The Effect of Visfatin on the Functioning of the Porcine Pituitary Gland: An In Vitro Study.

Visfatin (VIS), also known as nicotinamide phosphoribosyltransferase (NAMPT), is the rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Recently, VIS has been also recognized as an adipokine. Our previous study revealed that VIS is produced in the anterior and posterior lobes of the porcine pituitary. Moreover, the expression and secretion of VIS are dependent on the phase of the estrous cycle and/or the stage of early pregnancy. Based on this, we hypothesized that VIS may regulate porcine pituitary function. This study was conducted on anterior pituitary (AP) glands harvested from pigs during specific phases of the estrous cycle. We have shown the modulatory effect of VIS in vitro on LH and FSH secretion by porcine AP cells (determined by ELISA). VIS was also found to stimulate cell proliferation (determined by Alamar Blue) without affecting apoptosis in these cells (determined using flow cytometry technique). Moreover, it was indicated that VIS may act in porcine AP cells through the INSR, AKT/PI3K, MAPK/ERK1/2, and AMPK signaling pathways (determined by ELISA or Western Blot). This observation was further supported by the finding that simultaneous treatment of cells with VIS and inhibitors of these pathways abolished the observed VIS impact on LH and FSH secretion (determined by ELISA). In addition, our research indicated that VIS affected the mentioned processes in a manner that was dependent on the dose of VIS and/or the phase of the estrous cycle. Thus, these findings suggest that VIS may regulate the functioning of the porcine pituitary gland during the estrous cycle.

Molecular Action of Tamoxifen in the Ovaries of Rats with Mammary Neoplasia.

Tamoxifen (TAM) is a drug commonly used in patients with breast cancer. The anticancer effect of TAM occurs via its ability to antagonize estrogen-dependent growth of mammary epithelial cells. Previously, we demonstrated that TAM prevented the chemotherapy-induced loss of ovarian follicular reserves in both cancer-free rats and rats with cancer. Such follicular loss is a main cause of infertility in young women treated for cancer. The current study was undertaken to discover the molecules and intracellular pathways involved in the action of TAM in the ovaries of rats with mammary tumors. To meet this goal we used transcriptomic (RNA-Seq) and proteomic (2D-DIGE/MS) approaches. TAM inhibited the expression of genes and lncRNAs involved in ovarian steroidogenesis. Moreover, TAM altered the expression of genes related to primordial follicle activation or arrest. In addition, proteomic screening indicated the importance of basic metabolic processes in the ovarian actions of TAM. Although simple extrapolation of these data to humans is not possible, the results of this study emphasize the need to explore the ability of TAM to affect ovarian function in women undergoing cancer treatment.

Knock-down of aryl hydrocarbon receptor (AhR) affects the lncRNA-mediated response of porcine granulosa cells (AVG-16 cell line) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Recently, we found that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) - the most toxic dioxin - affected multiple cellular processes in AhR-knocked-down granulosa cells, including the expression of genes and the abundance of proteins. Such alterations may imply the involvement of noncoding RNAs in the remodeling of intracellular regulatory tracks. The aims of the current study were to examine the effects of TCDD on the expression of lncRNAs in AhR-knocked-down granulosa cells of pigs and to indicate potential target genes for differentially expressed lncRNAs (DELs). In the current study, the abundance of AhR protein in porcine granulosa cells was reduced by 98.9% at 24 h after AhR targeted siRNA transfection. Fifty-seven DELs were identified in the AhR-deficient cells treated with TCDD mostly after 3 h (3 h: 56, 12 h: 0, 24 h: 2) after the dioxin treatment. This number was 2.5 times higher than that of intact TCDD-treated granulosa cells. The high number of DELs identified in the early stages of the TCDD action may be associated with a rapid defensive response of cells to harmful actions of this persistent environmental pollutant. In contrast to intact TCDD-treated granulosa cells, AhR-deficient cells were characterized by a broader representation of DELs enriched in GO terms related to the immune response and regulation of transcription and cell cycle. The obtained results support the notion that TCDD may act in an AhR-independent manner. They increase our knowledge on the intracellular mechanism of TCDD action and may in the future contribute to better coping with detrimental consequences of human and animal exposure to TCDD.

Tamoxifen decreases ovarian toxicity without compromising cancer treatment in a rat model of mammary cancer.

BACKGROUND: Premenopausal women diagnosed with breast cancer often face aggressive chemotherapy resulting in infertility. Tamoxifen (TAM) is a selective estrogen receptor modulator that was previously suggested as a protective agent against chemotherapy-induced ovarian failure. In the current study, we examined mechanisms of the protective action of TAM in the ovaries of tumor-bearing rats treated with the chemotherapy drug cyclophosphamide (CPA). RESULTS: TAM prevented CPA-induced loss of ovarian follicular reserves. The protective TAM effect in the rat ovary partially resulted from decreased apoptosis. In addition, transcriptomic and proteomic screening also implicated the importance of DNA repair pathways as well as cell adhesion and extracellular matrix remodeling in the protective ovarian actions of TAM. CONCLUSIONS: Tamoxifen shielded the ovary from the side effects of chemotherapy without lessening the tumoricidal actions of mammary cancer treatment.

The involvement of CYP1A2 in biodegradation of dioxins in pigs.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most harmful chemicals showing resistance to biodegradation. The majority of TCDD effects is mediated by the aryl hydrocarbon receptor (AhR) pathway. TCDD binding to AhR results in the activation of cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP1B1) involved in dioxin biodegradation. The goal of the study was to explore the potential role of CYP1A2 in the metabolism of TCDD. We investigated a molecular structure of CYP1A2 and the binding selectivity and affinity between the pig CYP1A2 and: 1/ DiCDD or TCDD (dioxins differing in toxicity and biodegradability) or 2/ their selected metabolites. pCYP1A2 demonstrated higher affinity towards DiCDD and TCDD than other pCYP1 enzymes. All dioxin-pCYP1A2 complexes were found to be stabilized by hydrophobic interactions. The calculated distances between the heme oxygen and the dioxin carbon nearest to the oxygen, reflecting the hydroxylating potential of CYP1A2, were higher than in other pCYP1 enzymes. The distances between the heme iron and the nearest dioxin carbon exceeded 5 Å, a distance sufficient to allow the metabolites to leave the active site. However, the molecular dynamics simulations revealed that two access channels of CYP1A2 were closed upon binding the majority of the examined dioxins. Moreover, the binding of dioxin metabolites did not promote opening of channel S-an exit for hydroxylated products. It appears that the undesired changes in the behavior of access channels prevail over the hydroxylating potential of CYP1A2 towards TCDD and the favorable distances, ultimately trapping the metabolites at the enzyme's active site.

Effects of neonatal methoxychlor exposure on the ovarian transcriptome in piglets.

Methoxychlor (MXC) is a man-made organochlorine insecticide capable of disrupting endocrine functions due to its mixed steroidal properties (estrogenic, anti-estrogenic and/or anti-androgenic). Retarded follicle development was recently reported in neonatal pigs treated with MXC. The goal of the current study was to better understand the mechanism of MXC action in the ovary of newborn piglets. By employing RNA-Seq we studied the expression of protein coding (mRNA) and long non-coding RNA (lncRNA) transcripts in the ovary of the MXC-treated piglets. Piglets were injected (sc) daily with MXC (100 mg/kg body weight) or corn oil (controls) between postnatal Days 1 and 10 (n = 3 piglets/group). The ovaries excised from 11-day-old piglets were processed for total RNA isolation and subsequent RNA sequencing. Four hundred sixty differentially expressed genes (DEGs) and 143 differentially expressed lncRNAs (DELs) were identified in the ovaries of MXC-treated piglets (P-adjusted < 0.05; abs(log2FC) > 1). Functional enrichment analysis showed that MXC altered the expression of genes associated with intracellular and membrane transport, intra-ovarian signaling as well as cell-cell junction and communication. Moreover, positive and negative correlations determined between the identified DEGs and DELs suggest that some lncRNAs may mediate the MXC action in the ovary. The results support the hypothesis that MXC-induced changes in the expression of genes involved in neonatal ovarian folliculogenesis increase the risk of fertility problems in adults.

Transcriptional profiling of Chinese hamster ovary (CHO) cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a man-made chemical compound contaminating the environment. An exposure of organisms to TCDD results in numerous disorders. The main mechanism of TCDD action involves the induction of the aryl hydrocarbon receptor (AhR) pathway followed by the increase in the expression and activity of cytochrome P450 family 1 (CYP1) enzymes. The main aim of the present study was to identify, by means of RNA sequencing, transcripts involved in the mechanism of TCDD action in Chinese hamster ovary (CHO) cells, known to not express CYP1A1 enzyme. The CHO cells were treated with TCDD for 3, 12 or 24 h, and total RNA was isolated and sequenced. Thirty six (padjusted < 0.05) or six (padjusted < 0.05, log2FC ≥ 1.0/log2FC≤-1.0) differentially expressed genes (DEGs) were identified in TCDD-treated cells depending on the assumed statistical criteria. The dioxin up- and downregulated the expression of genes associated with ovarian follicle functions, development, cardiovascular system, signal transduction, inflammation and carcinogenesis. TCDD did not affect the expression of any of 522 miRNAs which were identified in the cells. The expression of CYP1A1, CYP1A2 and CYP1B1 was demonstrated neither in control nor in TCDD-treated CHO cells, although the respective genes were found in the cell genome. Twenty two other CYP enzymes were identified in CHO cells, however their expression was also not affected by TCDD.

The immune status, oxidative and epigenetic changes in tissues of turkeys fed diets with different ratios of arginine and lysine.

In the present experiment, it was assumed that the appropriate dietary ratio of arginine (Arg) to lysine (Lys) can improve the immune status and growth performance of turkeys. The aim of this study was to evaluate the effects of two inclusion rates of Arg relative to Lys in turkey diets with Lys content consistent with National Research Council (NRC) recommendations or 10% higher on the immune status of birds and indicators of protein and DNA damage due to oxidation, nitration or epigenetic changes. Another goal was to determine which dietary Arg:Lys ratio stimulates the immune response of turkeys vaccinated against Ornithobacterium rhinotracheale. The experiment was performed on 576 turkeys randomly assigned to four groups with two levels of Lys (low = NRC recommendation or high = NRC + 10%) and two levels of Arg (95% or 105% Arg relative to the content of dietary Lys). It was found that the Lys content of turkey diets should be 10% higher than that recommended by the NRC and combined with the higher Arg level (105% of Lys). Although the above Arg:Lys ratio did not improve the growth performance of birds, it stimulated their immune system and reduced protein nitration as well as protein and DNA oxidation.

Transcriptomic profiles of the ovaries from piglets neonatally exposed to 4-tert-octylphenol.

The environmental pollutants with hormonal activities may influence steroid-mediated processes in neonatal ovaries and increase the incidence of reproductive disorders. The aim of the current study was to examine effects of 4-tert-octylphenol (OP), a non-ionic surfactant widely used in a variety of industrial applications which has been reported to mimic the 17ß-estradiol activity, on the expression of protein-coding (mRNAs) and long non-coding (lncRNAs) transcripts in neonatal ovaries of the pig. By employing RNA-Seq we aimed to gain insights into regulatory networks underlying the OP effects on the follicular development in pigs. Piglets were injected (sc) daily with OP (100 mg/kg bw) or corn oil (controls) between postnatal Days 1 and 10 (n = 3/group). Ovaries were excised from the 11-day-old piglets and total cellular RNA was isolated and sequenced. Two hundred three differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change ≥1.0) and 23 differentially expressed lncRNAs (DELs; P-adjusted < 0.05 and log2 fold change ≥ 1.0) were identified in OP-treated piglet ovaries. The DEGs were assigned to Gene Ontology terms, covering biological processes, molecular functions and cellular components, which linked the DEGs to functions associated with movement of cell or subcellular component, regulation of plasma membrane bounded cell projection assembly as well as hydrolase and endopeptidase activity. In addition, STRING analysis demonstrated the strongest interactions between genes related to negative regulation of endopeptidase activity. Some correlations between DEGs and DELs were also found, revealing that the OP action on the ovary may be partially executed via the changes in the lncRNA expression. These results suggest that neonatal exposure of pigs to OP induces changes in the ovarian transcriptomic profile associated with genes encoding serine protease inhibitors and involved in steroid synthesis as well as genes linked to intracellular and membrane transport. We suggest that the changes in the mRNA and lncRNA expression in the ovaries of OP-treated piglets may disturb ovarian cellular function, including steroidogenesis, proliferation and apoptosis.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the transcriptome of aryl hydrocarbon receptor (AhR) knock-down porcine granulosa cells.

BACKGROUND: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical, adversely affecting reproductive processes. The well-characterized canonical mechanism of TCDD action involves the activation of aryl hydrocarbon receptor (AhR) pathway, but AhR-independent mechanisms were also suggested. By applying RNA interference technology and Next Generation Sequencing (NGS) we aimed to identify genes involved in the mechanism of TCDD action in AhR knock-down porcine granulosa cells. METHODS: Porcine granulosa cells were transfected with small interfering RNAs targeting mRNA of AhR. After transfection, medium was exchanged and the AhR knock-down cells were treated with TCDD (100 nM) for 3, 12 or 24 h, total cellular RNA was isolated and designated for NGS. Following sequencing, differentially expressed genes (DEGs) were identified. To analyze functions and establish possible interactions of DEGs, the Gene Ontology (GO) database and the Search Tool for the Retrieval of Interacting Genes (STRING) database were used, respectively. RESULTS: The AhR gene expression level and protein abundance were significantly decreased after AhR-targeted siRNAs transfection of the cells. In TCDD-treated AhR knock-down cells we identified 360 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change [log2FC] ≥ 1.0). The functional enrichment analysis of DEGs revealed that TCDD influenced the expression of genes involved, among other, in the metabolism of vitamin A, follicular development and oocyte maturation, proliferation and differentiation as well as inflammation, stress response, apoptosis and oncogenesis. The three-time point study demonstrated that TCDD-induced changes in the transcriptome of AhR knock-down porcine granulosa cells were especially pronounced during the early stages of the treatment (3 h). CONCLUSIONS: TCDD affected the transcriptome of AhR knock-down porcine granulosa cells. The molecules involved in the AhR-independent action of TCDD were indicated in the study. The obtained data contribute to better understanding of molecular processes induced by xenobiotics in the ovary.

Proteomic changes of aryl hydrocarbon receptor (AhR)-silenced porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment and affecting human/animal health and reproduction. Intracellular TCDD action usually involves the activation of aryl hydrocarbon receptor (AhR). The aim of the current study was to examine TCDD-induced changes in the proteome of AhR-silenced porcine granulosa cells. The AhR-silenced cells were treated with TCDD (100 nM) for 3, 12 or 24 h. Total protein was isolated, labeled with cyanines and next, the samples were separated by isoelectric focusing and SDS-PAGE. Proteins of interest were identified by MALDI-TOF/TOF mass spectrometry (MS) analysis and confirmed by western blotting and fluorescence immunocytochemistry. The AhR-targeted siRNA transfection reduced the granulosal expression level of AhR by 60-70%. In AhR-silenced porcine granulosa cells, TCDD influenced the abundance of only three proteins: annexin V, protein disulfide isomerase and ATP synthase subunit beta. The obtained results revealed the ability of TCDD to alter protein abundance in an AhR-independent manner. This study offers a new insight into the mechanism of TCDD action and provide directions for future functional studies focused on molecular effects exerted by TCDD.

Temporal changes in the transcriptomic profile of granulosa cells of pigs treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) compound is an environmental chemical adversely affecting reproductive processes. Intracellular TCDD effects are mediated via aryl hydrocarbon receptor (AhR). The aim of the current study was to identify genes linking the AhR pathway with phenotypic consequences of TCDD action in granulosa cells of pigs. By applying multifactorial analysis, with TCDD and incubation time as factors, it was possible to determine temporal changes induced by TCDD in the cell transcriptome. Among the identified 144 differentially expressed genes (DEGs; Padjusted<0.05, log2 fold change (FC)≥1), 111 DEGs were classified as sustained genes (FC values changing between 3 and 24 h). Eighty six DEGs were classified as early genes and only nine as late genes (FC changes observed between 3 and 12 h or 12 and 24 h, respectively). The sustained gene category included genes related to TCDD mechanism of action (AHR, ARNTL, CYP1A1), cell proliferation (TGFß3), follicular development and ovulation (PTGS2) as well as stress response (NR3C1). The early gene category contained DEGs associated with cell proliferation (DUSP4, TAB1) and cellular response to stress (DHX34). The CYP1A1 gene was the only DEG classified as an early, late and sustained gene. The multifactorial approach allowed for statistically analyzing TCDD-induced changes over time in the gene expression in granulosa cells of pigs. Changes over time in the granulosal transcriptome profile indicated the involvement of stress related molecules in the cellular response to TCDD and TCDD effects on ovulation. The TCDD effects were particularly evident during the early stage of action by this compound.

Flutamide-induced alterations in transcriptional profiling of neonatal porcine ovaries.

BACKGROUND: Androgens are involved in the regulation of ovarian development during fetal/neonatal life. Environmental chemicals displaying anti-androgenic activities may affect multiple signal transduction pathways by blocking endogenous androgen action. The aim of the current study was to examine effects of the anti-androgen flutamide on the expression of coding transcripts and long non-coding RNAs (lncRNAs) in neonatal porcine ovaries. By employing RNA-Seq technology we aimed to extend our understanding of the role of androgens in neonatal folliculogenesis and examine the impact of the anti-androgen flutamide on ovarian function. METHOD: Piglets were subcutaneously injected with flutamide (50 mg/kg BW) or corn oil (controls) between postnatal days 1 and 10 (n = 3/group). Ovaries were excised from the 11-day-old piglets and total cellular RNAs were isolated and sequenced. RESULTS: Flutamide-treated piglet ovaries showed 280 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change ≥1.0) and 98 differentially expressed lncRNAs (DELs; P-adjusted < 0.05 and log2FC ≥ 1.0). The DEGs were assigned to GO term, covering biological processes, molecular functions and cellular components, which linked the DEGs to functions associated with cellular transport, cell divisions and cytoskeleton. In addition, STRING software demonstrated strongest interactions between genes related to cell proliferation. Correlations between DEGs and DELs were also found, revealing that a majority of the genes targeted by the flutamide-affected lncRNAs were associated with intracellular transport and cell division. CONCLUSIONS: Our results suggest that neonatal exposure of pigs to flutamide alters the expression of genes involved in ovarian cell proliferation, ovarian steroidogenesis and oocyte fertilization, which in turn may affect female reproduction in adult life.

Is CYP1B1 involved in the metabolism of dioxins in the pig?

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most difficult to biodegradate and the most toxic dioxin congener. Previously, we demonstrated in silico the ability of pig CYP1A1 to hydroxylate 2,7-dichlorodibenzo-p-dioxin (DiCDD), but not TCDD. To increase our knowledge concerning the low effectiveness of TCDD biodegradability, we analyzed in silico the binding selectivity and affinity between pig CYP1B1 and the two dioxins by means of molecular modeling. We also compared the effects of TCDD and DiCDD on CYP1B1 gene expression (qRT-PCR) and catalytic (EROD) activity in porcine granulosa cells. It was found that DiCDD and TCDD were stabilized within the pig CYP1B1 active site by hydrophobic interactions. The analysis of substrate channel availability revealed that both dioxins opened the exit channel S, allowing metabolites to leave the enzyme active site. Moreover, DiCDD and TCDD increased the CYP1B1 gene expression and catalytic activity in porcine granulosa cells. On the other hand, TCDD demonstrated higher than DiCDD calculated affinity to pig CYP1B1, hindering TCDD exit from the active site. The great distance between CYP1B1's heme and TCDD also might contribute to the lower hydroxylation effectiveness of TCDD compared to that of DiCDD. Moreover, the narrow active site of pig CYP1B1 may immobilize TCDD molecule, inhibiting its hydroxylation. The results of the access channel analysis and the distance from pig CYP1B1's heme to TCDD suggest that the metabolizing potential of pig CYP1B1 is higher than that of pig CYP1A1. However, this potential is probably not sufficiently high to considerably improve the slow TCDD biodegradation.

Identification and characterization of long non-coding RNAs in porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

BACKGROUND: Long non-coding RNAs (lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics. The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, results in reproductive defects in many species including pigs. The aims of the study were to identify and characterize lncRNAs in porcine granulosa cells as well as to examine the effects of TCDD on the lncRNA expression profile in the cells. RESULTS: One thousand six hundred sixty-six lncRNAs were identified and characterized in porcine granulosa cells. The identified lncRNAs were found to be shorter than mRNAs. In addition, the number of exons was lower in lncRNAs than in mRNAs and their exons were longer. TCDD affected the expression of 22 lncRNAs (differentially expressed lncRNAs [DELs]; log2 fold change  ≥ 1, P-adjusted < 0.05) in the examined cells. Potential functions of DELs were indirectly predicted via searching their target cis- and trans-regulated protein-coding genes. The co-expression analysis revealed that DELs may influence the expression of numerous genes, including those involved in cellular response to xenobiotics, dioxin metabolism, endoplasmic reticulum stress and cell proliferation. Aryl hydrocarbon receptor (AhR) and cytochrome P450 1A1 (CYP1A1) were found among the trans-regulated genes. CONCLUSIONS: These findings indicate that the identified lncRNAs may constitute a part of the regulatory mechanism of TCDD action in granulosa cells. To our knowledge, this is the first study describing lncRNAs in porcine granulosa cells as well as TCDD effects on the lncRNA expression profile. These results may trigger new research directions leading to better understanding of molecular processes induced by xenobiotics in the ovary.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the proteome of porcine granulosa cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment. The exposure of living organisms to TCDD may result in numerous disorders, including reproductive pathologies. By employing two-dimensional fluorescence difference gel electrophoresis we aimed to identify proteins potentially involved in the mechanism of TCDD action and toxicity in porcine granulosa cells. The porcine granulosa cells were treated with TCDD (100 nM) for 3, 12 or 24 h, and afterwards, cytoplasmic proteins were isolated and labeled with cyanines. Next, samples were separated by isoelectric focusing and SDS-PAGE. Proteins of interest were identified by MALDI-TOF/TOF MS analysis. A total of 75 differentially expressed protein spots (p < 0.05 and fold change ≥2.0) were found in granulosa cells treated with TCDD. After 3, 12 and 24 h of TCDD treatment, we were able to identify 29, 34 and 12 spots, respectively. Functional analysis showed that cytoskeletal proteins formed the largest class of proteins significantly affected by TCDD in all time points. We also demonstrated that most of the identified proteins were associated with the "structural constituent of cytoskeleton" and "chaperone binding" Gene Ontology categories. Based on the analysis of the porcine granulosa cell proteome, we demonstrated that TCDD may affect the ovarian follicle fate by the rearrangement of the cytoskeleton and extracellular matrix as well as the modulation of proteins important for the cellular response to stress. The results of the current study present an extended insight into the TCDD mechanism of action in porcine granulosa cells, providing new directions for future functional studies.

Transcript variations, phylogenetic tree and chromosomal localization of porcine aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) genes.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and/or different activation pathways.

Transcriptional profiling of porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment. An exposure of living organisms to TCDD may result in numerous disorders, including reproductive pathologies. The aim of the current study was to examine the effects of TCDD on the transcriptome of porcine granulosa cell line AVG-16. By employing next-generation sequencing (NGS) we aimed to identify genes potentially involved in the mechanism of TCDD action and toxicity in porcine granulosa cells. The AVG-16 cells were treated with TCDD (100 nM) for 3, 12 or 24 h, and afterwards total cellular RNA was isolated and sequenced. In TCDD-treated cells we identified 141 differentially expressed genes (DEGs; padjusted < 0.05 and log2 fold change ≥1.0). The DEGs were assigned to GO term, covering biological processes, molecular functions and cellular components. Due to the large number of genes with altered expression, in the current study we analyzed only the genes involved in follicular growth, development and functioning. The obtained results showed that TCDD may affect ovarian follicle fate by influencing granulosa cell cycle, proliferation and DNA repair. The demonstrated over-time changes in the quantity and quality of genes being affected by TCDD treatment showed that the effects of TCDD on granulosa cells changed dramatically between 3-, 12- and 24-h of cell culture. This finding indicate that timing of gene expression measurement is critical for drawing correct conclusions on detailed relationships between the TCDD-affected genes and resulting intracellular processes. These conclusions have to be confirmed and extended by research involving proteomic and functional studies.

Structural-functional adaptations of porcine CYP1A1 to metabolize polychlorinated dibenzo-p-dioxins.

Polychlorinated dibenzo-p-dioxins (PCDDs) are widespread by-products of human industrial activity. They accumulate in tissues of animals and humans, exerting numerous adverse effects on different systems. In living organisms, dioxins are metabolized by enzymes of the cytochrome P450 family, including CYP1A1. Particular dioxin congeners differ in their toxicity level and ability to undergo biodegradation. Since the molecular mechanisms underlying dioxin susceptibility or resistance to biodegradation are unknown, in the present study the molecular interactions between five selected dioxins and porcine CYP1A1 protein were investigated. It was found that the ability of a dioxin to undergo CYP1A1-mediated degradation is associated mainly with the number and position of chlorine atoms in the dioxin molecule. Among all examined congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) demonstrated the highest affinity to CYP1A1 and, at the same time, the greatest distance to the active site of the enzyme. Interestingly, in contrast to other dioxins, the binding of the TCDD molecule to the porcine CYP1A1 active site resulted in a rapid and continuous closure of substrate channels. All the information may help to explain the extended half-life of TCDD in living organisms as well as its high toxicity.

Utilization of physiological and taxonomic fluorescent probes to study Lactobacilli cells and response to pH challenge.

pH stress is recognized as an important feature for Lactobacillus in relation to lifestyle and commercial utility. Hence, this study aims to investigate the cell function of Lactobacilli cells subjected to pHs between 7.0 and 2.0. For this purpose, the Lactobacilli isolates of vegetable origin were first hybridized with fluorescent oligonucleotide rRNA probes for detecting Lactobacillus species. Then, cells were exposed to pH stress and labelled with fluorescent probes, carboxyfluorescein diacetate (CFDA) and propidium iodine (PI), which provided the insight into esterase activity and membrane integrity of cells. Among isolates, fluorescence in situ hybridization (FISH) enabled us to specifically detect L. plantarum and L. brevis. Interestingly, FCM analysis revealed that at pHs between 7.0 and 4.0 the cell membrane was intact, while after the exposure at pH 3.0, and 2.0 became perturbed or impaired. Finally, L. brevis and L. plantarum differed from each other in fluorescence labeling behaviour and culturability. However, the results showed that the same standard protocol for labeling enables discrimination of subpopulations of tested species. Depending on the species, the substantial culturability loss was observed at pH 3.0 and 2.0. These results suggest that the taxonomic and physiological fluorescent probes could be suitable for in situ detection of specific bacteria and rapid assessment of the physiological status of cells.