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Am J Physiol Regul Integr Comp Physiol 283: R1155-R1163, 2002. First published June 20, 2002; doi:10.1152/ajpregu.00015.2002
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Vol. 283, Issue 5, R1155-R1163, November 2002

Glucocorticoids, thyroid hormones, and iodothyronine deiodinases in embryonic saltwater crocodiles

Caroline A. Shepherdley1, Christopher B. Daniels2, Sandra Orgeig2, Samantha J. Richardson3, Barbara K. Evans1, and Veerle M. Darras4

1 Department of Zoology, University of Melbourne, 3010, Victoria; 2 Department of Environmental Biology, Adelaide University, 5005, South Australia; 3 Department of Biochemistry and Molecular Biology, University of Melbourne, 3010, Victoria, Australia; and 4 Laboratory of Comparative Endocrinology, Zoological Institute, K. U. Leuven, B-3000 Leuven, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the relationship between glucocorticoids, thyroid hormones, and outer ring and inner ring deiodinases (ORD and IRD) during embryonic development in the saltwater crocodile (Crocodylus porosus). We treated the embryos with the synthetic glucocorticoid dexamethasone (Dex), 3,3',5-triiodothyronine (T3), and a combination of these two hormones (Dex + T3). The effects of these treatments were specific in different tissues and at different stages of development and also brought about changes in plasma concentrations of free thyroid hormones and corticosterone. Administration of Dex to crocodile eggs resulted in a decrease in 3,3',5,5'-tetraiodothyronine (T4) ORD activities in liver and kidney microsomes, and a decrease in the high-Km rT3 ORD activity in kidney microsomes, on day 60 of incubation. Dex treatment increased the T4 ORD activity in liver microsomes, but not kidney microsomes, on day 75 of incubation. Dex administration decreased T3 IRD activity in liver microsomes. However, this decrease did not change plasma-free T3 concentrations, which suggests that free thyroid hormone levels are likely to be tightly regulated during development.

reptile; development; outer ring deiodination; inner ring deiodination; liver; kidney


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THYROID HORMONES AND GLUCOCORTICOIDS have important roles during development. Both thyroid hormone and glucocorticoid concentrations in plasma increase around the time of birth in mammals (16, 48), hatching in birds (18, 41), metamorphosis in anurans (23, 40, 46), early in fish development (6, 7, 17, 25), and during smoulting in salmonid fishes (38, 39). During early development, thyroid hormones are of maternal origin. In eutherian mammals, they are provided by placental transfer (45), and in the case of egg-laying vertebrates, they are deposited in the egg yolk (20). Later in development, embryos synthesize and release thyroid hormones from their own developed thyroid gland. Glucocorticoids of maternal origin are also received across the placenta by eutherian embryos (33). Glucocorticoids are synthesized and released from the developed adrenal gland late in development. Another important event in development is the onset of the regulation of these hormones through the hypothalamo-pituitary-adrenal (HPA) axis or the hypothalamo-pituitary-thyroid (HPT) axis.

The peak in plasma thyroid hormones during the final stages of development is characterized by an increase in 3,3',5,5'-tetraiodothyronine (T4), mediated by the HPT axis, and a concurrent increase in 3,3',5-triiodothyronine (T3), which is largely the result of an increase in deiodination of T4 to T3 and a decrease in T3 degradation. Deiodination is the removal of iodine from iodothyronine molecules. Outer ring deiodination converts T4 into T3 and reverse T3 (rT3) into 3,3'-diiodothyronine (T2). Inner ring deiodination converts T4 into rT3 and T3 into T2. T3 is responsible for most of the biological effects of thyroid hormones and is the ultimate ligand for the thyroid hormone receptor (31). Therefore, the conversion of T4 to T3 is an important step in the regulation of thyroid hormone bioactivity. Outer ring deiodinase (ORD) activities are detectable already during embryonic stages and have a general increase in activity toward the end of development (mammals: 1, 13, 19, 28, 30, 51; birds: 2, 5; amphibians: 10, 12). In contrast to ORD activities, inner ring deiodinase (IRD) activity is highest during early developmental stages but generally declines toward the end of embryonic development (mammals: 28, 29; birds: 2, 5, 43; amphibians: 12).

During development, glucocorticoids affect deiodination. In the last days before spontaneous labor in the sheep, there is a coincident rise in plasma cortisol and T3 in the fetus. Cortisol administration increases T4-to-T3-converting activity in the liver of the fetal lamb (51) and therefore may contribute to the increase in type I ORD and increase in T3 plasma levels at the end of gestation. In cultured fetal mouse liver cells, the addition of hydrocortisone induced type I ORD activity (32). In the embryonic chicken, endogenous corticosterone levels have been observed to rise late in development (34), around which time there is also an increase in the conversion of T4 to T3 (8). Administration of glucocorticoids [dexamethasone (Dex) or corticosterone] or ACTH to embryonic chickens increased plasma T3, decreased rT3 concentrations, and decreased, or did not alter, plasma T4 concentrations (4, 9). These treatments also increased conversion of T4 to T3 in the liver (4, 9, 15) and decreased hepatic type III deiodinase (4). Interaction between corticosterone and thyroid hormones has also been demonstrated during amphibian metamorphosis. Corticosteroids increased the net production of T3 by stimulating ORD activity (T3 production) and by inhibiting IRD activity (T3 degradation) in premetamorphic tadpoles. This increased the whole body concentration of T3 in the tadpoles and led to premature metamorphosis (11).

It was therefore postulated that glucocorticoids have a role in increasing the activity of ORD and decreasing the activity of IRD enzymes during development of the saltwater crocodile. This study had three aims: 1) to measure corticosterone and free thyroid hormones in the blood plasma of the saltwater crocodile embryo, 2) to investigate the control of thyroid hormones and glucocorticoids by the HPT and HPA axis, respectively, and 3) to investigate the relationship between glucocorticoids, thyroid hormones, and deiodinases in the embryonic crocodile. These aims were tested by treating the embryos with the synthetic glucocorticoid Dex, T3, and a combination of these two hormones (Dex + T3). We hypothesized that Dex treatment would decrease endogenous corticosterone concentrations through interaction with the HPA axis and that T3 treatment would decrease endogenous free T4 concentrations through interaction with the HPT axis. We also hypothesized that Dex treatment would increase T4 ORD and rT3 ORD activities in liver and kidney microsomes and decrease T3 IRD activity in liver microsomes. As deiodinase activities in the liver and kidney are responsible for contributing to circulating concentrations of thyroid hormones, we hypothesized that changes in deiodinase activities in these tissues in response to glucocorticoid treatment would decrease plasma free T4 concentrations and increase plasma-free T3 concentrations.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Incubation of eggs. Crocodile eggs were purchased from a commercial crocodile farm, Crocodylus Park, Darwin, Northern Territory, Australia. Eggs were from two clutches and were incubated at Crocodylus Park for 40 days, then transferred to Adelaide University, South Australia. On arrival, eggs were weighed and placed in plastic storage boxes filled with moistened vermiculite substrate. Eggs from both clutches were placed in each box, half buried in substrate, 1 to 2 cm apart. Box lids were left slightly ajar to allow air to circulate. The storage boxes were placed in an incubator set at 32°C. Eggs were sprayed every 2 to 3 days with MilliQ water to maintain moisture levels. At day 40 of incubation, the mean weight of eggs in clutch 1 was 121.90 ± 0.62 g and the mean weight of eggs in clutch 2 was 132.67 ± 0.47 g.

Experimental design. The relationship between glucocorticoids, thyroid hormones, and deiodinases in embryonic saltwater crocodiles was investigated by injection into the eggs of solutions containing T3 (Sigma Chemicals, Sydney, Australia), Dex (Sigma Chemicals), Dex + T3, and control injections consisting mostly of isotonic saline. The doses of these treatments given to saltwater crocodile embryos were based on Dex and T3 treatment of snapping turtle eggs (Chelydra serpentina) (26) and chicken eggs (4, 9, 50) and were scaled up according to differences in body masses of embryos. Therefore, in crocodile embryos, the treatments consisted of two 50-µl injections of 5 µg T3 (dissolved in 0.1 M NaOH and diluted 1:50 with 0.15 M NaCl, pH 7.5), two 50-µl injections of 50 µg Dex (dissolved in 0.15 M NaCl, pH 7.5), and two 50-µl injections of 50 µg Dex + 5 µg T3 combined (each prepared as above). Control injections were two 50-µl injections of 0.1 M NaOH diluted 1:50 with 0.15 M NaCl, pH 7.5.

The four treatment groups consisted of six eggs per group. Eggs were randomly assigned to treatment groups, the only stipulation being that each group contained four eggs from clutch 2 and two eggs from clutch 1. This was to account for possible clutch variation in embryo size and response to treatments. Injection of treatments took place 24 and 48 h before embryos were euthanized for organ collection. The effects of the treatments were studied at three time points: animals were euthanized on day 60 (75% incubation), day 68 (85% incubation), and day 75 (94% incubation). One group of six eggs was allowed to develop through to 1 day after hatching and did not receive any hormone treatments.

Injection protocol. Eggs were removed from the incubator, and the top surface was swabbed with ethanol. A small hole was drilled in the shell above the embryo using a dental drill and 50 µl of treatment solution was injected into the egg through the hole. The hole was sealed by pipetting a drop of candle wax over the hole. Eggs were returned to the incubator. Twenty-four hours later, the wax was removed, and another 50 µl of hormone solution was injected. The hole was resealed and the eggs were returned to the incubator. At day 58, it was necessary to remove ~100 µl of egg fluid before the injection of 50 µl of treatment. This was rarely necessary at subsequent injection times, as the volume of egg albumen decreased.

On the day following the second injection, the eggs were opened with scissors and the embryos were removed from the shell. Embryos were injected intraperitoneally with 0.2 ml Lethabarb anesthetic (Arnolds of Reading, Sydney, Australia), and blood was immediately sampled from the postoccipital venous sinus using a heparinized syringe. Liver and kidney tissues were collected, weighed, frozen in liquid nitrogen, and stored at -80°C until used for the preparation of microsomes. Blood was stored at 4°C until centrifugation at 850 g for 5 min. Plasma was removed and stored at -20°C.

Corticosterone radioimmunoassay. The free fraction of corticosterone is probably more physiologically important than the total concentration, as defined by the free hormone hypothesis (22). However, an assay for the measurement of free corticosterone was unavailable. Therefore, total corticosterone concentrations were measured in this study. Corticosterone was extracted from blood plasma (100 µl) with 1 ml AR grade ethanol by mixing for 1 min on a vortex mixer. Tubes were centrifuged for 5 min at 1,400 g to separate the precipitate from the supernatant. Two-hundred microliters of these sample extracts and 50 µl of corticosterone standards in the range of 0.5 to 16 ng/ml were transferred into duplicate assay tubes. Samples and standards were evaporated under a stream of air. Radioactive tracer (100 µl) consisting of ~6,000 counts/min (cpm) [1,2,6,7-3H]corticosterone (Amersham) and corticosterone antibody (100 µl) (Endocrine Sciences) were added. Tubes were briefly vortexed and allowed to equilibrate at 4°C overnight. Unbound steroid was removed by addition of 500 µl of cold (4°C) 5 mg/ml charcoal (Norit A) and 0.5 mg/ml dextran (T-70). After being briefly vortexed, the mixture was kept on ice for 15 min followed by centrifugation at 1,400 g at 4°C for 15 min. For each of the tubes, 150 µl of the supernatant was transferred to scintillation tubes and 2.5 ml Aqueous Counting Scintillant (Amersham) was added. To measure the total amount of radioactivity, 100 µl of tracer was added to 2.5 ml of scintillant. The radioactivity in each tube was counted using a scintillation counter (Packard, TriCarb 2100TR) for 5 min. Nonspecific binding of the tracer was on average 0.63%, and maximum binding of the antibody was 30%. The sensitivity of the assay was 0.1 ng/ml, and recovery of tritiated corticosterone after extraction was 92%.

Free T4 and free T3 radioimmunoassays. There were insufficient volumes of plasma samples to measure both total and free thyroid hormones. Measurement of free T3 and free T4 during development should represent the physiologically relevant fraction of thyroid hormones, as defined by the free hormone hypothesis (22). Free thyroid hormone radioimmunoassays were carried out using Coat-a-Count kits (Diagnostic Products). It is a solid-phase radioimmumoassay for the measurement of nonprotein-bound T4 and T3 levels in human plasma. These assay kits were validated for use with crocodile blood plasma. Charcoal (Sigma) was used to strip the plasma of endogenous thyroid hormones. Charcoal-stripped crocodile plasma was mixed with T3 or T4 standards and assayed to determine the accuracy of the assays by comparing linear relationships between expected and observed free T3 and free T4 concentrations. The recovery of standards from stripped plasma was 93% (free T3) and 103% (free T4). These results were compared with the standard curve, showing good parallelism. In both assays, the sample volumes suggested by the manufacturer (100 µl for free T3 and 50 µl for free T4) were not large enough to easily measure free T3 and free T4 in crocodile plasma. Presumably, this was because the levels of free thyroid hormones in crocodile plasma are much lower than in human plasma. Therefore, the volume of saltwater crocodile plasma sample used in the assays was increased. This was also done in parallel with human serum samples as a control. Increasing the volume of human serum and crocodile plasma to 200 µl in both assays still gave accurate measurements of free T3 and free T4, showing good parallelism to the standard curve. Results obtained with crocodile plasma were corrected for the use of a larger sample volume. Except for this modification of the crocodile plasma sample volume, the assays were conducted as per the manufacturer's instructions. Standards in the range of 1 to 100 pg/ml for the free T4 assay (50 µl) and in the range of 0.5 to 42 pg/ml for the free T3 assay (100 µl), and crocodile plasma samples, were added to duplicate tubes. Occasionally, only single-sample determinations were made due to insufficient sample volume. One milliliter of tracer ([125I]T4 corresponding to ~36,000 cpm or [125I]T3 corresponding to ~40,000 cpm) was added, and the tubes were vortexed briefly and incubated at 37°C for 1 h for the free T4 assay and 3 h for the free T3 assay. The tubes were decanted thoroughly, and the radioactivity remaining was counted in a gamma counter (Wallac, 1270 Rackgamma II) for 10 min. For both assays, maximum binding of the antibody was 50%. For the free T4 assay, the sensitivity of the assay was 0.1 pg/ml and the nonspecific binding of the tracer was 1.6%. For the free T3 assay, the sensitivity of the assay was 0.2 pg/ml and the nonspecific binding of the tracer was 1.3%.

Deiodinase assays. The characteristics of deiodinases in saltwater crocodile tissues have been recently investigated (36). Due to the small amounts of embryonic liver and kidney able to be collected, the deiodinase assay conditions in this study were based on the optimal deiodinase assay conditions found for juvenile crocodile tissue microsomal preparations.

Microsome preparation and protein determination. Microsomal fractions were prepared as previously described (36). Total protein concentrations of the microsomal fractions were determined by a Bio-Rad protein assay as previously described (36).

Radioiodinated iodothyronines. Radioactive iodothyronines were prepared in the laboratory as previously described (36).

Measurement of high-Km ORD activity using rT3 as substrate and low-Km ORD activity using T4 as substrate. Assays for the measurement of high-Km rT3 ORD and low-Km T4 ORD were carried out as previously described (36). Each sample was tested in duplicate, and all assays included blanks for measurement of nonenzymatic degradation of tracer. The incubation temperature was chosen based on the recent study of the characterization of deiodinases in tissues of the juvenile saltwater crocodile (36).

The specific reaction conditions for the rT3 ORD activity assays were as follows: the substrate concentration was 0.0625 nM 3',5'-[125I]rT3 (corresponding to 50,000 cpm) + 5 µM rT3 with 10 mM dithiothreitol (DTT). Incubation was for 30 min at 32°C. The final protein concentrations in the assays were 0.1 mg protein/ml for liver microsomes and 0.4 mg protein/ml for kidney microsomes.

The specific reaction conditions for the T4 ORD activity assays were as follows: the substrate concentration was 0.0625 nM 3',5'-[125I]T4 (corresponding to 50,000 cpm) + 1 nM T4. Liver microsomes were incubated with 10 nM T3 (to reduce interference of T3 IRD activity in liver microsomes) and 15 mM DTT for 120 min at 32°C. Kidney microsomes were incubated with 20 mM DTT for 120 min at 25°C. All microsomal samples were diluted to a final protein concentration of 1 mg/ml. A second substrate mixture was prepared containing a tracer (0.0625 nM 3',5'-[125I]T4) and a high substrate concentration of 100 nM T4 and incubated with liver and kidney microsomal samples. This was to test for the possibility of both low-Km and high-Km enzymes being present in each tissue.

Measurement of IRD activity using T3 as substrate. Assays for the measurement of T3 IRD were carried out as previously described (36). The specific reaction conditions for the T3 IRD activity assays were as follows: microsome samples from livers of saltwater crocodiles from days 60 and 68 were diluted to a final protein concentration of 0.1 mg/ml. The substrate concentration was 0.19 nM 3'-[125I] T3 (corresponding to 150,000 cpm) + 10 nM T3 and 50 mM DTT. Incubation was for 60 min at 35°C. Liver microsomal samples from day 75 of incubation and from hatchlings were diluted to a final protein concentration of 1 mg/ml. The substrate concentration was 0.19 nM 3'-[125I]T3 (corresponding to 150,000 cpm) + 1 nM T3 and 50 mM DTT. Incubation was for 60 min at 35°C.

Statistical analysis. Plasma concentrations of corticosterone and free thyroid hormones during development were analyzed using a one-way ANOVA with a Tukey-Kramer posttest. Comparisons between plasma hormone concentrations and deiodinase activities in liver and kidney microsomes in the control groups, and those of treatment groups, were analyzed using a one-way ANOVA with a Dunnett's posttest. Significance was assumed at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of hormone treatments on plasma corticosterone concentrations. The average concentrations of plasma corticosterone on days 60, 68, and 75 of incubation in animals receiving control injections, and at 1 day posthatching, are presented in Fig. 1, top. Plasma corticosterone increased significantly between days 68 and 75 (P < 0.01) and decreased significantly between day 75 and hatching (P < 0.05). Plasma corticosterone concentrations in embryos receiving hormone treatments are also presented in Fig. 1, top. T3 increased corticosterone levels compared with the control treatment on day 60 (P < 0.05) and on day 68 (P < 0.01) but not on day 75. Treatment of embryos with Dex suppressed plasma corticosterone on day 75 (P < 0.05) as did treatment with Dex + T3 (P < 0.05).


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  		Fig. 1.   Plasma concentrations of corticosterone (ng/ml ± SE; top), free 3,3',5-triiodothyronine (T3; pg/ml ± SE; middle), and free 3,3',5,5'-tetraiodothyronine (T4; pg/ml ± SE; bottom) on days 60, 68, and 75 of incubation in embryonic saltwater crocodiles receiving the control injections and at 1 day posthatching (black bars). dagger P < 0.05; dagger dagger P < 0.01; dagger dagger dagger P < 0.001 vs. the previous time point. Plasma concentrations of corticosterone (top), free T3 (middle), and free T4 (bottom) on days 60, 68, and 75 of incubation following 2 treatments of 5 µg T3 (striped bars), 50 µg dexamethasone (Dex; spotted bars), and Dex (50 µg) + T3 (5 µg) (open bars). *P < 0.05; **P < 0.01 vs. controls.

Effects of hormone treatments on plasma free T3 concentrations. Free T3 levels in plasma on day 60 of incubation were not measured due to lack of sample. Moreover, free T3 was not previously detectable by our assay before day 67 in embryonic crocodiles (CA Shepherdley, SJ Richardson, and BK Evans, unpublished observations). The concentrations of free T3 in plasma on days 68 and 75 in embryos receiving the control injections, and at 1 day after hatching, are presented in Fig. 1, middle. Plasma free T3 levels after hatching were significantly higher than on day 75 (P < 0.001). Plasma free T3 concentrations in embryos receiving hormone treatments are also presented in Fig. 1, middle. Treatment with T3 significantly increased free T3 levels in the plasma on days 68 and 75 of incubation (P < 0.05). Dex treatment did not affect the concentration of free T3 in plasma on days 68 or 75. The concentration of plasma free T3 was significantly increased by treatment with Dex + T3 only on day 75 (P < 0.05).

Effects of hormone treatments on plasma free T4 concentrations. The average free T4 concentrations on days 60, 68, and 75 in animals receiving the control injections, and at 1 day posthatching, are presented in Fig. 1, bottom. Plasma free T4 levels after hatching were significantly higher than on day 75 (P < 0.001). Plasma free T4 concentrations in embryos receiving hormone treatments are also presented in Fig. 1, bottom. Treatment with T3 significantly reduced free T4 concentrations in plasma on day 68 (P < 0.05). Treatment with Dex significantly reduced free T4 concentrations in plasma on days 68 (P < 0.01) and 75 (P < 0.05). Treatment with Dex + T3 also significantly reduced free T4 concentrations in plasma on days 68 (P < 0.01) and 75 (P < 0.05).

Effects of hormone treatments on rT3 ORD activity in liver and kidney microsomes. On days 60 and 68 of development, treatment with T3 did not affect rT3 ORD activity in the liver. However, on day 75, treatment with T3 increased rT3 ORD activity (P < 0.05) (Fig. 2A). Treatment of embryos with Dex or Dex + T3 did not have any effect on rT3 ORD activity in liver microsomes on days 60, 68, or 75 of development (Fig. 2A).


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  		Fig. 2.   Reverse (r)T3 outer ring deiodinase (ORD) activity in liver (A) and kidney microsomes (B) (pmol rT3 deiodinated · mg protein-1 · min-1 ± SE) on days 60, 68, and 75 of incubation in animals receiving the control injections (black bars) and following 2 treatments of 5 µg T3 (striped bars), 50 µg Dex (spotted bars), and Dex (50 µg) + T3 (5 µg) (open bars). *P < 0.05; **P < 0.01 vs. controls.

Treatment of embryos with T3 did not have any effect on rT3 ORD activity in kidney microsomes (Fig. 2B). On day 60, kidney microsomal rT3 ORD activity was reduced by treatment with Dex (P < 0.05) (Fig. 2B). Treatment with Dex + T3 also reduced kidney microsomal rT3 ORD activity on day 60 (P < 0.01) (Fig. 2B).

Effects of hormone treatments on T4 ORD activity in liver and kidney microsomes. Treatment of embryos with T3 did not have any significant effects on T4 ORD activity in liver microsomes (Fig. 3A). Treatment with Dex reduced liver microsomal T4 ORD activity on day 60 of incubation (P < 0.01) (Fig. 3A). On day 68 of incubation, Dex treatment had not changed T4 ORD activity from that of control levels. On day 75 of incubation, treatment with Dex significantly increased T4 ORD (P < 0.01). Treatment with Dex + T3 reduced T4 ORD activity on day 60 of incubation (P < 0.01) but not on days 68 and 75 (Fig. 3A).


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  		Fig. 3.   T4 ORD activity in liver (A) and kidney microsomes (B) (fmol T4 deiodinated · mg protein-1 · min-1 ± SE) on days 60, 68, and 75 of incubation in animals receiving the control injections (black bars) and following 2 treatments of 5 µg T3 (striped bars), 50 µg Dex (spotted bars), and Dex (50 µg) + T3 (5 µg) (open bars). *P < 0.05; **P < 0.01 vs. controls.

Treatment with T3 significantly increased kidney microsomal T4 ORD activity on days 60 (P < 0.05) and 68 (P < 0.05) but had no effect on day 75 (Fig. 3B). Treatment with Dex significantly decreased T4 ORD activity in kidney microsomes only on day 60 (P < 0.05) (Fig. 3B). Treatment of embryos with Dex + T3 had no effect on kidney microsomal T4 ORD on days 60, 68, and 75 of development (Fig. 3B).

Liver microsomes incubated with the tracer and the high-T4 substrate concentration of 100 nM had ~50% of the activity measured compared with assays using a low-T4 substrate concentration of 1 nM T4 on days 60 and 68 (data not shown). The high substrate tracer demonstrated 40% of the activity on day 75 and at hatching, hence both low- and high-Km enzymes were present in this tissue (data not shown). Incubation of the tracer together with a high-T4 substrate concentration of 100 nM inhibited all deiodination activity in kidney microsomes, indicating the presence of only a low-Km enzyme (data not shown).

Effects of hormone treatments on T3 IRD activity in liver microsomes. Treatment of embryos with T3 had no effect on T3 IRD activity in liver microsomes on days 60, 68, or 75 of development (Fig. 4). Treatment with Dex suppressed T3 IRD activity on days 60 (P < 0.01) and 68 (P < 0.05) (Fig. 4). Treatment with Dex + T3 suppressed T3 IRD activity on day 60 of incubation (P < 0.01) but had no effect on days 68 and 75 (Fig. 4).


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  		Fig. 4.   T3 inner ring deiodinase activity in liver microsomes (fmol T3 deiodinated · mg protein-1 · min-1 ± SE) on days 60, 68, and 75 of incubation in animals receiving the control injections (black bars) and following 2 treatments of 5 µg T3 (striped bars), 50 µg Dex (spotted bars), and Dex (50 µg) + T3 (5 µg) (open bars). *P < 0.05; **P < 0.01 vs. controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasma corticosterone and free thyroid hormones in embryonic and hatchling saltwater crocodiles. Similar to the situation in other vertebrates, corticosteroids in the plasma of embryonic saltwater crocodiles increased during development. However, plasma corticosterone declined at hatching. Compared with the developmental profile of plasma corticosterone in the American alligator (Alligator mississippiensis), another Crocodilian species, the decrease in plasma corticosterone late in development is unique to saltwater crocodiles. In alligator embryos, plasma corticosterone levels of males and females increased from the last third of incubation, continued to rise until hatching (days 62 to 70 of incubation), and did not decline until 3 wk after hatching (21). The decrease in plasma corticosterone before hatching in the present study could be due to rapid metabolism of glucocorticoids, a decrease in the rate of synthesis or a decrease in secretion, or a decrease in binding proteins in blood. Plasma corticosteroids in bird embryos show a decrease during (47) or preceding hatching (18). The plasma binding capacity for corticosterone and corticosteroid binding globulin (CBG) concentrations have been shown to decrease after days 15 or 16 of incubation (hatching at day 21) in the embryonic chicken, resulting in increased free corticosterone in the blood (14, 37). This result suggested that higher levels of free glucocorticoids in blood were required and were being rapidly used by tissues in the last stages of in ovo development. It still remains to be determined whether there is a CBG equivalent in reptiles and what percentage of plasma corticosterone is bound to this and other plasma binding proteins, such as albumin.

Successful development also depends on precisely controlled changes in plasma thyroid hormone levels. We hypothesized that, as in other vertebrates, free thyroid hormones would increase in plasma during development. This was observed during the last 20 days of development, the most significant increase occurring between day 75 of incubation and hatching. The concentrations of plasma free T3 and free T4 1 day posthatching were 22 times greater and 10 times greater, respectively, than free T3 and free T4 levels in plasma of juvenile (6 mo old) saltwater crocodiles (Shepherdley et al., unpublished observations). The magnitude of the increases in free thyroid hormones late in development was clearly an indication of the many developmental processes relying on thyroid hormones to regulate gene transcription.

Effects of hormone treatment on rT3 ORD, T4 ORD, and T3 IRD activities in liver and kidney microsomes. In the embryonic chicken, glucocorticoid administration has been related to changes in plasma thyroid hormone concentrations and deiodinase activities, particularly in the liver. Twenty-four hours following a single Dex treatment in the chicken embryo, hepatic high-Km rT3 ORD activity (type I) was increased (4). We hypothesized that two Dex injections given to saltwater crocodile embryos over a 48-h period would increase rT3 ORD activity in liver microsomes. This effect on rT3 ORD activity in liver microsomes was not demonstrated with Dex or Dex + T3 treatment. Treatment with Dex and Dex + T3 suppressed rT3 ORD in kidney at day 60, but no other effects were observed on days 68 or 75.

We hypothesized here that low-Km T4 ORD would increase in liver and kidney microsomes following glucocorticoid treatment, as has been shown recently for type II deiodinase activity in embryonic chicken brain (44). The effects of glucocorticoids on a low-Km T4-to-T3-converting enzyme in peripheral tissues have not been well studied, as a distinction has not always been made between high- and low-Km ORD activities in previous studies. In the present study, we attempted to measure specifically the low-Km T4 ORD activity in liver microsomes. However, this was very difficult. Results of T4 ORD activity in liver microsomes also included some contribution of a high-Km ORD enzyme because it was not possible to completely inhibit high-Km ORD activity in these assays. Additionally, the presence of very high levels of a low-Km IRD activity in liver microsomes (particularly on days 60 and 68 of incubation) may have also interfered with the assay, despite the addition of 10 nM T3 in an attempt to reduce low-Km IRD interference. Therefore, the results of T4 ORD assays in liver microsomes are difficult to interpret. Dex and Dex + T3 treatments decreased T4 ORD activity in liver microsomes at day 60. It is possible that glucocorticoids could have a role in decreasing T4 ORD activity in liver microsomes by speeding up maturation of the deiodinases, as T4 ORD activity in liver microsomes in untreated crocodile embryos decreased during the last 20 days of development (35). However, Dex and Dex + T3 treatments stimulated T4 ORD activity in liver microsomes at day 75. Therefore, Dex treatment earlier in development (i.e., around days 60 and 68) could have been inhibiting the low-Km ORD present, and treatment later in development (i.e., day 75) could have been increasing the activity of the high-Km enzyme. However, an increase in high-Km ORD due to Dex treatment was not demonstrated in the rT3 ORD assays for measuring high-Km ORD activity. In contrast to T4 ORD activity in liver microsomes, the results from assays using kidney microsomes appeared to show the presence of only low-Km T4 ORD activity. Dex treatment decreased T4 ORD activity in kidney microsomes at day 60, and T4 ORD activity had a tendency to remain low in kidney microsomes later in development. T4 ORD activity measured in kidney microsomes in the present experiments was due to exclusively low-Km ORD activity. As for liver, glucocorticoids may play a role in decreasing T4 ORD activity in kidney microsomes by speeding up maturation of the deiodinases, as T4 ORD in kidney microsomes in untreated crocodile embryos was also observed to decrease during the last 20 days of development (35).

Glucocorticoid treatment in embryonic chickens resulted in a decrease in hepatic T3 IRD activity (4, 9). This inhibition of T3 IRD activity has recently been shown to parallel a decrease in hepatic type III deiodinase mRNA levels (42), indicating that the decrease in T3 IRD activity in response to Dex treatment is regulated predominantly at the transcriptional level (44). In the present study, a decrease in T3 IRD activity in liver microsomes was demonstrated on days 60 and 68, after Dex treatment. The combined Dex + T3 treatment had a similar result on day 60.

Effects of hormone treatment on corticosterone, free T4, and free T3 concentrations in plasma. Treatment with T3 increased plasma corticosterone concentrations on days 60 and 68. In the postnatal rat, where significant development occurs during the first few weeks postpartum, hyperthyroidism induced by T4 administration resulted in a significant rise in both total corticosterone concentration and the capacity of CBG to bind corticosterone (3). Thyroid hormone administration also induced an increase in corticotropin-releasing hormone mRNA synthesis (3). This could explain the increase in plasma corticosterone in response to T3 treatment in saltwater crocodile embryos.

In the present study, treatment with Dex had a powerful suppressive effect on free T4 concentrations in the plasma of saltwater crocodile embryos. The lack of change in rT3 ORD activity in liver and kidney microsomes in response to Dex treatment suggested that the decrease in plasma-free T4 concentrations on days 68 and 75 following Dex and Dex + T3 treatment was not due to increased conversion of T4 to T3 by a high-Km ORD. Interestingly, Darras et al. (4) recorded a decrease in plasma total T4 4 h after glucocorticoid administration in the chicken embryo, which did not correspond to the increase in type I ORD in liver, which occurred later. These authors suggested that the decrease in plasma T4 in chicken embryos following glucocorticoid treatment was due to lower thyroidal T4 secretion due to decreased thyrotropin (TSH) secretion, as indicated by decreased plasma alpha -subunit levels and a decrease in the calculated TSH index. Although these parameters were not measured in the saltwater crocodile embryos, it is possible that glucocorticoid treatment could be having a similar effect. Glucocorticoids are also observed to have a suppressive effect on TSH in postnatal mammals (24, 27, 49). Again, the decrease in plasma free T4 levels does not seem to be related to changes in deiodinase activities.

In embryonic chickens receiving glucocorticoid treatment, plasma total T3 concentrations increase, corresponding to the decrease in T3 IRD activity (4, 9). In the embryonic crocodiles, there was no increase in plasma free T3 concentrations following Dex or Dex + T3 treatments that corresponded with decreases in T3 IRD activity. The increase in plasma free T3 concentrations between day 75 and hatching occurred when T3 IRD activity had almost completely diminished. It is possible therefore that plasma free T3 levels are tightly controlled during development. However, this remains a topic for further investigation.

These experiments also demonstrated that both the HPA axis and HPT axis were functioning. Treatment with Dex and Dex + T3 significantly decreased endogenous corticosterone concentrations on day 75. Hence, Dex appeared to be mimicking the actions of endogenous glucocorticoids in the embryonic saltwater crocodiles by affecting the HPA axis. Treatment with T3 increased circulating free T3 concentrations on days 68 and 75 and decreased free T4 concentrations significantly on day 68 but not on day 75. Changes in circulating plasma thyroid hormone concentrations probably resulted from negative feedback regulation by the HPT axis.

In summary, the effects of hormone treatment on free thyroid hormones and corticosterone in plasma and deiodinase activities in saltwater crocodile embryos varied depending on the stage of development and the tissue type. Dex treatment had inhibiting effects on T4 ORD activity in liver and kidney microsomes and on rT3 ORD activity in kidney microsomes around day 60 of incubation. The stimulating effect of Dex treatment on T4 ORD activity in liver microsomes occurred during the later stages of development in this study, on day 75, suggesting specific timing of the responses of the deiodinases to glucocorticoids. A decrease in free T4 was not related to changes in rT3 ORD or T4 ORD activities and was possibly produced by changes in TSH secretion. Glucocorticoid treatment had a suppressive action on T3 IRD activity in liver microsomes, even though there was not a corresponding increase in free T3 concentrations in plasma, suggesting that free T3 levels are tightly regulated during development.


    ACKNOWLEDGEMENTS

The authors thank L. Sullivan, Dr. S. Munns, Dr. P. Wood, and J. Griffiths from Adelaide University and W. Van Ham, F. Voets, and L. Noterdaeme from the K. U. Leuven for technical assistance. Crocodile eggs were maintained at Adelaide University under permit from the National Parks and Wildlife (South Australia, Australia) (#Q-20168). Animal surgery was performed under permit from the Adelaide University Animal Ethics Committee (S/45/00).


    FOOTNOTES

This research was funded by Australian Research Council grants to C. B. Daniels and S. J. Richardson.

Address for reprint requests and other correspondence: C. A. Shepherdley, Dept. of Biochemistry and Molecular Biology, Univ. of Melbourne, 3010, Victoria, Australia (E-mail: sjrich{at}unimelb.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpregu.00015.2002

Received 11 January 2002; accepted in final form 17 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 283(5):R1155-R1163
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