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1.
Int J Mol Sci ; 20(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671654

RESUMO

Leptin, the first discovered adipokine, has been connected to various physiological and pathophysiological processes, including cancerogenesis. Increasing evidence confirms its influence on prostate cancer cells. However, studies on the effects of leptin on the proliferation and apoptosis of the androgen-sensitive LNCaP line of prostate cancer cells brought conflicting results. Therefore, we performed studies on the effects of high LEP concentration (1 × 10-6 M) on gene expression profile, change of selected signaling pathways, proliferation and apoptosis of LNCaP cells. RTCA (real-time cell analyzer) revealed inhibitory effect of LEP on cell proliferation, but lower LEP concentrations (10-8 and 10-10 M) did not affect cell division. Moreover, flow cytometry with a specific antibody for Cleaved PARP-1, an apoptosis marker, confirmed the activation of apoptosis in leptin-exposed LNCaP line of prostate cancer cells. Within 24 h LEP (10-6 M) increases expression of 297 genes and decreases expression of 119 genes. Differentially expressed genes (DEGs) were subjected to functional annotation and clusterization using the DAVID bioinformatics tools. Most ontological groups are associated with proliferation and apoptosis (seven groups), immune response (six) and extracellular matrix (two). These results were confirmed by the Gene Set Enrichment Analysis (GSEA). The leptin's effect on apoptosis stimulation was also confirmed using Pathview library. These results were also confirmed by qPCR method. The results of Western Blot analysis (exposure to LEP 10 min, 1, 2, 4 and 24 h) suggest (after 24 h) decrease of p38 MAPK, p44-42 mitogen-activated protein kinase and Bcl-2 phosphorylated at threonine 56. Moreover, exposure of LNCaP cells to LEP significantly stimulates the secretion of matrix metallopeptidase 7 (MMP7). Obtained results suggest activation of apoptotic processes in LNCaP cells cultured at high LEP concentration. At the same time, this activation is accompanied by inhibition of proliferation of the tested cells.

2.
Int J Mol Sci ; 20(16)2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31443152

RESUMO

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte-granulosa cells-are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h-before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: "Cell morphogenesis", "cell differentiation" and "cell development" were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.

3.
Int J Mol Sci ; 20(14)2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295879

RESUMO

Oviductal epithelial cells (OECs) actively produce stimulating and protecting factors, favoring survival and viability of gametes and early embryos. The oviduct participates in the initial reproductive events, which strongly depends on adhesion. The analysis of differential gene expression in OECs, during long-term in vitro culture, enables recognition of new molecular markers regulating several processes, including "biological adhesion". Porcine oviducts were stained with hematoxylin and eosin, as well as with antibodies against epithelial markers. Then, OECs were long-term in vitro cultured and after 24 h, 7, 15, and 30 days of culture were subjected to transcriptomic and proteomic assays. Microarrays were employed to evaluate gene expression, with Matrix-assisted laser desorption/ionization-time of light (MALDI-TOF) mass spectrometry applied to determine the proteome. The results revealed proper morphology of the oviducts and typical epithelial structure of OECs during the culture. From the set of differentially expressed genes (DEGs), we have selected the 130 that encoded proteins detected by MALDI-TOF MS analysis. From this gene pool, 18 significantly enriched gene ontology biological processes (GO BP) terms were extracted. Among them we focused on genes belonging to "biological adhesion" GO BP. It is suggested that increased expression of studied genes can be attributed to the process of intensive secretion of substances that exhibit favorable influence on oviductal environment, which prime gametes adhesion and viability, fertilization, and early embryo journey.

4.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067669

RESUMO

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the "transforming growth factor ß receptor signaling pathway", "response to protein stimulus", and "response to organic substance" were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.


Assuntos
Mitocôndrias/metabolismo , Oócitos/metabolismo , Oogênese/genética , Transcriptoma , Animais , Hipóxia Celular/genética , Células Cultivadas , Feminino , Técnicas de Maturação in Vitro de Oócitos , Mitocôndrias/genética , Oócitos/citologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Suínos , Fator de Crescimento Transformador beta/metabolismo
5.
DNA Cell Biol ; 38(6): 549-560, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31120353

RESUMO

Proper course of folliculogenesis and oogenesis have an enormous impact on female fertility. Both processes take place in the ovary and involve not only the maturing germ cell, but also few types of somatic cells that assist the ovarian processes and mediate the dialog with the oocyte. These cells, granulosa and theca, are heavily involved in essential reproductive processes, such as ovulation, fertilization, and embryo implantation. In this study, we have used the expressive microarray approach to analyze the transcriptome of porcine granulosa cells, during short-term in vitro culture. We have further selected differentially expressed gene ontologies, involved in cell proliferation, migration, adhesion, and tissue development, namely, "cell-cell adhesion," "cell motility," "cell proliferation," "tissue development," and "tissue migration" to screen them for the possibility of discovery of new markers of those processes. A total of 303 genes, expression of which varied significantly in different culture periods and belonged to the analyzed ontology groups, were detected, of which 15 that varied the most (between 0 and 48 h of culture) were selected for validation. As the validation confirmed the transcriptomic patterns, 10 genes of biggest changes in expression (CAV1, IGFBP5, ITGB3, FN1, ITGA2, LAMB1, POSTN, FAM83D, KIF14, and CDK1) were analyzed, described, and referred to the context of the study, with the most promising new markers and further proof for the viability of the currently recognized ones detailed. Overall, the study provided valuable insight into the molecular functioning of in vitro granulosa cell cultures.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Células da Granulosa/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Células da Granulosa/citologia , Células da Granulosa/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Suínos
6.
Biomed Res Int ; 2019: 6545210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834271

RESUMO

The physiological processes that drive the development of ovarian follicle, as well as the process of oogenesis, are quite well known. Granulosa cells are major players in this occurrence, being the somatic element of the female gamete development. They participate directly in the processes of oogenesis, building the cumulus-oocyte complex surrounding the ovum. In addition to that, they have a further impact on the reproductive processes, being a place of steroid sex hormone synthesis and secretion. It is known that the follicle development creates a major need for angiogenesis and blood vessel development in the ovary. In this study, we use novel molecular approaches to analyze markers of these processes in porcine granulosa cultured primarily in vitro. The cells were recovered from mature sus scrofa specimen after slaughter. They were then subjected to enzymatic digestion and culture primarily for a short term. The RNA was extracted from cultures in specific time periods (0h, 24h, 48h, 96h, and 144h) and analyzed using expression microarrays. The genes that exhibited fold change bigger than |2|, and adjusted p-value lower than 0.05, were considered differentially expressed. From these, we have chosen the members of "angiogenesis," "blood vessel development," "blood vessel morphogenesis," "cardiovascular system development," and "vasculature development" for further selection. CCL2, FGFR2, SFRP2, PDPN, DCN, CAV1, CHI3L1, ITGB3, FN1, and LOX which are upregulated, as well as CXCL10, NEBL, IHH, TGFBR3, SCUBE1, IGF1, EDNRA, RHOB, PPARD, and SLITRK5 genes whose expression is downregulated through the time of culture, were chosen as the potential markers, as their expression varied the most during the time of culture. The fold changes were further validated with RT-qPCR. The genes were described, with special attention to their possible function in GCs during culture. The results broaden the general knowledge about GC's in vitro molecular processes and might serve as a point of reference for further in vivo and clinical studies.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Células da Granulosa/citologia , Neovascularização Fisiológica/genética , Folículo Ovariano/crescimento & desenvolvimento , Animais , Vasos Sanguíneos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Células da Granulosa/metabolismo , Humanos , Morfogênese/genética , Oócitos/crescimento & desenvolvimento , Oogênese/genética , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Cultura Primária de Células , Biossíntese de Proteínas/genética , Suínos
7.
Adv Clin Exp Med ; 28(6): 737-746, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30843677

RESUMO

BACKGROUND: Galanin-like peptide (Galp) and alarin (Ala) are 2 new members of the galanin peptide family. Galanin (Gal), the "parental" peptide of the entire family, is known to regulate numerous physiological processes, including energy and osmotic homeostasis, reproduction, food intake, and secretion of adrenocortical hormones. Galp and Ala are known to regulate food intake. In the rat, Galp mRNA has been found in the brain, exclusively in the hypothalamic arcuate nucleus (ARC) and median eminence, which are involved in the regulation of energy homeostasis. Alarin-like immunoreactivity is present in the locus coeruleus (LC) and the ARC of rats and mice. OBJECTIVES: The aim of the study was to investigate the expression of Ala, Galp and their receptors in the organs of the hypothalamo-pituitary-adrenal (HPA) axis of the rat. MATERIAL AND METHODS: The expression of the examined genes was measured in different models of adrenal growth of the rat in vivo (postnatal ontogenesis, compensatory adrenal growth, adrenocortical regeneration, adrenocorticotropic hormone (ACTH) administration). The expression was evaluated using the Affymetrix® microarray system or quantitative polymerase chain reaction (qPCR). RESULTS: The expression of Ala gene was observed in each organ of the HPA axis (the hypothalamus, hypophysis and adrenal gland). The elevated level of expression of this gene was observed in the pituitary of 2-day rats, while very low levels of Ala mRNA were observed in the adrenals. Galp mRNA expression was observed only in the hypothalamus and the hypophysis during postnatal ontogenesis. The expression of Gal receptors was demonstrated in the hypothalamus, the hypophysis and the adrenal gland. In different compartments of the adrenal glands of adult, intact male and female rats, the expression of Ala, Galp and galanin receptor 1 (Galr1) genes was negligible, but the expression of galanin receptor 2 (Galr2), galanin receptor 3 (Galr3) and neurotrophic receptor tyrosine kinase 2 (Ntrk2) genes was noticeable. CONCLUSIONS: The examined genes showed different expression levels within the studied HPA axis; some of them were neither expressed in the hypothalamus or the pituitary gland, nor in the adrenal gland.


Assuntos
Glândulas Suprarrenais/metabolismo , Peptídeo Semelhante a Galanina/genética , Hipotálamo/metabolismo , Hipófise/metabolismo , Animais , Feminino , Peptídeo Semelhante a Galanina/metabolismo , Sistema Hipotálamo-Hipofisário , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Sistema Hipófise-Suprarrenal , Ratos , Reação em Cadeia da Polimerase em Tempo Real
8.
Mol Med Rep ; 19(3): 1705-1715, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30628715

RESUMO

Granulosa cells (GCs) have many functions in the endocrine system. Most notably, they produce progesterone following ovulation. However, it has recently been proven that GCs can change their properties when subjected to long­term culture. In the present study, GCs were collected from hyper­stimulated ovarian follicles during in vitro fertilization procedures. They were grown in vitro, in a long­term manner. RNA was collected following 1, 7, 15 and 30 days of culture. Expression microarrays were used for analysis, which allowed to identify groups of genes characteristic for particular cellular processes. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to validate the obtained results. Two ontological groups characteristic for processes associated with the development and morphogenesis of the heart were identified during the analyses: 'Heart development' and 'heart morphogenesis'. The results of the microarrays revealed that the highest change in expression was demonstrated by the lysyl Oxidase, oxytocin receptor, nexilin F­actin binding protein, and cysteine­rich protein 3 genes. The lowest change was exhibited by odd­skipped related transcription factor 1, plakophilin 2, transcription growth factor­ß receptor 1, and kinesin family member 3A. The direction of changes was confirmed by RT­qPCR results. In the present study, it was suggested that GCs may have the potential to differentiate towards other cell types under long­term in vitro culture conditions. Thus, genes belonging to the presented ontological groups can be considered as novel markers of proliferation and differentiation of GCs towards the heart muscle cells.


Assuntos
Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem da Célula/genética , Folículo Ovariano/citologia , Células Cultivadas , Feminino , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Humanos , Morfogênese/genética , Folículo Ovariano/metabolismo , Ovulação/genética , Progesterona/genética , Proteína-Lisina 6-Oxidase/genética , Receptores de Ocitocina/genética
9.
Int J Mol Sci ; 20(1)2018 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-30587792

RESUMO

The growth and development of oocyte affect the functional activities of the surrounding somatic cells. These cells are regulated by various types of hormones, proteins, metabolites, and regulatory molecules through gap communication, ultimately leading to the development and maturation of oocytes. The close association between somatic cells and oocytes, which together form the cumulus-oocyte complexes (COCs), and their bi-directional communication are crucial for the acquisition of developmental competences by the oocyte. In this study, oocytes were extracted from the ovaries obtained from crossbred landrace gilts and subjected to in vitro maturation. RNA isolated from those oocytes was used for the subsequent microarray analysis. The data obtained shows, for the first time, variable levels of gene expression (fold changes higher than |2| and adjusted p-value < 0.05) belonging to four ontological groups: regulation of cell proliferation (GO:0042127), regulation of cell migration (GO:0030334), and regulation of programmed cell death (GO:0043067) that can be used together as proliferation, migration or apoptosis markers. We have identified several genes of porcine oocytes (ID2, VEGFA, BTG2, ESR1, CCND2, EDNRA, ANGPTL4, TGFBR3, GJA1, LAMA2, KIT, TPM1, VCP, GRID2, MEF2C, RPS3A, PLD1, BTG3, CD47, MITF), whose expression after in vitro maturation (IVM) is downregulated with different degrees. Our results may be helpful in further elucidating the molecular basis and functional significance of a number of gene markers associated with the processes of migration, proliferation and angiogenesis occurring in COCs.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Oócitos/metabolismo , Transcriptoma , Animais , Movimento Celular/genética , Células do Cúmulo/metabolismo , Células do Cúmulo/patologia , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Técnicas de Maturação in Vitro de Oócitos , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/crescimento & desenvolvimento , Oócitos/patologia , RNA/genética , RNA/metabolismo , Suínos , Regulação para Cima
10.
Histochem Cell Biol ; 2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30382374

RESUMO

The human ovarian granulosa cells (GCs) surround the oocyte and form the proper architecture of the ovarian follicle. The ability of GCs to proliferate and differentiate in the conditions of in vitro culture has been proven. However, there is still a large field for extensive investigation of molecular basics, as well as marker genes, responsible for these processes. This study aimed to find the new marker genes, encoding proteins that regulate human GCs in vitro capability for proliferation and differentiation during long-term primary culture. The human follicular GCs were collected from hyper-stimulated ovarian follicles during IVF procedures and transferred to a long-term in vitro culture. The culture lasted for 30 days, with RNA samples isolated at days 1, 7, 15, 30. Transcriptomic analysis was then performed with the use of Affymetrix microarray. Obtained results were then subjected to bioinformatical evaluation and sorting. After subjecting the datasets to KEGG analysis, three differentially expressed ontology groups "cell differentiation" (GO:0030154), "cell proliferation" (GO:0008283) and "cell-cell junction organization" (GO:0045216) were chosen for further investigation. All three of those ontology groups are involved in human GCs' in vitro lifespan, proliferation potential, and survival capability. Changes in expression of genes of interest belonging to the chosen GOs were validated with the use of RT-qPCR. In this manuscript, we suggest that VCL, PARVA, FZD2, NCS1, and COL5A1 may be recognized as new markers of GC in vitro differentiation, while KAT2B may be a new marker of their proliferation. Additionally, SKI, GLI2, FERMT2, and CDH2 could also be involved in GC in vitro proliferation and differentiation processes. We demonstrated that, in long-term in vitro culture, GCs exhibit markers that suggest their ability to differentiate into different cells types. Therefore, the higher expression profile of these genes may also be associated with the induction of cellular differentiation processes that take place beyond the long-term primary in vitro culture.

11.
Int J Mol Sci ; 19(8)2018 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-30081524

RESUMO

Gonadotropin-inducible ovarian transcription factor-1 (Giot1) belongs to a family of fast-responsive genes, and gonadotropins rapidly induce its expression in steroidogenic cells of ovaries and testes of rats. Gonadal Giot1 gene expression is regulated by cyclic adenosine monophosphate (cAMP) -dependent protein kinase A pathway, with essential role of orphan nuclear receptor NR4A1 transcription factor (nuclear receptor subfamily 4, group A, member 1). A recent study reports that Giot1 is also expressed in adrenals, however, the mechanism of its regulation in adrenal gland is yet to be identified. Therefore, the aim of this study was to characterise the changes in Giot1 gene expression in male and female rat adrenals using wide range of in vivo and in vitro experimental models. Special emphasis was directed at the Giot1 gene regulation by ACTH and gonadotropin. In our study, we found that ACTH rapidly stimulates Giot1 expression both in vivo and in vitro. However, gonadotropin does not affect the adrenal Giot1 gene expression, presumably due to the low expression of gonadotropin receptor in adrenals. Both testosterone and estradiol administered in vivo had inhibitory effect on Giot1 gene expression in the adrenals of post-gonadectomized adult rats. Further, our studies revealed that the intracellular mechanism of Giot1 gene regulation in rat adrenals is similar to that of gonads. As in the case of gonads, the expression of Giot1 in adrenal gland is regulated by cAMP-dependent signaling pathway with essential role of the NR4A1 transcription factor. The results of our studies suggest that Giot1 may be involved in the regulation of rat adrenocortical steroidogenesis.


Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Gonadotropina Coriônica/farmacologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Gonadotropinas/farmacologia , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética
12.
Theriogenology ; 121: 122-133, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30145542

RESUMO

The processes underlying maturation of mammalian oocytes are considered crucial for the oocytes ability to undergo monospermic fertilization. The same factors of influence are suggested to impact the development of sex associated characteristics, allowing sex differentiation to progress during embryonic growth. The primary aim of the study was to analyze the gene ontology groups involved in regulation of porcine oocytes' response to endogenous stimuli. The results obtained would indicate potential genes influencing sex differentiation. Additionally, they could help to determine new genetic markers, expression profile of which is substantially regulated during porcine oocytes' in vitro maturation. To achieve that, porcine oocytes were collected for analysis before and after in vitro maturation. Pigs were used as they are a readily available model that presents significant similarity to humans in terms of physiology and anatomy. Microarray analysis of oocytes, before and after in vitro maturation was performed and later validated by RT-qPCR. We have particularly detected and analyzed genes belonging to gene ontology groups associated with hormonal stimulation during maturation of the oocytes, that exhibited significant change in expression (fold change ≥ |2|; p < 0.05) namely "Female sex differentiation" (CCND2, MMP14, VEGFA, FST, INHBA, NR5A1), "Response to endogenous stimulus" (INSR, ESR1, CCND2, TXNIP, TACR3, MMP14, FOS, AR, EGR2, IGFBP7, TGFBR3, BTG2, PLD1, PHIP, UBE2B) and "Response to estrogen stimulus" (INSR, ESR1, CCND2, IHH, TXNIP, TACR3, MMP14). Some of them were characteristic for just one of the described ontologies, while some belonged into multiple ontological terms. The genes were analyzed, with their relation to the processes of interest explained. Overall, the study provides us with a range of genes that might serve as molecular markers of in vitro maturation associated processes of the oocytes. This knowledge might serve as a reference for further studies and, after further validation, as a potentially useful knowledge in assessment of the oocytes during assisted reproduction processes.

13.
Artigo em Inglês | MEDLINE | ID: mdl-29883755

RESUMO

Ribosome-associated noncoding (ranc) RNAs are a novel class of short regulatory RNAs with functions and origins that have not been well studied. In this present study, we functionally characterized the molecular activity of Saccharomyces cerevisiae transfer RNA (tRNA)-derived fragments (tRFs) during protein biosynthesis. Our results indicate ribosome-associated tRFs derived from both 5' (ranc-5'-tRFs) and 3'-part of tRNAs (ranc-3'-tRFs) have regulatory roles during translation. We demonstrated five 3'-tRFs and one 5'-tRF associate with a small ribosomal subunit and aminoacyl-tRNA synthetases (aa-RSs) in yeast. Furthermore, we discovered that four yeast aa-RSs interact directly with yeast ribosomes. tRFs interactions with ribosome-associated aa-RSs correlate with impaired efficiency of tRNA aminoacylation.

14.
Int J Mol Sci ; 19(4)2018 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-29642441

RESUMO

Compensatory adrenal growth evoked by unilateral adrenalectomy (hemiadrenalectomy) constitutes one of the most frequently studied in vivo models of adrenocortical enlargement. This type of growth has been quite well characterized for its morphological, biochemical, and morphometric parameters. However, the molecular basis of compensatory adrenal growth is poorly understood. Therefore, the aim of this study was to investigate the rat adrenal transcriptome profile during the time of two previously described adrenocortical proliferation waves at 24 and 72 h after unilateral adrenalectomy. Surgical removal of the left adrenal or a sham operation was accomplished via the classic dorsal approach. As expected, the weight of the remaining right adrenal glands collected at 24 and 72 h after hemiadrenalectomy increased significantly. The transcriptome profile was identified by means of Affymetrix® Rat Gene 2.1 ST Array. The general profiles of differentially expressed genes were visualized as volcano plots and heatmaps. Detailed analyzes consisted of identifying significantly enriched gene ontological groups relevant to adrenal physiology, by means of DAVID and GOplot bioinformatics tools. The results of our studies showed that compensatory adrenal growth induced by unilateral adrenalectomy exerts a limited influence on the global transcriptome profile of the rat adrenal gland; nevertheless, it leads to significant changes in the expression of key genes regulating the circadian rhythm. Our results confirm also that regulation of compensatory adrenal growth is under complex and multifactorial control with a pivotal role of neural regulatory mechanisms and a supportive role of other components.


Assuntos
Glândulas Suprarrenais/crescimento & desenvolvimento , Adrenalectomia/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Glândulas Suprarrenais/química , Glândulas Suprarrenais/cirurgia , Animais , Ritmo Circadiano , Modelos Animais de Doenças , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Masculino , Tamanho do Órgão , Ratos
15.
Int J Mol Sci ; 19(4)2018 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-29596348

RESUMO

The oral mucosal tissue is a compound structure composed of morphologically and physiologically different cell types. The morphological modification involves genetically determined lifespan, which may be recognized as the balance between cell survival and apoptosis. Although the biochemical processes and pathways in oral mucosa, with special regards to drug transport, delivery, and metabolism, are well known, the cellular physiological homeostasis in this tissue requires further investigation. The porcine buccal pouch mucosal cells (BPMCs) collected from 20 pubertal crossbred Landrace gilts, were used in this study. Immediately after recovery, the oral mucosa was separated micro-surgically, and treated enzymatically. The dispersed cells were transferred into primary in vitro culture systems for a long-term cultivation of 30 days. After each step of in vitro culture (IVC), the cells were collected for isolation of total RNA at 24 h, 7, 15, and 30 days of IVC. While the expression was analyzed for days 7, 15, and 30, the 24th hour was used as a reference for outcome calibration. The gene expression profile was determined using Affymetrix microarray assays and necessary procedures. In results, we observed significant up-regulation of SCARB1, PTGS2, DUSP5, ITGB3, PLK2, CCL2, TGFB1, CCL8, RFC4, LYN, ETS1, REL, LIF, SPP1, and FGER1G genes, belonging to two ontological groups, namely "positive regulation of metabolic process", and "regulation of homeostatic process" at 7 day of IVC as compared to down-regulation at days 15 and 30. These findings suggest that the metabolic processes and homeostatic regulations are much more intense in porcine mucosal cells at day 7 of IVC. Moreover, the increased expression of marker genes, for both of these ontological groups, may suggest the existence of not only "morphological lifespan" during tissue keratinization, but also "physiological checkpoint" dedicated to metabolic processes in oral mucosa. This knowledge may be useful for preclinical experiments with drugs delivery and metabolism in both animals and humans.


Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Homeostase , Mucosa Bucal/metabolismo , Animais , Células Cultivadas , Células Epiteliais/citologia , Humanos , Mucosa Bucal/citologia , Suínos
16.
Biomed Res Int ; 2018: 2863068, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29546053

RESUMO

The cumulus-oocyte complexes (COCs) growth and development during folliculogenesis and oogenesis are accompanied by changes involving synthesis and accumulation of large amount of RNA and proteins. In this study, the transcriptomic profile of genes involved in "oocytes RNA synthesis" in relation to in vitro maturation in pigs was investigated for the first time. The RNA was isolated from oocytes before and after in vitro maturation (IVM). Interactions between differentially expressed genes/proteins belonging to "positive regulation of RNA metabolic process" ontology group were investigated by STRING10 software. Using microarray assays, we found expression of 12258 porcine transcripts. Genes with fold change higher than |2| and with corrected p value lower than 0.05 were considered as differentially expressed. The ontology group "positive regulation of RNA metabolic process" involved differential expression of AR, INHBA, WWTR1, FOS, MEF2C, VEGFA, IKZF2, IHH, RORA, MAP3K1, NFAT5, SMARCA1, EGR1, EGR2, MITF, SMAD4, APP, and NR5A1 transcripts. Since all of the presented genes were downregulated after IVM, we suggested that they might be significantly involved in regulation of RNA synthesis before reaching oocyte MII stage. Higher expression of "RNA metabolic process" related genes before IVM indicated that they might be recognized as important markers and specific "transcriptomic fingerprint" of RNA template accumulation and storage for further porcine embryos growth and development.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Oócitos/crescimento & desenvolvimento , Oogênese/genética , RNA/genética , Animais , Células do Cúmulo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/metabolismo , RNA/biossíntese , Suínos , Transcriptoma/genética
17.
Mol Med Rep ; 17(4): 6163-6173, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29436637

RESUMO

Nicotinamide phosphoribosyltransferase (Nampt), also termed visfatin, catalyses the rate­limiting step in the nicotinamide adenine dinucleotide (NAD) salvage pathway. In addition to its intracellular function (iNampt), extracellular Nampt (eNampt) also affects numerous intracellular signalling pathways. The current study investigated the role of Nampt in the regulation of the hypothalamic­pituitary­adrenal (HPA) axis in rats. At 1 h after intraperitoneal administration of eNampt (4 µg/100 g) in adult male rats, serum adrenocorticotropic hormone(ACTH) and aldosterone levels remained unchanged, while corticosterone levels were notably elevated compared with the control group, as determined by ELISA. The results of reverse transcription­quantitative polymerase chain reaction (RT­qPCR) demonstrated that, in the hypothalami of eNampt­treated rats, the mRNA expression levels of Fos proto­oncogene, which is also termed c­Fos, were not significantly different compared with the control group; however, the mRNA expression levels of proopiomelanocortin (POMC) were markedly increased in the pituitary gland of eNampt­treated rats compared with the control group. Furthermore, in hypothalamic explants, ELISA results demonstrated that the addition of the eNampt protein exhibited no effect on corticotropin­releasing hormone (CRH) release into the incubation medium and prevented potassium ion­induced CRH release. Additionally, the eNampt­induced increase in ACTH output by pituitary gland explants was not statistically significant, compared with the control group. However, RT­qPCR indicated that exposure of pituitary gland explants to eNampt and CRH increased the levels of POMC mRNA expression; the effect of eNampt, but not CRH, was inhibited by FK866, which is a specific Nampt inhibitor. In primary rat adrenocortical cell cultures, eNampt exhibited no effect on basal aldosterone or corticosterone secretion, while increases in aldosterone and corticosterone levels in response to ACTH were retained. To assess the potential role of iNampt in the regulation of adrenal steroidogenesis, experiments involving a specific Nampt inhibitor, FK866, were performed. Exposure of cultured cells to FK866 notably lowered basal aldosterone and corticosterone output compared with the control group, and completely eliminated the response of cultured cells to ACTH. The results of the present study indicated that the injected eNampt may have increased the corticosterone serum levels by acting at the pituitary level. In addition, iNampt may exert a tonic stimulating effect on the secretion of aldosterone and corticosterone from rat adrenocortical cells, as normal iNampt levels were required to retain the response of cultured rat adrenocortical cells to ACTH. Thus, these data suggest an important physiological role of both iNampt and eNampt in the regulation of the HPA axis activity in the rat.


Assuntos
Sistema Hipotálamo-Hipofisário/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/sangue , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Aldosterona/sangue , Aldosterona/metabolismo , Animais , Células Cultivadas , Corticosterona/sangue , Corticosterona/metabolismo , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Masculino , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Ratos
18.
Oncol Rep ; 39(1): 182-192, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29115533

RESUMO

Results of studies on the expression of leptin and its receptors in the human prostate gland and human prostate cell lines are contradictory. Regarding this, we carefully reinvestigated this issue using human normal prostate (PrEC, PrSC, PrSMC) and prostate cancer (DU145, LNCaP, PC3) cell lines. Expression of leptin receptor isoforms was assessed by qPCR while the effects of leptin on cell proliferative activity was determined by real-time cell analyzer (RTCA). Expression of the leptin receptor variant 1 was not detected in LNCaP and PrSMC cell lines, but it was found in the remaining cell lines. In contrast, in all examined cell lines, isoforms 1-3 and 2 and 4 of the leptin receptor were found. The expression of isoforms 3 and 6 of the leptin receptor was observed in PC3, PrEC, PrSMC and PrSC cell lines, but not in LNCaP and DU145 cells. Expression of the leptin receptor isoforms 4-6 and 5 was not demonstrated in any of the tested cell lines. We also studied the effects of leptin on the expression of its receptor isoforms in all tested cell lines. At a wide range of concentrations, leptin did not change the expression of leptin receptor variant 1 in the DU145, PrEC and PC3 cell lines. In contrast, in the PrSC cell line, leptin significantly increased the expression of this gene. In all prostate cell lines tested, leptin did not alter the expression levels of variants 1-3 of the leptin receptor isoforms. Leptin did not alter the expression of isoforms 2 and 4 of the leptin receptor in the PC3 and LNCaP cell lines. In the DU145 and PrEC cell lines, leptin inhibited expression of these receptor isoforms while an opposite effect was noted in the PrSC cells. Leptin did not affect the expression level of variants 3 and 6 of its receptor in the PrEC and PC3 cell lines. However, in PrSMC cells, leptin inhibited the expression of variants 3 and 6 of its receptor, while in the PrSC cell line this cytokine significantly increased their expression levels. As assessed by RTCA, leptin stimulated the proliferative activity of DU145 cells, but inhibited this activity in LNCaP cells. At all concentrations tested, leptin did not change the proliferation rate of the PC3, PrEC and PrSMC cells. In contrast, leptin notably stimulated the proliferative activity of the PrSC (prostate stromal cell) cell line. Thus, our study demonstrated that in all tested human normal prostate and prostate cancer cell lines, transcription variants 4, 5 and 6 of the leptin receptor were not expressed. Leptin receptor transcription variants 1, 2 and 3 showed differential expression, which was present in the PC3, PrEC and PrSC cell lines. Our data also suggest that the stimulatory effects of leptin on proliferative activity of the studied cell lines require the expression of leptin receptor variant 1 in the affected cells.


Assuntos
Expressão Gênica , Leptina/metabolismo , Neoplasias da Próstata/genética , Receptores para Leptina/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Isoformas de RNA/genética
19.
Int J Mol Sci ; 18(12)2017 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-29232835

RESUMO

Because of the deep involvement of granulosa cells in the processes surrounding the cycles of menstruation and reproduction, there is a great need for a deeper understanding of the ways in which they function during the various stages of those cycles. One of the main ways in which the granulosa cells influence the numerous sex associated processes is hormonal interaction. Expression of steroid sex hormones influences a range of both primary and secondary sexual characteristics, as well as regulate the processes of oogenesis, folliculogenesis, ovulation, and pregnancy. Understanding of the exact molecular mechanisms underlying those processes could not only provide us with deep insight into the regulation of the reproductive cycle, but also create new clinical advantages in detection and treatment of various diseases associated with sex hormone abnormalities. We have used the microarray approach validated by RT-qPCR, to analyze the patterns of gene expression in primary cultures of human granulosa cells at days 1, 7, 15, and 30 of said cultures. We have especially focused on genes belonging to ontology groups associated with steroid biosynthesis and metabolism, namely "Regulation of steroid biosynthesis process" and "Regulation of steroid metabolic process". Eleven genes have been chosen, as they exhibited major change under a culture condition. Out of those, ten genes, namely STAR, SCAP, POR, SREBF1, GFI1, SEC14L2, STARD4, INSIG1, DHCR7, and IL1B, belong to both groups. Patterns of expression of those genes were analyzed, along with brief description of their functions. That analysis helped us achieve a better understanding of the exact molecular processes underlying steroid biosynthesis and metabolism in human granulosa cells.


Assuntos
Técnicas de Cultura de Células/métodos , Perfilação da Expressão Gênica/métodos , Células da Granulosa/citologia , Redes e Vias Metabólicas , Esteroides/biossíntese , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células da Granulosa/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
20.
Int J Mol Sci ; 18(12)2017 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-29232894

RESUMO

Proper maturation of the mammalian oocyte is a compound processes determining successful monospermic fertilization, however the number of fully mature porcine oocytes is still unsatisfactory. Since oocytes' maturation and fertilization involve cellular adhesion and membranous contact, the aim was to investigate cell adhesion ontology group in porcine oocytes. The oocytes were collected from ovaries of 45 pubertal crossbred Landrace gilts and subjected to two BCB tests. After the first test, only granulosa cell-free BCB⁺ oocytes were directly exposed to microarray assays and RT-qPCR ("before IVM" group), or first in vitro matured and then if classified as BCB⁺ passed to molecular analyses ("after IVM" group). As a result, we have discovered substantial down-regulation of genes involved in adhesion processes, such as: organization of actin cytoskeleton, migration, proliferation, differentiation, apoptosis, survival or angiogenesis in porcine oocytes after IVM, compared to oocytes analyzed before IVM. In conclusion, we found that biological adhesion may be recognized as the process involved in porcine oocytes' successful IVM. Down-regulation of genes included in this ontology group in immature oocytes after IVM points to their unique function in oocyte's achievement of fully mature stages. Thus, results indicated new molecular markers involved in porcine oocyte IVM, displaying essential roles in biological adhesion processes.


Assuntos
Regulação para Baixo , Redes Reguladoras de Genes , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/citologia , Animais , Apoptose , Adesão Celular , Diferenciação Celular , Proliferação de Células , Feminino , Oócitos/metabolismo , Oogênese , Suínos
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