Seminal Plasma Induces Overexpression of Genes Associated with Embryo Development and Implantation in Day-6 Porcine Blastocysts

Abstract

The infusion of boar seminal plasma (SP) before artificial insemination (AI) positively alters the expression of endometrial genes and pathways involved in embryo development. This study aimed to determine which transcriptome changes occur in preimplantation embryos in response to SP infusions during estrus. Postweaning estrus sows received 40-mL intrauterine infusions of either SP (N = 6) or BTS extender (control group; N = 6) 30 min before each of two post-cervical AIs. On Day 6, embryos were surgically collected and analyzed for differential gene expression. Microarray analysis of embryos revealed 210 annotated genes, differentially expressed (p-value < 0.05 and fold change </> 2) in SP-blastocysts, compared to controls. Most of these genes were associated with biological, cellular, metabolic and developmental processes. The pathways enriched among the upregulated genes related to signal transduction, cellular processes and the endocrine system. Among altered genes involved in these pathways, the SP-group showed a conspicuous overexpression of ApoA-I, CDK1, MAPK1, SMAD2, PRKAA1 and RICTOR, with reported key roles in embryo development, implantation, or progression of pregnancy. In conclusion, the results demonstrate that SP infusions prior to AI upregulates the expression of embryo development related genes in Day 6 pig embryos.

Keywords: embryo; embryo transfer; pig; preimplantation; seminal plasma; transcriptome.

Figure 1

Hierarchical clustering analysis comparing differentially expressed genes for “seminal plasma (SP) vs. BTS-embryos”. Rows and columns represent samples and genes, respectively. Colors correspond to the expression levels of the detected genes upregulated (red) and downregulated (green). Each vertical line represents single gene and each row represents single sample (SPS or BTS). Upper branches indicate relationships among different genes and left branches indicate differences among samples.

Figure 2

Distribution of differentially expressed genes in embryos derived from seminal plasma-infused sows within biological processes (A) cellular components (B) and molecular functions (C). The distribution of upregulated and downregulated genes within each of biological process, cellular component and molecular function is illustrated in the right-hand side figures (numbers within parentheses are enrichment scores).

Figure 3

Schematic representation of biological processes and KEGG pathways found when comparing differentially expressed genes in embryos derived from sows infused with seminal plasma, relative to control embryos (BTS-group). The analysis of overrepresented functional categories was performed using the Cytoscape v3.0.0 application ClueGo v2.0.3. The following databases were used: GO subgroups in biological process (circles) and KEGG pathways (triangles). Terms are functionally grouped based on shared genes (kappa score), and are shown in different colors. The size of the nodes indicates the degree of significance, where the biggest nodes correspond to highest significance. The most significant term defines the name of the group. The following ClueGo parameters were used: biological process database (BP; date: 28.03.2019); GO three levels, 2–5 (first level = 0); minimum number of genes, 2; minimum percentage of genes, 2; GO term fusion; GO term connection restriction (kappa score), 0.4; GO term grouping, initial group size of 2 and 50% for group merge. The resulting network representation was manually adapted after removing unnecessary terms.

Figure 4

Validation of microarray results by real time quantitative PCR (RT-qPCR). The γ-axis represents the fold change between the seminal plasma and BTS groups. Asterisk indicates significant differences between seminal plasma and BTS groups by RT-qPCR analysis.

Figure 5

Schematic representation of the experimental design. Experimental groups: Sows infused with seminal plasma (SP) or BTS extender (Control) 30 min before each of two artificial inseminations (AI). Day 6 blastocysts were collected and pooled within each group. Pooled embryos (n = 10/tube) were subjected to transcriptomic analysis (microarrays).