Genes encoding proteins regulating fatty acid metabolism and cellular response to lipids are differentially expressed in porcine luminal epithelium during long-term culture

Fertility is a complicated phenomenon success of which is influenced by many factors. It is known that oocytes mature gradually, with only oocytes maintained under excellent conditions are able to progress past the MII stage [1]. Many studies focus on factors determining the reproductive capacity of oocytes, expression of their genes and in vitro maturation. It is obvious that oocytes play a key role here, but if it were not for the mechanisms surrounding the ovum, it would be impossible to obtain the right conditions for fertilization [2]. Among many factors, the epithelium lining the lumen of the fallopian tubes is very important for the development of the oocyte and its subsequent fertilization [3]. The oviductalepithelium is characterized by the presence of ciliary cells, supporting the movement of cumulus-oocyte complexes towards the uterus. By interacting with the semen, the epithelium of the fallopian tube allows the sperm to acquire the ability to fertilize [4]. The transport of spermatozoa through the female reproductive tract seemsto be an especially important stage [5]. So far, the exact molecular mechanisms of these changes have not been discovered. Hence, understanding the metabolism of oviduct epithelial cells and the level of expression of individual groups of genes seems to be a way to deepen the knowledge about the broadly understood reproduction.

The aim of our research was to maintain the culture of epithelial cells of the porcine fallopian tube in primary culture. We carried out this culture over a long term, lasting up to 30 days. We have made observations of biochemical and morphological changes occurring during the cell culture. We analyzed the expression profile of genes responsible for regulating the metabolism of fatty acids and cellular response to lipids. As there are reports of the negative effects of non-esterified fatty acids (NEFA) on the reproductive system [5,6], we decided to study their metabolism in oviductal epithelial cells (OECs).

The research related to animal use has been complied with all the relevant national regulations and instructional policies for the care and use of animals. Bioethical Committee approval no. 83/2012/DNT.

Whole transcriptome profiling with Affymetrix microarrays allows us to analyze the gene expression changes between 7, 15 and 30 days of porcine oviductal epithelial cell culture. Using Affymetrix® Porcine Gene 1.1 ST Array Strip, we have examined the expression of 12257 transcripts. Genes with fold change higher than abs (2) and with corrected p-value lower than 0.05 were considered as differentially expressed. This set of genes consists of 2533 different transcripts.

DAVID (Database for Annotation, Visualization and Integrated Discovery) software was used for extraction of gene ontology biological process terms (GO BP) that contain differently expressed transcripts. Up and down regulated gene sets were subjected to the DAVID search separately and only gene sets with adj. p-value lower than 0.05 were selected. The DAVID software analysis showed that the differently expressed genes belonged to 657 Gene ontology terms. In this paper, we focused on 166 genes that belong to “cellular lipid metabolic process”, “cellular response to lipid” and “fatty acid beta-oxidation”GO BP terms. These sets of genes were subjected to hierarchical clusterization procedure and presented as heatmaps (Fig. 1). The gene symbols, fold changes in expression, Entrez gene IDs and corrected p-values of these genes were shown in table 1.

Figure 1

The enrichment of each GO BP term was calculated as a z-score and shown on the circle diagram (Fig. 2).

Figure 2

The chosen GO BP terms contain 166 differently expressed genes. Therefore, we calculated the mean fold change ratio value of each gene between 7, 15 and 30 days of culture. Based on that criteria, we chose the 10 most downregulated and 10 most upregulated genes for further analysis.

In Gene Ontology database, genes that form one particular GO can also belong to other GO term categories. For this reason, we explored the gene in tersections between the selected GO BP terms. The relation between those GO BP terms was presented as circle plot (Fig. 3) as well as heatmap (Fig. 4).

Figure 3

Figure 4

STRING interaction network was generated among the differentially expressed genes belonging to each of the selected GO BP terms. Using such prediction method provided us with a molecular interaction network formed between protein products of the studied genes (Fig. 5).

Figure 5

Finally, we investigated the functional interactions between the chosen genes with REACTOME FIViz app to the Cytoscape 3.6.0 software. The results were shown in (Fig. 6).

Figure 6

Increasing food demand observed nowadays is connected with the necessity of more efficient acquisition of raw animal materials. Attempts are being made to increase the reproductive capacity of various animal species. Most often, this is achieved through introduction of more caloric diets. The metabolic pathways associated with fat management are also changing. Increasingly, the cause of metabolic disorders is attributed to elevated NEFA levels in the blood. It has also been shown that high values of these fatty acids may have an effect on the deterioration of reproductive rates in animals and humans [10, 11, 12].Studies using in vitro cultured bovine OECs have shown that increased NEFA levels may adversely affect the physiology of these cells, but not their morphology. The consequence of these physiological changes may be a decrease in the reproductive capacity of dairy cattle [13]. Recent studies have described the role of fatty acids in the function of sperm in the oviducts of hens [14]. The important role of fatty acids in sperm viability has been proven in this study. On the other hand, other studies carried out on in vitro cultured porcine oocytes showed a significant improvement in both nuclear and cytoplasmic maturation of oocytes after treatment with 100 μ ALA (alpha-linolenic acid) [15].

In our research we focused on genes related to the widely understood metabolism of fatty acids. The results of this study showed genes belonging to three ontological groups, each of which was associated with lipids. Not all these genes are closely related to the reproductive system in pigs, including the fallopian tube. Nevertheless, some of these genes are linked to animal reproductive aspects.

In our in vitro OEC culture studies, we demonstrated significant changes in expression of genes belonging to three main ontological groups: “cellular lipid metabolic processes”, “cellular response to lipid” and “fatty acid beta-oxidation”. The last-mentioned group included two genes: PPARD and ACADL, which showed up-regulation after in vitro culture (IVC). The PPARD gene (peroxisome proliferator activated receptor delta) participates in fat metabolism, but also has a role in differentiation towards adipocytes [16].

In “Cellular lipid metabolic processes” and “fatty acid beta-oxidation” the general trend of expression was increasing, as the genes showed up-regulation after the first day of culture. The “cellular response to lipid” group, on the other hand, showed more differentiated expression pattern, with a slight down-regulation predominance after the first day of the experiment.

The “cellular response to lipid” group was represented by the following genes: MUC1, CYP24A1, KLF4, IL24, SNAI2, CXCL10, which all showed down-regulation. The remaining 3 genes from this group (PPARD, TNC, ABCA10) showed up-regulation. The above mentioned PPARD gene is a member of all three described groups of genes. The remaining 12 genes form the “cellular lipid metabolic processes” GO, and showed increased (LIPG, ARSK, ACADL, FADS3, P2RX7, ACSS2, PPARD, KITLG), as well as decreased expression (SPTLC3, ERBB3, KLF4, CRABP2).

The CRABP2 gene (cellular retinoic acid binding protein 2), belonging to the family of lipoproteins, plays a very important role in the metabolism of retinoic acid (RA) and its intracellular transport. Studies conducted on mouse embryos have shown that both CRABP1 and CRABP2 transcripts show specific distribution patterns and play an important role in the embryo development processes [17].

Expression of the SPTLC3 gene (serine palmitoyl transferase long chain base subunit 3) leads to the formation of the serine palmitoyl transferase subunit, which takes part in the biosynthesis of sphingolipids [18].

The MUC1 gene expression is responsible for the formation of mucin proteins, which are characteristic for epithelia. Mucins are responsible for creating a barrier on the surface of the epithelium and are also involved in intracellular signal transmission [19]. MUC1 is considered to be one of the epithelial markers of porcine embryo pluripotent cells [20]. A decrease in MUC1 gene expression in the endometrium is responsible for a decrease in the rate of embryo implantation in humans [21].

The KITLG gene (KIT ligand), exhibiting increased expression in the OEC in vitro culture, is related to the development of, among others, stem cell or germ cell populationin reproductive organs, and its mutations may lead to porcine infertility [22].Its role is also described in maintaining the viability of primary germ cells. Studies of porcine uteri suggest that it also has a role in uterine intracellular communication [23].

The family of ABC transporters, to which belongs the ABCA1 gene (ATP binding cassette subfamily A member 1) shows an involvement in the metabolism of steroids and fetal development, which is important in gametogenesis and folliculogenesis [24].

The bovine oviduct epithelial cells (BOECs) were examined for the effect of increased NEFA concentration. These studies showed that elevated NEFAs had an effect on the metabolism of BOECs. Additionally, the study suggests that the fallopian tube can significantly influence the conditions of early embryo development [25].

Significant changes in gene expression observed in our in vitro cultured OECs may indicate their potential role as markers of OECs development in pigs. Thanks to advanced molecular analyses we can get closer to a deeper understanding of the mechanisms controlling the changes in the epithelium of the fallopian tube and its correlation with the reproductive cells, both male and female.