Abstract 129: Smyd1 is an Essential Regulator of Adaptive Response to Glucose Starvation in Cardiomyocytes

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Junco S Warren ◽  
Amira Sabry ◽  
Keiko Cawley ◽  
Aman Makaju ◽  
Marta W Szulik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Survival ◽  
Adaptive Response ◽  
Target Genes ◽  
Mrna Level ◽  
Glucose Deprivation ◽  
Cardiac Metabolism ◽  
Mrna Levels ◽  
Glucose Starvation ◽  
Metabolic Remodeling

Smyd1, a muscle-specific histone methyltransferase, has been implicated in global metabolic remodeling in cardiac hypertrophy and failure. We previously showed that cardiac-specific ablation of Smyd1 in mice led to metabolic perturbations prior to overt cardiac dysfunction, suggesting that Smyd1 positively regulates cardiac metabolism. However, the role of Smyd1 in adaptive response to nutritional stress (NS) in cardiomyocytes is largely unknown. Here, we found that glucose deprivation-induced NS led to upregulation of Smyd1 in cultured rat neonatal ventricular myocytes (NRVMs) (FC=1.87, p<0.05), which was associated with the increased mRNA level of PGC-1α, a key regulator of mitochondrial energetics (FC=2.71, p<0.05). Strikingly, siRNA-mediated knockdown of Smyd1 (Smyd1-KD) in NRVM prior to glucose starvation led to extensive cell death not observed in control NRVMs (scrambled siRNA), suggesting that Smyd1 is required for cell survival in NS. To elucidate the mechanism how Smyd1 is involved in adaptive response to NS, we performed unbiased proteomic and metabolomic screening of Smyd1-KD NRVMs. Bioinformatic analysis of proteins and metabolites that were differentially expressed in Smyd1-KD NRVM revealed that oxidative phosphorylation was the most perturbed metabolic pathway in Smyd1-KD NRVMs, concomitant with a reduction in mitochondrial substrates (BCAAs; pyruvate; lactate, all p<0.05). Gene expression analyses using RT-PCR and RNA-seq in Smyd1-KD NRVMs further identified PGC-1α and Perm1 (the muscle-specific PGC-1α and ESRR induced regulator) as potential downstream targets of Smyd1 in regulation of cardiac energetics (FC=-1.92 and -1.66, respectively, both p<0.05). Consistent with downregulation of Perm1, the known Perm1-target genes (Tfb1m; Ctp1b; Glut4; Myl2) were all downregulated at the mRNA levels in Smyd1-KD NRVMs (p<0.05). Lastly, Smyd1-KD NRVMs exhibited accelerated loss of mitochondrial membrane potential during hypoxia, revealing an increased vulnerability to metabolic stress. Taken together, these results show that Smyd1 is an essential regulator of adaptive response and cell survival during metabolic insults, presumably through regulating PGC-1α/Perm1 gene expression.

scholarly journals Treatment with PPARαAgonist Clofibrate Inhibits the Transcription and Activation of SREBPs and Reduces Triglyceride and Cholesterol Levels in Liver of Broiler Chickens

PPAR Research ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Lijun Zhang ◽  
Chunyan Li ◽  
Fang Wang ◽  
Shenghua Zhou ◽  
Mingjun Shangguan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Broiler Chickens ◽  
Target Genes ◽  
Nuclear Protein ◽  
Mrna Level ◽  
Control Group ◽  
Mrna Levels ◽  
Protein Levels ◽  
Cholesterol Levels ◽  
Regulation Mechanisms

PPARαagonist clofibrate reduces cholesterol and fatty acid concentrations in rodent liver by an inhibition of SREBP-dependent gene expression. In present study we investigated the regulation mechanisms of the triglyceride- and cholesterol-lowering effect of the PPARαagonist clofibrate in broiler chickens. We observed that PPARαagonist clofibrate decreases the mRNA and protein levels of LXRαand the mRNA and both precursor and nuclear protein levels of SREBP1 and SREBP2 as well as the mRNA levels of the SREBP1 (FASNandGPAM) and SREBP2 (HMGCRandLDLR) target genes in the liver of treated broiler chickens compared to control group, whereas the mRNA level ofINSIG2, which inhibits SREBP activation, was increased in the liver of treated broiler chickens compared to control group. Taken together, the effects of PPARαagonist clofibrate on lipid metabolism in liver of broiler chickens involve inhibiting transcription and activation of SREBPs and SREBP-dependent lipogenic and cholesterologenic gene expression, thereby resulting in a reduction of the triglyceride and cholesterol levels in liver of broiler chickens.

Abstract 3: The Histone Methyltransferase Smyd1 is a Novel Transcriptional Regulator of Cardiac Fatty Acid Metabolism in Mice

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Junko S Warren ◽  
Dane W Barton ◽  
Mickey Miller ◽  
Li Wang ◽  
James Cox ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
Fatty Acid Metabolism ◽  
Cardiac Dysfunction ◽  
Acid Metabolism ◽  
Transcriptional Start Site ◽  
Cardiac Metabolism ◽  
Histone Methyltransferase ◽  
Tamoxifen Treatment ◽  
Mrna Levels

Epigenetic control of metabolism in the healthy and diseased heart remains poorly understood. We recently demonstrated that chromatin-bound Smyd1, a muscle-specific histone methyltransferase, is significantly upregulated in a mouse model of pressure overload-induced heart failure (HF) and that inducible, cardiac-specific Smyd1 knock-out (Smyd1-KO) mice develop cellular hypertrophy and fulminate HF. Bioinformatic analysis of transcripts differentially regulated in these mice revealed that cardiac metabolism was the most perturbed biological function in the heart. However, it was not clear whether alterations in cardiac metabolism were a direct consequence of Smyd1 deletion or were secondary to developed HF. Here we hypothesized that Smyd1 directly regulates cardiac metabolism; the effects of which should be detectable in Smyd1-KO mice before overt cardiac dysfunction. To test this hypothesis we performed unbiased metabolomic analysis of Smyd1-KO mice using GC/MS and MS/MS (n=9 control, n=10 KO) combined with targeted gene expression analysis. Our results showed significant changes in the metabolic profile of Smyd1-KO mice at the earliest time point (3 weeks after tamoxifen treatment) in which Smyd1 protein expression was significantly reduced while cardiac function remained normal. The most profound difference, in energetics-associated pathways in these mice, was found in fatty acid β-oxidation, manifested by the decreased myocardial content of carnitine and free fatty acids and downregulation of their transporters, OCTN2 and CD36. In addition, mRNA levels of the PPAR-α complex (PPAR-α;RXR-α;PGC-1α), an established regulator of fatty acid β-oxidation, and its target genes (CPT1b;CD36;Acox1;MCAD) were significantly reduced in Smyd1-KO mice prior to the onset of cardiac dysfunction (all p<0.05). To identify whether Smyd1 directly controls gene expression of PPAR-α, we examined the PPAR-α loci using chromatin-immunoprecipitation followed by qPCR and observed significant binding of Smyd1 upstream of the PPAR-α transcriptional start site. Overall, this study identifies Smyd1 as a novel regulator of fatty acid metabolism and suggests that Smyd1 controls cardiac energetics directly by regulating gene expression of PPAR-α.

scholarly journals A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function

2019 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Gene Knockout ◽  
Mrna Levels ◽  
Beta Cell Line ◽  
Human Beta Cell

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

Combining Bevacizumab with knocked-down β-catenin reduces VEGF-A and Slug mRNA in HepG2 but not in Caco-2 cell lines

2021 ◽  
Vol 21 ◽  
Author(s):  
Reem Mebed ◽  
Yasser BM Ali ◽  
Nahla Shehata ◽  
Nadia El-Guendy ◽  
Nahla Gamal ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Target Genes ◽  
Cultured Cells ◽  
Mrna Level ◽  
Negative Control ◽  
The Other ◽  
Signalling Pathways ◽  
Mrna Levels ◽  
Protein Expressions ◽  
The Difference

Background: Bevacizumab (Bev) resistance is hypothesized to be overcome by combination with inhibitors of other signalling pathways. Objective: We aimed to study the effect of combining Bev with knocked down β-catenin (Bev-β-cat-siRNA) on the expression of VEGF-A, Slug, NFКB and its two target genes c-Flip and FasR in HepG2. Expression of VEGF-A and Slug was also studied in Caco-2 cells. Methods: Cultured cells were divided into six groups 1) cells treated with Bev only 2) cells treated with β-catenin-siRNA 3) cells treated with Bev-β-cat-siRNA 4) cells treated with negative control 5) cells treated with Bev-negative control and untreated cells. Expressions were assessed using qPCR and western blotting. Results: Bev-β-cat-siRNA significantly reduced the mRNA level of VEGF-A, which was initially increased in response to Bev alone in HepG2 but not in Caco-2. Additionally, Bev-β-cat-siRNA significantly decreased Slug mRNA level compared to Bev only treated HepG2 cells. In contrast, VEGF-A and Slug mRNA levels in Bev only group were remarkably lower than Bev-β-cat-siRNA in Caco-2 cells. Distinct β-catenin and Slug protein expressions were noticed in HepG2 and Caco-2 cells. On the other hand, Bev-β-cat-siRNA remarkably reduced the level of NFКB, FasR and c-Flip compared to Bev only treated HepG2 cells although the difference was not statistically significant. Conclusion: We conclude that, combining Bevacizumab with knocked down β-catenin reduce the expression of VEGF-A and Slug in HepG2 but not in Caco-2 cells.

scholarly journals Differential Gene Expression of Steroid 5α-Reductase 2 in Core Needle Biopsies from Malignant and Benign Prostatic Tissue1

1997 ◽  
Vol 82 (7) ◽  
pp. 2210-2214
Author(s):  
Catarina Bjelfman ◽  
Torbjörn G. Söderström ◽  
Einar Brekkan ◽  
Bo Johan Norlén ◽  
Lars Egevad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Mrna Level ◽  
Transrectal Ultrasound ◽  
Mrna Levels ◽  
Prostate Hyperplasia ◽  
Noncancerous Tissue ◽  
Core Needle ◽  
Total Rna ◽  
Prostate Diseases

Androgens are implicated in the development of prostate cancer (CAP) and benign prostate hyperplasia. The conversion of testosterone to the more potent metabolite dihydrotestosterone by prostate-specific steroid 5α-reductase type 2 (5α-red2) is a key mechanism in the action of androgens in the prostate and is important in the promotion and progression of prostate diseases. Manipulation of the turnover of androgens is thus fundamental in the pharmacological treatment strategy. We have developed a sensitive solution hybridization method for quantification of the gene expression of 5α-red2 in core needle biopsies of the prostate. The 5α-red2-specific messenger RNA (mRNA) levels were measured in 50 human prostate transrectal ultrasound-guided core biopsies obtained from 31 outpatients (median age 72, range 57–88 yr) undergoing biopsy for diagnostic purposes. Significant differences were observed in the gene expression of 5α-red2 between cancerous and noncancerous tissue. In the 14 biopsies judged cancerous, the median 5α-red mRNA levels were 3.5 amol/ng total RNA compared with 12.0 amol/ng total RNA in the biopsies showing no cancer (P = 0.0018). The median 5α-red2 mRNA level in noncancerous tissue was thus 3.4 times higher than in the cancerous specimens.

Developmental expression of pIgR gene in sheep mammary gland and hormonal regulation

2002 ◽  
Vol 69 (1) ◽  
pp. 13-26 ◽  
Author(s):  
AURORE RINCHEV-ALARNOLD ◽  
LUCETTE BELAIR ◽  
JEAN DJIANE
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Hormonal Regulation ◽  
Mrna Level ◽  
Immunohistochemical Analysis ◽  
Developmental Expression ◽  
Secretory Iga ◽  
Mrna Levels ◽  
Immunoglobulin Receptor ◽  
Pregnancy And Lactation

Secretory IgA found in external secretions are constituted by polymeric IgA (pIgA) bound to the extra-cellular part of the polymeric immunoglobulin receptor (pIgR). The receptor mediates transcytosis of pIgA across epithelial cells. The aim of the present study was to analyse the evolution of pIgR expression in the sheep mammary gland during the development of the mammary gland and to analyse its hormonal regulation. Gene expression of the pIgR was analysed in sheep mammary gland during pregnancy and lactation. By Northern Blot analysis, we observed that low levels of pIgR mRNA are expressed until day 70 of pregnancy. Accumulation of pIgR mRNA started during the third part of pregnancy and intensified 3 d after parturition to reach highest levels during established lactation (day 70). In situ hybridization analysis was used to confirm the increase in pIgR gene expression per mammary epithelial cell. In order to examine the hormonal regulation of the pIgR expression, virgin ewes were hormonally treated. Treatment with oestradiol and progesterone increased pIgR mRNA levels slightly. Subsequent addition of glucocorticoids induced a significant accumulation of pIgR mRNA in the mammary gland of the treated animals. Immunohistochemical analysis was performed to verify that the increase of pIgR mRNA level was associated with enhancement of the pIgR protein in mammary cells. No increase of pIgR mRNA levels were observed if PRL secretion was blocked by bromocryptine injections throughout the hormonal procedure. In conclusion, the present experiments suggest that the enhancement of pIgR levels during lactation result from combined effects of both prolactin and glucocorticoids.

scholarly journals Review of: Metalloproteinase axes increase β-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

2007 ◽  
Vol 10 (8) ◽  
Author(s):  
D. S. Salomon
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Target Genes ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Specific Effects ◽  
Primary Mouse ◽  
Ductal Elongation

Citation of original article:C. V. Hojilla, I. Kim, Z. Kassiri, J. E. Fat, H. Fang, R. Khokha. Journal of Cell Science 2007; 120(6): 1050–1060.Abstract of the original article:Multiple cancers exhibit mutations in β-catenin that lead to increased stability, altered localization or amplified activity. β-Catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter β-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased β-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3−/− MEFs being more sensitive and Timp3−/− MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3−/− MECs had higher dephosphorylated β-catenin levels and increased β-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of β-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated β-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3−/− mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.

198 PROGESTERONE, ESTROGEN (ER-α AND ER-β), AND OXYTOCIN RECEPTOR GENE EXPRESSION IN CANINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 248
Author(s):  
A. A. P. Derussi ◽  
A. C. S. Castilho ◽  
R. W. A. Souza ◽  
R. Volpato ◽  
C. R. F. Guaitolini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Hormone Receptors ◽  
Target Genes ◽  
Receptor Gene ◽  
Rna Extraction ◽  
Amplification Efficiency ◽  
Mrna Levels ◽  
Lh Surge ◽  
Total Rna

The aim of this study was to compare the mRNA levels of hormone receptor for progesterone (PR), oestrogen α (ER-α), oestrogen β (ER-β), and oxytocin (OTR) in canine morulae and blastocysts. Ten healthy mature bitches were inseminated based on monitoring vaginal cytology and progesterone concentration. The first insemination was performed on Day 2 after the preovulatory LH surge (progesterone 4 ng mL–1), and the second was performed 48 h later. All females were submitted to ovariohysterectomy (OVH), and the oviduct as well the uterurs were flushed with PBS solution to obtain the embryos. The females were divided into two groups: Group A (n = 5), morulae were collected 8 days after the LH surge and Group B (n = 5), blastocysts were collected 12 days after the LH surge. The pools (n = 10) of embryos (5 embryos/pool) were stored in RNAlater® (Ambion, Life Technologies, USA) at –80°C. The samples were analysed together. The RNA later was removed used PBS calcium free and the total RNA extraction was performed using the Qiagen RNeasy micro-kit (Hildesheim, Germany). Before reverse-transcription (RT) reaction, the total RNA was treated with DNase I Amplification Grade (Invitrogen Life Technologies, Carlsbad, CA, USA). The gene expression of target genes was assessed by real-time RT-qPCR, using SuperScript III for RT and power SYBR Green PCR Master Mix (Applied Biosystems, USA) for cDNA for PCR. The primers for target genes were designed using the software Primer Express® (Applied Biosystems, USA). The gene expression of target genes was normalized by HPRT gene and the relative abundance of mRNA was determined by the ΔΔct method corrected by amplification efficiency using Pffafl’s equation. The means of mRNA relative abundance were compared by t-test. The PR mRNA expression only in blastocysts is similar to the results obtained by Hou et al. (1997) in rat embryos. It is believed that the absence of PR in the early stages of cleavage is due to the indirect action of progesterone by growth factors produced by the maternal reproductive tract (2). Apparently, ER-β action does not occur in the embryo canine phases analysed; however, the action of ER-α seems related to the deployment signal as seen by Hou et al. (1996) in rats. Similarly to findings in the literature, OTR expression decreased in canine embryonic development. This receptor was produced by blastocysts while present in the uterus, which may represent an incidental mechanism to the embryo control of endometrial receptivity, such as also to prevent the development of endometrial luteolytic mechanism. The variation in hormone receptors gene expression in canine embryos can be influencing the transition from morula to blastocyst. In addition, a hormonal influence on these structures can occur in different ways.

scholarly journals Differential regulation of γ-glutamylcysteine synthetase heavy and light subunit gene expression

1997 ◽  
Vol 326 (1) ◽  
pp. 167-172 ◽  
Author(s):  
Jiaxin CAI ◽  
Zong-Zhi HUANG ◽  
Shelly C. LU
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Steady State ◽  
Mrna Level ◽  
Rat Hepatocytes ◽  
Mrna Levels ◽  
Subunit Gene ◽  
Subunit Mrna ◽  
Glutamylcysteine Synthetase ◽  
Steady State Mrna Level

γ-Glutamylcysteine synthetase (GCS) is the rate-limiting enzyme in the biosynthesis of glutathione and is composed of a heavy and a light subunit. Although the heavy subunit is enzymically active alone, the light subunit plays an important regulatory role by making the holoenzyme function more efficiently. In the current study we examined whether conditions which are known to influence gene expression of the heavy subunit also influence that of the light subunit, and the mechanisms involved. Treatment of cultured rat hepatocytes with hormones such as insulin and hydrocortisone, or plating hepatocytes under low cell density increased the steady-state mRNA level of the heavy subunit only. Treatment with diethyl maleate (DEM), buthionine sulphoximine (BSO) and t-butylhydroquinone (TBH) increased the steady state mRNA level and gene transcription rates of both subunits. These treatments share in common their ability to induce oxidative stress and activate nuclear factor κB (NF-κB). Treatment with protease inhibitors 7-amino-1-chloro-3-tosylamido-2-heptanone (TLCK) or L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) had no influence on the basal NF-κB and GCS subunit mRNA levels, but blocked the activation of NF-κB by DEM, BSO and TBH, and the increase in GCS heavy subunit mRNA level by BSO and TBH. On the other hand, the DEM-, BSO- and TBH-induced increase in GCS light-subunit mRNA level was unaffected by TLCK and TPCK. Thus only the heavy subunit is hormonally regulated and growth sensitive, whereas both subunits are regulated by oxidative stress. Signalling through NF-κB is involved only in the oxidative-stress-mediated changes in the heavy subunit gene expression.

scholarly journals Regulation of pituitary inhibin/activin subunits and follistatin gene expression by GnRH in female rats

2011 ◽  
Vol 210 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Petra Popovics ◽  
Zoltan Rekasi ◽  
Alan J Stewart ◽  
Magdolna Kovacs
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Inhibin B ◽  
Female Rats ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Short Term ◽  
Α Subunit ◽  
Long Term Treatment

Pituitary inhibin B, activin B, and follistatin are local regulators of FSH. Activin B is a homodimeric molecule (βB–βB), while inhibin B contains an α and a βB subunit. The regulation of gene expression of α, βB, and follistatin by local and endocrine hormones was examined in pituitaries from female rats and in perifused pituitary cells by RT-PCR. Ovariectomy (OVX) induced an elevation in the mRNA level of α and βB subunits and follistatin. Short-term (4 h) treatment of pituitary cells with GnRH decreased both the inhibin α and the inhibin/activin βB subunit mRNA levels, while long-term treatment (20 h) with 100 nM GnRH stimulated the expression of both subunits. In contrast, the mRNA level of follistatin was elevated after the short-term GnRH treatment. Long-term exposure of pituitary cells to estradiol and inhibin B suppressed the mRNA expression of βB and had no effect on the expression of α subunit and follistatin. Our results demonstrate that the increased expressions of inhibin/activin subunits and follistatin in the post-OVX period can be induced by the lack of gonadal negative feedback, resulting in a high GnRH environment in the pituitary. This study reports for the first time that GnRH administered in high doses and for a long period stimulates the gene expression of inhibin/activin subunits and thereby may contribute to the stimulatory effect of OVX on the expression of these genes.

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