Abstract
L-ascorbic acid (Vitamin C) can enhance the meiotic maturation and developmental competence of porcine oocytes, but the underlying molecular mechanism remains obscure. Here we show the role of ascorbic acid in regulating epigenetic status of both nucleic acids and chromatin to promote oocyte maturation and development in pigs. Supplementation of 250 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P) during in vitro maturation significantly enhanced the nuclear maturation (as indicated by higher rate of first polar body extrusion and increased Bmp15 mRNA level), reduced level of reactive oxygen species, and promoted developmental potency (higher cleavage and blastocyst rates of parthenotes, and decreased Bax and Caspase3 mRNA levels in blastocysts) of pig oocytes. AA2P treatment caused methylation erasure in mature oocytes on nucleic acids (5-methylcytosine (5 mC) and N6-methyladenosine (m6A)) and histones (Histone H3 trimethylations at lysines 27, H3K27me3), but establishment of histone H3 trimethylations at lysines 4 (H3K4me3) and 36 (H3K36me3). During the global methylation reprogramming process, levels of TET2 (mRNA and protein) and Dnmt3b (mRNA) were significantly elevated, but simultaneously DNMT3A (mRNA and protein), and also Hif-1α, Hif-2α, Tet3, Mettl14, Kdm5b and Eed (mRNA) were significantly inhibited. Our findings support that ascorbic acid can reprogram the methylation status of not only DNA and histone, but also RNA, to improve pig oocyte maturation and developmental competence.
Highlights
L-ascorbic acid (Vitamin C), a water-soluble antioxidant and electron donor, can be synthesized in the liver of many species, except for guinea pigs, human and other primates[1]
2-phosphate sesquimagnesium salt hydrate (AA2P) into the in vitro maturation (IVM) system to culture pig cumulus-oocyte complexes (COCs) for 44 h showed that the rate of the first polar body (PB1) extrusion was significantly higher in 250 μM ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P) group (84.8%, n = 430) in comparison to the control, 100 μM, 500 μM and 750 μM groups (Fig. 1A and B)
Ascorbic acid has been reported to play an important role in many biological processes, through acting as an electron donor, suppressing oxidative stress, and regulating epigenetic modifications at DNA and histone levels[3]
Summary
L-ascorbic acid (Vitamin C), a water-soluble antioxidant and electron donor, can be synthesized in the liver of many species, except for guinea pigs, human and other primates[1]. Studies confirmed that ascorbic acid regulates Tet, JmjC domain containing enzymes and HIF hydroxylases, to modulate dynamically the epigenetic status of DNA/histone methylation and HIF-1α activity[6,9,12,13,14]. Mammalian oocyte development is coordinated by a complex molecular network, and dynamic epigenetic methylation regulation on DNA and histones is crucial for both oocyte meiosis[15,16] and early embryo development[17,18,19]. We aimed to understand how ascorbic acid improves the maturation and developmental competence of porcine oocytes enclosed with cumulus cells, with a special focus on epigenetic regulation. We showed that through regulating global epigenetic modifications at DNA, RNA and histone levels, supplementation of ascorbic acid during in vitro maturation can benefit the meiotic maturation and subsequent development of pig oocytes
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