RESUMO
Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.
Assuntos
Núcleo Celular/enzimologia , Metilação de DNA , Ectogênese , Epigênese Genética , Peroxirredoxinas/metabolismo , Zigoto/enzimologia , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Metilação de DNA/efeitos dos fármacos , Ectogênese/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Feminino , Fertilização in vitro , Peróxido de Hidrogênio/toxicidade , Masculino , Camundongos Endogâmicos ICR , Microscopia Confocal , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Zigoto/citologia , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimentoRESUMO
Single embryo culture is useful for assessing the developmental competence of an embryo in detail. Recently, a device made of poly(dimethylsiloxane) (PDMS), which is biocompatible and nontoxic, has been widely used for culture various types of cells. However, PDMS plates are porous, causing the serious osmolality increment of the medium (over 600 mOsm/kg from Day 4 to Day 7). Here, we report that curing the PDMS under low pressure (LP-PDMS) greatly reduced the porosity, resulting in a constant osmolality of the medium. The blastocyst rate of single bovine embryos cultured with LP-PDMS microwell (MW) plates was the same as that of group-cultured embryos (25 embryos/50 µl droplet; control, P>0.05). These results indicate that MWs on a plate made of PDMS cured under low pressure can be successfully used for individual embryo culture.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Resistência a Medicamentos , Etoposídeo/farmacologia , Feminino , Humanos , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Distribuição TecidualRESUMO
Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.
Assuntos
Fertilização in vitro/métodos , Animais , Animais Geneticamente Modificados , Criopreservação , Feminino , Fertilidade , Lasers , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos , Espermatozoides , Zona PelúcidaRESUMO
Linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Delta12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Delta12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were approximately 10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained approximately 20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases.