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ISSN 2052-1472 (online)

Reproduction Abstracts (2014) 1 P058 | DOI: 10.1530/repabs.1.P058

O-GlcNAcylation in pig embryos during preimplantation development

Mihiro Shibutani, Takashi Miyano & Masashi Miyake

Kobe University, Kobe, Japan.

Protein modification with O-linked β-N-acetylglucosamine (O-GlcNAcylation) is essential for eukaryotic cells. There are many reports concerned with O-GlcNAcylateion, but little is known about it in preimplantation development. The objective of this study was to examine the presence of O-GlcNAcylation and the role of O-GlcNAc cycling in pig preimplantation development using parthenogenetic diploids. In-vitro matured oocytes were subjected to an electro-stimulation and cytochalacin B treatment to obtain parthenogenetic diploids. Diploids were cultured to 144 h after electro-stimulation. At first, mRNA expression of glutamine-fructose-6-phosphate transaminase (GFPT) that is the rate limiting enzyme for production of UDP-GlcNAc (substrate for O-GlcNAcylation), O-GlcNAc transferase (OGT) that catalyzes O-GlcNAcylation of proteins, and O-GlcNAcase (OGA) that removes O-GlcNAc from O-GlcNAcylated proteins was examined in diploids at various stages using RT-PCR. Secondly, O-GlcNAcylated proteins were detected by immunostaining using anti-O-GlcNAc antibodies (CTD110.6 and RL2). Finally, effect of inhibition of O-GlcNAc cycling on the preimplantation development was examined. Diploids were cultured for 144 h in PZM3 containing an OGA inhibitor, PUGNAc (0-300 μM) and observed every 24 h. GFPT and OGT mRNA were detected in diploids from 2-cell to expanded blastocyst stage. OGA mRNA was also observed in diploids at all preimplantation stages, but not 4-cell. O-GlcNAcylated proteins were detected throughout preimplantation development. Furthermore, almost all diploids cultured with 300 μM PUGNAc did not develop beyond the 4-cell stage. These results suggest that O-GlcNAcylation and O-GlcNAc cycling are present in preimplantation development and inhibition of OGA results in developmental arrest of embryos at the 4-cell stage in pigs.

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