| Because of closing to human on genetics,physiology and anatomy, pig is considered to be an ideal animal material with broad applications in basic research of medicine and biology, creation of disease models and donor organs for human xenotransplantation, and exploration of mechanisms of reproduction and development Porcine embryo production and manipulation acts as a key step to realize the above mentioned potential benefits of pigs. However, effciency of porcine embryonic technology is still very low, which is generally due to our insufficient understanding of the mechanisms underlying preimplantation embryonic development. MOF (males absent on the first) is crucial to dosage compensation in fly, and acts specifically to catalyze acetylation of histone H4 lysine 16 (H4K16ac) as one histone acetyltransferase of MYST family, playing essential roles during mammalian development. However, little is known about MOF gene in porcines so far. Therefore, in order to evaluate the impact of MOF on preimplantation development of porcine embryos, we attempted firstly to clone MOF gene and explore its spatio-temporal expression patterns in porcine oocytes, early parthenogenetic embryos and other tissues, and to investigate the function in pig embryonic develoment by RNAi technology.Experiment Ⅰ aimed to clone and analyse MOF sequence. Porcine MOF (pMOF) of 1471bp long was obtained and submited to GenBank. We found that pMOF located in the third autosome, and the length of its CDS is 1377bp coding 458 animo acids; As for amino acid sequence, pMOF showed 99.34% and 98.25% homology to human and mouse, and for nucleotide sequence pMOF dispayed 92.88% and 88.96% similiarity to human and mouse homologous.Experiment Ⅱ was designed to explore the expression pattern of MOF in porcine organs or tissues. Results of quatitative real-time PCR (qPCR) showed that pMOF is expressed dominantly in oocytes, but at low levels in sperm and other organs such as heart, liver, spleen, lung, kidney, muscle and brain.Experiment Ⅲ aimed to explore spatio-temporal expression patterns of MOF and H4K16ac during the embryo development. We found that mRNA abundance of pMOF was decreasing from pronuclear stage to 8-cell stage and thereafter still keeping at low level to blastocyst stage, based on qPCR results; but according to immunostaining results, pMOF protein remained highly expressed during the whole preimplantation development period. For the expression of H4K16ac, a target of pMOF, we found that the level of H4K16ac was constantly low from pronuclear to morula stage, but became quite high in blastocysts.Experiment IV was to investigate whether the pMOF was a maternal gene or not in pigs. After porcine embryonic genemo activation, we detected the expression level of pMOF after 4-cell and 8-cell were treated with Amanitin for 24h.The results dispalyed that the mRNA level of pMOF was not decrease after the inhibition of transcription. Then we supposed that pMOF was a maternal gene.Experiment V was to evaluate the impact of MOF on preimplantation development of porcine embryos. We finally tried to knockdown pMOF by cytoplasmic injection of siRNA into porcine MII oocytes, and the results demonstrated that the rate and the quality of blastocysts were all declined significantly in test group, as the total cell number decreased, the rate of y-H2AX modification, closely related to DNA double-strand break and repair, increased and blastomere severely underwent apoptosis. Reduction of development competency of embryos after pMOF knockdown was shown to be closely associated with decreasing expression of H4K16ac.In all, cDNA of pMOF was obtained, and the spatio-temporal expression patterns of MOF and H4K16ac during the embryo development was revealed for the first time; we proposed that pMOF is essential to preimplantational development of porcine parthenogenetic embryos by regulating H4K16ac. |
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