145 CHARACTERIZATION OF THE PROTEIN ARGININE METHYLTRANSFERASE-DIMETHYLARGININE DIMETHYLAMINOHYDROLASE-NITRIC OXIDE AXIS DURING PORCINE EMBRYO DEVELOPMENT

Abstract

Transcriptional deep sequencing analysis by Bauer et al. (2010) revealed a significant increase in expression of the arginine transporter SLC7A1 in in vitro–cultured porcine blastocysts compared with those cultured in vivo and this was corrected through supplemental arginine. This indicates an important role for arginine during porcine embryo development. Arginine is the precursor for nitric oxide (NO) production and previous work in mice and cattle has shown decreased development when embryos were cultured with a nitric oxide synthase (NOS) inhibitor. The NOS activity is inhibited by monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) that are released during degradation of proteins methylated by protein arginine methyltransferases (PRMT). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for degrading MMA and ADMA in the cell. Therefore, the goal of this study was to investigate whether this PRMT-DDAH-NO axis exists in pre-implantation porcine embryos. To this end, expression of PRMT1, PRMT3, PRMT5, DDAH1 and endothelial NOS (NOS3) was analysed at different stages of embryonic development using real-time quantitative RT-PCR. In addition, the effect of supplemental arginine (1.69 mM) on the expression of the aforementioned genes was investigated. Production of NO in porcine embryos was also visualised using 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM-DA). In vitro–fertilized porcine embryos were collected at the 4-cell and blastocyst stages. The RNA was isolated from pools of 18 to 20 embryos and cDNA, was synthesised using Superscript III (Invitrogen, Carlsbad, CA, USA). Real-time PCR analysis was performed and the mean fold change in gene expression from the reference gene YWHAG was analysed by t-test after a log transformation. Expression of PRMT3 and PRMT5 was significantly higher (P < 0.05) in blastocysts versus 4-cell embryos. Expression of PRMT1, however, was higher in 4-cell embryos (P < 0.05). The expression of DDAH1 was detected in 4-cell embryos, but DDAH1 became undetectable by the blastocyst stage. Previous microarray analysis in our laboratory by Whitworth et al. (2005 Biol. Reprod. 72(6), 1437–1451) also revealed a significant up-regulation of DDAH2 expression at the 4-cell stage versus blastocysts. Expression of NOS3 was undetectable in the 4-cell and blastocyst; however, NO was detected in 4-cell and blastocyst stage embryos by using DAF-FM-DA. This suggests that a different NOS may be acting in the porcine embryo. Addition of arginine did not have a significant effect on expression of the analysed genes. These results suggest that PRMT-DDAH regulated NO production may play a role during porcine embryo development. Understanding the PRMT-DDAH-NO axis and its regulation during embryonic development will further our ability to tailor in vitro culture so that it more appropriately mimics that of an in vivo environment.

Funding was provided by NIH U42 RR18877.