Main

In the immature animal, catecholamines secreted by the fetal adrenal play a key role in the initiation and coordination of cardiovascular, respiratory, and metabolic responses, which are essential for intrauterine survival and the transition to extrauterine life (1–4). Impaired or altered adrenal function may reduce the ability of the fetus or newborn to mount an appropriate hormonal or metabolic response to intra- or extrauterine stress. In the adult adrenal medulla, splanchnic innervation plays a key role in the synthesis and secretion of catecholamines and ENK peptides (5–7). Previously, we have demonstrated that catecholamines and ENK-containing peptides are localized in the chromaffin cells of the adrenal medulla of the sheep fetus (8). We have also shown the total content and the proportion of small molecular mass forms of ENK-containing peptides in the fetal adrenal increase with advancing gestational age (9) and have suggested that the onset of splanchnic innervation may play a role in the regulation of ENK synthesis. During fetal life, endogenous ENK-containing peptides have been implicated in the regulation the cardiovascular responses to stress by modulating the actions of catecholamines at their receptor target sites (10).

Reduced placental delivery of essential substrates in utero, results in fetal growth restriction and is associated with increased perinatal morbidity and mortality, and impaired function of various body systems (11). In human fetuses, cordocentesis studies have demonstrated that intrauterine hypoxemia and malnutrition are associated with an increase in plasma catecholamines (12). In the sheep, the surgical removal of endometrial caruncles produces restriction of placental weight (PR) and hence fetal growth in subsequent pregnancies. This restriction of fetal growth is associated with chronic fetal hypoxemia and hypoglycemia (13,14). In addition, PR results in elevation of fetal plasma noradrenaline and adrenaline concentrations in response to chronic and further acute episodes of hypoxia (13,15). Recently, we have shown that PR reduces the expression of PNMT mRNA in the adrenal gland of the fetal sheep at term (i.e. 140-145 d of gestation), whereas, there was no change in the expression of ProENK A mRNA (16). These data suggest that the functional development of the adrenal medulla is altered as a consequence of restriction of placental growth and function. It is unknown, however, whether the underlying cause of the altered fetal adrenal responses are initiated early in gestation before the onset of splanchnic innervation of the adrenal. The aims of this study were to investigate the effect PR on fetal adrenal growth and the pattern of localization of catecholamine-synthesizing enzymes and ENK-containing peptides in the of adrenal gland in the midgestation sheep fetus. We also measured the total amount and molecular mass forms of ENK-containing peptides in the adrenal gland of the midgestation PR sheep fetus.

METHODS

Animals and tissue collection. All procedures were approved by the University of Adelaide Standing Committee on Animal Ethics in Animal Experimentation. Seventeen Border-Leicester X Merino pregnant ewes were used in this study. Eight sheep were operated on before pregnancy, and all visible endometrial caruncles were removed under general anesthesia and aseptic conditions, as described previously (17–19). At 90 d of gestation, the ewes were killed with an overdose of sodium pentobarbitone, and the fetal sheep were delivered through a hysterotomy incision. Placental weight, fetal body weight, and fetal organ weights were determined in all animals. Left and right adrenals were removed rapidly from control (n = 9) and PR (n = 8) fetuses and weighed; the were randomized equally between the two protocols. Adrenals were either immersed in fixative (0.1 M phosphate buffer containing 4% paraformaldehyde, pH 7.4) (control: n = 5 left, n = 4 right; PR: n = 4 left, n = 4 right adrenals) and embedded in paraffin for histologic analyses or frozen (control: n = 4 left, n = 5 right; PR: n = 4 left, n = 4 right adrenals) in liquid nitrogen and stored at -80°C before extraction for determination of the ENK-containing peptides.

Immunocytochemistry. We used a modification of the peroxidase anti-peroxidase immunocytochemical technique (20) to localize catecholamine-synthesizing enzymes and ENK-containing peptides in midglandular sections (6 µm) of adrenal glands from fetal sheep as described previously (21,22). The primary antibodies used were raised in rabbits to bovine adrenal DβH (EC 1.14.17.1) (1:2000), bovine adrenal PNMT (EC 2.1.1.28) (1:2000), and synthetic Leu-ENK (1:500). Specificity of the antisera has been determined previously to show that preabsorption of each antibody (i.e. DβH, PNMT, ENK) with the appropriate protein enzyme or peptide completely abolished specific immunocytochemical staining (21,23). In each staining run, and adrenal gland from a 130-d fetal sheep was used as a positive control for each of the antibodies (i.e. DβH, PNMT, and Enk peptide. For all adrenals used in this study, on an adjacent adrenal section, the primary antisera was replaced with normal rabbit serum at dilutions equivalent to those for each antibody (i.e. 1:500, 1:2000, and 1:2000) and used as a negative control.

Morphometric analyses and quantification of immunocytochemical staining. The morphometric analyses were carried on midglandular sections of adrenal glands stained with hematoxylin and eosin. Adrenal cortical cells have a high lipid content and therefore stain more intensely with eosin and thus can be easily distinguished from adrenal medullary cells which stain more intensely with hematoxylin. Adrenal measurements were made using a video generated image analysis system (Videometric 150, Oncor Inc., Gaithersburg, MD) as described previously (24,25). The image analysis program was used to calculate total adrenal gland area (the area enclosed by the adrenal capsule) and area of the adrenal medulla (the area enclosed by the outer border of adrenomedullary cells). The area of the adrenal cortex was calculated by subtracting the area of the adrenal medulla from the total area of the adrenal gland. As the adrenal sections used for morphometry were selected from the midglandular region they included the central vein or its major tributaries. The areas occupied by the adrenal venous or sinusoidal vessels were included in the morphometric analyses.

Quantification of the intensity of immunocytochemical staining (grains/unit area) was carried out for each of the antibodies used in the study (i.e. anti-DβH, anti-PNMT, anti-ENK) on the midglandular sections of adrenal gland from control and PR fetuses by image analysis using the Videometric 150 system as described elsewhere (24,25). This system permits the investigator to select the spectral composition of any desired color and establish limits of its true brilliance and intensity. These parameters can be saved for equivalent measurements in successive sessions. The light microscope video image is automatically scanned for the presence of the defined color, and the quantification (intensity) of staining (number of pixels) is presented as the number of grains per unit area of the image. In this study, the color limits were set manually to detect the brown immunocytochemical reaction product and to eliminate an unstained background signal. Triplicate measurements were made of the amount of staining for each antibody in adrenal sections from all fetuses.

ENK determination. The content of ENK-containing peptides was determined in extracts of individual adrenal glands after gel filtration chromatography and RIA, as described previously (9,26). Adrenal glands (n = 17) were homogenized separately (1:10, wt/vol) in ice-cold acetic acid (1 mol/L containing HCl (20 mmol/L) (extraction solution) using an Ultra-Turax 10N homogenizer. An adrenal extract was reconstituted in extraction solution and centrifuged at 5000 × g at 4°C for 30 min. The sample was filtered through a Millipore filter (0.45 µm; Waters Associates, Bedford, MA) and loaded onto the precalibrated Sephadex G75 column (2.5 × 75 cm; Pharmacia Fine Chemicals, Uppsala, Sweden) and eluted with extraction solution at a flow rate of 10 mL/h. The column was calibrated using the following molecular mass markers: dextran blue (void volume; V0), cytochrome c (12.4 kD), IGF-I (7.5 kD), peptide B (3.7 kD, proteolytic cleavage peptide from the ProENK A precursor) and Met-ENK (575 Da). Thirty fractions (10 mL) were collected and lyophilized before enzymatic digestion (N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin 1 mg/fraction for 6 h then carboxypeptidase B 0.05 mg/fraction for 2 h). The fractionated samples were then oxidized with H2O2, lyophilized, and assayed for Met-o-Enk.

Statistics. All data are expressed as mean ± SEM. Data were analyzed using SuperANOVA (Abacus, Berkeley, CA) on a Macintosh IIci computer (Apple, Cupertino, CA). A one-way ANOVA was used, to determine the effect of placental restriction on fetal adrenal weight, adrenal morphometric measurements, adrenal protein content, the intensity of immunocytochemical staining (DβH, PNMT, and ENK), the molecular mass profile of ENK-containing peptides, and total amount of ENK-containing peptides. The Student-Newman Kuels test was used post-ANOVA to identify significant differences between mean values. A probability of p < 0.05 was considered to be significant.

RESULTS

Fetal outcome and organ weights. Placental weight was reduced significantly (p < 0.05) in the PR group (237.9 ± 39.5 g) when compared with the control group (479.1 ± 36.9 g). Fetal body weight was reduced significantly in the PR group (564.7 ± 41.6 g) when compared with the control group (721.2 ± 22.8 g). Although fetal organ weights (brain, liver, spleen, heart, lung) were significantly decreased in the PR group, only fetal spleen weight remained significantly reduced when organ weights were expressed in relation to fetal body weight (Table 1). The relative weight of the fetal brain per kg of body weight was increased significantly in the PR group (Table 1).

Table 1 The effect of placental restriction on selected fetal organ weights expressed as gram of tissue or per kilogram of fetal body weight
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Adrenal growth. There was no significant difference in the combined adrenal weight in the PR fetuses (129.0 ± 7.7 mg/pair) when compared with the control fetuses (131.1 ± 9.4 mg/pair). Total adrenal protein content was not significantly different in PR fetuses (0.93 ± 0.13 mg/adrenal) compared with control fetuses (1.16 ± 0.12 mg/adrenal). The adrenal to body weight ratio was significantly greater, however, in PR fetuses (0.238 ± 0.013 × 10-3 mg/g) when compared with control fetuses (0.181 ± 0.012 × 10-3 mg/g). There were no significant differences in the total adrenal area, the area of the adrenal cortex or adrenal medulla in PR group when compared with control fetuses (Fig. 1).

Figure 1
figure 1

Effect of placental restriction, on total area of the fetal adrenal gland and areas of the fetal adrenal cortex and adrenal medulla. Data are from control (filled bars; n = 9 animals) and PR (open bars; n = 8 animals) fetuses and represent the mean ± SEM from each animal. Morphometric measurements were made from three midglandular adrenal sections from each animal.

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Immunocytochemistry. In control fetuses at 90 d of gestation, intense positive staining for DβH and ENK-containing peptides was observed throughout the adrenal medulla. PNMT was localized in the peripheral rim of cells of the adrenal medulla adjacent to the adrenal cortex and in the cells surrounding the central adrenal vein (Fig. 2, A-C). In PR fetuses, the staining for DβH and ENK-containing peptides in the adrenal was less intense and not all adrenomedullary cells were stained. PNMT was localized only in the peripheral rim of cells of the adrenal medulla adjacent to the adrenal cortex and again not all cells were stained (Fig. 2, D-F). For each enzyme (DβH, PNMT) and for the ENK-containing peptides the amount of staining was determined using the image-analysis system, and data are consistent with the microscopic observations (Fig. 3). There was a significant decrease (p < 0.05) in the amount of staining for from the PR fetuses when compared with controls (Fig. 3). A 130-d fetal adrenal was used as a positive control for the immunocytochemistry staining, and we observed intense positive staining for DβH throughout the adrenal medulla, whereas staining for PNMT and ENK-containing peptides was localized in the peripheral rim of cells of the adrenal medulla adjacent to the adrenal cortex (Fig. 4, A-C). We observed no detectable staining in the adrenal cortex or capsule and no staining was detected in the negative controls (Fig. 4, D-F). These data on the pattern of localization of DβH, PNMT, and ENK in the adrenal gland at 130 d are entirely consistent with those from our previous study (8).

Figure 2
figure 2

The effect of placental restriction on DβH, PNMT, and ENK immunostaining in sections of adrenal glands from fetal sheep at 90 d of gestation. To illustrate the zones within the adrenal, a bar at the top of panels A and D identifies the demarcation of the adrenal medulla (am) and adrenal cortex (ac) and is representative for the subsequent panels below. The peripheral-rim adrenomedullary cells are adjacent to and interdigitate with the unstained adrenocortical cells. The scale bar represents 100 µm. (A) Immunocytochemical localization of DβH in an adrenal gland from a control fetal sheep at 90 d of gestation. Positive staining for DβH (1:2000; dark cells) is observed throughout the central and peripheral-rim adrenomedullary cells, and no staining is observed in the adrenal cortex (ac). (B) Immunocytochemical localization of PNMT in an adrenal gland from a control fetal sheep at 90 d of gestation. Positive staining for PNMT (1:2000; dark cells) is observed in the peripheral-rim adrenomedullary cells, and no staining is observed in the adrenal cortex (ac) or central adrenomedullary cells. (C) Immunocytochemical localization of ENK in an adrenal gland from a control fetal sheep at 90 d of gestation. Positive staining for ENK (1:500; dark cells) is observed throughout the central and peripheral-rim adrenomedullary cells, and no staining is observed in the adrenal cortex (ac). (D) Immunocytochemical localization of DβH in an adrenal gland from a PR fetal sheep at 90 d of gestation. Sparse staining for DβH (1:2000; dark cells) is observed throughout the central and peripheral-rim adrenomedullary cells, and no staining is observed in the adrenal cortex (ac). (E) Immunocytochemical localization of PNMT in an adrenal gland from a PR fetal sheep at 90 d of gestation. Staining for PNMT (1:2000; dark cells) is observed in the peripheral-rim adrenomedullary cells and not all peripheral-rim cells are stained. No staining for PNMT is observed in the adrenal cortex (ac) or central adrenomedullary cells. (F) Immunocytochemical localization of ENK in an adrenal gland from a PR fetal sheep at 90 d of gestation. Sparse staining for ENK (1:500; dark cells) is observed in central and peripheral-rim adrenomedullary cells, and no staining is observed in the adrenal cortex (ac).

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Figure 3
figure 3

Effect of placental restriction on the intensity (arbitrary units; grains) of immunostaining for DβH, PNMT, and ENK in sections from 90-d fetal sheep adrenal glands, measured using an image analysis system. The quantification (intensity) of staining (number of pixels) is presented as the number of grains per unit area of the image. Data are from control (filled bars; n = 9 animals) and PR (open bars; n = 8 animals) fetuses and represents the mean ± SEM of from each animal. Measurements were made from three midglandular adrenal sections from each animal. Asterisks identify significant differences (p < 0.05) between control and PR groups.

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Figure 4
figure 4

The localization of DβH, PNMT, and ENK immunostaining in sections of adrenal glands from fetal sheep at 130 d of gestation has been used as positive controls (panels A-C). On an adjacent adrenal section the primary antisera has been replaced with normal rabbit serum and shown as a representative example of a negative control. To illustrate the zones within the adrenal, a bar at the top of panels A and D identifies the demarcation of the adrenal medulla (am) and adrenal cortex (ac) and is representative for the subsequent panels below. The peripheral-rim adrenomedullary cells are adjacent to and interdigitate with the unstained adrenocortical cells. The scale bar represents 100 µm. (A) Immunocytochemical localization of DβH in an adrenal gland from a control fetal sheep at 130 d of gestation. Positive staining for DβH (1:2000; dark cells) is observed in the peripheral-rim adrenomedullary cells, and no staining is observed in the adrenal cortex (ac). (B) Immunocytochemical localization of PNMT in an adrenal gland from a control fetal sheep at 130 d of gestation. Positive staining for PNMT (1:2000; dark cells) is observed in the peripheral-rim adrenomedullary cells, and no staining is observed in the adrenal cortex (ac) or central adrenomedullary cells. (C) Immunocytochemical localization of ENK in an adrenal gland from a control fetal sheep at 130 d of gestation. Positive staining for ENK (1:500; dark cells) is observed in the peripheral-rim adrenomedullary cells, and no staining is observed in the adrenal cortex (ac). (D) The DβH antisera has been replaced with normal rabbit serum (1:2000). No immunostaining is observed in an adrenal gland from a fetal sheep at 130 d of gestation. (E) The PNMT antisera has been replaced with normal rabbit serum (1:2000). No immunostaining is observed in an adrenal gland from a fetal sheep at 130 d of gestation. (F) The ENK antisera has been replaced with normal rabbit serum (1:500). No immunostaining is observed in an adrenal gland from a fetal sheep at 130 d of gestation.

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Content and molecular mass profile of ENK-containing peptides in the fetal adrenal. The total adrenal content of ENK-containing peptides was significantly reduced in the PR fetuses (103.4 ± 18.6 ng/adrenal) when compared with the control fetuses (243.6 ± 24.8 ng/adrenal) (Fig. 5).

Figure 5
figure 5

Effect of placental restriction on the total amount of Met-ENK (ng/adrenal) in whole adrenal gland extracts from fetal sheep at 90 d of gestation. Data are from control (filled bars; n = 9 animals) and PR (open bars; n = 8 animals) fetuses and represents the mean ± SEM in each group. Asterisks identify significant differences (p < 0.05) between the control and PR groups.

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A representative molecular mass profile of adrenal ENK-containing peptides from a control and PR fetus is shown in Figure 6. The Met-o-Enk-ir was distributed in four main peaks, which corresponded to the following molecular mass ranges: greater than 12, 7 to less than 12, less than 7 to 3 and less than 3 kD. The proportion of Met-o-Enk-ir in each of the molecular mass ranges was calculated as a percentage of the total Met-o-Enk in each adrenal gland extract (Fig. 6). The molecular mass profile of ENK-containing peptides was not altered significantly in the PR fetuses compared with control fetuses (Figs. 6 and 7).

Figure 6
figure 6

Effect of placental restriction on the molecular mass profile of ENK-containing peptides from whole adrenal gland extracts from a control (panel A) and a PR fetal sheep (panel B). The Sephadex G-75 column was calibrated using the following molecular mass markers: dextran blue (void volume; V0), cytochrome c (CytoC; 12.4 kD), IGF-I (7.5 kD), peptide B (3.7 kD, proteolytic cleavage peptide from the precursor, ProENK A) and Met-ENK (575 D).

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Figure 7
figure 7

Effect of placental restriction on the molecular mass profile of ENK-containing peptides from whole adrenal gland extracts from fetal sheep at 90d gestation, where the proportion of immunoreactive Met-ENK was found in the following molecular mass ranges: >12k Da, 7-12 kD, 3-7 kD, and <3 kD. The proportion of immunoreactive Met-ENK in each of these molecular mass ranges was calculated as a percentage of the total amount of immunoreactive Met-ENK in each adrenal gland. Data are from control (filled bars; n = 9 animals) and PR (open bars; n = 8 animals) fetuses and represent the mean ± SEM of each group.

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DISCUSSION

In the present study, we have investigated the effects of placental restriction on adrenal medullary development in the midgestation sheep fetus. By 90 d of gestation, we have shown that there is a relative increase in fetal adrenal weight and also sparing of fetal brain weight in the PR fetuses. We also observed decreases in liver, spleen, heart, and lung weight. Thus at 90 d of gestation, there is an asymmetric effect of placental restriction on the growth of specific fetal organs. This is consistent with other models of fetal growth restriction, such as the single uterine artery ligation model, where there is also maintained fetal brain and adrenal growth (27).

In previous studies using this model of placental restriction, we have found that the fetuses are chronically hypoxic and hypoglycemic from the earliest age at which fetal blood samples have been collected at around 100 d of gestation (15). It is therefore likely that the fetal growth restriction measured in the present study at 90 d of gestation is most likely caused by a decrease in the placental transfer of oxygen and/or glucose delivery to the fetus. It is interesting to note that the fetus has already adapted to the restriction of placental function at this early stage of gestation by maintaining brain and adrenal growth.

In the PR fetus, although adrenal growth and total protein content of the adrenal are maintained, we observed a marked decrease in the immunostaining for the catecholamine synthetic enzymes (DβH and PNMT). In the fetal sheep at term, we have shown previously that placental restriction is associated with a significant reduction in PNMT mRNA and PNMT immunoreactivity in the fetal adrenal medulla (16). Interestingly, in these studies fetal adrenal PNMT mRNA was significantly correlated with mean arterial Po2. Taken together, the data from both studies suggests that fetal adrenal may respond directly to changes in fetal arterial Po2 from as early as 90 d of gestation and that placental restriction and chronic hypoxemia result in a specific suppression of PNMT mRNA levels and a concomitant decrease in the PNMT-containing region of the fetal adrenal. A lack of PNMT enzyme would suggest that the fetal adrenal is compromised in its ability to synthesize and/or secrete adrenaline. In fact, Oyama et al. (27) have shown adrenal medullary responses to delivery were not sustained in the newborn PR sheep. Our recent studies have shown that, in PR fetuses, there was significant inverse correlation between arterial Po2 and fetal plasma noradrenaline concentrations (15). In contrast, we found that a decrease in arterial Po2 was associated with a relative suppression of circulating adrenaline concentrations in the placentally restricted fetuses. We speculate that with increasing gestational age and a decreasing fetal Po2, the hypoxic drive to the fetal adrenal increases in the PR fetus. This is associated with a suppression of adrenaline synthesis, and the ability of the fetus to mount an adequate adrenal catecholamine response to subsequent stimuli may be diminished.

There is good evidence to demonstrate that glucocorticoids also play a critical role in adrenal medullary development and in the regulation of PNMT gene expression and PNMT enzyme activity. In the mouse, knockout of the glucocorticoid receptor showed the complete loss of adrenaline-synthesizing cells, demonstrating that glucocorticoids are essential for the development of fetal adrenal medulla (28). We have previously demonstrated that circulating cortisol concentrations are similar in the normally grown and growth-restricted fetal sheep between 100 and 125 d gestation (29). These data would suggest that the PR fetus does not have impaired fetal adrenal cortical function and that it is unlikely that the decrease in adrenal PNMT staining is due to a lack of adrenal glucocorticoids.

Secretion of catecholamines in response to stimuli such as acute hypoxia is essential for the survival of the fetus (30) and neonate (4), and an impairment in the fetal adrenal's capacity to synthesize and secrete catecholamines has potentially serious consequences for the growth-restricted fetus. Despite our current findings, we have shown previously that catecholamine concentrations are elevated in the circulation of the growth-restricted fetus during late gestation (13,15). Infusion of the sympathomimetic amine, tyramine, results in a significantly greater catecholamine response in PR compared with control fetuses (15). This suggests that the sympathetic neurones rather than the adrenal medulla are likely to be the source of the elevated fetal plasma catecholamine concentrations in the PR fetus. It may be that although fetal adrenal development in the PR fetus is impaired and/or altered, the fetus adapts by activation of catecholamine synthesis and secretion from developing sympathetic neurones. Clearly further studies are required to determine the relative contributions of the fetal adrenal and sympathetic neurones to circulating catecholamines in the PR fetus.

In the PR fetus, we observed fewer adrenomedullary cells which were positively stained for ENK, a decrease in the amount of immunostaining as quantified by the image analysis system and a similar quantitative decrease in the total amount of ENK-containing peptides in the adrenal of the PR fetus. As there was no change in adrenal weight or in the relative area of the adrenal medulla we conclude that there must be a reduction in the number of adrenomedullary cells synthesizing ProENK A.

In the present study, we observed a similar degree of diminution of immunostaining of catecholamine synthetic enzymes (DβH and PNMT) as well as the ENK-containing peptides in the fetal adrenal of the PR fetus. In contrast to these findings, we found that there was no change in expression of ProENK A mRNA in the adrenal of the PR fetus at around 140 d of gestation (16). It is possible that, at term, ENK-containing adrenomedullary cells increase expression of ProENK A to maintain ENK-containing peptide synthesis. There is little information on factors which regulate ProENK A synthesis in the adrenal gland during development. In the adult adrenal medulla, studies have shown that glucocorticoids play a key role in ProENK A synthesis (31). Previously, we have found that ENK immunoreactivity and the amount of ENK-containing peptide content of the term fetal adrenal were increased in the hypophysectomized fetal sheep after ACTH replacement therapy and an elevation in fetal plasma cortisol concentrations (26). Therefore, it may be the increase in fetal plasma cortisol concentrations in the PR fetal circulation which occurs after 125 d of gestation that maintains ProENK A gene expression in the fetal adrenal medulla at term.

Although we observed changes in the pattern of localization of ENK immunostaining and total amount of ENK peptides in the PR fetus, there were no changes in the molecular mass profile of the ENK-containing peptides in this group. Indeed, we observed the expected gestational age change in the molecular mass profile of ENK-containing peptides. i.e. an increase in the proportion of the smaller molecular mass forms (>3 kD) of the ENK-containing peptides (9), which suggests that there is an increase in the processing of ProENK A in adrenal medulla of the PR fetus. These data suggest that although fewer adrenal medullary cells are synthesizing ProENK A-derived peptides, the processing enzymes are still present and capable of the cleaving ProENK A in the typical pattern observed at this stage of development.

In summary, in the adrenal of the growth-restricted fetal sheep at 90 d of gestation, we observed a significant decreases in DβH, PNMT, and ENK immunostaining despite the maintenance of adrenal cortical and medullary growth. In addition, there was a 50% decrease in adrenal ENK content, whereas the molecular mass profile of ENK-containing peptides was not altered in the PR fetuses. In conclusion, these data suggest that PR may alter the synthetic capacity and/or secretion of catecholamines and ENK-containing peptides from the fetal adrenal medulla. Therefore, whereas adrenal growth is maintained in the PR fetus, the functional capabilities of the fetal adrenal medulla are clearly altered and may be significantly impaired.