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  • Author: Michal Jeseta x
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Amino acids metabolism and degradation is regulated during porcine oviductal epithelial cells (OECs) primary culture in vitro – a signaling pathways activation approach

Abstract

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.

Open access
Ontology groups representing angiogenesis and blood vessels development are highly up-regulated during porcine oviductal epithelial cells long-term real-time proliferation – a primary cell culture approach

Abstract

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.

Open access
Fatty Acids Related Genes Expression Undergo Substantial Changes in Porcine Oviductal Epithelial Cells During Long-Term Primary Culture

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
Does Porcine Oocytes Maturation in Vitro is Regulated by Genes Involved in Transforming Growth Factor Beta Receptor Signaling Pathway?

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
Positive Regulation of Macromolecule Metabolic Process Belongs to the Main Mechanisms Crucial for Porcine Oocytes Maturation

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
Epithelium morphogenesis and oviduct development are regulated by significant increase of expression of genes after long-term in vitro primary culture – a microarray assays

Abstract

The correct oviductal development and morphogenesis of its epithelium are crucial factors influencing female fertility. Oviduct is involved in maintaining an optimal environment for gametes and preimplantation embryo development; secretory oviductal epithelial cells (OECs) synthesize components of oviductal fluid. Oviductal epithelium also participates in sperm binding and its hyperactivation. For better understanding of the genetic bases that underlay porcine oviductal development, OECs were isolated from porcine oviducts and established long-term primary culture. A microarray approach was utilized to determine the differentially expressed genes during specific time periods. Cells were harvested on day 7, 15 and 30 of in vitro primary culture and their RNA was isolated. Gene expression was analyzed and statistical analysis was performed. 48 differentially expressed genes belonging to “tube morphogenesis”, “tube development”, “morphogenesis of an epithelium”, “morphogenesis of branching structure” and “morphogenesis of branching epithelium” GO BP terms were selected, of which 10 most upregulated include BMP4, ARG1, SLIT2, FGFR1, DAB2, TNC, EPAS1, HHEX, ITGB3 and LOX. The results help to shed light on the porcine oviductal development and its epithelial morphogenesis, and show that after long-term culture the OECs still proliferate and maintain their tube forming properties.

Open access
Expression Changes in Fatty acid Metabolic Processrelated Genes in Porcine Oocytes During in Vitro Maturation

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
Genes encoding proteins regulating fatty acid metabolism and cellular response to lipids are differentially expressed in porcine luminal epithelium during long-term culture

Abstract

Among many factors, the epithelium lining the oviductal lumenis very important for the development of the oocyte and its subsequent fertilization. The oviductal epithelium is characterized by the presence of ciliary cells, supporting the movement of cumulus-oocyte complexes towards the uterus. By interacting with the semen, the epithelium of the fallopian tube makes the sperm acquire the ability to fertilize. So far, the exact molecular mechanisms of these changes have not been known. Hence, understanding the metabolism of oviduct epithelial cells and the level of expression of individual groups of genes seems to be a way to deepen the knowledge about the broadly understood reproduction.

In our research, we decided to culture oviductal epithelial cells (OECs) in vitro for a long period of time. After 24h, 7, 15 and 30 days, the OECs were harvested, with their RNA isolated. Transcriptomic changes were analyzed using microarrays. The “cellular response to lipid” group was represented by the following genes: MUC1, CYP24A1, KLF4, IL24, SNAI2, CXCL10, PPARD, TNC, ABCA10, while the genes belonging to the “cellular lipid metabolic processes” were: LIPG, ARSK, ACADL, FADS3, P2RX7, ACSS2, PPARD, KITLG, SPTLC3, ERBB3, KLF4, CRABP2. Additionally, PPARD and ACADL were members of the “fatty acid beta-oxidation” ontology group. Our study describes genes that are not directly related to fertility processes. However, significant changes in their expression in in vitro cultured OECs may indicate their usefulness as markers of OECs’ physiological processes.

Running title: Fatty acids changes in porcine oviductal epithelial cells in in vitro cultivation

Open access
“Cell cycle process”, “cell division” and “cell proliferation” belong to ontology groups highly regulated during long–term culture of porcine oviductal epithelial cells

Abstract

Morphological and biochemical changes in the cells surrounding the oocyte seem to be extremely important in an effective fertilization process. Thanks to advanced cell culture techniques, as well as biochemical and bioinformatics analyses, we can partly imitate the phenomena occurring in the living organism. Previous studies showed a possibility of short – and long – term OEC in vitro cultivation, during which these cells have shown to have significant proliferation and expression of genes responsible for differentiation. Our research was aimed at maintaining a culture of porcine oviduct epithelial cells and analyzing their gene expression profile. The study employed cross-bred gilts at the age of about 9 months, obtained from commercial herds. With the use of Affymetrix® Porcine Gene 1.1 ST Array Strip, we have examined the expression of 12257 transcripts. Genes with fold change higher than abs (2) and with corrected p-value lower than 0.05 were considered as differentially expressed. We chose 20 genes with the most marked expression (10 up – regulated, 10 down – regulated) for further investigation in the context of literature sources. These genes belonged to three ontological groups: “cell cycle process”, “cell division” and “cell proliferation”. The results obtained from these studies may be the basis for further molecular analyses.

Open access
Selected aspects of endometritis – pyometra complex in dogs – current troubles and treatment perspectives

Abstract

Pyometra is the most common gynecological disease in female dogs. It usually occurs in middle age female dogs, usually about two months after the completion of heat. This disease is the accumulation of purulent fluid inside the uterus. Etiology of pyometra is not fully understood. It is assumed, that pyometra is a result of hormonal disorders in the endometrium combined with bacterial superinfection. The diagnosis is based on the interview, clinical examination, additional laboratory tests and ultrasound or x-ray of the abdomen. There are two treatments: ovariohysterectomy and conservative treatment with pharmacological agents for example prostaglandin, aglepriston, antibiotics with a broad spectrum of action. Currently conducted molecular studies have a large influence on the development of the present knowledge on the pathogenesis and course of pyometra, whose conclusions may be used to change the current therapeutic protocols.

Open access