Differential Gene Expression Profile in Bovine Blastocysts Resulting from Hyperglycemia Exposure During Early Cleavage Stages1

Abstract

To understand the compromised survival of embryos derived from assisted reproductive techniques, transcriptome survey of early embryonic development has shown the impact of in vitro culture environment on gene expression in bovine or other living species. However, how the differentially expressed genes translate into developmentally compromised embryos is unresolved. We therefore aimed to characterize transcriptomic markers expressed by bovine blastocysts cultured in conditions that are known to impair embryo development. As increasing glucose concentrations has been shown to be stressful for early cleavage stages of mammalian embryos and to decrease subsequent blastocyst survival, in vitro-matured/fertilized bovine zygotes were cultured in control (0.2 mM) or high-glucose (5 mM) conditions until the 8- to 16-cell stage, and then transferred to control media until they reached the blastocyst stage. The concentration of 5 mM glucose was chosen as a stress treatment because there was a significant effect on blastocyst rate without the treatment's being lethal as with 10 mM. Microarray analysis revealed gene expression differences unrelated to embryo sex or hatching. Overrepresented processes among differentially expressed genes in treated blastocysts were extracellular matrix signalling, calcium signaling, and energy metabolism. On a pathophysiological level, higher glucose treatment impacts pathways associated with diabetes and tumorigenesis through genes controlling the Warburg effect, i.e., emphasis on use of anaerobic glycolysis rather than oxidative phosphorylation. These results allowed us to conclude that disruption of in vitro preattachment development is concomitant with gene expression modifications involved in metabolic control.

Metabolic stress induces modifications in the epigenetic program of preimplantation bovine embryos

“…DNA methylation at sub‐telomeric regions was negatively correlated with telomere length and telomere recombination frequencies (Vera, Canela, Fraga, Esteller, & Blasco, ), suggesting that the sub‐telomeric hypomethylation observed in glucose‐treated bovine embryos mimics a cancer‐like methylation status that promotes rapid DNA replication. This hypothesis is consistent with our previous report that embryos challenged by high glucose concentration adapt to their environment through the activation of Warburg metabolism, which is often present in cancer cells (Cagnone et al, ).…”

Metabolic stress induces modifications in the epigenetic program of preimplantation bovine embryos

“…DNA methylation at sub‐telomeric regions was negatively correlated with telomere length and telomere recombination frequencies (Vera, Canela, Fraga, Esteller, & Blasco, ), suggesting that the sub‐telomeric hypomethylation observed in glucose‐treated bovine embryos mimics a cancer‐like methylation status that promotes rapid DNA replication. This hypothesis is consistent with our previous report that embryos challenged by high glucose concentration adapt to their environment through the activation of Warburg metabolism, which is often present in cancer cells (Cagnone et al, ).…”

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism

“…LDHA is a gene coding for the main enzyme of anaerobic conversion of pyruvate into lactate, which produces NAD+ and stimulates glycolysis. Similar to our results, it has been reported that hyperglycaemia in bovine embryos increases LDHA expression, 14 suggesting that enhanced expression of metabolic genes results in an ‘un‐quiet’ embryo that is less viable 13 …”

“…LDHA is a gene coding for the main enzyme of anaerobic conversion of pyruvate into lactate, which produces NAD+ and stimulates glycolysis. Similar to our results, it has been reported that hyperglycaemia in bovine embryos increases LDHA expression, 14 suggesting that enhanced expression of metabolic genes results in an 'un-quiet' embryo that is less viable. 13 The mitochondrial constitution seems to be critical to ensure proper developmental competence, as in vitro embryo production negatively affects mitochondrial number and function in murine and bovine embryos.…”

Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos

“…This result was in agreement with previous investigations that female embryo have higher glucose consumption than male embryos. Higher utilization of glucose ultimately stimulates the glycolysis, which disrupts the oxidative phosphorylation by altering the mitochondrial membrane potential and subsequently leads to the activation of apoptosis by the overproduction of ROS [22]. Our results also revealed that X-BLs have a significantly higher cell apoptotic ratio and caspase-3 expression than Y-BL group (Figure 4B,C).…”

Metabolic stress induces modifications in the epigenetic program of preimplantation bovine embryos

“…DNA methylation at sub‐telomeric regions was negatively correlated with telomere length and telomere recombination frequencies (Vera, Canela, Fraga, Esteller, & Blasco, ), suggesting that the sub‐telomeric hypomethylation observed in glucose‐treated bovine embryos mimics a cancer‐like methylation status that promotes rapid DNA replication. This hypothesis is consistent with our previous report that embryos challenged by high glucose concentration adapt to their environment through the activation of Warburg metabolism, which is often present in cancer cells (Cagnone et al, ).…”

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism

“…LDHA is a gene coding for the main enzyme of anaerobic conversion of pyruvate into lactate, which produces NAD+ and stimulates glycolysis. Similar to our results, it has been reported that hyperglycaemia in bovine embryos increases LDHA expression, 14 suggesting that enhanced expression of metabolic genes results in an ‘un‐quiet’ embryo that is less viable 13 …”

“…LDHA is a gene coding for the main enzyme of anaerobic conversion of pyruvate into lactate, which produces NAD+ and stimulates glycolysis. Similar to our results, it has been reported that hyperglycaemia in bovine embryos increases LDHA expression, 14 suggesting that enhanced expression of metabolic genes results in an 'un-quiet' embryo that is less viable. 13 The mitochondrial constitution seems to be critical to ensure proper developmental competence, as in vitro embryo production negatively affects mitochondrial number and function in murine and bovine embryos.…”

Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos

“…This result was in agreement with previous investigations that female embryo have higher glucose consumption than male embryos. Higher utilization of glucose ultimately stimulates the glycolysis, which disrupts the oxidative phosphorylation by altering the mitochondrial membrane potential and subsequently leads to the activation of apoptosis by the overproduction of ROS [22]. Our results also revealed that X-BLs have a significantly higher cell apoptotic ratio and caspase-3 expression than Y-BL group (Figure 4B,C).…”

Metabolic stress induces modifications in the epigenetic program of preimplantation bovine embryos

“…DNA methylation at sub‐telomeric regions was negatively correlated with telomere length and telomere recombination frequencies (Vera, Canela, Fraga, Esteller, & Blasco, ), suggesting that the sub‐telomeric hypomethylation observed in glucose‐treated bovine embryos mimics a cancer‐like methylation status that promotes rapid DNA replication. This hypothesis is consistent with our previous report that embryos challenged by high glucose concentration adapt to their environment through the activation of Warburg metabolism, which is often present in cancer cells (Cagnone et al, ).…”

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism

“…LDHA is a gene coding for the main enzyme of anaerobic conversion of pyruvate into lactate, which produces NAD+ and stimulates glycolysis. Similar to our results, it has been reported that hyperglycaemia in bovine embryos increases LDHA expression, 14 suggesting that enhanced expression of metabolic genes results in an ‘un‐quiet’ embryo that is less viable 13 …”

“…LDHA is a gene coding for the main enzyme of anaerobic conversion of pyruvate into lactate, which produces NAD+ and stimulates glycolysis. Similar to our results, it has been reported that hyperglycaemia in bovine embryos increases LDHA expression, 14 suggesting that enhanced expression of metabolic genes results in an 'un-quiet' embryo that is less viable. 13 The mitochondrial constitution seems to be critical to ensure proper developmental competence, as in vitro embryo production negatively affects mitochondrial number and function in murine and bovine embryos.…”

Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos

“…This result was in agreement with previous investigations that female embryo have higher glucose consumption than male embryos. Higher utilization of glucose ultimately stimulates the glycolysis, which disrupts the oxidative phosphorylation by altering the mitochondrial membrane potential and subsequently leads to the activation of apoptosis by the overproduction of ROS [22]. Our results also revealed that X-BLs have a significantly higher cell apoptotic ratio and caspase-3 expression than Y-BL group (Figure 4B,C).…”