New gene markers involved in regulation of granulosa cells development and differentiation towards endodermal and epithelial tissues – a new insight into the stemness specificity of ovarian follicular cells

The ovarian follicle consists of different types of cells, such as of oocytes (OCs), theca cells, ovarian surface epithelial cells and granulosa cells (GCs). GCs not only are fundamental for folliculogenesis and oogenesis, but they are also involved in cell communication. Releasing exosomes, small vesicles secreting miRNAs, and forming gap-junctions, they exchange nutrients and metabolites with OCs. The bidirectional stimulation allowed by gap-junctions promotes communication and reciprocal maturation stimulation between OCs and GCs, enhancing folliculogenesis and oogenesis [1].

Ovarian follicular cells show stem-like potential as they show changes in phenotype during long-term in vitro cultures and the ability to differentiate towards multiple cell lineages. For example, studies have demonstrated the ability of GCs under specific culture conditions, to express genes characteristic for heart morphogenesis but also osteogenic differentiation [2,3,4]. Understanding the process of differentiation of GCs towards different cell lineages and the molecular pathways underlying this mechanism is fundamental to uncover other possible stem cell markers of GCs [5].

The follicular epithelium is much more dynamic than the epithelium in the rest of the body. The follicular epithelium expands as the follicle grows. The number of layers of granulosa, forming the follicular epithelium, grow around the oocyte. Subsequently, the follicular epithelium expands laterally. However, the understanding of the follicular epithelial dynamics, which may disrupt if the follicle becomes atretic, remains poorly understood [6].

The endoderm is one of the three main germ layers and, after gastrulation, it primarily gives rise to the respiratory and digestive tracts together with being part of many glands, including thyroid, thymus, pancreas, and liver [7]. It is hypothesized that in vitro endoderm stem cells may give rise to a multitude of tissues. However, this type of stem cell is not yet fully characterized. Moreover, during embryonic development the endoderm forms the extraembryonic endoderm of the yolk sac where primordial germ cells are found. In vertebrates, these cells migrate to the gonad rudiments and arise outside the gonads.

The present study reports the transcriptomic profile of porcine GCs during their short-primary culture during 168 h. The main goal of presented study has been identify genes group response for processes associated with epithelium and endoderm development.

The Affymetrix® Porcine Gene 1.1 ST Array Strip for the microarray gene expression was employed for the analysis of porcine granulosa cells. The array method allowed us to study the gene expression of 27,558 transcripts at 0, 48, 96 and 144h of in vitro porcine granulosa cell culture. We selected genes with more than 2-fold changes and corrected p-values less than 0.05 for downstream analysis. A total of 3380 differentially expressed genes (DEGs) were identified according to the above criteria. Microarray gene expression analysis began by subjecting the list of DEGs to DAVID software, which showed that the genes can be assigned to 775 GO BP, 33 GO MF and 125 GO CC gene ontology terms. The focus of the study was on genes involved in regulation of granulosa cells differentiation towards endodermal and epithelial tissues. The DAVID software indicated the following GO BP terms, which cover the above processes: “developmental process”, “cellular developmental process”, “regulation of cell differentiation”, “epithelium migration”, “epithelial cell proliferation”, “epithelial cell migration”, “epithelial cell differentiation”, “epithelial cell development”, “endodermal cell differentiation”, “endoderm formation”, “endoderm development” and “epithelium development”. The 360 genes involved in those processes were clustered using hierarchical clustering and presented as heatmaps (Fig. 1). 205 genes were upregulated while 155 genes were downregulated. The 6 genes, their symbols, fold changes and corrected p-values are shown in table 1.

FIGURE 1

z-scores analysis reveals whether molecular function is more likely to be decreased (negative value) or increased (positive value). The z-scores were presented as segments of inner circles in the figure 2. Expression of most genes was increased (red dots) in all ontological groups. The z-scores of the GO BP terms had positive values, indicating the processes described by these GO BP terms were upregulated. The expression pattern did not change at any of the analyzed time points. Considering the above, the subsequent analysis was based only on 48h/0h comparison.

FIGURE 2

The interaction between selected ontological groups was examined and and are presented in as dendrogram (Fig. 3). Clusters contain functionally related genes based on their expression pattern. The middle circle represents a logarithm of fold change (logFC) of differentially expressed genes assigned to the studied GO terms. The GO terms are shown as the outer ring. The genes whose expression is downregulated form clusters marked by blue part of the middle circle and analogously, red indicates upregulated genes. Clusters of the same color over the entire width of the outer circle represent genes that are unique for a specific GO term. Clusters of different colors on the cross section of outer circle show sets of genes which are likely to be functionally related. The dendrogram showed that many genes belong simultaneously to “cellular developmental process”, “developmental process” and “regulation of cell differentiation”. The genes that are unique for a specific GO term belong mainly to “developmental process”.

FIGURE 3

In the gene ontology database, single genes may belong to many ontological terms. For this reason, we used plots with visualization of logFC values and relationship between genes and selected GO BP terms (Fig. 4). The relationship was also presented as a heatmap (Fig. 5). The strongest upregulated genes from examined GO BP terms included, among others: ITGA2-integrin, alpha 2; FN1-fibronectin 1; LAMB1-laminin, beta 1; LOX-lysyl oxidase; POSTN-periostin, osteoblast specific factor. The most downregulated genes are: HSD17B1-hydroxysteroid (17−beta) dehydrogenase 1; DAPL1-death associated protein–like 1; CXCL10-chemokine (C−X−C motif) ligand 10.

FIGURE 4

FIGURE 5

Finally, we used ReactomeFIViz app for investigation of functional interactions between proteins encoded by DEGs belonging to selected GO BP terms. Among the most significantly enriched functional interaction networks was FI networks for “positive regulation of cell proliferation” (Fig. 6).

FIGURE 6

Genes involved in the following processes: “developmental process”, “cellular developmental process”, “regulation of cell differentiation”, “epithelium migration”, “epithelial cell proliferation”, “epithelial cell migration”, “epithelial cell differentiation”, “epithelial cell development”, “endodermal cell differentiation”, “endoderm formation”, “endoderm development” and “epithelium development” were validated (Fig. 7). The main aim of the study was to validate the expression directions of the selected genes. In the case of 6 genes, the direction of expression was confirmed by the RT-qPCR method at individual intervals.

FIGURE 7

Several recent studies have suggested that every tissue and organ contain a population of cells with stem-like properties, including the ovary [13,14,15]. Stem cells were found both in mouse and human ovaries, and they were observed to be involved in the processes of oogenesis and folliculogenesis. Specifically, granulosa cells (GCs) show programmed stem cell properties during in vitro culture [16,17]. These cells surround the oocytes and are fundamental for the functioning of the reproductive system and the maintenance of pregnancy releasing hormones. As these stem cells sustains the formation of the oocyte and the follicle in the postnatal mammalian ovary, the belief that females have a determined number of primordial follicles formed in the embryonic period was suggested to be wrong [18]. The great plasticity of GCs, showing stem cells properties, has been suggested as a therapeutic intervention for regenerative medicine and transplantology. Multiple studies present the ability of the GCs to go through transdifferentiation, by which already differentiated cells can be reprogrammed towards a different cell lineage [19].

Further understanding of the molecular basis underlying these processes and of the cells in the reproductive system may reveal the functioning of determined clinical diseases, potentially leading to therapies and treatments of diverse conditions, such as infertility. Microarray analyses identified a group of genes with altered levels of expression. Some of these affected genes are associated with cell development and differentiation towards endodermal and epithelial tissues.

Epithelial tissue shows polarization. In many cases, neoplastic transformation is accompanied by the loss of the polarized phenotype typical of epithelial cells. Apical transport of protein takes place in epithelial tissue in two ways: direct and indirect pathways. Proteins transported indirectly are first directed to the basolateral surface. After proteins undergo endocytosis and are directed further to the apical surface by the transcryptotic pathway [20]. One of the proteins that play an important role in indirect protein transport is the MAL protein family, which has been detected in many normal epithelial cells isolating from the respiratory system, gastrointestinal tract, genitourinary tract and endocrine glands. Specific changes in MAL (T-cell differentiation protein) and MAL2 (T-cell differentiation protein 2) expression have been observed in certain types of cancer[21]. Lak et al. suggest that overexpression of MAL2 may promote the proliferation of breast cancer cells, while its knockout may reduce the proliferation of breast cancer cancer cells [22]. A study by Zhou et al. suggest that tumor tissue cells and breast cancer cell lines showed decreased expression of ITM2A (integral membrane of 2A protein). On the other hand, overexpression of this gene significantly inhibited the proliferation of breast cancer cells, which qualifies ITM2A as a prognostic marker [23]. Moreover, the ITM2A gene, encoding a porcine protein that is highly homologous to the integrated 2A (ITM2A) membrane protein of humans and mice, was herby downregulated. The expression of this gene is observed in fat, spleen, lungs, muscles, liver, small intestine, large intestine and kidneys [24]. ITM2A was also observed to have a therapeutic potential for epithelial ovarian cancer, as it acts as a chemosensitizer and induces cell cycle arrest [25].

The experimental gene overexpression also had an inhibitory effect on the proliferation of breast cancer, DAB1 (DAB protein adaptor 1) [26].This gene encodes for an adaptive protein necessary for intracellular Reelin signal transmission, which, in turn, controls the migration and differentiation of postmitotic neurons in the brain development process [27]. Moreover, this gene plays important roles in cell morphogenesis, differentiation and development, specifically in neuronal differentiation of granulosa cells [28].

Next downregulated gene is NEBL (nebulette). This gene encodes a nebulin-like protein that binds to actin and is highly expressed in the myocardium, playing an important role in Z-disk assembly [29]. It is abundant in the heart muscle, especially in the myocardium, where it assembles cardiac myofibril, combining sarcomeric actin with desmin fibers in sarcomeres [30,31].

Another gene showing downregulated expression in the presented studies is DAPL1 (death associated protein-like 1). This gene is involved in the early stages of epithelial differentiation, but also in the processes of apoptosis. Increased levels of expression of DAPL1 were observed in studies on uterine inflammation in dairy cows, analyzing levels of its transcripts in the endometrium [32]. It may show a different level of expression depending on the tissue [33]. Moreover, Chen et al. proved that DAPL1 is an important factor regulating steroidogenesis [34]. Zhang et al. noted that DAPL1 is less expressed in breast cancer cells than in other healthy tissues. Low expression of this gene was correlated with poor prognosis of survival in patients with breast cancer [35].

Finally, HSD17B1 was the most downregulated gene. Its product enzyme catalyzes the conversion of estrone to estradiol, and genetic variations of this gene are associated with the risk of breast and endometrial cancer, as well as modifying the susceptibility to endometriosis [36,37]. Its expression is observed in ovaries, placenta, testis, endometrium, malignant and normal breast epithelium, and prostatic cancer cells [38,39].

The changes in expression during long-term primary in vitro cultures of genes belonging to ontological groups associated with endodermal and epithelial tissues formation, differentiation and migration suggest that these genes response for endoderm and epithelial lineages formation. This study serves as a basic molecular reference for the potential further research on the protein level elucidating the functional meaning of several gene markers and on the mechanisms underlying GCs differentiation toward the lineages of interest.