Genes regulating biochemical pathways of oxygen metabolism in porcine oviductal epithelial cells during long-term IVC

Oxygen is an indispensable element of aerobic metabolism, playing a crucial role in cell respiration and influencing energy production. Oxygen atmospheric concentration is approximately 21%; however, mammalian cells are generally adapted to much lower oxygen levels. The oxygen supply is determined by the metabolic activity and functional status of organs and tissues, ranging from 2–9% in vivo [1,2]. An imbalance between the production of reactive oxygen species (ROS) and their detoxification leads to oxidative stress, resulting in cellular damage. The overproduction of ROS accompanied by impaired antioxidant system in cells could contribute to inflammation and senescence. The most destructive effect of ROS is their ability to cause single- and double-strand DNA breaks. Prolonged generation of ROS may have profound effects on epigenetic regulation of gene expression due to their interaction with several chromatin-modifying enzymes [2]. Moreover, ROS can lead to failure of membrane fluidity and function, as well as activation of apoptosis induced by lipid peroxidation of cell membrane or organelles [3]. Free radical-induced damage caused by oxidative stress plays a significant role in the pathogenesis of many diseases [4, 5, 6, 7].

Oxygen metabolism has an important role in the normal functioning of reproductive system and in the pathogenesis of infertility in females. Oxidative stress seems to be responsible for the initiation or development of reproductive organ diseases, including polycystic ovary syndrome, endometriosis, preeclampsia, etc. [8, 9, 10]. However, disturbances in oxygen metabolism may take place outside of pathological conditions. Ovulation is considered to be a major stressor for surrounding cells, causing trauma-induced inflammation and unbalanced ROS levels, leading to local oxidative stress [11,12].

Animal oviducts and human Fallopian tubes represent a part of the female reproductive tract subjected to periodic exposure to free radicals. Whereas ROS contained in follicular fluid mainly affect the fimbriae, the backward flow of menstrual debris through the tubes, called menstrual reflux, may influence the oxygen tension along the entire uterine tube lumen. Exposure to free radicals, accompanied by local oxidative stress and inflammation, brings a risk of malignant transformation of Fallopian tube secretory epithelial cells, leading to high grade serous ovarian carcinoma (HGSC).The proper functioning of cellular antioxidant system helps to decrease the negative effects of local stressors, such as pelvic inflammation or retrograde menstruation-derived Fe³⁺, and may contribute to prevention of cancer development [11,13].

Moreover, it has become clear that the oxygen levels influence fertilization and pre-implantation development of the embryo. The oviductal epithelial cells play an important rolein these processes by providing appropriate microenvironment within the uterine tube. Oviductal secretory fluid protects gametes from environmental stress to ensure embryo quality and positive pregnancy outcome [14,15]. The activity of antioxidant enzymes in oviductal epithelial cells is crucial for oxidative stress avoidance and contributes to the maintenance of low oxygen concentration within the uterine tube. Multiple meta-analyses have demonstrated improved pregnancy rates and live birth rates at 5% oxygen in the case of in vitro fertilization. While the mechanisms underlying the reduced developmental efficiency of embryos cultured at higher oxygen concentrations are not well understood, oxidative stress is supposed to be the potential cause of suboptimal embryo development.

However, a majority of clinical embryology studies have been performed at atmospheric oxygen concentrations, which may be explained by the additional cost associated with reducing the oxygen level in the culture environment [16].

Here, using the microarray approach, we aimed to investigate whether the atmospheric oxygen level could affect the expression of genes related to oxygen metabolism in porcine oviductal epithelial cells during long-term in vitro culture.

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 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 consisted 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 differentially expressed transcripts. Up and down regulated gene sets were subjected to DAVID searches separately, with only gene sets with adj. p-value lower than 0.05 selected. The DAVID software analysis showed that the differently expressed genes belonged to 657 Gene ontology terms. In this paper, we focused on 90 genes that belong to “cellular response to oxidative stress” and “cellular response to oxygen-containing compound” 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 presented 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 90 differently expressed genes. Therefore, we calculated the mean fold change of each gene between 7, 15 and 30 days of culture. Based on that criteria, we choose 10 most downregulated and 10 most upregulated genes to further analysis.

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

Figure 3

Figure 4

STRING-generated interaction network among differentially expressed genes belonging to each of selected GO BP terms. Using such prediction method provided us with a molecular interaction network formed between protein products of studied genes (Fig. 5). Finally, we investigated the functional interactions between the chosen genes with the REACTOME FIViz app to the Cytoscape 3.7.2 software. The results were shown in (Fig. 6).

Figure 5

Figure 6

Despite the essential role of oxygen for aerobic organisms, it may cause harmful reactions such as oxidative injury and senescence in living cells. ROS, produced by hyperoxic conditions, appear to be directly associated with the aging process. Cells cultivated under hyperoxic conditions show characteristics of cellular senescence, including growth arrest, lipofusin accumulation and protein oxidation [23,24]. All this leads to mitotic arrest, appearing to be caused by ROS-triggered malfunction of the cell cycle machinery [25].

Given the important role of maintaining the balance between the production of ROS and antioxidant defence in the proper functioning of reproductive system, in this study we have analysed the expression of genes related to oxygen metabolism in porcine oviductal epithelial cells during long-term culture. The genes belonging to “cellular response to oxidative stress” and “cellular response to oxygen-containing compound” GO BP terms were differently expressed. However, most of the genes belonging to the former ontology group were upregulated after the 7 days days of culture, when compared with the first day. This may be due to the cell exposure to atmospheric oxygen tension, which is much higher than in oviduct, and may cause excessive ROS accumulation in culture medium in vitro [26].

Here, we would like to focus on eight genes belonging to both analyzed groups: CCNA2, CDK1, PRDX3, MDM2, FAS, TPM1, CST3 and ARG1. The first two genes - CCNA2 and CDK1–code proteins necessary for cell cycle progression through checkpoints. Cyclins, such as CCNA2, form complexes with cyclin dependent kinases (CDK), making the transition to the next phase of cell cycle possible. This promotes cell growth, unless inhibited by tumor suppressors, such as p53 or CDK-inhibitors (CD-Ki’s), such as p21 or p27. Over expression of cell cycle inducers, such as the CDK’s and cyclins, and under-expression of inhibiting factors are frequently found in tumors. Thus, Horree et al. reported a significantly higher expression of cyclin A in tubal metaplasia compared to the normal endometrium [27]. CDK1gene encodes a protein binding to all cyclins, which results in the phosphorylation of the Rb protein and the expression of genes regulated by E2F transcription factors. In the absence of CDK1, the embryos fail to develop into the morula and blastocyst stages. Therefore, CDK1 has been suggested to be the only essential cell cycle CDK [28].

In our study, CCNA2 and CDK1 genes exhibited the similar pattern of expression during the culture – they were up-regulated after 7 and 14 days and down-regulated at D30. We suggest that down-regulation of CCNA2 and CDK1 could be associated with the signs of cellular senescence resulting from longterm culturing. Furthermore, we have also observed down-regulation of theARG1 gene, which has been reported to possess anti-inflammatory properties, at D15-D30 [29].

The next analyzed gene, PRDX3, encodes an antioxidant enzyme that modulates cellular response to reactive oxygen species. PRDX3 enzyme is localized exclusively in mitochondria. It has been demonstrated that PRDX3 functions in parallel with mitochondrial glutathione peroxidases to protect mitochondria against oxidative injury by scavenging H₂O₂ [30,31]. The study of Bonfim et al. revealed that bovine blastocysts under high oxygen partial pressure exhibited elevated ROS accumulation and increased expression of PRDX1 and PRDX3,confirming the correlation between oxidative stress level and expression of the aforementioned genes [32].

We have also observed up-regulation of the MDM2 gene, which encodes an oncoprotein targeting the p53 tumor suppressor for proteasomal degradation [33,34]. Due to the ability to inhibit p53 activity, MDM2 promotes cell survival and cell cycle progression.

TPM1 is the next gene belonging to both studied ontology groups, which was upregulated after D7 of culture. TPM1plays a key role in cytoskeleton remodelling and stress fibre formation in response to oxidative stress [35]. On the other hand, there have been studies demonstrating that TPM1 functions as a tumour suppressor gene in chosen tumour cell lines [36]. In our recent research, we have found that TPM1 was involved in cell migration mechanisms and up-regulated in porcine oocytes before in vitro maturation(IVM) as compared with post-IVM expression analysis [37].

Interestingly, the obtained data revealed upregulation of genes encoding the proteins, which ensure the optimal conditions for spermatozoa transfer through the female reproductive tract, as well as fertilization. These genes are cell surface death receptor (FAS) and cystatin (CST3). The former is involved in the processes ensuring that spermatozoa in the tubal sperm reservoir escape phagocytosis by polymorphonuclear leukocytes. This may be due to the ability of the Fas–FasL system to eliminate cytotoxic T lymphocytes and NK cells to prevent them from attacking spermatozoa or the early embryo in the oviduct [38]. The second gene - CST3 – encodes a protein prominently expressed in the female reproductive tract, including the epithelial lining, particularly at times around ovulation.CST3 may help spermatozoa enter the upper reproductive tract by enhancing their motility [39].

In conclusion, our study revealed that atmospheric oxygen affects the expression of genes involved in oxygen metabolism. The most of the “cellular response to oxidative stress” genes were upregulated during the culture. However, we did not observe any main trend in changes within the “cellular response to oxygen-containing compound” ontology group, where the gene expression levels were changed in various manners.