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The umbilical cord is an unlimited source of mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC). MSC obtained from the umbilical cord can be differentiated into different types of mesodermal cells, e.g. chondrocytes, osteocytes, adipocytes, and myocytes. It is also worth mentioning that there are reports of MSC differentiation into endo and ectodermal cells. The immunosuppressive properties of MSCs can protect against graft versus host disease as well as prevent rejection after transplantation. Umbilical cord stem cells can be frozen and then stored in liquid nitrogen for many years. In this work, we focused on the use of preclinical and clinical umbilical cord stem cells in disease entities such as type I diabetes, chronic renal failure, and multiple sclerosis. Furthermore, the anti-cancer properties of Wharton’s jelly cells are described. Running title: Umbilical cord stem cells in humans

Umbilical cord is a waste material, and therefore does not raise ethical concerns related to its use for research and medicine. Stem cells from umbilical cord have a significant advantage over cells from other sources. First, the umbilical cord is an infinite source of stem cells, because it can be taken theoretically during each delivery. Secondly, acquisition of umbilical cord is a non-invasive, safe procedure for mother and child. Thirdly, the transplantation of umbilical cord stem cells is associated with a lower risk of infection and a less-frequent “graft versus host” reaction. In this work, the authors present a historical background of research on the cell from its discovery to modern times characterized by highly advanced methods of obtaining stem cells from umbilical cord and from other sources.

Resveratrol is a phytoalexin that naturally occurs in grapes, blueberries, cranberries, peanuts and many other plants. Although resveratrol inhibits carcinogenesis in all three stages, its clinical application is restricted due to poor pharmacokinetics. The methylated analogues of resveratrol have been found to have higher bioavailability and cytotoxic activity than that of the prototupe compound. Among the various methoxy derivatives of resveratrol, 3,4,5,4&prime, tetrametoxystilbene (DMU-212) is suggested to be one of the strongest activators of cytotoxicity and apoptosis. DMU-212 has been shown to exert anti-tumor activity in DLD-1 and LOVO colon cancer cells. Since colorectal cancer is the third most common cause of cancer-related deaths worldwide, the development of new anticancer agents is nowadays of high significance. The aim of the present study was to assess the anticancer activity of 4&prime, hydroxy-3,4,5-trimetoxystilbene (DMU-281), the metabolite of DMU-212, in DLD-1 and LOVO cell lines. We showed for the first time the cytotoxic activity of DMU-281 triggered via cell cycle arrest at G2/M phase and apoptosis induction accompanied by the activation of caspases-9, -8, -3/7. Furthermore, DMU-281 has been found to change the expression pattern of genes and proteins related to intrinsic as well as extrinsic apoptosis. Since the activation of these pathways of apoptosis is still the most desired strategy in anticancer research, DMU-281 seems to provide a promising approach to the treatment of colon cancer.

The morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the group of compound processes responsible for proper oocyte transport and successful fertilization. The cellular interactions between cumulus-oocyte complexes (COCs) and oviductal epithelial cells (OECs) are crucial for this unique mechanism. In the present study we have analyzed angiogenesis and blood vessel development processes at transcript levels. By employing microarrays, four ontological groups associated with these mechanisms have been described. Differentially expressed genes belonging to the “angiogenesis”, “blood circulation”, “blood vessel development” and “blood vessel morphogenesis” GO BP terms were investigated as a potential markers for the creation of new blood vessels in cells under in vitro primary culture conditions.

A wide variety of mechanisms controlling oligomerization are observed. The dynamic nature of protein oligomerization is important for bioactivity control. The oocyte must undergo a series of changes to become a mature form before it can fully participate in the processes associated with its function as a female gamete. The growth of oocytes in the follicular environment is accompanied by surrounding somatic cumulus (CCs) and granulosa cells (GCs). It has been shown that oocytes tested before and after in vitro maturation (IVM) differ significantly in the transcriptomic and proteomic profiles. The aim of this study was to determine new proteomic markers for the oligomerization of porcine oocyte proteins that are associated with cell maturation competence. The Affymetrix microarray assay was performed to examine the gene expression profile associated with protein oligomerization in oocytes before and after IVM. In total, 12258 different transcriptomes were analyzed, of which 419 genes with lower expression in oocytes after IVM. We found 9 genes: GJA1, VCP, JUP, MIF, MAP3K1, INSR, ANGPTL4, EIF2AK3, DECR1, which were significantly down-regulated in oocytes after IVM (in vitro group) compared to oocytes analyzed before IVM (in vivo group). The higher expression of genes involved in the oligomerization of the protein before IVM indicates that they can be recognized as important markers of biological activation of proteins necessary for the further growth and development of pig embryos.

The ovary is part of the reproductive system, possessing very important functions in the reproduction process (ovum and embryo transfer, providing a suitable environment for sperm capacitation, etc.). There are two types of cells in the fallopian tubes: alveolar and secretive cells. These study shows the metabolic processes in pig oviductal epithelial cells associated with the activation of signaling pathways of amino acids metabolism and degradation during long-term in vitro culture. Oviductal epithelial cells from 45 colonies in the anestrous phase of the estrous cycle have been utilized in this study. RNA extract from the OEC primary cultures was pooled after 24h, 7days, 15 days and 30 days from the beginning of culture and the transcriptome investigated by Affymetrix® Porcine Gene 1.1 ST. From the whole transcript that consisted of 2009 different genes, 1537 were upregulated and 995 were downregulated after 7 days of culture, 1471 were upregulated and 1061 were downregulated after 15 days of culture and 1329 were upregulated and 1203 were downregulated after 30 days of culture. The results of these studies provide, for the first time, information on the activation of metabolic pathways of amino acids such as valine, leucine, isoleucine, cysteine, and methionine in the investigated tissue. They also indicate genes that may be OECs-specific genetic markers that are expressed or upregulated during long-term in vitro culture.

In modern medical research, stem cells are one of the main focuses, believed to be able to provide the solution to many currently unsolvable medical cases. However, their extraordinary potential for differentiation creates much obstacles in their potential application in clinical environment, without understanding the whole array of molecular mechanisms that drive the processes associated with their development and maturation. Because of that, there is a large need for studies that concern the most basic levels of those processes. Progenitor stem cells are a favorable target, as they are relatively lineage committed, making the amount of signaling required to reach the final form much lower. Their presence in the adult organism is also an advantage in their potential use, as they can be extracted without the need for storage from the moment of pre-natal development or birth. Epithelial tissues, because of their usual location or function, exhibit extraordinary level of plasticity and proliferative potential. That fact makes them one of the top candidates for use in applications such as tissue engineering, cell based therapies, regenerative and reconstructive medicine. The potential clinical application, however, need to be based on well developed methods, in order to provide an effective treatment without causing major side effects. To achieve that goal, a large amount of research, aiming to analyze the molecular basics of proliferation and differentiation of epithelial stem cells, and stem cells in general, needs to be conducted.

Mammalian epithelial and epithelial-like cells are significantly involved in various processes associated with tissue development, differentiation and oncogenesis. Because of that, high number of research is focused on identifying cells that express stem-like or progenitor characteristics. Identifying such cells and recognizing their specific markers, would open new clinical opportunities in transplantology and oncology. There are several epithelia characterized by their ability to rapidly proliferate and/or differentiate. Due to their function or location they are subject to cyclic changes involving processes of apoptosis and regeneration. Literature presenting well-structured studies of these types of epithelia was analyzed in order to compare various results and establish if epithelial cells’ migrative and proliferative ability indicates their stemness potential. Endometrial, ovarian, oviductal and oral mucosal epithelia were analyzed with most of the publications delivering relatively unified results. The ability to rapidly proliferate/differentiate usually indicated the presence of some kind of stem/stem-like/progenitor cells. Most of the papers focused on pinpointing the exact location of these kind of cells, or analyzing specific markers that would be used for their future identification. There have also been substantial proportion of research that focused on discovering growth factors or intercellular signals that induced proliferation/differentiation in analyzed epithelia. Most of the research provided valuable insights into the modes of function and characteristics of the analyzed tissue, outlining the importance of such study for the possible clinical application of in vitro derived cell cultures.

The mammalian oocytes undergo significant biochemical and structural modifications during maturation both in vitro and in vivo. These changes involve chromatin reorganization and modification within metabolic status of cytoplasmic organelles. After oocytes’ successful maturation the substantially increased storage of RNA was observed. Moreover, the early embryo interaction with maternal endometrial tissue after fertilization is up to now considered as the main marker of proper embryo implantation and early growth. In this study, we first investigated the expression profile of genes involved in blood vessel formation and blood circulation in porcine oocytes before and after in vitro maturation.The cumulus-oocyte complexes were collected from pubertal Landrace gilts and classified as before in vitro maturation (in Vivo) or after in vitro maturation (in Vitro). The RNA was isolated from these two experimental groups and analyzed using Affymetrix microarrays.We found an increased expression of genes involved in ontological groups such as “blood circulation” (TPM1, ECE1, ACTA2, EPHX2, EDNRA, NPR2, MYOF, TACR3, VEGFA, GUCY1B3), “blood vessel development” (ANGPTL4, CYR61, SEMA5A, ID1, RHOB, RTN4, IHH, ANGPT2, EDNRA, TGFBR3, MYO1E, MMP14), and “blood vessels morphogenesis” (ANGPT2, as well as other common transcripts) in in Vivo group as compared to decreased expression of these genes in in Vitro group of oocytes.It has been suggested that investigated genes undergo significant expression before in vitro maturation, when enhanced storage of large amount of RNA takes place. Creating templates for synthesis of proteins is required for formation of fully mature gametes and early embryo growth. Therefore we hypothesized that the processes of vascularization and/or angiogenesis reach a high activity in immature oocytes and are distinct from achievement of maturational stage by oocytes in pigs.

The morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the compound process responsible for proper oocytes transport and successful fertilization. However, the main mechanisms which regulated this process are still not entirely known. Moreover, the OECs metabolism, which may be identified as the “cellular activity” marker, is poorly recognized. In this study we investigated the fructose and mannose metabolic pathway in porcine OECs primary long-term cultured in vitro.In our study, we employ a primary long term in vitro culture (IVC) and microarray approach (the Affymetrix microarray were used for analysis of transcriptomic profile of OECs) for expression levels analysis.We found that from the whole analyzed transcriptome, 1537 genes were upregulated and 995 were down regulated after 7 days of culture, 1471 genes were upregulated and 1061 were downregulated after 15 days of culture and 1329 genes were upregulated and 1203 were downregulated after 30 days of culture. Moreover, the differential expression of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 at 24 hours, 7 day, 15 day and 30 day, was also observed.We suggested that fructose and mannose metabolism may be important molecular bio-marker of porcine OECs capability in in vitro model. The metabolic profile is significantly accompanied by cells proliferation in vitro. The transcriptomic profile of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 expression may be identified as “fingerprint” of fructose and mannose metabolism in OECs as well as involved in cellular in vitro developmental capacity in pigs.

Mammalian cumulus-oocyte complexes (COCs) reach full developmental capability during folliculogenesis and oogenesis. It is well recognized that only gametes achieving MII stage after in vivo or in vitro maturation (IVM) are successfully fertilized by a single spermatozoon. Although the process of oocyte nuclear and/or cytoplasmic maturation in pigs is well determined, there exist many differences that promote these processes in vivo and in vitro. Therefore, this study aimed to investigate the differences in RNA expression profiles between porcine oocytes before and after IVM using microarray and real-time quantitative polymerase chain reaction (RT-qPCR) assays. Experiments were performed on oocytes isolated from 55 pubertal crossbred Landrace gilts. The oocytes were analyzed both before and after IVM and only Brilliant Cresyl Blue (BCB)-positive gametes were used for subsequent microarray analysis (Affymetrix) and RT-qPCR analysis. The microarray assay, which measures expression of 12,258 transcripts, revealed 419 differentially expressed transcripts in porcine oocytes, from which 379 were downregulated and 40 were upregulated before IVM compared to those analyzed after IVM. After DAVID analysis, we found eight different transcripts, including IHH, BMP1, WWTR1, CHRDL1, KLF10, EIF2AK3, MMP14, and STC1. Their expression is related to the "bone development" ontology group and was further subjected to hierarchical clusterization. Using RT-qPCR analysis, we confirmed the results of the microarray assay, showing increased expression of the eight genes in oocytes before IVM compared to oocytes after maturation in vitro. It has been suggested that "bone development" belongs to one ontological group involving genes substantially upregulated in porcine oocytes before IVM. We suggest that the gamete mRNA expression profile before IVM may comprise stored transcripts, which are templates for protein biosynthesis following fertilization. We also hypothesize that these mRNAs may be a specific "fingerprint" of folliculogenesis and oogenesis in pigs.

SummaryMammalian oocyte maturation is achieved when oocytes reach metaphase II (MII) stage, and accumulate mRNA and proteins in the cytoplasm following fertilization. It has been shown that oocytes investigated before and after in vitro maturation (IVM) differ significantly in transcriptomic and proteomic profiles. Additionally, folliculogenesis and oogenesis is accompanied by morphogenetic changes, which significantly influence further zygote formation and embryo growth. This study aimed to determine new transcriptomic markers of porcine oocyte morphogenesis that are associated with cell maturation competence. An Affymetrix microarray assay was performed on an RNA template isolated from porcine oocytes before (n = 150) and after (n = 150) IVM. The brilliant cresyl blue (BCB) staining test was used for identification of cells with the highest developmental capacity. DAVID (Database for Annotation, Visualization, and Integrated Discovery) software was used for the extraction of the genes belonging to a cell morphogenesis Gene Ontology group. The control group consisted of freshly isolated oocytes. In total, 12,000 different transcripts were analysed, from which 379 genes were downregulated and 40 were upregulated in oocytes following IVM. We found five genes, SOX9, MAP1B, DAB2, FN1, and CXCL12, that were significantly upregulated in oocytes after IVM (in vitro group) compared with oocytes analysed before IVM (in vivo group). In conclusion, we found new transcriptomic markers of oocyte morphogenesis, which may be also recognized as significant mediators of cellular maturation capacity in pigs. Genes SOX9, MAP1B, DAB2, FN1, and CXCL12 may be involved in the regulation of the MII stage oocyte formation and several other processes that are crucial for porcine reproductive competence.

Maturation of cumulus–oocyte complexes (COCs) is crucial for further successful monospermic fertilization, embryo growth, and implantation. All these events are accompanied by proliferation and differentiation of cumulus cells. The migration of COCs to the oviduct after ovulation and the interaction between female gametes and/or embryos with maternal tissues are still poorly recognized on the molecular level. This study was aimed to first demonstrate the mRNA expression profile of cell migration markers during different stages of porcine oocytes maturation and developmental capability in vitro. The COCs were collected from a total of 45 pubertal crossbred Landrace gilts, brilliant cresyl blue (BCB) stained, and analyzed before (n = 150) or after (n = 150) in vitro maturation (IVM). Using the Affymetrix® Porcine Gene 1.1 ST Array, the expression profile of 12,258 porcine transcripts was examined. We found nine genes involved in cell migration mechanisms, that is, PLD1, KIT, LAMA2, MAP3K1, VEGFA, TGFBR3, IN...

SummaryThe mammalian oocytes maturation is the compound process that involves morphological and molecular changes. These modifications include storage of macromolecules, which are crucial for proteins biosynthesis during periimplantation stages of embryo development. This study was aimed to investigate the genes expression profile encoding macromolecules important for regulation of proper porcine oocytes maturation.The porcine oocytes were collected from large ovarian follicles and analyzed both before and after in vitro maturation (IVM). Additionally, to check the developmental competence status, brilliant crezyl blue test (BCB) was performed. The obtained cDNA was used for biotin labeling and fragmentation by AffymetrixGeneChip® WT Terminal Labeling and Hybridization (Affymetrix). The preliminary analysis of the scanned chips was performed using AffymetrixGeneAtlasTM Operating Software. The created CEL files were imported into downstream data analysis software.In results, we found expression of 419 different genes, 379 genes were down-regulated and 40 genes were up-regulated in relation to the oocyte transcriptome before in vitro procedure. We observed up-regulation of all genes involved in “positive regulation of macromolecule metabolic process” before IVM as compared to transcriptional profile analyzed after IVM.In conclusion, we suggested that genes encoding proteins involved in macromolecule metabolism are important for achieving of porcine oocytes maturational stage. Moreover, the “activity of macromolecules metabolism” is much more increased in immature oocytes.

Summary The oocyte growth and development in follicular environment are substantially accompanied by surrounding somatic cumulus (CCs) and granulosa cells (GCs). During these processes, the mammalian gametes reach full maturational stage and may be further successfully fertilized by single spermatozoon. These unique mechanisms are regulated by expression of clusters of genes and their biochemical signaling pathways. In this article we described differential expression pattern of transforming growth factor beta (TGFB) gene superfamily in porcine oocytes before and after in vitro maturation (IVM). We performed Affymetrix® microarray assays to investigate the TGFB-related genes expression profile in porcine immature oocytes and gametes cultured for 44h in vitro. In results we found 419 different genes, 379 genes with lower expression, and 40 genes characterized by increased RNA profile. Moreover, significant up-regulation of 6 genes belonging to TGFB signaling pathway such as: TGFBR3, SMAD4, FOS, KLF10, ID1, MAP3K1 in immature porcine oocytes (before IVM), was also observed. It may be suggested that genes involved in TGFB-related signaling pathway are substantially regulated before IVM. Furthermore, these genes may play a significant role during early stages of nuclear and/or cytoplasmic porcine oocytes maturation. The investigated transcripts may be also recommended as the markers of oocytes maturational capability in pigs.

Summary Cell death plays an important role in maintaining the homeostasis of multicellular organisms. It can occur in a controlled manner by apoptosis or autophagy. Cell death which occurs regardless of regulatory factors include necrosis, mitotic catastrophe or oncosis. Apoptosis and necrosis are cellular process that leads to cell death. However their mechanisms are different, although factors triggering them can be similar. Necrosis and apoptosis have many different characteristics in terms of biochemistry and morphology. There are two main pathways of apoptosis induction signal: receptor - dependent and mitochondrial. The outsider apoptotic pathway is induced by external factors stimulating membrane receptors having an intracellular domain called death domain. Mitochondrial apoptotic pathway is activated by increased concentration of reactive oxygen species (ROS), DNA damage, disorders electrolyte transport and an increase in the concentration of the calcium ions in the cytoplasm. In response to stress-factors, mitochondrial channels are opened, so that is released into the cytoplasm cytochrome C. This work is aimed at an overall description of exchanged processes.

Summary Resveratrol (RSV) is one of the polyphenols - metabolites common in plants,however it does not occur in animals. It occurs mainly in grape skin (Vitisvinifera), peanuts (Arachis hypogeal) and in the roots of (Polygonumcupidatum) a traditional Chinese curative plant. RSV has a preventive property against the most serious diseases of modern world such as cancer, neurodegenerative diseases and cardiovascular diseases. Due to pleiotropy, RSV is currently the main object of many research teams′ interest, which is shown by the significant number of publications devoted to this subject. Animal and human conducted studies have shown very low bioavailability of RSV (approx. 2%), which is the result of rapid biotransformation to sulphate and to a lesser extent, to the glucuronide conjugates as well. The studies on the improvement of RSV bioavailability, which have beencarried out for many years, have contributed to the synthesis of the analogues of more chemopreventive and more desirable pharmacokinetic properties. In order to enhance antiproliferative activity and RSV bioavailability, series of methyl analogues were synthesized and this will be described later in more detail. An example of such a derivative is DMU-212 (3,4,4’5-tetramethoxystilbene).

The ovary is a complex endocrine gland that is responsible for sex steroid production in mammals. The basic functional unit of the ovary is the follicle, which contains the oocyte (OC), and surrounding cells, including granulosa cells (GCs) and theca cells, which support OC development. The process of formation and development of a follicle is known as folliculogenesis, during which the proliferation and differentiation of the GCs are required for proper formation of the follicle. In this review article, we present an overview of the molecular aspects of oogenesis and folliculogenesis, with subsequent description of mammalian ovulation. Moreover, we describe how growth, proliferation, and differentiation of GCs is regulated on a molecular level across mammalian species. Based on recent scientific reports, we depict possible biomedical applications and propose directions for future research using mammalian GCs in primary culture.

Progesterone (P4) and estradiol (E2) play a significant role in mammalian reproduction. Our study demonstrated that separated porcine cumulus cells (CCs) and/or granulosa cells (GCs) might proliferate in vitro during short-term, real-time primary culture. The GCs were analyzed according to gene expression of the progesterone receptor (nuclear form) (pgr), progesterone receptor membrane component 1 (pgrmc1), and estrogen-related receptor beta 3 (esrrb3) in relation to two housekeeping genes: actb and pbgd. GCs were cultivated in medium with the E2. Both pgr/actb and pgr/pbgd revealed higher expression between 24 and 168 h of IVC of prolonged E2 treatment and at 48 h of IVC after acute E2 administration. The pgrmc1/actb and pgrmc1/pbgd displayed increased expression after prolonged E2 treatment between 24 and 120 h of IVC. The highest level of esrrb3/actb at 120 and 144 h, as well as esrrb3/pbgd at 120 h, in untreated controls as compared to the hormone-stimulated group, was observed. We suggest that E2 significantly influences the upregulation of pgr, pgrmc1, and esrrb3 expression in porcine GCs during real-time cell proliferation. Since esrrb3 expression is stimulated by E2 in both an acute and prolonged manner, estradiol may be recognized as a potential estrogen receptor agonist in GCs.

The cumulus-oocyte complexes (COCs) growth and development during folliculogenesis and oogenesis are accompanied by changes involving synthesis and accumulation of large amount of RNA and proteins. In this study, the transcriptomic profile of genes involved in “oocytes RNA synthesis” in relation to in vitro maturation in pigs was investigated for the first time. The RNA was isolated from oocytes before and after in vitro maturation (IVM). Interactions between differentially expressed genes/proteins belonging to “positive regulation of RNA metabolic process” ontology group were investigated by STRING10 software. Using microarray assays, we found expression of 12258 porcine transcripts. Genes with fold change higher than 2 and with corrected p value lower than 0.05 were considered as differentially expressed. The ontology group “positive regulation of RNA metabolic process” involved differential expression of AR, INHBA, WWTR1, FOS, MEF2C, VEGFA, IKZF2, IHH, RORA, MAP3K1, NFAT5, SMARCA1, EGR1, EGR2, MITF, SMAD4, APP, and NR5A1 transcripts. Since all of the presented genes were downregulated after IVM, we suggested that they might be significantly involved in regulation of RNA synthesis before reaching oocyte MII stage. Higher expression of “RNA metabolic process” related genes before IVM indicated that they might be recognized as important markers and specific “transcriptomic fingerprint” of RNA template accumulation and storage for further porcine embryos growth and development.

The cumulus-oocyte complexes (COCs) growth and development during folliculogenesis and oogenesis are accompanied by changes involving synthesis and accumulation of large amount of RNA and proteins. In this study, the transcriptomic profile of genes involved in “oocytes RNA synthesis” in relation to in vitro maturation in pigs was investigated for the first time. The RNA was isolated from oocytes before and after in vitro maturation (IVM). Interactions between differentially expressed genes/proteins belonging to “positive regulation of RNA metabolic process” ontology group were investigated by STRING10 software. Using microarray assays, we found expression of 12258 porcine transcripts. Genes with fold change higher than |2| and with corrected p value lower than 0.05 were considered as differentially expressed. The ontology group “positive regulation of RNA metabolic process” involved differential expression of AR, INHBA, WWTR1, FOS, MEF2C, VEGFA, IKZF2, IHH, RORA, MAP3K1, NFAT5, SMARCA1, EGR1, EGR2, MITF, SMAD4, APP, and NR5A1 transcripts. Since all of the presented genes were downregulated after IVM, we suggested that they might be significantly involved in regulation of RNA synthesis before reaching oocyte MII stage. Higher expression of “RNA metabolic process” related genes before IVM indicated that they might be recognized as important markers and specific “transcriptomic fingerprint” of RNA template accumulation and storage for further porcine embryos growth and development.

Proper oocyte maturation in mammals produces an oocyte capable of monospermic fertilization and embryo preimplantation. The cumulus-oocyte complexes (COCs), surrounding an oocyte, play a significant role in oocyte maturation. During this process, when the COCs undergo cumulus expansion wherein tightly compact cumulus cells (CCs) form a dispersed structure, permanent biochemical and molecular modifications occur in the maturing oocytes, indicating that the gene expression between immature and mature oocytes differs significantly. This study focuses on the genes responsible for the cellular components of morphogenesis within the developing oocyte. Brilliant cresyl blue (BCB) was used to determine the developmental capability of porcine oocytes. The immature oocytes (GV stage) were compared with matured oocytes (MII stage), using microarray and qRT-PCR analysis to track changes in the genetic expression profile of transcriptome genes. The data showed substantial upregulation of genes influencing oocyte’s morphology, cellular migration and adhesion, intracellular communication, as well as plasticity of nervous system. Conversely, downregulation involved genes related to microtubule reorganization, regulation of adhesion, proliferation, migration and cell differentiation processes in oocytes. This suggests that most genes recruited in morphogenesis in porcine oocytein vitro,may have cellular maturational capability, since they have a higher level of expression before the oocyte’s matured form. It shows the process of oocyte maturation and developmental capacity is orchestrated by significant cellular modifications during morphogenesis.

The key mechanisms responsible for achievement of full reproductive and developmental capability in mammals are the differentiation and transformation of granulosa cells (GCs) during folliculogenesis, oogenesis, and oocyte maturation. Although the role of 17 beta-estradiol (E2) in ovarian activity is widely known, its effect on proliferative capacity, gap junction connection (GJC) formation, and GCs-luteal cells transformation requires further research. Therefore, the goal of this study was to assess the real-time proliferative activity of porcine GCs in vitro in relation to connexin (Cx), luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), and aromatase (CYP19A1) expression during short-term (168 h) primary culture. The cultured GCs were exposed to acute (at 96 h of culture) and/or prolonged (between 0 and 168 h of culture) administration of 1.8 and 3.6 μM E2. The relative abundance of Cx36, Cx37, Cx40, Cx43, LHR, FSHR, and CYP19A1 mRNA was measured. We conclude that the proliferation capability of GCs in vitro is substantially associated with expression of Cxs, LHR, FSHR, and CYP19A1. Furthermore, the GC-luteal cell transformation in vitro may be significantly accompanied by the proliferative activity of GCs in pigs.

Resveratrol (3,4',5-trans-trihydroxystilbene) is a naturally occurring phytoalexin which shows a wide spectrum of biological activity. Although the anticancer effects of resveratrol have been revealed, its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. The structural alteration of the stilbene motif of resveratrol has been found to be a promising strategy for generation of several synthetic analogues with improved pharmacokinetic parameters. The substitution of hydroxyl groups of resveratrol for methoxy groups may increase the molecule stability, making it less susceptible to phase II conjugation reactions in vivo. The structure-activity studies have revealed that introduction of additional methoxy groups resulted in an increased cytotoxic properties of resveratrol analogues. Furthermore, the methoxy groups at positions 3,5- and 3,4,5- of the trihydroxystilbene scaffold have been identified as crucial for pro-apoptotic activity of the compound. To date few reports have demonstrated the antiproliferative effect of DMU-212 (3,4,4',5-tetramethoxystilbene) in transformed fibroblasts, prostate, liver, colon, cervical, breast and ovarian cancer cells. It has been shown that DMU-212 is biotransformed to four metabolites: 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214), 4'-hydroxy-3,4,5-trimethoxystilbene (DMU-281), 4-hydroxy-3,5,4'-trimethoxystilbene (DMU-291), and 3-hydroxy-4,5,4'-trimethoxystilbene (DMU-807). However, if these metabolites retain the biological activity of DMU-212 remains unknown. Therefore, we evalueted the cytotoxic activity of these four metabolites of DMU-212 and the induction of apoptosis in ovarian A-2780 and SKOV-3 cancer cell lines. CYP1A1 and CYP1B1 are known to catalyze the conversion of estradiol to 4-hydroxyestradiol. Hence, the compounds such as resveratrol or DMU-212, which are structurally similar to the endogenous oestrogen estradiol, have been suggested to undergo metabolism by these enzymes. Surprisingly, DMU-212 has been shown to entirely inhibit CYP1B1 in ovarian A-2780 cancer cell line. Since CYP1B1 has been suggested to be involved in DMU-212 biotransformation, it can be hypothesised that one or more metabolites of DMU-212 regulate the expression of CYP1B1 by the feedback inhibition. To have an insight into DMU-212 metabolites' role in the expression of isoenzymes of CYP1 family we assessed CYP1A1 and CYP1B1 mRNA and protein levels in metabolites-treated A-2780 and SKOV-3 cancer cells. The inhibitory effect of four metabolites of DMU-212 was investigated in ovarian A-2780 and SKOV-3 cancer cell lines by MTT assays. The induction of apoptosis was assayed by Cell Death Detection ELISAPLUS test, Caspase-Glo® 3/7 luminescent test and flow cytometry analysis. The expression of CYP1A1 and CYP1B1 mRNA and protein levels was investigated by RT-qPCR and Western blott, respectively. From among four metabolites of DMU-212, DMU-214 exhibited the highest cytotoxic activity (IC50 A-2780=13.26±2.11 nM; IC50 SKOV-3=29.21±3.17nM). Furthermore, DMU-214 has been revealed to exert more pronounced inhibitory effects than the prototype compound, DMU-212. In addition, we observed that the stronger cytotoxic activity of DMU-214 evoked its greater pro-apoptotic effect. DMU-214 caused an increase in nucleosomes level in A-2780 and SKOV-3 cells, EF= 15.89 and 7.91, respectively. Exposure of either cell line to DMU-214 resulted in a statistically significant decrease in CYP1A1 and CYP1B1 mRNA and protein levels as compared to control. We showed for the first time that DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cell lines. Since DMU-214 has been observed to decrease expression of CYP1A1 and CYP1B1 in A-2780 and SKOV-3 cells our results provide a new insight into the concept of their feedback inhibition by the compound tested. Citation Format: Sylwia Borys, Malgorzata Kucinska, Dorota Trzybulska, Mariusz Kaczmarek, Marcin Wierzchowski, Marek Murias, Jadwiga Jodynis-Liebert, Hanna Piotrowska. Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 40.

The porcine model is often used in clinical trials. The pig has many fundamental anatomic, physiological and nutritional similarities to humans. Additionally, the European Medicines Agency (EMA) demands the use large animals in clinical studies. Oral mucosa has received special attention due to its regenerative properties. Oral tissue is composed of several types of cells including fibroblasts and keratinocytes. The porcine oral mucosa/buccal pouch mucosa has a cellular structure with defined proliferation and differentiated capability. In this study, we investigated the expression pattern of porcine buccal pouch mucosal cell proliferation and differentiation markers such as Ki-67, proliferating cell nuclear antigen (PCNA), and involucrin. We observed a clear monolayer culture of spindle-shaped, porcine buccal pouch mucosal cells during 168 h of real-time in vitro culture. The RTCA assays revealed parametric and progressive increases in proliferation after 72 h of IVC. We found an altered proliferation index (PI) in the replicated groups of experiments except through the 144-168 h proliferation period. The RT-qPCR results demonstrated a significant increase in Ki-67 and PCNA expression after 48, 120, and 168 h of IVC as compared to other culture periods (P0.001). The involucrin mRNA displayed increased expression after 168 h of IVC as compared to other periods. We observed a lack of PCR product at 24 h in the case of Ki-67 and both before IVC (0h) and after 24 h of IVC for PCNA mRNA. When we analyzed the three transcripts together, we found the highest expression of involucrin during each of the culture periods. It has been suggested that Ki-67, PCNA, and involucrin may be successfully used as markers of porcine buccal pouch mucosal cell proliferation and differentiation capability in vitro.

For normal folliculogenesis and oogenesis to occur many intrinsic and extrinsic factors are needed, i.e. positive feedback of hormone secretion and local ovarian-follicular growth factors distribution. During follicle formation, granulosa cells (GCs) change their morphology and physiological properties. The factors needed for GCs to differentiate within each layer are transforming growth factor beta (TGFB) and insulin-like growth factor (IGF), as well as the activation and modification of biochemical pathways involved in folliculogenesis. Physiological alterations occur when GC genes are characterized by several differences in their gene expression profile. Studies in recent years indicate a variety of processes involved in follicle morphology and biochemical remodeling during growth and development. It was demonstrated that IGFs play a central role in the differentiation of GCs both in vivo and in vitro. Moreover, the primary role of FSH and LH in the formation of the ovarian follicle, was also described. Our review article characterizes the most important pathways involved in the differentiation of GCs and the effect of various factors on gene expression in GCs during folliculogenesis.