Transcriptional and post-transcriptional regulation of FSH receptor in rat granulosa cells by cyclic AMP and activin

1997 ◽  
Vol 153 (3) ◽  
pp. 465-473 ◽  
Author(s):  
M Tano ◽  
T Minegishi ◽  
K Nakamura ◽  
S Karino ◽  
Y Ibuki ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Receptor Expression ◽  
Northern Blot Hybridization ◽  
Fsh Receptor ◽  
Mrna Levels ◽  
Mrna Transcript ◽  
Receptor Mrna ◽  
Degradation Rates ◽  
Mrna Transcripts ◽  
In Cells

Abstract The effect of FSH on the induction of FSH receptors in granulosa cells is believed to be mediated, at least in part, by the cAMP second messenger system. We examined the effect of activin and cAMP on FSH receptor expression in this culture system. Steady-state levels of FSH receptor mRNA, analyzed by Northern blot hybridization, increased 3·5-fold in response to 24-h incubation with activin and 1·7-fold with 12-h incubation with 8-bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP; 0·2 mm). We have investigated whether 8-Br-cAMP- and/or activin-induced increases in FSH receptor mRNA levels are the result of increased transcription and/or altered mRNA stability. The rates of FSH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, increased 3-fold in cells treated with activin and 1·5-fold in cells treated with 8-Br-cAMP for 2 h. To examine the degradation rates of FSH receptor mRNA transcripts, granulosa cells were preincubated with 8-Br-cAMP, activin, or medium alone for 6 h. After the preincubation period, 5 μm actinomycin-D or 200 μm 5,6-dichloro-1-β-ribofuranosyl benzimidazole were added to arrest new RNA synthesis. The decay curves for the 2·4 kb FSH receptor mRNA transcript in granulosa cells were not significantly different in the absence or presence of 8-Br-cAMP. Activin, on the other hand, significantly altered the slope of the FSH receptor mRNA decay curve and increased the half-life of the 2·4 kb FSH receptor mRNA transcript. These data provide evidence that cAMP induces FSH receptor mRNA levels by stimulating the transcription rate and that activin increases FSH receptor mRNA levels both by stimulating transcription rates and by stabilizing the FSH receptor mRNA transcripts. Journal of Endocrinology (1997) 153, 465–473

227. FSH receptor expression in small human ovarian follicles

2005 ◽  
Vol 17 (9) ◽  
pp. 88
Author(s):  
J. H. Quennell ◽  
J. L. Stanton ◽  
P. R. Hurst
Keyword(s):  
Real Time ◽  
Granulosa Cells ◽  
Real Time Pcr ◽  
In Situ Hybridisation ◽  
Receptor Expression ◽  
Follicle Development ◽  
Fsh Receptor ◽  
Relative Expression ◽  
Receptor Mrna

Follicle-stimulating hormone (FSH) is pivotal in ovarian follicle development; the granulosa cells are the targets of FSH action in the ovary via FSH receptors. Granulosa cell growth and division mark initial follicle recruitment. The acquisition of FSH receptors on granulosa cells is regarded as a key event in hormone responsiveness and consequently follicle development. Due to the low abundance of FSH receptors and low expression of its mRNA it has been difficult to definitively characterise FSH receptor expression patterns. Here, localisation of FSH receptor in different follicle populations has been assessed with in situ hybridisation and real-time PCR of laser microdissected samples. We have used non-radioactive in situ hybridisation to investigate FSH receptor mRNA on a wide range of follicle stages. Biopsies from healthy fertile women (28–33 years) were frozen, embedded and cryosectioned at 10 µm. DIG-labelled RNA probes were designed to detect all splice variants. Hybridised probes were detected with NBT/BCIP in a colorimetric reaction. Secondly, follicles of different morphometric stages were isolated with a laser microscope. RNA extraction, reverse transcription and real-time PCR were used to confirm RNA presence and quantify relative expression. All follicle stages (from primordial to large antral) showed the presence of FSH receptor mRNA in their granulosa cells; sense controls were negative. Observations from real-time PCR indicate FSH receptor mRNA is present in all follicle stages observed and relative expression levels increase over early follicle development. These results challenge the existing doctrine that FSH receptor is absent in the smallest follicles. This suggests initial follicle recruitment may involve gonadotrophins. The use of sensitive molecular techniques will be crucial in elucidating this further.

Ontogeny of FSH receptor messenger ribonucleic acid transcripts in relation to FSH secretion and testicular function in sheep

1997 ◽  
Vol 18 (2) ◽  
pp. 113-125 ◽  
Author(s):  
T A Yarney ◽  
M H Fahmy ◽  
M R Sairam ◽  
H Khan ◽  
E A Macdonald
Keyword(s):  
Sertoli Cell ◽  
Receptor Gene ◽  
Pubertal Development ◽  
Receptor Expression ◽  
Fsh Receptor ◽  
Testicular Function ◽  
Messenger Ribonucleic Acid ◽  
Receptor Mrna ◽  
Mrna Transcripts ◽  
Fsh Receptor Gene

ABSTRACT The role of alternative splicing of the FSH receptor gene in the generation of FSH receptor proteins and testicular function remains an enigma. To address this issue, this investigation was conducted to determine variations in the expression of alternate FSH receptor mRNA transcripts in relation to changes in FSH release, hormone binding activity and testicular function during pubertal development of ram lambs from two genotypes of sheep (Romanov and a cross between Booroola × DLS) with different sexual precocity. Serum 17β-estradiol and testosterone concentrations were used as indices of Sertoli cell and testicular function. The results indicated that increases in Sertoli cell and testicular function normally seen during pubertal development are accompanied by age-dependent reductions in concentration of functional FSH receptors, as determined by binding of iodinated FSH to testicular membrane preparations. During the course of these changes, FSH release was either maintained at a steady level in Romanov lambs or it was gradually reduced in the Booroola × DLS cross, thus indicating that the testis had become more responsive to hormonal signal. This acquisition of heightened sensitivity was also associated with contrasting changes in the level of expression of FSH receptor mRNA transcripts. For both genotypes of sheep, 5 distinct species of mRNA transcripts of approximately 1·1, 1·5, 2·0, 2·5 and 6·5 kb were highly expressed from 11 to 22 weeks of age. Amongst these transcripts, the 1·1 kb molecular species was the most abundant. Specific probing for a previously cloned transcript called 151A1 representing the first 4 exons of the FSH receptor gene revealed a paradoxical increase in the level of expression from 11 weeks up to a maximum at 18-22 weeks of age for both genotypes. Collectively, the results indicated that contrasting changes in the production of specific alternatively spliced mRNA transcripts may mediate changes in FSH receptor expression which apparently accounts for the augmentation in sensitivity and function of the testis during pubertal development. Furthermore, the data provide the first important indication that the novel truncated transcript (151A1), which predictably encodes a soluble protein of either intra- or extracellular fate, could be physiologically relevant.

scholarly journals Phospholipase Cβ3 Mediates LH-Induced Granulosa Cell Differentiation

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2857-2869 ◽  
Author(s):  
Francesc X. Donadeu ◽  
Cristina L. Esteves ◽  
Lynsey K. Doyle ◽  
Catherine A. Walker ◽  
Stephanie N. Schauer ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Granulosa Cells ◽  
Inositol Phosphate ◽  
Ex Vivo ◽  
Receptor Expression ◽  
Target Cells ◽  
Down Regulation ◽  
Protein Levels ◽  
Prostaglandin Endoperoxide Synthase ◽  
In Cells

Previous studies showed that under certain conditions LH can stimulate not only adenylate cyclase (AC) but also phospholipase Cβ (PLCβ) signaling in target cells; however, the physiological involvement of PLCβ in LH-induced ovarian follicular cell differentiation has not been determined. To address this, ex vivo expression analyses and specific PLCβ targeting were performed in primary bovine granulosa cells. Expression analyses in cells from small (2.0–5.9 mm), medium (6.0–9.9 mm), and ovulatory-size (10.0–13.9 mm) follicles revealed an increase in mRNA and protein levels of heterotrimeric G protein subunits-αs, -αq, -α11, and -αi2 in ovulatory-size follicles, simultaneous with a substantial increase in LH receptor expression. Among the four known PLCβ isoforms, PLCβ3 (PLCB3) was specifically up-regulated in cells from ovulatory-size follicles, in association with a predominantly cytoplasmic location of PLCB3 in these cells and a significant inositol phosphate response to LH stimulation. Furthermore, RNA interference-mediated PLCB3 down-regulation reduced the ability of LH to induce hallmark differentiation responses of granulosa cells, namely transcriptional up-regulation of prostaglandin-endoperoxide synthase 2 and down-regulation of both aromatase expression and estradiol production. Responses to the AC agonist, forskolin, however, were not affected. In addition, PLCB3 down-regulation did not alter cAMP responses to LH in granulosa cells, ruling out a primary involvement of AC in mediating the effects of PLCB3. In summary, we provide evidence of a physiological involvement of PLCβ signaling in ovulatory-size follicles and specifically identify PLCB3 as a mediator of LH-induced differentiation responses of granulosa cells.

scholarly journals Heat stress diminishes gonadotropin receptor expression and enhances susceptibility to apoptosis of rat granulosa cells

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 463-472 ◽  
Author(s):  
Takashi Shimizu ◽  
Izumi Ohshima ◽  
Manabu Ozawa ◽  
Satoko Takahashi ◽  
Atsushi Tajima ◽  
...  
Keyword(s):  
Heat Stress ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Follicular Development ◽  
Receptor Expression ◽  
Female Rats ◽  
Aromatase Activity ◽  
Mrna Levels ◽  
Gonadotropin Receptor ◽  
Estradiol Levels

Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 °C, 50% RH; heat stress: 35 °C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.

Water deprivation upregulates ANG II AT1 binding and mRNA in rat subfornical organ and anterior pituitary

1997 ◽  
Vol 273 (1) ◽  
pp. E156-E163 ◽  
Author(s):  
G. L. Sanvitto ◽  
O. Johren ◽  
W. Hauser ◽  
J. M. Saavedra
Keyword(s):  
Paraventricular Nucleus ◽  
Median Eminence ◽  
Anterior Pituitary ◽  
Water Deprivation ◽  
Subfornical Organ ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Ang Ii ◽  
At1 Receptors ◽  
Receptor Mrna

We studied angiotensin II (ANG II) receptor subtype expression in selected brain nuclei and pituitary gland after water deprivation by in vitro receptor autoradiography using 125I-labeled [Sar1]ANG II and by in situ hybridization using 35S-labeled AT1A, AT1B, and AT2 receptor-specific riboprobes. In control rats we found binding to AT1 receptors in the subfornical organ, paraventricular nucleus, median eminence, and anterior pituitary; AT1A mRNA expression in the subfornical organ and paraventricular nucleus; and AT1B mRNA expression in the anterior pituitary. No receptor mRNA was found in the median eminence. AT1 receptors and AT1A receptor mRNA levels were increased in the subfornical organ, and, in the anterior pituitary, AT1 receptors and AT1B receptor mRNA were increased, only after 5 days of water deprivation. No significant changes occurred after 1 or 3 days of water deprivation, and no regulation of ANG II receptor expression was detected in other brain areas. Our results show that prolonged water deprivation selectively regulates AT1 receptor expression and AT1A and AT1B receptor mRNA levels in the subfornical organ and anterior pituitary, respectively, supporting a role for these receptors during sustained dehydration.

scholarly journals Endotoxin suppresses rat hepatic low-density lipoprotein receptor expression

1996 ◽  
Vol 313 (3) ◽  
pp. 873-878 ◽  
Author(s):  
Wei LIAO ◽  
Mats RUDLING ◽  
Bo ANGELIN
Keyword(s):  
Time Course ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Expression ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Receptor Mrna

Endotoxin induces hyperlipidaemia in experimental animals. In the current study, we investigated whether endotoxin alters hepatic low-density lipoprotein (LDL) receptor expression in rats. Endotoxin treatment suppressed hepatic LDL receptor expression in a dose- and time-dependent manner. Eighteen hours after intraperitoneal injection of increasing amounts of endotoxin, LDL receptor and its mRNA levels were determined by ligand blot and solution hybridization respectively. LDL receptor expression was inhibited by about 70% at a dose of 500 μg/100 g body weight. However, LDL receptor mRNA levels were markedly increased in all endotoxin-treated groups at this time point (by 83–136%; P < 0.001). Time-course experiments showed that LDL receptor expression was already reduced by 48% 4 h after endotoxin injection and was maximally reduced (by 63–65%) between 8 and 18 h. Changes in hepatic LDL receptor mRNA showed a different pattern. By 4 h after endotoxin injection, LDL receptor mRNA had decreased by 78% (P < 0.001). However, by 8 h after endotoxin injection, LDL receptor mRNA had returned to levels similar to controls, and 18 and 24 h after endotoxin injection, they were increased by about 60% (P < 0.05). Separation of plasma lipoproteins by FPLC demonstrated that endotoxin-induced changes in plasma triacylglycerols and cholesterol were due to accumulation of plasma apolipoprotein B-containing lipoproteins among very-low-density lipoprotein, intermediate-density lipoprotein and LDL. It is concluded that endotoxin suppresses hepatic LDL receptor expression in vivo in rats.

scholarly journals Inhibitory effect of retinoic acid on the development of immature porcine granulosa cells to mature cells

2000 ◽  
Vol 25 (1) ◽  
pp. 53-61 ◽  
Author(s):  
M Hattori ◽  
K Takesue ◽  
N Nishida ◽  
Y Kato ◽  
N Fujihara
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Inhibitory Effect ◽  
Immature Stage ◽  
Mrna Levels ◽  
Lh Receptor ◽  
Receptor Mrna ◽  
Immature Cells

The present study investigated the effect of retinoic acid (RA) on the differentiation of granulosa cells prepared from porcine ovaries. The granulosa cells were precultured for 15 h, then cultured for 48 h with FSH and further treated for 24 h with LH in order to induce their transformation into luteal cells. After the cells had been exposed to 1 microM retinoids (RA, retinal and retinol) for 87 h, analysis of the LH receptor mRNA expression, an indicator of granulosa cell differentiation, was carried out by using semiquantitative RT-PCR. The results showed that there was a decrease in LH receptor mRNA levels, and that RA had a more potent effect on these levels than the other two retinoids. When cells were exposed to RA in the immature stage (before the addition of FSH) or the early stage of development (0-24 h after the addition of FSH), expression of LH receptor mRNA was greatly diminished. When the immature cells were cultured for 15 h with RA, then washed and cultured for 48 h with FSH and for 24 h with LH, the expression of LH receptor mRNA was not reversed. In the differentiated cells (24 h after the addition of FSH), however, RA no longer had any inhibitory effect. When the immature cells were exposed to RA, FSH-induced expression of c-fos mRNA was markedly decreased. In contrast, expression of c-jun and activating transcription factor-4 mRNAs remained constant. However, the expression of c-fos mRNA was not decreased by forskolin. The results indicate that RA is a potent inhibitor in the immature stage of porcine granulosa cell differentiation, probably through decreased expression of FSH receptor, but that RA does not inhibit differentiation in the mature stage of the cells.

scholarly journals Modulation of glomerular endothelin and endothelin receptor gene expression in aminonucleoside-induced nephrosis.

1995 ◽  
Vol 5 (8) ◽  
pp. 1585-1590
Author(s):  
T Nakamura ◽  
I Ebihara ◽  
M Fukui ◽  
S Osada ◽  
Y Tomino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Receptor Gene ◽  
Mrna Level ◽  
Experimental Period ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Puromycin Aminonucleoside ◽  
Receptor Mrna ◽  
Etb Receptor ◽  
Receptor Gene Expression

This study assessed glomerular endothelin (ET)-1, ET-3, and ET-receptor A and B mRNA levels in puromycin aminonucleoside (PAN)-induced nephrosis. During the nephrotic stage, 8 days after PAN injection, ET-1 and ETB receptor mRNA were elevated by 2.8 +/- 0.8-fold (P < 0.01) and 2.4 +/- 0.9-fold (P < 0.01), respectively, as compared with controls. These mRNA levels decreased to control levels by Day 20, when the nephrosis was in remission. In contrast, glomerular ETA receptor mRNA levels did not change in PAN nephrosis or control rats during the experimental period. ET-3 mRNA was not detected in the glomeruli of PAN nephrosis or control rats. Additionally, plasma ET concentration and glomerular ET production were measured in PAN nephrosis and control rats by radio-immunoassay. Eight days after PAN injection, ET-1 levels in plasma and glomeruli were not significantly altered in rats with PAN-induced nephrosis (glomeruli, 104.68 +/- 16.46 pg/mg of protein versus 98.24 +/- 13.68 pg/mg of protein; plasma, 2.68 +/- 1.10 versus 2.52 +/- 0.98 pg/mL). The administration of methylprednisolone to PAN rats resulted in the rapid disappearance of proteinuria and partially attenuated the increased ET-1 and ETB receptor gene expression in the glomeruli. These data indicate that glomerular ET-1 and ETB receptor expression in PAN nephrosis in increased at the mRNA level and that methylprednisolone treatment results in an attenuated increase.

Abstract 553: Prostaglandin E2 EP3 Receptor Downregulates COX2 Expression in the Medullary Thick Ascending Limb (mTAL) Induced by Hypertonic NaCl Intake

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Shoujin Hao ◽  
Carlos P Vio ◽  
Carlos Cespedes ◽  
Mariana Quiroz-Munoz ◽  
Nicholas R Ferreri
Keyword(s):  
Protein Expression ◽  
Tap Water ◽  
Receptor Expression ◽  
Free Access ◽  
Thick Ascending Limb ◽  
Mrna Levels ◽  
Mrna Accumulation ◽  
Cox 2 ◽  
Ep3 Receptor ◽  
In Cells

We recently showed that a novel negative feedback mechanism involving PGE 2 acting on EP3 receptors regulates cyclooxygenase-2 (COX-2) expression in the thick ascending limb (TAL) induced by the selective COX-2 inhibitors, celecoxib and rofecoxib. In the present study we tested the hypothesis that inhibition of EP3 facilitates COX-2 expression in the TAL induced by ingestion of hypertonic NaCl or exposure of mTAL cells to hypertonic media. COX-2 protein expression in the outer medulla (OM) increased 2.2±0.3 fold in mice given free access to 1% NaCl in the drinking water for 3 days compared with tap water; p<0.05. The increase was associated with a 4-fold elevation in COX-2 mRNA accumulation (tap water: 0.5±0.1 vs 1% NaCl: 1.9±0.4; p<0.05), and higher PGE 2 production by freshly isolated mTAL tubules (tap water: 87.6±9.4 vs 1% NaCl: 203.3±19.3 pg/mg protein; p<0.05). EP3 mRNA levels also increased approximately 2-fold in OM of mice ingesting 1% NaCl (tap water: 0.7±0.2 vs 1% NaCl: 1.3±0.2). Administration of a selective EP3 receptor antagonist (L-798106: 20μg/kg/day) for 2 days increased COX-2 mRNA accumulation in mTAL tubules (L-798106: 2.1±0.1 fold change vs control; p<0.05), and the elevation in COX-2 protein expression induced by 1% NaCl was increased an additional 50% in mice given L-798106. COX-2 mRNA accumulation in primary cultures of mTAL cells increased 2-fold in response to media made hypertonic by addition of NaCl (400 mosmol/kg H 2 O), compared with cells incubated in isotonic media. L-798106 increased COX-2 mRNA 2-fold in isotonic media and 4-fold in cells exposed to 400 mosmol/kg H 2 O. PGE 2 production by mTAL cells increased approximately 4-fold in response to challenge with 400 mosmol/kg H 2 O for 9 hr, and was inhibited in cells transiently transfected with a lentivirus shRNA construct (EGFP-C2-ex5) to silence COX-2 (286.1±14.8 to 64.3±5.2 pg/mg protein; p<0.05). Collectively, the data suggest that local hypertonicity in the mTAL is associated with an increase in COX-2 expression concomitant with elevated EP3 receptor expression, which attenuates COX-2 activity in this segment of the nephron. Moreover, PGE 2 signaling via EP3 receptors in the TAL may be part of a mechanism that regulates mTAL COX-2 expression and function in response to diverse stimuli.

Ontogeny and effect of cortisol on vasopressin-1b receptor expression in anterior pituitaries of fetal sheep

2003 ◽  
Vol 284 (1) ◽  
pp. R51-R56 ◽  
Author(s):  
Sharla F. Young ◽  
Jennifer L. Smith ◽  
Jorge P. Figueroa ◽  
James C. Rose
Keyword(s):  
Receptor Expression ◽  
Late Gestation ◽  
Mrna Levels ◽  
Fetal Sheep ◽  
Fetal Plasma ◽  
Protein Levels ◽  
Receptor Mrna ◽  
Naturally Occurring ◽  
V1b Receptor

Corticotroph responsiveness to arginine vasopressin (AVP) increases during late gestation in fetal sheep. The mechanism of this increase in AVP responsiveness is currently unknown but could be related to an increase in vasopressin type 1b (V1b) receptor expression in the pituitary during development. To determine if there are ontogenic changes in V1b receptor expression that may help explain the changes in ACTH responses to AVP, we studied pituitaries from three groups of fetal sheep [100, 120, or 140 days gestational age (dGA)]. V1b receptor mRNA and protein significantly decreased by 140 dGA. Peak V1b mRNA levels were detected at 100 dGA, while peak V1b protein levels were detected at 120 dGA. The reduction in V1b receptor expression in late gestation may be due to the naturally occurring peripartum increase in fetal plasma cortisol because cortisol infusion at 122–130 dGA decreased V1b receptor mRNA. Thus there is a marked decrease in the expression of the V1b receptor in the pituitary during fetal development, leaving the role of the V1b receptor in increasing AVP responsiveness uncertain.

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