Differential expression analysis of gene-level counts was carried out in R version 3.6.1 using the edgeR package (Robinson et al. 2010). The genes were filtered to exclude genes that were assigned less than 10 reads for all the samples in one experimental group. The genes remaining in the analysis were subjected to differential expression testing. Tagwise dispersion estimates were obtained based on the trended dispersions, and statistical comparisons were performed using a generalised linear model followed by likelihood ratio tests. The P value was adjusted for multiple testing to obtain the false discovery rate (FDR) value using the default Benjamini-Hochberg method (Benjamini and Hochberg 1995). The results were considered statistically significant at false discovery rate (FDR) ≤ 0.05.
Pathway over-representation analysis was carried out using R-package clusterProfiler (Yu et al. 2012) and KEGG Pathway database annotations. Pathway analyses were carried out separately for up- and down-regulated genes which were differentially expressed at FDR ≤ 0.05.
cDNA was synthesized using a mixture of oligo(dT) and random primers (FIREScript RT cDNA synthesis kit, Solis BioDyne, Tartu, Estonia). q-PCR was performed in triplicates for each biological sample and the cDNA products were amplified using the EvaGreen assay system (Solis BioDyne, Tartu, Estonia). The following program was used for amplification: 95 °C for 15 min, followed by 40 cycles of 95 °C for 20 s, 62 °C for 20 s and 72 °C for 20 s. A dissociation curve was performed to verify the purity of the PCR product. q-PCR data was analysed using a comparative CT method and the relative expression of RNA was calculated based on the 2-ΔΔCTmethod (Winer et al. 1999). Beta-2-microglobulin (B2M) and TATA-binding protein (TBP) genes were used for normalisation. Primers were designed using Primer-BLAST and the preference was given to the sequences that spanned exon-exon junction. Primer sequences for all genes can be found in Table 1.
Statistical analyses of qPCR-derived gene expression results were carried out in R version 3.6.1. For each gene of interest, a linear mixed model was fitted using the lmer function from the lme4 package (Bates et al. 2015). For each model, treatment and time were included as interacting fixed effect terms and experimental batch was included as a random effect to account for the collection of samples from three experimental batches. Inter-group comparisons at specific timepoints were performed as post-hoc pairwise comparisons of estimated marginal means with Tukey correction for multiple testing, for which the emmeans function from the emmeans package was used. All graphs were generated using ggplot2 version 3.2.0 (Wickham 2009), except for the Venn diagram, for which the GOplot package version 1.02 was used (Walter et al. 2015).
Cytokeratins are important and major intermediate filaments of epithelial cells while Vimentin is the intermediate filament expressed in fibroblasts (Zeisberg and Neilson 2009). We used antibodies against cytokeratin and vimentin to evaluate if the primary cell culture we had established consisted mostly of epithelial cells, without fibroblast contamination. Incubation of the isolated cells with anti-Cytokeratin antibody showed a strong positive signal (Fig. 2a), whereas staining with anti-Vimentin antibody produced a fade signal which confirmed the presence of epithelial cells mostly.
Sequencing of the mRNA libraries yielded 6.2 ± 1.2 million reads (mean ± SD) per sample. After filtering for read quality, 98.7 ± 0.2% of the reads remained, out of which 96.3 ± 0.2% aligned to the B. taurus genome assembly (ARS-UCD1.2) at least once. Read counts were summarized at the gene level, and after filtering to remove genes considered not to be expressed in any of the groups, 10,636 genes remained and were subsequently tested for differential expression.
Non-contact co-culture model samples completely segregated from both control and contact co-culture model samples on the first principal component axis but exhibited considerably more noticeable intra-group variation compared to the other two groups (Fig. 3a). Contact co-culture with spermatozoa induced only minor changes to gene expression profiles of BOECs, with nine genes upregulated and one gene down-regulated (Fig. 3b). Complete list of differentially expressed (DE) genes in contact co-culture can be found in Supplementary Data (Table SI). The most upregulated gene in response to contact co-culture was DHRS3, while the only downregulated gene was RANBP3 (Fig. 4a). Surprisingly, the non-contact co-culture treatment induced more extensive gene expression changes in BOECs (52 genes upregulated, and 56 genes downregulated; Fig. 3b). List of DE genes in non-contact co-culture can be found in supplementary data (Table SII). The topmost upregulated genes in response to non-contact co-culture were metabolic enzyme genes, including the mono-oxidases CYP1A1 and CYP1B1, as well as TXNRD1, DHRS3, and PTGS2 (Fig. 4b).
Four upregulated genes (PLAU, TNFRSF11B, SCIN, and DHRS3) were common to both contact and non-contact co-culture (Fig. 4c). Of the four common upregulated genes, DHRS3 exhibited a higher degree of upregulation in contact co-culture (log2FC = 1.5) compared to non-contact co-culture (log2FC = 0.92). The other six DE genes in response to contact co-culture were unique to this treatment and interestingly included ATF3, a known negative regulator of PTGS2 (Hellmann et al. 2015).
To identify pathways that were over-represented among genes that were differentially expressed in BOECs in response to non-contact co-culture with spermatozoa, pathway over-representation analyses was conducted separately for significantly up-regulated and down-regulated genes based on KEGG pathway annotations for B. taurus.
Among up-regulated genes, six pathways were significantly enriched at FDR < 0.05, of which the most pertinent were retinol metabolism (bta00830), steroid hormone biosynthesis (bta00140), ovarian steroidogenesis (bta04913), and cytokine-cytokine receptor interaction (bta04060) (Table 2). The topmost enriched pathways also featured some of the most strongly upregulated genes: five featured CYP1A1, four featured CYP1B1, and two featured PTGS2 (Fig. 4d). No significantly enriched pathways were detected among down-regulated genes.
To examine how rapidly the BOECs respond to the stimuli of spermatozoa by changing the gene expression, we performed q-PCR analysis of four genes of interest for both contact and non-contact co-culture models after 30, 90, 150, and 240 min of co-culture. Specifically, we examined the expression of one gene shown to be up-regulated by RNA-seq in both treatments (DHRS3), two up-regulated genes only in response to non-contact co-culture (CYP1B1 and PTGS2), and one up-regulated gene only in response to contact co-culture (ATF3).
DHRS3 showed increased expression in response to both contact and non-contact co-culture (Fig. 5a), but was stronger in response to contact, in which a statistically significant increase in expression was detectable starting from 30 min (P = 0.02) and a highly prominent increase observed at 240 min. In contrast, a significant increase in DHRS3 in response to non-contact culture was not observed until 240 min had elapsed. DHRS3 expression was analysed following the removal of a prominent outlier in the non-contact group (2nd replicate, 90 min).
Interestingly, the expression of CYP1B1 was prominently increased in response to non-contact after 90 min (P < 0.001), but the expression returned to the level of control samples at 240 min (Fig. 5b). Similarly DHRS3, CYP1B1 expression showed a weaker increase in response to contact co-culture which peaked at 150 min (P = 0.04) and subsided afterwards.
PTGS2 expression was similarly increased at 30 min in response to non-contact (P = 0.003), but conversely showing a sustained increase in expression levels of PTGS2 until the final timepoint. In contact co-culture, PTGS2 showed a modest increase in response to spermatozoa observable at 90 min (P = 0.02), but this increase in the expression levels of the gene was not sustained until the final timepoint (Fig. 5c).
ATF3 expression (Fig. 5d) was increased at 30 min in response to both contact (P < 0.001) and non-contact co-culture (P < 0.001). However, the expression of ATF3 was not increased at any other subsequent timepoint in non-contact co-culture but remained significantly increased in response to contact co-culture until and including 150 min timepoint (P = 0.04) before subsiding.
While the capacity of spermatozoa to directly exert changes in maternal physiology has been known for some time (Fazeli 2011; Fazeli et al. 2004; Kodithuwakku et al. 2007; López-Úbeda et al. 2015) the possible mechanisms of communication between