Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium

Abstract

The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.

Keywords: BSA; PF4; defined media; embryo; in vitro; microarray; porcine; transcriptome.

Figure 1

Representation of the experimental design conducted during the study. The samples consisted of pools of 10 blastocysts classified as ZP-covered and expanded. A total of three experimental groups were used, one of which consisted of surgically in vivo-obtained embryos (IVV group) and the other two consisted of embryos produced in vitro; the first one was cultured with undefined culture medium consisting of North Carolina State University (NCSU)-23 supplemented with 0.4 mg/mL bovine serum albumin (BSA group), and the second was cultured with a defined culture medium consisting of NCSU-23 supplemented with 0.3 mg/mL polyvinyl alcohol (PVA) and 100 ng/mL platelet factor 4 (PF4 group). The RNA was extracted from the pools and processed for microarrays. A total of 7 arrays were performed for the IVV group while 5 were performed for each in vitro group. IVC: in vitro culture

Figure 2

Distribution of the analyzed samples according to unsupervised analysis. (A) Principal component analysis obtained from the microarray results. (B) Hierarchical clustering analysis comparing the differentially expressed genes (|FC|</>1.5 and adjusted p value < 0.05) for the three experimental groups.

Figure 3

Differentially expressed genes (DEGs) represented by a volcano plot of each pairwise comparison. (A) Comparison between in vitro embryos cultured in defined medium (PF4: platelet factor 4) versus in vivo-derived embryos (IVV). (B) Comparison between in vitro embryos cultured in undefined medium (BSA: bovine serum albumin) versus IVV embryos. (C) Comparison between PF4 and BSA embryos. (D) Venn diagram comparison showing the cross-analysis of the DEG obtained from each in vitro group comparison against the IVV group.

Figure 4

Classification of differentially expressed genes (DEG) organized by gene ontology (GO)-terms. The lists of DEG were obtained after comparing the in vitro embryos cultured in defined media (PF4: platelet factor 4) versus the in vivo-derived embryos (IVV), and the in vitro embryos cultured in undefined media (BSA: bovine serum albumin) versus the IVV embryos. In each forest plot, the X-axis represents the percentage of DEG out of the total number of genes belonging to each category. Green bars represent the percentages of downregulated DEG, while the red bars indicate the percentages of upregulated DEG. Next to each bar, the number of upregulated (red bar) or downregulated (green bar) DEG, belonging to that GO-term is indicated. ES: enrichment score.

Figure 5

Representation of the different pathways considered altered (p value < 0.05) obtained from the (A) upregulated differentially expressed genes (upregulated pathways) and from (B) the downregulated differentially expressed genes (downregulated pathways). The enrichment values for each pathway are indicated by a circle in the case of the comparison between in vitro embryos cultured in defined medium (PF4: platelet factor 4) and in vivo-derived embryos (IVV) and by a square in the case of the comparison between in vitro embryos cultured in undefined medium (BSA: bovine serum albumin) and IVV embryos. The size of the symbol indicates the percentage of affected genes out of the total number of genes belonging to that pathway. The color of the symbol indicates the p-value of each pathway.

Figure 6

Heat maps of the most prominent pathways representing the expression of each differentially expressed gene in each experimental group: in vivo-derived embryos (IVV), in vitro embryos cultured in defined medium (PF4: platelet factor 4) and in vitro embryos cultured in undefined medium (BSA: bovine serum albumin). * Indicates a gene considered differentially expressed in the case of the PF4 versus IVV comparison but not in the BSA versus IVV comparison. ** Indicates a gene considered differentially expressed in the BSA versus IVV comparison but not in the PF4 versus IVV comparison. Genes without any symbol indicate that they were considered differentially expressed in both comparisons. Genes surrounded by a box were selected for validation by q-PCR.

Figure 7

Heat maps grouping differentially expressed genes by specific functions at the embryonic level or as markers of damage for the three experimental groups: in vivo-derived embryos (IVV), in vitro embryos cultured in defined medium (PF4) and in vitro embryos cultured in undefined medium (BSA). * Indicates a gene considered differentially expressed in the case of the PF4 (PF4: platelet factor 4) versus IVV comparison but not in the BSA (BSA: bovine serum albumin). versus IVV comparison. ** Indicates a gene considered as differentially expressed in the BSA versus IVV comparison but not in the PF4 versus IVV comparison. Genes without any symbol indicate that they were considered differentially expressed in both comparisons. Genes surrounded by a box were selected for validation by q-PCR.

Figure 8

Comparison of the fold changes obtained by microarray and q-PCR analyses for the different validated genes in the experiment. The black circles represent genes where the significance obtained with the q-PCR was accordingly to those obtained by the microarray. White circles represent genes where the significance obtained with q-PCR differs from those obtained by the microarray.