Arginine increases development of in vitro-produced porcine embryos and affects the protein arginine methyltransferase–dimethylarginine dimethylaminohydrolase–nitric oxide axis

Abstract

Culture systems promote development at rates lower than the in vivo environment. Here, we evaluated the embryo’s transcriptome to determine what the embryo needs during development. A previous mRNA sequencing endeavor found SLC7A1, an arginine transporter, to be up-regulated in in vitro compared to in vivo cultured embryos. We added different concentrations of arginine to our culture medium to meet the needs of the embryo. Increasing arginine from 0.12 to 1.69 mM improved the number of embryos that developed to the blastocyst stage. These blastocysts also had more total nuclei compared to controls and specifically, more trophectoderm nuclei. Embryos cultured in 1.69 mM arginine had lower SLC7A1 levels and had higher abundance of messages involved with glycolysis (HK1, HK2, and GPT2) and decreased expression of genes involved with blocking the TCA cycle (PDK1) as well as the pentose phosphate pathway (TALDO1). PRMT1, PRMT3 and PRMT5 expression throughout development was not affected by arginine. DDAH1 and DDAH2 message was found to be differentially regulated through development and DDAH2 protein was localized to the nuclei of blastocysts. Arginine has a positive effect on preimplantation development and may be affecting the NO-DDAH-PRMT axis.

Pharmacologic treatment of donor cells induced to have a Warburg effect‐like metabolism does not alter embryonic development in vitro or survival during early gestation when used in somatic cell nuclear transfer in pigs

“…After electric pulse fusion, fused embryos were fully activated with 200 μM thimerosal for 10 min in the dark and 8 mM dithiothreitol for 30 min. Embryos were then incubated in our in house culture media MU1 (Redel, Tessanne, Spate, Murphy, & Prather, ) with a histone deacetylase inhibitor 0.5 μM Scriptaid, for 14–16 hr in the normal (atmospheric 20%) oxygen incubator. The next day, the SCNT embryos were moved into new MU1 culture media (without Scriptaid) and placed in a low (5%) oxygen incubator.…”

Glycine supplementation in vitro enhances porcine preimplantation embryo cell number and decreases apoptosis but does not lead to live births

“…For example, high rates of loss occur for many mammalian embryos, with up to 80% of in vitro‐matured and inseminated oocytes failing to progress to the blastocyst stage (Watson et al, ). Recent adaptations have been made to culture systems that improve embryo development and subsequent in vivo development (Bauer et al, ,; Spate et al, ; Lee et al, ; Redel et al, ; Spate et al, ), but these improvements have not kept up with the demands associated with the increasing use of nuclear transfer and genetic editing technology to produce animal models for human clinical applications, such as for xenotransplantation and cystic fibrosis (Lai et al, ; Rogers et al, ), and to protect agricultural animal production by creating pigs resistant to the porcine reproductive and respiratory syndrome virus (Whitworth et al, ).…”

Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs

“…The embryos used in this experiment were cultured in an arginine optimized pyruvate, lactate, glutamine based PZM3 medium, MU1 (Redel et al 2015; Yoshioka et al 2002) and cultured in MU2 (Spate et al 2015). Embryos that formed morula and blastocysts on day 5 of culture were transferred to the recipient gilts.…”

“…Zygotes were cultured in MU2 (MU1 supplemented with the phosphopeptide mimetic that triggers PDK1 phosphorylation, PS48 (5 ng/ml) (Stemgent, Inc, Cambridge, MA) until embryo transfer. MU1 and MU2 have been described previously (Redel et al 2015) (Spate et al 2015). The embryos, 60 into one recipient and 75 into a second recipient, were surgically transferred into the ampullary-isthmic junction of the oviduct of the surrogate (Lee et al 2013).…”