RESUMO
Inflammation in the reproductive tract has become a serious threat to animal fertility. Recently, the role of peroxisome proliferator-activated receptor gamma (PPARγ) in the context of reproduction and the inflammatory response has been highlighted, but the role of PPARß/δ has not been fully elucidated. The aim of the present study was to investigate the in vitro effect of PPARß/δ ligands (agonist: L-165,041 and antagonist: GSK 3787) on the transcriptome profile of porcine endometrium during LPS-induced inflammation in the mid-luteal and follicular phases of the oestrous cycle (days 10-12 and 18-20, respectively) using the RNA-Seq method. During the mid-luteal phase of the oestrous cycle, the current study identified 145 and 143 differentially expressed genes (DEGs) after treatment with an agonist or antagonist, respectively. During the follicular phase of the oestrous cycle, 55 and 207 DEGs were detected after treatment with an agonist or antagonist, respectively. The detected DEGs are engaged in the regulation of various processes, such as the complement and coagulation cascade, NF-κB signalling pathway, or the pathway of 15-eicosatetraenoic acid derivatives synthesis. The results of the current study indicate that PPARß/δ ligands are involved in the control of the endometrial inflammatory response.
Assuntos
Endométrio , Inflamação , Lipopolissacarídeos , PPAR delta , PPAR beta , Animais , Feminino , Suínos , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , PPAR delta/genética , PPAR delta/metabolismo , PPAR beta/metabolismo , PPAR beta/genética , Inflamação/induzido quimicamente , Fenoxiacetatos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , TranscriptomaRESUMO
Plastics have become an integral part of our daily lives. In the environment, plastics break down into small pieces (<5 mm) that are referred to as microplastics. Microplastics are ubiquitous and widespread in the environment, and all living organisms are exposed to their effects. The present study provides new insights into the potential effects of polyethylene terephthalate (PET) microplastics on organisms via extracellular vesicle (EV)-mediated communication. The study demonstrated that serum-derived EVs are able to transport plastic particles. In addition, PET microplastics alter the content of miRNA in EVs. The identified differentially regulated miRNAs may target genes associated with lifestyle diseases, such as cardiovascular or metabolic diseases, and carcinogenesis. This work expands our understanding of PET microplastics' effects on organisms via EV-mediated communication and identifies directions for further research and strategies.
Assuntos
Microplásticos , Poluentes Químicos da Água , Microplásticos/toxicidade , Plásticos/toxicidade , Polietilenotereftalatos , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/análise , ComunicaçãoRESUMO
Inflammation in the female reproductive system is one of the most common causes of reproductive dysfunction such as infertility, delay of the reproductive cycle and a reduction in reproductive efficiency. In this study, we aimed to investigate the effect of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of selected inflammatory mediators: nuclear factor kappa (NF-κB), interleukin (IL)-1ß, IL-6, IL-4, IL-10, leukemia inhibitory factor (LIF) and toll-like receptor 4 (TLR4) in the porcine endometrium treated in vitro with lipopolysaccharide (LPS) on days 10-12 and 18-20 of the estrous cycle. In addition, two experimental protocols were applied to evaluate the role of PPARγ agonists in ongoing and developing inflammation. Endometrial slices were incubated in vitro in the presence of LPS (to induce inflammation) and PPARγ agonists, prostaglandin J2 or pioglitazone (natural or synthetic, respectively). The study showed that PPARγ agonists decreased the expression of pro-inflammatory (NF-κB, TLR4, IL-6) and increased the abundance of anti-inflammatory mediators (IL-10) in the inflamed endometrium of pigs. These findings indicate anti-inflammatory properties of the tested ligands.
Assuntos
PPAR gama , Doenças dos Suínos , Animais , Anti-Inflamatórios/farmacologia , Endométrio/metabolismo , Feminino , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/veterinária , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Ligantes , Lipopolissacarídeos/toxicidade , NF-kappa B/metabolismo , Suínos , Doenças dos Suínos/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Inflammation is a biological response of the immune system, which can be triggered by many factors, including pathogens. These factors may induce acute or chronic inflammation in various organs, including the reproductive system, leading to tissue damage or disease. In this study, the RNA-Seq technique was used to determine the in vitro effects of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of genes and long non-coding RNA, and alternative splicing events (ASEs) in LPS-induced inflammation of the porcine endometrium during the follicular phase of the estrous cycle. Endometrial slices were incubated in the presence of LPS and PPARγ agonists (PGJ2 or pioglitazone) and a PPARγ antagonist (T0070907). We identified 169, 200, 599 and 557 differentially expressed genes after LPS, PGJ2, pioglitazone or T0070907 treatment, respectively. Moreover, changes in differentially expressed long non-coding RNA and differential alternative splicing events were described after the treatments. The study revealed that PPARγ ligands influence the LPS-triggered expression of genes controlling the DNA damage response (GADD45ß, CDK1, CCNA1, CCNG1, ATM). Pioglitazone treatment exerted a considerable effect on the expression of genes regulating the DNA damage response.
Assuntos
RNA Longo não Codificante , Tiazolidinedionas , Animais , Dano ao DNA , Endométrio/metabolismo , Feminino , Inflamação/metabolismo , Ligantes , Lipopolissacarídeos/metabolismo , PPAR gama/metabolismo , Pioglitazona/efeitos adversos , Prostaglandina D2/metabolismo , RNA Longo não Codificante/metabolismo , Suínos , Tiazolidinedionas/efeitos adversosRESUMO
The current study was conducted with the aim to investigate effects of PPARγ ligands on synthesis of nuclear receptor κB (NF-κB) and selected cytokines (IL-1ß, IFNγ, TNFα, IL-4, IL-10, LIF) in the pig myometrium on days 14-15 of the estrous cycle (late-luteal phase) and days 14-15 of the gestational period (beginning of embryonic implantation). The myometrial slices were incubated in vitro for 6 h in medium containing PPARγ ligands, agonists: 15d-prostaglandin J2 or pioglitazone, and antagonist - T0070907. The mRNA transcript and protein abundances were evaluated in tissues and culture medium. During the estrous cycle, PPARγ ligands did not have an effect on the mRNA transcript abundance of the immune response mediators used for treatments. The IL-10 protein abundance in the tissue was less when there was inclusions of pioglitazone in the medium, while the treatment with T0070907 resulted in a larger abundance of NF-κB, IL-1ß (in the tissue) and IL-4 (in tissue and culture media). During the gestational period, pioglitazone or PGJ2 suppressed mRNA IFNγ and IL-10 transcript and protein abundances (in the tissue and culture media), whereas there was an enhanced NF-κB protein abundance (in the tissue). Treatment with T0070907 had diverse effects (e.g., for NFκB inhibited mRNA transcript abundance or enhanced protein abundance). The observed changes are related mainly in tissues from pregnant animals. Responses to PPARγ antagonist are indicative of the possible involvement of PPARγ-independent factors as well as ligand-independent activation of the receptor, ligand selectivity/functionality or tissue receptivity to the factors evaluated.
Assuntos
Imunidade/fisiologia , Miométrio/metabolismo , PPAR gama/metabolismo , Suínos/fisiologia , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipoglicemiantes/farmacologia , PPAR gama/genética , Pioglitazona/farmacologia , Gravidez , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Piridinas/farmacologia , Técnicas de Cultura de TecidosRESUMO
Female fertility depends greatly on the capacity of the uterus to recognize and eliminate microbial infections, a major reason of inflammation in the endometrium in many species. This study aimed to determine the in vitro effect of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the transcriptome genes expression and alternative splicing in the porcine endometrium in the mid-luteal phase of the estrous cycle during LPS-stimulated inflammation using RNA-seq technology. The endometrial slices were incubated in vitro in the presence of LPS and PPARγ agonists-PGJ2 or pioglitazone and antagonist-T0070907. We identified 222, 3, 4, and 62 differentially expressed genes after LPS, PGJ2, pioglitazone, or T0070907 treatment, respectively. In addition, we detected differentially alternative spliced events: after treatment with LPS-78, PGJ2-60, pioglitazone-52, or T0070907-134. These results should become a basis for further studies explaining the mechanism of PPARγ action in the reproductive system in pigs.
Assuntos
Endométrio/efeitos dos fármacos , Inflamação/metabolismo , PPAR gama/agonistas , Pioglitazona/farmacologia , Prostaglandina D2/análogos & derivados , Processamento Alternativo/efeitos dos fármacos , Animais , Benzamidas/farmacologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Perfilação da Expressão Gênica , Inflamação/induzido quimicamente , Inflamação/genética , Lipopolissacarídeos , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Prostaglandina D2/farmacologia , Piridinas/farmacologia , SuínosRESUMO
PROBLEM: Cytokines are immune response mediators that play an important role in the regulation of reproductive functions. An association between cytokines and peroxisome proliferator receptors (PPARs) has been reported in various tissues, including the endometrium. The present study aimed to evaluate the impact of PPARα ligands on the expression of nuclear factor kappa B (NF-κB) and cytokines (interleukin [IL]-1ß, IL-4, IL-6, IL-8, IL-10, and LIF) in the porcine endometrium in different reproductive stages. METHODS OF STUDY: Endometrial slices were collected from gilts on days 10-12 or 14-16 of the estrous cycle and pregnancy. Endometrial tissue explants were incubated in vitro in the presence or absence of PPARα agonist WY-14643 and antagonist MK886. Expression of mRNA and protein for NF-ĸB and selected cytokines was evaluated by real-time PCR and immunoblot. RESULTS: PPARα agonist WY-14643 decreased the mRNA expression of NF-κB in most of the analyzed stages (excluding days 10-12 of the estrous cycle), but increased the expression of NF-κB protein (excluding days 14-16 of pregnancy). The WY-14643 increased expression of IL-1ß and IL-6 proteins, and the mRNA expression of IL-8 and LIF, decreased IL-4 expression, and did not affect the mRNA and protein expression of IL-10. CONCLUSION: The obtained results demonstrate that PPARα is involved in the regulation of NF-κB and cytokine expression in the porcine endometrium. PPARα ligands exert a varied influence on immune system components, which could be attributed to differences in the receptivity of porcine endometrial tissue during the reproductive cycle.
Assuntos
Endométrio/metabolismo , PPAR alfa/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Endométrio/patologia , Ciclo Estral , Feminino , Humanos , Imunidade , Mediadores da Inflamação/metabolismo , NF-kappa B/metabolismo , PPAR alfa/antagonistas & inibidores , Gravidez , Primeiro Trimestre da Gravidez , Pirimidinas/farmacologia , SuínosRESUMO
The peroxisome proliferator-activated receptor (PPAR) ß/δ belongs to a group of nuclear receptors that act as transcription factors. PPAR ß/δ plays a significant role in the regulation of female reproductive processes. It has been demonstrated that PPARß/δ is expressed in mouse, rat and porcine endometrium during the estrous cycle and pregnancy. The current study aimed to investigate the effect of selected PPARß/δ ligands on the expression of nuclear factor kappa (NF-κB) and selected cytokines - interleukin (IL)-1ß, IL-6, IL-8, IL-4, IL-10, leukemia inhibitory factor (LIF), in the porcine endometrium on days 10-12 and 14-16 of the estrous cycle (mid- and late-luteal phases corresponding to the full activity and luteolysis of the corpus luteum, respectively) and pregnancy (maternal recognition of pregnancy and beginning of implantation, respectively). Endometrial slices were incubated in vitro in the presence of PPARß/δ agonist L-165,041 (1 or 10⯵M) or antagonist GW9662 (10⯵M). The expression of mRNA and protein of the immune response mediator in the tissues was determined by real-time PCR and Western Blot. In general, the PPARß/δ agonist inhibited endometrial NF-κB mRNA expression during all analyzed reproductive stages, but it did not change protein expression. In turn, the PPARß/δ antagonist increased NF-κB protein levels on days 10-12 of the estrous cycle or pregnancy. The presence of the PPARß/δ agonist stimulated mRNA expression of LIF, IL-1ß and IL-8 and decreased the expression of IL-6. The presence of PPARß/δ ligands had a varied effect on protein expression in different stages on the analyzed period. The obtained results indicate that PPARß/δ regulates the expression of endometrial NF-κB and selected cytokines in pigs. The effects of PPARß/δ ligands on immune response mediators varied subject to the reproductive status of females and could be associated with differences in endometrial receptivity.
Assuntos
Citocinas/biossíntese , Endométrio/metabolismo , PPAR delta/metabolismo , Suínos/metabolismo , Animais , Ciclo Estral , Feminino , Regulação da Expressão Gênica , Ligantes , NF-kappa B/metabolismoRESUMO
PROBLEM: Cytokines, mediators of the immune response, are involved in the regulation of female reproductive processes during the estrous cycle and pregnancy. The present study aimed to investigate the effect of selected peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of nuclear factor kappa B (NF-κB) and selected cytokines, such as interleukin (IL)-1ß, -4, -6, -8, -10, and the leukemia inhibitory factor, in the porcine endometrium on days 10-12 and 14-16 of the estrous cycle (mid- and late luteal phase, respectively) or pregnancy (maternal recognition of pregnancy and beginning of implantation, respectively). METHOD OF STUDY: Endometrial slices were incubated in vitro in the presence of PPARγ agonists, 15-deoxy-Δ12, 14-prostaglandin J2 or rosiglitazone, and PPARγ antagonist T0070907. mRNA and protein levels in tissues were determined by real-time PCR and Western blot. RESULTS: On days 10-12 of the estrous cycle and days 14-16 of pregnancy, PPARγ ligands enhanced the expression of NF-κB, mRNA cytokines, and/or proteins. During the late luteal phase of the estrous cycle (days 14-16) and maternal recognition of pregnancy (days 10-12), PPARγ ligands inhibited the expression of NF-κB, and they differentially affected the expression of mRNA and proteins of cytokines. CONCLUSION: Our results indicate that PPARγ is engaged in the endometrial synthesis of NF-κB and selected cytokines in pigs. The influence of PPARγ ligands on the tested components of the immune system varied subject to the physiological status of females, and it could be associated with differences in endometrial receptivity.
Assuntos
Endométrio/patologia , PPAR gama/agonistas , RNA Mensageiro/genética , Suínos/imunologia , Trofoblastos/fisiologia , Animais , Benzamidas/farmacologia , Células Cultivadas , Citocinas/metabolismo , Implantação do Embrião , Endométrio/imunologia , Feminino , Ligantes , Ciclo Menstrual/genética , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , PPAR gama/antagonistas & inibidores , Gravidez , Piridinas/farmacologia , Rosiglitazona/farmacologiaRESUMO
Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1ß or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1ß 10 ng/ml or LIF 50 ng/ml. After maturation for 44-46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1ß did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1ß decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1ß in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.
Assuntos
Proteínas do Ovo/metabolismo , Embrião de Mamíferos/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Interleucina-1beta/farmacologia , Fator Inibidor de Leucemia/farmacologia , Metáfase/fisiologia , Oócitos/metabolismo , Animais , Proteínas do Ovo/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização in vitro/veterinária , Fármacos Gastrointestinais/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Metáfase/efeitos dos fármacos , Nucleoplasminas/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , SuínosRESUMO
BACKGROUND: The effect of hormonal estrus induction on maternal effect (MATER - maternal antigen that embryo requires, ZAR-1 - zygote arrest 1, and BMP15 - bone morphogenetic protein 15) and apoptosis-related genes expression (BCL-2 and BAX) in porcine cumulus-oocyte complexes (COCs) and selected follicular parameters was investigated in this study. METHODS: Gilts were divided into three groups: (I) with natural estrus; (II) stimulated with PMSG/hCG; and (III) with PMSG/hCG + PGF2alpha. Analysis of maternal effect and apoptosis-related transcripts expression in COCs, and progesterone synthesis pathway genes expression (P450scc and 3betaHSD) in granulosa cells was performed by qPCR. BMP15 protein expression in follicular fluid (FF) was analyzed by western blot. Oocyte nuclear maturation was assessed by aceto-orcein staining. Progesterone (P4) and estradiol (E2) concentrations in FF and serum were measured by ELISA. Data were analyzed with the one-way ANOVA and Bonferroni post-test or Kruskal-Wallis test and Dunns post-test. RESULTS: The highest expression of MATER, ZAR-1, and BMP15 genes was found in COCs recovered from gilts treated with PMSG/hCG when compared to PMSG/hCG + PGF2alpha-stimulated or non-stimulated gilts. Hormonal treatment did not affect the BMP15 protein expression in FF, but increased the expression of genes participating in P4 synthesis in granulosa cells. The higher percentage of immature oocytes was found in PMSG/hCG-treated when compared to the non-stimulated gilts. The expression of BCL-2 and BAX mRNA, and BCL-2/BAX mRNA ratio was significantly higher in COCs derived from PMSG/hCG-treated when compared to PMSG/hCG + PGF2alpha-treated or non-stimulated subjects. The level of P4 in serum was similar in animals from all experimental groups, while its concentration in FF was greater in gilts subjected to PMSG/hCG treatment than in PMSG/hCG + PGF2alpha-stimulated and non-stimulated gilts. The concentration of E2 did not differ in the serum or FF between the control group and the hormonally stimulated groups. CONCLUSIONS: Hormonal induction of estrus affected maternal effect gene transcripts levels in COCs and and oocyte nuclear maturation. The inclusion of PGF2alpha into the stimulation protocol enabled maintaining of physiological concentration of P4 in FF. Additionally, both hormonal treatments seem to be beneficial for apoptosis prevention through increasing BCL-2/BAX transcript ratio.
Assuntos
Células do Cúmulo/efeitos dos fármacos , Fármacos para a Fertilidade Feminina/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Indução da Ovulação/veterinária , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sus scrofa/fisiologia , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína Morfogenética Óssea 15/genética , Proteína Morfogenética Óssea 15/metabolismo , Gonadotropina Coriônica/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Dinoprosta/farmacologia , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Estro/efeitos dos fármacos , Feminino , Gonadotropinas Equinas/farmacologia , Recuperação de Oócitos/veterinária , Oócitos/citologia , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Progesterona/sangue , Progesterona/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
This study was undertaken to determine the effect of the presence of embryos in the uterine horn on peroxisome proliferator activated receptors (PPARs; A, D, G) gene expression in the reproductive tissues of gilts subjected to a surgical procedure. The uterus consisted of one intact horn connected to the uterine corpus and the second horn detached from the uterine corpus but connected with the contiguous ovary. The gilts were hormonally stimulated and divided into two groups: the first group, inseminated (pregnant) and the second group (cyclic), with surgical procedure but not inseminated. The animals of both groups were slaughtered on day 14 of pregnancy or on day 14 of the oestrous cycle, respectively. PPARs mRNA abundance in the endometrium and the corpus luteum (CL) was analysed by quantitative real-time PCR. During pregnancy, PPARA and PPARD µmRNA abundance in the porcine endometrium was significantly higher in the horn containing embryos than in the contralateral horn, where embryos were absent. The endometrial PPARG1 mRNA abundance did not differ between the two horns during pregnancy and the oestrous cycle, but a higher level of the transcript was observed during pregnancy when compared to the oestrous cycle. In the CL, there were no significant differences in PPARA and PPARDµ mRNA abundance between horns in pregnant or cyclic sows. However, there was a significant increase of PPARA and PPARD transcript level in the CL from cyclic compared with pregnant sows. The results of our study suggest that PPARA and PPARD have regulatory functions in early pregnancy, and they indicate that increased levels of endometrial gene expression are correlated with the presence of embryos in the uterine horn. Higher levels of PPARA and PPARD expression in the porcine CL on day 14 of the oestrous cycle than on day 14 of pregnancy suggest that both forms are involved in the regulation of CL functions.
Assuntos
Ciclo Estral , Receptores Ativados por Proliferador de Peroxissomo , Animais , Endométrio , Prenhez/metabolismo , RNA Mensageiro , Suínos , ÚteroRESUMO
The aim of the present study was to examine the effects of interferon gamma (INFγ) or interleukin 6 (IL-6) on gene expression of PPARs in the porcine endometrium on day 14 of the estrous cycle and pregnancy. Endometrial tissue (200-210 mg), after 18 h of pre-incubation, was incubated for 6 or 12 h in the presence of INFγ (5 or 50 ng/ml) or IL-6 (1 or 10 ng/ml). Gene expression was analyzed by quantitative real time RT-PCR. During the estrous cycle, neither INFγ nor IL-6 affected PPARα and PPARß/δ transcript levels in the endometrium of the cyclic pigs incubated for 6 or 12 hours. The presence of INFγ (5 ng/ml) significantly (p<0.05) increased PPARγ1 gene expression in the tissue incubated for 12 h. During pregnancy, INFγ (50 ng/ml) significantly (p<0.05) enhanced PPARα and PPARß/δ mRNA levels in the endometrium incubated for 6 h, whereas IL-6 (1 or 10 ng/ml) did not change their expression at any incubation time. The effect of both cytokines on PPARγ1 transcript level differed and was dependent on the incubation time. We observed an inhibitory (after 6 h of incubation, p<0.0001) and a stimulatory (after 12 h of incubation, p<0.05) effect of INFγ (5 ng/ml) or IL-6 (10 ng/ml) on PPARγ1 gene expression. The present study indicates that INFγ and IL-6 modulate PPARs gene expression in the porcine endometrium during the estrous cycle and pregnancy. The effect depends on the reproductive status of animals and the length of in vitro incubation of endometrial tissue with the treatments.
Assuntos
Endométrio/metabolismo , Ciclo Estral/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-6/farmacologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Sus scrofa/metabolismo , Análise de Variância , Animais , Feminino , Técnicas In Vitro , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Lysophosphatidic acid (LPA) belongs to the group of lipid messengers, which act via lysophosphatidic acid receptor 3 coupled to G-proteins. The participation of LPA3 in reproductive biology was revealed in mice and has not been studied in gilts. The present study was performed to evaluate the gene expression of LPA3 by a quantitative real-time PCR technique in the endometrium during different stages of pregnancy (days 6-30) and corresponding days of the estrous cycle (days 2-20) as well as in periimplantation period in pigs with surgically detached uterine horns. Based on the most conserved segments of human and rodent LPA3 we obtained a product containing 619bp (GenBank: EF137953), which exhibited high homology with human and rodents sequences. The highest transcript level was noted on days 10-12 of gestation in comparison to remaining periods and during pregnancy on days: 6-7, 8-9, 10-12 and 13-14 in comparison with the corresponding days of the estrous cycle. Higher mRNA level was noted in the horn containing embryos compared to the contralateral horn, where embryos did not develop. The results imply the important role of receptor LPA3 during early pregnancy.
Assuntos
Implantação do Embrião , Endométrio/metabolismo , Estro , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Sequência de Bases , Primers do DNA , DNA Complementar , Feminino , Humanos , Reação em Cadeia da Polimerase , Gravidez , RNA Mensageiro/genética , Receptores de Ácidos Lisofosfatídicos/química , Homologia de Sequência do Ácido Nucleico , SuínosRESUMO
Oxytocin (OT) is involved in the stimulation of secretion of anterior pituitary hormones in females during the periovulatory and periparturient periods. In the present study we examined the role of OT in control of ACTH, beta-endorphin, LH and PRL secretion in vitro from dispersed anterior pituitary cells collected from gilts during the luteal (Days 10-12; n=6) and follicular (Days 18-20; n=5) phases of the estrous cycle. Isolated anterior pituitary cells (1 x 10(6)/ml) were transferred into 24-well plates, separately for each animal, and were pre-incubated for three days at 37 degrees C in atmosphere of 5% CO(2) and 95% air. The cells which attached to the dishes were incubated (3.5 h, 37 degrees C) in McCoy's medium in the absence (control) or in the presence of the following factors: CRH alone (10(-10), 10(-9), 10(-8), 10(-7) M), OT alone (10(-8), 10(-7), 10(-6) M), LVP alone (10(-7) M), OT (10(-7) M) plus CRH (10(-9) M) and LVP (10(-7) M) plus CRH (10(-9) M) for studying ACTH and beta-endorphin secretion; OT alone (10(-8), 10(-7), 10(-6) M), GnRH alone (100 ng/ml), CRH alone (10(-9) M), OT (10(-7) M) plus GnRH (100 ng/ml) and OT (10(-7) M) plus CRH (10(-9) M) for studying LH and PRL secretion. Concentrations of the studied hormones in media were analyzed by RIA. Oxytocin alone increased ACTH (at doses 10(-7), 10(-6) M), beta-endorphin (at dose 10(-8) M), LH (at dose 10(-8) M) and PRL (at doses 10(-7), 10(-6) M) secretion by pituitary cells isolated only from luteal-phase gilts. None of the studied hormone concentrations in the medium was increased in response to OT when pituitary cells of follicular-phase gilts were examined. Oxytocin in combination with CRH exerted an additive effect on beta-endorphin secretion during the luteal phase. Summarizing, in the present study the stimulatory effect of oxytocin on ACTH, beta-endorphin, LH and PRL secretion by pituitary cells isolated from gilts during the luteal phase was demonstrated. However, the cells collected from follicular-phase gilts appeared to be unresponsive to OT. Moreover, interaction between OT and CRH in affecting beta-endorphin secretion was shown. These results suggest that OT may be transiently involved in the modulation of anterior pituitary hormone secretion in cyclic pigs.
Assuntos
Ciclo Estral/fisiologia , Ocitocina/fisiologia , Adeno-Hipófise/metabolismo , Hormônios Liberadores de Hormônios Hipofisários/fisiologia , Hormônios Adeno-Hipofisários/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Animais , Células Cultivadas , Feminino , Hormônio Luteinizante/metabolismo , Prolactina/metabolismo , Suínos , beta-Endorfina/metabolismoRESUMO
OBJECTIVE: Pioglitazone is a member of the thiazolidinediones (TZDs), insulin-sensitizing agents used to treat type 2 diabetes. The aim of this study was to define the effect of pioglitazone on the expression of genes related to carbohydrate and lipid metabolism in subcutaneous fat obtained from type 2 diabetic patients. RESEARCH DESIGN AND METHODS: Forty-eight volunteers with type 2 diabetes were divided into two groups treated for 12 weeks with placebo or pioglitazone (30 mg/day). The expression of several genes was quantified by real-time RT-PCR. RESULTS: Pioglitazone treatment increased the expression of genes involved in glycerol-3-phosphate synthesis. The mRNA expression of PEPCK-C and glycerol-3-phosphate dehydrogenase (GPDH) increased (P < 0.01) in patients treated with pioglitazone. There was no difference in glycerol kinase (GyK) mRNA levels. The expression of genes that regulate fatty acid availability in adipocytes, including lipoprotein lipase (LPL) and acetyl-CoA synthetase (ACS), was higher (P < 0.01) in pioglitazone-treated patients. Pioglitazone stimulated (P < 0.0001) expression of c-Cbl-associated protein (CAP), whereas tumor necrosis factor-alpha, leptin, resistin, angiopoietin like-4, and 11-beta-hydroxysteroid dehydrogenase type 1 (11beta HSD 1) were not affected by pioglitazone. The baseline peroxisome proliferator-activated receptor (PPAR)-gamma1 mRNA was significantly correlated with mRNA for LPL, CAP, ACS, 11beta HSD 1, GyK, fatty acid synthase, leptin, and GPDH, whereas PPAR-gamma2 mRNA was correlated with CAP, PEPCK-C, leptin, and GPDH. CONCLUSIONS: Treatment with pioglitazone increased body weight, and this is associated with upregulation of some, but not all, genes previously demonstrated as "TZD responsive" in subcutaneous fat. The results suggest that TZDs might increase body weight through the upregulation of genes facilitating adipocyte lipid storage in vivo.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Metabolismo dos Lipídeos , PPAR gama/genética , Tiazolidinedionas/uso terapêutico , Composição Corporal/efeitos dos fármacos , Primers do DNA , Ácidos Graxos/metabolismo , Feminino , Humanos , Lipídeos/genética , Masculino , Pessoa de Meia-Idade , Pioglitazona , Placebos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The direct effects of alpha- and beta-adrenergic agents on PRL and beta-endorphin (beta-END) secretion in vitro by porcine pituitary cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomised (OVX) one month before slaughter. Ovariectomised gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2) and (3) oestradiol benzoate (EB; 2.5 mg/100 kg b.w.) at 30-36 h (OVX+EB I) and 60-66 h (OVX+EB II) before slaughter, respectively; and (4) progesterone (P4; 120 mg/100 kg b.w.) for 5 consecutive days before slaughter (OVX+P4). Isolated anterior pituitary cells were submitted to 3.5 h incubation in the presence of GnRH, alpha- and beta-adrenergic agonists [phenylephrine (PHEN) and isoproterenol (ISOP), respectively], or alpha- and beta-adrenergic blockers [phentolamine (PHENT) and propranolol (PROP), respectively]. The culture media were assayed for PRL (exp. I) and beta-endorphin-like immunoreactivity (beta-END-LI) (experiment II). In experiment I, GnRH did not influence PRL release by pituitary cells in all experimental groups. Some of tested doses of adrenergic agonists, PHEN and ISOP, increased PRL release from pituitary cells of OVX gilts, but not from those of OVX+EB I animals. In the OVX+EB II group, PHEN alone, but ISOP with PROP, potentiated PRL secretion by the cells. In OVX+P4 animals, PHEN alone or in combination with PHENT and also ISOP alone or with PROP enhanced PRL output from the cells. In experiment II, addition of GnRH increased beta-END-LI release from pituitary cells only in the OVX+EB II group. PHEN and PHENT potentiated beta-END-LI secretion by pituitary cells in OVX+EB II and OVX+P4 groups, while ISOP and PROP increased beta-END-LI secretion by the cells of OVX and OVX+EB II animals. In turn, in the OVX+EB I group, effect of PHENT and PROP on PRL secretion by pituitary cells was inhibitory. In conclusion, our results suggest that adrenergic agents can modulate PRL and beta-END secretion by porcine pituitary cells in a manner dependent on the hormonal status of gilts.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Fármacos para a Fertilidade Feminina/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Isoproterenol/farmacologia , Fenilefrina/farmacologia , Hipófise/efeitos dos fármacos , Prolactina/biossíntese , Suínos/metabolismo , beta-Endorfina/biossíntese , Animais , Técnicas de Cultura de Células , Estradiol/sangue , Feminino , Hormônio Luteinizante/sangue , Ovariectomia , Fentolamina/farmacologia , Hipófise/citologia , Hipófise/metabolismo , Progesterona/sangue , Propranolol/farmacologiaRESUMO
The effects of separate and simultaneous recombinant bovine (rb) activin A and buserelin administration on the FSH release and pituitary FSH beta subunit gene expression in vivo were examined in ovariectomised, estradiol pretreated rats. The animals received a single injection of either rb activin A (50 ng), buserelin (1 micro g) or activin/buserelin (50 ng+1 micro g/0.1 ml PBS) into the jugular vein and were killed 30 min, 1, 3 and 5h later. Activin A stimulated FSH release and effect appeared 1h after injection (168% increase of controls) reaching a maximum at 3h (437% of controls). Activin A and buserelin exerted their effects with a distinct time courses: activin's stimulation was not so rapid when compared with buserelin. The simultaneous administration of rb activin A and buserelin amplified FSH release (118, 309, 1006 and 779% of controls). The low dose of activin A was sufficient to elevate FSH beta mRNA level as early as 3 and 5h after administration (170 and 140%, respectively). Activin plus buserelin stimulation resulted in a higher (340 and 360% of controls) FSH beta gene expression than after their separate administration. These results suggest that activin and buserelin may act independently and synergistically in the regulation of FSH release and beta subunit mRNA level.