69 Phosphorylation of mechanistic target of rapamycin in porcine blastocyst-stage embryos is dependent on the concentration of glutamine in the medium

Abstract

Supplementation of glutamine to porcine embryo culture medium improves blastocyst development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the phosphorylation and activation of mechanistic target of rapamycin (MTOR) to support rapid cellular proliferation. The objective of this study was to determine if phosphorylation of MTOR in porcine blastocysts was dependent upon the concentration of glutamine in the medium and presence of leucine, another activator of MTOR. Presumptive zygotes (n=732 per treatment across 6 replicates) were split into four groups and cultured in 0, 1, 3.75, or 10mM GlutaMAX, an L-glutamine alternative, with or without 0.2mM leucine. On Day 6, percentages of embryos developed to the blastocyst stage were recorded. Blastocyst-stage embryos (n=100 per treatment) were collected for immunoblotting to detect total MTOR and phosphorylated (Ser2448) MTOR (pMTOR) with 3 replicates per target. To assess MTOR and lysosomal colocalization as another indicator of MTOR activation, blastocyst-stage embryos (n=15 per treatment) were fixed, probed with antibodies against MTOR and lysosomal associated membrane protein 1 (LAMP1), and imaged on a confocal microscope. Data were analysed by using the GLM procedure in SAS 9.4 (SAS Institute Inc.), and differences between means were detected by using one-way ANOVA followed by a least significant difference test with P<0.05 declared significant. Compared with the other treatments, culture in 0mM GlutaMAX resulted in decreased blastocyst development with (26.2±4.8%) or without (23.2±3.0%) leucine. Embryos cultured in 3.75mM GlutaMAX had increased development to the blastocyst stage (+Leu: 49.7±4.2%; −Leu: 46.2±2.6%) compared with culture in 1mM (+Leu: 39.5±3.5%; −Leu: 37.8±2.1%). In the presence of leucine, culture in 0mM GlutaMAX resulted in decreased pMTOR abundance compared with 3.75 or 10mM GlutaMAX. In the absence of leucine, embryos cultured in 0mM GlutaMAX had decreased abundance of total MTOR compared with all other groups, and pMTOR abundance was decreased in embryos cultured in 0 or 1mM compared with 3.75 or 10mM GlutaMAX. Furthermore, embryos cultured in 0mM GlutaMAX had decreased colocalization of MTOR and LAMP1 compared with those cultured in 3.75 or 10mM GlutaMAX. Therefore, glutamine supplementation is sufficient to prompt MTOR phosphorylation in porcine embryos. Further analyses are examining the phosphorylation status of MTOR downstream targets and effects of inhibiting enzymes involved in glutaminolysis. Funding was provided by the United States Department of Agriculture, National Institute of Food and Agriculture (2019-67011-29543) and Food for the 21st Century at the University of Missouri.