BACKGROUND: During the last years, embryo development was studiedin vitroin different culture conditions and oxygen (O2) concentrations. Higher developmental blastocyst rates were obtained with embryos cultured under a physiological O2 tension (5%), respect to those cultured under atmospheric O2 conditions (20%) (Belli et al., 2020a). Still, the mechanisms responsible for this, during the preimplantation embryogenesis remain unclear, but mitochondria are believed to play an important role. This study aimed to evaluate the effect of physiologic or atmospheric O2 tension on mouse blastocyst’s ultrastructure. METHODS: In vivo, blastocyst were flushed out of the uterus after natural fertilization (controls). In vitro fertilization (IVF) was performed using KSOM medium and embryos were then cultured under an O2 tension of 5% and 20% for 5 days. After collection, blastocysts were subjected to the standard preparative for transmission electron microscopy (TEM) or used to assess mitochondrial function by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, and ATP levels (Belli et al., 2019; 2020b; Bianchi et al., 2015). RESULTS: In general, the trophoblast cells (TE) formed a single, continuous layer of flattened cuboidal cells. Both inner cell mass (ICM) and TE showed extensive regions of less dense, granular cytoplasm in all the groups. Microvilli were distributed on the apical surface, projecting toward the zona pellucida. Nuclei were delimited by integral nuclear membranes and contained dispersed euchromatin with patches of heterochromatin. Isolated mitochondria and vacuoles were numerous. Our results showed that in vitro culture under 5% and 20% O2 increased vacuolated mitochondria, cytoplasmic vacuolization, and the presence of multi-vesicular bodies. Also, blastocysts generated by IVF showed a lower heterochromatincontent, an increased density of residual bodies, and high levels of glycogen in the cytoplasm. Furthermore, IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, and lower ATP content. CONCLUSIONS: In conclusion, our data support the interpretation that pre-implantation embryos are vulnerable to the environmental disturbances determined by high O2tension, and therefore optimal O2 exposure or the addition of an antioxidant in extended culture may be a crucial factor for improved embryo development in vitro.

Mouse blastocyst cultured under different oxygen concentration showed altered ultrastructure

Manuel Belli;Maria Grazia Palmerini;Serena Bianchi;Guido Macchiarelli
2021

Abstract

BACKGROUND: During the last years, embryo development was studiedin vitroin different culture conditions and oxygen (O2) concentrations. Higher developmental blastocyst rates were obtained with embryos cultured under a physiological O2 tension (5%), respect to those cultured under atmospheric O2 conditions (20%) (Belli et al., 2020a). Still, the mechanisms responsible for this, during the preimplantation embryogenesis remain unclear, but mitochondria are believed to play an important role. This study aimed to evaluate the effect of physiologic or atmospheric O2 tension on mouse blastocyst’s ultrastructure. METHODS: In vivo, blastocyst were flushed out of the uterus after natural fertilization (controls). In vitro fertilization (IVF) was performed using KSOM medium and embryos were then cultured under an O2 tension of 5% and 20% for 5 days. After collection, blastocysts were subjected to the standard preparative for transmission electron microscopy (TEM) or used to assess mitochondrial function by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, and ATP levels (Belli et al., 2019; 2020b; Bianchi et al., 2015). RESULTS: In general, the trophoblast cells (TE) formed a single, continuous layer of flattened cuboidal cells. Both inner cell mass (ICM) and TE showed extensive regions of less dense, granular cytoplasm in all the groups. Microvilli were distributed on the apical surface, projecting toward the zona pellucida. Nuclei were delimited by integral nuclear membranes and contained dispersed euchromatin with patches of heterochromatin. Isolated mitochondria and vacuoles were numerous. Our results showed that in vitro culture under 5% and 20% O2 increased vacuolated mitochondria, cytoplasmic vacuolization, and the presence of multi-vesicular bodies. Also, blastocysts generated by IVF showed a lower heterochromatincontent, an increased density of residual bodies, and high levels of glycogen in the cytoplasm. Furthermore, IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, and lower ATP content. CONCLUSIONS: In conclusion, our data support the interpretation that pre-implantation embryos are vulnerable to the environmental disturbances determined by high O2tension, and therefore optimal O2 exposure or the addition of an antioxidant in extended culture may be a crucial factor for improved embryo development in vitro.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11697/170331
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