ABSTRACT
The objective of this study was to investigate the effects of inhibiting the epidermal growth factor receptor (EGFR) pathway on meiosis blockage and resumption, mRNA expression of genes involved in oocyte maturation and cumulus expansion, and embryo development. Bovine cumulus-oocyte complexes (COCs) were cultured for 15 h in the presence of the EGFR inhibitor (AG1478) and follicular hemisections (FHS). Most of the oocytes (89.3%) remained at the germinal vesicle (GV) stage when cultured in the presence of FHS and 5 µM AG1478. The inhibitory effect was reversible as most oocytes (83.8%) completed meiosis after additional 20 h maturation. Embryo development to the blastocyst stage was similar (P > 0.05) between FHS and 5 µM AG1478 treated (39.3%) and control (41.1%) groups. In cumulus cells, mRNA abundance of early growth response protein 1 (EGR1), tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) genes, and phosphorylated extracellular regulated kinase (p-ERK1/2) protein were lower in COCs treated with AG1478 plus FHS compared with FHS alone (P < 0.05). In granulosa cells of FHS, AG1478 treatment reduced transcript levels of PGR and ADAMTS1 (P < 0.05). The inhibitory effect of AG1478 on meiotic progression was not reverted by treatment with angiotensin II (ANG II) or prostaglandins (PGF2α or PGE2). This study demonstrates that inhibition of EGFR in the presence of FHS is a reliable approach to promote reversible arrest of bovine oocytes at the GV stage.
Subject(s)
ErbB Receptors/antagonists & inhibitors , Gene Expression Regulation/drug effects , In Vitro Oocyte Maturation Techniques/veterinary , Oocytes/drug effects , Ovarian Follicle , Quinazolines/pharmacology , Tyrphostins/pharmacology , Angiotensin II/pharmacology , Animals , Cattle , Cell Cycle Checkpoints/drug effects , Cumulus Cells , Dinoprostone/pharmacology , Embryo Culture Techniques/methods , Embryo Culture Techniques/veterinary , Female , Follicle Stimulating Hormone/pharmacology , Granulosa Cells , In Vitro Oocyte Maturation Techniques/methods , Meiosis/drug effects , Oocytes/physiology , Quinazolines/administration & dosage , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/physiology , Tyrphostins/administration & dosageABSTRACT
The Grb14 protein is a member of the Grb7 protein family. This protein family acts by binding to tyrosine kinase receptors, promoting cell proliferation and differentiation. There is evidence of the involvement of tyrosine kinase factors in the bovine oocyte maturation process. However, Grb14 has not been studied for bovine cumulus-oocyte complexes (COCs). The aim of the present study was to characterize Grb14 mRNA expression in bovine COCs during follicular development. Furthermore, we demonstrated that the expression of Grb14 mRNA is not regulated by estradiol. mRNA expression of Grb14 was assessed in 480 COCs from follicles of different sizes (1-3, 4-6, 6-8 or >8 mm) by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Grb14 mRNA expression decreased in COCs throughout follicular growth (P < 0.05). The role of estradiol in the expression of Grb14 mRNA in COCs was studied. Grb14 mRNA abundance did not differ in COCs cultured in the presence or absence of 17ß-estradiol or fulvestrant. In conclusion, we showed that Grb14 mRNA is downregulated in COCs during antral follicle development, a finding that suggests a role for Grb14 in oocyte competence.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cattle , Cumulus Cells/metabolism , Oocytes/cytology , Oocytes/metabolism , Ovarian Follicle/metabolism , Animals , Cell Proliferation/drug effects , Cells, Cultured , Cumulus Cells/cytology , Cumulus Cells/drug effects , Estradiol/pharmacology , Estrogens/pharmacology , Female , Oocytes/drug effects , Oogenesis/drug effects , Oogenesis/physiology , Ovarian Follicle/cytology , Ovarian Follicle/drug effects , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.
