Seminal Plasma Modifies the Transcriptional Pattern of the Endometrium and Advances Embryo Development in Pigs

Abstract

Background: Seminal plasma (SP) promotes sperm survival and fertilizing capacity, and potentially affects embryo development, presumably via specific signaling pathways to the internal female genital tract. Objectives: This study evaluated how heterologous SP, infused immediately before postcervical artificial insemination (AI) affected embryo development and the transcriptional pattern of the pig endometria containing embryos. Materials and Methods: Postweaning estrus sows (n = 34) received 40-mL intrauterine infusions of either heterologous pooled SP or Beltsville Thawing Solution (BTS; control) 30 min before AI of semen extended to 10% of homologous SP. Embryos (all sows) and endometrium samples (3 sows/group) were removed during laparotomy 6 days after the infusion of SP or BTS to morphologically evaluate the embryos to determine their developmental stage and to analyze the endometrial transcriptome using microarrays (PORGENE 1.0 ST GeneChip array, Affymetrix) followed by qPCR for further validation. Results: Embryo viability was equal between the groups (~93%), but embryo development was significantly (P < 0.05) more advanced in the SP-treated group compared to control. A total of 1,604 endometrium transcripts were differentially expressed in the SP group compared to the control group. An enrichment analysis showed an overrepresentation of genes and pathways associated with the immune response, cytokine signaling, cell cycle, cell adhesion, and hormone response, among others. Conclusions: SP infusions prior to AI positively impacted the preimplantation embryo development and altered the expression of the endometrial genes and pathways potentially involved in embryo development.

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

Figure 1

Embryos collected 6 days after the onset of estrus. (A) Compacted morulae. (B) Full blastocysts. (C–E) Expanded, hatching and hatched blastocysts, respectively. (F) Degenerated embryos. Note that most of the embryos have a developmental stage that is inappropriate for the day of the collection. Inset shows some unfertilized oocytes.

Figure 2

Histological representative images of the endometrium. Microphotographs of endometria 6 days after BTS (control, A) or heterologous seminal plasma (SP, B) infusions prior to AI, depicting conspicuous differences in mucosal edema, vascular congestion and peri-vascular infiltration of immune cells (see inset in B, white arrows; inset in A shows lack of peri-vascular infiltration of immune cells). Arrows, uterine glands; bv, blood vessels; ms, mucosal stroma. Bar: 100 μm.

Figure 3

Functional distribution (KEGG database) of transcripts. The differentially expressed (P < 0.05) genes represented correspond to the distal (A) or proximal (B) regions of the uterine horn endometrium 6 days after heterologous seminal plasma (SP) or BTS (control) infusions prior to insemination.

Figure 4

Schematic representation of selected altered transcripts potentially involved in embryo kinetics in distal and proximal regions of the SP treated endometrium relative to the BTS (control) 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 subgroup biological process shown as circles and KEGG pathways shown as triangles. Terms are functionally grouped based on shared genes (kappa score) and are shown in different colors. The sizes of the nodes indicate the degree of significance, where the biggest nodes correspond to the highest significance. The most significant term defines the name of the group. The following ClueGo parameters were used: GO tree levels, 2–5 (first level = 0); minimum number of genes, 3; minimum percentage of genes, 5; P-value correction, Benjamini-Hochberg, terms with P < 0.05, 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 was modified; that is, some redundant and non-informative terms were deleted, and the network was manually rearranged.

Figure 5

Hierarchical cluster analysis of gene expression patterns. The selected transcriptional profiles (DAVID database) were obtained from the distal (A) or proximal (B) regions of the uterine horn endometrium collected 6 days after heterologous seminal plasma (SP) or BTS (control) infusions prior to insemination. Colors correspond to relative RNA abundance for the detected genes, each of which is represented by one vertical bar. Red color indicates upregulated expression and green downregulated expression while black indicates the mean value.

Figure 6

Correlation of fold change determined by microarray platform and real time PCR of 15 genes (Rho, Spearman coefficient).

Figure 7

Embryonic developmental stage at collection. Developmental stages of embryo recovery 6 days after heterologous seminal plasma (SP; black bars; N = 16 sows) or BTS (Control; gray bars; N = 18 sows) infusions prior to insemination. Thirty minutes before each postcervical insemination (1.5 × 10⁹ sperm/dose in 40 mL of BTS diluent), sows were infused with 40 mL of heterologous SP or BTS (control). At day 6 after the first insemination, the sows were subjected to laparotomy to collect the embryos and to evaluate their quality and developmental stage. A total of 362 and 379 viable embryos were collected in sows from the SP and BTS groups, respectively. Numbers in parentheses are the number of embryos for each stage. ^a,bDifferent letters within each embryonic developmental stage represent significant differences (P < 0.05; Fisher's exact test).

Figure 8

Total cell number of morulae, full blastocysts and peri-hatching blastocysts recovered 6 days after the postcervical infusion of heterologous seminal plasma (SP; black bars; N = 16 sows) or BTS (Control; gray bars; N = 18 sows) prior to insemination. Numbers in parentheses are the number of embryos counted for each stage.