Thyroid development in altricial Ring doves, Streptopelia risoria

CTENERALAND

COMPARATIVE

ENDOCRINOLOGY

Thyroid Development

58,

243-251 (1985)

in Altricial

F. M. ANNE

MCNABE

Ring Doves, Stepfopelia AND MEI-FANG

rdsoria

CHENG

Department of Biology, Virginia Polytechnic Institute and State,University, Blacksburg, Virginia 24061, and Institute of Animal Behavior, Rutgers University, Newark, New Jersey 07102 Accepted July 20, 1984 The development of thyroid function in altricial Ring doves was assessed by measuring thyroid hormone concentrations in the serum, hormone content of the thyroid glands and hepatic 5’-monodeiodinase activity. Thyroid function is low at hatching and increases during the first eight days whiIe the nestlings are ectothermic and completely dependent on parental care. The rate of increase of serum hormone concentrations slows after Day 8; hormone concentrations are stable by Day 15 and for the remainder of the nestling and early fledgling periods while locomotor ability, feeding self-sufficiency and thermoregulatory ability are maturing. Increases in serum hormone concentrations precede increases in thyroidal hormone content. T,/T, ratios in serum are much higher than those of stored hormones in the thyroid. Deiodination of T4 to T3 is important in T, production throughout development but most so in the early nestling stages. 0 1985 Academic Press. ICIC.

Ring doves are altricial, that is, they are hatched in a poorly developed condition (naked, blind, and incapabIe of coordinated locomotor activity), require complete parental care, are ectothermic initially, and acquire thermoregulatory control during the latter part of the nestling period. Histological comparisons of the thyroid glands of ring doves (altricial) and Japanese quail (precocial) suggest the doves have very low thyroid activity prior to hatching in comparison to the precocial quail (McNabb and McNabb, 1977). Since the thyroid is inherently involved in the control of metabolism in homeothermic vertebrates, it seems likely that the ectothermic part of the nestling period in altricial young is characterized by low thyroid activity. Recently, one of us has demonstrated that thyroid activity (as indicated by radioiodine uptake and serum hormone concentrations) is low in early dove nestlings and that there are marked differences in the pattern of thyroid development in altricial doves vs precocial quail (McNabb et al., 1984). In the only other published study of posthatching thyroid development in altricial birds, Dawson and Allen (1960) concluded

that the establishment of homeothermy in vesper sparrows was not limited by the functional state of the thyroid glands. This conclusion was based on measurements of thyroid epithelial cell height which peaked on the fifth day, a day or two before the establishment of homeothermy. However, serum hormone concentrations were not measured, so the extent to which their measurements reflected hormone release. as well as hormone production and storage, cannot be evaluated. The objectives of this study were to describe more completely the pattern of thyroid development in altricial ring doves, and to examine the components that determine that pattern, from the day before hatching through the time of fledging. V& measured serum thyroid hormone concentrations, hormone content of the thyroi gland, and hepatic capacity for peripheral triiodothyronine (TX) production in relation to age, body growth, and body temperature. METHODS AND MATERIALS Ring doves were obtained from the breeding colony at the Institute of Animal Behavior. Rutgers Univer243 QO16-6480185 $1.50 Copyright All ri&ts

0 1985 by Academic Press, inc. of reproduction in any form reserved.

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sity, and sampled on a single day. Maintenance conditions for the colony have been described previously (Cheng, 1979). Each bird was removed from the nest, its body temperature (Ts) was measured as cloaca1 temperature to the nearest O.l”C (Tele-Thermometer with thermistor probe; Yellow Springs Instrument Corp., Yellow Springs, Ohio), body weight (W,) was determined to the nearest 0.1 g, and blood was drawn by cardiac puncture. Thyroids were removed, weighed to the nearest 0.1 mg (thyroid weight: W,), and stored on ice. Livers were removed, weighed to the nearest 0.1 mg and a sample of liver was weighed, stored in three volumes of buffer (see deiodinase methods below), then frozen in liquid nitrogen. Embryos within 1 day of hatching were sampled as were chicks except that Ts was not measured. Samples were analyzed at Virginia Polytechnic Institute and State University. Serum hormone concentrations were analyzed by a double antibody radioimmunoassay (RIA) as described by McNabb and Hughes (1983) verified for use on dove serum by the methods described previously (McNabb et al., 1981). Primary antibodies and carrier immunoglobulin were purchased from Antibodies, Inc. (Davis, Calif.); second antibody produced in sheep was provided by Dr. Larry Cogburn, University of Delaware. To determine thyroidal hormone content, each pair of thyroid glands was homogenized in 250 ~1 of a Pronase digestion medium and incubated at 37°C for 24 hr. The digestion medium was modified from the method of Dunn (1975); Tris (0.1 M), glutathione (5 mlM), thiouracil (2.5 mM), Triton X-100 (1% of final volume) with five times the thyroid weight of Pronase (Calbiochem-Behring, San Diego, Calif.) added just before homogenization. After incubation an equal volume of 95% ethanol was added to the tubes, they were vortexed, extracted at - 20°C for 24 hr, then centrifuged at 13,500g for 5 min. The supernatant was pipetted off the precipitated proteins and stored frozen for analysis. Supematants were analyzed for T, and T, content by the double antibody RIA described previously (McNabb and Hughes, 1983) using standards prepared in an ethanol concentration equivalent to that in the samples. Precision and accuracy of the RIA in the ethanolbased system was comparable to that previously reported (McNabb and Hughes, 1983). To determine the digestion efficiency of Pronase for releasing stored thyroid hormones, we prelabeled thyroid glands in vivo with 1251.After different digestion times, the labeled protein and hormones in 250+1 samples of homogenate were separated by column chromatography on Sephadex G/25 Superfine (Sigma Chemical Co., St. Louis, MO.) using 0.021 M NaOH. The optimal digestion time considering thyroidal protein digestion as well as preservation of released hormones was -24 hr. At this time, 75% of the label in thyroidal protein had been released by Pronase digestion. Studies in which

high-specific activity labeled T, (750 &iip.g; Industrial Nuclear Co., St. Louis, MO.) and T, (1200 t&i/ kg; Amersham) were added to homogenates before incubation indicated ethanol extraction recoveries of 100.0% of the labeled T, and 95.5% of the labeled T,. The enzymatic activity of 5’-monodeiodinase in liver tissue, with abundant substrate available, was determined by a modification of the method of Harris et al. (1978). Weighed samples of liver tissue in three volumes (voliwt) of buffer [O. 1 M morpholinopropanesulfonic acid (MOPS), 10 mM EDTA, pH 7.41 were homogenized in a glass/Teflon coaxial tissue grinder. Duplicate 50-~1 samples of homogenate plus 25 ng T, in 10 ~1 of buffer were incubated, in the presence of 1.0 mi!4 dithiothreitol, in 6 x 50-mm glass tubes at 37°C for 1 hr. After incubation, the reaction was stopped by the addition of 70 ~1 of ice-cold ethanol, stored for 16 hr at - 20°C than centrifuged at 1OOOgfor 20 min at 4°C. Blanks were treated as above except that T, was added immediately before the addition of the ethanol. The concentration of T, in the blanks was subtracted from sample T, values. Recovery of [12sI]T, added to homogenate just before the addition of ethanol was determined for each set of samples and used to calculate an extraction factor used to correct T, values. Protein concentration of the homogenate was determined by Biorad Protein Assay (Biorad Laboratories, Richmond, Calif.), with bovine serum albumin (Sigma) as the standard. Statistical comparisons were made by Duncan’s new multiple range test. Values of P s 0.05 were considered indicative of statistically significant differences.

RESULTS

Serum concentrations of both T, and T, (Fig. 1) were low at hatching, rose during the first half of the nestling period (values at Day 8 significantly greater than perinatal values), and stabilized before the age of fledging at 20-22 days. There was close correspondence between the hormone concentrations of the Rutgers colony doves which were sampled on a single day and the animals from Virginia Tech which were sampled between 1 and 3 PM EDST, as available, over a longer period of time (see values from McNabb et al. (1984) in Fig. 1 of this study; rearing conditions equivalent in both locations). Body and thyroid growth rates also were consistent with those re-

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1. Serum thyroid hormone concentrations during development in ring doves. Solid circles are means i 2 SE for doves from the Rutgers colony. E, embryos 1 day prior to hatching; H, nestlings within 12 hr after hatching. The results of Duncan’s new multiple range test are presented bt’low each graph. Ages joined by solid lines do not differ significantly. N values for both panels are at the top of the graph. For comparison, data from the Virginia Tech colony (McNabb et al., 1984) are shawn; small open circles are individual values, large open circle is the means + 2 SE for adults. FIG.

ported in the same study. Data from the present study extend and support the previous data indicating that thyroid and body growth are proportional from hatching to adulthood (i.e., constant W,iW, ratio). Mean serum T,/T, ratios (Fig. 2) doubled during the first 7-8 days of the nestling period because the increase in serum T, concentration was proportionally much greater than the increase in T4 concentrations. However, because of high variability, the ratios were not significantly higher than those of the perinatal period until the time of fledging, During the early nestling period mean TB

of dove nestlings tended to increase but individual values were highly variable: 37.0 rt l.O”C at hatching, 39.2 & 1.2”C at Day 7 (values expressed as X k 2 SE). The nestlings were fed regularly and brooded almost continuously, by the parents, for the first 6 days but by Day 8 the parents spent most of the time off the nest. We did not attimpt to correlate Tn of nestlings with parental attentiveness. The TB of dove nestlings was 41.8 + O.l”C by Day 15, increased significantly to 42.4 k 0.4”C by Day’21 and did not change significantly between Days 21 and 28. The hormonal content of the thyroid

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FIG. 2. Serum T,/T, ratios during development in ring doves. Solid circles are means + 2 SE for doves from the Rutgers colony. E, embryos 1 day prior to hatching, H, nestlings within 12 hr after hatching. N values are above the graph. The result of Duncan’s new multiple range test are presented below the graph. Ages joined by solid lines do not differ significantly. For comparison, data from the Virginia Tech colony (McNabb et al., 1984) are shown; small open circles are individual values, the large open circle is the mean k 2 SE for adults.

gland (Fig. 3) was very low through Day 3, then increased markedly until Day 22 (a 52fold increase in T, content; a 33-fold increase in T, content). The quantity of hormone, per milligram of thyroid tissue, increased 8.9-fold for T, and 5.6fold for T, from hatch to Day 22. The rise in gland hormone content (Fig. 3) was delayed in time compared to the rise in serum thyroid hormone concentrations (Fig. 1). Mean T,/T, ratios in the thyroid gland were highest in the early nestling stages (Fig. 3). The ratio decreased during the remainder of the nestling/fledgling period because T, content of the gland increases faster than T, content. T,/T, ratios in the gland (Fig. 3) are about i/lo those in the serum (Fig. 2). Peripheral 5’-monodeiodinase activity of the liver (Fig. 4) was low at the time of hatching, then rose significantly to peak at Day 3. Although mean values tended to decrease after Day 3 there were no further significant changes in deiodinase activity when expressed on a whole-body basis (Fig. 4, top). On a weight-specific basis, deiodinase activity decreased significantly after Day 3 (Fig. 4, bottom).

DlSClJSSlON

Thyroid Function in Relation and Development

to Growth

In altricial doves thyroid activity is low at hatching and for the first few days of the nestling period. All the variables we have studied, as well as histological studies of embryonic thyroids (McNabb and McNabb, 1977), support this generality; serum hormone concentrations, hormonal content of the thyroid gland, peripheral T,-to-T, conversion (this study), and thyroidal ‘*‘I uptakes (McNabb et al., 1984). This low thyroid activity seems consistent with the limited physical development and ectothermic nature of altricial hatchlings. Most of the increase in serum concentrations of both hormones occurs between hatching and about Day 8. During this time the parents provide warmth by almost continuous brooding and provide nourishment in the form of regurgitated crop milk. Body growth is very rapid (McNabb et al., 1984) consistent with the pattern typical of altricial young (Ricklefs, 1973, 1979) which are not expending energy on thermoregulation.

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FIG. 3. Hormonal content of the thyroid glands during development in ring doves. Values are the mean + 2 SE. N values that apply to all panels are at the top of the graph. E, embryos 1 day prior to hatching; H, nestlings within 12 hr after hatching. The results of Duncan’s new multiple range test are presented at the bottom of each panel. Ages joined by solid lines do not differ significantly.

Breitenbach and Baskett (1967) describe 6to S-day dove nestlings as just beginning to show thermoregulatory responses. On the basis of serum T, concentrations equal to those of adults, and serum T,/T, ratios greater than those of adults, thyroid activity appears to be relatively high during the time when nestlings are gaining physiological maturity., This period is initiated at about Day 8 when parental care becomes intermittent. From this point onward the young are attaining coordinated locomotor ability, developing thermoregulatory responses to cooling and acquiring full plumage. TB increases and stabilizes at a level

characteristic of adults by the time of fledging. The correlation of these developmental and physiological changes with t-e]atively high serum T, concentrations and T,/T, ratios is suggestive of the thyroid playing a key role. This idea is consistent with receptor studies (Bellabarba and Lehoux, 198 1; Weirich and McNabb, 1984) that suggest T, is the metabohcahy active hormone in birds as it is in mammals (Onpenheimer, 1979). However, a direct role for T, in some of these events cannot be ‘excluded at this time. All the studsees necessary (as judged by standard’ criteria for’ receptor studies; Hechter, 19783 to exclude

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FIG. 4. Hepatic 5’-monodeiodinase activity during development in ring doves. Values are the mean +- 2 SE. N values are at the top of the graph. E, embryos 1 day prior to hatching; H, nestlings within 12 hr after hatching. The results of Duncan’s new multiple range test are given for each panel. Ages joined by solid lines do not differ significantly.

the possibility of T, receptors in birds have not been done, and T, receptors have been documented in at least one vertebrate order (see work on amphibians by Galton, 1980). Historically a number of studies have suggested equipotency of the two hormones in birds (see review by Ringer, 1976; Almeida and Thomas, 1980). In these studies, the goitrogens used were assumed to suppress all T,-to-T, conversion. However, King et al. (1977) have presented evidence that such suppression is only partial, so apparent T, potency in birds may have been due to T, conversion to metabolically active T,. Recently Chandola et al. (1982) have suggested that while T, is important in the control of metabolic events, T, may act as a true hormone with respect to plumage and reproductive influences during seasonal cycles in adult birds. Thus, while T, and TX/T, ratios seem to best fit with key

developmental effects in doves in our study, additional work is needed to resolve whether T, also is playing a direct role. Hormone

Content of the Thyroid Gland

The pattern of thyroid hormonal content alone does not determine the serum hormone pattern during development. The rise in serum concentrations of T, and T, precedes the period when there are marked increases in thyroidal stores of both hormones. This suggests a balance favoring hormone release with very little hormone storage during the early nestling period. Thyroidal lzsI uptakes indicate thyroid activity is increasing during this time (McNabb et al., 1984). We speculate that hormone turnover may be low due to the ectothermic condition of early nestlings. Low turnover and a thyroidal balance favoring hormone release would promote the

THYROID

DEVELOPMENT

rise in serum T, and T, concentrations that occurs in the first 8 days. By the time serum hormone concentrations and T,/T4 ratios stabilize after about Day 15 the hormone content of the thyroid is substantial, indicating there have been increases in hormone production and storage. Although hormone turnover also may be high (because the nestlings have become metabolically more active) thyroidal capacity is sufficient to maintain stable serum hormone concentrations as well as substantial hormone stores in the thyroid. The relatively high thyroidal T,/T, ratio of early dove nestlings may reflect the effects of relatively low iodine stores, a condition known to shift hormone production toward T, in mammals (Studer et al., 1974). Thyroidal radioiodine uptake studies support this idea; uptake rates at 2-3 days of age are
IN

249

DOVES

and consequently have low T3/Td ratios in the thyroid. The thyroidal T,IT, ratios in developing doves are similar to those seen during development in precocial quail (unpublished studies in our laboratory) and in adult ducks, pigeons, and quail (Astier, 1975) but are about l/lo those reported for chicken embryos (Daugeras et al., 1976). Peripheral

Deiodination

of T4 to T3

Comparison of T3/T4 ratios in the serum with those in the thyroid (serum lo-fold higher) suggests T,-to-T, conversion is a significant factor at all developmental stages. When hepatic 5’-monodeiodinase activity is considered on either a wholebody or weight-specific basis it is seen to increase significantly for the first 3 days after hatching. However, by Day 8 this activity has again decreased when considered in proportion to body weight. Some caution is needed in the interpretation of deiodinase activity as measured in this study. Since abundant substrate and cofactor were available, these values probably exceed in vivs activity. At the itz vitro rates we measured, the liver alone could generate more than tfie amount of T, per 24 hr needed to achieve the serum concentrations observed in early nestling stages, while it could generate only a fraction of the amount per 24 hr needed to supply serum concentrations from Days 8 through 28. These calculations and the weight-specific expression of deiodinase activity, are consistent with the suggestion that peripheral T&o-T, conversion is most important in early nestlings. klowever, intrathyroidal T,-to-T, conversion during hormone release may also change with development and play a role in the pattern of serum T,/T, ratios. Additional studies that measure tissue substrate (T4) concentrations and cofactor availability, and attempt to estimate actual T,-toaT, conversion rates are needed. The increase in peripheral T, generation in doves occurs much later than that seen in precocial avian development. Chicken

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MCNABBANDCHENG

embryos deiodinate T, primarily to reverseT, (rTJ by inner ring monodeiodination until shortly before hatching, then shift to T, production by 5’-monodeiodination (Borges et al., 1980). This shift from rT, to T, production and the importance of peripheral T, production in the increased T,/ T, ratios during the perinatal period has been revealed by developmental studies of the metabolism of labeled hormones (Borges et al., 1980), of serum concentrations of T,, T,, and rT, (Thommes and Hylka, 1977) and of the effects of inhibitors of peripheral deiodination on serum hormone concentrations (Decuypere et al., 1982). In contrast to this pattern in chickens, in doves, peripheral production of T, appears to be highest and to have the greatest effect on serum T,/T, ratios during early posthatching life. Altricial

vs Precocial Development

This study complements and extends our previous study (McNabb et al., 1984) showing that the development of thyroid function in altricial doves occurs much later and is distinctly different in pattern from that characteristic of precocial species. Thus the functional development of the thyroid during embryonic life in doves (this study: McNabb and McNabb, 1977) is very low in comparison to that in precocial species (see, for example, for chickens, Thommes and Hylka, 1977; quail, McNabb et al., 1981). The perinatal peak in serum thyroid hormones, that occurs in precocial birds (see references above), is lacking in altricial doves (this study; McNabb et al., 1984). During the first 8 days after hatching thyroid functional development in doves proceeds steadily and relatively rapidly (this study; McNabb et al., 1984). In contrast, after hatching in precocial species, serum hormone concentrations decrease and stabilization of thyroid function at adult levels occurs slowly (see McNabb et al.,

1984 for a more detailed comparison the altricial and precocial patterns).

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ACKNOWLEDGMENTS This study was supported by NIH Grant ROlAM28216 to F.M.A.M., NSF Grant BNS 8121495 to M.-EC. and NLH Research Scientist Development Award MH 70897 to M.-EC. We thank Caroline Desidario, Thomas Hughes, and Sam Dicken for technical assistance.

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Decuypere, E., Kuhn, E. R., Clijmans, B., Nouwen, E. J., and Michels, H. (1982). Prenatal peripheral monodeiodination in the chick embryo. Gen. Comp. Endocrinoi. 47, 15-17. Dunn, A. D. (1975). Iodine metabolism in the Ascidian, Molgula manhattensis. Gem. Comp. Endocrinol. 25, 83-95. Galton, V. A. (1980). Binding of thyroid hormones in vivo by hepatic nuclei of Rana catesbeiana tadpoles. Endocrinology (Baltimore) 106, 859-866. Harris, A. R. C., Fang, S.-L., Prosky, J., Braverman, L. E., and Vagenakis, A. G. (1978). Decreased outer ring monodeiodination of thyroxine and reverse triiodothyronine in the fetal and neonatal rat. Endocrinology (Baltimore) 103, 2216-2222. Hechter, 0. (1978). The receptor concept: Predjudice, prediction and paradox. In “Hormone Receptors” (D. M. Klachko, L. R. Forte, and J. M. Franz, eds.), Vol. 96. Plenum, New York. King, D. B., King, C. R., and Eshleman, J. R. (1977). Serum triiodothyronine levels in the embryonic and post-hatching chicken, with particular reference to feeding-induced changes. Gen. Comp. Endocrinol. 31, 216-223. McNabb, F. M. A., and Hughes, T. E. (1983). The role of serum binding proteins in determining free thyroid hormone concentrations during development in quail. Endocrinology (Baltimore) 113, 957-963.

McNabb, E M. A., and McNabb, R. A. (1977). Thyroid development in precocial and altricial avian embryos. Auk 94, 736-742. McNabb, R M. A., Stanton, E W., and Dicken, S. G. (1984). Fost-hatching thyroid development and body growth in precocial vs altricial birds. Camp. Biochem. Physiol., A 78, 629-635.

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McNabb, R. A., Stouffer, R. L., and McNabb, E M. A. (1972). Thermoregulatory ability and the thyroid gland: Their development in embryonic Japanese quail (Coturnix coturnix japonica). Comp. Biochem. Physiol. A 43, 187-193. Oppenheimer, J. H. (1979). Thyroid hormone action at the cellular level. Science (Washington, D.C.) 203,971-979.

Ricklefs, R. E. (1973). Patterns of growth in birds. IT. Growth rate and mode of development. ibis 115, 177-201. Ricklefs, R. E. (1979). Patterns of growth in birds. 1% A comparative study of development in the starling, common tern and Japanese quail. Auk 96, 10-30. Ringer, R. K. (1976). Thyroids. In “Avian Physiology” (P. D. Sturkie, ed.), pp. 349-358. Springer-Verlag, New York. Studer, H., Kohler, I-I., and Burgi, H. (1974). Iodine deficiency. In “Handbook of Physiology,” Section 7 “Endocrinology,” Vol. 3; “Thyroid” (R. 0. Greep, and E. B. Astwood, eds.), pp. 303328. Amer. Physiol. Sot., Washington, D.C. Thommes, R. C., and Hylka, V. W. (1977). Plasma iodothyronines in the embryonic and immediate post-hatch chick. Gen. Comp. Endocrinol. 32; 417-422.

Trunnell, J. B., and Wade, P (1955). Factors governing the differentiation of the chick embryo thyroid: II. Chronology of the synthesis of iodinated compounds studied by chromatographic analysis. J. Clin. Endocrinol. Metab. 15, 107-117. Weirich, R. T., and McNabb, E M. A. (1954). Nu* clear receptors for L-triiodothyronine in quail liver. Gen. Comp. Endocrinol. 53, 90-99.