Survival and growth of developing rats during centrifugation at 2G

We studied the effects of 2G hypergravity on the survival, body mass and growth of postnatal rats (Rattus norvegicus). Nursing litters comprised of either neonatal (Postnatal day [P]7) or pre-weanling (P14) rats and their mothers were exposed to 16 days of continuous centrifugation. All of the offspring survived and gained body mass, indicating that mothers nursed their young. Following the onset of centrifugation, neonatal and pre-weanling rats showed a reduction in growth relative to age-matched environmental controls (EC). At the completion of testing, body mass of the hypergravity (HG) groups was significantly less than that of controls (p<0.05). Over the course of the test, the HG-exposed P7 group showed an overall 55% gain in body mass as compared to a 71% increase in controls, while the HG-exposed P14 group showed a 62% increase relative to 75% in controls. Neonatal offspring (P7) gained body mass during centrifugation, but at significantly slower rates as compared to EC controls (p<0.05). In contrast, growth rates of pre-weanling (P14) rats were not reduced relative to controls, possibly related to the initiation of weaning, around P18 in the rat. These findings raise key issues relevant to studies of nursing mammals reared in altered gravity.     

Thyroid hormones modulate ornithine decarboxylase in the immature rat cerebellum

In this study, we measured ornithine decarboxylase (ODC) activity as a potential parameter to evaluate the response of the developing rat brain to thyroid hormones. In cerebellum, neonatal hyperthyroidism (40 micrograms thyroxine/100 g body weight daily from birth) increased ODC activity at 2 and 5 days of age and then accelerated its developmental decline. Conversely, ODC activity was decreased in 2- and 5-day-old hypothyroid rats (propylthiouracil to the mother), but it was not significantly different from normal thereafter. No significant differences were observed in the forebrain following either treatment. In hypothyroid rat cerebellum, a single injection of triiodothyronine (T3, 100 micrograms/100 g 18 h before sacrifice) increased significantly ODC activity at all ages. A dose-response study showed that 0.5 micrograms T3/100 g is sufficient to obtain maximal stimulation. Finally, administration of antiserum against rat growth hormone had no significant effect on ODC response to T3. These results show that ODC is a useful marker of thyroid state and tissue response in the neonatal rat cerebellum.     

Dominant role of thyrotropin-releasing hormone in the hypothalamic-pituitary-thyroid axis

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the beta isoforms of TH receptors (TRbeta KO), or both (double KO). TRbeta knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.     

Maternal reproductive experience enhances early postnatal outcome following gestation and birth of rats in hypergravity

A major goal of space life sciences research is to broaden scientific knowledge of the influence of gravity on living systems. Recent spaceflight and centrifugation studies demonstrate that reproduction and ontogenesis in mammals are amenable to study under gravitational conditions that deviate considerably from those typically experienced on Earth (1 x g). In the present study, we tested the hypothesis that maternal reproductive experience determines neonatal outcome following gestation and birth under increased (hyper) gravity. Primigravid and bigravid female rats and their offspring were exposed to 1.5 x g centrifugation from Gestational Day 11 either through birth or through the first postnatal week. On the day of birth, litter sizes were identical across gravity and parity conditions, although significantly fewer live neonates were observed among hypergravity-reared litters born to primigravid dams than among those born to bigravid dams (82% and 94%, respectively; 1.0 x g controls, 99%). Within the hypergravity groups, neonatal mortality was comparable across parity conditions from Postnatal Day 1 through Day 7, at which time litter sizes stabilized. Maternal reproductive experience ameliorated neonatal losses during the first 24 h after birth but not on subsequent days, and neonatal mortality was associated with changes in maternal care patterns. These results indicate that repeated maternal reproductive experience affords protection against neonatal losses during exposure to increased gravity. Differential mortality of neonates born to primigravid versus bigravid dams denotes gravitational load as one environmental mechanism enabling the expression of parity-related variations in birth outcome.     

Identification of genes mediating thyroid hormone action in the developing mouse cerebellum

Despite the indispensable role thyroid hormone (TH) plays in brain development, only a small number of genes have been identified to be directly regulated by TH and its precise mechanism of action remains largely unknown, partly because most of the previous studies have been carried out at postnatal day 15 or later. In the present study, we screened for TH-responsive genes in the developing mouse cerebellum at postnatal day 4 when morphological alterations because of TH status are not apparent. Among the new candidate genes selected by comparing gene expression profiles of experimentally hypothyroid, hypothyroid with postnatal thyroxine replacement, and control animals using oligoDNA microarrays, six genes were confirmed by real-time PCR to be positively ( orc1l, galr3, sort1, nlgn3, cdk5r2 , and zfp367 ) regulated by TH. Among these, sort1 , cdk5r2, and zfp367 were up-regulated already at 1 h after a single injection of thyroxine to the hypothyroid or control animal, suggesting them to be possible primary targets of the hormone. Cell proliferation and apoptosis examined by BrdU incorporation and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed that hypothyroidism by itself did not enhance apoptosis at this stage, but rather increased cell survival, possibly through regulation of these newly identified genes.     

In a hypergravity environment neonatal survival is adversely affected by alterations in dam tissue metabolism rather than reduced food intake.

Exposure of rat dams to hypergravity during pregnancy is associated with increased pup mortality, reduced food intake, and decreased rates of glucose oxidation and lipogenesis in mammary tissue. We hypothesized that increased pup mortality is due to changes in maternal metabolism and not to reduced food intake of dams. Effects of hypergravity on rate of glucose oxidation and lipogenesis in mammary, liver, and adipose tissue were measured in rat dams centrifuged at 2.0 G [hypergravity (HG)], kept at 1.0 G (control), or fed to match the intake of HG rats (pair fed) from gestation day 11 (G11) until G21 or postpartum day 3 (P3). Body weight, percent body fat, metabolizable energy, and nitrogen balance were significantly less in HG dams compared with controls (P<0.05); however, these factors were not different between HG and pair-fed dams. By P3, 100% of control and pair-fed pups survived, while only 49% of HG pups survived. At G21, rates of glucose oxidation and lipogenesis in mammary and adipose tissue were less in HG than in control and pair-fed dams (P<0.1 and P<0.05). In liver, at G21, the rate of lipogenesis was greater in HG than control and pair-fed dams (P<0.01); at P3, lipogenesis was greater in control than HG and pair-fed dams (P<0.05). Gene expression of ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase increased in liver from pregnancy to lactation in control and pair-fed dams but not HG dams. Thus reduced food intake and body mass due to hypergravity exposure cannot explain the dramatic decrease in HG pup survival.     

Impaired serum thyrotropin response to hypothyroidism in mice with disruption of neuromedin B receptor

Neuromedin B (NB), a neuropeptide highly concentrated in pituitary, has been proposed to be an inhibitor of thyrotropin (TSH) secretion. Previous study showed that mice with disruption of neuromedin B receptor (NBR-KO) have higher TSH release in response to thyrotropin-releasing hormone (TRH), although TSH seems to have decreased bioactivity. Here we examined in NBR-KO mice the response of TSH to thyroid hormone (TH) deprivation, obtained by methimazole treatment, or excess, obtained by acute and chronic TH administration. In response to hypothyroidism NBR-KO mice exhibited a lower magnitude increase in serum TSH compared to wild-type (WT) mice (1.7 vs. 3.3-times increase compared to euthyroid values, respectively, P<0.001). One hour after a single T4 injection (0.4 microg/100 g BW), WT and NBR-KO hypothyroid mice presented similar degree of serum TSH reduction (54%, P<0.05). However, 3 h after T4 administration, WT mice presented serum TSH similar to hypothyroid baseline, while NBR-KO mice still had decreased serum TSH (30% reduced in comparison to hypothyroid baseline P<0.05). T3 treatment of euthyroid mice for 21 days, with progressively increasing doses, significantly reduced serum TSH similarly in WT and NBR-KO mice. Also, serum T4 exhibited the same degree of suppression in WT and NBR-KO. In conclusion, disruption of neuromedin B receptor did not interfere with the sensitivity of thyroid hormone-mediated suppression of TSH release, but impaired the ability of thyrotroph to increase serum TSH in hypothyroidism, which highlights the importance of NB in modulating the set point of the hypothalamus-pituitary-thyroid axis at hypothyroidism.     

Thyrotropin (TSH) binding activities in bovine thyroid tissue: possible role of adenosine 3',5'-monophosphate dependent phosphorylation of thyroid plasma membranes in TSH receptor degradation

The relationship between thyroid plasma membrane phosphorylation and thyrotropin (TSH) receptor degradation was investigated by using bovine thyroid tissues. By fractionation of thyroid cytosol (105,000 X g supernatant of thyroid homogenate) in a continuous sucrose density gradient centrifugation, three different TSH binding activities were separated. During the incubation of thyroid plasma membranes, TSH binding activities were spontaneously released in vitro. By fractionation of the fraction containing released TSH binding activities in the same sucrose density gradient centrifugation, three different TSH binding activities were isolated. These peaks of TSH binding activity corresponded to the peaks of TSH binding activity obtained in cytosol fraction. Adenosine 3',5'-monophosphate (cyclic AMP) enhanced the release of TSH binding activities from the plasma membranes in vitro. After fractionation on a sucrose density gradient centrifugation of the supernatant of the plasma membranes which were preincubated with cyclic AMP, three different peaks of TSH binding activity were identified. These peaks corresponded to the peaks obtained in spontaneously released TSH binding activity. In this case, however, the amount of small molecule TSH binding activities was predominant compared to that of large molecule TSH binding activity. During the incubation of the plasma membranes with [r-32P]-ATP and with cyclic AMP, phosphorylated soluble proteins were released. The profile of the phosphorylated soluble proteins in the sucrose density gradient centrifugation showed three different peaks which corresponded to the peaks of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA⁺RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T₃-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T₄ intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)     

Mild iodine deficiency in pregnancy in Europe and its consequences for cognitive and psychomotor development of children: A review

Despite the introduction of salt iodization programmes as national measures to control iodine deficiency, several European countries are still suffering from mild iodine deficiency (MID). In iodine sufficient or mildly iodine deficient areas, iodine deficiency during pregnancy frequently appears in case the maternal thyroid gland cannot meet the demand for increasing production of thyroid hormones (TH) and its effect may be damaging for the neurodevelopment of the foetus. MID during pregnancy may lead to hypothyroxinaemia in the mother and/or elevated thyroid-stimulating hormone (TSH) levels in the foetus, and these conditions have been found to be related to mild and subclinical cognitive and psychomotor deficits in neonates, infants and children. The consequences depend upon the timing and severity of the hypothyroxinaemia. However, it needs to be noted that it is difficult to establish a direct link between maternal iodine deficiency and maternal hypothyroxinaemia, as well as between maternal iodine deficiency and elevated neonatal TSH levels at birth. Finally, some studies suggest that iodine supplementation from the first trimester until the end of pregnancy may decrease the risk of cognitive and psychomotor developmental delay in the offspring.     

Effect of perinatal thyroid hormone deficiency on expression of rat hippocampal conventional protein kinase C isozymes

Thyroid hormone (TH) is essential for the proper development of mammalian central nervous system. TH deficiency during critical period of brain development results in permanent cognitive and neurological impairments. Hippocampus is a structure involved in various memory processes that are essential for creating new memories, and lesions to hippocampus result in impaired learning and memory. Protein kinase C (PKC) isoforms play an important role in many types of learning and memory, and deletion of specific PKC genes results in deficits in learning. In the present study, we used real-time PCR and Western blot to investigate the conventional PKC expression in developing rat hippocampus with different thyroid status, trying to establish a correlation between TH deficiency and conventional PKC expression in developing rat hippocampus. We found that PKCβI and PKCγ expression decreased significantly both in mRNA and protein levels in hypothyroid group compared with the normal controls, and thyroxine replacement could restore it. As for PKCα, we did not find any difference between different thyroid status. Though the expression of PKCβII also decreased in the TH deficiency group, the change was not significant. Taken together, our data indicate TH deficiency can cause hippocampal PKCβ1 and PKCγ downregulation during rat brain development. Since there are other PKC isoforms in the rat brain, whether these change is related to impaired learning and memory of perinatal hypothyroid rats requires further researches.     

Influence of cold exposure and thyroid hormones on regulation of adrenal catecholamines.

Since thyroid hormones influence urinary excretion of catecholamines after exposure to cold, the effects of hyper- and hypo-thyroidism on adrenal tyrosine hydroxylase (TH) (EC 1.14.16.2), phenylethanolamine-N-methyl transferase (PNMT) (EC 2.1.1.28), and serum dopamine-beta-hydroxylase (DbetaH) (EC 1.14.17.1) of rats of 23 and 4 degrees C were studied. TH changes resembled the urinary excretion pattern at 4 degrees C in being higher after 8 days than after 1 day of exposure, and in declining as acclimation occurred. At 23 degrees C, TH activity of hypothyroid rats was significantly higher than in euthyroid or hyperthyroid animals, and after 1 day at 4 degrees C the value increased even more. While in the hypothyroid animals at 4 degrees C the concentration of adrenal catecholamines was less, the epinephrine to norepinephrine ratio was higher than at 23 degrees C. Very high TH activity with a decline in catecholamine concentration suggests that the capacity of TH had been exceeded. PNMT activity was significantly elevated in this group. TH activity was not decreased in the hyperthyroid group at 23 degrees C, and was increased after 8 days at 4 degrees C, suggesting that circulating thyroid hormones have no direct inhibitory effect on TH. Serum DbetaH was elevated after exposure to 4 degrees C, regardless of thyroid hormonal status. The activation of adrenal TH in hypothyroid rats at 23 degrees C and of TH, PNMT, and serum DbetaH at 4 degrees C is probably the result of increased activity of the sympathetic nervous system.     

Effect of hypothyroidism on the biogenesis of free mitochondria in the cerebral hemispheres and in cerebellum of rat during postnatal development

The effect of propylthiouracil-induced neonatal hypothyroidism on some aspects of the biogenesis of free (non-synaptosomal) mitochondria in the cerebral hemispheres and in the cerebellum of developing rat has been studied. The results obtained show that in hypothyroid rats mitochondrial DNA synthesis is delayed, mitochondrial RNA synthesis is not affected and cytochrome aa₃ content of mitochondria is lower than in controls. Furthermore ultrathin sections of 14-and 21-day old hypothyroid rat cerebella show mitochondria with an altered ultrastructural organization and large intracristal spaces.     

Regulation of Five Tubulin Isotypes by Thyroid Hormone During Brain Development

Nucleic acid probes derived from the 3' noncoding region of five tubulin cDNAs were used to study the effects of thyroid hormone deficiency on the expression of the mRNAs encoding two alpha (alpha 1 and alpha 2)- and three beta (beta 2, beta 4, and beta 5)-tubulin isotypes in the developing cerebral hemispheres and cerebellum. The content of alpha 1, which markedly declines during development in both brain regions, is maintained at high levels in the hypothyroid cerebellum, whereas it is decreased in the cerebral hemispheres. The alpha 2 level also declines during development and is decreased in both regions by thyroid hormone deficiency, but only during the two first postnatal weeks. Thyroid hormone deficiency slightly increases at all stages the beta 2 level in the cerebellum, whereas a decrease is observed at early stages in the cerebral hemispheres. The beta 5 level seems to be independent of thyroid hormone in the cerebral hemispheres, whereas it decreases at early stages in the hypothyroid cerebellum. Finally, the expression of the brain-specific beta 4 isotype is markedly depressed by thyroid hormone deficiency, particularly in the cerebellum. These data suggest that the genes encoding the tubulin isotypes are, directly or not, differently regulated by thyroid hormone during brain development. This might contribute to abnormal neurite outgrowth seen in the hypothyroid brain and therefore to impairment in brain functions produced by thyroid hormone deficiency.     

A Modest Protective Effect of Thyrotropin against Bone Loss Is Associated with Plasma Triiodothyronine Levels

Background

The independent skeletal effect of thyrotropin (thyroid stimulating hormone, TSH) has been suggested in animal studies. However, clinical data on the association between bone loss and variations in TSH levels is inconsistent. This study aimed to investigate the relationship between TSH levels and bone mineral density (BMD).

Methods

We conducted a cross-sectional study with 37,431 subjects (33,052 cases with euthyroidism and 4,379 cases with subclinical thyroid dysfunction) aged over 35 years. We performed thyroid function tests and measured BMD at the lumbar spine, femur neck, and total hip.

Results

Levels of TSH and T3 were positively correlated in women (r = 0.076, P = 0.001) and uncorrelated in men. In both men and women, TSH levels correlated positively and T3 levels correlated negatively with BMD at all skeletal sites in age and body mass index adjusted analyses. BMD increased steadily with TSH levels from the subclinical hyperthyroid to subclinical hypothyroid range in subjects with T3 levels in the highest tertile (119.5–200.0 ng/dL), but was no longer significant in subjects with lower plasma T3 levels.

Conclusions

The variations in TSH levels within the euthyroid and subclinical range were positively correlated with BMD in healthy men and women. The negative effect of T3 on BMD appears to be compensated for by increased TSH in subjects with plasma T3 levels in the upper normal range.     

Impaired growth hormone secretion in neonatal hypothyroid rats: hypothalamic versus pituitary component

In 10-day-old rats made hypothyroid by giving dams propylthiouracil (PTU) in the drinking water since the day of parturition, simultaneous radioimmunoassay (RIA) determinations of basal and stimulated growth hormone (GH) secretion, hypothalamic GH-releasing hormone (GHRH)-like immunoreactivity (LI) content, immunocytochemical localization of somatotrophs, and hypothalamic GHRH-LI-positive structures were performed. The frequency of somatotrophs was also determined. One-day-old hypothyroid rats, whose mothers had been given PTU since the 14th day of pregnancy, were also used for comparison. In 10-day-old hypothyroid rats, pituitary and plasma GH levels and the number of somatotrophs were considerably lower and plasma TSH levels were significantly higher than those in age-matched control rats; however, GHRH-LI titers in the mediobasal hypothalamus and the morphology of GHRH-LI-positive structures were unaltered. In 1-day-old rats the only alteration present, in addition to elevated plasma TSH levels, was a clear-cut decrease in plasma GH levels. An acute challenge with GHRH (20 ng/100 g body wt, sc) or clonidine (15 micrograms/100 g body wt, sc) induced a clear-cut rise in plasma GH levels 15 min postinjection in 10-day-old control rats but failed to do so in age-matched hypothyroid rats. Both compounds failed to rise plasma GH in both hypothyroid and control 1-day-old rats. Taken together these data indicate that in neonatal and infant rats deprivation of thyroid hormones acts primarily to depress pituitary somatotroph function and that possible changes in GHRH-secreting structures represent a later postnatal event.     

Elements in erythrocytes of population with different thyroid hormone status

The contents of elements K, Ca, Fe, Cu, Zn, Se, and Rb in erythrocytes of 78 cases with different thyroid hormone status have been measured by proton-induced X-ray emission and neutron activation analysis. According to the status of thyroid hormones T₃, T₄, TSH, FT₃, and FT₄ detected by radioimmunoassay, the experiment subjects were divided into four groups (i.e., hyperthyroid, hypothyroid, critical [one of thyroid hormones was abnormal], and normal). Elements contents and hormones levels of four groups were analyzed by one-way analysis of variance and correlation using an SPSS/PC statistical package. The results showed that the Se contents of four groups were not significantly different (p<0.05). Zn content of hypothyroid group was significantly higher than those of hyperthyroid and critical groups. The Zn content of the normal group was higher than that of the hypothyroid group and lower than that of the hyperthyroid and critical groups. In the hyperthyroid group, there were significant correlations between elements contents and thyroid hormones levels (except TSH), but not between elements contents and levels of thyroid hormones. However, in the hypothyroid group, relatively strong correlations have been found between elements contents and thyroid hormones levels, especially between Zn and the T₃/T₄ ratio, and between Zn and TSH.