Influencia de la producción in vitro de embriones bovinos sobre marcas epigenéticas y expresión de genes

Abstract

RESUMEN: El uso de las tecnologías de reproducción asistida (TRAs) en la moderna producción bovina, es una importante herramienta para mejorar los niveles de producción en leche y carne. Una de las TRAs frecuentemente utilizada en bovinos es la producción in vitro de embriones. Sin embargo, los embriones producidos in vitro defieren de aquellos producidos in vivo en muchos aspectos, incluyendo el desarrollo competente. En este trabajo se evaluó la influencia de la producción in vitro de embriones sobre los perfiles de metilación del DNA y expresión génica de genes relacionados con fenómenos epigenéticos en el desarrollo embrionario temprano en bovinos Bos indicus. El presente estudio analizó la metilación del DNA en dos secuencias satélites, la secuencia satélite BovineTestis I (BTS) y Bos taurus satelite alfa I (BTSαI) y la abundancia relativa de transcriptos relacionados con metilación del DNA (DNMT1, DNMT3A), impronta (IGF2, IGF2R) y la pluripotencia (POU5F1) en embriones producidos in vivo e in vitro. No hubo diferencia estadística significativa en los niveles de expresión de los genes DNMT3A, IGF2R y POU5F1 entre los embriones producidos in vivo vs los producidos in vitro. Sin embargo los resultados evidenciaron diferencias entre los embriones producidos in vivo vs los producidos in vitro, con reducciones significativas (p< 0.05) en los niveles de transcripción de los genes DNMT1, IGF2 en condiciones de cultivo in vitro.

ABSTRACT: The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in several aspects, including developmental competence. This study investigated the effects of in vitro embryo production on the profiles of DNA methylation and gene expression of epigenetics related genes in early embryo development in Bos indicus. Here, we analyzed the DNA methylation status in two satellite sequences, i.e. ‘bovine testis satellite I’ (BTS) and ‘Bostaurus alpha satellite I’ (BTαS), and the relative abundance of transcripts related to DNA methylation (DNMT1 and DNMT3A), imprinting (IGF2 and IGF2R) and pluripotency (POU5F1) in embryos produced in vitro and in vivo. Relative transcriptabundance for DNMT3A, IGF2R and POU5F1 was not significantly different between blastocysts produced in vivo vs in vitro. However, our results evidence that differences continue to be found between in vitro cultured and in vivo embryos, as the transcript levels of DNMT1 and IGF2 were significantly reduced (p <0.05) by the in vitro culture conditions. The ability of bovine embryos to develop to the blastocyst stage, to implant and generate healthy offspring depends greatly on the competence of the oocyte.Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesize and store substantial amounts of mRNA. Higher developmental competence of bovine oocytes has been associated with both the expression of a cohort of developmental genes, and the concentration of sex steroids in the follicular fluid. We studied differences in the expression of FST in cumulus cells and OCT-4 and MATER in oocytes and the influence of the follicular P4 and E2 concentration on the competence of bovine oocytes retrieved 30 minutes (Group I) or 4 hours (Group II) after slaughter. There were no significant differences between cleavage rates (72 hpi: hours postinsemination) between both groups (63.5% versus 69.1%). However, blastocyst (168 hpi) and hatching (216 hpi) rates were higher (P < 0.05) in Group II compared to Group I (21.3% versus 30.7% and 27.6% versus 51.5% respectively). Group II oocytes exhibited the highest MATER and OCT-4 abundance (P < 0.05). Follicular estradiol concentration was not different between both groups while progesterone concentration was significantly lower (P ≤ 0.05) in Group II follicles. These results indicate that retrieving COC’s 4 hours after slaughter could increase bovine in vitro developmental competence, which is linked to higher levels of oocyte MATER and OCT-4 transcripts and lower follicular progesterone concentration. Moreover, the results of the present study contribute to the identification of factors involved in the developmental competence of immature oocytes