69 Can Bovine Sperm Interaction with the Oviduct Cells After Artificial Insemination Affect the Transcriptome Profile of the Oviduct?

Abstract

During their transit through the female genital tract, spermatozoa bind to oviduct cells, where they are maintained alive for a long period. Although some studies have been carried out to evaluate the effects of the oviduct on spermatozoa, less is known about the effect of spermatozoa on the oviduct. The aim of this study was to evaluate the effect of sperm on the transcriptome profile of the bovine oviduct after fixed-time AI (FTAI). Nellore cows submitted to FTAI were randomly inseminated using 8 × 10⁶ frozen–thawed spermatozoa (sperm group, n = 9) or saline solution (control group, n = 9). Eighteen hours after FTAI, the cows were slaughtered, and the cells from oviducal isthmus were collected and stored in liquid nitrogen until transcriptome analysis. For RNA extraction, 3 pools of 3 oviducts/cows from each group were formed. Total RNA was extracted and the RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The transcription profiling from each group was performed by HiSEqn 2500 (Illumina Inc., San Diego, CA, USA). After sequencing, the data were analysed by edgeR software, and differential gene expression analysis between sperm and control group was performed using the generalized linear model (P = 0.05). The P-value was adjusted by false discovery rate (FDR) Benjamini-Hochberg method, to avoid false positives. Moreover, using mRNA expression levels (log fold change and P-values) as coefficients, functional enrichment analysis was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. A total of 9,374 genes were found to be expressed in bovine oviducal isthmus, of which 219 genes were differentially expressed between sperm and control group. Differentially expressed genes genes were classified according to 3 major GO classifications, with 10 gene categories in molecular function, 3 gene categories in cellular component, and zero categories in biological process. In the molecular function category, genes related to calcium ion binding (n = 113), molecular transducer activity (n = 111), and receptor activity (n = 111), were predominant. For cellular component, the highest number of genes (n = 176) was related to extracellular space. Moreover, KEGG assignments were used to classify the functional annotations of the pathways genes to further understand the biological functions of the expressed genes. It was shown that steroid hormone biosynthesis, arachidonic acid metabolism, calcium signalling, cytokine-cytokine receptor interaction, neuroactive ligant-receptor interaction, extracellular matrix-receptor interaction, cell adhesion molecules, coagulation and complement cascades, ovarian steroidogenesis, protein digestion and absorption and mineral absorption pathways had genes with higher or lower expression than the median of gene expression out of the tested category. The results suggested that bovine sperm affect the transcriptome profile of the bovine oviduct after FTAI. Moreover, based on the functional enrichment analysis, an effect of the sperm-oviduct interaction on some pathways analysed by GO and KEGG was identified.

This research was financially supported by FAP-DF, FAPESP, CNPq.