Nucleotide, ribonucleotide and ribonucleoside binding belongs to differentially expressed genes in porcine epithelial oviductal cells during longterm primary cultivation

The oviduct (uterine tube or Fallopian tube) of the domestic mammals plays a crucial role in providing an optimal microenvironment for final gamete maturation and transport, fertilization, and early embryo development [1]. Moreover, these main functions of the oviduct are highly based on its contractibility, the capacity of secreting oviductal fluid into the lumen and other oviductal components include hormones, growth factors and their receptors [2]. The characteristic feature of oviduct epithelial cells (OECs) are cilia, which facilitate the transport of the oocyte and/or the embryo. The rest of the cells in the OECs are secretory cells, that produce fluid secreted into the fallopian tube. The histological architecture of oviducts undergo substantial modification as response to reproductive cycle during lifespan of females.

Establishment and characterization of in vitro cell cultures (IVC) may be one of the most prospective methods of tissue and organ regeneration, with a large probability for application as grafts in regenerative medicine. However, despite the fact that the stem-like specificity of cells cultured primarily in vitro is well known, the cellular physiology as well as the molecular properties require further development. Long-terms primary cell cultures seem to be a very good tool for detailed characterization of different cell populations by recognition about the transcriptomic and proteomic profile and the changes that occur during cultivation.

Recent experiments indicated that the gene expression profile changes within the OECs was also accompanied by significant changes in cellular proliferation capability in vitro. The results presented in previous studies are a valuable contribution for better understanding and elucidating many biochemical pathways and morphological/metabolomic factors existing in epithelial oviductal cells. Thus, employing a microarray approach, the molecular level processes identified were angiogenesis and blood vessels development [3], carbohydrates [4] and amino acids [5] metabolism. Furthermore, and plasticity of porcine cells associated with reproduction processes [6,7].

The gene expression profile in OECs primary cultured for a long-time using microarray assays is demonstrated in this study, as part of a project aimed at the molecular characterization of this cell population. Affymetrix microarray assays identified the regulatory peptides and enzymes involved in nucleotide, ribonucleotide and ribonucleoside binding pathways. In this study we demonstrated the transcripts expression variability of genes belonging to “adenyl nucleotide binding”, “adenyl ribonucleotide binding”, “ribonucleotide binding”, “ribonucleoside binding” gene ontology biological process terms (GO BP) during OECs long-term primary culture in vitro.

Whole transcriptome profiling by Affymetrix microarray allowed the analyzation of gene expression changes between 7, 15 and 30 days of porcine oviductal epithelial cells culture. Through use of Affymetrix® Porcine Gene 1.1 ST Array Strip, we examined expression of 12257 transcripts. Genes with fold change higher than abs (2) and wit corrected p-value lower than 0.05 were considered as differentially expressed. This set of genes consisted of 2533 different transcripts. The first detailed analysis was based on GO BP identification of differentially expressed genes belonging to the significantly enrichment GO BP terms.

DAVID (Database for Annotation, Visualization and Integrated Discovery) software was used for extraction of gene ontology biological process term (GO BP) that contains differently expressed transcripts. Up- and down-regulated gene sets were subjected to DAVID searching separately and only the gene sets which had an adj. p-value lower than 0.05 were selected. The analysis of the DAVID software showed that there were 657 differentially expressed genes belonging to the Gene ontology terms. In this paper, we focused on 222 genes that belong to “adenyl nucleotide binding”, “adenyl ribonucleotide binding”, “ribonucleotide binding”, “ribonucleoside binding” GO BP terms. These sets of genes were subjected to the hierarchical clusterization procedure and presented as heatmaps (Fig. 1). The gene symbols, fold changes in expression, Entrez gene IDs and corrected p-values of those genes are also shown in table1.

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

Chosen GO BP terms contain 222 differentially expressed genes. Therefore, we calculated the mean value of mean of fold change ratio of each gene between 7, 15 and 30 days of culture. Based on that criteria we chose the 10 most down-regulated and the 10 most up-regulated genes to further analysis.

In Gene Ontology database genes that formed one particular GO group can also belong to other different GO term categories. By this reasoning, we explored the gene intersections between 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-generated an interaction network among differentially expressed genes belonging to each of the selected GO BP terms. Using such a prediction method provided a molecular interaction network formed between protein products of studied genes (Fig. 5).

Figure 5

The morphological modifications of oviducts in response to the female reproductive cycle are well established [12]. However, detailed characterization at the molecular level remains a unresolved. Establishment and depiction of primary cell cultures in vitro appears to be a good approach for comprehensive elucidating many biochemical pathways existing in ovaries. Therefore, the goal of the present project is to describe molecular changes in OECs during primary in vitro long-term culture, through employing microarray approach. Previous studies have shown various aspects of OECs’ morphological modifications and transcriptomic profile changes during in vitro cultivation [3, 4, 5]. The emphasis of the present study, is on groups of genes associated with nucleotide/ribonucleotide binding mechanism.

From the whole transcript profile after the microarray assay, during long-term in vitro cell culture, the lowest transcript levels were observed for OASL, 2′-5′-oligoadenylate synthetases (OASs) family member. OASs proteins belong to a group of IFN-induced antiviral proteins that are pivotal in the host defense mechanism against viral infection [13,14]. 2’-5’-oligoadenylate synthetase like (OASL) does not have the typical 2’-5’-OAS activity (to catalyze the synthesis of 2′-5′-oligoadenylates (2-5A) from ATP for viral RNA degradation), but have double-stranded RNA binding ability, and thus demonstrates antivirus activity [15,16]. Two homologs of human OSAL (Oasl1 and Oasl2) have been identified in mice [17]. Provided by Evsikov et al. [18], analysis of the cDNA library of Mus musculus fully grown oocytes (FGOs) reported Oasl family members transcripts are expressed. Thus, the results suggest important role of OASs family to also activate molecular pathways underlying the oocyte-to-embryo transition. Other studies by Yan et al. [19], employ targeted deletion of one of the Oasl genes, Oasl1d, which causes reduced fertility in females but not in males, demonstrating that members of this family are maternal effect genes, suggesting that OASL proteins may suppress the IFN/OAS/RNase L-mediated mRNA degradation, protecting oocyte [19]. A similar expression pattern was observed for another OASs family member, 2’-5’-oligoadenylate synthetase 1 (OAS1). Talukder et al. [20] described increased interferon-stimulated genes expression (including OAS1) in medium after co-culture of bovine oviduct epithelial cells with zygote up to embryos had reached the 16-cell stage.

The PIM family of serine/threonine kinase proteins consists of three, highly homologous, isoforms PIM1, PIM2 and PIM3. The PIM genes’ expression is induced by a variety of cytokines, growth factors and mitogens [21]. The Pim1, as pro-survival kinase, has been implicated in tumorigenesis at different levels, especially in the control of cancer cells proliferation, migration and apoptosis [22]. These proteins have been labeled as “weak oncogenes” given that their overexpression induces hyperproliferation but no tumors in many cases [23]. Subsequent studies by Jiménez-García et al. [24] have described generation of two conditional PIM1 and PIM2 transgenic mice that overexpress PIM1 or PIM2 in mammary gland, uterus and ovary to characterize the proto-oncogenic role of PIM1/PIM2 in female hormone-dependent tissues. PIM1/2 overexpression induced hyperproliferation and tumors in the female reproductive system and evoked a high inflammatory response and stem markers [24]. Other research highlighted the role of Pim1 in ovarian cancer (OC) development [25]. Currently, it appears that Pim1 is overexpressed in OC and is correlated with the poor overall survival of patients. Moreover, Pim1 may significantly enhance glycolysis in OC cells. In conclusion, these results suggest that silencing or overexpressing Pim1 can suppress or promote OC cell proliferation [25]. PIM1 transcripts expression significantly decreases over the time of cultivation. Thus decreasing activity, cell viability, because the PIM family of proteins has been implicated in the regulation of apoptosis, metabolism, cell cycle, homing and migration.

In comparison, the presence of numerous genes, within the analyzed ontological groups, with a different mRNA expression pattern during the culture was demonstrated. We observed a growing transcript levels for subsequent measurement points during long-term cultivation. Definitely the highest upward trend was shown by ACTA2, a gene belonging to all GO terms of interest. Expression of α-smooth muscle actin (α-SMA) (encoded by the gene ACTA2) is involved in vascular contractility and blood pressure homeostasis [26]. Studies with mesenchymal stem cells (MSC) emphasized the role of Rho GTPases, Rac1 and Cdc42 in TGFβ-induced α-SMA expression in MSC [27]. A murine oviductal cell line (MOE^LOW) was also developed and then continuously passaged in culture to mimic cellular aging (MOE^HIGH) [28]. The MOE^HIGH cellular model during molecular analyses exhibited a downregulation of Acta2 transcript levels.

A member of the ATP-binding cassette (ABC) family, ABCA1 also showed increased expression in cells during long-term cultivation, and was included in all discussed GO BP terms. It is well established that ABC family of proteins play a key role in controlling cellular and total body lipid homeostasis [29,30]. ABCA1 is responsible for mediating of cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I and to lipoprotein particles (e.g., high-density lipoproteins (HDL)) transport [31]. Transcriptomic analysis of the bovine oviduct epithelium exhibit lower ABCA1 transcript levels in the ipsilateral side of the ampulla [32]. The promoter hypermethylation of ABCA1, both in ovarian cancer cell lines and ovarian cancer patient samples, was associated with poor prognosis in ovarian cancer patients [33]. Other investigators described expression of ABC family members (including ABCA1) in mouse spermatozoa in the seminiferous tubules and epididymis [34]. Furthermore, authors have shown that ABCA1 antibody treatment of immature spermatozoa reduces cholesterol efflux to lipid acceptors and inhibits in vitro fertilization. The findings suggest that these transporters play a role the process of sterol efflux which renders spermatozoa capable of interacting and fertilizing an oocyte [34].

The current results characterize a large scale the transcriptomic profile of this cell population during long-term in vitro culture. The results presented in this paper indicate potential markers whose decreased expression during the cultivation may affect the reduction of activity, cell viability.