Perfil transcricional da criotolerância de embriões produzidos in vitro

Abstract

Embryo cryopreservation is an assisted reproductive technology that allows the storage of in vitro produced or in vivo derived embryos for long periods while are not commercialized or transferred. It is considered as one of the most challenging areas within the biotechnologies of reproduction. The reduced pregnancy rate of the cryopreserved embryos associated with na increase demand of qualified technician to handle this biotechnology could explain the reduced number of embryos being cryopreserved. Embryo cryosurvival is a complex process involving dynamic structural and molecular changes. Embryo transcriptional profile information after cryopreservation is still lacking. The aim of this work was to establish the transcriptome of in vitro produced bovine blastocysts with high and low cryosurvival. Immature oocytes (n= 3926) were recovered from slaughterhouse-derived ovaries and in vitro matured, fertilized and culture under standard conditions. Cleavage and embryo production were recorded on day three and seven after fertilization. Expanded blastocysts (n= 894) with exceptional quality (grade I only) were vitrified by the cryotop method. Vitrified blastocysts were warmed, and embryo re- expansion and hatching were recorded at 6 and 12 h after warming, respectively. A rigorous morphological evaluation was done by experienced technician considering: a) blastocoele re- expansion; b) hatching; c) inner cell mass and trofoectoderm integrity and organization; and d)color and extrusion of blastomeres. After morphological evaluation, embryos were classified as high cryosurvival (HC: blastocysts with the blastocoele completely re-expanded followed by hatching 12 h after warming and without or with mild and few signs of degeneration); re- expanded (blastocysts with the blastocoele completely re-expanded but not followed by hatching 12 h after warming and with mild and few signs of degeneration), and non-viable (low cryosurvival - LC: blastocysts without the blastocoele completely re-expanded followed by hatching 12 h after warming and with severe and frequently signs of degeneration) after cryopreservation. In order to increase accuracy of molecular differences among phenotypes, only HC and LC blastocysts (n= 3, total of 123 blastocysts per group, pool of 41 /sample) after cryopreservation and submitted to total RNA extraction (PicoPure, Applied Biosystems®). Extracted RNA was evaluated through 2100-Bioanalyzer (Agilent Technologies®). Only samples with RNA integrity ≥ 7.7 were submitted to through standard protocol of NEBNext Ultra II library preparation (New England Biolabs®) and RNAseq (Illumina®). Data were cleaned (seqyClean v1.3.12), mapped (Tophat v.2.0.8), reads counted (HTSeq-count v0.5.4p2), and differential expression analysis was performed (DESEq v1.12.1 from R/Bioconductor). For gene enrichment analysis, ingenuity pathway analysis (IPA) was used. Cleavage and blastocyst production rates were 73.2 % (2873/3926) and 29.0 % (1137/3926), respectively. The re-expansion (89.2 vs. 73.4%) and hatching (23.7 vs. 8.4) rates were higher (P<0.05) at 12h compared with 6h after warming, respectively. A mean 17.8 millions of reads per sample were used on different expression analysis. Blastocysts group samples were clearly separated with pronounced group individualization at two- and three-dimensional principal componente analysis and heat map clustering sustaining the phenotype characterization of the model (high vs. low cryosurvival). A total of 9422 genes were identified, 114 were differently expressed genes (DEG; FDR: P<0.05), with 27 and 84 genes up-regulated in HC and LC, respectively. At the enrichment analysis of the DGE (FDR: P<0.1) the top five biological functions identified were cellular movement (66 molecules), cellular development (82 molecules), cell grown and proliferation (79 molecules), and cell death and survival (80 molecules). At IPA prediction analysis, the biological functions of organismal injury, cell death and survival, cell grow and proliferation were predicted to be more activated (z-score ≥ +2), whereas cellular movement and cell-to-cell signaling were predicted to be more inhibited (z-score ≤ -2). The present work provided a comprehensive analysis of the transcriptional profile of bovine embryo cryosurvival and elucidated the involvement of a great number of noble biological processes molecules in the embryo development resumption after cryopreservation. Organismal injury, cellular movement, cell death and survival, cell growth and proliferation were crucial biological functions for embryo cryosurvival.