2 results
Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone
- Gustavo Bruno Mota, Ingrid Oliveira e Silva, Danielle Kaiser de Souza, Flavia Tuany, Michele Munk Pereira, Luiz Sergio de Almeida Camargo, Alzira Amélia Martins Rosa e Silva
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The aim of this study was to evaluate the dose–response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus–oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus–oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P < 0.05) than the DCM control. Insulin concentrations of 1, 10, or 100 ng/ml decreased the relative levels of GDF9 and HSP70-1 transcripts in oocytes at the end of IVM (P < 0.05). The transcripts levels of PRDX1 decreased (P < 0.05) only when 10 or 100 ng/ml insulin was added to the DCM medium. No difference in levels of GLUT1 transcripts (P > 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.
Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue
- Gustavo Bruno Mota, Ribrio Ivan Tavares Pereira Batista, Raquel Varella Serapião, Mariana Cortes Boité, João Henrique Moreira Viana, Ciro Alexandre Alves Torres, Luiz Sergio de Almeida Camargo
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The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus–oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 °C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB− (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB− group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB− (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB− (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB− groups. Despite the relative expression of MATER in holding control, BCB+ and BCB− were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.