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Endocrinology Vol. 147, No. 12 5988-5994
Copyright © 2006 by The Endocrine Society

Developmental Control of Iodothyronine Deiodinases by Cortisol in the Ovine Fetus and Placenta Near Term

Alison J. Forhead, Katrina Curtis, Ellen Kaptein, Theo J. Visser and Abigail L. Fowden

Department of Physiology, Development, and Neuroscience (A.J.F., K.C., A.L.F.), University of Cambridge, Cambridge CB2 3EG, United Kingdom; and Department of Internal Medicine (E.K., T.J.V.), Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Dr. A. J. Forhead, Department of Physiology, Development, and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom. E-mail: ajf1005{at}cam.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Preterm infants have low serum T4 and T3 levels, which may partly explain the immaturity of their tissues. Deiodinase enzymes are important in determining the bioavailability of thyroid hormones: deiodinases D1 and D2 convert T4 to T3, whereas deiodinase D3 inactivates T3 and produces rT3 from T4. In human and ovine fetuses, plasma T3 rises near term in association with the prepartum cortisol surge. This study investigated the developmental effects of cortisol and T3 on tissue deiodinases and plasma thyroid hormones in fetal sheep during late gestation. Plasma cortisol and T3 concentrations in utero were manipulated by exogenous hormone infusion and fetal adrenalectomy. Between 130 and 144 d of gestation (term 145 ± 2 d), maturational increments in plasma cortisol and T3, and D1 (hepatic, renal, perirenal adipose tissue) and D3 (cerebral), and decrements in renal and placental D3 activities were abolished by fetal adrenalectomy. Between 125 and 130 d, iv cortisol infusion raised hepatic, renal, and perirenal adipose tissue D1 and reduced renal and placental D3 activities. Infusion with T3 alone increased hepatic D1 and decreased renal D3 activities. Therefore, in the sheep fetus, the prepartum cortisol surge induces tissue-specific changes in deiodinase activity that, by promoting production and suppressing clearance of T3, may be responsible for the rise in plasma T3 concentration near term. Some of the maturational effects of cortisol on deiodinase activity may be mediated by T3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREMATURITY IS ASSOCIATED with a range of postnatal clinical complications, such as respiratory distress syndrome and necrotizing enterocolitis (1), which are due to structural and functional immaturity of tissues of the preterm infant. In normal pregnancy, prepartum increments in circulating glucocorticoids and thyroid hormones have an important role in the maturation of fetal tissues near term (2). Premature infants, however, often have low serum T4 and T3 levels (3), which may, in part, account for the immaturity of their tissues and physiological systems. For most of gestation, thyroid hormones in both human and ovine fetuses are characterized by high circulating levels of T4 and rT3 and a low concentration of T3, in comparison with values in postnatal life (4, 5). This is due to relatively high type III (inner ring) deiodinase (D3) activity in fetal and placental tissues, which favors conversion of T4 to rT3 and maintains a high rate of T3 clearance (4). In addition, the activities of type I and II (outer ring) deiodinases (D1 and D2), which are responsible for the metabolism of T4 to T3 and rT3 to diiodothyronine (T2), are relatively low in the fetus for most of gestation (4).

In the fetus near term, there are changes in thyroid hormone metabolism that promote production, and reduce clearance, of T3 (4, 5, 6). In fetal sheep, increased production rate of T3 is induced, at least in part, by a developmental rise in hepatic D1 activity and therefore increased deiodination of T4 to T3, rather than to rT3 (7). Thus, there is a prepartum surge in plasma T3 and a progressive postnatal decline in rT3 concentration in the developing offspring (8). Similar profiles in circulating thyroid hormone concentrations and hepatic D1 activity have been reported in the fetuses of other mammalian species and in the chick embryo (9, 10). The rise in circulating T3 close to term is known to have an important role in the maturation of the fetus in preparation for extrauterine life (11, 12).

In fetal sheep, there is a close temporal relationship between the prepartum increments in plasma cortisol and T3 (8), and several studies have shown a role for glucocorticoids in the control of thyroid hormone metabolism near term. An exogenous infusion of cortisol in immature sheep fetuses causes an increase in plasma T3 concentration, and the normal prepartum T3 surge is abolished by fetal adrenalectomy (13, 14). The cortisol-induced changes in plasma T3 close to term may be due, in part, to an increase in D1 activity in the fetal liver. Wu et al. (15) have shown that a supraphysiological dose of cortisol up-regulates hepatic D1 activity in immature sheep fetuses. However, the role of cortisol in the developmental control of deiodinase activities in other fetoplacental tissues is unknown. Furthermore, the extent to which T3 can influence tissue deiodinase activity directly is unknown. Therefore, the aim of the present study was to investigate the effects of cortisol and T3 on tissue deiodinase activities in sheep fetuses during late gestation. Plasma cortisol and T3 concentrations in utero were manipulated experimentally by fetal adrenalectomy and exogenous hormone infusion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Forty Welsh Mountain sheep fetuses of known gestational age were used in this study; 24 were singletons and 16 were twin fetuses, and there were 23 male and 17 female fetuses. The number of fetuses in each experimental group and their gestational age at surgery and tissue collection are shown in Table 1Go. The ewes were housed in individual pens and maintained on 200 g kg–1 concentrates with free access to hay, water, and a salt-lick block. Food, but not water, was withheld for 18–24 h before surgery. All surgical and experimental procedures were in accordance with the U.K. Animals (Scientific Procedures) Act 1986.


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  		TABLE 1. Number and gestational ages of fetuses used in the experimental groups

 
Surgical procedures
All surgical operations were carried out under halothane anesthesia (1.5% in O2-N2O) with positive pressure ventilation. Between 116 and 120 d of gestation (term 145 ± 2 d), eight fetuses were adrenalectomized (AX) in utero using surgical techniques described previously (16) (Table 1Go). Fetal adrenalectomy abolishes the prepartum rise in plasma cortisol in utero; the low concentration of plasma cortisol in the AX fetus is derived from placental transfer of cortisol from the maternal circulation (17). For three of the AX fetuses, the unoperated twin acted as a control intact fetus. In 23 of the remaining intact fetuses, intravascular catheters were inserted into the femoral artery and vein at between 115 and 118 d (18) (Table 1Go). All catheters were exteriorized through the flank of the ewe and secured in a plastic bag sutured to the skin. The catheters were flushed daily with heparinized saline solution [100 IU heparin ml–1 0.9% saline (wt/vol)] from the day after surgery. At surgery, all fetuses were administered 100 mg ampicillin iv (Penbritin; Beecham Animal Health, Brentford, UK) and 2 mg gentamicin iv (Frangen-100; Biovet, Mullingar, Ireland). The ewes were given antibiotics im (procaine penicillin, Depocillin; Mycofarm, Cambridge, UK) on the day of surgery and for 3 d thereafter.

Experimental procedures
Twenty-three intact fetuses were infused iv with saline (0.9% NaCl, 3 ml d–1, n = 8), cortisol (2–3 mg kg–1 d–1, n = 10, Efcortelan; Glaxo, Ware, UK) or T3 (8–12 µg kg–1 d–1, n = 5; Sigma, Poole, UK) for 5 d before delivery for tissue collection at 129–131 d (130 ± 0.2 d, Table 1Go). The doses of cortisol and T3 infused were calculated to mimic the plasma concentrations normally observed in the immediate prepartum period (19). Daily arterial blood samples (4 ml) were taken immediately before and throughout the infusion. The eight AX and remaining seven untreated intact fetuses were delivered for collection of tissue and blood samples at 141–146 d of gestation (144 ± 0.3 d); two untreated fetuses were delivered at 130 d of gestation (Table 1Go).

All of the fetuses were delivered by cesarean section under general anesthesia (20 mg kg–1 sodium pentobarbitone iv). At delivery, 10-ml blood samples were taken by venepuncture of the umbilical artery, and a number of tissues were collected from the fetuses after the administration of a lethal dose of barbiturate (200 mg kg–1 sodium pentobarbitone). Samples of fetal liver, kidney, cerebral cortex, perirenal adipose tissue (PAT), and placenta were immediately frozen in liquid nitrogen and stored at –80 C until analysis. At delivery, there was no evidence of adrenal remnants in any of the AX fetuses.

Biochemical analyses
Plasma hormone concentrations.
All blood samples were immediately placed into tubes containing either EDTA or EGTA and glutathione (for catecholamine determination in the infused fetuses) and were centrifuged for 5 min at 1000 x g and 4 C. The plasma aliquots were stored at –20 C (EDTA) or –80 C (EGTA and glutathione) until analysis. Plasma cortisol concentration was measured by RIA validated for use with ovine plasma as described previously (20). The lower limit of detection was 3.5 nM, and the interassay coefficient of variation was 12%.

Plasma T3 and T4 concentrations were also measured by RIA using a commercial kit validated for ovine plasma (21) (ICN Biomedicals, Thame, UK). The lower limits of detection were 0.11 nM for T3 and 9.8 nM for T4. The interassay coefficients of variation were 10% for both assays. Plasma rT3 concentrations were measured by RIA (22); the lower limit of detection was 0.01 nM and the samples were measured in a single assay in which the intraassay coefficient of variation was 3–4%.

Plasma catecholamine concentrations (adrenaline and noradrenaline) were measured by HPLC (23). The lower limits of detection were 0.30 nM for noradrenaline and 0.38 nM for adrenaline; the interassay coefficients of variation were 6 and 7%, respectively.

Tissue D1 activity.
Tissue D1 activities in liver and renal cortex were determined in the Cambridge laboratory by a radiometric enzyme assay adapted from Clarke et al. (24). This assay measured the release of 125I– by outer ring deiodination of 125I-labeled rT3. All reagents were obtained from Sigma unless otherwise specified. Samples of fetal liver (100 mg ml–1) and renal cortex (500 mg ml–1) were homogenized by hand in ice-cold 120 mM sodium phosphate buffer containing 1 mM EDTA (pH 7.0) (P120E1). The homogenates were centrifuged at 800 x g and 4 C for 10 min and the supernatants frozen at –80 C until analysis. Tissue protein content was measured in the supernatant by the method of Lowry et al. (25).

On the day of each D1 assay, outer-ring-labeled 125I-rT3 (PerkinElmer Life Sciences, Boston, MA) was purified by chromatography on Sephadex LH-20 (Fluka, Gillingham, UK). Appropriate dilutions of homogenate were incubated in duplicate for 15 min at 37 C with 2.5 µM rT3, including 105 cpm 125I-rT3, in 100 µl P120E1 containing 10 mM dithiothreitol (P120E1D10). The reaction was terminated by the addition of 200 µl horse serum and 200 µl ice-cold 10% trichloroacetic acid (TCA). The tubes were centrifuged at 1500 x g and 4 C for 10 min to separate the precipitated protein (including unreacted 125I-rT3) into a pellet, and the free 125I– radioactivity in a 50-µl sample of supernatant was measured using a {gamma}-counter (Cobra II Auto-{gamma}; Packard Bioscience, Pangbourne, UK). An additional set of samples was incubated with horse serum and 10% TCA before the addition of 125I-rT3 and dithiothreitol. This provided a blank value that was subtracted from the counts obtained from each sample. Tissue D1 activity was expressed as picomoles of I– released per minute per milligram protein.

Cerebral, placental, and PAT D1 activities were measured in the Rotterdam laboratory using somewhat different conditions (26). Tissue samples were homogenized (100 mg ml–1) using a Polytron homogenizer in P100E2D10 and were incubated at appropriate dilutions for 30 min at 37 C with 0.1 µM 125I-rT3 in 100 µl P100E2D10. Parallel incubation mixtures also included 0.1 mM 6-n-propyl-2-thiouracil (to inhibit D1 activity). The mixtures were processed essentially as described above with further purification of 125I– from the final supernatants by elution over Sephadex LH-20 minicolumns with 0.1 M HCl. Deiodinase activities were corrected for nonenzymatic deiodination using blank samples in the absence of homogenate.

Tissue D2 activity.
Tissue D2 activities in PAT, cerebral cortex, and placenta were measured in the Rotterdam laboratory using a radiometric enzyme assay as described previously (26). This assay measured the release of 125I– by outer ring deiodination of 125I-labeled T4. Tissue samples were homogenized (100 mg ml–1) using a Polytron homogenizer in P100E2D10. An aliquot of homogenate was kept on ice before assay on the day of preparation.

On the day of assay, outer ring-labeled 125I-T4 (Amersham Biosciences, Little Chalfont, UK) was purified on a Sephadex LH-20 minicolumn. Appropriate dilutions of homogenate were incubated for 60 min at 37 C with 1 or 500 nM T4, including 125I-T4 (105 cpm), and 0.5 µM T3 (to saturate any D3 activity) in 100 µl P100E2D10. Blank incubations were carried out in the absence of homogenate. The reaction was terminated by the addition of 100 µl 5% BSA solution and 500 µl 10% TCA. The samples were centrifuged, and radioiodide was purified from the supernatants on Sephadex LH-20 minicolumns. Deiodinase activity was corrected for nonenzymatic deiodination in the blank samples. The difference in fractional deiodination between incubations with 1 and 500 nM T4 represented low Km D2 activity, and was expressed as femtomoles of I– released per minute per milligram protein. Tissue protein content was measured by the Bradford method (Bio-Rad, Veenendaal, The Netherlands).

Tissue D3 activity.
Tissue D3 activities in liver, renal cortex, PAT, cerebral cortex, and placenta were determined in the Rotterdam laboratory by radiometric enzyme assay and HPLC as described previously (26). This assay measured the generation of radiolabeled T2 and 3'-iodothyronine by inner ring deiodination of 125I-labeled T3. Tissue samples were homogenized (100 mg ml–1) using a Polytron homogenizer in P100E2D10. Homogenates were stored at –80 C before analysis.

Appropriate dilutions of homogenate were incubated in duplicate for 60 min at 37 C with 1 nM T3, including 125I-T3 (105 cpm), in P100E2D10. Blank incubations were carried out in the absence of homogenate. The reaction was terminated by the addition of 100 µl ice-cold methanol. The samples were centrifuged, and 100 µl of the supernatant were added to 100 µl 0.02 M ammonium acetate (pH 4). This mixture was applied to a 4.6 x 250 mm Symmetry C18 column connected to an Alliance HPLC system (Waters, Etten-Leur, The Netherlands), and eluted with a gradient of acetonitrile in 0.02 M ammonium acetate at a flow rate of 1.2 ml min–1. Radioactivity in the eluate was measured using a Radiometric A-500 flow scintillation detector (Packard, Meriden, CT), and the conversion of 125I-T3 to 125I-T2 and 125I-3'-iodothyronine was corrected for nonenzymatic deiodination. Tissue D3 activity was expressed as femtomoles of I– released per minute per milligram protein, in which protein content was measured by the Bradford method.

Statistical analyses
All values are presented as mean ± SEM and were log10 transformed for statistical analysis where appropriate. No significant differences were observed between the hormonal or enzyme data from the untreated and saline-infused fetuses at 130 d in the present or previous studies (21), and therefore, the data were combined. Significant differences in plasma hormone concentrations and tissue deiodinase activities between the infused fetuses were assessed by two-way ANOVA for repeated measures and one-way ANOVA, respectively; both analyses were followed by the Student-Newman-Keuls post hoc test. Plasma hormone concentrations and tissue deiodinase activities were compared between intact fetuses at 130 and 144 d of gestation, and between intact and AX fetuses at 144 d of gestation, by Student’s unpaired t test. Differences where P < 0.05 were regarded as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma hormone concentrations
Significant increments in plasma cortisol and T3 were observed in the intact control fetuses between 130 and 144 d of gestation (P < 0.05, Fig. 1Go). However, there was no difference in plasma T4 or rT3 between the fetuses of these two groups (Table 2Go).


Figure 1
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  		FIG. 1. Mean (± SEM) plasma concentrations of cortisol (A) and T3 (B) in the sheep fetuses of each experimental group. Within each infusion day, columns with different letters are significantly different from each other. *, Significant difference from control (saline-infused and control) fetuses at 130 d; {dagger}, significant difference from control fetuses at 144 d; {ddagger}, significant difference from d 0 (P < 0.05). Number of fetuses indicated in parentheses.

 

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  		TABLE 2. Mean (± SEM) plasma concentrations of T4, rT3, noradrenaline, and adrenaline and placental D2 activity in sheep fetuses from each treatment group

 
Fetal adrenalectomy prevented the normal prepartum increases in plasma cortisol and T3 concentrations (Fig. 1Go). At 144 d of gestation, plasma cortisol and T3 concentrations in the AX fetuses were significantly lower than those observed in the intact fetuses (P < 0.001) and similar to those seen in the saline-infused and untreated fetuses at 130 d of gestation (Fig. 1Go). Fetal AX had no effect on plasma T4 (Table 2Go). Plasma rT3 was higher in the AX fetuses, compared with the intact fetuses at 144 d, but this was not statistically significant (P = 0.062, Table 2Go).

Before infusion, basal plasma concentrations of cortisol, T4, T3, rT3, noradrenaline, and adrenaline were similar in all groups of infused fetuses at 124–126 d of gestation (Fig. 1Go and Table 2Go). No significant changes in plasma cortisol, thyroid hormone or catecholamine concentrations were observed over the 5 d of saline infusion (Fig. 1Go and Table 2Go). An exogenous infusion of cortisol increased significantly plasma cortisol and T3 concentrations from baseline (P < 0.05 in both cases) to values significantly greater than those seen in the fetuses infused with saline (P < 0.05 in both cases, Fig. 1Go). On the fifth day of infusion, plasma cortisol and T3 in the cortisol-infused fetuses were similar to the concentrations normally seen in mature fetuses near term (Fig. 1Go). Plasma T3 but not cortisol concentration was increased significantly by exogenous T3 infusion; on the fifth day of infusion, plasma T3 in the T3-infused fetuses was significantly greater than baseline (P < 0.05) and the value seen in the fetuses infused with saline (P < 0.05, Fig. 1Go). There were no significant changes in plasma rT3, noradrenaline or adrenaline in the fetuses infused with cortisol or T3 for 5 d (Table 2Go). A significant decrease in plasma T4 concentration was observed between d 0 and 5 in the T3, but not cortisol, infused fetuses (P < 0.05, Table 2Go).

Tissue D1 and D2 activities
Maturational increments in hepatic, renal, and PAT D1 activities were observed in the control fetuses between 130 and 144 d of gestation (P < 0.01, Fig. 2Go). These changes were prevented by fetal AX; in the AX fetuses, hepatic, renal, and PAT D1 activities were significantly lower than in the intact fetuses near term (P < 0.001, Fig. 2Go).


Figure 2
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  		FIG. 2. Mean (± SEM) activities of liver D1 (A), kidney D1 (B), PAT D1 (C), and cerebral D2 (D) of the sheep fetuses of each experimental group. Columns with different letters are significantly different from each other. *, Significant difference from control (saline-infused and untreated) fetuses at 130 d; {dagger}, significant difference from control fetuses at 144 d (P < 0.05). Number of fetuses indicated in parentheses.

 
The increase in plasma cortisol induced by exogenous cortisol infusion was associated with significant increments in D1 activities in these tissues (Fig. 2Go). On the fifth day of treatment, hepatic, renal, and PAT D1 activities in the cortisol-infused fetuses were significantly greater than in the fetuses infused with saline (P < 0.05, Fig. 2Go). Furthermore, tissue D1 activities in the liver, kidney, and PAT in the cortisol-infused fetuses at 130 d of gestation were similar to those seen in the mature fetuses near term (Fig. 2Go). Five days of T3 infusion caused a significant increase in D1 activity in the fetal liver but not kidney (P < 0.05, Fig. 2Go). Hepatic D1 activity in the T3-infused fetuses was significantly greater than that seen in the saline-infused fetuses (P < 0.05) but did not reach the value seen in the fetuses infused with cortisol (Fig. 2AGo). In the T3-infused fetuses, PAT D1 activity was increased to the level seen in the fetuses infused with cortisol, but because of wide interanimal variation, the mean value was not significantly different from that observed in the saline-infused fetuses (Fig. 2CGo).

In the cerebral cortex, D2 activity decreased in the control fetuses between 130 and 144 d of gestation (P < 0.05, Fig. 2DGo). However, there were no significant differences in cerebral D2 activity between the infused fetuses or between the intact and AX fetuses at 144 d (Fig. 2DGo). No significant difference in placental D2 activity was observed between the treatment groups (Table 2Go). Tissue D1 activities in the placenta and cerebral cortex were negligible, and, relative to D1, D2 activity was negligible in PAT (data not shown).

Tissue D3 activity
In the intact fetuses between 130 and 144 d of gestation, D3 activity decreased in the kidney and placenta and increased in the cerebral cortex (P < 0.05 in all cases, Fig. 3Go). After fetal AX, the ontogenic changes in D3 activity were abolished in the placenta and cerebral cortex and attenuated in the kidney of the fetus (Fig. 3Go). In all three tissues, a significant difference in D3 activity was observed in the AX fetuses, compared with intact fetuses at 144 d (P < 0.05, Fig. 3Go).


Figure 3
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  		FIG. 3. Mean (± SEM) activities of D3 in kidney (A), placenta (B), and cerebral cortex (C) of the sheep fetuses of each experimental group. Columns with different letters are significantly different from each other. *, Significant difference from control (saline-infused and untreated) fetuses at 130 d; {dagger}, significant difference from control fetuses at 144 d (P < 0.05). Number of fetuses indicated in parentheses.

 
Exogenous cortisol infusion caused significant reductions in renal and placental D3 activity (P < 0.05, Fig. 3Go). Renal and placental D3 activities in the cortisol-infused fetuses were significantly lower than those observed in the fetuses infused with saline (P < 0.05) and were similar to values seen in the intact fetuses near term (Fig. 3Go). An infusion of T3 alone also suppressed D3 activity in the renal cortex of the fetus (P < 0.05) but had no effect on placental D3 activity (Fig. 3Go).

No significant difference was observed in cerebral D3 activity between the groups of infused fetuses (Fig. 3Go). Tissue D3 activity in liver and PAT was negligible (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates, for the first time, that cortisol is an important physiological regulator of several iodothyronine deiodinases in the ovine fetus and placenta during late gestation. Prevention of the endogenous prepartum cortisol surge by fetal adrenalectomy abolished the normal up-regulation of hepatic, renal, and PAT D1 activities and down-regulation of renal and placental D3 activities toward term. In addition, an exogenous infusion of cortisol, at a stage of gestation when plasma cortisol levels are normally low, caused 2- to 4-fold increments in hepatic, renal, and PAT D1 and decrements in renal and placental D3, such that the level of tissue activity in the cortisol-infused fetuses resembled that seen in mature fetuses near term with similar plasma cortisol concentrations. The cortisol-dependent changes in tissue deiodinase activity were accompanied by a significant increase in circulating T3 concentration. Therefore, in the sheep fetus, the prepartum cortisol surge increases plasma T3, at least in part, by stimulation of outer ring deiodinase activities in the liver, kidney, and PAT and suppression of inner ring deiodinase activities in the kidney and placenta (Fig. 4Go).


Figure 4
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  		FIG. 4. Summary diagram to show the effects of cortisol on thyroid hormone deiodination in the ovine fetus and placenta.

 
The comprehensive findings of the current study extend those of Wu et al. (15) to show that physiological changes in plasma cortisol concentration influence deiodinase enzyme activities, in a tissue-specific manner, before birth. In particular, up-regulation of D1 enzyme activity in the fetal liver and kidney is likely to make a major contribution to the increase in T3 production observed in sheep fetuses near term (6). Premature induction of hepatic D1 activity has been demonstrated previously in the chick embryo after administration of ACTH or glucocorticoids (27, 28). Furthermore, in vitro studies using fetal mouse liver in an organ culture system have shown a dose-dependent stimulation of D1 activity by physiological concentrations of cortisol (29).

In the present study, the ovine placenta and fetal kidney were found to be major sites of D3 activity. Moreover, cortisol-induced suppression of D3 in these tissues helps to explain the decreased clearance of T3 to T2 observed previously near term and in response to exogenous cortisol administration (6, 30). A fall in placental D3 activity toward term has been reported in other species, including man (31, 32, 33). In the embryos of avian and reptilian species, D3 activity in the liver appears to be an important determinant of T3 clearance, in a similar way to placental D3 in mammalian species. Gene expression and enzyme activity of hepatic D3 are reduced by ACTH or glucocorticoid administration in the chick and crocodile embryos (34, 35).

The gestational and cortisol-induced changes in tissue deiodinase activity in utero are likely to have important implications for the control of local, as well as circulating, concentrations of T3. For instance, the developmental rise in cerebral D3 seen in the sheep fetus toward term may be an important modulator of T3 exposure during brain development. Indeed, an inverse relationship between D3 and tissue T3 concentration has been observed in brain tissue of human fetuses (36). In the present study, although the gestational change in cerebral D3 was abolished by adrenalectomy, exogenous cortisol and T3 infusions had no effect on cerebral D3 activity; this may be due to the responsiveness of the tissue to these hormones at the gestational age studied.

The mechanisms by which cortisol influences tissue deiodinase activity in utero remain unclear. It may act directly at the level of gene transcription and/or mRNA stability, although the presence of a glucocorticoid response element has not yet been reported for any of the deiodinase genes. Alternatively, other cortisol-dependent factors may be involved in the developmental regulation of deiodinase activity in utero. For instance, T3 is not only a product and substrate of the deiodinase enzymes but can also directly affect their gene expression and activity. In the present study, raising plasma T3 concentrations alone in immature sheep fetuses increased hepatic D1 and decreased renal D3 activities, although not to the same extent as that seen in cortisol-infused fetuses and mature fetuses near term. In adult rats, hepatic D1 activity is suppressed by thyroidectomy and promoted by infusions of T3 or T4 (37). Indeed, a thyroid hormone response element has been identified on the human type I deiodinase gene (38). In vitro studies using hepatocytes from adult rats and fetal mice have also shown synergistic interactions between glucocorticoids and T3 in the control of D1 gene expression (29, 39, 40). Therefore, in the sheep fetus, the prepartum cortisol surge stimulates T3 production and reduces T3 clearance by tissue-specific effects on deiodinase activity. In turn, increased circulating and local concentrations of T3 may help to maintain T3 bioavailability by promoting stimulation of hepatic D1 and inhibition of renal D3 enzyme activities in the fetus near term. Overall, the cortisol-induced rise in plasma T3 appears to contribute to the ontogenic changes in both T3 production and clearance by a positive feedback mechanism.

The regulation of tissue deiodinase enzyme activity by cortisol is consistent with the other known maturational effects of cortisol in utero (2). The cortisol-induced activation of D1, and inhibition of D3, enzymes appears to be essential for the endocrine production of T3 over the prepartum period. Thus, some of the maturational effects attributed to the prepartum cortisol surge, such as maturation of the fetal lungs, may be achieved via the concomitant increase in circulating T3 (2, 41). Furthermore, up-regulation of hepatic, renal, and PAT D1 activities, and local generation of T3 toward term, are likely to be important for the onset of maturational processes specific to these tissues. In the fetal liver, these include stimulation of gluconeogenic enzyme activity (12) and changes in somatotrophic gene expression (19, 42), both of which have been shown to be dependent on the prepartum rise in plasma T3. Indeed, the developmental changes in hepatic D1 activity and T3 production coincide with increased binding capacity of nuclear thyroid receptors in the ovine fetal liver (43). In fetal PAT, which is primarily brown adipose tissue (44), the prepartum rise in plasma T3 has an important role in the activation of nonshivering thermogenesis, which is vital for survival in the extrauterine environment (45). Indeed, compared with rodents, the predominant expression of D1 rather than D2 in PAT of the sheep fetus and newborn goat (46) may be responsible for the greater thermogenic ability of newborn PAT in these species. Circulating and local concentrations of T3 may mediate the maturational effects of cortisol on uncoupling protein-1 content seen previously in PAT of fetal sheep (47). Therefore, maturational changes in PAT and hepatic function in the sheep fetus near term may be regulated by both the endocrine actions of circulating T3 and the paracrine actions of locally generated T3.

Overall, the present findings contribute to the understanding of the mechanisms of fetal maturation and the consequences of prematurity. Premature offspring that do not experience the prepartum surge in plasma cortisol may have immature deiodinase enzyme activity and thus fail to show the normal increase in circulating T3 over the prepartum period. Indeed, preterm infants often experience transient hypothyroidism associated with tissue immaturity and poor adaptation to the extrauterine environment (3). Cortisol-induced changes in deiodinase activity in fetal and placental tissues near term may be an important mechanism to ensure normal thyroid hormone metabolism and physiological maturation of the offspring over the perinatal period.


   Acknowledgments
 
The authors are grateful to the members of the Department of Physiology, Development, and Neuroscience who assisted with this study.


   Footnotes
 
This work was supported by the Biotechnology and Biological Sciences Research Council and the Isaac Newton Trust, Cambridge, UK.

Disclosure statement: The authors have nothing to disclose.

First Published Online September 7, 2006

Abbreviations: AX, Adrenalectomized; D1, type I (outer ring) deiodinase; D2, type II (outer ring) deiodinase; D3, type III (inner ring) deiodinase; PAT, perirenal adipose tissue; T2, diiodothyronine; TCA, trichloroacetic acid.

Received May 26, 2006.

Accepted for publication August 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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