Long-Term In Vitro Culture Alters Gene Expression Pattern of Genes Involved in Ontological Groups Representing Cellular Processes

Abstract

The oviduct provides an optimal environment for the final preparation, transport, and survival of gametes, the fertilization process, and early embryonic development. Most of the studies on reproduction are based on in vitro cell culture models because of the cell's accessibility. It creates opportunities to explore the complexity of directly linked processes between cells. Previous studies showed a significant expression of genes responsible for cell differentiation, maturation, and development during long-term porcine oviduct epithelial cells (POECs) in vitro culture. This study aimed at establishing the transcriptomic profile and comprehensive characteristics of porcine oviduct epithelial cell in vitro cultures, to compare changes in gene expression over time and deliver information about the expression pattern of genes highlighted in specific GO groups. The oviduct cells were collected after 7, 15, and 30 days of in vitro cultivation. The transcriptomic profile of gene expression was compared to the control group (cells collected after the first day). The expression of COL1A2 and LOX was enhanced, while FGFBP1, SERPINB2, and OVGP1 were downregulated at all selected intervals of cell culture in comparison to the 24-h control (p-value < 0.05). Adding new detailed information to the reproductive biology field about the diversified transcriptome profile in POECs may create new future possibilities in infertility treatments, including assisted reproductive technique (ART) programmes, and may be a valuable tool to investigate the potential role of oviduct cells in post-ovulation events.

Keywords: RNA processing; cell adhesion; cell migration; intercellular communication; reproductive biology.

Figure 1

Volcano plots of differentially expressed genes in the d7, d15, and d30 experimental groups compared to the h24 control group. Cut-off values shown on the graph as orange dashed lines were obtained based on parameters |fold change| = 2; p-value = 0.05. Red dots represent genes with downregulated expression, while green dots—upregulated expression. The exact number of differentially expressed genes in each comparison is shown in the upper part of the graph. The five most overexpressed genes are labeled with their names. Two biological replicates were performed for each experiment.

Figure 2

(A) Principal component analysis (PCA) plot of the first two components of the filtered microarray data set. (B) Venn diagrams show genes with upregulated (left panel) and downregulated (right panel) expression shared among different analyzed groups.

Figure 3

Bubble plot showing differentially expressed gene sets in DAVID GO BP DIRECT database. Each column represents corresponding gene sets in comparison groups d7 vs. h24; d15 vs. h24; d30 vs. h24, respectively. Red bubbles represent downregulated expression and green bubbles represent upregulated expression gene sets. The size of the bubble reflects the number of different genes in a particular GO term. Higher bubble transparency means closer proximity of p-value to 0.05. The parameters used for the cut-off criteria were p-value with correction < 0.05, number of genes in set >2.

Figure 4

Heatmap of expressed gene clusters in 24 h control group and d7, d15, and d30 experimental groups. Leftmost heat map shows scaled levels of gene expression in each study group (green = 2, black = 0, red = −2). The middle heatmap shows the fold change of the gene expression in comparison to the 24 h control group. The right heatmap highlights genes belonging to the four most significantly enriched ontological groups (black = present, white = absent).

Figure 5

(A) Clustering of differentially expressed genes. Centroid values are shown as black lines. Each line represents an individual gene, where the color corresponds to the level of correlation to the centroid values according to the color scale. (B) Heatmap of differentially expressed genes in all analyzed groups divided into five gene clusters. Leftmost heat map illustrates mean expression of genes grouped in five clusters in 24 h control group and in each study group: d7, d15, d30 (green = 2, black = 0, red = −2). The rightmost heatmap shows the fold change of the gene expression compared to the 24 h control group.

Figure 6

Bubble plot representing enrichment of genes in GO groups divided among five gene clusters. The size of the bubble corresponds to the number of genes assigned to the GO BP terms.