Pituitary-thyroid axis reactivity to hyper- and hypothyroidism in the perinatal period: ontogeny of regulation of regulation and long-term programming of responses.

To evaluate the role of perinatal thyroid status in the development of pituitary-thyroid axis regulation, we administered triiodothyronine to newborn rats for the first five days postpartum to achieve hyperthyroidism, or propylthiouracil perinatally to rat dams and pups from gestational day 17 through postnatal day 5 to achieve hypothyroidism. Plasma T4, T3, and TSH levels were determined from birth through 50 days postpartum. Administration of exogenous T3 produced the expected immediate suppression of plasma T4 and TSH, with recovery toward normal values beginning within days of discontinuing the T3 regimen. Plasma T3 values were markedly elevated during the period in which T3 was being given, but subsequently became subnormal, with deficits persisting into young adulthood. With the PTU regimen, plasma T4 and T3 levels were markedly suppressed through postnatal day 10, rose over the ensuing two weeks, but nevertheless showed significant deficits into adulthood. TSH levels in the immediate neonatal period were subnormal in the PTU group, despite the marked lowering of circulating thyroid hormones; TSH then rose dramatically to levels four times normal, subsiding to control values by the end of the first month. These results suggest that a critical period exists in which regulation of pituitary-thyroid axis function is programmed. During this phase, TSH secretion can be suppressed by excess thyroid hormones, but cannot be increased by hormone deficiencies. Perhaps more importantly, perinatal thyroid status "programs" its own future reactivity, so that early hypothyroidism results in reduced T4 and T3 levels in adulthood, despite normal levels of TSH.     

Alcohol dehydrogenase activity in the developing chick embryo

Before day 9 of incubation, chick embryos contain no measurable alcohol dehydrogenase (ADH) activity. Following day 9 of incubation, chick embryo liver ADH activity increases as a linear function of liver mass. A single dose of ethanol given at the start of incubation is cleared only slowly prior to day 9 of incubation but is completely cleared by day 13. Chick embryo liver ADH has two detectable isozymes throughout development. The percentage contribution of each isozyme to total ADH activity does not change significantly during development. The Km apparent of chick liver ADH is significantly increased shortly after hatching relative to the Km apparent of embryonic ADH. Ethanol exposure during incubation has no effect on the development of ADH activity or isozyme distribution.     

Synthesis of a lens-specific antigen (delta-crystallin) in rudimentary chicken adenohypophysis

The synthesis of two lens-specific proteins, delta- and beta-crystallins, by adenohypophyseal anlage of 4-day chick embryos was studied by the immunofluorescence technique in conjunction with autoradiography. Isolated anlages were incubated for 16 hours in a culture medium containing 14c-leucine. The synthesis was determined with the use of an unlabelled carrier, extract of chick lens, as well as of antisera against delta- and beta-crystallins. 14C-Leucine incorporation was found to occur only in delta-crystalline precipitation line rather than in beta-crystallin line. This evidence attests to the synthesis of delta-crystalline by the chick embryo adenohypophyseal anlage. The results are in agreement with the previously obtained immunohistochemical data on delta-crystalline localization in cells of the developing adenohypophysis.     

Vitamin A economy of the developing chick embryo and of the freshly hatched chick

1. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.     

Role of the thyroid in the involution of the mesonephric Malpighian corpuscles in the chick embryo

The mesonephros of the chick embryo normally begins to regress during the second half of embryonic life. Experimental methods, such as adenohypophysis grafting, hypophysectomy or use of antithyroid drugs, which stimulate or depress the thyroid function of the embryo, modified accordingly the regressive processes occurring in the mesonephric Malpighian corpuscles, particularly at the level of the glomerular basement laminae. These results as well as the known sensitivity of the mesonephros to thyroxine and the concordance between the steps of embryonic thyroid development and the mesonephric modifications show that the thyroid normally plays a major determining role in this phenomenon.     

Preliminary investigation of the use of high frequency ultrasound imaging in the chick embryo

BACKGROUND: The purpose of this study was to investigate the feasibility of using high frequency ultrasound to study the chick embryo in a noninvasive and longitudinal fashion. METHODS: A total of 10 SPF White Leghorn chick embryos (GDs 11-17; Hamburger and Hamilton stage 37-43) were consecutively examined with a GE Logiq 400 ProSeries ultrasound unit using an 11-MHz small parts ultrasound probe. Access for ultrasound visualization of the embryos was accomplished by opening a 2-3-cm window either in the air cell over the blunt end of the egg or laterally over the embryo-dependent side of the egg. Warmed ultrasound coupling gel was used for imaging, and thermal regulation was maintained with infant heel warmers. The ultrasound images were recorded directly on digital video using a Sony TRV 900 DV camcorder. The images were directly converted to jpeg and mjeg2 files for further analysis. RESULTS: Effective visualization of each embryo was possible on each day of the study period. The embryos were best visualized through the opening made in the air cell at the blunt end of the egg. The extent of the anatomic survey of the chick embryo was dependent upon the position of the embryo in the egg relative to the opening in the air cell. Doppler color flow mapping studies were obtained of the embryonic and extraembryonic circulation. CONCLUSIONS: This preliminary investigation clearly shows the feasibility of high frequency ultrasound imaging to study chick embryo development in a longitudinal and noninvasive fashion. Further studies are presently ongoing regarding earlier embryo development, as well as to determine the stability and dynamics of the methodology.     

Lipid and carbohydrate metabolism in euthyroid and hypothyroid chick embryo (Gallus domesticus).

1. The effect of propylthiouracil (PTU)-induced hypothyroidism on carbohydrate and lipid metabolism was studied in the chick embryo. 2. A single dose of PTU (250 micrograms/embryo) was administered on day 11 and embryos sacrificed on day 20 of incubation. 3. Thyroid glands were significantly enlarged (6 fold) by PTU administration. 4. Increased thyroid weight was associated with growth retardation and decreased plasma thyroxine levels. 5. Plasma glucose level was lower and phospholipids were significantly higher in the hypothyroid embryo. 6. Liver lipid concentrations in the control and hypothyroid embryos were not different but were significantly higher in both groups when compared to previously reported values in the young chick. 7. In contrast to PTU treatment after hatching, liver glycogen levels were not increased in the hypothyroid chick embryo. This was attributed to the high lipid nutrient condition of the chick embryo since a high lipid diet in the young chick decreased hepatic glycogen accumulation significantly.     

Degradation and biological inactivation of thyrotropin releasing hormone (TRH): regulation of the membrane-bound TRH-degrading enzyme from rat anterior pituitary by estrogens and thyroid hormones

Thyrotropin releasing hormone (TRH, pyroGlu-His-Pro-NH2) is important in the regulation of adenohypophyseal hormone secretion and also serves important functions in extrahypothalamic brain areas, indicating that it is involved in neurotransmission and other forms of cellular communication. This hypothesis is strengthened by the observation that TRH is rapidly inactivated by a heterogeneously distributed ecto-enzyme which exhibits a high degree of substrate specificity. Moreover, in the rat, the activity of the membrane-bound TRH-degrading enzyme of the anterior pituitary is found to be stringently controlled by thyroid hormones and estrogens. In contrast, the activity of the TRH-degrading brain enzyme is neither influenced by thyroid hormones nor estrogens. These data indicate that the TRH-degrading brain enzyme serves specialized functions for the transmission of TRH signals and apparently represents the peptidergic equivalent to acetylcholine esterase, whereas the membrane-bound adenohypophyseal TRH-degrading enzyme itself fulfills a biologically important control function within feedback-regulatory mechanisms.     

Thyroid hormone response to thyrotropin releasing hormone after cold treatment during pre- and postnatal development in the domestic fowl

The purpose of the present study was to compare the effect of periodic cooling during the establishment of a functional pituitary-thyroid axis at days 11-14 of incubation and at other developmental stages, on the subsequent thyroid hormone response to thyrotropin releasing hormone (TRH). In the first and second experiment chick embryos were cooled for 6 hr/day to 30 degrees C from day 11 till 14 and from day 15 till 18 respectively, whereas control groups were incubated throughout at 37.8 degrees C. In both experiments the thyroxine (T4) response upon TRH in 19 day-old embryos was higher in the previously cold treated embryos, according to the percentages of increase. However, the higher T4 response in the cold treated animals disappeared in 1 or 7 day-old chicks hatched from the 2nd experiment, but remained present in chicks of the same ages in the 1st experiment. In a third experiment the T4 response to TRH injection immediately and 3 and 8 days after a temperature treatment (25 degrees C or 12 degrees C) for one week on four weeks old broiler chickens was found to be similar in both temperature groups. In all experiments there was a concomitant triiodothyronine (T3) increase after TRH injection, but differences between experimental groups were observed at days 15 and 19 of incubation and immediately after the postnatal temperature treatment. As an overall conclusion the results indicate that cold treatment only during the establishment of the hypothalamo-hypophysial control of thyroid function can have a long lasting effect by enhancing the T4 response to TRH injection.     

Histological changes in the pituitary-thyroid axis during spontaneous and artificially-induced metamorphosis of larvae of the flounder Paralichthys olivaceus

Summary Histological changes in the pituitary TSH cells and in the thyroid gland of flounder (Paralichthys olivaceus) larvae during spontaneous or artificially induced metamorphosis were studied. Activity of the immunoreactive TSH cells (IrTSH cells) gradually increased during premetamorphosis, reaching the highest level in prometamorphic larvae, and the cells were degranulated in metamorphic climax. The IrTSH cells were most inactive at the post-climax stage. The thyroid gland was morphologically the most active in metamorphic climax when the degranulation occurred in the pituitary IrTSH cells, and appeared inactive at post-climax. A few weeks after metamorphosis, both the IrTSH cells and the thyroid gland appeared to be activated again in the benthic, juvenile flounder. Administration of thyroxine or thiourea revealed negative feedback regulation of the pituitary-thyroid axis in flounder larvae. These results indicate that activation of the pituitary-thyroid axis induces metamorphosis in the flounder.     

Tryptophan 2,3-dioxygenase in chick embryo hepatocytes: studies in ovo and in culture

Tryptophan dioxygenase is a hemoprotein in its active form, which has a relatively low affinity for heme. From previous studies in rats, the ratio of holoenzyme/total enzyme activity of tryptophan dioxygenase has been proposed to reflect the size of a "free" heme pool in hepatocytes. Chick embryo hepatocytes in ovo and in culture are other systems that have proven useful for study of hepatic heme metabolism and its control. Heretofore, there have been few studies of tryptophan dioxygenase activity in chick embryo hepatocytes. As part of studies on hepatic heme metabolism, using two different assays, we have measured tryptophan dioxygenase activity and percentage of heme saturation of the enzyme in chick embryo livers cells in ovo and in culture. One method of assay relies on endogenous formamidase to generate the final product, kynurenine, which is measured directly, whereas the other method uses a chemical hydrolysis step to form kynurenine which is further diazotized prior to measurement. The latter method is shown to be preferable for studies with chick embryo hepatocytes. In addition, we show that (i) tryptophan dioxygenase activity is present and can be increased by tryptophan and phenobarbital-like drugs in chick embryo hepatocytes in ovo; (ii) total enzyme activity falls markedly in cultured hepatocytes despite the presence of high concentrations of glucocorticoids in the culture medium; and (iii) under all conditions studied thus far in the cultures, the enzyme is nearly saturated with heme. Results are discussed in relation to regulation of heme metabolism in chick embryo hepatocytes.     

In ovo electroporations of HH stage 10 chicken embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology. With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function. By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function. Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements. Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive. This video demonstrates first how to make a window in the eggshell to manipulate the embryo. Next, the embryo is visualized with a dilute solution of India ink injected below the embryo. A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. Finally, the egg is sealed with tape and placed back into an incubator for further development. Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.     

Nonresponsiveness of the thyroid gland to goitrogen in the early neonatal period in the rat: light- and electron-microscopic observations.

The thyroid response of fetal and neonatal rats to propylthiouracil (PTU) as a goitrogen was studied with observation of the thyroid glands by light and electron microscopy. On day 19 of gestation and on days 1, 3, 5 and 8 after birth, fetal and neonatal rats were given a subcutaneous injection of PTU and were autopsied 2 days later. PTU induced conspicuous goiters in fetal rats but did not in neonatal rats aged up to day 5 after birth. Beyond that age, PTU again induced goiters. Histologically, the follicular cell height in goitrous thyroid glands was significantly increased. Ultrastructurally, follicular cells in goitrous thyroid glands often had colloid droplets and lysosomes. It seems that nonresponsiveness of the thyroid glands in early neonatal rats to goitrogen is due to a temporary decline of the pituitary activity of thyrotropin secretion. About 5 days or more after birth, the pituitary-thyroid system begins to operate again in response to goitrogen.     

Reactions of hypothalamic ascorbic acid, serum ceruloplasmin and the adenohypophysis to oestradiol: inhibition by L-thyroxine

Four weeks' administration of oestradiol benzoate to male and female rats in doses of 1 mg twice a week leads to adenohypophyseal hyperplasia and to an increase in the thyroxine-binding capacity of the adenohypophyseal proteins in vitro. At the same time, serum polyphenol oxidase (ceruloplasmin) activity rises and the hypothalamic ascorbic acid concentration falls. The simultaneous administration of L-thyroxine (0.1 mg/rat/per day) or dried thyroid (but not D-thyroxine) significantly inhibits these changes (adenohypophysis, ceruloplasmin) or completely suppresses them (hypothalamic ascorbic acid). L-thyroxine evidently blocks the action of oestradiol in the adenohypophysis, the liver and the hypothalamus; the significance of this inhibition is discussed in relation to dopaminergic modulation of the adenohypophyseal reaction to oestradiol.     

Differentiation of the thyroid in the hypophysectomized chick embryo

The influence of hypophyseal hormones on the normal development of the thyroid gland was investigated by decapitating chick embryos at a time before the hypophysis has begun to form. Thyroids from decapitated embryos were compared with thyroids of normal embryos from shortly after decapitation to the time when the embryo should hatch. The thyroids were examined for ultrastructural differences and for accumulation of thyroglobulin. Thyroxine levels in the blood were also examined. It was concluded that the thyroids in decapitated embryos develop normally up to about 12 days of development, by which time morphogenesis and differentiation of the gland is essentially complete. The rates of thyroid growth and accumulation of thyroglobulin are diminished after 12 days of development. The amount of thyroxine in the blood is decreased by about one-half. Thyroxine was found in the yolk of unincubated eggs at levels sufficient to sustain blood thyroxine levels throughout embryonic development. We conclude that hormones that are synthesized by the embryonic hypophysis affect thyroid development only through their influence on generalized growth and metabolic activity.     

Dopaminergic control of adenohypophyseal growth after oestrogens: negative results with L-DOPA, RO-4-4602, alpha-methyl-DOPA, apomorphine, haloperidol, pyridoxine and cyproheptadine.

Chronic oestrogen treatment augments adenohypophyseal weight and the thyroxine-binding capacity of adenohypophyseal proteins in rats. Since these reactions are both inhibited by ergocornine and ergoline derivatives (as well as by the thyroid hormones, testosterone, anti-oestrogens and antiandrogens), and are potentiation by perphenazine and the dopaminergic neurone blocker Pimozide, the peroral effectiveness of various other substances presumed to act in the region of the dopaminergic or serotoninergic neurones of the hypothalamus was tested. L-DOPA (10 mg/rat/day) did not modify the adenohypophyseal reaction, either alone or combined with the DOPA-decarboxylate inhibitor Ro-4-4602 (1 mg/rat/day). alpha-Methyl-DOPA (10 mg/rat/day), apomorphine (3 mg/rat/day), haloperidol (0.2 mg/rat/day), pyridoxine (20 mg/rat/day) and cyproheptadine (1 mg/rat/day) were likewise ineffective.     

Maturation of the pituitary-thyroid axis during the perinatal period.

To clarify the maturation process of the pituitary-thyroid axis during the perinatal period, thyrotropin (TSH) response to thyrotropin releasing hormone (TRH) and serum thyroid hormone levels were examined in 26 healthy infants of 30 to 40 weeks gestation. A TRH stimulation test was performed on 10 to 20 postnatal days. Basal concentrations of serum thyroxine (T4), free thyroxine (free T4) and triiodothyronine (T3) were positively correlated to gestational age and birth weight (p less than 0.001-0.01). Seven infants of 30 to 35 gestational weeks demonstrated an exaggerated TSH response to TRH (49.7 +/- 6.7 microU/ml versus 22.1 +/- 4.8 microU/ml, p less than 0.001), which was gradually reduced with gestational age and normalized after 37 weeks gestation. A similar decrease in TSH responsiveness to TRH was also observed longitudinally in all of 5 high responders repeatedly examined. There was a negative correlation between basal or peak TSH concentrations and postconceptional age in high responders (r = -0.59 p less than 0.05, r = -0.66 p less than 0.01), whereas in the normal responders TSH response, remained at a constant level during 31 to 43 postconceptional weeks. On the other hand, there was no correlation between basal or peak TSH levels and serum thyroid hormones. These results indicate that (1) maturation of the pituitary-thyroid axis is intrinsically controlled by gestational age rather than by serum thyroid hormone levels, (2) hypersecretion of TSH in preterm infants induces a progressive increase in serum thyroid hormones, and (3) although there is individual variation in the maturation process, the feedback regulation of the pituitary-thyroid axis matures by approximately the 37th gestational week.     

Development of 1,25-dihydroxycholecalciferol receptor in the duodenal cytosol of chick embryo.

The development of 1,25-(OH)2D3 receptor in the duodenal cytosol of chick embryo was studied by the sucrose density gradient analysis. The binding profile for 1,25-(OH)2D3 in the cytosol of vitamin D-deficient chick duodenum on the sucrose density gradient revealed 3 binding components, and the sedimentation constant was estimated as 2.5, 3.5 and 5.5S respectively. The 3.5S binding component has high affinity and low capacity for 1,25-(OH)2D3 and is thought to be 1,25-(OH)2D3 receptor. During the development of chick embryo, the 3.5S binding component was not detected in 13-day embryonic duodenum, it appeared on 15th day of incubation and then gradually increased to the level of vitamin D-deficient chick on 19th day of incubation. The 5.5S binding component was specific for 25-OH-D3 and it was found even in 13-day embryo, but it did not show any significant change during development. On the other hand, the 2.5S component was not specific for either 1,25-(OH)2D3 or 25-OH-D3. However, it was main binding component in early stages of development and decreased during development. From these results, it is suggested that the receptor for 1,25-(OH)2D3 is available a few days before hatching and the inability to produce CaBP in the duodenum of chick embryo could not be ascribed to the absence of the receptor.     

Tissue interactions in the induction of anterior pituitary: role of the ventral diencephalon, mesenchyme, and notochord.

Rathke's pouch, the epithelial primordium of the anterior pituitary, differentiates in close topographical and functional association with the ventral diencephalon. It is still not known whether the ventral diencephalon acts as the initial inducer of pituitary development. The roles of the adjacent mesenchyme and notochord, two other tissues located in close proximity to Rathke's pouch, in this process are even less clear. In this report we describe an in vitro experimental system that reproduces the earliest steps of anterior pituitary development. We provide evidence that the ventral diencephalon from 2- to 4-day-old chick embryos is able to function as an inducer of pituitary development and can convert early chick embryonic head ectoderm, which is not involved normally in pituitary development, into typical anterior pituitary tissue. This induction is contact-dependent. In our experimental system, there is a requirement for the supporting action of mesenchyme, which is independent of the mesenchyme source. Transplantation of the notochord into the lateral head region of a six-somite chick embryo induces an epithelial invagination, suggesting that the notochord induces the outpouching of the roof of the stomodeal ectoderm that results in formation of Rathke's pouch and causes the close contact between this ectoderm and the ventral diencephalon. Finally, we demonstrate that the ventral diencephalon from e9.5-e11.5 mouse embryos is also an efficient inducer of anterior pituitary differentiation in chick embryonic lateral head ectoderm, suggesting that the mechanism of anterior pituitary induction is conserved between mammals and birds, using the same, or similar, signaling pathways.