The role of semen and seminal plasma in inducing large-scale genomic changes in the female porcine peri-ovulatory tract

Abstract

Semen modifies the expression of genes related to immune function along the porcine female internal genital tract. Whether other pathways are induced by the deposition of spermatozoa and/or seminal plasma (SP), is yet undocumented. Here, to determine their relative impact on the uterine and tubal transcriptomes, microarray analyses were performed on the endocervix, endometrium and endosalpinx collected from pre-ovulatory sows 24 h after either mating or artificial insemination (AI) with specific ejaculate fractions containing spermatozoa or sperm-free SP. After enrichment analysis, we found an overrepresentation of genes and pathways associated with sperm transport and binding, oxidative stress and cell-to-cell recognition, such as PI3K-Akt, FoxO signaling, glycosaminoglycan biosynthesis and cAMP-related transcripts, among others. Although semen (either after mating or AI) seemed to have the highest impact along the entire genital tract, our results demonstrate that the SP itself also modifies the transcriptome. The detected modifications of the molecular profiles of the pre/peri-ovulatory endometrium and endosalpinx suggest an interplay for the survival, transport and binding of spermatozoa through, for instance the up-regulation of the Estrogen signaling pathway associated with attachment and release from the oviductal reservoir.

Figure 1

Differential expression (up- and down-regulation) of annotated genes in the mucosa of the internal genital tract of sows after Mating or artificial deposition of the sperm-peak portion P1 (P1-AI); sperm-free seminal plasma of the whole ejaculate (SP-Ejac) or of pooled P1 (SP-P1). All treatments were compared to Control (AI with 50 mL of BTS). The numbers represent the number of differentially expressed genes (p-value < 0.05). Endocervix (EndoCvx), endometrium (distal: DistEndom or proximal: ProxEndom) and endosalpinx from 4 different segments; the utero-tubal junction (UTJ), isthmus (Isth), ampulla (Amp) and infundibulum (Inf).

Figure 2

Venn diagrams depicting common genes among mucosal segments of the internal genital tract of the sow, after the various treatments: M (A), P1-AI (B), SP-P1 (C) and SP-Ejac (D).

Figure 3

Schematic representation of common altered transcripts among all mucosal tissues collected from the M group. The analysis of overrepresented functional categories was performed using the Cytoscape v3.0.0 application ClueGo v2.0.3. 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 following ClueGo parameters were used: GO tree levels, 3–6 (first level = 0); minimum number of genes, 1; P-value correction, Benjamini-Hochberg, terms with P < 0.05, GO term connection restriction (kappa score), 0.4; GO term grouping, initial group size of 1. The resulting network was modified; that is, some redundant and non-informative terms were deleted and the network manually rearranged.

Figure 4

Schematic representation of selected altered transcripts in the mucosae of segments of the internal genital tract of the sows in P1-AI group; Endocervix (EndoCvx: (A)), Endometrium (DisEndom: (B), ProEndom: (C)), Utero-tubal Junction (UTJ: (D)), Isthmus (Isth: (E)), Ampulla (Amp: (F)) and Infundibulum (Inf: (G)). The analysis of overrepresented functional categories was performed using the Cytoscape v3.0.0 application ClueGo v2.0.3. 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 following ClueGo parameters were used: GO tree levels 3–6 (first level = 0); minimum number of genes 1; minimum percentage of genes 8, 5P-value correction, Benjamini-Hochberg, terms with P < 0.05, GO term connection restriction (kappa score) 0.4; GO term grouping, GO term fusion; initial group size of 3. The resulting network was modified; that is, some redundant and non-informative terms were deleted and the network manually rearranged.

Figure 5

Schematic representation of CAMP-related transcripts in the mucosa of the segments of the sow oviduct [Utero-tubal junction (UTJ: (A)), Isthmus (Isth: (B)), Ampulla (Amp: (C)) and Infundibulum (Inf: (D))] after mating (M: Elipse); Artificial insemination of the sperm-peak fraction (P1-AI: Triangle), seminal plasma from the sperm-peak fraction (SP-P1: Hexagon); or seminal plasma from the entire ejaculate (SP-Ejac: V). The analysis of overrepresented functional categories was performed using the Cytoscape v3.0.0 application ClueGo v2.0.3. 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 following ClueGo parameters were used: GO tree levels, 1–4 (first level = 0); minimum number of genes, 1; P-value correction, Benjamini-Hochberg, terms with P < 0.05, GO term connection restriction (kappa score), 0.4; GO term grouping, initial group size of 1. The resulting network was modified; that is, some redundant and non-informative terms were deleted and the network manually rearranged.