Different pre-implantation phenotypes of bovine blastocysts produced in vitro

in Reproduction

Correspondence should be addressed to I Hue; Email: isabelle.hue@inra.fr
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Embryo transfer in cattle is performed with blastocysts produced in vivo or in vitro using defined media. However, outdated systems such as those that use serum and co-culture remain of interest for research purposes. Here, we investigated the effect of additional culture time on in vitro-produced embryos. Specifically, we compared embryos that formed a blastocoel at different times after fertilisation to those that stayed in culture for up to two additional days with respect to their development in vivo after temporary transfer to oestrus-synchronised recipients. A pre-transfer set (D6, D6+1, D6+2, D7, D7+1, D8) was examined using microarray analyses and correlated with a post-transfer set that included two different days of transfer (D6-T6, D6+2-T8, D7+1-T8, D8-T8). All surviving conceptuses reached primitive-streak stages and filamentous sizes similarly to in vivo (D18) or in vitro controls (D7/T7). The recovery rate differed between D6 and D8 embryos that were immediately transferred (58 vs 25%). With an intermediate survival rate (33%), the D6 embryos with two additional days in culture produced nine times more IFN-tau (IFNT) at D18 than the D6 embryos that were immediately transferred. At the end of culture, D6 and D6+2 embryos displayed the highest number of gene expression differences. Despite a mortality of 40–60%, no signature was detectable in any of the transferred groups that would account for the embryos’ fates. Initially reputed to be beneficial in producing more blastocysts, our culture system of B2 medium plus serum and co-culture generated blastocysts that were distinct from those developed in vivo (D7).

Supplementary Materials

    • Supplementary Figure 1: Principal-components analysis (PCA) of all samples (n=21: 7 groups, 3 replicates per group). In the three panels are the projections based on gene expression data from the microarray analysis on 3 main axes (1-2, 1-3, and 2-3). In red, green and pink: the D6, D7, and D8 blastocysts. In yellow, blue, and light green: the D6+1, D7+1 and D6+2 embryo groups.
    • Supplementary Table 1: Oligonucleotides used for bovine gene quantification by real time RT-qPCR. Seven pairs were designed de novo. Eight originated from previous reports: 1 (Sakurai et al. 2013), 2 (Khan et al. 2012), 3 (Bertolini et al. 2002), 4 (Nuttinck et al. 2008). (*) refers to primer sequences being adapted to the bovine NM accession numbers; (N) indicates the nucleotide that has been changed as compared to the original report.
    • Supplementary Tables 2 : Lists of enriched genes and their associated functions, in every paired comparison of the design (n=9). Provided are: i) the size of the gene set indexed in the hallmark or homemade database for each function, ii) the total number of genes enriched in each comparison (|NES|>1.8; FDR<0.05; NES: Normalised Enrichment Score, as provided by the software from the Broad Institute), iii) the number of genes similarly enriched in the three replicates (->), iv) the number of unique gene IDs per comparison, after we merged all the functions, and v) the gene IDs shared across comparisons, for each of the enriched functions. In bold, the enriched functions that were shared across all groups. In Supp. Table 2: the D6/D7/D8-forming blastocysts; Supp. Table 3a and 3b: the blastocysts that spent extra days in culture (+1/+2); Supp. Table 4: blastocysts which, overall, spent the same time in culture (7 days); Supp. Table 5a and 5b: blastocysts that were transferred in vivo at D8, plus D8 vs D6 (no NES at FDR <0.05 for the D7+1 vs D8 comparison, which is thus not shown); Supp. Table 6: the in vivo vs in vitro-produced embryos assessed with respect to metabolic sensors (NES=-1.4; FDR<0.05). Corresponding gene lists in the seven Supp. Tables from 2-bis to 6-bis are (.xls) files accessible at https://data.inra.fr/privateurl.xhtml?token=00c06d2c-f4c6-446d-bfe5-8af5e7759038.
    • Supplementary Tables 3: Lists of enriched genes and their associated functions, in every paired comparison of the design (n=9). Provided are: i) the size of the gene set indexed in the hallmark or homemade database for each function, ii) the total number of genes enriched in each comparison (|NES|>1.8; FDR<0.05; NES: Normalised Enrichment Score, as provided by the software from the Broad Institute), iii) the number of genes similarly enriched in the three replicates (->), iv) the number of unique gene IDs per comparison, after we merged all the functions, and v) the gene IDs shared across comparisons, for each of the enriched functions. In bold, the enriched functions that were shared across all groups. In Supp. Table 2: the D6/D7/D8-forming blastocysts; Supp. Table 3a and 3b: the blastocysts that spent extra days in culture (+1/+2); Supp. Table 4: blastocysts which, overall, spent the same time in culture (7 days); Supp. Table 5a and 5b: blastocysts that were transferred in vivo at D8, plus D8 vs D6 (no NES at FDR <0.05 for the D7+1 vs D8 comparison, which is thus not shown); Supp. Table 6: the in vivo vs in vitro-produced embryos assessed with respect to metabolic sensors (NES=-1.4; FDR<0.05). Corresponding gene lists in the seven Supp. Tables from 2-bis to 6-bis are (.xls) files accessible at https://data.inra.fr/privateurl.xhtml?token=00c06d2c-f4c6-446d-bfe5-8af5e7759038.
    • Supplementary Tables 4: Lists of enriched genes and their associated functions, in every paired comparison of the design (n=9). Provided are: i) the size of the gene set indexed in the hallmark or homemade database for each function, ii) the total number of genes enriched in each comparison (|NES|>1.8; FDR<0.05; NES: Normalised Enrichment Score, as provided by the software from the Broad Institute), iii) the number of genes similarly enriched in the three replicates (->), iv) the number of unique gene IDs per comparison, after we merged all the functions, and v) the gene IDs shared across comparisons, for each of the enriched functions. In bold, the enriched functions that were shared across all groups. In Supp. Table 2: the D6/D7/D8-forming blastocysts; Supp. Table 3a and 3b: the blastocysts that spent extra days in culture (+1/+2); Supp. Table 4: blastocysts which, overall, spent the same time in culture (7 days); Supp. Table 5a and 5b: blastocysts that were transferred in vivo at D8, plus D8 vs D6 (no NES at FDR <0.05 for the D7+1 vs D8 comparison, which is thus not shown); Supp. Table 6: the in vivo vs in vitro-produced embryos assessed with respect to metabolic sensors (NES=-1.4; FDR<0.05). Corresponding gene lists in the seven Supp. Tables from 2-bis to 6-bis are (.xls) files accessible at https://data.inra.fr/privateurl.xhtml?token=00c06d2c-f4c6-446d-bfe5-8af5e7759038.
    • Supplementary Tables 5: Lists of enriched genes and their associated functions, in every paired comparison of the design (n=9). Provided are: i) the size of the gene set indexed in the hallmark or homemade database for each function, ii) the total number of genes enriched in each comparison (|NES|>1.8; FDR<0.05; NES: Normalised Enrichment Score, as provided by the software from the Broad Institute), iii) the number of genes similarly enriched in the three replicates (->), iv) the number of unique gene IDs per comparison, after we merged all the functions, and v) the gene IDs shared across comparisons, for each of the enriched functions. In bold, the enriched functions that were shared across all groups. In Supp. Table 2: the D6/D7/D8-forming blastocysts; Supp. Table 3a and 3b: the blastocysts that spent extra days in culture (+1/+2); Supp. Table 4: blastocysts which, overall, spent the same time in culture (7 days); Supp. Table 5a and 5b: blastocysts that were transferred in vivo at D8, plus D8 vs D6 (no NES at FDR <0.05 for the D7+1 vs D8 comparison, which is thus not shown); Supp. Table 6: the in vivo vs in vitro-produced embryos assessed with respect to metabolic sensors (NES=-1.4; FDR<0.05). Corresponding gene lists in the seven Supp. Tables from 2-bis to 6-bis are (.xls) files accessible at https://data.inra.fr/privateurl.xhtml?token=00c06d2c-f4c6-446d-bfe5-8af5e7759038.
    • Supplementary Tables 6: Lists of enriched genes and their associated functions, in every paired comparison of the design (n=9). Provided are: i) the size of the gene set indexed in the hallmark or homemade database for each function, ii) the total number of genes enriched in each comparison (|NES|>1.8; FDR<0.05; NES: Normalised Enrichment Score, as provided by the software from the Broad Institute), iii) the number of genes similarly enriched in the three replicates (->), iv) the number of unique gene IDs per comparison, after we merged all the functions, and v) the gene IDs shared across comparisons, for each of the enriched functions. In bold, the enriched functions that were shared across all groups. In Supp. Table 2: the D6/D7/D8-forming blastocysts; Supp. Table 3a and 3b: the blastocysts that spent extra days in culture (+1/+2); Supp. Table 4: blastocysts which, overall, spent the same time in culture (7 days); Supp. Table 5a and 5b: blastocysts that were transferred in vivo at D8, plus D8 vs D6 (no NES at FDR <0.05 for the D7+1 vs D8 comparison, which is thus not shown); Supp. Table 6: the in vivo vs in vitro-produced embryos assessed with respect to metabolic sensors (NES=-1.4; FDR<0.05). Corresponding gene lists in the seven Supp. Tables from 2-bis to 6-bis are (.xls) files accessible at https://data.inra.fr/privateurl.xhtml?token=00c06d2c-f4c6-446d-bfe5-8af5e7759038.
    • Supplementary Tables 7 are also (.xls) files that are accessible at https://data.inra.fr/privateurl.xhtml?token=00c06d2c-f4c6-446d-bfe5-8af5e7759038. In Supp. Table 7: the gene lists corresponding to the shared enrichments from Figure 2 and in Supp. Table 8 the lists of the differentially expressed probes (DEP; Table 1) as well as the analyses performed on the DEGs from the in vivo vs in vitro contrast.
    • Supplementary Tables 8 are also (.xls) files that are accessible at https://data.inra.fr/privateurl.xhtml?token=00c06d2c-f4c6-446d-bfe5-8af5e7759038. In Supp. Table 7: the gene lists corresponding to the shared enrichments from Figure 2 and in Supp. Table 8 the lists of the differentially expressed probes (DEP; Table 1) as well as the analyses performed on the DEGs from the in vivo vs in vitro contrast.
    • Supplementary Tables 9
    • Supplementary Tables 10

 

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