Melatonin reduces apoptotic cells, SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts.

Effects of adding different concentrations of melatonin (10-7 , 10-9 and 10-11  M) to maturation (Experiment 1; Control, IVM + 10-7 , IVM + 10-9 , IVM + 10-11 ) and culture media (Experiment 2; Control, IVC + 10-7 , IVC + 10-9 , IVC + 10-11 ) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10-9  M) from Experiments 1-2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM + 10-9 , IVC + 10-9 , IVM/IVC + 10-9 ). In Experiment 1, maturated oocytes from Control and IVM + 10-9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC + 10-7 (43.5%; 56.7%) and IVC + 10-9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC + 10-9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC + 10-9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM/IVC + 10-9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC + 10-9 treatment also had a higher mRNA expression of antioxidant gene (SOD2) compared to the Control, as well as the heat shock protein (HSPB1) compared to the IVM + 10-9 . Reactive oxygen species production was greater in the IVM/IVC + 10-9 treatment group. In conclusion, the 10-9  M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.

Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production.