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Open access

Joanna Budna, Piotr Celichowski, Sandra Knap, Maurycy Jankowski, Magdalena Magas, Mariusz J. Nawrocki, Piotr Ramlau, Andrzej Nowicki, Magdalena Rojewska, Błażej Chermuła, Michal Jeseta, Paweł Antosik, Dorota Bukowska, Małgorzata Bruska, Maciej Zabel, Michał Nowicki and Bartosz Kempisty

Abstract

The process of reproduction requires several factors, leading to successful fertilization of an oocyte by a single spermatozoon. One of them is the complete maturity of an oocyte, which is acquired during long stages of folliculogenesis and oogenesis. Additionally, the oviduct, composed of oviductal epithelial cells (OECs), has a prominent influence on this event through sperm modification and supporting oocyte’s movement towards uterus. OECs were isolated from porcine oviducts. Cells were kept in primary in vitro culture for 30 days. After 24h and on days 7, 15 and 30 cells were harvested, and RNA was isolated. Transcript changes were analyzed using microarrays. Fatty acids biosynthetic process and fatty acids transport ontology groups were selected for analysis and described. Results of this study indicated that majority of genes in both ontology groups were up-regulated on day 7, 15 and 30 of primary in vitro culture. We analyzed genes involved in fatty acids biosynthetic process, including: GGT1, PTGES, INSIG1, SCD, ACSL3, FADS2, FADS1, ACSS2, ALOX5AP, ACADL, SYK, ACACA, HSD17B8, FADS3, OXSM, and transport, including: ABCC2, ACSL4, FABP3, PLA2G3, PPARA, SYK, PPARD, ACACA and P2RX7. Elevated levels of fatty acids in bovine and human oviducts are known to reduce proliferation capacity of OECs and promote inflammatory responses in their microenvironment. Most of measured genes could not be connected to reproductive events. However, the alterations in cellular proliferation, differentiation and genes expression during in vitro long-term culture were significant. Thus, we can treat them as putative markers of changes in OECs physiology.

Open access

Joanna Budna, Piotr Celichowski, Artur Bryja, Michal Jeseta, Maurycy Jankowski, Dorota Bukowska, Paweł Antosik, Andrzej Nowicki, Klaus P. Brüssow, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty

Abstract

Mammalian oocytes undergo compound processes of nuclear and cytoplasmic maturation that allow them to reach MII stage. Only fully mature, oocyte can be successfully fertilized by a single spermatozoon. Fatty acids, apart from their role in cellular metabolism, inflammation and tissue development, have positive and detrimental effects on oocyte maturation, fertilization, blastocyst cleavage rate and embryo development in mammals. Using microarrays, we have analyzed the expression changes in fatty acids- -related genes during in vitro maturation of porcine oocytes. The oocytes were recovered from ovaries of 45 pubertal crossbred Landrace gilts and subsequently subjected to BCB test. For further analyses, only granulosa cell-free BCB+ oocytes were used and divided into two groups. The first one, described as “before IVM”, was directly exposed to molecular assays, the second one, described as “after IVM”, was first in vitro matured and then subjected to a second BCB test. Oocytes, if classified as BCB+, were then passed to corresponding molecular analyses. We found significant down-regulation of genes involved in fatty acid metabolic process, such as: ACSL6, EPHX2, FADS2, PTGES, TPI1, TBXAS1, NDUFAB1, MIF, ACADSB and DECR1 in porcine oocytes analyzed after IVM, in comparison to those analyzed before IVM. In conclusion, apart from poor data available concerning analyzed genes in relation to reproductive events, significant changes in their expression point to their potential role as an oocyte developmental competence markers in pigs. Introducing molecular diagnostics of oocytes could be the prospective tool for selection of best gametes, leading to improved outcomes of in vitro fertilization.

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, 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

Sylwia Borys-Wójcik, Ievgenia Kocherova, Piotr Celichowski, Małgorzata Popis, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Michał Nowicki and Bartosz Kempisty

Abstract

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.

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

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

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.

Open access

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

Abstract

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.