Effects of follicle stimulating hormone (FSH) and amphiregulin (AREG) on meiosis dynamics and embryo production of bovine oocytes cultured in vitro

In vivo, the maturation of the cumulus oocyte complex (COC) is triggered by the ovulatory peak of LH, which induces germinal vesicle breakdown (GVBD) and progression of nuclear maturation to metaphase II (MII), in vitro FSH provides this process. The LH surge induces the secretion of epidermal growth factor (EGF)-like peptides, amphiregulin (AREG), epiregulin (EREG) and betacelulin (BTC) that bind to EGFR leading to Cx43 phosphorylation through the MAPK/ERK pathway closuring of gap junctions communications prevents the transfer of cGMP and cAMP from cumulus cells to the oocyte. The decrease of cGMP levels augments the activity of phosphodiesterase 3 (PDE3) on cAMP, which is hydrolyzed and in low concentrations fails to inhibit via phosphorylation the maturation promoting factor (MPF), thus leading to meiosis resumption. This study aimed to compare meiosis dynamics in oocytes cultured with FSH or AREG and embryo production, testing the hypothesis that the direct stimulation with AREG speeds up nuclear maturation and improves blastocyst rates. The COCs were aspirated from bovine ovaries obtained at a slaughterhouse, and submitted to IVM for 20, 22 and 24 hours in TCM 199 containing Earle’s salts, L-glutamine, NaHCO3, supplemented with BSA (0.4%), amikacin (75µg/mL), pyruvate (22µg/mL) and 10-1 UI/ml of r-hFSH or 100ƞg/mL of AREG. At the end of the IVM, oocytes were fixed and stained with Hoechst 33342 to assess meiotic stage, or submitted to IVF after 24 hours of cultured. Data were transformed to arcsine and groups compared by the Student t test, considering values of P<0.05 as significant. AREG promoted progression to MII at equivalent rates from 20 to 24 hours of culture (75.35%; 72.06% and 77.41% respectively), whereas FSH promoted MII at a lower rate at 20 hours (24.57%) increasing at 22 and 24 hours (64.52% and 65.76% respectively), presenting statistical difference between AREG and FSH after 20 hours of culture (P=0,0198). Embryo production after 24 hours of maturation with AREG or FSH promoted equivalent blastocyst rates (31% vs 31.33% respectively). The present data demonstrated that AREG was able to progress meiosis and more rapidly than FSH however, when tested on embryo production AREG and FSH group were similar. Although, no differences were observed on embryo production, the achievement of an in vitro system able to mimic physiological mechanisms has an important role on research related to oocyte physiology.