Autophagy Influences Maternal mRNA Degradation and Apoptosis in Porcine Parthenotes Developing <i>In Vitro</i>

Abstract

Abstract. Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig. Key words: Apoptosis, Autophagy, LC3, Maternal gene, 3-MA, Rapamycin (J. Reprod. Dev. 58: [576][577][578][579][580][581][582][583][584] 2012) A utophagy is an intracellular, bulk degradation process in which a portion of the cytoplasm is sequestered in an autophagosome and subsequently degraded upon fusion with a lysosome [1][2][3]. Autophagy plays a critical role during fertilization [4] and an essential role in differentiation and development, as well as in the cellular response to stress. Our previous study was the first to report that mitochondrial stress influences autophagy in porcine parthenotes developing in vitro [5]. However, the role of autophagy in early porcine parthenotes is still poorly understood.A balance between the formation and degradation of cellular proteins is required for cell survival. In mammals, protein degradation is accelerated shortly after fertilization and is apparent by the early two-cell stage in mice [6] and the four-cell stage in pigs. Early embryogenesis may rely on maternal protein stores as nutrients. After fertilization, maternal proteins in oocytes are...

Toxic effects of HT-2 toxin on mouse oocytes and its possible mechanisms

“…This was used as a template to evaluate DNMTs mRNA expression by qRT-PCR. White bar control; light gray bar oocytes cultured in HT-2 toxin (color figure online) parthenotes (Xu et al 2012) that develop in vitro. However, another study also showed that low concentration of rapamycin induced autophagy during in vitro maturation contributes to enhancing the nuclear and cytoplasmic maturation of porcine oocytes (Song et al 2014).…”

Efectos de los RNAm maternos sobre la maduración del ovocito y el desarrollo embrionario temprano en mamíferos

“…Las proteínas DICER y AGO2 son de origen materno y actúan en el procesamiento de los microRNAs (51) . Ovocitos prior to the activation of the embryonic genome (120) . The work of Xu et al (120) does not indicate whether this process of autophagy acts after the action of the miRNAs, only shows that by adding autophagy inhibitor 3-metiladedina to early embryonic cultures, the expression of maternal mRNA as c-mos, gdf9 and bmp15 remains still in embryonic stages of 4 cells.…”

“…Ovocitos prior to the activation of the embryonic genome (120) . The work of Xu et al (120) does not indicate whether this process of autophagy acts after the action of the miRNAs, only shows that by adding autophagy inhibitor 3-metiladedina to early embryonic cultures, the expression of maternal mRNA as c-mos, gdf9 and bmp15 remains still in embryonic stages of 4 cells. It would be interesting to find out whether autophagocityc vacuoles are comprised with the maternal mRNA debris.…”

“…ATG5, es una proteína materna, relacionada con los procesos de degradación por autofagia en los lisosomas; embriones derivados de ovocitos de ratones atg5-/-no pasan más allá del estadio de 4 a 8 células (119) . Esto coincide con lo encontrado en el modelo porcino, donde se demostró que el proceso de degradación de RNAm maternos está relacionado con el proceso de autofagia en embriones tempranos previo a la activación del genoma embrionario (120) . El trabajo de Xu et al (120) no indica si este proceso de autofagia actúa después de la acción de los miRNAs, sólo muestra que al adicionar el inhibidor de autophagia 3-metiladedina a cultivos embrionarios tempranos, la expresión de RNAm maternos como c-mos, gdf9 y bmp15 permanece aún en estadios embrionarios de 4 células.…”

“…Esto coincide con lo encontrado en el modelo porcino, donde se demostró que el proceso de degradación de RNAm maternos está relacionado con el proceso de autofagia en embriones tempranos previo a la activación del genoma embrionario (120) . El trabajo de Xu et al (120) no indica si este proceso de autofagia actúa después de la acción de los miRNAs, sólo muestra que al adicionar el inhibidor de autophagia 3-metiladedina a cultivos embrionarios tempranos, la expresión de RNAm maternos como c-mos, gdf9 y bmp15 permanece aún en estadios embrionarios de 4 células. Sería interesante dilucidar si las vacuolas autofagocíticas se conforman con los detritos de RNAm maternos.…”