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  • Author: Michal Jeseta x
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The genes regulating maintenance of cellular protein location are differentially expressed in porcine epithelial oviductal cells during longterm in vitro cultivation

Abstract

The oviduct is a part of female reproductive tract that is essential for successful fertilization and early embryo development. It is lined with epithelium consisting of two types of cells: ciliated and secretory. The primary function of ciliated oviductal epithelial cells (OECs) is to support the transport of gametes and embryos through the ovary, whereas secretory OECs produce components of the oviductal fluid. Undoubtedly, the oviductal epithelium plays a major part in the early aspects of pregnancy development, from providing an optimal environment for gametes and embryos to supporting fertilization. Therefore, our aim was to gain a better insight into the genetic changes underlying function of these cells. We have harvested OECs from crossbred gilts (n=45), at the age of about nine months and which displayed two regular estrous cycles, and established long-term primary culture of porcine OECs. Microarray analysis was utilized to determine differentially expressed genes during day 1, 7, 15 and 30 of cultivation, with our results revealing54 differentially expressed genes belonging to three ontology groups: „maintenance of location”, „maintenance of protein location” and „maintenance of protein location in cell”. Since the biochemistry and morphology of epithelial cells may change during long term cultivation, we conclude that our results are a reflection of these changes and help to shed a light on porcine OECs properties in in vitro environment.

Running title: Maintenance of cellular protein location in porcine epithelial oviductal cells

Open access
Endocrine disruptive compounds and male reproduction

Abstract

Endocrine disruptors (EDs) are chemical substances that affect physiological processes in the organism via hormonal regulation. The EDs are present in the environment and objects of everyday use. They are often detected in food, particularly released from packaging of canned food, but also from plastic water bottles, and they are also found in cosmetics and fertilizers. They are commonly detected in children's toys, banknotes, receipts and many more objects. Permanent and long-term utilization of EDs has harmful effects on human reproductive health mainly by interference with sex hormone synthesis and mechanism of action. The endocrine disruptors show many negative effects on male reproductive system. Any change during synthesis or activity of sex hormones can cause abnormal reproduction, including developmental anomalies of the sexual system, disruption of testicular development or deterioration of sperm quality. Mainly the impact on the development of testicles in prenatal and early postnatal period can be crucial for reproductive health in males. This review provides an overview of the EDs and their possible impact on reproductive health in males with focus on sperm quality and development of testicles.

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Current Topics in Assisted Reproduction in the Czech Republic

Abstract

Assisted reproduction is a very dynamic part of reproductive gynaecology with fast changes in therapeutic approaches resulting from intensive research supported by private or public companies. This progress brings also new questions regarding ethical, legal or therapeutic issues. At the 26th Czech-Slovak symposium of assisted reproduction in Brno, several interesting topics of assisted reproduction were presented. During two days, more than 350 specialists discussed current topics related to gynaecology, embryology, andrology, genetics and sexology. From this fruitful discussion we chose the following topics that we consider to be the most important: (1) “Soft” versus “standard” stimulation protocols for IVF (2) Use of exogenous and endogenous progestins in IVF stimulation protocols (3) Cryopreservation and transplantation of ovarian tissue - state of the art and situation in the Czech Republic (4) Selection of spermatozoa with fragmented DNA: practical recommendations (5) Artificial activation of oocytes (8) Access of single and lesbian women to assisted reproduction technologies and possibility of their reproduction in the Czech Republic. Panel conclusions were presented at the end of the conference, which had great attendance, invoked lively commentaries and produced some definitive consensus. Certain issues remained inconclusive and these matters will be the subject of further discussion in future. This is a summary of the most important theses from this field in the Czech Republic in 2017.

Open access
Genes regulating programmed cell death are significantly upregulated in porcine immature oocytes

Abstract

Correct maturation of the oocyte is crucial for further fertilization and embryogenesis. It comprises of both nuclear and cytoplasmic maturation, during which the proteins, nutrients and mRNAs are assembled. Cumulus cells are connected with the oocyte via gap-junctions, which enable bi-directional transfer of molecules, forming cumulus-oocyte complex (COC). The expression pattern in CCs is thought to resemble the genes expressed in the oocyte. The CCs surrounding the gamete of high developmental competence have an increased expression of apoptotic markers. Therefore, our aim in this study was to determine whether any apoptosis-related genes are upregulated in porcine oocytes before or after IVM. We isolated COCs from 45 pubertal crossbred gilts, performed brilliant cresyl blue (BCB) staining and analyzed the gene expression pattern in oocytes before and after IVM with the use of microarray analysis. The results include 419 differentially expressed transcripts, 25 of which belong to „regulation of apoptosis” and „regulation of cell death” GO BP terms. This set of genes includes BCLAF1, EIF2AK3, KLF10, MIF, MAP3K1, NOTCH2, TXNIP and APP, all of which have been upregulated in immature porcine oocytes. Our results suggest that they play part in porcine oocyte maturation and could be used as potential markers of female gamete’s developmental competence. This knowledge could serve as a basis to improve ART in pigs.

Open access
Endocrine disruptors: General characteristics, chemical nature and mechanisms of action. A review.

Abstract

Over recent decades, different types of industrially manufactured chemicals have become widespread environmental contaminants with potential to interfere with the synthesis, secretion, transport, binding or elimination of natural hormones in the body. These chemical substances were named endocrine disruptors (EDs). The main route of exposure to EDs is the ingestion of contaminated food and water. EDs are very dangerous, because they have long half-life, stay present in the environment for years and may concentrate at great distances from the site where were produced. The group of EDs is heterogeneous and contains industrial lubricants, solvents, plastics, plasticizers, pesticides, fungicides, drugs, but also natural chemicals. The mechanisms of EDs action are difficult to predict, many substances act by interfering with the estrogen receptors (ER), androgen receptor (AR), thyroid receptors (TRs) and aryl hydrocarbon receptor (AhR), but they can also influence hormone synthesis or can have effect on epigenetic mechanisms. Further research is necessary to improve knowledge about EDs and their metabolites, and to identify endocrine-disruptive potential of chemicals, those replacing current EDs before they are widely distributed.

Open access
Resveratrol and Its Analogues – Is It a New Strategy of Anticancer Therapy?

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).

Open access
Genes regulating biochemical pathways of oxygen metabolism in porcine oviductal epithelial cells during long-term IVC

Abstract

Oxygen metabolism has an important role in the normal functioning of reproductive system, as well as the pathogenesis of female infertility. Oxidative stress seems to be responsible for the initiation or development of reproductive organ diseases, including polycystic ovary syndrome, endometriosis, preeclampsia, etc. Given the important role of maintaining balance between the production of ROS and antioxidant defence in the proper functioning of reproductive system, in the present study we aimed to analyse the expression of genes related to oxygen metabolism in porcine oviductal epithelial cells during long-term in vitro culture. The oviducts were collected from 45 crossbred gilts at the age of approximately nine months that displayed at least two regular oestrous cycles. The oviductal endothelial cells were isolated by enzymatic digestion to establish long-term primary cultures. Gene expression changes between 7, 15 and 30 daysof culturewere analysed with the use ofwhole transcriptome profiling by Affymetrix microarrays. The most of the “cellular response to oxidative stress” genes were upregulated. However, we did not observe any main trend in changes within the “cellular response to oxygen-containing compound” ontology group, where the gene expression levels were changed in various manner.

Running title: Oxygen metabolism in porcine oviductal epithelial cells

Open access
Analysis of expression of genes responsible for regulation of cellular proliferation and migration – microarray approach based on porcine oocyte model

Abstract

The formation of mammalian oocytes begins in the ovary during fetal development. The proper development of oocytes requires close communication with surrounding somatic cells, the substances they emit allow proper maturation of oocytes. Somatic cumulus (CC) cells and oocytes form cumulus-oocyte (COC) complexes.

In this study, the Affymetrix microarray analysis was used to investigate changes in gene expression occurring in oocytes before and after in vitro maturation (IVM). The aim of the study was to examine oocyte genes involved in two ontological groups, “regulation of cell migration” and “regulation of cell proliferation” discovered by the microarray method.

We found a reduced expression of all 28 genes tested in the ontological groups: ID2, VEGFA, BTG2, CCND2, EDNRA, TGFBR3, GJA, LAMA2, RTN4, CDK6, IHH, MAGED1, INSR, CD9, PTGES, TXNIP, ITGB1, SMAD4, MAP3K1, NOTCH2 , IGFBP7, KLF10, KIT, TPM1, PLD1, BTG3, CD47 and MITF. We chose the most regulated genes down the IVM culture, and pointed out those belonging to two ontological groups.

Increased expression of the described genes before IVM maturation may indicate the important role of these genes in the process of ovum maturation. After the maturation process, the proteins produced by them did not play such an important role. In summary, the study provides us with many genes that can serve as molecular markers of oocyte processes associated with in vitro maturation. This knowledge can be used for detailed studies on the regulation of oocyte maturation processes.

Running title: Genes regulating cellular migration and proliferation in porcine oocytes

Open access
Magnetic-Activated Cell Sorting in Combination with Swim-Up Efficiency Improve Effectivity of Spermatozoa Separation

Abstract

Correct selection of spermatozoa before their using for an assisted reproductive techniques is one of the crucial step in therapy of human infertility. It was previously reported that male factor plays a major role in infertility. Basic semen analyses and standard methods for sperm selection in many cases does not eliminate sufficiently proportion of spermatozoa with genetics defects. Magnetic Activated Cell Sorting (MACS) is a selection method which reduce apoptotic sperm and improve sperm and embryo quality. The aim of our study was the comparison of swim-up method and MACS and their combination. We tested swim-up and MACS alone and treatment of spermatozoa in combination when was is first swim-up and second MACS and vice versa. In this study we evaluated sperm concentration, motility and their DNA integrity before and after separtion methods. On the basis of our results we recommend to use swim-up before MACS method. This approach brings better results in the sperm selection - lower proportion of spermatozoa with fragmented DNA and also it brings better gain of total spermatozoa usable for next IVF or ICSI methods.

Open access
Recent Findings of the Types of Programmed Cell Death

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.

Open access