences the ratio of enzymes regulating steroidogenesis in the human fetal adrenal gland. In summary, it would appear that regulation of fetal adrenal growth and maturation is extremely complex and may involve the collaborative actions of the fetal pituitary and placenta and perhaps other fetal tissues capable of producing and secreting growth factors. Moreover, growth may be modulated by cAMP-independent mechanisms, including protein kinase C. Because of the different profile of A and C kinases in definitive and cortical cells, at least during early gestation, as well as the apparent exclusivity of the adult adrenal to ACTH, it is possible that growth in these two different cellular compartments of the fetal adrenal is regulated differently. Indeed, the marked growth and vascularization of the fetal cortex that occur throughout gestation do not appear to be associated with a concomitant growth of the •
B
\
C
0
E
F
definitive zone, which occurs only very late in gestation and continues into the perinatal period, a time during which there is marked remodeling of the fetal cortex. In addition, because several of the studies on growth of the human fetal adrenal have been performed on tissue obtained primarily during early gestation, the responsivity of the gland to mitogens during late gestation is poorly understood. Thus, the possibility that mitogenic responsivity to trophic factors changes with advancing gestation cannot be excluded. Indeed, in the baboon, the patterns of fetal adrenal DHA/DHAS production, as well as responsivity to pituitary peptides both in vitro (22, 143145) and in vivo (83, 146), are markedly different at midgestation and term.
IV. Regulation of Fetal Adrenal Steroidogenesis A. Substrate for fetal adrenal steroid production 1. Low density lipoprotein (LDL) pathway. Based primarily on in vitro studies utilizing human fetal adrenal tissues obtained early in gestation, it is apparent that cholesterol derived from circulating LDL produced in the fetal liver is a major substrate for fetal adrenal steroidogenesis (147-156). Thus, the quantity of DHAS and cortisol synthesized by human fetal adrenal cells in culture was increased as a function of the concentration of LDL present in the culture medium (Fig. 4 and Refs. 148 and 156). Consistent with these results are the earlier observations of Solomon et al. (60) who demonstrated using in situ perfusion that neither circulating acetate nor free cholesterol were efficient precursors for human
*
*C
20 -
•
Arbit
o
Vol. 11, No. 1
PEPE AND ALBRECHT
156
1
•
FETAL ZONE
NEOCORTEX
m
n
ANENCEPHALIC
FlG. 3. Protein kinase-C content in human fetal adrenal tissues. Protein (100 fig/lane) was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted using an antibody directed against protein kinase-C and [125I] protein-A. Visualization of the bands corresponding to protein kinaseC was accomplished by autoradiography. The autoradiograph is shown at the top, and the bars are the mean results from densitometric analysis of the 80K bands from two experiments; the individual results are presented by the dots. Lane A, neocortex; lane B, fetal zone; lane C, neocortex; lane D, fetal zone; lane E, anencephalic adrenal (31 weeks); lane F, anencephalic adrenal (40 weeks). The arrow indicates the location of radioactive protein (80,000) mol wt standard. [Reproduced with permission from W. E. Rainey et ai: J Clin Endocrinol Metab 67:908,1988 (140). © The Endocrine Society.]
100
200
300 400 LDL /xg/ml
500
600
FIG. 4. Cortisol and DHAS (DS) secretion by human fetal adrenal tissue on the sixth day of culture in the presence of ACTH plus LDL in concentrations ranging from 0-500 fig/ml The results presented are the mean ± SE of five replicates and are expressed as micrograms of steroid per mg tissue protein/24 h. [Reproduced with permission from B. R. Carr et al: Endocrinology 106:1854,1980 (148). © The Endocrine Society.]
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
February, 1990
FETAL ADRENAL DEVELOPMENT
fetal steroidogenesis. Similar conclusions have been deduced from in vivo studies in baboons (63) and rhesus monkeys (64) in which less than 5% of placental progesterone and pregnenolone secreted into the fetus were utilized for cortisol production between mid and late gestation. Pregnenolone does not appear to be a quantitatively significant precursor in the human as well, because the total amount of this precursor estimated to be used for steroidogenesis is less than 1% of the fetal production rate of DHAS (154, 155). In addition, pregnenolone sulfate, although present in much higher concentrations, is inefficiently converted to DHAS in the human fetal adrenal (154, 155, 157, 158). To evaluate the mechanism(s) by which LDL-cholesterol is utilized for steroidogenesis, Carr and colleagues (148-150) examined the uptake and degradation of radiolabeled LDL by human fetal adrenal tissue fragments cultured in the presence or absence of LDL and/or ACTH. Fetal adrenal LDL uptake was a saturable process, presumably mediated by a population of high-affinity low-capacity binding sites. Moreover, LDL degradation was inhibited by chloroquinone, an inhibitor of lysosomal enzymes. Carr et al. (148-150) have suggested, therefore, that LDL metabolism by the human fetal adrenal must occur by a process similar to that postulated (159) for other tissues, namely adsorptive endocytosis mediated by binding to cell surface receptors in which the fate of the internalized molecule is dependent upon lysosomal degradation. ACTH appears to be important in the regulation of this process. Thus, degradation of LDL by fetal adrenal tissue fragments (150, 160) was stimulated 3-fold when ACTH was added to the culture medium (Fig. 5). The degradation of LDL appears essential to ensuring a continuous supply of cholesterol to meet the
157
large demand for precursor required for DHAS and cortisol synthesis. To investigate the nature and regulation of the receptors involved in the endocytosis of LDL, the binding of LDL to membrane fractions prepared from adrenals of normal and anencephalic human fetuses was also determined (160, 161). Regardless of tissue source, binding was of high affinity with the apparent dissociation constant (kd) approximating 24 ng LDL-protein/ml. However, compared to values in tissue from anencephalics, not only were binding and degradation greater in normal fetuses, but only in normal tissue were the latter processes stimulated by ACTH (Fig. 6). Although there was substantial binding of high density lipoprotein (HDL) by the human fetal adrenal (162), HDL was not degraded, neither uptake nor degradation was modified by ACTH, and HDL did not support steroidogenesis efficiently. Collectively, these findings confirm that LDL-cholesterol is a major precursor for fetal adrenal steroidogenesis, that availability of precursor is regulated by specific membrane receptors on the adrenal cell that must be internalized and degraded, and that the concentration of receptors as well as uptake and internalization of ligand are ACTH-dependent processes. Because no more than 20% of cholesterol in the fetus is derived from the maternal compartment (163), Simpson et al. (154, 155) have suggested that the majority of circulating LDL-cholesterol must arise from de novo synthesis within the fetus. Carr and Simpson (152) have demonstrated that there is extensive cholesterol production by human fetal liver, testes, and adrenal. Considering the size of the fetal liver and the high rate of de novo cholesterol production within the fetal hepatocytes, the liver has the capacity to meet the needs of the fetal adrenals to maintain optimal rates of steroidogenesis.
700 _ A.
-
B
[i25ijjodo LDL DEGRADATION
600 -
FIG. 5. Effect of ACTH on the uptake and degradation of [125I]iodo-LDL by human fetal adrenal tissue. Tissue fragments were preincubated for 72 h in medium containing ACTH and LDL [500 jug LDL-protein ml" 1 (A)] or ACTH alone (B). Each value presented is the mean and SE (n = 4). [Reproduced with permission from B. R. Carr et al.: Endocrinology 107:1034, 1980 (150). © The Endocrine Society.]
500-
400[ 1 2 5 l]iodo LDL TISSUE UPTAKE
& S. 1
300200-
LPPS LDL +ACTH
LPPS
LPPS
LPPS
LDL
LDL
LDL
LPPS +ACTH
LPPS
+ACTH
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
•
LPPS +ACTH
LPPS
PEPE AND ALBRECHT
158
150
FlG. 6. Specific binding capacity (left panel) and the effect of ACTH on the rate of degradation (right panel) of [125I] iodo-LDL in anencephalic and normal human fetal adrenal tissue. The data bars represent the mean ± SE of specific binding of [125I]iodo-LDL (ng mg"1 protein) to membrane fractions or the percentage of the rate of degradation observed with control adrenal tissue fragments. [Reproduced with permission from B. R. Carr et al.\ J Clin Endocrinol Metab 53:406, 1981 (160). © The Endocrine Society.]
1500r p
Vol. 11, No. 1 Printed in U.S.A.
Regulation of the Primate Fetal Adrenal Cortex* GERALD J. PEPE AND EUGENE D. ALBRECHT Department of Physiology (G.J.P.), Eastern Virginia Medical School, Norfolk, Virginia 23501; and Department of Obstetrics and Gynecobgy and Department of Physiology (E.D.A), University of Maryland School of Medicine, Baltimore, Maryland 21201
I. Physiological Significance of the Fetal Adrenal II. Fetal Adrenal Development III. Regulation of Fetal Adrenal Growth A. Role of fetal pituitary B. POMC-derived peptides C. ACTH D. Placental factors E. Fetal growth factors IV. Regulation of Fetal Adrenal Steroidogenesis A. Substrate for fetal adrenal steroid production 1. LDL pathway 2. De novo biosynthesis B. Enzyme regulation of fetal adrenal steroidogenesis C. Multifactorial regulation of fetal adrenal steroidogenesis 1. Role/effect of ACTH and other peptides 2. Protein kinase A and protein kinase C pathways 3. Role of calcium 4. Peptide growth factors 5. Modulating effect of estrogen V. Placental Metabolism of Cortisol: Relationship to Maturation of the Fetal Adrenal Gland VI. Summary
enzymes in the fetal brain, retina, pancreas, and gastrointestinal tract (12-17) that normally are associated with late intrauterine life. It is well known, from the elegant work of Liggins and colleagues (7), that ablation of the fetal adrenal in sheep prevents parturition, whereas infusion of cortisol or ACTH1 to the fetus induces premature delivery. Although evidence that the fetal adrenal of the human or nonhuman primate participates in the initiation of labor appears less convincing (5, 8, 18-20), maturation of fetal adrenal steroidogenic enzyme systems that permit de novo synthesis of cortisol (5, 21, 22) must occur, as in sheep (23, 24), to ensure neonatal adrenocortical self-sufficiency in the perinatal period. In addition to these purported roles for fetal adrenal cortisol, it is well established that the fetal adrenal in primates, including the human, is important to the synthesis and secretion of androgen precursors essential to the production of estrogen by the placenta (25-32). It is apparent, therefore, that the fetal adrenal is important to several physiological homeostatic mechanisms and maturational events operative during fetal development. Thus, aberrations in pituitary-adrenal function in fetal life could have important consequences on fetal development and the onset of delivery. An understanding of the factors regulating growth, maturation, and hormonogenesis of the fetal adrenal, therefore, appears to be a prerequisite to formulating methods for reducing the incidence of abnormal human pregnancy
I. Physiological Significance of the Fetal Adrenal
I
N MOST mammalian species, products of the fetal adrenal gland appear to play an important role in regulating maturation of various organ systems in the fetus (1-5), providing the fetus homeostatic mechanisms to respond to stress (6), and initiating and/or participating in the cascade of events culminating in the birth of a newborn (7-9). Thus, cortisol, presumably of fetal adrenal origin, is one of the chemical messengers involved in the stimuli to lung maturation (3,4), deposition of glycogen in the liver (10, 11), and induction of several
1 The following abbreviations have been used in this article: ACTH, adrenocorticotropic hormone; CLIP, corticotropin-like intermediate lobe peptide; CRF, corticotropin releasing hormone; DHA, dehydroepiandrosterone; DHAS, dehydroepiandrosterone sulfate; MSH, melanocyte stimulating hormone; POMC, proopiomelanocortin; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor I; FGF, fibroblast growth factor; IGF-I, insulin-like growth factor I; IGF-II, insulinlike growth factor II; PDGF, platelet-derived-growth factor; TGF«, transforming growth factor a; TGF/3, transforming growth factor /?; 11/3-HSD, 11/3-hydroxysteroiddehydrogenase; 3/3-HSD, 3/?-hydroxysteroid dehydrogenase; HMG CoA, 3-hydroxy-3-methyl-glutaryl coenzyme A; HST, hydroxysteroid-sulfotransferase; P45011/?, 11/3-hydroxylase; P450i7a, 17a-hydroxylase; P450C2i, 21-hydroxylase; P4508CC, P450-cholesterol side chain cleavage; HDL, high density lipoprotein; LDL, low density lipoprotein; TPA, 12-O-tetracanoyl phorbol 13-acetate.
Address request for reprints to: Dr. Gerald J. Pepe, Department of Physiology, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, Virginia 23501-1980. * This work was supported by National Institutes of Health Grant HD-13294. 151
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
PEPE AND ALBRECHT
152
and thus neonatal morbidity and mortality. This review will attempt to summarize the present status of our understanding of the regulation of the fetal adrenal in humans and the more extensively studied nonhuman primate models of human pregnancy, namely the baboon and rhesus monkey. Attention will be focused on developing the perspective that fetal adrenal development is influenced by factors of uteroplacental origin and perhaps integrated with and/or directed by metabolic systems in the trophoblast and/or decidua. II. Fetal Adrenal Development During the course of gestation in humans (33, 34) and nonhuman primates (21, 35-38), the fetal adrenal gland not only undergoes extensive anatomical and biochemical changes but also exhibits a remarkable rate of growth, primarily during the final third of intrauterine development (Fig. 1). In the baboon and rhesus monkey, and presumably the human, increased weight is associated with marked growth of the inner zone (fetal cortical zone) which comprises between 80 and 90% of the gland during the majority of gestation. According to McClellan and Brenner (39), fetal cortical cells reach maximal size late in gestation with a marked increase in the number and size of the mitochondria and an expansion of smooth endoplasmic reticulum accounting for the majority of the increase in cytoplasmic volume. In addition, there is no apparent evidence of lipid droplet depletion or imminent cell death or necrosis with advancing gestation. Of particular interest, vascularization within the fetal zone is extensive and clearly exceeds that in the outer, adulttype cortical zone throughout gestation (39).
Vol. 11, No. 1
While there appears to be minimal maturation of the outer cortex during the majority of intrauterine development, by late gestation there is the initiation of substantial development of the adult-type definitive zone or neocortex in humans (40-42), baboons (21), and rhesus monkeys (39). Thus, by day 150 of gestation in the rhesus monkey (term = day 165) a presumptive zona glomerulosa and differentiation of cells into a zona fasciculata are evident (39). It is well established that the activity of the enzyme A5-3/3-hydroxysteroid dehydrogenase-isomerase (3/3HSD) is minimal during most of intrauterine development in the fetal adrenal of humans (43-47), baboons (21), and rhesus monkeys (43, 48, 49), as has been demonstrated in the fetal adrenal of the sheep (23, 50). Moreover, based on studies of human fetal adrenals obtained on weeks 18-24 of gestation and cultured in the absence of serum additives, the activity of 3/3-HSD is only 3-fold higher in the outer adult-type definitive tissue as compared with that in fetal cortex (51, 52). Consequently, throughout most of intrauterine development, the primate fetal adrenal has a limited capacity to synthesize A4-ketosteroids, such as cortisol, from precursors such as pregnenolone (53). In contrast, the activities of the enzymes catalyzing hydroxylations at steroid carbons 17, 21, and 11 (21, 54-56), as well as messenger RNAs (mRNAs) for the 17- and 21-hydroxylases (57-59), appear to be present earlier in gestation. These findings suggest that the baboon and monkey fetal adrenal have the capacity to synthesize cortisol from placental progesterone, as has been suggested in humans (60-62). However, based on in vivo studies in the baboon (63) and rhesus monkey (64), less than 5% of the cortisol meas1 900
FlG. 1. Changes in the DHAS concentrations (mean ± SE) in fetal plasma and in weight of fetal adrenals in the fetus of the rhesus monkey from midgestation to term. [Reproduced with permission from M. Seron-Ferre et al.\ J Clin Endocrinol Metab 57:1173, 1983 (37). © The Endocrine Society.]
E
2000
£
1500
O
GESTATIONAL AQE DAYS
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
February, 1990
FETAL ADRENAL DEVELOPMENT
ured in fetal plasma during gestation is derived from circulating progesterone. Thus, at least during early to midgestation, the fetus must obtain the majority of its cortisol from the maternal circulation. Indeed, in humans (65, 66), baboons (67), and rhesus monkeys (68, 69) there is extensive transplacental passage of maternal cortisol into the fetal compartment throughout gestation. Very late in gestation in the baboon (21), rhesus monkey (49, 70), and presumably the human (43-45, 71), there is a marked increase in fetal adrenal 3/3-HSD activity. Increased enzyme activity was associated with a rise in the serum concentrations of cortisol in the baboon fetus (67), and in umbilical artery obtained at select times during human gestation (72). A rise in fetal serum cortisol was also demonstrable in chronically catheterized rhesus monkeys studied longitudinally over the course of gestation (48). The increase in circulating cortisol in the baboon (67) and human (72) fetus late in gestation reflects the ontogenesis of fetal adrenal production and secretion and is not the result of placental transfer of maternal cortisol (65, 67) or alterations in the MCR of cortisol (73) due to changes in the concentrations of plasma binding proteins (74, 75) or patterns of fetal tissue metabolism (76). Moreover, studies in humans at midgestation (77), rhesus monkeys at term (69), and baboons at midgestation (78) and term (73) indicate that the ability of the primate fetus to utilize circulating cortisone as a precursor for the production of cortisol is also negligible. Thus, near term the primate fetal adrenal develops the capacity to synthesize cortisol, and there is an apparent increase in fetal cortisol concentrations due primarily to increased production by the fetal adrenal. It is apparent, based on the enzymatic profile of the fetal adrenal throughout most of intrauterine development, that quantitatively the major steroid products of the human (79-81) and nonhuman primate fetal adrenal (37, 48, 82-84) are the A5-hydroxysteroid androgen dehydroepiandrosterone (DHA) and its sulfurylated derivate DHA sulfate (DHAS). In the fetal rhesus monkey (Fig. 1), the serum levels and daily production rates of DHAS, which is formed primarily by the fetal cortical tissue, increase steadily during gestation and parallel fetal cortical growth (37). III. Regulation of Fetal Adrenal Growth A. Role of fetal pituitary Although the growth and differentiation of the definitive and fetal cortical zones of the human and nonhuman primate fetal adrenal are striking features of intrauterine development, our understanding of the factors regulating these processes is incomplete. It is well established that at mid to late gestation fetal adrenal size and weight in anencephalic human (41) and rhesus monkey (31) fetuses
153
are considerably reduced when compared with normal. Moreover, fetal adrenal atrophy and reduced steroidogenesis occur after treatment with glucocorticoids late in gestation in humans (81), rhesus monkeys (85, 86), and baboons (82, 87, 88). Collectively, these findings suggest that factors of fetal pituitary origin are important to fetal adrenal growth at this time in gestation. Indeed, fetal adrenal weight (13 ± 3 mg/100 g BW) in rhesus monkeys in which the fetal pituitary was removed on day 115 of gestation (term = day 165) was 85% lower than that (88 ± 16 mg/100 g BW) of sham-operated intact fetuses (89). Similar observations have been made in the fetal lamb (7) in which the maturation of immature fetal fascicular cells was prevented by removal of the fetal pituitary (90). Because treatment of hypophysectomized fetal lambs with ACTH reversed most of the effects of pituitary ablation, it appears that in this species fetal pituitary ACTH is critical to fetal adrenal development during the second half of gestation. While this may also be true in the human and nonhuman primate, it has not been unequivocally established, due in part to the absence of in vivo studies in nonhuman primate models and the inherent limitations with in vivo studies that have been performed in humans. For example, in studies of human anencephalics administered ACTH either during the second or third trimester (40, 91, 92) or in the immediate postnatal period (93-95) the number of patients was small, and for ethical reasons appropriate controls were limited. B. POMC-derived peptides Studies of Silman and colleagues (96, 97) indicate that there are several POMC-derived peptides in addition to ACTH present in the human fetal pituitary gland. Similar observations have been made in the fetal rhesus monkey in which desacetyl as well as mono and diacetyl a-MSH are specifically localized in the neurointermediate lobe by day 65 of gestation (98). Moreover, there appears to be a relative increase in the mono and diacetyl forms of a-MSH during the second half of gestation in the fetal rhesus monkey (98). A peptide consisting of XMSH with a carboxy terminal extension of 15 additional residues has also been isolated from the pituitary of the adult rat (99). This material, denoted X3-MSH, has the capacity to modulate adrenal function in the rat by stimulating pathways different from those responsive to ACTH!_24. In addition, compounds resembling a-MSH and corticotropin-like intermediate lobe peptide (CLIP) are quantitatively the dominant hormones in the human fetus after week 20 of gestation and may only be replaced by ACTH just before parturition (96). Based on these observations, Silman et al. (96) have suggested that several POMC-derived pituitary peptides, other than
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
154
PEPE AND ALBRECHT
ACTH, may serve to drive the fetal cortical zone of the adrenal during the course of pregnancy. In contrast, it was suggested that ACTH may be important only to the definitive zone. Thus, Silman et al. (96) have proposed that the switch in pituitary peptide synthesis to increased ACTH just before parturition may be a central mechanism in the chain of events leading to adrenal maturation and neonatal adrenocortical self-sufficiency. This hypothesis is interesting, because MSH and CLIP are thought to be synthesized primarily in the pars intermedia (100), a structure that is present in rudimentary form in normal human fetuses (101), and which disappears after birth. Indeed, a-MSH is not present in the anterior lobe of either the fetal or adult rhesus monkey pituitary gland (98). Moreover, a pars intermedia is only found occasionally in anencephalic fetuses (102), and there is evidence that when present, it is structurally (103) and functionally (104) abnormal. More recently, the studies of Facchinetti et al. (105) suggest that by the end of the second trimester, the processing of pituitary POMC in normal human fetuses is similar to that in the adult, and the functional activity of the anterior lobe appears to prevail over that of the "fetus-related" neurointermediate lobe around week 25 of gestation. C. ACTH Several investigators have examined the effects of ACTH on fetal adrenal maturation in vitro. After culture of collagenase-dispersed human fetal adrenal cells obtained early in gestation, there is marked proliferation of the adult-type definitive cells and an apparent involution and subsequent disappearance of the large fetal cortical cells (106-108). Under the culture conditions employed by Kahri and co-workers (106, 107), addition of ACTH increased the rate of growth and the cytoplasmic size (i.e. hypertrophy) of the definitive cells over an 8-day period but had no apparent effect on fetal cortical cells. Roos (109) demonstrated that ACTH enhanced growth and protein concentration of cultured fetal adrenal cells maintained for more than 10 days in 10%, but not in 20% fetal calf serum (FCS), while ACTH enhanced corticosteroid secretion in 10% and 20% FCS. These findings were interpreted to indicate that factors in fetal serum may modulate the effect of ACTH on growth of adrenal cells. In contrast to the stimulatory effects of ACTH, it has been well established that ACTH inhibits mitosis of adult human and bovine adrenocortical cells in culture (110-112), and ACTH has been reported to have inhibitory effects on human fetal definitive and cortical cells cultured for 6 days (113, 114). Clearly, therefore, the role of ACTH per se on growth (hyperplasia) and differentiation of fetal adrenal cortical cells appears tenuous. Rather, ACTH or other POMC-
Vol. 11, No. 1
derived peptides of fetal pituitary origin may interact with additional factors either produced in the adrenal itself or in other fetal and/or perhaps placental tissues to modulate adrenal growth during most of intrauterine development. In human anencephalic fetuses, the fetal adrenal gland develops normally up to 14-17 weeks of gestation and involutes sometime before week 20 (115-117), supporting the suggestion that extrapituitary factors perhaps of maternal and/or placental origin are important to adrenal maturation at least early in gestation. However, because the involution and/or remodeling of fetal cortical cells only ensues after delivery in neonates with normal pituitary function, it appears that placental and/or maternal factors may actually modulate fetal cortical development throughout the course of pregnancy. D. Placental factors
Several growth factors or oncogene-related peptides that may be important to fetal adrenal growth have been isolated from the human placenta, including transforming growth factors [TGFs a (118) and 0 (119)], fibroblast growth factor, [FGF (120)], platelet-derived-growth factor [PDGF (121)], placental GH (122), epidermal growth factor [EGF (121)], insulin-like growth factor I [IGF-I (123)], IGF-II (124), and nerve growth factor (125). In addition, a 34-kilodalton (KDa) growth factor exhibiting receptor binding specificity and antigenic structure quite different from placental FGF, TGFa, TGF/3, and PDGF, has recently been isolated from brush-border membrane preparations of human term placenta (126, 127). Simonian and Gill (113) and Crickard et al. (128) demonstrated that EGF and FGF have the capacity to stimulate growth of the human fetal adrenal in culture, although the mitogenic effect of these growth factors appeared greater on fetal definitive than on fetal cortical cells. The concentration of EGF required to elicit half-maximal stimulation of cell proliferation of both zones was considerably lower than the concentration of EGF required to saturate adrenal EGF receptors, suggesting that only a small percentage of occupied EGF binding sites is required to effect a maximal proliferative response. Of particular interest, definitive but not fetal cortical cells responded to EGF and FGF when the concentration of FCS in the medium was reduced from 10% to 1% (Fig. 2). This implies that EGF and FGF may interact with other factors present in serum to promote mitogenesis, particularly in the fetal cortex. Apparently ACTH is not one of the factors, because it attenuated the mitogenic effects of FGF and EGF on both definitive and cortical cells (113, 128). The mitogenic effects of EGF observed in vitro may also occur in vivo. Thus, in a preliminary report, Addiego et al. (129) demonstrated that fetal ad-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
February, 1990 2.5
FETAL ADRENAL DEVELOPMENT
factors involved, their regulation, secretion into the fetus, and potential interactions with the POMC-derived fetal pituitary peptides, particularly ACTH in vivo, remain to be elucidated.
- DEFINITIVE ZONE
2.0 (~"]i0% SERUM
m o
•
E. Fetal growth factors
i% SERUM
5 1-5
1.0 FETAL ZONE
0.5
ll fl CONTROL EGF
155
FGF CONTROL EGF
FGF
FIG. 2. Relationship between FGF, EGF, and serum concentration on culture growth. Separated human fetal adrenal cells were plated at 6000 cells per 3.5-cm dish in the presence of 1% or 10% fetal calf serum. EGF (20 ng/ml) or FGF (100 ng/ml) were added every other day. On day 9, the cells were trypsinized and counted. The results are expressed as the mean of duplicate plates; SD did not exceed 10% of the mean. [Reproduced with permission from K. Crickard et al.: J Clin Endocrinol Metab 53:790, 1981 (128). © The Endocrine Society.]
renal weight and maturation in rhesus monkeys were significantly increased after intraamniotic injection of EGF over a 7-day period at midgestation. Whether other factors similarly administered would be equally effective remains to be determined. Simonian et al. (130) have recently shown that an aqueous extract of media from cultures of human placenta stimulated proliferation of human definitive zone cells in culture. The effects of this extract of placentalderived mitogenic factor(s), as with purified EGF and FGF, were inhibited by concomitant treatment with ACTH. Branchaud and colleagues (131) have also demonstrated that conditioned media from cultures of human placenta (PM) stimulated fetal adrenal cell proliferation. According to these workers, PM was 2 to 5 times more potent than EGF or FGF, effective in the absence of serum, and not inhibited by ACTH. Based on these observations, plus the fact that conditioned PM is partially heat resistant, Branchaud et al. (131) have proposed that the active components in PM are not EGF, FGF, or TGFa. Moreover, because adrenal cortical growth in the presence and absence of exogenous EGF was inhibited by TGF/3, apparently TGF/3 is not one of the active components of PM. It is apparent, therefore, that the placenta may be a source of a variety of growth factors capable of modulating fetal adrenal mitogenesis. However, the requisite
Although the placenta has the capacity to provide the fetus with growth factors, it has recently become apparent that several fetal tissues, including the adrenal, have the biochemical machinery to produce peptide growth factors. Using in situ hybridization histochemistry, Han et al. (132) have shown that mRNAs for IGF-I and IGFII were measurable in 14 organs of the human fetus including the kidney, liver, and adrenal. These workers more recently demonstrated (133) that mRNA for IGFII in the fetal adrenal was present at concentrations 200fold greater than the mRNA for IGF-I. Of particular interest, all of the hybridizing regions appeared to be comprised predominantly of fibroblasts or other cells of mesenchymal origin. As pointed out by Han et al. (132), because these cells are widely distributed and anatomically integrated they are ideally located for the production of growth factors that may exert autocrine and/or paracrine effects on nearby target tissues, as originally suggested by D'Ercole and colleagues (134). Thus, Branchaud et al. (131) have shown that conditioned media of human fetal liver, lung, and kidney have the capacity to stimulate fetal adrenal mitogenesis, although at a rate lower than that of placental media. Voutilainen and Miller (135) demonstrated that the expression of the mRNA for IGF-II in the human fetal adrenal can be stimulated by concomitant treatment with ACTH. DiBlasio et al. (136) have also shown that the human fetal adrenal expresses message for basic FGF (bFGF); expression is increased by ACTH. These observations are of particular interest in light of previous studies (113, 128) demonstrating mitogenic effects of FGF on human fetal adrenal definitive and cortical cells in culture. What remains to be explained is the apparent inhibition of adrenal mitogenesis clearly exhibited by ACTH in vitro (113). Recently, McAllister and Hornsby (137) demonstrated that the phorbol ester 12-O-tetradecanoyl phorbol 13acetate (TPA), which activates protein kinase C by substituting for the physiological activator diacylglycerol (138), is also a mitogen for fetal human adrenal definitive zone cells in culture. Because IGF-I has the capacity to activate protein kinase C via increased hydrolysis of glycosyl-phosphatidyl inositols (139), factors capable of stimulating IGF-I or protein kinase C directly in the fetal adrenal may have profound effects on growth and differentiation. Rainey et al. (140) have demonstrated the presence of protein kinase C in the human fetal adrenal
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
and indicate that both the activity and content of this molecule are more abundant in the fetal than the definitive zone (Fig. 3). Interestingly, protein kinase C activity and content are very low in fetal adrenal tissues of anencephalics. The potential impact of protein kinase C as a promoter of fetal adrenal growth takes on additional import in light of recent observations by McAllister and Hornsby (141, 142), indicating that protein kinase C activation profoundly influences the ratio of enzymes regulating steroidogenesis in the human fetal adrenal gland. In summary, it would appear that regulation of fetal adrenal growth and maturation is extremely complex and may involve the collaborative actions of the fetal pituitary and placenta and perhaps other fetal tissues capable of producing and secreting growth factors. Moreover, growth may be modulated by cAMP-independent mechanisms, including protein kinase C. Because of the different profile of A and C kinases in definitive and cortical cells, at least during early gestation, as well as the apparent exclusivity of the adult adrenal to ACTH, it is possible that growth in these two different cellular compartments of the fetal adrenal is regulated differently. Indeed, the marked growth and vascularization of the fetal cortex that occur throughout gestation do not appear to be associated with a concomitant growth of the •
B
\
C
0
E
F
definitive zone, which occurs only very late in gestation and continues into the perinatal period, a time during which there is marked remodeling of the fetal cortex. In addition, because several of the studies on growth of the human fetal adrenal have been performed on tissue obtained primarily during early gestation, the responsivity of the gland to mitogens during late gestation is poorly understood. Thus, the possibility that mitogenic responsivity to trophic factors changes with advancing gestation cannot be excluded. Indeed, in the baboon, the patterns of fetal adrenal DHA/DHAS production, as well as responsivity to pituitary peptides both in vitro (22, 143145) and in vivo (83, 146), are markedly different at midgestation and term.
IV. Regulation of Fetal Adrenal Steroidogenesis A. Substrate for fetal adrenal steroid production 1. Low density lipoprotein (LDL) pathway. Based primarily on in vitro studies utilizing human fetal adrenal tissues obtained early in gestation, it is apparent that cholesterol derived from circulating LDL produced in the fetal liver is a major substrate for fetal adrenal steroidogenesis (147-156). Thus, the quantity of DHAS and cortisol synthesized by human fetal adrenal cells in culture was increased as a function of the concentration of LDL present in the culture medium (Fig. 4 and Refs. 148 and 156). Consistent with these results are the earlier observations of Solomon et al. (60) who demonstrated using in situ perfusion that neither circulating acetate nor free cholesterol were efficient precursors for human
*
*C
20 -
•
Arbit
o
Vol. 11, No. 1
PEPE AND ALBRECHT
156
1
•
FETAL ZONE
NEOCORTEX
m
n
ANENCEPHALIC
FlG. 3. Protein kinase-C content in human fetal adrenal tissues. Protein (100 fig/lane) was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted using an antibody directed against protein kinase-C and [125I] protein-A. Visualization of the bands corresponding to protein kinaseC was accomplished by autoradiography. The autoradiograph is shown at the top, and the bars are the mean results from densitometric analysis of the 80K bands from two experiments; the individual results are presented by the dots. Lane A, neocortex; lane B, fetal zone; lane C, neocortex; lane D, fetal zone; lane E, anencephalic adrenal (31 weeks); lane F, anencephalic adrenal (40 weeks). The arrow indicates the location of radioactive protein (80,000) mol wt standard. [Reproduced with permission from W. E. Rainey et ai: J Clin Endocrinol Metab 67:908,1988 (140). © The Endocrine Society.]
100
200
300 400 LDL /xg/ml
500
600
FIG. 4. Cortisol and DHAS (DS) secretion by human fetal adrenal tissue on the sixth day of culture in the presence of ACTH plus LDL in concentrations ranging from 0-500 fig/ml The results presented are the mean ± SE of five replicates and are expressed as micrograms of steroid per mg tissue protein/24 h. [Reproduced with permission from B. R. Carr et al: Endocrinology 106:1854,1980 (148). © The Endocrine Society.]
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
February, 1990
FETAL ADRENAL DEVELOPMENT
fetal steroidogenesis. Similar conclusions have been deduced from in vivo studies in baboons (63) and rhesus monkeys (64) in which less than 5% of placental progesterone and pregnenolone secreted into the fetus were utilized for cortisol production between mid and late gestation. Pregnenolone does not appear to be a quantitatively significant precursor in the human as well, because the total amount of this precursor estimated to be used for steroidogenesis is less than 1% of the fetal production rate of DHAS (154, 155). In addition, pregnenolone sulfate, although present in much higher concentrations, is inefficiently converted to DHAS in the human fetal adrenal (154, 155, 157, 158). To evaluate the mechanism(s) by which LDL-cholesterol is utilized for steroidogenesis, Carr and colleagues (148-150) examined the uptake and degradation of radiolabeled LDL by human fetal adrenal tissue fragments cultured in the presence or absence of LDL and/or ACTH. Fetal adrenal LDL uptake was a saturable process, presumably mediated by a population of high-affinity low-capacity binding sites. Moreover, LDL degradation was inhibited by chloroquinone, an inhibitor of lysosomal enzymes. Carr et al. (148-150) have suggested, therefore, that LDL metabolism by the human fetal adrenal must occur by a process similar to that postulated (159) for other tissues, namely adsorptive endocytosis mediated by binding to cell surface receptors in which the fate of the internalized molecule is dependent upon lysosomal degradation. ACTH appears to be important in the regulation of this process. Thus, degradation of LDL by fetal adrenal tissue fragments (150, 160) was stimulated 3-fold when ACTH was added to the culture medium (Fig. 5). The degradation of LDL appears essential to ensuring a continuous supply of cholesterol to meet the
157
large demand for precursor required for DHAS and cortisol synthesis. To investigate the nature and regulation of the receptors involved in the endocytosis of LDL, the binding of LDL to membrane fractions prepared from adrenals of normal and anencephalic human fetuses was also determined (160, 161). Regardless of tissue source, binding was of high affinity with the apparent dissociation constant (kd) approximating 24 ng LDL-protein/ml. However, compared to values in tissue from anencephalics, not only were binding and degradation greater in normal fetuses, but only in normal tissue were the latter processes stimulated by ACTH (Fig. 6). Although there was substantial binding of high density lipoprotein (HDL) by the human fetal adrenal (162), HDL was not degraded, neither uptake nor degradation was modified by ACTH, and HDL did not support steroidogenesis efficiently. Collectively, these findings confirm that LDL-cholesterol is a major precursor for fetal adrenal steroidogenesis, that availability of precursor is regulated by specific membrane receptors on the adrenal cell that must be internalized and degraded, and that the concentration of receptors as well as uptake and internalization of ligand are ACTH-dependent processes. Because no more than 20% of cholesterol in the fetus is derived from the maternal compartment (163), Simpson et al. (154, 155) have suggested that the majority of circulating LDL-cholesterol must arise from de novo synthesis within the fetus. Carr and Simpson (152) have demonstrated that there is extensive cholesterol production by human fetal liver, testes, and adrenal. Considering the size of the fetal liver and the high rate of de novo cholesterol production within the fetal hepatocytes, the liver has the capacity to meet the needs of the fetal adrenals to maintain optimal rates of steroidogenesis.
700 _ A.
-
B
[i25ijjodo LDL DEGRADATION
600 -
FIG. 5. Effect of ACTH on the uptake and degradation of [125I]iodo-LDL by human fetal adrenal tissue. Tissue fragments were preincubated for 72 h in medium containing ACTH and LDL [500 jug LDL-protein ml" 1 (A)] or ACTH alone (B). Each value presented is the mean and SE (n = 4). [Reproduced with permission from B. R. Carr et al.: Endocrinology 107:1034, 1980 (150). © The Endocrine Society.]
500-
400[ 1 2 5 l]iodo LDL TISSUE UPTAKE
& S. 1
300200-
LPPS LDL +ACTH
LPPS
LPPS
LPPS
LDL
LDL
LDL
LPPS +ACTH
LPPS
+ACTH
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 May 2015. at 05:18 For personal use only. No other uses without permission. . All rights reserved.
•
LPPS +ACTH
LPPS
PEPE AND ALBRECHT
158
150
FlG. 6. Specific binding capacity (left panel) and the effect of ACTH on the rate of degradation (right panel) of [125I] iodo-LDL in anencephalic and normal human fetal adrenal tissue. The data bars represent the mean ± SE of specific binding of [125I]iodo-LDL (ng mg"1 protein) to membrane fractions or the percentage of the rate of degradation observed with control adrenal tissue fragments. [Reproduced with permission from B. R. Carr et al.\ J Clin Endocrinol Metab 53:406, 1981 (160). © The Endocrine Society.]
1500r p
