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.

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.

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.

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.

Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 349-354 ◽  
Author(s):  
Yong-Hai Li ◽  
Rui-Hua Liu ◽  
Li-Hong Jiao ◽  
Wei-Hua Wang
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Early Embryo ◽  
Antral Follicles ◽  
Nuclear Maturation ◽  
17Β Estradiol ◽  
Epidermal Growth ◽  
Cytoplasmic Maturation ◽  
Pronuclear Formation

This study was conducted to examine the effect of epidermal growth factor (EGF) and 17β-estradiol (E2) on nuclear and cytoplasmic (male pronuclear formation and early embryo development) maturation of porcine oocytes. Oocytes were aspirated from antral follicles and cultured in modified TCM-199 medium supplemented with 0.57 mM cysteine, 10 IU/ml eCG, 10 IU/ml hCG, with or without EGF and/or E2. In vitro fertilisation of matured oocytes was performed in a modified Tris-buffered medium (mTBM) with frozen-thawed ejaculated spermatozoa. Oocytes were transferred to NCSU-23 supplemented with 0.4% bovine serum albumin at 6 h after in vitro fertilisation. Significantly higher (p < 0.05) rates of nuclear maturation, pronuclear formation and cleavage (91.7%, 65.2% and 37.3%, respectively) were observed when oocytes were cultured in the medium containing both EGF (10 ng/ml) and E2 (1 μg/ml) than in the medium supplemented with either EGF or E2 or without both. Intracellular glutathione concentration in the oocytes cultured in the medium containing both E2 and EGF was also significantly higher (12.1 pmol per oocyte) than that of oocytes cultured in the medium with E2 or EGF alone or without both. These findings suggested that EGF and E2 have a synergestic effect on both nuclear and cytoplasmic maturation of porcine oocytes.

scholarly journals Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

2014 ◽  
Vol 35 (2) ◽  
pp. 468-478 ◽  
Author(s):  
Tristan T. Eifler ◽  
Wei Shao ◽  
Koen Bartholomeeusen ◽  
Koh Fujinaga ◽  
Stefanie Jäger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Specificity Factor

Transcriptional cyclin-dependent kinases (CDKs) regulate RNA polymerase II initiation and elongation as well as cotranscriptional mRNA processing. In this report, we describe an important role for CDK12 in the epidermal growth factor (EGF)-induced c-FOS proto-oncogene expression in mammalian cells. This kinase was found in the exon junction complexes (EJC) together with SR proteins and was thus recruited to RNA polymerase II. In cells depleted of CDK12 or eukaryotic translation initiation factor 4A3 (eIF4A3) from the EJC, EGF induced fewer c-FOS transcripts. In these cells, phosphorylation of serines at position 2 in the C-terminal domain (CTD) of RNA polymerase II, as well as levels of cleavage-stimulating factor 64 (Cstf64) and 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF73), was reduced at the c-FOS gene. These effects impaired 3′ end processing of c-FOS transcripts. Mutant CDK12 proteins lacking their Arg-Ser-rich (RS) domain or just the RS domain alone acted as dominant negative proteins. Thus, CDK12 plays an important role in cotranscriptional processing of c-FOS transcripts.

scholarly journals Stimulation of ovarian follicle growth after AMPK inhibition

Reproduction ◽  
2017 ◽  
Vol 153 (5) ◽  
pp. 683-694 ◽  
Author(s):  
Xiaowei Lu ◽  
Song Guo ◽  
Yuan Cheng ◽  
Jae-hong Kim ◽  
Yi Feng ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Follicle Growth ◽  
Compound C ◽  
Old Mice ◽  
Stimulation Of

Previous studies showed that the protein kinase B (Akt)–mammalian target of rapamycin (mTOR) and Hippo signaling Yes-associated protein (YAP) pathways play important roles in promoting follicle growth. Additionally, other studies demonstrated that 5′ adenosine monophosphate-activated protein kinase (AMPK) is an upstream regulatory element of mTOR and YAP. Here, we used AMPK inhibitor (Compound C) toin vitrocultured ovaries from 10-day-old mice followed byin vivografting into adult hosts or toin situtreated ovaries of 3-week-old mice by intrabursal injection followed by gonadotropin stimulation. We found that the phosphorylation of ovarian mTOR and downstream proteins (ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4B (eIF4B)) was upregulated following Compound C administration, whereas tuberous sclerosis complex 2 (TSC2) phosphorylation was downregulated. Additionally, treatment with Compound C increased hypoxia-inducible factor 1-alpha (Hif1a), vascular endothelial growth factor A (Vegfa), VEGF receptor 2 (Vegfr2) and connective tissue growth factor (Ctgf) mRNA levels. Furthermore, treatment of 10-day-old mice with Compound C promoted the growth of preantral and antral follicles accompanied by enhanced angiogenesis.In situintrabursal injection with Compound C, followed by controlled ovarian hyperstimulation, increased the number of ovulated oocytes in 3-week-old mice, and these oocytes could be successfully fertilized, leading to the delivery of healthy pups. Our results demonstrated that treatment with AMPK inhibitor resulted in the activation of the mTOR signaling pathway, increases inCtgfexpression in mouse ovaries, stimulation of follicle development and promotion of ovarian angiogenesis for ovary growth.

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