189 Effects of epidermal growth factor and progesterone on oocyte meiotic resumption and expression of maturation-related transcripts in bovine oocytes from medium-size antral follicles

2020 ◽  
Vol 32 (2) ◽  
pp. 223
Author(s):  
L. G. Barrozo ◽  
F. T. G. Bezerra ◽  
L. R. F. M. Paulino ◽  
A. W. B. Silva ◽  
J. R. V. Silva
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mrna Expression ◽  
Cyclin B1 ◽  
Medium Size ◽  
Mrna Levels ◽  
Control Medium ◽  
Oocyte Growth ◽  
Antral Follicles ◽  
Epidermal Growth

The aims of this study were to evaluate the effects of epidermal growth factor (EGF) and progesterone (P4) on maturation and expression transcripts for GDF9, CCNB1, H1FOO, cMOS, PARN, and eIF4E after prematuration of cumulus-oocyte complexes (COCs) from antral follicles. Bovine COCs (3-6mm) were aspirated and pre-matured for 20h in control medium [TCM-199 containing 5.0mgmL−1 LH, 0.5mgmL−1 FSH, 0.4% bovine serum albumin, cilostamide (10μM) and follicular hemisections] alone or supplemented with EGF (10ngmL−1), P4 (100 µM), or both EGF (10ngmL−1) and P4 (100 µM). After that, COCs were matured for 24h in the same medium, without EGF, P4, cilostamide, and follicular hemisections. Oocyte diameters were evaluated with the software Nis Elements (Nikon Instruments Inc.). To evaluate meiotic progression, the oocytes were fixed in 4% paraformaldehyde and transferred to 0.5% Triton X-100. The chromatin configuration during meiosis was assessed by 10μgmL−1 bisbenzimide (Hoechst 33342) and analysed under an epi-fluorescent inverted microscope (DMI4000B; Leica). Oocytes were classified according to the nuclear maturation stage as germinal vesicle, metaphase I, anaphase I, telophase I, and metaphase II. To evaluate mRNA expression, oocytes were stored in micr-centrifuge tubes at −80°C until RNA extraction. RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, mRNA for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH (housekeeping gene) was quantified by real-time PCR and analysed by Kruskal-Wallis test. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test (P<0.05). The results showed that prematuration of COCs in the presence of P4 and both EGF and P4 promoted an increase in oocyte diameter compared with the control or EGF treatment alone. The presence of cilostamide inhibited early meiotic resumption, benefiting oocyte capacitation, but the presence of EGF, P4, or EGF and P4 together in the prematuration medium did not influence meiosis resumption rates. The presence of EGF or P4 in prematuration medium increased the mRNA levels for cMOS in oocytes (P<0.05). The H1FOO mRNA levels in oocytes cultured with EGF and P4 increased significantly compared with oocytes cultured in EGF alone (P<0.05). In contrast, mRNA levels for cyclin B1 in oocytes cultured with P4 were higher than those cultured in the presence of EGF alone (P<0.05). In addition, levels of mRNA for eIF4E showed a significant reduction in oocytes cultured with P4 compared with those pre-matured with EGF or both EGF and P4. The EGF treatment reduced the levels of mRNA for GDF9 compared with control medium. The mRNA levels of PARN did not differ significantly between treatments. In conclusion, EGF, P4, and EGF and P4 combined did not influence oocyte growth and meiotic resumption. However, EGF or P4 increased the mRNA expression of cMOS, whereas EGF reduced the levels of transcripts for GDF9.

183 Effects of epidermal growth factor and progesterone on invitro oocyte growth, meiotic resumption, and expression of maturation-related transcripts in oocytes from bovine small antral follicles

2020 ◽  
Vol 32 (2) ◽  
pp. 219
Author(s):  
J. R. V. Silva ◽  
F. T. G. Bezerra ◽  
L. R. F. M. Paulino ◽  
B. R. Silva ◽  
A. W. B. Silva
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Bovine Serum ◽  
Cyclin B1 ◽  
Oocyte Diameter ◽  
Mrna Levels ◽  
Control Medium ◽  
Oocyte Growth ◽  
Antral Follicles ◽  
Epidermal Growth

Oocytes from small antral follicles are not able to undergo nuclear maturation because they still need to accumulate mRNA and proteins. We hypothesised that invitro growth of cumulus-oocyte complexes (COCs) of these follicles in the presence of epidermal growth factor (EGF) and progesterone (P4) increase the levels of transcripts and have a positive impact on maturation. This study aimed to evaluate the effects of EGF and P4 on growth, maturation, and expression of mRNAs for growth and differentiation factor 9 (GDF9), cyclin B1, oocyte-specific linker histone (H1FOO), quinase cMOS, poly(A) ribonuclease (PARN), and initiation factor 4E (eIF4E) after growth and prematuration of COCs. Bovine COCs (n=400) were aspirated from antral follicles (1-3mm) and cultured for 48h in control medium (TCM-199 plus 5.0mgmL−1 LH, 0.5mgmL−1 FSH, and 5% fetal bovine serum) alone or with EGF (10ngmL−1), P4 (100 µM), or both EGF and P4. After growth, the COCs were pre-matured for 20h in TCM-199 containing 5.0mgmL−1 LH, 0.5mgmL−1 FSH, 0.4% bovine serum albumin, and 10μM cilostamide. Then, COCs were matured invitro in the same medium used for prematuration, but without cilostamide. Oocyte diameter and meiotic progression were evaluated after growth, prematuration, and maturation periods. After prematuration, oocytes were stored until RNA extraction. Total RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, quantification of mRNAs for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH was performed by real-time PCR. The delta-delta-cycle threshold (ΔΔCT) method was used to normalise the data. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test. Differences in expression of mRNAs were analysed by Kruskal-Wallis test (P<0.05). The results showed that, compared with time 0, an increase in oocyte diameter was observed after 48h of culture for COCs in all groups (P<0.05), except for those cultured in control medium alone or with only P4. However, when compared with control medium, no effects of EGF, P4, or both were seen after the growth and prematuration periods. After prematuration, a higher percentages of oocytes at the germinal vesicle stage was observed for COCs cultured with P4 compared with those cultured in TCM-199 (P<0.05). After IVM, the rate of meiosis resumption was significantly reduced in oocytes cultured with P4 (P<0.05). The levels of mRNA for cMOS, H1FOO, and cyclin B1 in oocytes cultured with P4 were higher than those cultured in the control medium (P<0.05). The mRNA levels for eIF4E in oocytes cultured with both EGF and P4 were increased (P<0.05) compared with those cultured with EGF. The levels of transcripts for PARN in oocytes cultured with both EGF and P4 were higher than those in COCs cultured with only P4. In contrast, mRNA levels of GDF9 did not show differences between treatments. In conclusion, P4 inhibited oocyte meiotic resumption and increased mRNA levels for cMOS, H1FOO, and cyclin B1 in COCs after culture and prematuration invitro.

Effects of epidermal growth factor and progesterone on oocyte meiotic resumption and the expression of maturation-related transcripts during prematuration of oocytes from small and medium-sized bovine antral follicles

2020 ◽  
Vol 32 (14) ◽  
pp. 1190
Author(s):  
Francisco Taiã G. Bezerra ◽  
Laís R. F. M. Paulino ◽  
Bianca R. Silva ◽  
Anderson W. B. Silva ◽  
Ana L. P. Souza Batista ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cyclin B1 ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Oocyte Diameter ◽  
Mrna Levels ◽  
Eukaryotic Translation ◽  
Antral Follicles ◽  
Epidermal Growth

This study evaluated the effects of epidermal growth factor (EGF) and progesterone (P4) on growth, the resumption of meiosis and expression of eukaryotic translation initiation factor 4E(eIF4E), poly(A)-specific ribonuclease (PARN), oocyte-specific histone H1 (H1FOO), oocyte maturation factor Mos (cMOS), growth differentiation factor-9 (GDF9) and cyclin B1 (CCNB1) mRNA in oocytes from small and medium-sized antral follicles after prematuration and maturation invitro. Oocytes from small (<2.0mm) and medium (3.0–6.0mm) antral follicles were cultured in medium containing EGF (10ng mL–1), P4 (100 µM) or both. After culture, growth rate, resumption of meiosis and eIF4E, PARN, H1FOO, cMOS, GDF9 and CCNB1 mRNA levels were evaluated. P4 increased cMOS, H1FOO and CCNB1 mRNA levels after the culture of oocytes from small antral follicles, and EGF increased CCNB1 mRNA levels in these oocytes. In the medium-sized antral follicles, P4 alone or in combination with EGF increased oocyte diameter after prematuration invitro. In these oocytes, the presence of either EGF or P4 in the culture medium increased cMOS mRNA levels. In conclusion, P4 increases cMOS, H1FOO and CCNB1 mRNA levels after the culture of oocytes from small antral follicles. P4 and the combination of EGF and P4 promote the growth of oocytes from medium-sized antral follicles, and both EGF and P4 increase cMOS mRNA levels.

SRC-homology 2 domain-containing phosphatase 2 (SHP2) in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9540-9540
Author(s):  
Rafael Rosell ◽  
Masaoki Ito ◽  
Jordi Codony-Servat ◽  
Ana Giménez-Capitán ◽  
Mireia Serra-Mitjans ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mrna Expression ◽  
Mrna Levels ◽  
Wild Type ◽  
Src Homology 2 ◽  
Src Homology ◽  
Epidermal Growth ◽  
Src Homology 2 Domain ◽  
Egfr Mutant

9540 Background: Epidermal growth factor (EGFR)-mutant lung adenocarcinomas (LUADs) display impaired phosphorylation of extracellular signal-regulated kinase (ERK) and SRC-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. However, the function of SHP2 in early EGFR-mutant LUADs and EGFR wild-type LUADs has not been reported. We posit that SHP2 mRNA expression could be a predictive marker in resected EGFR-mutant LUADs versus EGFR wild-type patients (pts). Methods: We examined 267 resected LUADs from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1 and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction (PCR). EGFR mutant cell lines were investigated for their activity of SHP2. Results: Among the 267 enrolled pts, 100 (37.3%) were EGFR-mutant LUADs. Five-year recurrence-free survival (RFS) and overall survival (OS) were lower for EGFR-mutant LUADs with high SHP2 mRNA levels (hazard ratio = 1.83 and 2.28, respectively. p = 0.03 and p = 0.04). However, SHP2 was not associated with RFS nor OS in the 167 wild-type EGFR LUADs. In EGFR-mutant cells, RMC-4550 (SHP2 inhibitor) plus erlotinib showed synergism via inhibition of AKT (S473) and ERK1/2 (T202/Y204). While erlotinib translocates SHP2 (Y542) into the nucleus, either RMC-4550 alone, or in combination with erlotinib, relocalizes SHP2 into the cytoplasm membrane, limiting AKT and ERK activation. Conclusions: High SHP2 mRNA is related to shorter RFS and OS in EGFR-mutant LUADs, but not in EGFR wild-type LUADs. The findings indicate that the addition of SHP2 inhibitors could improve adjuvant therapy in EGFR-mutant LUADs.

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.

scholarly journals Growth factor gene expression in kidney of murine polycystic kidney disease.

1993 ◽  
Vol 3 (7) ◽  
pp. 1378-1386
Author(s):  
T Nakamura ◽  
I Ebihara ◽  
I Nagaoka ◽  
Y Tomino ◽  
S Nagao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Kidney Disease ◽  
Epidermal Growth Factor ◽  
Mrna Expression ◽  
Mrna Levels ◽  
Cystic Lesions ◽  
Tgf Beta ◽  
Polycystic Kidney ◽  
Igf I ◽  
Epidermal Growth

The DBA/2FG-pcy mouse has a form of slowly progressive kidney disease that appears similar in many respects to that seen in the autosomal dominant form of human polycystic kidney disease. The aim of this study was to examine the mRNA expression of growth-related proteins in kidney obtained from DBA/2FG-pcy mice and control DBA/2 mice at 8, 16, and 30 wk of age. The mRNA levels encoding for proliferating cell nuclear antigen (PCNA), transforming growth factor (TGF)-beta, platelet-derived growth factor (PDGF)-A and PDGF-B chains, insulin-like growth factor (IGF)-I, and basic fibroblast growth factor (bFGF) were increased with the progression of cystic lesions in the kidneys of DBA/2FG-pcy mice. At 30 wk of age, mRNA levels of PCNA, TGF-beta, PDGF-A and PDGF-B chains, IGF-I, and bFGF were increased 5.4-fold, 4.8-fold, 4.4-fold, 3.8-fold, 3.7-fold, and 4.6-fold, respectively, compared with those of control DBA/2 mice. In contrast, mRNA levels for epidermal growth factor in kidney of DBA/2FG-pcy mice decreased with age as compared with those of DBA/2 mice. These results suggest that decreased epidermal growth factor mRNA expression and increased expression of PCNA, TGF-beta, PDGF-A and PDGF-B chains, IGF-I, and bFGF mRNA may contribute to the progression of cystic lesions in DBA/2FG-pcy mice.

Emergence of salivary gland cell lineage diversity suggests a role for androgen-independent epidermal growth factor receptor signaling

1995 ◽  
Vol 108 (6) ◽  
pp. 2205-2212
Author(s):  
E.M. Durban ◽  
P.G. Nagpala ◽  
P.D. Barreto ◽  
E. Durban
Keyword(s):  
Salivary Gland ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Cell Lineage ◽  
Mrna Levels ◽  
Receptor Signaling ◽  
Receptor Mrna ◽  
Epidermal Growth ◽  
Lineage Diversity

Diversity of cell lineages within glandular organs is generated postnatally by differentiation of committed progenitor cells. Fundamental regulatory aspects of this process are not understood. The mouse submandibular salivary gland (SSG) served as model to assess the role of epidermal growth factor (EGF) receptor signaling during emergence of cell lineage diversity. Temporal fluctuations in EGF receptor mRNA levels coincident with crucial differentiative cell lineage transitions were revealed by RNase protection analyses. Between days 2 and 5, when proacinar cells are maturing and striated duct cells emerge, EGF receptor mRNA levels were highest and all differentiating cells exhibited EGF receptor immunoreactivity. EGF receptor mRNA levels then declined sharply and immunoreactivity became confined to ductal cells. At day 11 in male mice, and days 11 and 16 in females, a second increase in EGF receptor mRNA was detected coincident with emergence of granular convoluted tubule (GCT) cells. With completion of androgen-dependent GCT cell differentiation at the onset of puberty, EGF receptor mRNA levels and intensity of immunoreactivity decreased. Androgen effects on EGF receptor mRNA or immunoreactivity could not be detected. These temporally distinct patterns of EGF receptor expression suggest that this signaling pathway is a mechanism of potential importance in emergence of cell lineage diversity in a glandular organ.

scholarly journals Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis

1999 ◽  
Vol 277 (4) ◽  
pp. L684-L693 ◽  
Author(s):  
Christine L. Zanella ◽  
Cynthia R. Timblin ◽  
Andrew Cummins ◽  
Michael Jung ◽  
Jonathan Goldberg ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Direct Interaction ◽  
Mrna Levels ◽  
Binding Studies ◽  
Therapeutic Implications ◽  
Asbestos Fibers ◽  
Epidermal Growth

We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c- fos/c- jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR ( P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c- fos but not of c- jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c- fos and the development of apoptosis. The ability to block asbestos-induced elevations in c- fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.

Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model

2002 ◽  
Vol 282 (1) ◽  
pp. G156-G164 ◽  
Author(s):  
Bohuslav Dvorak ◽  
Melissa D. Halpern ◽  
Hana Holubec ◽  
Catherine S. Williams ◽  
Debra L. McWilliam ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mrna Expression ◽  
Necrotizing Enterocolitis ◽  
Rat Model ◽  
Transforming Growth Factor ◽  
Gastrointestinal Disease ◽  
Neonatal Rat ◽  
Epidermal Growth ◽  
Neonatal Rat Model

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of prematurely born infants. Maternal milk plays an important protective role against NEC development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to examine the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. Newborn rats were artificially fed either with growth factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/ml of EGF (RMS+EGF). Experimental NEC was induced by exposure to asphyxia and cold stress. Development of NEC was evaluated by gross and histological scoring of damage in the ileum. Ileal EGF receptor (EGF-R), EGF, and transforming growth factor-α mRNA expression was assessed by RT competitive-PCR, and the EGF-R was localized by immunohistochemistry. EGF supplementation of formula reduced the incidence and severity of NEC in rats (13/16 RMS vs. 4/13 RMS+EGF). Ileal EGF-R mRNA expression was markedly increased in the RMS group compared with RMS+EGF. Enhanced EGF-R expression in the RMS group was localized predominantly in the epithelial cells of injured ileum. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC.

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