96 EFFECT OF MOF GENE ON PREIMPLANTATION DEVELOPMENT OF PIG PARTHENOGENETIC EMBRYOS

Abstract

The Mof gene (males absent on the first) is crucial to X-chromosome dosage compensation in the fly. It acts specifically to catalyse acetylation of histone H4 lysine 16 (H4K16ac) as one histone acetyltransferase of the MYST family, playing essential roles during mammalian development. However, little is known about Mof gene in pigs. The present study was designed to explore effects of Mof on pre-implantation development of pig parthenogenetic embryos obtained as reported by Cao et al. (2012 Zygote 20, 229–236). Immunofluorescent staining was performed to examine protein expression level of porcine Mof (pMof) and H4K16ac, and fluorescent intensity was measured by Image J software (NIH, Bethesda, MD, USA). Data are presented as mean ± standard error, and statistical analyses of the fluorescent intensity value, embryo development rate, and quality were performed using ANOVA with SPSS software (version 15.0, SPSS Inc., Chicago, IL, USA), and P-value <0.05 was considered significant. First, the coding sequence (CDS) of the pMof gene was cloned and the spatio-temporal expression patterns of the CDS were determined in pig oocytes, early embryos, and other tissues. A 1471-bp-long cDNA of pMof was obtained with 99.34% and 98.25% amino acid sequence homology and 92.88% and 88.96% nucleotide sequence homology to the human and mouse MOF homologues. We observed that pMof is expressed predominantly in oocytes and early embryos but at low levels in sperm and other organs. We found that pMof decreased from pronuclear to 8-cell stages and remained low until the blastocyst stage based on RT-qPCR results (n = 10 for each stage embryos). In contrast, pMof protein expression as examined by immunofluorescent staining (n = 15 for each stage embryos) remained high throughout the pre-implantation development period. After porcine embryonic genome activation, pMOF remained detectable in 4-cell (n = 10) and 8-cell (n = 10) embryos despite amanitin treatment for 24 h. Thus, the mRNA level of MOF was not decreased after transcription inhibition suggesting that MOF is a maternal gene. To assess functional significance, we examined the expression of H4K16ac, a target of pMof, and found that the level of H4K16ac was constantly low from pronuclear to morula stage, but increased dramatically in blastocysts. When we knocked down pMOF by cytoplasmic injection of siRNA into porcine MII oocytes, rate and cell number of blastocysts declined significantly [blastocyst rate: uninjected (n = 228) v. negative-siRNA (n = 220) v. MOF-siRNA (n = 230) = 65.8 ± 3.75% v. 57.5 ± 4.30% v. 46.3 ± 5.72%; cell numbers: uninjected v. negative-siRNA v. MOF-siRNA = 84.73 ± 5.25 v. 77 ± 5.50 v. 55.08 ± 6.56]. A marker for DNA double-strand breaks and repair, γ-H2AX, increased in parallel to more apoptotic cells. Knockdown of MOF reduced H4K16ac. Overall, pMof is highly conserved among human, mouse, and pig; pMof is essential to pre-implantational development of pig parthenogenetic embryos involved in regulating H4K16ac.

Research was supported by NSFC (31272442).