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Articles |
-Inhibin1
a Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507
b Department of Obstetrics/Gynecology, McGill University Faculty of Medicine, Montreal, Canada H3A 1A1
c Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, The Center for Studies in Reproduction, The University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibin levels decline with advancing development. It is possible, therefore, that the subunit of inhibin provides a good marker of fetal zone cellular function and that the changes in circulating fetal -inhibin with advancing pregnancy reflect ontogenetic changes in fetal adrenal cortical zone-specific cell function. However, it remains to be determined whether the fetal adrenal is a major source of circulating -inhibin in the fetus and whether -inhibin is expressed in the fetal, definitive, and/or transitional zones. Therefore, the current study compared fetal serum -inhibin levels with immunocytochemical localization of -inhibin in baboon fetal adrenals obtained on Days 60 (early), 100 (mid), and 165 or 182 (late) of gestation (term averages Day 184) from animals untreated or treated with betamethasone, which we previously demonstrated suppressed fetal pituitary ACTH and adrenal weight. Fetal serum -inhibin levels (mean ± SE) were greater (p < 0.05) at mid (5863 ± 730 µl eq/ml) than at late (3246 ± 379) gestation and were reduced (p < 0.05) by betamethasone. The inhibin subunit was expressed in abundant quantities in the fetal adrenal cortex, but not in medulla, throughout gestation. At mid and late gestation, -inhibin was expressed throughout the fetal adrenal cortex but most intensely in the innermost area of fetal zone cells. By late gestation, the fetal adrenal exhibited a gradient of -inhibin expression. Thus, the outermost definitive zone cells were devoid of -inhibin, the transitional zone exhibited a relatively low -inhibin content, and fetal zone cells continued to exhibit extensive expression of -inhibin. Betamethasone diminished the intensity of -inhibin expression throughout the fetal adrenal cortex. These results indicate that the fetal adrenal fetal zone is a significant source of circulating -inhibin in the baboon fetus and that -inhibin provides a good marker to study the developmental regulation of fetal zone-specific adrenocortical function.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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hydroxylase/17–20 lyase (P45017OH) enzyme and thus are a major source of the C19-steroids dehydroepiandrosterone (DHA) and DHA sulfate. In contrast, the cells of the transitional zone [1] emerge later in gestation and express both the P45017OH and 
5-3ß-hydroxysteroid dehydrogenase-isomerase (3ß-HSD) enzymes for synthesis of cortisol (see [2, 3] for review), while the definitive zone expresses the 3ß-HSD and P450 18-hydroxylase enzymes for aldosterone. We recently demonstrated that ACTH receptor mRNA was expressed in high levels within the definitive/transitional zones of the baboon fetal adrenal gland at late gestation [4] and that expression of the ACTH receptor as well as 3ß-HSD mRNA in these cells was dependent upon fetal pituitary ACTH [5, 6]. In contrast, ACTH receptor mRNA levels in cells of the fetal zone were reduced between mid and late gestation [4, 7], a decline that was associated with a decrease in basal as well as ACTH-stimulated DHA production [8, 9]. We proposed, therefore, that there is a divergence in ACTH receptor-mediated adrenocortical zone-specific steroidogenesis within the primate fetal adrenal gland during the second half of gestation [4, 7].
Although 3ß-HSD is an excellent marker of definitive/transitional zone development, a marker for the fetal zone has not been reported. Recently, we [10] demonstrated that serum immunoreactive inhibin (i-inhibin) concentrations in the baboon fetus were greater at midgestation than at term. Moreover, i-inhibin in baboon [11] and human [12] fetal blood has been identified as free subunit (-inhibin) whereas dimeric inhibin, a heterodimer of and ß subunits joined together by disulfide bonds, dominated in maternal blood. Recent studies confirmed that the fetal adrenal of baboons [10] and humans [13, 14] expressed the mRNA for the inhibin subunit but relatively little mRNA for the ß subunit [13–16]. It is possible, therefore, that -inhibin provides a good marker of fetal zone cellular function and that the changes in circulating fetal serum -inhibin levels with advancing pregnancy reflect ontogenetic changes in fetal adrenal cortical zone-specific cell function. However, it remains to be determined whether the fetal adrenal is a major source of circulating -inhibin and whether -inhibin is expressed in cortical cells of the fetal, definitive, and/or transitional zones. Therefore, using immunocytochemistry and an antibody directed against the human subunit of inhibin, the current study was designed to determine which cells of the baboon fetal adrenal express -inhibin at early, mid, and late gestation. Moreover, the present study also determined fetal serum -inhibin concentrations and fetal adrenal inhibin -subunit expression in animals in which fetal pituitary ACTH and fetal adrenal growth were suppressed by the administration of betamethasone [5, 6], to confirm whether the fetal adrenal is a major source of fetal -inhibin and whether this peptide is regulated by ACTH.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum and fetal adrenals used in this study were obtained on Day 100 [5] and Day 165 [6] of gestation (term averages Day 184) from baboons untreated or treated with betamethasone and/or ACTH as part of previously published studies [5, 6]. Animal care and use [17] were in accordance with USDA regulations and the NIH Guide for the Care and Use of laboratory animals (Publication 85–23,1985). The experimental protocols and sampling have been described previously [5, 6] and were approved by the Institutional Animal Care and Use Committees of the Eastern Virginia Medical School and the University of Maryland School of Medicine.
On Days 100 or 165 of gestation, baboons were anesthetized with halothane-nitrous oxide, and the fetuses were delivered by cesarean section. After obtaining an umbilical arterial blood sample for analysis of -inhibin, the fetal adrenals were removed and weighed, and one gland was fixed in 4% buffered formalin and embedded in paraffin. To examine the role of the fetal adrenal at midgestation, baboons were either left untreated (n = 15) or treated (n = 8) with 1.5 mg, 3.0 mg, or 4.5 mg betamethasone (Celestone Soluspan, Schering Corp, Chicago, IL) administered to the mother daily between Days 60 and 99 of gestation [5]. Six additional animals and 4 of the betamethasone (3.0 mg)-treated animals also received ACTH (25 µg/0.1 ml saline; Cortrosyn, Organon Inc., West Orange, NJ), which was administered to the fetus daily on Days 95–99 of gestation [5].
To examine the role of the fetal pituitary-adrenal axis in late (Day 165) gestation, baboons were left untreated (n = 11) or treated with betamethasone administered both to the fetus (0.6 mg; n = 4) or the mother (6 mg) and fetus (0.6 mg; n = 4) every other day between Days 150 and 164 of gestation [6]. Fetal adrenals were also obtained from 3 untreated baboons on Day 182 of gestation.
Inhibin RIA
Fetal serum -inhibin concentrations in baboons of mid and late gestation treated with betamethasone and/or ACTH were determined by the RIA procedure of McLachlan et al. [18] as previously described [19]. The lower limit of detection for this assay is 1000 µl equivalents (eq)/ml. Serum -inhibin was also analyzed in 4 untreated control baboons not previously studied [10]. Because the -inhibin values in these untreated baboon fetuses of mid (4000–4400 µl eq/ml) and late (4250–4850 µl eq/ml) gestation were within the range of values (mid = 2450–11640 µl eq/ml; late = 1000–4600 µl eq/ml) previously reported in a contemporaneously studied group of baboons [10], values were combined with those of the previous study for statistical purposes.
Immunocytochemistry
Four-micrometer paraffin-embedded sections of baboon fetal adrenals were heated at 37°C overnight and then at 55°C for 30 min; this was followed by deparaffinization with xylene and treatment with graded ethanols. Endogenous peroxidase was inactivated with 0.3% H2O2 (adrenals) or 3% H2O2 (placentae) in methanol for 10 min. The tissues were incubated with 5% normal goat serum in phosphate-saline buffer, pH 7.4 (PBS) for 30 min at room temperature and then overnight at 4°C with 1.0 µg/ml of the R-1 mouse monoclonal antibody against the human subunit of inhibin [11] in the absence or presence of the 32-amino acid peptide antigen (100:1 molar ratio, antigen:antibody). After incubation with biotinylated goat anti-mouse IgG and avidin, and biotinylated horseradish peroxidase (Vector Laboratories, Burlingame, CA), sections were incubated with H2O2, imidazole, and diaminobenzidine, counterstained with hematoxylin, and examined by light microscopy using an Optiphot 2 microscope (Nikon Instruments, Melville, NY). The expression of 3ß-HSD protein, a marker of cells of the definitive and transitional zones [1–3] was determined as described previously [5] on 4-µm sections of paraffin-embedded sections using a polyclonal antibody to rabbit antihuman 3ß-HSD (generously supplied by Dr. Ian Mason, University of Edinburgh, Scotland). The width of the cell layer expressing 3ß-HSD was quantified [5] using a Video Based Image 1 Analysis System (Universal Imaging Corp, West Chester, PA).
Statistical Analyses
Data were analyzed by linear regression or by ANOVA with post-hoc comparison of the means by the Neuman-Keuls multiple-comparison test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibin levels (overall mean ± SE; Fig. 1) were greater (p < 0.05) at midgestation (5863 ± 730 µl eq/ml) than at term (3246 ± 379). Administration of betamethasone between Days 60 and 99 of gestation, which decreased fetal adrenal weight (108 ± 12 mg vs. 41 ± 6 mg; [5]), also reduced (p < 0.05) fetal -inhibin (3310 ± 129), by approximately 50% (Fig. 1). Similarly, betamethasone administration to the fetus or mother and fetus in late gestation, which decreased fetal adrenal weight (378 ± 21 vs. 183 ± 20; [6]) also reduced fetal -inhibin (1587 ± 312), although the latter was not statistically significant because values approached the lower limits of sensitivity of the inhibin assay (1000 µl eq/ml). Thus, in 6 of the betamethasone-treated baboons of late gestation, fetal inhibin levels were below or only slightly above the minimal level of detection.
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ACTH treatment of the fetus of untreated baboons at midgestation, which increased fetal adrenal weight by 50% [5], also increased (p < 0.05) fetal serum -inhibin concentrations by approximately 50% (Table 1). Although administration of ACTH to the fetus of betamethasone-treated baboons restored fetal adrenal weights to normal [5] and also enhanced fetal -inhibin concentrations by approximately 40% compared to betamethasone alone, the latter was not significant due to the variation in responsivity to ACTH. However, linear regression analysis indicated that there was a significant (r = 0.72) correlation between fetal adrenal weight and fetal serum -inhibin concentrations (p < 0.01) at midgestation in control and betamethasone-treated baboons treated or not treated with ACTH.
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Regardless of the in vivo treatment protocol, serum -inhibin concentrations were similar in male (n = 17) and female (n = 15) fetuses of mid (
= 6251 ± 954, n = 10; 
7907 ± 1218, n = 9) and late ( 2947 ± 514, n = 7; = 3182 ± 671, n = 6) gestation.
Immunocytochemical analyses demonstrated that the -inhibin was expressed in relatively abundant quantities in fetal adrenals of untreated baboons on Days 60 (not shown), 100, 165, and 182 of gestation (Fig. 2). Preincubation of the monoclonal anti--subunit antibody R-1 with the 32-amino acid peptide antigen completely eliminated any adrenal tissue labeling (Fig. 2D). Also, -inhibin was not detected in sections of a baboon placenta obtained on Day 100 of gestation (not shown), a time when the placenta is not secreting inhibin [10], thus confirming tissue specificity of the R-1 antibody.
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Inhibin subunit was present throughout the fetal adrenal cortex on Days 60 (not shown) and 100 (Fig. 2B). The most intensely labeled cells on Day 100 were usually found in the innermost area of the fetal zone and were often adjacent to medullary cells, which were essentially devoid of the subunit at all gestational ages (Fig. 2A). On Day 100, -inhibin was detected in the narrow rim of fetal cortical cells adjacent to the capsule, in which 3ß-HSD (not shown) was expressed.
Late in gestation, there was clearly a gradation in -inhibin expression in the fetal adrenal gland (Fig. 2, A and F). Thus, a significant proportion of the outer layer of cortical cells that expressed 3ß-HSD (Fig. 2E) and are presumed to be the definitive zone cells were devoid of -inhibin (Fig. 2, A and F). In contrast, 3ß-HSD-positive cells (Fig. 2E) further away from the capsule and presumed to comprise the transitional zone expressed -inhibin (Fig. 2, A and F), but to a relatively low extent. The cells of the fetal zone exhibited the highest expression of the subunit near term, particularly in the innermost area of the fetal zone.
The relationship between the expression of -inhibin and 3ß-HSD (Fig. 2) in fetal adrenal cortical cells at mid and late gestation is summarized in Table 2. At midgestation, -inhibin was expressed throughout the relatively small 30- to 40-µm layer of 3ß-HSD-positive cells. Although the layer of 3ß-HSD-positive cells increased to 200–300 µm by Days 165–182 of gestation, only the inner 150- to 250-µm layer of these 3ß-HSD-positive cells expressed -inhibin.
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Compared to the marked expression of -inhibin in adrenals of untreated baboons, betamethasone administration at mid (not shown) and late (Fig. 2C) gestation greatly diminished -inhibin immunoexpression throughout the fetal adrenal cortex. In the adrenal glands of fetuses treated with betamethasone and ACTH at midgestation, however, -inhibin expression and adrenal weights were comparable to respective values in untreated controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibin protein was expressed by the baboon fetal adrenal throughout the course of gestation and that the fetal zone cells were the primary source of -inhibin expression as measured by immunocytochemistry. Therefore, it is possible that the decline in fetal serum -inhibin concentrations with advancing baboon gestation reflects a decrease in -inhibin formation/secretion by individual cells of the fetal zone. Although the latter remains to be determined by quantitative approaches such as in situ hybridization, we suggest that -inhibin may serve as a useful marker to study the developmental change in fetal adrenal zone-specific function, e.g., the decline in ACTH-dependent DHA synthesis [8, 9] by cells of the fetal zone that we recently reported between mid and late baboon gestation. We have also shown both in vitro [20] and in vivo [21] that estrogen can act directly on fetal cortical cells to decrease ACTH-stimulated DHA production at midgestation. Whether the apparent role of estrogen on ACTH-dependent fetal adrenal development also involves modulation of zone-specific cellular expression of -inhibin remains to be determined.
The current study also showed that both fetal serum -inhibin concentrations and fetal adrenal -inhibin expression were reduced at mid and late gestation by treatment with betamethasone, which, as we [5, 6] previously demonstrated, decreased fetal adrenal weight by suppressing fetal pituitary proopiomelanocortin/ACTH expression. Although betamethasone may elicit effects on tissues other than the pituitary, supplementation of control or betamethasone-treated fetuses of midgestation with ACTH increased/restored fetal adrenal weight [5], as well as fetal adrenal -inhibin expression and fetal -inhibin levels. Collectively, these findings plus the observation that fetal -inhibin levels were correlated at midgestation with fetal adrenal weight support our previous suggestion [10] that the fetal adrenal gland is a significant source of -inhibin measured in fetal baboon serum. The same probably occurs in the human since the human fetal adrenal also expresses mRNA for -inhibin early in gestation [13–15] and circulating fetal i-inhibin has been identified as predominately the free subunit [11, 12].
It also appears that expression of -inhibin by the primate fetal adrenal is dependent upon ACTH. ACTH, as well as cAMP, have been shown to up-regulate inhibin subunit mRNA in human fetal and adult adrenal cells in culture [13, 15], and ACTH receptor mRNA was expressed by the baboon fetal adrenal during the course of gestation in a zone-specific biphasic pattern [4, 7] that was dependent upon the secretion of ACTH by the fetal pituitary [5, 6]. Moreover, the promoter region of the -inhibin gene contains cAMP as well as phorbol ester response elements [22–24]. Although a glucocorticoid response element proximal to the promoter region has also been identified [22], the ability of ACTH to restore fetal adrenal -inhibin expression in long-term betamethasone-treated baboons of the present study renders unlikely the possibility of a direct glucocorticoid inhibition of transcription of the subunit of inhibin.
The cellular distribution of -inhibin appeared to change with development of fetal cortical cells. Thus, although -inhibin was expressed throughout the adrenal cortex, including the narrow layer of 3ß-HSD-positive definitive zone cells adjacent to the capsule at midgestation, -inhibin was not expressed in the outermost layer of 3ß-HSD-positive cells adjacent to the capsule late in gestation. However, -inhibin was expressed in 3ß-HSD-positive cells of the developing transitional zone, although at a level that appeared lower than that in fetal zone cells. It remains to be determined whether the changes in cellular expression of -inhibin in the developing definitive and transitional zones are a consequence of, and/or participate in, the differentiation and maturation of the primate fetal adrenal cortex. Although in vitro studies would suggest that inhibin does not exert a direct effect on adrenal function, subunit may serve to limit production of activins (dimers of ß subunits), which have been shown to stimulate ACTH-dependent cortisol production and inhibit fetal adrenal cell proliferation [15].
We [11] and others [25] have found no sex difference in the umbilical cord blood concentrations of -inhibin of term human fetuses, and in the present study this observation is extended to the midgestation baboon fetus as well. Although Massa et al. [26] reported a higher concentration of i-inhibin in human male than female fetuses at 26–28 wk gestation, many of the mothers were being "treated with corticosteroids and/or thyroid-stimulating hormone-releasing hormone to accelerate fetal lung maturation" [26]; the sex of the fetuses being treated was not reported. Recently, Wallace et al. [27] observed the presence of dimeric inhibin-B in human male but not in female fetuses. The concentration of inhibin-B compared to -inhibin in either the human or baboon fetus is apparently not sufficient to present as a sex-difference in the RIA that measures free subunits as well as inhibin A and inhibin B.
In summary, the current study provides evidence that the fetal adrenal was a significant source of -inhibin in the baboon fetus throughout gestation and that -inhibin expression was maintained by ACTH. Immunocytochemical analyses confirmed that fetal zone cortical cells were the dominant site of -inhibin expression by the baboon fetal adrenal gland and that -inhibin may provide a good marker to study the developmental regulation of fetal zone-specific adrenocortical function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gerald J. Pepe, Department of Physiological Sciences, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501–1980. FAX: 757 624 2269; gjp{at}borg.evms.edu
Accepted: February 22, 1999.
Received: September 23, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase/17,20-lyase, and 3ß-hydroxysteroid dehydrogenase isomerase steroidogenic enzymes in human and rhesus monkey fetal adrenal glands: reappraisal of functional zonation. J Clin Endocrinol Metab 1993; 77:1184–1189.[Abstract]-subunit(s). J Clin Endocrinol Metab 1995; 80:3173–3179.[Abstract]-inhibin gene. Mol Cell Endocrinol 1990; 68:121–128.[CrossRef][Medline]-, ßA- and ßB-inhibin/activin genes. Identification of transcription factor AP-2-binding sites in the 5'-flanking regions by DNase I footprinting. Eur J Biochem 1994; 226:751–764.[Medline]This article has been cited by other articles:
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  F. Beuschlein, B. D. Looyenga, S. E. Bleasdale, C. Mutch, D. L. Bavers, A. F. Parlow, J. H. Nilson, and G. D. Hammer Activin Induces x-Zone Apoptosis That Inhibits Luteinizing Hormone-Dependent Adrenocortical Tumor Formation in Inhibin-Deficient Mice Mol. Cell. Biol., June 1, 2003; 23(11): 3951 - 3964. [Abstract] [Full Text] [PDF]
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 R. B. Billiar, N. C. Zachos, M. G. Burch, E. D. Albrecht, and G. J. Pepe Up-Regulation of {alpha}-Inhibin Expression in the Fetal Ovary of Estrogen-Suppressed Baboons Is Associated with Impaired Fetal Ovarian Folliculogenesis Biol Reprod, June 1, 2003; 68(6): 1989 - 1996. [Abstract] [Full Text] [PDF]
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 J. C. Achermann, M. Ito, B. L. Silverman, R. L. Habiby, S. Pang, A. Rosler, and J. L. Jameson Missense Mutations Cluster within the Carboxyl-Terminal Region of DAX-1 and Impair Transcriptional Repression J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3171 - 3175. [Abstract] [Full Text] [PDF]
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