Semen induces post-coital inflammation of the endometrium in several species. Post-coital inflammation is proposed to alter the endometrial environment of early pregnancy, mediate embryonic development and modulate the maternal immune response to pregnancy. In cattle, it is common for pregnancies to occur in the absence of whole semen due to the high utilization of artificial insemination. Here, we have utilized a cell culture system to characterize semen-induced expression of inflammatory mediators in bovine endometrial cells and test the efficacy of transforming growth factor beta as the active agent in mediating any such change. We hypothesize that seminal plasma-derived transforming growth factor beta increases the expression of inflammatory mediators in bovine endometrial cells. Initially, we describe a heat-labile cytotoxic effect of seminal plasma on BEND cells, and a moderate increase in IL1B and IL6 expression. In addition, we show that transforming growth factor beta is present in bovine semen and can increase the expression of endometrial IL6, whereas blocking transforming growth factor beta in semen ameliorates this effect. However, intra-uterine infusion of seminal plasma, sperm or transforming growth factor beta did not alter the endometrial expression of inflammatory mediators. We conclude that bovine semen can modulate endometrial gene expression in vitro, which is partially due to the presence of transforming growth factor beta. It is likely that additional, unidentified, bioactive molecules in semen can alter the endometrial environment. Characterizing bioactive molecules in bovine semen may lead to the development of additives to improve artificial insemination in domestic species.
Supplemental Figure 1. Effect of seminal plasma on viability of BEND cells supplemented with estradiol. Cell viability was assessed using the MTT assay. BEND cells were cultured in the presence of 0 (A), 0.1 (B) or 1 nM (C) estradiol for 24 h before addition of seminal plasma (SP). BEND cells were then treated with medium alone (0) or 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, or 20% v/v SP for 24 h in medium supplemented with estradiol. Data are presented as mean fold-change of estradiol-free medium alone treated cells ± SEM. Experiments were replicated in four independent assays. Data were log transformed and analyzed using the generalized linear mixed model and pairwise comparisons were made between medium alone and each seminal plasma treatment within a given estradiol concentration. *, represents P ≤ 0.05 compared to medium alone within estradiol concentration.
Supplemental Figure 2. Concentration of TGFβ in bull semen. Seminal plasma of 33 individual bulls was evaluated for TGFβ-1 (A, C, E) and TGFβ-2 (B, D, F) content by commercial ELISA specific for human TGFβ. Bioactive (A, B) and total (C, D) TGFβ concentrations are reported for each individual bull in ng/mL. Absolute content of TGFβ was calculated using the concentration of total TGFβ and ejaculate volume. Human and bovine TGFβ-1 and TGFβ-2 share 95% and 98% homology respectively based on NCBI protein-blast analysis, while the homology between human and bovine TGFβ-3 is low and was not assessed here with human kits.
Supplemental Figure 3. Effect of transforming growth factor β and estradiol on BEND cell viability. Cell viability was assessed using the MTT assay following culture in the presence of 0 (A, D), 0.1 (B, E) or 1 nM (C, F) estradiol for 24 h before addition of rhTGFβ. Cells were subsequently exposed to either medium alone (0), rhTGFβ-1 (1, 10 or 100 ng/mL), or rhTGFβ-2 (0.1, 1 or 10 ng/mL) for 24 h in medium supplemented with estradiol. Data are presented as mean fold-change of estradiol-free medium alone treated cells ± SEM. Experiments were replicated in four independent assays. Data were log transformed and analyzed using the generalized linear mixed model and pairwise comparisons were made between each treatment and appropriate medium alone within an estradiol concentration.