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Open access

Marta Bukowska, Anna Bogacz, Marlena Wolek, Przemysław Ł. Mikołajczak, Piotr Olbromski, Adam Kamiński and Bogusław Czerny

Summary

Introduction: Blood brain barrier and multidrug resistance phenomenon are subjects of many investigations. Mainly, because of their functions in protecting the central nervous system (CNS) by blocking the delivery of toxic substances to the brain. This special function has some disadvantages, like drug delivery to the brain in neurodegenerative diseases

Objective: The aim of this study was to examine how natural and synthetic substances affect the expression levels of genes (Mdr1a, Mdr1b, Mrp1, Mrp2, Oatp1a4, Oatp1a5 and Oatp1c1) that encode transporters in the blood-brain barrier.

Methods: cDNA was synthesized from total RNA isolated from rat hippocampus. The expression level of genes was determined using real-time PCR (RT-PCR) method.

Results: Our findings showed that verapamil, as a synthetic substance, caused the greatest reduction of mRNA level of genes studied. The standardized extract of Curcuma longa reduced the expression level for Mrp1 and Mrp2, whereas the increase of mRNA level was observed for Mdr1b, Oatp1a5 and Oatp1c1.

Conclusions: These results suggests that herbal extracts may play an important role in overcoming the blood brain barrier during pharmacotherapy.

Open access

Marta Rybska, Sandra Knap, Maurycy Jankowski, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Michał Nowicki, Maciej Zabel, Bartosz Kempisty and Jędrzej M. Jaśkowski

Abstract

Folliculogenesis is the process of ovarian follicle formation,, taking presence during foetal period. During the follicular development, oogoniums undergo meiosis and oocytes are formed. In the ovaries of new born sows, primary and secondary follicles are present and, 90 days after birth, tertiary follicles appear. During development in the ovarian follicles growth of granulosa cells and differentiation of the thecal cells can be observed. A cavity filled with follicular fluid appears. Granulosa cells are divided into: mural cells and corona radiata, which together with the oocyte form the cumulus oophorus. Corona radiata cells, mural layers and oolemma contact each other by a network of gap junctions. Secreted from the pituitary gland, FSH and LH gonadotropin hormones act on receptors located in granular and follicular cells. In the postnatal life tertiary follicles and Graafian follicles are formed. When the follicle reaches a diameter of 1 mm, further growth depends on the secretion of gonadotropins. Mature ovarian follicles produce: progestins, androgens and oestrogens. The growth, differentiation and steroidogenic activity of ovarian follicles, in addition to FSH and LH, is also affected by prolactin, oxytocin, steroid and protein hormones, numerous proteins from the cytokine and interleukin family, metabolic hormones like insulin, glucocorticoids, leptin, thyroid hormones and growth hormones. Despite numerous studies, many processes related to folliculogenesis have not been discovered Learning the mechanisms regulating reproductive processes would allow to easily distinguish pathological processes and discover more and more genes and mechanisms of their expression in cells that build ovarian follicles.

Open access

Marta Rybska, Sandra Knap, Maurycy Jankowski, Blanka Borowiec, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Michał Nowicki, Maciej Zabel, Bartosz Kempisty and Jędrzej M. Jaśkowski

Abstract

Ovarian cysts remain to be one of the most common and serious problems in reproduction of farm animals, as well as humans. Apart from causing the fall in reproductive potential of the ovaries, occupying the place in which folliculogenesis and oogenesis occur, they also cause hormone imbalances, by preventing corpus luteum formation, hence lowering the amount of steroid hormone production. While singular cysts rarely affect fertility, hormone fluctuations that are associated with their presence promotes their multiplication, which usually has more adverse effects. While the cysts are easily detectable in humans, possessing distinct echography while examined by ultrasound, multiple factors prevent widespread use of effective detection methods among large herds of farm animals. Because of lack of noticeable symptoms of early stages of such malignancies, they rarely get detected before the animal stops to exhibit symptoms of heat. That causes scientific research to be focused on not only methods of detection, but also the ways to negate the effects of ovarian cysts and bring the affected specimen back to reproductive potential. Despite that, high costs of diagnosis and treatment, cause them to be uncommon on commercial farms. As lack of fertility eliminates animals from breeding purposed herds, ovarian cysts persist as a cause of large losses of the animal husbandry business. Continuous research, focused on natural examples of ovarian cysts should be conducted, in order to improve methods of detection, prevention, treatment and recovery from the effects of ovarian cysts.

Open access

Marta Rybska, Sandra Knap, Katarzyna Stefańska, Maurycy Jankowski, Agata Chamier-Gliszczyńska, Małgorzata Popis, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Bartosz Kempisty and Jędrzej M. Jaśkowski

Abstract

The superfamily of transforming growth factors β (TGF-β) consists of cytokines that are crucial in regulating the organism’s biological functions and includes three isoforms of TGF-β protein, Anti-Müllerian Hormone (AMH), inhibin A and B, activins, 20 bone morphogenetic proteins (BMP1-20) and 9 growth factors (GDF1-9). Their signal transduction pathway involves three types of membrane receptors that exhibit a serine/threonine kinase activity, as well as the Smad proteins. After ligand binding, the Smad proteins are phosphorylated and translocated to the nucleus, where they interact with transcription factors and affect gene expression. TGF-β family members are involved in cell growth and differentiation, as well as chemo-taxis and apoptosis, and play an important role during an inflammation. Defects in TGF-β proteins or in their signalling pathway underlie many severe diseases, such as systemic lupus, systemic scleroderma, bronchial asthma, atherosclerosis, hyperthyroidism or cancer. These factors are also crucial in mammal reproductive functions, as they are involved in folliculogenesis, steroidogenesis, ovulation, maternal-embryo interaction, embryo development and uterine decidualization. Their defects result in issues with fertility. This review focuses on the relevance of TGF-β family members in a mammal reproduction with an emphasis on three TGF-β isoforms, inhibins A and B, GDF-9 and their signal transduction pathway.

Open access

Marta Rybska, Sandra Knap, Maurycy Jankowski, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Michał Nowicki, Maciej Zabel, Bartosz Kempisty and Jędrzej M. Jaśkowski

Abstract

The pig is a polyestrous animal in which the ovarian cycle lasts about 21 days and results in ovulation of 10-25 oocytes. Ovum reaches 120-150 μm in diameter, with the surrounding corona radiata providing communication with the environment. The zona pellucida is composed of glycoproteins: ZP1, ZP2, ZP3. In the course of oogenesis, RNA and protein accumulation for embryonic development occurs. Maternal mRNA is the template for protein production. Nuclear, cytoplasmic and genomic maturity condition the ability of the ovum to undergo fertilization. There are several differences in protein expression profiles observed between in vitro and in vivo conditions. Oogenesis is the process of differentiating female primary sex cells into gametes. During development gonocytes migrate from the yolk sac into the primary gonads with TGF-1, fibronectin, and laminin regulating this process. Cell cycle is blocked in dictyotene. Primary oocyte maturation is resumed before each ovulation and lasts until the next block in metaphase II. At the moment of penetration of the sperm into the ovum, the metaphase block is broken. The oocytes, surrounded by a single layer of granular cells, form the ovarian follicle. The exchange of signals between the oocyte and the cumulus cells done by gap-junctions, as well as various endo and paracrine signals. The contact between the corona radiata cells provides substances necessary for growth, through the same gap junctions. Studies on follicular cells can be used to amplify the knowledge of gene expression in these cells, in order to open way for potential clinical applications.

Open access

Joanna Budna, Piotr Celichowski, Paresto Karimi, Wiesława Kranc, Artur Bryja, Sylwia Ciesiółka, Marta Rybska, Sylwia Borys, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Klaus P. Brüssow, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty

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.

Open access

Wiesława Kranc, Piotr Celichowski, Joanna Budna, Ronza Khozmi, Artur Bryja, Sylwia Ciesiółka, Marta Rybska, Sylwia Borys, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Klaus P. Brüssow, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty

Summary

The 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.

Open access

Artur Bryja, Marta Dyszkiewicz-Konwińska, Maurycy Jankowski, Piotr Celichowski, Katarzyna Stefańska, Agata Chamier-Gliszczyńska, Małgorzata Popis, Katarzyna Mehr, Dorota Bukowska, Paweł Antosik, Małgorzata Bruska, Maciej Zabel, Michał Nowicki and Bartosz Kempisty

Abstract

The oral mucosa is a compound tissue composed of several cells types, including fibroblasts and keratinocytes, that are characterized by different morphology, as well as biochemical and metabolomic properties. The oral mucosal cells are the most important factors mediated between transport and drugs delivery. The changes in cellular ion homeostasis may significantly affect the bioavailability of administrated drugs and their transport across the mucous membrane. Therefore we investigated the expression profile of genes involved in ion transport and homeostasis in porcine buccal pouch mucosal cells.

The oral mucosa was separated surgically and isolated enzymatically. The cells were examined during long-term in vitro culture (IVC). The cultured cells were collected at 7, 15 and 30 days of IVC and subsequently transferred to RNA isolation and next, the gene expression profile was measured using Affymetrix microarray assays.

In the results, we can extract genes belonging to four ontology groups: “ion homeostasis”, “ion transport”, “metal ion transport”, and “inorganic ion homeostasis”. For TGFB1 and CCL2, we observed up-regulation after 7 days of IVC, down-regulation after 15 days of IVC and upregulation again after 30 days of IVC. The ATP13A3, ATP1B1, CCL8, LYN, STEAP1, PDPN, PTGS2, and SLC5A3genes showed high activity after day 7 of IVC, and in the days 15 and 30 of IVC showed low activity.

We showed an expression profile of genes associated with the effects of ion influence on the porcine normal oral mucosal cell development in IVC. These studies may be the starting point for further research into oral diseases and will allow for the comparison of the gene expression profile of normal and disease altered cells.

Open access

Artur Bryja, Marta Dyszkiewicz-Konwińska, Maurycy Jankowski, Piotr Celichowski, Katarzyna Stefańska, Agata Chamier-Gliszczyńska, Blanka Borowiec, Katarzyna Mehr, Dorota Bukowska, Paweł Antosik, Małgorzata Bruska, Maciej Zabel, Michał Nowicki and Bartosz Kempisty

Abstract

The mucous membrane is composed of two layers. The layer of stratified squamous epithelium and the underlying layer of the connective tissue. The epithelium is composed of keratinocytes that are in different stages of differentiation, depending on their localization. In our research, after isolation of primary in vitro cultured buccal pouch mucosal cells, we observed keratinocytes in various stages of differentiation and fibroblasts. These cells, depending on the ionic dynamics, may be subject to different morphological and biochemical transformations. Understanding the expression profile of the normal oral mucosal tissue is important for further research into the effects of biomaterials on the mucosal cells, their growth, proliferation, and differentiation.

The porcine buccal pouch mucosal cells were used in this study. The oral mucosa was separated surgically and isolated enzymatically. The cells were in vitro cultured for 30 days, and after each step of in vitro culture (7 days, 15 days, 30 days), samples were collected for isolation of total RNA. The gene expression profile was measured using Affymetrix microarray assays.

In results, we observed genes belonging to two ontology groups: cation homeostasis and cation transport. These genes were up-regulated after 7 days of in vitro culture as compared to down-regulation after 15 and 30 days of in vitro culture. These results suggested that dynamic growth, proliferation and cell adhesion are more intense in the first 7 days of in vitro culture. We also observed, for the first time, the expression of ATP13A3 in porcine oral mucosal cells.

Open access

Maurycy Jankowski, Marta Dyszkiewicz-Konwińska, Joanna Budna, Sandra Knap, Artur Bryja, Sylwia Borys, Wiesława Kranc, Magdalena Magas, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Klaus P. Brüssow, Marie Machatkova, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty

Abstract

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.