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 10 mM GlutaMAX, an L-glutamine alternative, with or without 0.2 mM 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 0 mM GlutaMAX resulted in decreased blastocyst development with (26.2 ± 4.8%) or without (23.2 ± 3.0%) leucine. Embryos cultured in 3.75 mM GlutaMAX had increased development to the blastocyst stage (+Leu: 49.7 ± 4.2%; −Leu: 46.2 ± 2.6%) compared with culture in 1 mM (+Leu: 39.5 ± 3.5%; −Leu: 37.8 ± 2.1%). In the presence of leucine, culture in 0 mM GlutaMAX resulted in decreased pMTOR abundance compared with 3.75 or 10 mM GlutaMAX. In the absence of leucine, embryos cultured in 0 mM GlutaMAX had decreased abundance of total MTOR compared with all other groups, and pMTOR abundance was decreased in embryos cultured in 0 or 1 mM compared with 3.75 or 10 mM GlutaMAX. Furthermore, embryos cultured in 0 mM GlutaMAX had decreased colocalization of MTOR and LAMP1 compared with those cultured in 3.75 or 10 mM 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.