Volume 8 (2020): Issue 1 (March 2020)

The growth and development of the oocyte is essential for the ovarian follicle. Cumulus cells (CCs) - a population of granulosa cells - exchange metabolites, proteins and oocyte-derived paracrine factors with the oocyte through gap junctions, to contribute to the competency and health of the oocyte. This bi-directional communication of the cumulus-oocyte complex could be better understood through the micro-analysis of a porcine oocyte gene expression before in vitro maturation (IVM) and after. Additionally, the study of the somatic and gamete cells differentiation capability into neuronal lineage would be promising for future stem cell research as granulosa cells are easily accessible waste material from in vitro fertilization (IVF) procedures. Therefore, in this study, the oocytes of 45 pubertal Landrace gilts were isolated and the protein expression of the COCs were analyzed through micro-analysis techniques. Genes belonging to two ontological groups: neuron differentiation and negative regulation of cell differentiation have been identified which have roles in proliferation, migration and differentiation. Twenty identified porcine oocyte genes (VEGFA, BTG2, MCOLN3, EGR2, TGFBR3, GJA1, FST, CTNNA2, RTN4, MDGA1, KIT, RYK, NOTCH2, RORA, SMAD4, ITGB1, SEMA5A, SMARCA1, WWTR1 and APP) were found to be down-regulated after the transition of IVM compared to in vitro. These results could be applied as gene markers for the proliferation, migration and differentiation occurring in the bi-directional communication between the oocyte and CCs.

Running title: Differentiation and neurogenesis in oocyte cells

Coronary artery bypass graft (CABG) is the surgical method most commonly used to treat coronary artery disease (CAD). The vessels that are used in CABG are usually the internal thoracic artery (ITA) and the saphenous vein (SV). Transplant patency is one of the most important factors affecting transplant success. In this study, we used an expressive microarray method, approved by RT-qPCR, for transcriptome analysis of arterial and venous grafts. In the search for potential molecular factors, we analyzed gene ontologies of different expression based on the muscular system. Among interesting groups, we distinguished muscle cell proliferation, muscle contraction, muscle system process, regulation of smooth muscle cell proliferation and smooth muscle cell proliferation. The highest increase in gene expression was observed in: ACTN2, RBPMS2, NR4A3, KCNA5, while the smallest decrease in expression was shown by the P2RX1, KCNH2, DES and MYOT genes. Particularly noteworthy are the ACTN2 and NR4A3 genes, which can have a significant impact on vascular patency. ACTN2 is a gene that can affect the formation of atherosclerotic plaques, while NR4A3 occurs in 4 of the 5 ontological groups discussed and can affect the inflammatory process in the blood vessel. To summarize, the presented study provided valuable insight into the molecular aspects characterizing the vessels used in CABG, and in particular identified genes that may be the target for further studies on duct patency.

Running title: CABG grafts’ molecular analysis of ‘muscle system process’

Coronary artery bypass grafting (CABG), together with percutaneous coronary intervention (PCI), are both still the most efficient procedures for myocardial revascularization to treat advanced coronary artery disease (CAD). Donor blood vessels used in CABG are usually the internal thoracic artery (ITA) and saphenous vein (SV). The importance of inflammation and inflammatory pathways in graft patency is well established. Nevertheless, not all molecular mechanisms underlying the inflammatory process appear to be clear. Employing the expressive microarray approach to analyze the transcriptome of both venous and arterial grafts, five GO BP terms has been selected: “cellular response to interferon-gamma”, “inflammatory response”, “interferon-gamma-mediated signaling pathway”, “response to interferon-gamma” and “positive regulation of inflammatory response”. This study aimed to evaluate potential molecular factors that could be characteristic markers for both SV and ITA conduits.

Running title: Aortocoronary conduits may show a different inflammatory response

The growth and maturation of the oocyte is a dynamic process which requires a variable supply of hormones, growth factors and energy. These needs are met partially by the surrounding somatic cells and the cumulus-oocyte complex, which communicate bi-directionally via gap junctions. Identifying and analyzing protein expression in the oocyte can provide insight in its development and growth. Further, like bone marrow stem cells, if relevant marker genes are found in oocytes, there is a potential for the oocyte to be manipulated into becoming hemopoietic stem cells. In this study, porcine oocytes were isolated and subjected to microarray analysis to compare the oocyte gene expression in vivo and in vitro maturation (IVM). Genes identified belonged to both ‘hemopoietic or lymphoid organ development’(GO:0048534) and ‘immune system development’ (GO:0002520), and the markers can be used to identify several activities such as cell migration, neurogenesis and proliferation. The following are the identified genes and all were downregulated after IVM to varying degrees: ID2, VEGFA, TGFBR3, INHBA, CDK6, BCL11A, MYO1E, ITGB1, EGR1, NOTCH2, SPTA1, KIT and TPD52. Our results should provide new markers to further investigate oocyte development and growth regulation.

Running title: Markers of hemopoietic organ development

In vitro models represent an alternative technique to in vivo or ex vivo studies in the drug development process. Cell-based assays are used to measure the level of proliferation and toxicity, as well as activation of signalling pathways and changes in morphology in cultivated cells. The studies conducted in vitro are aimed to estimate the newly synthesised drugs’ ability to permeate biological barriers and exert their therapeutic or cytotoxic effects. However, more than half of all studied drugs fail in the second or third phase of clinical trials due to a lack of confirmed efficacy. About a third of drugs fail because of safety issues, such as unacceptable levels of toxicity. To reduce attrition level in drug development, it is crucial to consider the implementation of translational phenotypic assays as well as to decipher various molecular mechanisms of action for new molecular entities. In this review, we summarise the existing cell-based methods most frequently used in the studies on drugs, taking into account their advantages and drawbacks.

Running title: Cell-based approaches in drug development

Mucous membranes appear in various parts of the whole body performing similar functions. However, they differ based on where the mucosa is located. It functions as a barrier in such systems as: respiratory, urogenital and digestive . In this study we will be focusing strictly on the oral mucosa. Keratinocytes and fibroblasts, which mainly form the structure of the oral mucosa, are subjected to numerous factors. Being one of the million parts that build the animal organism, they are involved in various processes. In this study, we will try to confirm that in the in vitro culture of oral mucosa cells, the expression of our selected genes undergoes significant changes which are tied to such processes as: homeostasis, chemotaxis and organic/inorganic response of the organism. For this study, 20 pubertal crossbred Landrace gilts were used. After slaughter, samples of buccal pouch mucosa were obtained and transported to the laboratory. The excised tissue was prepared and processed due to protocols. The final pellet was resuspended in supplemented DMEM. Once the cultures attained 70–80% confluency, they were passaged. Total RNA from each pooled sample was subjected to two rounds of sense cDNA amplification. The cDNA was processed on microarrays. Analysis of the scanned arrays was performed. The files were imported into downstream data analysis software. The DAVID analysis showed that differently expressed genes belongs to 56 Gene ontology groups. In this paper we focused on “cellular divalent inorganic cation homeostasis”, “chemical homeostasis”, “chemotaxis”, “homeostatic process” and “response to organic substance” GO BP terms. These sets of genes were subjected to hierarchical clusterization procedure. In summary, the data we collected showed primarily changes in gene expression that occurred in the thirty-day cell culture of oral mucosa tissue. We assume that indicated genes could be new gene markers for studied processes.

Running title: Homeostasis in oral mucosa cells

The presence of CCs around the oocyte after ovulation is one of the key elements contributing to oocyte developmental competence. In the presented study, we used CCs from 12 patients aged 18-40 diagnosed with infertility. After harvesting cells on day 1, 7, 15 and 30 of culture, total RNA was isolated and transcriptomic analysis was performed. The DAVID software indicated the following GO BP terms: “cell junction organization”, “cell migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility”. Of the genes belonging to all ontological groups, the most downregulated were: SLC7A8, DFNB31, COL1A1, CDC42SE1, TGFBR3, HMGB1, with the most upregulated genes being: ANXA3, KIAA1199, HTR2B, VCAM1, DKK1.

While many studies focus on attempts to obtain fully competent oocytes, scientists still have difficulty attaining adequate results in vitro. Lack of adequate knowledge often results in low in vitro fertilization efficiency. Therefore, our research focuses on CCs cells, thanks to which the oocyte most likely acquires developmental competence. The main purpose of the study was to identify the potential molecular markers responsible for cell junction organization, migration, differentiation, morphogenesis and motility.

Running title: New markers of human cumulus oophorus cells cultured in vitro