Localization and thyroid hormone influenced expression of collagen II in ovarian tissue.

Collagen type II (Col II), one of the main components of the hyaline cartilage, is a member of the fibril-forming collagen family. Due to its amino acid composition, the extent of lysine hydroxylation of Col II is much higher than that of other fibril forming collagens. Since lysyl hydroxylase isoforms are less synthesized in hypothyroid ovarian tissue, Col II level is expected to be reduced here and contribute to the degradation of ovarian ECM in this condition. As there was no previous report, we have demonstrated Col II expression in rat ovary. Col2A1 mRNA shares significant part of the total collagens in ovary as shown by the relative expression of the major collagen genes present in this tissue. It has also been shown that Col II is down regulated in hypothyroid ovarian tissue and its expression is increased upon stimulation by thyroid hormone (T(3)). To know whether less Col II in hypothyroid ovarian tissue is due to less synthesis of the protein or its increased rate of degradation is also involved in it, we demonstrated the status of Collagen - degrading Matrix Metalloproteinases in this condition and found up regulation of MMP-1, -8 and -13 in hypothyroid rat ovary. The present study shows the reduced Col II expression in hypothyroid rat ovary, with the concomitant increase in Col II degradation. This information will be useful for further studies on reproductive disorders.     

Cystic fibrosis as a bowel cancer syndrome and the potential role of CK2

Thyroid hormones are major regulators of postnatal brain development. Thyroid hormones act through nuclear receptors to modulate the expression of specific genes in the brain. We have used microarray analysis to identify novel responsive genes in 14-day-old hypothyroid rat brains, and discovered that synaptosomal-associated protein of 25 kDa (SNAP-25) was one of the thyroid hormone-responsive genes. SNAP-25 is a presynaptic plasma membrane protein and an integral component of the vesicle docking and fusion machinery mediating secretion of neurotransmitters and is required for neuritic outgrowth and synaptogenesis. Using microarray analysis we have shown that SNAP-25 was down-regulated in the hypothyroid rat brain compared with the age-matched controls. Real-time RT-PCR and western blotting analysis confirmed that SNAP-25 mRNA and protein levels decreased significantly in the developing hypothyroid rat brain. Our data suggest that in the developing rat brain, SNAP-25 expression is regulated by thyroid hormone, and thyroid hormone deficiency can cause decreased expression of SNAP-25 and this may on some level account for the impaired brain development seen in hypothyroidism.     

Effects of neonatal hypothyroidism on rat brain gene expression.

To define at the molecular biological level the effects of thyroid hormone on brain development we have examined cDNA clones of brain mRNAs and identified several whose expression is altered in hypothyroid animals during the neonatal period. Clones were identified with probes prepared by subtractive or differential hybridization, and those corresponding to mRNAs altered in hypothyroidism were further studied by Northern blot analysis. Using RNA prepared from whole brains, no effect of hypothyroidism was found on the expression of the astroglial gene coding for glial fibrillary acidic protein. Among genes of neuronal expression, no significant alterations were found in the steady state levels of mRNAs coding for neuron-specific enolase, microtubule-associated protein-2, Tau, or nerve growth factor. N-CAM mRNA increased slightly in hypothyroid brains. In contrast a 2- to 3-fold decrease was found in the mRNA coding for a novel neuronal gene, RC3. This is the first neuronal gene known to be significantly altered at the mRNA level by thyroid hormone deprivation. The abundance of the mRNAs for the major myelin proteins proteolipid protein, myelin basic protein, and myelin-associated glycoprotein, expressed by oligodendrocytes, were also decreased in hypothyroid brains. Developmental studies on RC3 and myelin-associated glycoprotein expression indicated that the corresponding mRNAs accumulate in the brain of normal rats during the first 15-20 days of neonatal life. A similar accumulation occurred in hypothyroid brains, but at much reduced levels. The results demonstrate that thyroid hormone controls the steady state levels of particular mRNAs during brain development.     

Defining global gene expression changes of the hypothalamic-pituitary-gonadal axis in female sGnRH-antisense transgenic common carp (Cyprinus carpio)

Background

The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system.

Methodology/Principal Findings

In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified.

Conclusions/Significance

This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of teleost fish.     

Adult-onset hypothyroidism enhances fear memory and upregulates mineralocorticoid and glucocorticoid receptors in the amygdala

Hypothyroidism is the most common hormonal disease in adults, which is frequently accompanied by learning and memory impairments and emotional disorders. However, the deleterious effects of thyroid hormones deficiency on emotional memory are poorly understood and often underestimated. To evaluate the consequences of hypothyroidism on emotional learning and memory, we have performed a classical Pavlovian fear conditioning paradigm in euthyroid and adult-thyroidectomized Wistar rats. In this experimental model, learning acquisition was not impaired, fear memory was enhanced, memory extinction was delayed and spontaneous recovery of fear memory was exacerbated in hypothyroid rats. The potentiation of emotional memory under hypothyroidism was associated with an increase of corticosterone release after fear conditioning and with higher expression of glucocorticoid and mineralocorticoid receptors in the lateral and basolateral nuclei of the amygdala, nuclei that are critically involved in the circuitry of fear memory. Our results demonstrate for the first time that adult-onset hypothyroidism potentiates fear memory and also increases vulnerability to develop emotional memories. Furthermore, our findings suggest that enhanced corticosterone signaling in the amygdala is involved in the pathophysiological mechanisms of fear memory potentiation. Therefore, we recommend evaluating whether inappropriate regulation of fear in patients with post-traumatic stress and other mental disorders is associated with abnormal levels of thyroid hormones, especially those patients refractory to treatment.     

Tachykinins and ovarian function in mammals

Tachykinins are bioactive peptides whose presence has been demonstrated in endocrine glands, where they likely exert a paracrine modulatory activity on hormonal secretions. In the ovary, tachykinins have been shown to be present in nerve fibers, blood vessels, and in granulosa, luteal and interstitial cells. Tachykinin gene expression was shown in granulosa and luteal cells. Tachykinins have also been found in the follicular fluid. Substance P (SP) has been demonstrated to significantly affect the release of hormonal steroids by ovarian cells in vitro. While some authors found that SP stimulated the release of steroids, others found an inhibitory effect by the same tachykinin. Gonadotropins decrease tachykinin concentrations in the ovary. The neonatal treatment of rats with capsaicin, a drug that depletes SP in primary afferent neurons, resulted in a modest reduction in the reproductive success in rats. The experimental results listed in this review suggest that tachykinins are synthesized in the ovary, in the granulosa and luteal cells. Tachykinins are likely intraovarian modulators of the secretion of hormonal steroids. Their stores in the ovary are likely regulated by pituitary gonadotropins.     

Type-1 cannabinoid receptor expression in the frog, Rana esculenta, tissues: a possible involvement in the regulation of testicular activity

Endogenous cannabinoids and type-1 cannabinoid receptor (CB1) are widely produced and distributed in the central nervous system (CNS) and peripheral nerves in mammals. In addition, the detection of endocannabinoids and corresponding receptors in non nervous peripheral tissues indicates an involvement of the system in the control of a wide range of physiological activities, including reproduction. Recently, the existence of CB1 was also observed in lower vertebrates and in urochordate suggesting that the endocannabinoid system is phylogenetically conserved. Using RT-PCR, CB1 mRNA expression profiles were characterized in a wide range of tissues of the anuran amphibian, the frog, Rana esculenta. Besides a strong expression in the CNS, CB1 was also present in testis, kidney, liver, ovary, muscle, heart, spleen, and pituitary. The CB1 expression pattern has been characterized in both testis and CNS during the annual sexual cycle. In testis, CB1 is poorly expressed during the winter stasis of the spermatogenesis rising during the breeding season and resumption period. An expression profile mismatching to that observed in testis was detected in whole-brain preparations during the sexual cycle; in particular in the diencephalon, the encephalic area mainly involved in the control of reproductive functions. Furthermore, fluctuations inside isolated encephalic areas and spinal cord were observed all over the reproductive cycle. In conclusion, CB1 receptor is expressed in R. esculenta CNS and testis. As far as the gonad it concerns, our results suggest the involvement of the endocannabinoids in the control of reproductive function.     

Activity of the hypothalamo-pituitary ovarian axis in hypothyroid rats with or without triiodothyronine replacement

The hypothalamic pituitary ovarian axis in adult female rats with 131-I induced hypothyroidism was studied before and after triiodothyronine (T3) replacement. Forty days after 131-I, hypothyroid (H) rats showed irregular cycles with predominantly diestrous vaginal smears, atrophied and underweight ovaries, and decreased serum T3, T4, LH and estradiol (E2). T3 replacement restored normal cycles and ovary weight and increased serum E2 levels above control values, while LH levels remained below the limit of detection of the RIA. The GnRH stimulation test performed on the day that the rats exhibited diestrous vaginal smears elicited a greater increase in FSH than in LH in H rats and a greater increase in LH than in FSH in both H-T3 treated and control rats. The data suggest that the lack of thyroid hormones in adult female rats seems to produce a reversion of sexual hormones to a prepubertal pattern, while T3 treatment restored normal estrous cycles and ovarian function.     

Thyroid hormone regulation and cholesterol metabolism are connected through Sterol Regulatory Element-Binding Protein-2 (SREBP-2)

High affinity uptake of serum-derived low density lipoprotein (LDL) cholesterol is accomplished through the LDL receptor in the liver. In mammals, thyroid hormone depletion leads to decreased LDL receptor expression and elevated serum cholesterol. The clinical association in humans has been known since the 1920s; however, a molecular explanation has been lacking. LDL receptor levels are subject to negative feedback regulation by cellular cholesterol through sterol regulatory element-binding protein-2 (SREBP-2). Here we demonstrate that the SREBP-2 gene is regulated by thyroid hormone and that increased SREBP-2 nuclear protein levels in hypothyroid animals results in thyroid hormone-independent activation of LDL receptor gene expression and reversal of the associated hypercholesterolemia. This occurs without effects on other thyroid hormone-regulated genes. Thus, we propose that the decreased LDL receptor and increased serum cholesterol associated with hypothyroidism are secondary to the thyroid hormone effects on SREBP-2. These results suggest that hypercholesterolemia associated with hypothyroidism can be reversed by agents that directly increase SREBP-2. Additionally, these results indicate that mutations or drugs that lower nuclear SREBP-2 would cause hypercholesterolemia.     

Differential thyroid hormone-regulated rat thyrotropin beta gene expression detected by blot hybridization

In the rat, there is a single TSH beta-subunit gene represented by three exons interrupted by two introns. This gene contains two promoters which determines the synthesis of two mRNAs with 5'-untranslated regions that differ by 43 base pairs. This study evaluates the steady state levels of these TSH beta mRNAs in various thyroidal states. Blot hybridization analyses of pituitary mRNA with synthetic probes designed to detect either one or both TSH beta mRNAs were performed. One probe corresponds to 24 bases in the 5'-untranslated region of mRNA1 and a second corresponds to 25 nucleotides in the coding region and detects both mRNA1 and mRNA2. These studies indicate the presence of TSH beta mRNA species of indistinguishable size consistent with the presence of two TSH beta mRNAs that contain slightly different 5'-untranslated regions. Comparison of pituitary RNA obtained from normal and hypothyroid rats reveals that the shorter mRNA (mRNA2) is increased approximately 6- to 8-fold with hypothyroidism while the abundance of the longer mRNA (mRNA1) is relatively unchanged. Treatment of either normal or hypothyroid animals with T3 decreases the abundance of mRNA2 while again mRNA1 is relatively unaffected. Thus, although both mRNAs are detected, only one mRNA is dramatically altered by thyroidal status. Therefore, the single rat TSH beta gene is transcribed into two mRNAs via the use of alternative promoters of which only one is markedly regulated by thyroid hormones.