Results
Whole transcriptome profiling by Affymetrix microarray allowed us to analyze the expression changes in GCs, after 1, 7, 15 and 30 days of culture. By Affymetrix® Human HgU 219 Array, we examined the expression of 22480 transcripts. Genes with a fold change higher then abs (2) and with a corrected P-value lower than 0.05 were considered as differentially expressed. This set of genes consisted of 2278 different transcripts.
DAVID (Database for Annotation, Visualization and Integrated Discovery) software was used for extraction of the gene ontology biological process terms (GO BP). Up and down-regulated gene sets were subjected to the DAVID search separately and only gene sets of adj. P-values <0.05 were selected. The DAVID software analysis showed that differentially expressed genes belonged to 582 Gene Ontology Biological Process (GO BP) terms and 45 KEGG pathways. In this report, we focused on ‘osteoblast differentiation’ GO BP term. This set of genes was subjected to hierarchical clusterization procedure and presented as a heatmap (Fig. 1). The gene symbols, fold changes in expression, Entrez gene IDs and corrected P-values of these genes were shown in Table II.
STRING interaction network was generated among differentially expressed genes belonging to each of selected GO BP terms. Using such a prediction method provided us with a molecular interaction network formed between protein products of studied genes (Fig. 2). Finally, we investigated the functional interactions between chosen genes with REACTOME FIViz app to Cytoscape 3.6.0 software. The results were shown in Fig. 3.
RT-qPCR was conducted to validate the results obtained during microarray analysis. The outcomes were presented and compared in a form of a bar graph (Fig. 4).
As can be seen, the direction of changes in expression was confirmed in most examples. Nevertheless, the microarray approach, used to analyze the full transcriptome of the cells, is largely qualitative, which can be observed as validation of the results with quantitative RT-qPCR in two examples gives variable results. For SPP1 transcript expression level, microarray results indicate downregulation in 15 days of cell culture, while RT-qPCR results give a different direction of changes. A similar situation may be observed for RRAS1 mRNA levels. This might be due to the fact that the microarrays account for multiple available exons forming many variants of the expressed gene, which is not usually the case with RT-qPCR, as it probes for a specific gene sequence. Likewise, the scale of differences in transcript levels varied between both of the methods analysed.
Discussion
The process of MSC differentiation towards osteoblasts is regulated by a number of transcription factors, the list of which is still incomplete and systematically supplemented and updated (35,36). During the development of the skeleton, MSCs may give rise to two cell lines: i) Osteoblasts and ii) chondroblasts. The dual development potential of MSC is made possible due to expression of two basic bone formation regulators: Runx-2 (CBFA-core binding factor Ralpha/osteoblast-specific factor 2-OSF-2) and SOX-9. The further way of differentiation (towards osteoblasts or chondroblasts) is decided by the inclusion of further transcription factors. Osterix (OSX), and Runx-2 are some of the factors that facilitate the osteogenesis process (37,38). The increase in expression of OSX factor influences the increase of expression and secretion of osteoblast-specific proteins-collagen type I, osteopontin, sialoprotein and alkaline phosphatase (39). Other important factors regulating osteoblastogenesis are involved in the Wnt/β-catenin signalling pathway. Activation of Wnt pathway proteins stimulates osteoblast proliferation (36,38). Bone morphogenetic proteins (BMPs) are yet another important group of factors regulating the process of osteoblastogenesis. These proteins have a stimulating effect on osteoblast activity through BMP serine-threonine kinase receptors. Many other factors also contribute to the stimulation of osteoblast activity such as parathormone (PTH)-acting via insulin-like growth factor (IGF), dexamethasone, lectin etc. (40–43).
As mentioned above, the literature provides a number of factors that influence the differentiation of MSCs towards osteoblasts in vitro. Many authors indicate that the source of these stem cells is also the ovary. Kossowska-Tomaszczuk et al (21) were the first to suggest that GCs have the potential of stem cells (mesenchymal stem cells), due to the expression of markers characteristic for this type of cells. It was the first to prove that GCs can differentiate into osteoblasts in long-term in vitro culture under the influence of a suitable differentiating medium. GCs thus shed new properties and could be successfully used as a starting material for obtaining stable populations of osteoblasts used in regenerative medicine of skeletal-related disorders (21,44,45). The results of the presented studies confirm the possibility of differentiating GCs towards osteoblasts. We can observe that GCs is subject to such differentiation without differentiating factors constituting the supplement of the culture medium. We can, therefore, conclude that GCs undergoes a number of changes in gene expression during long-term in vitro culture, the effect of which is their entry into the pathway of osteoblast differentiation.
The presented studies also indicate that GCs may differentiate towards osteoblasts under long-term in vitro culture conditions. Moreover, it was shown that GCs express genes characteristic for this process, which can be considered genetic markers of differentiation of GCs towards osteoblasts. GCs in the presented studies were cultivated without the addition of supplements considered necessary for the process of cell differentiation towards osteoblasts. The basal medium did not contain any supplements such as dexamethasone, BMP-2, vitamin D3, ascorbic acid, β-glycerophosphate, valproic acid, which are considered to be key factors osteoblast differentiation (15,19,46). The results of the presented research on the potential of GCs differentiation towards osteoblasts are confirmed in the literature of recent years (21,23,44).
The ‘osteoblast differentiation’ ontological group defines a group of genes responsible for the biological process during which the differentiation of less specialized cells towards osteoblasts occurs. The included heat map presents a set of genes that are characteristic of the process of GCs differentiation towards osteoblasts.
As can be seen in the attached table, during the 30-day in vitro culture, the expression of 19 genes is changed, with SYNCRIP, MRC2, RRAS2 and TPM4 exhibiting the lowest expression, and LRRC17, COL1A1, BMP4, TWIST1, IGFBP5, GLI2, CTHRC1 showing the highest expression. This report is focused on the genes of the highest expression and their mutual relation.
Analyzing the relationships and interactions between the 19 genes of interest, we can only observe some between two pairs of genes (BMP4 and GLI2 and SPP1 and IGFBP5).
The highest expression from all genes has been demonstrated by LRRC17 (Leucine-rich repeat containing 17). It is not only one of the genes that regulate the osteoblastogenesis process, but also a gene that is expressed by the ovary. The above result, on one hand, can confirm that the obtained cells are cells derived from the ovary, but on the other hand can be a factor regulating the process of differentiation of GCs towards osteoblasts (47,48). It is a gene that is highly expressed in osteoblasts under physiological conditions (47). The high expression change of this gene during long-term in vitro GC culture indicates that these cells can spontaneously gain osteoblast properties.
Another very important gene demonstrating the differentiation of a given population towards osteoblasts is the expression of the collagen type I gene and protein. The expression of collagen type I is significantly increased after 30 days of GC in vitro culture. We can, therefore, suppose that the process of GCs differentiation towards osteoblasts is also regulated by typical factors of MSC osteoblastogenesis. COL1A1 (Collagen type I alpha 1 chain) is a gene providing inductions for the synthesis of a large molecular molecule called collagen type I. Collagens are a family of proteins that are part of most organs in the human body: They build cartilage, tendons, skin, sclera, and above all bones. Collagen molecules form long fibrils, connected by transverse bonds between them in intercellular spaces. Such structure and interactions between collagen fibres are called cross-linking, which results in the formation of very strong type 1 collagen fibres (49). The expression of the COL1A1 gene indicates that GCs have the potential to differentiate towards the bone tissue. Under physiological conditions, the COL1A1 gene is not expressed in GCs. Only the presence of collagen type I in the theca cells of the ovarian follicle has been proven (50,51). However, these are not the subject of the research presented. Only GCs building an internal layer of ovarian follicle were used in the study.
Osteogenesis is the process leading to the formation of bone tissue. It begins during the formation of the embryo and continues throughout the entire life of the organism by maintaining a balance between bone formation and resorption (52). Bone morphogenetic proteins belong to the superfamily of TGF-βs (Transforming growth factors β). The family of these proteins is responsible for the formation of bone and cartilage in vivo but also fulfils important roles in the female reproductive system (53,54). One of the strongest inductors in bone formation through osteoblast differentiation stimulation is BMP-4. Under physiological conditions, BMP-4 transduced signals through heterodimer formation of Type II and Type I cognate complex (BMPRIA and BMPRIB) through serine/threonine receptors. This leads to phosphorylation of Sma and Mad proteins playing an important role in the differentiation of cells derived from the mesenchymal line (55–57). The group of BMP proteins also plays a role in the regulation of follicular development and has an effect on GCs proliferation and steroidogenesis. Tanwar and Mcfarlane (58) indicate, in their mice studies, that BMP-4 is expressed in the ovary, uterus and oviduct epithelium. It has also been shown that BMP-4 acts as a paracrine/autocrine modulator of steroidogenesis of GCs cells. BMP-4 together with BMP-6, BMP-7 stimulates Smad-1 accumulation and release of estradiol (E2) stimulated by IGF (59,60). Other studies have shown that another types of BMP, above all BMP-15, are necessary for the proper functioning of the reproductive system of the female. It is BMP-15 that affects the proliferation and differentiation of GCs into individual layers within the follicle (61). The presented studies do not clearly indicate whether the expression of BMP-4 is associated with the the process of steroidogenesis occurring in vitro culture or whether this expression is associated with osteogenesis. In a broad sense, we can conclude that the presence of other genes involved in the BMP-4 osteogenesis process could confirm its occurrence in the culture. This conclusion is supported by the expression of the previously mentioned SPP1 gene (secreted phosphoprotein; osteopontin) (62). SPP1 is a gene responsible for the proper bone mineralization in the process of osteogenesis. Kim et al (63) suggested that SPP1 could be a marker of ovarian cancer. Under physiological conditions, the expression of SPP1 increases in the antral follicles (64) suggesting that SPP1 does not indicate a differentiation of GCs towards osteoblasts. Kulterer et al (65) proved that during the differentiation of MSCs towards osteoblasts, SPP1 and COL1A1 are expressed (66). Our research confirms this scheme; however, it should be emphasized that in the presented results P-values were <0.05 only after 7 days of culture, bringing their actual significance into question. In addition, another gene supporting the GCs' tendency for osteogenic differentiation in long-term in vitro is the GLI2 (GLI-Kruppel Family Member 2). This gene belongs to the family of zinc finger proteins. Under physiological conditions, the GLI family proteins play an important role in embryonic development. Abnormal operation of these genes causes development defects, eg: Mutations in the GLI2 gene cause defects in the development of the skeleton. In addition, GLI2 has been shown to play a large role in regulating BMP-2 protein expression during osteoblast differentiation (67). The presented research results confirm the relationship between the expression of GLI2 and BMP-4. During the differentiation of GCs towards osteoblasts in long-term in vitro culture, GLI2 may interact with BMP-4 in a positive manner (Figs. 2 and 3).
IGFBP-5 (Insulin-like growth factor-binding protein 5) is another factor involved in the process of osteoblast formation. This substance is detected during osteoblastogenesis. It is most intensively released during the first days of in vitro culture before mature osteoblasts arise. It is known that the amount of this factor decreases during long-term in vitro culture (68). In addition, IGFBP-5 produced by osteoblasts stimulates osteoclastogenesis and thus acts as an osteoblast-osteoclast coupling agent (69). Liu and Ling (70) proved that the rat ovary produces 5 types of IGFBP, including IGFBP-5 in GCs of atretic preantral follicles. In addition, the presented study results suggest that IGFBP5 interacts with the SPP1 gene described above (Fig. 2).
According to the results presented, growth factors such as BMP-4 or IGFBP5 interact with genes that, in addition to their role in the ovary, also play key roles in osteogenesis. Therefore, these factors may be potential regulators of the expression of the above genes, as well as the process of GC differentiation towards osteoblasts. As already mentioned, these genes play a key role in the process of osteoblast differentiation from mesenchymal stem cells. The results of the presented studies therefore suggest one of the probable ways of osteo-differentiation of GCs.
Another gene that is expressed by GCs after 30 days of in vitro culture is TWIST (Transcription Factor TWIST). Under physiological conditions, this gene is expressed in cells that express RUNX2 expression. As mentioned earlier, the RUNX2 gene is involved in the process of osteoblast formation. The basic function of TWIST is the inhibition of the RUNX2 function during skeletogenesis (71). The TWIST expression can, therefore, explain the lack of RUNX2 expression in the presented GCs after 30 days of culture.
Osteoblast differentiation from stem cells is possible thanks to CTHRC1. Wang et al (72) indicated that this gene, under physiological conditions, is involved in bone remodelling, and also shows the presence in osteocytes, bone matrix and periodontal ligament cells in rat. In presented research CTHRC1 has been activated during long-term in vitro culture of GCs.
In addition to gene expression changes observed during the course of the long-term in vitro culture, we have also observed significant changes of morphology. These changes, documented in Fig. 5, show that in the first days of culture the cells assume star-like shape, followed by their elongation and transition into fibroblast like shape in the later stages of culture. Similar results can be found in literature (22,44). This fact can be associated with the assumed loss of granulosa specific gene expression, caused by the absence of the physiological extracellular environment, and assumption of new, culture-specific phenotype. This process is also accompanied by upregulation of expression of non-granulosa specific growth factors (such as BMP4 and IGFBP5), which may further explain the assumption of new morphology.
Summing up the conducted research, it can be concluded that there is a lot of evidence for the possibility of GC differentiation towards osteoblasts. Obtaining stable cultures of differentiated osteoblasts derived from GCs may find wide application in the treatment of skeletal disorders. However, it needs to be noted that this is an entry level transcriptomic study, which only accounts for the gene changes observed in the granulosa cell culture and refers them to the available literature. Despite that, the presented research is an excellent “signpost” for further, more detailed studies, possibly including comparisons to osteoblasts cultured in similar conditions, or based on proteomic approaches (which are much better translatable to in vivo knowledge). Detailed analysis of pathways involved in the differentiation of GCs towards osteoblasts is required.