Prenatal betamethasone exposure results in pituitary-adrenal hyporesponsiveness in adult sheep

Fetal exposure to synthetic glucocorticoids in sheep results in increased fetal hypothalamic-pituitary-adrenal (HPA) activity persisting to one year of age. We aimed to determine the effects of single or repeated maternal or fetal betamethasone injections on offspring HPA activity at 2 and 3 yr of age and whether changes in adrenal mediators of steroidogenesis contribute to changes in pituitary-adrenal function. Pregnant ewes or their fetuses received either repeated intramuscular saline or betamethasone injections (0.5 mg/kg) at 104, 111, 118, and 124 days of gestation (dG) or a single betamethasone injection at 104 dG followed by saline at 111, 118, and 124 dG. Offspring were catheterized at 2 and 3 yr of age and given corticotrophin-releasing hormone + arginine vasopressin challenges. Adrenal tissue was collected for quantitative RT-PCR mRNA determination at 3.5 yr of age. In 2-yr-old offspring, maternal betamethasone injections did not alter basal ACTH or cortisol levels, but repeated injections elevated ACTH responses. At 3 yr of age, basal ACTH was elevated, and both basal and stimulated cortisol levels were suppressed by repeated maternal injections. Basal and stimulated cortisol-to-ACTH ratios and basal cortisol-to-cytochrome P-450 17α-hydroxylase (P450c17) mRNA ratios were suppressed by repeated injections. Repeated fetal betamethasone injections attenuated basal ACTH and cortisol levels in offspring at 2 but not 3 yr of age. Plasma changes were not associated with altered adrenal P450c17, ACTH receptor, β-hydroxysteroid dehydrogenase, or glucocorticoid receptor mRNA levels. These data suggest that maternal, but not fetal, betamethasone administration results in adrenal suppression in adulthood.

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Cytochrome P-450 17α-hydroxylase protein and mRNA in the testis of the testicular feminized (Tfm) mouse

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0110077 ◽

1993 ◽

Vol 11 (1) ◽

pp. 77-82 ◽

Cited By ~ 48

Author(s):

P J O'Shaughnessy ◽

L Murphy

Keyword(s):

Leydig Cells ◽

Enzyme Synthesis ◽

Mrna Levels ◽

Normal Numbers ◽

Rt Pcr ◽

Hydroxylase Activity ◽

Actin Mrna ◽

Polymerase Chain ◽

Cytochrome P 450 ◽

Pcr Conditions

ABSTRACT The testicular feminized (Tfm) mouse lacks functional androgen receptors and develops with a female external phenotype and internal testes. The testes of these animals contain normal, or close to normal, numbers of Leydig cells but secrete very low amounts of androgen due to a lack of 17α-hydroxylase activity. To determine whether this loss of activity is due to a lack of enzyme synthesis or a change in catalytic activity we have examined 17α-hydroxylase cytochrome P-450 (P-45017α) protein and mRNA levels in the testes of Tfm mice. Levels of P-45017α protein were measured by immunoblotting, while mRNA was measured following reverse transcription (RT) and amplification by the polymerase chain reaction (PCR). Conditions for RT-PCR were determined which allowed semiquantification of P-45017α mRNA relative to β-actin mRNA. In extracts of Tfm testes P-45017α protein was undetectable using antiserum against porcine P-45017α. In contrast, a protein of around 54 kDa was clearly detectable in extracts of control cryptorchid testes. Using RT-PCR, P-45017α mRNA was detectable in both control and [ill] testes but, expressed in terms of β-actin mRNA, levels of P-45017α mRNA in control testes were 40-fold higher than those in [ill] testes. If the total amount of RNA extracted from each testis is taken into account then P-45017α mRNA levels per testis were up to 400-fold higher in control testes. These results show that the reduced level of 17α-hydroxylase activity in [ill] testes is related to reduced protein synthesis. Previous results have shown that androgens reduce P-45017α mRNA levels in cultured Leydig cells. Results from this study suggest, however, that androgens are required to induce normal levels of P-45017α mRNA in Leydig cells.

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Glucocorticoids and serotonin alter glucocorticoid receptor mRNA levels in fetal guinea-pig hippocampal neurons, in vitro

Reproduction Fertility and Development ◽

10.1071/rd05043 ◽

2005 ◽

Vol 17 (7) ◽

pp. 743 ◽

Cited By ~ 14

Author(s):

P. Erdeljan ◽

M. H. Andrews ◽

J. F. MacDonald ◽

S. G. Matthews

Keyword(s):

Guinea Pig ◽

Glucocorticoid Receptor ◽

Hpa Axis ◽

Hippocampal Neurons ◽

In Situ Hybridisation ◽

Fetal Life ◽

Mrna Levels ◽

Glucocorticoid Receptor Mrna ◽

Synthetic Glucocorticoids

The hypothalamic–pituitary–adrenal (HPA) axis is susceptible to programming during fetal life. Such programming occurs, at least partially, at the level of the hippocampus. The hippocampus plays a central role in regulation of the HPA axis and release of endogenous glucocorticoids, via mediation of glucocorticoid negative feedback. Fetal exposure to synthetic glucocorticoids can permanently alter glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) levels within the hippocampus, and serotonin is thought to be involved in this process. In the present study, we hypothesised that dexamethasone, cortisol and serotonin exposure would modify GR mRNA expression within fetal guinea-pig hippocampal cultures. Cultures were derived from 40-day-old guinea-pig fetuses, and were exposed to 0, 1, 10 and 100 nm dexamethasone, cortisol or serotonin for 4 days. Expression of GR and MR mRNA was examined by in situ hybridisation followed by high-resolution silver emulsion autoradiography. Four-day exposure to dexamethasone (P < 0.05; 100 nm) or cortisol (P = 0.08; 100 nm) downregulated the expression of GR mRNA within neurons. There was no change in the expression of MR mRNA levels following cortisol treatment. Exposure to serotonin (100 nm) significantly increased GR mRNA levels in hippocampal neurons. We conclude that synthetic and endogenous glucocorticoids, as well as serotonin, can influence GR expression during hippocampal development and in this way may act to permanently programme HPA function.

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Knockout of the hsd11b2 Gene Extends the Cortisol Stress Response in Both Zebrafish Larvae and Adults

International Journal of Molecular Sciences ◽

10.3390/ijms222212525 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12525

Author(s):

Antonia Theodoridi ◽

Alberto Dinarello ◽

Lorenzo Badenetti ◽

Michail A. Pavlidis ◽

Luisa Dalla Valle ◽

...

Keyword(s):

Stress Response ◽

Adult Males ◽

Wild Type ◽

Good Tool ◽

Zebrafish Model ◽

Basal Cortisol ◽

Inactive Form ◽

Old Adult ◽

Cortisol Levels ◽

One Year

The Hsd11b2 enzyme converts cortisol into its inactive form, cortisone and regulates cortisol levels, in particular in response to stress. Taking advantage of CRISPR/Cas9 technology, we generated a hsd11b2 zebrafish mutant line to evaluate the involvement of this gene in stress response regulation. The absence of a functional Hsd11b2 affects survival of zebrafish, although homozygous hsd11b2−/− mutants can reach adulthood. Reproductive capability of hsd11b2−/− homozygous adult males is almost completely abrogated, while that of females is reduced. Interestingly, basal cortisol levels and glucocorticoid-dependent transcriptional activities are not affected by the mutation. In agreement with basal cortisol results, we also demonstrated that basal response to light (LMR-L/D) or mechanical (VSRA) stimuli is not significantly different in wild-type (hsd11b2+/+) compared to mutant larvae. However, after exposure to an acute stressor, the cortisol temporal patterns of synthesis and release are prolonged in both 5 days post fertilization larvae and one-year-old adult hsd11b2−/− zebrafish compared to wild-type siblings, showing at the same time, at 5 dpf, a higher magnitude in the stress response at 10 min post stress. All in all, this new zebrafish model represents a good tool for studying response to different stressors and to identify mechanisms that are induced by cortisol during stress response.

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Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

Current Molecular Medicine ◽

10.2174/1566524019666190308115624 ◽

2019 ◽

Vol 19 (2) ◽

pp. 120-126

Author(s):

J. Wei ◽

Y. Yu ◽

Y. Feng ◽

J. Zhang ◽

Q. Jiang ◽

...

Keyword(s):

Negative Correlation ◽

Hepg2 Cells ◽

Serum Levels ◽

Significant Negative Correlation ◽

Mrna Levels ◽

Rt Pcr ◽

Apolipoprotein M ◽

Protein Mass ◽

Protein Levels ◽

Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

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Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Applied Sciences ◽

10.3390/app11135776 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5776

Author(s):

Varvara G. Blinova ◽

Natalia S. Novachly ◽

Sofya N. Gippius ◽

Abdullah Hilal ◽

Yulia A. Gladilina ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Inflammatory Reactions ◽

Effector Cells ◽

Cycle Regulation ◽

Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line

Journal of Pharmacological and Toxicological Methods ◽

10.1016/s1056-8719(01)00112-5 ◽

2000 ◽

Vol 44 (3) ◽

pp. 513-517 ◽

Cited By ~ 3

Author(s):

S.J Roberts-Thomson ◽

N.A Holman ◽

F.J May ◽

W.-J Lee ◽

G.R Monteith

Keyword(s):

Plasma Membrane ◽

Calcium Atpase ◽

Breast Epithelial Cell ◽

Epithelial Cell Line ◽

Mrna Levels ◽

Human Breast ◽

Rt Pcr ◽

Pcr Assay ◽

Plasma Membrane Calcium Atpase ◽

Breast Epithelial Cell Line

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Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas

Acta Endocrinologica ◽

10.1530/eje.0.1470795 ◽

2002 ◽

pp. 795-802 ◽

Cited By ~ 35

Author(s):

F Fallo ◽

V Pezzi ◽

L Barzon ◽

P Mulatero ◽

F Veglio ◽

...

Keyword(s):

Real Time ◽

Secretory Activity ◽

Zona Glomerulosa ◽

Zona Fasciculata ◽

Mrna Levels ◽

Rt Pcr ◽

Pathophysiological Role ◽

Aldosterone Production

BACKGROUND: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. METHODS: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. RESULTS: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. CONCLUSIONS: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

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Relative Levels of mRNA Encoding Enamel Proteins in Enamel Organ Epithelia and Odontoblasts

Journal of Dental Research ◽

10.1177/154405910308201209 ◽

2003 ◽

Vol 82 (12) ◽

pp. 982-986 ◽

Cited By ~ 59

Author(s):

T. Nagano ◽

S. Oida ◽

H. Ando ◽

K. Gomi ◽

T. Arai ◽

...

Keyword(s):

Tooth Enamel ◽

Structural Proteins ◽

Enamel Organ ◽

Maturation Stage ◽

Mrna Levels ◽

Rt Pcr ◽

Enamel Protein ◽

Enamel Proteins ◽

Enamel Layer

Amelogenin, enamelin, sheathlin (ameloblastin/ amelin), enamelysin (MMP-20), and KLK4 (EMSP-1) are the major structural proteins and proteinases in developing tooth enamel. Recently, odontoblasts were reported to express amelogenin, the most abundant enamel protein. In this study, we hypothesized that odontoblasts express all enamel proteins and proteases, and we measured their relative mRNA levels in enamel organ epithelia and odontoblasts associated with porcine secretory- and maturation-stage enamel by RT-PCR, using a LightCycler instrument. The results showed that amelogenin mRNA in secretory-stage EOE is 320-fold higher than in odontoblasts beneath secretory-stage enamel, and over 20,000-fold higher than in odontoblasts under maturation-stage enamel. Similar results were obtained for enamelin and sheathlin. Enamelysin mRNA levels were equivalent in these two tissues, while KLK4 mRNA was higher in odontoblasts than in secretory-stage EOE. These results support the conclusion that odontoblasts are involved in the formation of the enamel layer adjacent to enamel-dentin junction.

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Steroidogenic enzymes mRNA expression profile and steroids production in bovine theca cells cultured in vitro and stimulated with angiotensin II

Ciência Rural ◽

10.1590/0103-8478cr20240254 ◽

2015 ◽

Vol 45 (4) ◽

pp. 704-710 ◽

Cited By ~ 2

Author(s):

Melânia Lazzari Rigo ◽

Andressa Minussi Pereira Dau ◽

Werner Giehl Glanzner ◽

Manoel Martins ◽

Renato Zanella ◽

...

Keyword(s):

High Performance ◽

Culture Media ◽

Mrna Levels ◽

Ang Ii ◽

Rt Pcr ◽

Steroidogenic Enzymes ◽

Theca Cells ◽

Mrna Profile ◽

Dose Response Effect

The main objective of this study was to detect the steroidogenic effects of Ang II in bovine theca cells in vitro. Bovine theca cells were obtained from follicles (larger than 10mm of diameter) collected from a local abattoir and submitted to different treatments in a sequence of experiments. In experiment 1, CYP17A1 mRNA profile was evaluated in LH- (10ng ml-1) and Ang II-treated (0.1µM) theca cells. In experiment 2, a dose-response effect of Ang II (0.001; 0.1 e 10µM) plus insulin (100ng ml-1) and LH (100ng ml-1) was evaluated on steroidogenesis of bovine theca cells. Experiment 3 explored the effects of saralasin (an antagonist of Ang II receptors) on steroid production and steroidogenic enzymes regulation in theca cells. After 24 hours, culture media from experiments 2 and 3 was collected to evaluate testosterone and androstenedione levels by High-Performance Liquid Chromatography. In parallel, mRNA levels of key steroidogenic enzymes (HSD3B2, CYP11A1, CYP17A1) and STAR were assessed by RT-PCR. There was no difference in testosterone and androstenedione production between treated and controls groups, as well as in mRNA levels of the evaluated genes. In conclusion, the results suggest that Ang II does not regulate steroidogenesis in bovine theca cells

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