Relative expression of mRNAs related to cavitation process in bovine embryos produced in vivo and in vitro

The objectives of this work were to identify and to evaluate possible differences on gene expression of aquaporins and Na/K-ATPases transcripts between embryos in vivo and in vitro produced. For each group, 15 blastocysts distributed in three pools were used for RNA extraction followed by amplification and reverse transcription. The resulting cDNAs were submitted to Real-Time PCR, using the GAPDH gene as endogenous control. It was not possible to identify AQP1 transcripts. Relative expression of AQP3 (1.33 ± 0.78) and AQP11 (2.00 ± 1.42) were not different in blastocysts in vitro and in vivo produced. Na/K-ATPase α1 gene (2.25 ± 1.07) was overregulated whereas Na/K-ATPase β2 transcripts 0.40 ± 0.30) did not differ among blastocysts produced in vitro from those produced in vivo. Transcripts for gene AQP1 are not present in bovine blastocysts. In vitro culture system does not alter expression of genes AQP3, AQP11 and Na/K-ATPase β2 genes, however, it affects expression of Na/K-ATPase α1.Os objetivos neste trabalho foram identificar e avaliar possíveis diferenças na expressão gênica de transcritos de Aquaporina e ATPases-Na/K presentes em embriões produzidos in vivo e in vitro. Para cada grupo, 15 blastocistos distribuídos em três conjuntos foram utilizados para a extração do RNA, seguida da amplificação e da transcrição reversa. Os DNAs complementares foram submetidos à reação em cadeia da enzima polimerase em tempo real, utilizando-se o gene GAPDH como controle endógeno. Não foi possível identificar transcritos de AQP1. A expressão relativa dos genes AQP3 (1,33 ± 0,78) e AQP11 (2,00 ± 1,42) não foi diferente em blastocistos produzidos in vitro e in vivo. O gene ATPase-Na/K α1 (2,25 ± 1,07) encontrou-se sobrerregulado, enquanto o gene ATPase-Na/K β2 (0,40 ± 0,30) não diferiu entre os blastocistos produzidos in vitro e aqueles produzidos in vivo. Transcritos para o gene AQP1 não estão presentes em blastocistos bovinos. O sistema de cultivo in vitro não influencia a expressão dos genes AQP3, AQP11 e ATPase-Na/K β2, porém altera a expressão do gene ATPase-Na/K α1

180 Glucose versus fatty acids: Different energy supplies for the bovine oocyte

Studies show that optimal concentrations of fatty acids (FA) and glucose are crucial for proper oocyte development, maturation, and further embryo quality support (Sutton McDowall et al. 2010 Reproduction 139, 685-95, https://doi.org/10.1530/REP-09-0345; Sutton McDowall et al. 2014 Theriogenology 82, 95-103, https://doi.org/10.1016/j.theriogenology.2014.03.011). A balance between metabolism of FA and glucose in oocytes needs to be maintained; however, it is not clear what the preferable pathway of energy production is and how it affects the oocyte. The aim of the experiment was to selectively block pathways of either glucose or FA metabolism during IVM of bovine oocytes to reveal changes within oocyte lipid droplets (LD) under crucial changes in energy metabolism. Cumulus-oocyte complexes were matured under standard conditions (Warzych et al. 2007Mol. Reprod. Dev. 74, 280-289; https://doi.org/10.1002/mrd.20610) without FA and glucose supplementation. The experimental groups were (1) control (IVM in basic medium), (2) group with inhibited glucose metabolism [supplementation with 1.5 µM iodoacetate (IO, inhibitor of glycolysis) and 150 µM dehydroepiandrosterone (DHEA, inhibitor of pentose phosphate pathway)], and (3) group with inhibited FA metabolism (150 µM etomoxir supplementation, ETO). Oocytes after 24h of IVM were stained with boron-dipyrromethene (BODIPY) 493/503 dye (lipid droplets) and 4′,6-diamidino-2-phenylindole (DAPI; chromatin) and analysed using a confocal microscope (LSM 880 AiryScan FAST; Zeiss). Obtained data were analysed using the Kruskal-Wallis test. The MII rate decreased to 60.5% for IO+DHEA (P&amp;lt;0.05) and to 78% for the ETO group compared with the control (83.8%). The average LD area (% of total oocyte area) significantly decreased in both experimental groups (IO+DHEA 6.72±2.5, ETO 6.28±3.2; P&amp;lt;0.01) compared with control (8.7±3.6). Total lipid content (intensity of the fluorescence), was significantly lower in experimental groups (IO+DHEA: 1.17×106±3×105, ETO: 7.1×104±2×104 vs. control: 1.68×106±4.6×105; P&amp;lt;0.01). With regard to the total number of LD, only in the IO+DHEA group were significantly fewer LD noted (987±343; P&amp;lt;0.05), whereas the ETO group did not differ significantly (1146±414) compared with control (1148±357). The ETO group had significantly lower total lipid content (P&amp;lt;0.01) and higher total LD number (P&amp;lt;0.05) compared with IO+DHEA. The area of lipid droplets did not differ between the experimental groups. These results show that blocking glucose metabolism strongly affects the nuclear maturation process of the oocyte, limiting the number of oocytes that reach the MII stage. A significant decrease in total lipid content in the ETO group may suggest strong utilisation of lipids during the maturation process, when de novo synthesis of lipids is blocked. Accumulation of lipids in LD before maturation allows this process to be passed even under the applied FA metabolism inhibitory conditions. In contrast, inhibition of glucose metabolism negatively affects oocyte development strictly after inhibition conditions, indicating the greater significance of glucose for proper oocyte maturation. Further studies are being conducted to investigate the effect of inhibitory systems on lipids in bovine cumulus cells and further embryos. Funding for this study was provided by National Science Centre, Poland (project no 2017/27/B/NZ9/00904). </jats:p

Growth hormone exerts no effect on the timing of the first zygotic cleavage in cattle

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