Ribosomal protein synthesis in 16 and 19 day gestation fetuses of hypothyroid mothers.

Thyroid hormones are transported across the placenta. Thyroid hormone receptors are present in the midgestation rat fetus and fetal tissues selectively accumulate thyroid hormones prior to the onset of fetal thyroid function. It has not been demonstrated adequately that maternal thyroid hormones are essential for early fetal physiologic functions and neurological development. The present study compares the effects of maternal thyroid hormone versus fetal thyroid hormone on the regulation of rRNA and ribosomal protein synthesis in the developing fetus. This was accomplished by first comparing 16-day gestation (just prior to the onset of fetal thyroid function) fetuses of control and hypothyroid mothers. Then 19-day gestation (fetal thyroids are functional) fetuses of control and hypothyroid mothers were compared as development of fetal thyroid function lags in hypothyroid mothers. Rats made hypothyroid (Tx) by radiothyroidectomy were given replacement doses of thyroxine (T4) until the day that they were placed with a male for mating. Control, Tx and growth hormone (GH)-treated Tx dams and their fetuses were sacrificed on either the 16th or 19th day of gestation. Ribosomes (r) were isolated from placentas and from fetal brains and livers and rRNA, total 14C-leucine incorporation, and 14C-leucine incorporation into ribosomal protein per microgram of rRNA were determined. On the 16th day of gestation, prior to the onset of fetal thyroid function, all three of the above metabolic parameters were reduced significantly below control levels in the placentas of Tx rats and in the brains and livers of their fetuses. This was true also for the fetal brains and livers of GH-treated Tx mothers. Development of fetal thyroid function lags in the Tx mother. rRNA, total 14C-leucine incorporation, and 14C-leucine incorporation into ribosomal protein per microgram of rRNA continue to be significantly depressed in these fetal tissues and placentas of Tx rats on the 19th day of gestation. In fact, brain protein synthesis falls further behind in fetuses of Tx dams at this gestational age when compared with control fetal brains. These data support the hypothesis that maternal thyroid hormones are important, by at least midgestation, for normal fetal physiologic development and support the concept that appropriate maturation of the fetal pituitary-thyroid plays a role in fetal brain protein synthesis and therefore neurological development.

Serum growth hormone levels in hypothyroid and GH-treated thyroidectomized rats and their progenies.

Growth hormone (GH) was measured in the sera of control, hypothyroid (thyroidectomized [Tx]) and GH-treated Tx rats and their fetuses on Days 19, 20, 21, and 22 of gestation and in their progenies on postnatal Days 1, 5, 30, and 75. Maternal endogenous serum GH increased dramatically between the 19th and 20th days of gestation and remained elevated through the 22nd day in control rats, but was depressed significantly in Tx and GH-treated Tx rats during this period. GH was not always detected in the sera of 19-day-old fetuses. On Day 20, GH was depressed in fetuses of Tx mothers as compared with those form controls or GH-treated Tx mothers. GH was elevated in sera of fetuses from GH-treated Tx rats over fetuses of control and Tx only rats on the 22nd day of gestation. In postnatal rats, those from GH-treated mothers continued to show elevated serum GH on Day 1 as compared with those from Tx only mothers. On postnatal Days 5 and 30, progenies of Tx mothers had significantly elevated GH as compared with progenies of control mothers. At 75 days of age, the GH levels of these progenies had normalized. We have shown previously that the hormonal secretions of the pituitary-thyroid axis are badly disrupted in the progenies of Tx and GH-treated Tx mothers and that even as adults these animals have tissue (brain and liver) deficits of active thyroid hormones. Although the onset of GH secretion is mildly delayed in fetuses of Tx but not GH-treated Tx mothers, the serum GH levels of both groups of progenies are elevated during most of the neonatal period through the time of puberty. It is, therefore, concluded that GH in the absence of adequate levels of thyroid hormones is ineffective in preventing many of the learning and memory deficits induced in the progenies of Tx mothers.

Tissue iodothyronine levels in fetuses of control and hypothyroid rats at 13 and 16 days gestation.

Some investigators have reported that there is minimal placental transport of thyroid hormones in humans and rats. Consequently, it was thought that thyroid hormones were not present in the fetal brain before fetal thyroid hormone synthesis and, hence, were not important for brain development before fetal thyroid hormonogenesis. Recently, however, thyroid hormones have been detected by 14 days postconception (dpc) in the rat fetus and by 11 dpc in the rat embryotrophoblast. Thyroid hormone receptors have been shown in the fetal rat by 14 dpc. The present experiments were designed to determine if T4, T3, and their metabolites can be detected in rat fetuses at 13 and 16 dpc and if iodothyronines are selectively accumulated in fetal brain and liver. Furthermore, one group of dams was radiothyroidectomized before breeding to ascertain the effect of maternal hypothyroxinemia on fetal tissue iodothyronine concentrations. Tissue iodothyronines were extracted and measured by HPLC. T4, T3, rT3, and 3,5-diiodothyronine were well within the limits of detection by this procedure at both fetal ages. The only possible source of these hormones is the mother. In addition, if maternal serum T4 levels are low, fetal tissue T4 and T3 levels are low. The presence of high intracellular T3 levels, even at 13 dpc, shows that 5'-monodeiodination occurs in the midgestational fetus. Intracellular hormone measurements show that T3, rather than rT3, is the predominant intracellular iodothyronine in the rat fetus. Both brain and liver selectively accumulate T4 and T3, supporting the observations of others that fetal thyroid hormone receptors are present in midgestation. The presence of thyroid hormones in fetal rat brain by 13 dpc coupled with the observation that hormone receptors are present by 14 dpc suggests that thyroid hormones do play a role in midgestational brain development. These data show that normal maternal serum thyroid hormone levels are important during midgestation to provide adequate thyroid hormones to the fetus.

Method for the quantitation of iodothyronines in body tissues and fluids using high-performance liquid chromatography.

The separation and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-, 3,3' and 3',5'-diiodo-L-tyronines, 3,5,3'-triiodo-L-thyronine (T3), reverse 3,3',5'-triiodo-L-thyronine and 3,3',5,5'-tetraiodo-L-thyronine (T4)] from animal tissues (brain, liver and serum) by a new high-performance liquid chromatographic (HPLC) method is described. Rats were infused with iso-osmotic sodium chloride containing 100 microM phloretin to block deiodination. The tissues were extracted using differential pH values to separate other amines from the amine containing iodothyroid hormones. Aliquots of tissue extracts (25-100 microliters) were reacted overnight with 5-dimethylaminonaphthalene-1-sulfonyl chloride and their iodotyrosine and iodothyronine content determined by HPLC utilizing fluorimetric detection. Resolution of the individual compound peaks was achieved by gradient elution with a 3.0 mM H3PO4 buffer. Greater sensitivity has been achieved (less than 1.0 pmol/g) utilizing fluorescence rather than ultraviolet absorbance for the quantitation of these iodinated compounds. The method is superior also to other methods in that recoveries, based on those of 125I-labelled T4 and T3, were 89-97%.

Distribution of free amino acids in streptozotocin-induced diabetic pregnant rats, their placentae and fetuses.

Amino acid levels in the non-pregnant streptozotocin (STZ)-induced diabetic rat have been shown to be abnormal. Our preliminary studies showed that placental transport, fetal serum levels and tissue uptake of the non-metabolizable amino acid alpha-amino isobutyric acid (AIB) were decreased in STZ-diabetic pregnant rats. In the present experiments, amino acid concentrations were measured in maternal (MS) and fetal (FS) sera and placentae (PL) by high performance liquid chromatography (HPLC) after triple extraction in 80% ethanol. Control (C), STZ-diabetic (D) and insulin-treated diabetic (DI) animals were studied at 22 days gestation. Pregnant diabetic rats had low serum levels of Gln, Lys, and Ser and insulin treatment corrected Gln and Ser but not Lys levels. Branched-chain amino acids did not show the large elevation characteristic of the non-pregnant diabetic rat. Placental levels of Tau, Gln, HPr, Thr and Lys were depressed in the diabetic animals and insulin treatment only partially improved these amino acid profiles. Placental amino acid levels did not always reflect maternal serum levels. Serum levels of most amino acids were lower in the fetus of the diabetic rat than in the fetus of the control rat. The notable exception was Ala which was higher in the fetuses of the diabetic animals. Insulin treatment of the mother did not correct many of the fetal amino acid levels even though maternal and fetal serum glucose levels at the time of autopsy were normal. The ability to maintain normal serum levels of many amino acids is impaired in the fetus of the diabetic rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Iodothyronine-5'-deiodinase activity in progenies of hypothyroid rats.

Iodothyronine-5'-deiodinase activity (I-5'DA) was measured in the progenies of control rats, hypothyroid (Tx) rats, and hypothyroid treated with ovine GH (Tx + GH) during gestation. The enzyme was measured in cerebral cortex and cerebellum at 22 days gestation and at 5, 10, 30 and 60 days postpartum. In addition, the pituitary I-5'-DA was assessed in the postnatal animals. The experiments were undertaken because the tissues of the progenies of rats that were hypothyroid during pregnancy appeared in many ways to resemble those of hypothyroid animals, even at ages when serum thyroxine (T4) and triiodothyronine (T3) levels were normal. It was found that the progenies of Tx mothers had low liver 5'-deiodinase activities. This is a likely cause of the low serum T3 levels with normal T4 levels seen in these progenies in the neonatal period. Cerebral and cerebellar 5'-deiodinase activities were low in these progenies during the thyroid hormone-dependent perinated period of brain development. The progenies of GH-treated Tx dams had higher enzyme activities than the progenies of untreated Tx dams. These pups from GH-treated Tx mothers have been shown previously to have significantly less neurological impairment than the progenies of untreated Tx mothers. As most of the brain intracellular T3 is produced in situ, a functional thyroid deficiency could result from such a 5'-deiodinase deficiency. As the deiodinase deficiency was still seen in the progenies of Tx mothers at 60 days of age, such a deficiency could explain why, even though serum T4 and T3 levels were normal, brain metabolism was in many ways characteristic of hypothyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioral testing of progenies of Tx (hypothyroid) and growth hormone-treated Tx rats: an animal model for mental retardation.

Virgin Sprague-Dawley Holtzman rats were rendered Tx (hypothyroid) by radiothyroidectomy and maintained on 1.0 microgram T4 (thyroxine) per 100 g BW until pregnant. One-half of these Tx animals were administered 0.5 IU of growth hormone (GH) during the last 10-11 days of gestation as GH secretion is especially deficient in Tx rats. Untreated, food restricted to the level consumed by the Tx-only rats, GH-treated euthyroid, and T4-treated until pregnant animals served as controls. The animals were allowed to go through parturition and each litter was reduced to no more than 6 pups by removing pups for tissue weights and protein analyses at 1 and 5 days of age. The pups were weaned at 22 days of age and 2 animals per litter were utilized for behavioral testing between 40 and 60 days of age. At the end of the behavioral testing period the 60-day-old offspring were sacrificed to obtain tissue weights and protein concentrations. The behavioral tests were based on the ability of the animals to learn a Lashley's type 3 enclosed alley maze and their spontaneous activity was measured in stabilimeter cages. The animals were fasted overnight on alternate days and then given a food reward upon traversing the maze. This allowed for 10 separate trials in both the Lashley maze and the stabilimeters over the 20-day period from 40 to 60 days of age. Our previous studies have shown the fetuses and progenies of Tx-only mothers to have multiple metabolic defects including reduced rates of protein synthesis and tissue protein concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of serum thyroxine, triiodothyronine, and thyrotropin in the progeny of hypothyroid rats.

Thyroid function in the progeny of hypothyroid rats was studied from 19 days of gestation until adulthood. Total T4, T3, and TSH were measured by RIA in the sera of control, surgically thyroidectomized (Tx), and GH-treated Tx pregnant rats and their progeny from the late fetal period through adulthood. By the 22nd day of gestation, the fetuses of Tx mothers had T4 and TSH levels significantly above those of their controls. This condition, whereby fetal serum T4 and TSH levels were elevated, was aggravated by GH treatment of the Tx mothers. The progeny of Tx- and Tx and GH-treated mothers also showed alterations in serum T4, T3, and TSH. While the T4 levels of the progeny of Tx mothers were significantly below those of th control progeny only at 30 days of age, T3 levels were significantly depressed at 1, 5, and 30 days of age. These same animals had lower serum TSH levels than the control progeny at 1 and 5 days of age and higher TSH levels at 30 and 75 days of age. Prenatal GH treatment of the mother did not consistently correct thyroid function of the progeny. As adults the progeny of the Tx mothers had normal serum concentrations of T4 and T3 but TSH was high. High serum TSH values were not seen in the progeny of Tx plus GH-treated mothers. These results suggest that maternal hypothyroidism during pregnancy may result in permanent alterations in the thyroidal response to TSH in their progeny.

Pituitary-thyroid function of fetuses of hypothyroid and growth hormone treated hypothyroid rats.

Maternal hypothyroidism induced by surgical thyroidectomy (Tx) of the rat resulted in significantly higher fetal serum levels of thyroid stimulating hormone (TSH) and thyroxine (T4) on day 22 of gestation. Surprisingly, administration of growth hormone (GH) to hypothyroid mothers increased further the fetal serum T4 and TSH. The in vitro uptake of 131I-T4 by erythrocytes was elevated significantly when incubated with serum from fetuses of both hypothyroid and hypothyroid GH-treated mothers. Although the plasma protein levels of hypothyroid mothers and their fetuses are decreased significantly as compared to controls this is not true of hypothyroid GH-treated mothers and their fetuses. The T4 levels of both groups of Tx mothers were significantly below that of controls. However, as in the case of their fetuses, the serum T4 of GH-treated hypothyroid mothers was elevated from that of Tx only animals. It is concluded that the pituitary-thyroid system of fetuses of hypothyroid mothers is activated excessively during late gestation, that considerable T4 can be transported from the fetus to the mother during this period and that these fetuses are in fact born in a hyperthyroid state which is aggravated by maternal treatment with GH.

The effects of growth hormone treatment of thyroid-deficient pregnant rats on maternal and fetal carbohydrate metabolism.

Maternal hypothyroidism in rats has been shown previously to result in alterations of maternal, placental, and fetal metabolism. Maternal treatment with 2 IU GH/day for three days prior to autopsy (on the 22nd day of pregnancy) corrected many of the observed alterations of carbohydrate metabolism in hypothyroidism. The maternal and fetal liver glycogen concentrations and the fetal serum glucose levels of the hypothyroid animals were elevated significantly by the GH treatment. In most cases, the utilization of a [1-14C]glucose tracer dose was returned to normal by GH treatment. These results suggest that the impairment of fetal metabolism occurring in maternal hypothyroidism may be due in part to insufficient maternal GH secretion. However, GH alone in the absence of sufficient thyroid hormones did not totally correct all of the observed fetal abnormalities.

A comparison of the effects of altered thyroid and parathyroid function on reproduction in the rat.

Mild maternal hypothyroidism produced by surgical thyroidectomy resulted in significantly reduced food intake along with retarded reproductive performance in rats. Hyperthyroidism increased food consumption above the control level along with an increase in fetal resorptions which was the only parameter of reproductive performance altered by the twenty-second day of gestation. In animals which underwent pregnancies, reproductive performance suffered a further decline in the second gestation due to increasing duration of the hypothyroidism. Food restriction alone failed to duplicate these adverse effects on fetal development. Serum gonadotropins (LH and FSH) were not significantly altered by mild maternal hypothyroidism of hypoparathyroidism. The hematocrit and total blood volume of hypothyroid but not hypoparathyroid rats was significantly reduced from normal. Maternal hypoparathyroidism alone did not affect fetal development grossly.

Hypoglycemia and glycogen deficits in fetuses of hypothyroid pregnant rats.

Maternal hypothyroidism, when induced by surgical thyroidectomy with parathyroid hormone replacement, results in fewer live fetuses and smaller fetuses at the 22nd day of gestation. The hypothyroid mother shows the ability to mobilize adequate amounts of glucose even at the expense of her own reserves but the supply of glucose to the fetus appears to be impaired. These fetuses have subnormal skeletal muscle and liver glycogen and are severely hypoglycemic. The impaired development of these fetuses may result from alterations of either transplacental carbohydrate transport or placentofetal carbohydrate metabolism.

Pituitary-thyroid function of rats in hypobaric oxygen-inert gas environments.

Rats (200-260 g) were exposed in sealed, recycling chambers continuously for 2-30 days to gas mixtures designed to maintain the same alveolar PO2 in the presence or absence of inert gas. Mixtures with inert gas (N2, He, or Ne) were at ground level; those without inert gas (100 percent O2) were in an altitude chamber. The O2 categories were: I-100 percent O2 at 747 torr; II-74 percent O2 + 26 percent inert and 566 torr 100 percent O2; III-47 percent O2 + 53 percent inert and 381 torr 100 percent O2; IV-21 percent O2 + 79 percent inert and 197 torr 100 percent O2. One of the two room-air controls was "restricted-fed" to the level of the lowest intake group. Measurements included body, pituitary, and thyroid weight, food and water intake, plasma volume and hematocrit, pituitary and plasma TSH, and plasma PBI. Severe depression in all variables and over 50 percent mortality was seen in I by day 4. All variables were depressed in II, but there was no mortality to 20 days. Pituitary-thyroid function appeared to be particularly sensitive to depression by hyperoxia, with plasma TSH levels reduced between 42 and 60 percent in II and III. No effect was attributable to the inert gas, whether it was N2, He, or Ne, nor was any specific effect traceable to the presence or absence of inert gas.

The effect of maternal hypothyroidism on maternal and fetal tissue glucose-1-14C incorporation in rats.

These experiments were conducted to help elucidate the mechanism for the impaired fetal development occurring during maternal hypothyroidism. The disposition of glucose was measured using glucose-1-14C. Maternal hypothyroidism depressed glucose utilization in thematernal-fetal system. Maternal net glycogen synthesis from the labeled glucose was impaired. However, while the fetal glycogen storage system may be capable of at least relatively normal glycogen synthesis, abnormally low levels of glycogen were measured. If, as the data suggest, enzymatic deficiencies do not exist, the low liver glycogen might be a result of the inability of the maternal-placental system to provide adequate substrate to the fetus.