Regulation of steady-state follistatin mRNA levels in rat granulosa cells in vitro

1992 ◽  
Vol 9 (2) ◽  
pp. 147-156 ◽  
Author(s):  
U. Michel ◽  
J. W. McMaster ◽  
J. K. Findlay
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Internal Standard ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Epidermal Growth

ABSTRACT The regulation of steady-state follistatin mRNA levels by different pituitary hormones and peptide factors was examined in granulosa cell cultures derived from diethylstilboestrol-treated immature rats. Cytosolic RNA from cell cultures was prepared by lysis and equal amounts of RNA from all samples were analysed with a solution—hybridization assay using a 32P-labelled antisense probe corresponding to a part of exon 5 together with a part of the 5′ end of exon 6 of the rat follistatin gene. In addition, a specific 35S-labelled probe for cyclophilin was used as an internal standard. The results show that 5 μg FSH/1 for 24 to 72 h stimulated steady-state follistatin mRNA levels, reaching levels 18·5-fold higher than controls. LH (0·2-100 μg/l) had only minor effects on follistatin mRNA levels in FSH-primed granulosa cells and prolactin, GH and IGF-I did not show any significant effects. Activin raised basal as well as FSH-stimulated steady-state follistatin mRNA levels up to ten- and twofold above controls respectively, whereas epidermal growth factor was found to inhibit FSH-stimulated follistatin mRNA levels in a dose-dependent manner. It is concluded that follistatin mRNA levels in granulosa cells are regulated by FSH rather than LH, and that the stimulation by FSH can be inhibited by epidermal growth factor but enhanced by activin. Activin alone was also capable of stimulating follistatin mRNA.

Augmentation by epidermal growth factor of basal and stimulated progesterone production by human luteinized granulosa cells

1989 ◽  
Vol 121 (2) ◽  
pp. 397-402 ◽  
Author(s):  
M. C. Richardson ◽  
S. C. Gadd ◽  
G. M. Masson
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Cultured Cells ◽  
Vitro Fertilization ◽  
Human Granulosa Cells ◽  
Culture Cells ◽  
Oocyte Collection ◽  
Epidermal Growth

ABSTRACT Human granulosa cells were prepared from follicular aspirates obtained during oocyte collection for in-vitro fertilization. Following several days in culture, cells were washed and then progesterone output was measured in 2-h incubations. After culture for 3 days, incubated cells responded well to human chorionic gonadotrophin (hCG) and prostaglandin (PG) E2 with similar levels of maximum response. Exposure of cultured cells to epidermal growth factor (EGF) for 2 days (days 3–5) led to substantial increases both in basal production and in responses to hCG and PGE2 during subsequent incubations. These effects of EGF were not accompanied by measurable increases in DNA levels in cultures over this time. Results may point to a possible paracrine role for EGF-like factors modulating the activity of cells forming the early corpus luteum. Journal of Endocrinology (1989) 121, 397–402

Steady-state level of epidermal growth factor (EGF) mRNA and effect of EGF on in vitro culture of caprine preantral follicles

2011 ◽  
Vol 344 (3) ◽  
pp. 539-550 ◽  
Author(s):  
Juliana Jales H. Celestino ◽  
Jamily B. Bruno ◽  
Márcia Viviane A. Saraiva ◽  
Rebeca M. P. Rocha ◽  
Ivina R. Brito ◽  
...  
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
In Vitro Culture ◽  
State Level ◽  
Steady State Level ◽  
Preantral Follicles ◽  
Epidermal Growth

343 EFFECTS OF EPIDERMAL GROWTH FACTOR SUPPLEMENTATION ON IN VITRO MATURATION AND GENE EXPRESSION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 279
Author(s):  
H.-J. Song ◽  
S.-H. Lee ◽  
G.-H. Maeng ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
In Vitro Maturation ◽  
Luteinizing Hormone Receptor ◽  
Control Group ◽  
Dependent Manner ◽  
Junction Protein ◽  
Epidermal Growth

Despite many efforts to improve canine in vitro maturation (IVM), the efficiency is still low compared to that of other mammalian species (Marie et al. 2004). Epidermal growth factor (EGF) has stimulatory effects on the resumption of oocyte maturation and cumulus expansion in vitro and on prei-mplantation embryonic development in mammals by either an autocrine or a paracrine pathway, or a combination of both systems (Paria et al. 2001 PNAS 98, 1047-1052). The present study investigated the effects of EGF supplementation on in vitro maturation and gene expression of canine oocytes. Oocytes were recovered by slicing ovaries recovered from 40 bitches after ovariohysterectomy at random stages of the estrous cycle. Cumulus-oocyte complexes (COCs) were matured in TCM-199 containing 10% FBS, 1 �g/mL FSH and LH, and EGF (0, 10, or 30 ng/mL) for 48 or 72 h at 39�C in a humidified atmosphere of 5% CO2 in air. In Experiment I (n = 2520 oocytes), the nuclear maturation status was assessed by fluorescence microscopy after bisbenzimide (Hoechst 33342) staining (10 �g/mL) at 0, 48, and 72 h of incubation. In Experiment II (n = 90 oocytes), expression of transcripts such as EGF receptor (EGFR), luteinizing hormone receptor (LHR), and gap junction protein (GJA5) were determined in 10 intact COCs each at 0, 48, and 72 h, respectively, by reverse transcription-polymerase chain reaction (RT-PCR). At 0 h 10-20% of the oocytes had undergone resumption of meiosis (GVBD<MII). After 48 h of IVM, rate of meiotic resumption for 0, 10, and 30 ng/mL EGF were 28, 35, and 30%, respectively. At 72 h of IVM, oocytes in the 10 ng/mL EGF group had resumed meiosis at a higher frequency (55%; P < 0.05) than in the 30 ng/mL EGF or the control group (39 and 42%, respectively). At 72 h of IVM, the frequency of maturation to the MII stage was significantly higher in the 10 ng/mL EGF group (9.6%) than in the 30 ng/mL EGF or the control group (4.2 and 3.3%, respectively). The expression of EGFR was significantly higher (P < 0.05) in 0 h oocytes than in the 48- or 72-h oocytes. Further EGFR expression levels were decreased in the presence of EGF in a dose dependent manner. Transcripts for LHR were detected at all maturation intervals and its expression patterns were not altered by supplementation with 10 ng/mL EGF. Expression of GJA5 was observed only after 48 h of IVM, and levels of expression were similar in oocytes supplemented with both 10 and 30 ng/mL EGF. In summary, our results indicate that supplementation of canine IVM medium with 10 ng/mL EGF had a positive influence on the progression of maturation to MII at 72 h. The effect may not be related to the alteration of mRNA expression of genes analyzed in the present study, due to the complex patterns regulating meiotic arrest in canine oocytes. This work was supported by Grant no. 204119-03-1-LG000 from ARPC, Republic of Korea.

249 EXPRESSION OF mRNA ENCODING EPIDERMAL GROWTH FACTOR-LIKE FACTORS IN BOVINE CUMULUS CELLS DURING IN VITRO MATURATION: EFFECTS OF TIME AND FOLLICLE-STIMULATING HORMONE

2011 ◽  
Vol 23 (1) ◽  
pp. 222
Author(s):  
E. S. Caixeta ◽  
M. F. Machado ◽  
P. Ripamonte ◽  
P. F. Lima ◽  
A. C. S. Castilho ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mrna Expression ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Mrna Abundance ◽  
Mrna Levels ◽  
Significant Difference ◽  
Epidermal Growth

Epidermal growth factor (EGF)-like family members [amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC)] have been shown to be important regulators of cumulus–oocyte complex (COC) maturation, particularly cumulus expansion. The aim of this study was to determine the temporal expression patterns of mRNA encoding EGF-like growth factors in bovine cumulus cells (CC) during COC in vitro maturation and to assess the effects of grading doses of FSH on EGF-like mRNA expression in CC. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries. In the first experiment, CC were separated from 20 COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 h with (10 ng mL–1) or without FSH. In the second experiment, pools containing 20 COC were matured for 12 h with grading doses of FSH (0, 0.1, 1, 10, and 100 ng mL–1). After culture, CC were mechanically separated and stored at –80°C. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed. Expression of target genes was assessed by real-time PCR and normalized by Cyclophilin (CYC-A). Relative quantification of mRNA abundance was determined by the Pfaffl equation. Effects of time of culture and FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer honestly significant difference test. Nonparametric analysis was used when data were not normally distributed. Differences were considered significant when P < 0.05. In the presence of FSH, AREG and EREG mRNA abundance was increased at 4 h of culture, whereas in the absence of FSH, AREG but not EREG mRNA levels were increased by 4 h of culture. The addition of FSH stimulated AREG mRNA expression from 4 to 16 h of culture. In contrast, BTC mRNA was more expressed in immature CC, decreased after 4 h of culture with FSH, and did not vary during maturation in the absence of FSH. In the dose–response experiment, AREG and EREG mRNA expression was stimulated by FSH starting from 10 ng mL–1 and did not increase from 10 ng mL–1 to 100 ng mL–1. Again in contrast, BTC mRNA expression was inhibited by FSH at 100 ng mL–1. In conclusion, the present data suggest that FSH differently regulates the expression of EGF-like factors during bovine COC maturation, although AREG and EREG are stimulated, BTC is inhibited by FSH. This work was supported by FAPESP.

Transcriptional down-regulation by epidermal growth factor of TRH receptor mRNA in rat pituitary cells

1995 ◽  
Vol 15 (1) ◽  
pp. 73-79 ◽  
Author(s):  
T Monden ◽  
M Yamada ◽  
S Konaka ◽  
T Satoh ◽  
H Ezawa ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mrna Level ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Dependent Manner ◽  
R Gene ◽  
Binding Studies ◽  
Epidermal Growth ◽  
Rat Pituitary Cells

ABSTRACT To gain insight into the mechanism underlying the epidermal growth factor (EGF)-induced changes in responsiveness to TRH and in the numbers of TRH receptors (TRH-Rs) in the pituitary, we investigated the transcriptional regulation by EGF of the TRH-R gene in GH4C1 cells. Northern blot analyses and binding studies revealed that EGF reduced both TRH binding and TRH-R mRNA levels in a dose- and time-dependent manner, while no significant changes were observed in β-actin mRNA levels. Addition of actinomycin D caused an acute increase in the basal TRH-R mRNA level, and the rate of decrease of the TRH-R mRNA was identical in control and EGF-treated groups, suggesting that the stability of the TRH-R mRNA was not significantly affected in EGF-treated cells. Incubation with cycloheximide also induced an increase in the basal TRH-R mRNA level and completely reversed the EGF-induced reduction of TRH-R mRNA levels. Furthermore, a nuclear run-on assay demonstrated that the rate of transcription of the TRH-R gene was significantly inhibited in cells treated with EGF. We conclude that (1) EGF decreases the expression of the TRH-R mRNA largely by reducing its rate of transcription, and this action requires the synthesis of new proteins, and (2) inhibitors of protein and RNA synthesis cause a significant increase in the basal TRH-R mRNA level, suggesting that there may be a short-lived protein suppressing the TRH-R mRNA level in the pituitary.

scholarly journals Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2375
Author(s):  
Dorota Gudanis ◽  
Damian Kaniowski ◽  
Katarzyna Kulik ◽  
Daniel Baranowski ◽  
Zofia Gdaniec ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Messenger Rna ◽  
Growth Factor Receptor ◽  
Living Cells ◽  
Cellular Systems ◽  
Mrna Levels ◽  
Epidermal Growth

Antisense DNA oligonucleotides, short interfering RNAs (siRNAs), and CRISPR/Cas9 genetic tools are the most useful therapeutic nucleic acids regulating gene expression based on the antisense specificity towards messenger RNA. Here, we present an effective novel strategy for inhibiting translation based on the antisense-controlled formation of an RNA quadruplex-duplex hybrid (QDH) between a G-rich RNA antisense oligoribonucleotide (Q-ASO) and specific mRNA, comprising two distant G-tracts. We selected epidermal growth factor receptor (EGFR) as a well-established target protein in anticancer therapy. The chemically modified, bi-functional anti-EGFR Q-ASO and a 56-nt long EGFR mRNA fragment, in the presence of potassium ions, were shown to form in vitro very stable parallel G-quadruplex containing a 28-nt long external loop folding to two duplex-stem structure. Besides, the Q-ASOs effectively reduced EGFR mRNA levels compared to the non-modified RNA and DNA antisense oligonucleotides (rASO, dASO). In addition, the hybridization specificity of Q-ASO comprising a covalently attached fluorescent tag was confirmed in living cells by visualization of the G4 green fluorescent species in the presence of other antisense inhibitors under competitive conditions. The results presented here offer novel insights into the potential application of Q-ASOs for the detection and/or alteration of (patho)biological processes through RNA:RNA quadruplex-duplex formation in cellular systems.

scholarly journals Role of FSH and epidermal growth factor (EGF) in the initiation of steroidogenesis in granulosa cells associated with follicular selection in chicken ovaries

Reproduction ◽  
2003 ◽  
pp. 683-691 ◽  
Author(s):  
AG Hernandez ◽  
JM Bahr
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Mrna Abundance ◽  
Dependent Manner ◽  
Preovulatory Follicle ◽  
Progesterone Secretion ◽  
Chain Cleavage ◽  
Epidermal Growth

In chicken ovaries, one small yellow follicle (SYF) is selected daily from a pool of follicles of similar size and becomes a preovulatory follicle. FSH induces follicular growth and steroidogenesis. Epidermal growth factor (EGF), an intraovarian hormone, suppresses granulosa cell differentiation. This study demonstrates that recruitment of SYFs into the hierarchy of preovulatory follicles is associated with a change in steroidogenic activity in granulosa cells regulated, at least in part, by FSH and EGF. Abundance of P450 side-chain cleavage (P450scc) mRNA was higher in the smallest preovulatory follicle (F6) compared with SYF, whereas FSH and EGF receptor (FSHr and EGFr, respectively) mRNA abundance was similar. FSH increased P450scc mRNA abundance and progesterone secretion and decreased FSHr mRNA in cultured granulosa cells, whereas EGF attenuated or suppressed P450scc mRNA and decreased FSHr mRNA abundance. None of the hormones influenced EGFr mRNA abundance. When used in combination, EGF attenuated or suppressed the stimulatory effect of FSH on the expression of P450scc mRNA and production of progesterone in a dose-dependent manner. The results indicate that (1) selection is associated with an increase in P450scc mRNA; (2) FSH stimulates expression of P450scc mRNA and progesterone secretion in granulosa cells of SYF; and (3) induction of P450scc mRNA and progesterone secretion by FSH is attenuated or blocked by EGF.

scholarly journals Epidermal growth factor increases the level of the cyclin-dependent kinase (CDK) inhibitor p21/CIP1 (CDK-interacting protein 1) in A431 cells by increasing the half-lives of the p21/CIP1 transcript and the p21/CIP1 protein

1999 ◽  
Vol 337 (3) ◽  
pp. 599-606 ◽  
Author(s):  
Lene E. JOHANNESSEN ◽  
Sigrun L. KNARDAL ◽  
Inger Helene MADSHUS
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Half Life ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
A431 Cells ◽  
Interacting Protein ◽  
Prolonged Incubation ◽  
Epidermal Growth

DNA synthesis was inhibited in A431 cells by epidermal growth factor (EGF) in a p21/CIP1-dependent manner [where CIP1 is cyclin-dependent kinase (CDK)-interacting protein 1]. When 1 or 10 nM EGF was added, the level of p21/CIP1 was increased to the same extent, and the protein level peaked after approx. 5 h of incubation. The increase in p21/CIP1 mRNA upon addition of EGF was rapid, and was enhanced in the presence of cycloheximide. The half-life of p21/CIP1 mRNA in EGF-treated A431 cells was increased approx. 2-fold; this is in contrast with the case in MCF-7 cells with normal p53, in which the half-life of p21/CIP1 mRNA was not increased upon addition of EGF. This increased stability accounts for most of the increase in mRNA levels observed in A431 cells during short incubation periods. Additionally, upon prolonged incubation of A431 cells with EGF, the half-life of the protein was also increased compared with that in untreated cells and in cells treated with EGF for short time periods. Nuclear run-on assays demonstrated only marginal stimulation of transcription by 10 or 1 nM EGF, or by 10 ng/ml tumour necrosis factor α. Our results indicate that the most important mechanisms by which EGF increases p21/CIP1 protein levels in A431 cells are post-transcriptional and post-translational stabilization.

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