scholarly journals Generation of Thyrotropin-Releasing Hormone Receptor 1-Deficient Mice as an Animal Model of Central Hypothyroidism

2004 ◽  
Vol 18 (6) ◽  
pp. 1450-1460 ◽  
Author(s):  
Roland Rabeler ◽  
Jens Mittag ◽  
Lars Geffers ◽  
Ulrich Rüther ◽  
Michael Leitges ◽  
...  
Keyword(s):  
Animal Model ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Pcr Analysis ◽  
Target Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Central Hypothyroidism ◽  
R1 Gene

Abstract To provide an animal model of central hypothyroidism, mice deficient in the TRH-receptor 1 (TRH-R1) gene were generated by homologous recombination. The pituitaries of TRH-R1−/− mice are devoid of any TRH-binding capacity, demonstrating that TRH-R1 is the only receptor localized on TRH target cells of the pituitary. With the exception of some retardation in growth rate, TRH-R1−/− mice appear normal, but compared with control animals they exhibit a considerable decrease in serum T3, T4, and prolactin (PRL) levels but not in serum TSH levels. In situ hybridization histochemistry and real-time RT-PCR analysis revealed that in adult TRH-R1−/− animals TSHβ-mRNA expression is not impaired whereas PRL mRNA and GH mRNA levels are considerably reduced compared with control mice. The numbers of thyrotropes, somatotropes, and lactotropes, however, are not affected by the deletion of the TRH-R1 gene. The mutant mice are fertile, and the dams nourish their pups well, indicating that TRH is not a decisive factor for suckling-induced PRL release. In situ hybridization and quantitative RT-PCR analysis, furthermore, revealed that, as in control animals, pituitary PRL-mRNA expression in TRH-R1−/− is considerably increased during lactation, albeit strongly reduced as compared with lactating control animals.

Estrogen Receptor-β mRNA Expression in Rat Ovary: Down-Regulation by Gonadotropins

1997 ◽  
Vol 11 (2) ◽  
pp. 172-182 ◽  
Author(s):  
Michael Byers ◽  
George G. J. M. Kuiper ◽  
Jan-Åke Gustafsson ◽  
Ok-Kyong Park-Sarge
Keyword(s):  
Estrogen Receptor ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Adult Rats ◽  
Mature Adult ◽  
Preovulatory Follicles

Abstract We have examined the expression and regulation of the two estrogen receptor (ERα and ERβ) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ERα and ERβ genes as single (∼6.5-kb) and multiple (ranging from ∼1.0-kb to ∼10.0-kb) transcripts, respectively. ERα mRNA is expressed at a level lower than ERβ mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ERβ mRNA levels were decreased. Examination of ERβ mRNA expression at the cellular level, by in situ hybridization, showed that ERβ mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ERβ mRNA was observed in a subset of corpora lutea, and that the decrease in ERβ mRNA during proestrous evening is attributable, at least in part, to down-regulation of ERβ mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ERα mRNA in the ovary. Although whole ovarian content of ERα mRNA was clearly detected by RT-PCR, no apparent modulation of ERα mRNA levels was observed during the estrous cycle. Examination of ERα mRNA expression at the cellular level, by in situ hybridization, showed that ERα mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ERβ mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ERβ mRNA. In situ hybridization for ERβ mRNA shows that small, growing, and preovulatory follicles express ERβ mRNA in the granulosa cells. The preovulatory follicles contain ERβ mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ERβ mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ERβ mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ERβ mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ERα and ERβ genes, although ERβ is the predominant form of estrogen receptor in the ovary, 2) ERβ mRNA is localized predominantly to the granulosa cells of small, growing, and preovulatory follicles, and 3) the preovulatory LH surge down-regulates ERβ mRNA. These results clearly implicate the physiological importance of ERβ in female reproductive functions.

scholarly journals Quantification of TGF-β1 mRNA along rat nephron in obstructive nephropathy

2001 ◽  
Vol 281 (3) ◽  
pp. F513-F521 ◽  
Author(s):  
Kyoichi Fukuda ◽  
Koji Yoshitomi ◽  
Taihei Yanagida ◽  
Masanori Tokumoto ◽  
Hideki Hirakata
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Proximal Tubules ◽  
Obstructive Nephropathy ◽  
Renal Tubules ◽  
Rt Pcr ◽  
Tgf Β1

Unilateral ureteral obstruction (UUO) leads to interstitial fibrosis of the obstructed kidney, and transforming growth factor-β1 (TGF-β1) is thought to play an important role in this process. Although increased TGF-β1 mRNA expression in the obstructed kidney has been demonstrated, the source of the increased TGF-β1 remains to be elucidated. To determine the precise localization of TGF-β1 in the obstructed kidney, we examined TGF-β1 mRNA expression using in situ hybridization and competitive RT-PCR in rats with UUO. In situ hybridization demonstrated that TGF-β1 mRNA expression was preferentially increased in tubular epithelial cells and to a lesser degree in infiltrating macrophages in obstructed kidneys. Quantitative analysis using competitive RT-PCR in microdissected nephron segments revealed that levels of TGF-β1 mRNA in obstructed kidneys relative to control kidneys increased significantly in proximal tubules, thick ascending limbs of Henle, and distal convoluted tubules, whereas those in glomeruli and collecting ducts did not change significantly. Of the tubular segments, the proximal tubules appeared to predominantly contribute to increased TGF-β1 mRNA. Our findings suggest that renal tubules, particularly proximal tubules, are the main contributors to increased TGF-β1 mRNA expression in obstructed kidneys and to the subsequent interstitial fibrosis.

scholarly journals Food-Dependent Cushing’s Syndrome: Characterization and Functional Role of Gastric Inhibitory Polypeptide Receptor in the Adrenals of Three Patients1

1998 ◽  
Vol 83 (12) ◽  
pp. 4514-4519
Author(s):  
M. C. Lebrethon ◽  
O. Avallet ◽  
Y. Reznik ◽  
F. Archambeaud ◽  
J. Combes ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Cushing’S Syndrome ◽  
Functional Role ◽  
Gastric Inhibitory Polypeptide ◽  
Cushing's Syndrome ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Adrenal Cells ◽  
Gip Receptor

In the present work, the presence of gastric inhibitory polypeptide (GIP) receptors and their functional role in the adrenal cells of three patients with food-dependent Cushing’s syndrome were studied. RT-PCR and in situ hybridization studies demonstrated the presence of GIP receptor in the adrenals of the three patients. The presence of this receptor was also demonstrated in two human fetal adrenals, but not in two normal adult human adrenals or in the adrenals of one patient with nonfood-dependent Cushing’s syndrome. Freshly isolated cells from patient adrenals responded in a dose-dependent manner to the steroidogenic action of both ACTH and GIP, whereas cells from normal adrenals responded only to ACTH. Treatment of cultured normal adrenal cells with ACTH, but not with GIP, increased the messenger ribonucleic acid (mRNA) levels of cholesterol side-chain cleavage cytochrome P-450, P450c17, and 3β-hydroxysteroid dehydrogenase, whereas both hormones enhanced these mRNAs in patients’ adrenal cells, although the effects of ACTH were greater than those of GIP. Moreover, pretreatment with ACTH enhanced the steroidogenic responsiveness of both normal and patient adrenal cells, whereas GIP caused homologous desensitization, and this was associated with a marked reduction of GIP receptor mRNA levels, as demonstrated by RT-PCR and in situ hybridization. Finally, both ACTH and GIP inhibited DNA synthesis in one patient’s adrenal cells, whereas in normal adrenal cells only ACTH had this effect. In conclusion, the present data demonstrate that ectopic expression of functional GIP receptors is the main cause of food-dependent Cushing’s syndrome.

scholarly journals Expression of Tandem P Domain K+ Channel, TREK-1, in the Rat Carotid Body

2006 ◽  
Vol 54 (4) ◽  
pp. 467-472 ◽  
Author(s):  
Y. Yamamoto ◽  
K. Taniguchi
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Carotid Body ◽  
Nerve Fibers ◽  
K Channel ◽  
Membrane Excitability ◽  
Rt Pcr ◽  
Glomus Cells ◽  
Afferent Nerve Endings

TREK-1 is one of the important potassium channels for regulating membrane excitability. To examine the distribution of TREK-1 in the rat carotid body, we performed RT-PCR for mRNA expression and in situ hybridization and immunohistochemistry for tissue distribution of TREK-1. RT-PCR detected mRNA expression of TREK-1 in the carotid body. Furthermore, in situ hybridization revealed the localization of TREK-1 mRNA in the glomus cells. TREK-1 immunoreactivity was mainly distributed in the glomus cells and nerve fibers in the carotid body. TREK-1 may modulate potassium current of glomus cells and/or afferent nerve endings in the rat carotid body.

Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium

2004 ◽  
Vol 287 (4) ◽  
pp. H1625-H1631 ◽  
Author(s):  
Katherine L. Tran ◽  
Xiangru Lu ◽  
Ming Lei ◽  
Qingping Feng ◽  
Qingyu Wu
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Mrna Expression ◽  
Rat Model ◽  
Left Ventricular ◽  
Biologically Active ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Neonatal Cardiomyocytes

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells ( r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.

Intragraft mRNA Expression of Cytokines and Growth Factors in Human Kidney Allograft Biopsies by In Situ RT-PCR Analysis

2005 ◽  
Vol 37 (2) ◽  
pp. 767-769 ◽  
Author(s):  
D. Kaminska ◽  
B. Tyran ◽  
O. Mazanowska ◽  
W. Letachowicz ◽  
A. Kochman ◽  
...  
Keyword(s):  
Growth Factors ◽  
Mrna Expression ◽  
Human Kidney ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Kidney Allograft

scholarly journals INTRAGRAFT MRNA EXPRESSION OF CYTOKINES AND GROWTH FACTORS IN HUMAN KIDNEY ALLOGRAFT BIOPSIES BY IN SITU RT-PCR ANALYSIS.

2004 ◽  
Vol 78 ◽  
pp. 551
Author(s):  
D Kamińska ◽  
B Tyran ◽  
O Mazanowska ◽  
W Letachowicz ◽  
A Kochman ◽  
...  
Keyword(s):  
Growth Factors ◽  
Mrna Expression ◽  
Human Kidney ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Kidney Allograft

scholarly journals Serum Leptin Concentrations and Expression of Leptin Transcripts in Placental Trophoblast with Advancing Baboon Pregnancy1

1999 ◽  
Vol 84 (7) ◽  
pp. 2543-2549
Author(s):  
Michael C. Henson ◽  
V. Daniel Castracane ◽  
Jennifer S. O’Neil ◽  
Terry Gimpel ◽  
Kenneth F. Swan ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Gestational Age ◽  
Venous Blood ◽  
Late Gestation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Messenger Ribonucleic Acid ◽  
Human Pregnancy ◽  
Villous Tissue

Leptin is a polypeptide hormone originally thought to be produced exclusively by adipocytes. Recently, however, both leptin messenger ribonucleic acid (mRNA) and leptin protein were identified in human placental trophoblast cells, suggesting a potential role in primate pregnancy. In the present study, venous blood samples were collected at 5-day intervals during gestation from baboons (Papio sp), an established model for the study of human pregnancy, as well as from nonpregnant baboons, and leptin concentrations were determined by RIA. Additionally, placental villous tissue was collected upon cesarean delivery at early (days 60–62; n = 5), mid (days 98–102; n= 5), and late (days 159–167; n = 5) gestation (term = ∼184 days), and leptin mRNA was quantitated by competitive RT-PCR. Finally, in situ hybridization was employed to localize transcripts to specific placental cell types. Results determined that maternal leptin levels (mean ± sem), which were dramatically greater (P &lt; 0.01) than those in nonpregnant cycling baboons (1.4 ± 0.1 ng/mL), increased (P &lt; 0.005) with gestational age from 63.6 ± 10.4 ng/mL on day 60 of gestation to 157.8 ± 16.1 near term. Levels declined to those found in cycling baboons by 15 days postdelivery. In contrast to maternal leptin concentrations, placental leptin mRNA decreased (P &lt; 0.02) with advancing pregnancy, as transcript abundance declined approximately 8-fold from early to late gestation. Maternal peripheral leptin concentrations were positively correlated (r = 0.66; P &lt; 0.001) whereas placental leptin mRNA levels were negatively correlated (r= −0.64; P &lt; 0.01) with gestational age. Expression of leptin mRNA transcripts, as evidenced by RT-PCR in villous tissue, was localized principally within syncytiotrophoblast by in situ hybridization. In summary, changes in maternal peripheral leptin concentrations and placental leptin mRNA abundance that occur commensurate with advancing gestational age may imply evolving roles for the polypeptide with advancing primate pregnancy. In this capacity, localization of leptin transcripts within the baboon syncytiotrophoblast suggests the potential for autocrine or paracrine interactions within this endocrinologically active tissue. Finally, both the similarities in leptin ontogeny in baboon and human pregnancy and the singular enhancement of maternal leptin levels inherent throughout baboon gestation emphasize the potential of this nonhuman primate model for the study of leptin action in the maternal-fetoplacental unit.

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