Coenzyme and cofactor metabolism belongs to biochemical processes significantly regulated in human granulosa cells collected after IVF during long-term primary in vitro culture

Ovarian follicles are formed during the process of folliculogenesis and oogenesis. The follicle consists of the oocyte, the follicular fluid and the oocyte‑associated cells: granulosa cells (GCs). GCs provide the microenvironment necessary for the development of the follicle and the maturation of the oocyte [1]. GCs also influence the development of the female gamete in a paracrine and autocrine manner [2]. Paracrine communication leads to the establishment of bidirectional cooperation affecting the hormonal production and the expression of genes associated with follicular development [3]. GCs are associated with the functioning of the reproductive system and the maintenance of pregnancy by participating in the synthesis of steroid hormones. The physiological and morphological changes occurring in GCs during the formation of the ovarian follicle have been well characterized. In contrasts, the deep molecular mechanisms and the gene interactions of GCs are still insufficiently described.

With a large probability for application as the grafts in regenerative medicine, the establishment and characterization of in vitro cell cultures (IVC) may be the most prospective methods of tissue and organ regeneration,. Some studies point to future potential of GCs in regenerative medicine and in transplantology. GCs containing follicular fluid after being transferred for utilization during routine in vitro fertilization procedures may be the source of stem cells. Several authors clearly indicate these previously unidentified properties of GCs. Oppositely, many articles suggest that GCs are subject to a transdifferentiation process [6,7]. During this process, under the influence of the appropriate factors, the mature cell is reprogrammed and it changes into another cell type [4,5]. Kossowska‑Tomaszczuk et al. [6,7] indicated that GCs have stem-like properties.

The aim of study is to furtherly characterize GCs at the molecular level and obtain more insight in the variability of the transcriptomic profile during longterm cell culture. Affymetrix microarray assays revealed the regulatory peptides and enzymes involved in the functioning of the mechanism of enzymatic reactions. The focus of the current study was to examine transcript variability of genes belonging to “coenzyme biosynthetic process”, “coenzyme metabolic process”, “cofactor biosynthetic process” and “cofactor metabolic process” gene ontology biological process terms (GO BP) during OECs longterm primary culture in vitro. It will delineate the transcriptomic profile variability for the genes (ELOVL5, ELOVL6 and GPAM) involved in enzymatic regulation of fatty acid metabolism.

Whole transcriptome profiling by Affymetrix microarray allows to analyze the gene expression changes of the oocyte after in vitro maturation in relation to freshly isolated oocyte before in vitro procedure (in Vivo). By Affymetrix® Porcine Gene 1.1 ST Array the expression of 22480 transcripts were examined. Genes were considered as differentially expressed when presented fold changes higher than abs (2) and corrected p value lower than 0.05. This set of genes consist of 2278 different transcripts. The first detailed analysis based on GO BP lead to the identification of differentially expressed genes belonging to the significantly enrichment GO BP terms.

The DAVID (Database for Annotation, Visualization and Integrated Discovery) software was used for the extraction of the genes belonging to the gene ontology biological process terms (GO BP). Up and down regulated gene sets were subjected to DAVID searching separately and only gene sets where adj. p values were lower than 0.05 were selected. The DAVID software analysis showed that differently expressed genes belong to 582 Gene ontology groups and 45 KEGG pathways. The genes of main focus were “coenzyme biosynthetic process”, “coenzyme metabolic process”, “cofactor biosynthetic process” and “cofactor metabolic process” GO BP terms. These sets of genes were subjected to hierarchical clusterization procedure and presented as heatmaps (Fig. 1). The symbols of genes, fold changes in expression, Entrez gene IDs and corrected p values of that genes are shown in table 1. The enrichments of each GO BP term were calculated as z-score and they are shown on a circle diagram (Fig. 2)

Figure 1

Figure 2

In the Gene Ontology database, genes that formed one particular GO group can also belong to other different GO term categories. By this meaning, the gene intersections between selected GO BP terms were explored and the relation between the GO BP terms are presented as circle plot (Fig. 3) as well as heatmap (Fig. 4).

Figure 3

Figure 4

The STRING software generated interaction networks among differentially expressed genes belonging to each of the selected GO BP terms. Through this method it was possible to observe the molecular interaction network formed between protein products of studied genes (Fig. 5). Finally, the functional interactions between chosen genes were investigated with REACTOME FIViz app to Cytoscape 3.6.0 software and the results are shown in figure 6.

Figure 5

Figure 6

GCs are known to be closely related to mammalian oocytes. GCs surround the oocyte and they form the proper architecture of the ovarian follicle. In physiological conditions, GCs support the development and the maturation of the oocyte, and the formation of ovarian follicles [12]. Moreover, they produce gap junctions that allow a bidirectional communication with the oocyte. Thus, in addition to the hormonal secretory function, GCs also play an important role in communication with oocytes. They are associated with the functioning of the reproductive system, as well as the maintenance of pregnancy through the participation in the synthesis of steroid hormones. After ovulation, when the follicle ruptures, the GCs fill the formed gap and transform into lutein cells, whose main purpose is to produce progesterone and maintain pregnancy. The resulting structure is called the corpus luteum [13]. Understanding the molecular mechanisms regulating this process is pivotal to completely understand the regulation of oogenesis and the ovarian function.

The physiological and morphological changes that occur in GCs during the formation of the ovarian follicle have been well characterized. However, the molecular mechanisms and gene interactions of GCs are still insufficiently described, Previous studies are an important introduction to a better understanding of all key mechanisms in this cell population [13]. In addition to indicating numerous molecular markers that are key to many physiological and biochemical processes occurring in these cells [13, 14, 15], the very high plasticity of GCs and their potential to differentiate into other cell populations were demonstrated[16, 17, 18]. In the present study, by employing primary in vitro long-term culture and microarray approach, groups of genes associated with enzymatic reactions were described.

Of all the genes showed on the heat maps in Fig. 1 and belonging to GO BP terms of interest, the greatest reduction in the level of mRNA expression has been observed in the case of the gene ELOVL5. To date, seven enzymes have been identified, termed ELOVL 1–7 (Elongation of very-long-chain fatty acid) they are suggested to perform the condensation reaction in the elongation cycle, [19]. Long-chain fatty acid elongase 5 (Elovl5) is the rate-controlling enzyme that catalyzes the elongation reaction of long-chain polyunsaturated fatty acids (PUFAs), including arachidonic acid, oleic acid, stearidonic acid, palmitic acid, linolenic acid and eicosapentaenoic acid. These fatty acids (FA) not only are an essential components of the cell membrane, but they also participate in many important signaling processes [20,21]. Accordingly, their proper elongation and desaturation of are essential to the maintenance of lipid homeostasis and the disruption of these processes may have devastating consequences. Studies using ovaries of slaughterhouse origin have revealed correlations between FA content in follicular fluid (FF) and the expression levels of several genes, including ELOVL5, in GCs[22]. These results confirmed the importance of lipid metabolism balance in the interaction between oocytes and maternal environment. A study by Warzych et al. confirmed the distinct roles of cumulus cells (CCs) and GCs in energy metabolism of the follicle. In fact, they showed different ELOVL5 transcript levels (GCs were characterized by significantly higher mRNA levels), regardless of the donor [23]. Almiñana et al., demonstrated presence of ELOVL5 transcripts in oviductal extracellular vesicles (OEVs), which contain oviductal secretions [24].

The detection of the reduced mRNA expression level of another ELOVL family member, ELOVL6 is an additional argument suggesting decreased activity of elongation very long chain fatty acids (VLCFA) in GCc during in vitro conditions. ELOVL6 protein, in contrast to ELOVL5, prefers saturated and mono-unsaturated fatty acids as substrates in the elongation pathway [25]. A studies provided by Annes et al. compared the expression of lipid-related genes and follicular fluid molecules between different bovine follicle sizes. It was demonstrated that ELOVL6 showed lower transcript levels in larger follicle-derived (≥6 mm) oocytes compared with the ≤2 mm group [26]. Another study provided a comprehensive description of stage-specific lipidome signatures in early bovine embryo development, via assessment the mRNA abundance of lipid metabolism-related genes (including both ELOVL5 and ELOVL6). When compared to earlier stages (immature oocytes, two-cell embryos, and eight- to 16-cell embryos), ELOVL5 and ELOVL6 mRNA quantity increased significantly at morula stage, followed by a decrease at blastocyst stage. The authors proved that both ELOVL family members overexpression preceded an increased abundance of a series of lipid species at blastocyst stage [27].

Significantly reduced GPAM transcript level also confirms the importance of changes at the molecular level in the enzymatic regulation of fatty acid metabolism. GPAM (Glycerol-3-phosphate acyltransferase mitochondrial) is a gene that encodes a mitochondrial enzyme which catalyzes the initial and committed step of glycerolipids synthesis and that prefers saturated fatty acids as its substrates for the catalyzed biosynthesis process [28]. Transcriptomic studies on molecular regulation of FA metabolism in 3–8 mm ovarian follicles of bovine have shown high GPAM transcript expression in theca (TH), GCs, and also in CCs [29]. Subsequent studies of Yu laboratory [30] showed that GPAM gene can promote the synthesis of triglycerides in the fat metabolism pathway of bovine embryonic fibroblast cells. Especially important conclusions provided by Marchan et al. indicated that analysis of GPAM expression in different cancer types revealed a significant association between high GPAM expression and reduced overall survival in ovarian cancer. The authors’ results clearly showed how GPAM levels influence intracellular lysophosphatidic acid (LPA) levels to promote cell migration and tumor growth [31].

Decreasing levels of gene expression involved in the functioning of the mechanism of enzymatic reactions occur over the course of long-term cell culture. The transcriptomic profile variability for genes (ELOVL5, ELOVL6 and GPAM) involved in enzymatic regulation of fatty acid metabolism illustrated a down regulation in these genes. It is likely that the reduced enzymatic activity over culture indicates lower expression levels compared to in vivo situation.