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1.
Sci Rep ; 13(1): 14133, 2023 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-37644083

RESUMEN

Angiogenesis is important for endometrial remodeling in mature females. The endometrium synthesizes high amounts of prostacyclin (PGI2) but the role of PGI2 in angiogenesis-related events in this tissue was not fully described. In the present study, porcine endometrial endothelial (pEETH) cells and/or a swine umbilical vein endothelial cell line (G1410 cells) were used to determine the regulation of PGI2 synthesis and PGI2 receptor (PTGIR) expression by cytokines and to evaluate the effect of PGI2 on pro-angiogenic gene expression, intracellular signaling activation, cell proliferation and migration, cell cycle distribution, and capillary-like structure formation. We found that IL1ß, IFNγ, and/or TNFα increased PGI2 secretion and PTGIR expression in pEETH cells. Iloprost (a PGI2 analogue) acting through PTGIR enhanced the transcript abundance of KDR, FGFR2, and ANGPT2 and increased proliferation of pEETH cells. This latter was mediated by PI3K and mTOR activation. In support, transfection of G1410 cells with siRNA targeting PGI2 synthase decreased pro-angiogenic gene expression and cell proliferation. Furthermore, iloprost accelerated the gap closure and promoted cell cycle progression. Intriguingly, the formation of capillary-like structures was inhibited but not completely blocked by iloprost. These findings point to a complex pleiotropic role of PGI2 in angiogenesis-related events in the porcine uterus.


Asunto(s)
Coagulantes , Epoprostenol , Femenino , Porcinos , Animales , Epoprostenol/farmacología , Iloprost/farmacología , Prostaglandinas I , División Celular , Endometrio
2.
Animals (Basel) ; 12(17)2022 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-36077955

RESUMEN

Although prostacyclin (PGI2) has been well described as a regulator of smooth muscle activity, limited data are available concerning its role in the myometrium of pigs. The present research aimed to examine profiles of PGI2 synthase (PTGIS) and PGI2 receptor (PTGIR) expression and 6-keto PGF1α (a PGI2 metabolite) concentrations in the myometrium of gilts throughout the estrous cycle and during early pregnancy using qPCR, Western blot, and/or ELISA methods. Furthermore, myometrial explants were exposed to iloprost (a stable PGI2 analog) to investigate the effect of PGI2 on the mRNA expression of factors engaged in smooth muscle contraction, nutrient transport, prostaglandin synthesis and action, and inflammatory response. PTGIS mRNA expression was greater in cyclic than in pregnant gilts on days 11-12 after estrus and was accompanied by greater concentrations of 6-keto PGF1α detected in cyclic than in pregnant animals on days 11-20. Iloprost stimulated fatty acid transporters and contractility-related calponin 1 and caldesmon 1 mRNA expression and decreased interleukin 1ß and tumor necrosis factor transcript abundance. The obtained results indicate a physiologically relevant role of PGI2 during the estrous cycle in the porcine myometrium with its importance for regulating the expression of contractility-, nutrient transport- and inflammatory response-related factors.

3.
Anim Reprod Sci ; 221: 106583, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32882581

RESUMEN

Supplementation of progesterone (P4) in pregnant gilts increases concentrations of circulating P4 and stimulates the secretory activity of the endometrium. In this study, there was examination of the consequences of exogenous P4 administration on luteal P4 content and the expression of genes related to the corpus luteum (CL) function. Gilts with gonadotropin-induced estrus were administered daily injections of corn oil (n = 8) or P4 (n = 8) on days 3 through 10 after insemination. The animals were slaughtered on day 12 of pregnancy to obtain corpora lutea for real-time polymerase chain reaction analyses of selected genes and for enzyme immunoassay of P4. Injections with P4 had no effect on the concentration of P4 and the relative abundance of mRNA transcripts of cholesterol transport-related proteins, steroidogenic enzymes, and receptors for luteotropic factors in the luteal tissue. The abundance of prostaglandin (PG) endoperoxide synthase 2, PGI2 synthase, PGI2 receptor, fibroblast growth factor 2, peroxisome proliferator-activated receptor γ, and tumor necrosis factor α receptor type I transcripts increased after P4 treatment. In contrast, the relative abundance of angiopoietin 2 mRNA decreased in response to P4 administration. In summary, P4 supplementation in pregnant gilts does not affect luteal steroidogenesis but modulates the abundance of factors related to vascular function. Given that the endometrium is the main target tissue for P4, an indirect uterine-mediated effect of exogenous P4 on CL function is likely.


Asunto(s)
Cuerpo Lúteo/efectos de los fármacos , Preñez , Progesterona/farmacología , Progestinas/farmacología , Porcinos/fisiología , Animales , Cuerpo Lúteo/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Embarazo
4.
Theriogenology ; 152: 36-46, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32361305

RESUMEN

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor family of ligand-dependent transcription factors. PPARs have been shown to be important regulators of female reproductive functions, including conceptus development and placenta formation. This study examines the effect of PPARß/δ and PPARγ agonists and antagonists on (1) the synthesis of prostaglandin (PG) E2, interleukin (IL) 6, interferon (IFN) γ, and tumor necrosis factor (TNF) α and (2) the mRNA expression of genes encoding nutrient transporters and/or binding proteins in Day 15 conceptus trophoblast cells. The study also examines whether PPAR agonist-modulated IL6, IFNγ, and TNFα secretion is mediated via mitogen-activated protein kinase (MAPK) pathways. Trophoblast cells were exposed to L-165,041 (a PPARß/δ agonist) or rosiglitazone (a PPARγ agonist) in the presence or absence of GSK3787 (a PPARß/δ antagonist) or GW9662 (a PPARγ antagonist) or in the presence or absence of U0126 (a MAPK inhibitor). Rosiglitazone stimulated PGE synthase and IFNG mRNA expression in trophoblast cells and enhanced PGE2 concentrations in the incubation medium. Moreover, cells treated with rosiglitazone exhibited increased abundance of the solute carrier organic anion transporter family member 2A1 (SLCO2A1, a PG transporter) and of fatty acid binding protein (FABP) 5 transcripts. All these effects were abolished by the addition of GW9662, which indicates that the action of rosiglitazone is PPARγ-dependent in the studied cells. L-165,041 inhibited TNFα synthesis and decreased the mRNA expression of FABP3 and IL6 in trophoblast cells. However, this effect was not abolished by the addition of GSK3787 into the incubation medium, suggesting that L-165,041 action is independent of PPARß/δ. The inhibitory effect of L-165,041 on TNFα concentration and the stimulatory effect of rosiglitazone on IFNγ accumulation in the medium were not observed in the presence of the MAPK inhibitor, suggesting that the action of both agonists may be mediated by MAPKs. In conclusion, PPARß/δ and PPARγ agonists are differentially involved in the trophoblast expression of genes related to conceptus development and implantation in pigs. Furthermore, L-165,041 and rosiglitazone may have PPAR-dependent and -independent effects in conceptus trophoblast cells.


Asunto(s)
Benzamidas/farmacología , Citocinas/metabolismo , Dinoprostona/metabolismo , Fenoxiacetatos/farmacología , Rosiglitazona/farmacología , Sulfonas/farmacología , Trofoblastos/efectos de los fármacos , Anilidas/farmacología , Animales , Butadienos/farmacología , Proteínas Portadoras , Células Cultivadas , Citocinas/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Nitrilos/farmacología , PPAR delta/agonistas , PPAR delta/antagonistas & inhibidores , PPAR gamma/agonistas , PPAR gamma/antagonistas & inhibidores , PPAR-beta/agonistas , PPAR-beta/antagonistas & inhibidores , Embarazo , Porcinos , Trofoblastos/metabolismo
5.
Theriogenology ; 127: 88-101, 2019 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-30677596

RESUMEN

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are important regulators of glucose and fatty acid metabolism, apoptosis, angiogenesis, cell proliferation and differentiation, and immune response. Their possible role in the female reproductive tract was demonstrated. In the present study, cultured luminal epithelial (LE) and stromal (ST) cells of the porcine endometrium were used to examine (1) the effect of conceptus exposed medium (CEM) on mRNA and protein expression and DNA binding activity of PPARA, PPARD, and PPARG isoforms, and (2) the effect of PPARA, PPARD, and PPARG agonists on the expression of selected genes, apoptosis, and cell proliferation. The addition of CEM stimulated PPARA expression and DNA binding activity of this isoform in LE and ST cells (P < 0.05). Increased expression of PPARD mRNA in the presence of CEM was detected in ST cells (P < 0.05), while the concentration of PPARG transcripts decreased in response to CEM in both cell types (P < 0.05). LE and ST cells of the pig endometrium possess PPARA, PPARD, and PPARG proteins, with clear nuclear staining visible predominately in ST cells. In LE cells, activation of PPARG with 15-deoxy-Δ12,14-prostaglandin(PG)J2 down-regulated the expression of genes encoding amino acid transporter 1 (SLC38A1), leukemia inhibitory factor (LIF) and enzymes involved in PG synthesis (P < 0.05). In ST cells, activation of PPARD isoform with both agonists used (L-165,041 and cPGI2) and PPARG isoform with 15-deoxy-Δ12,14-PGJ2 increased vascular endothelial growth factor A (VEGFA) mRNA expression (P < 0.05). Moreover, GW9578 (PPARA agonist) and 15-deoxy-Δ12,14-PGJ2 stimulated glucose transporter 1 (SLC2A1) gene expression in ST cells. 15-deoxy-Δ12,14-PGJ2 was also effective in up-regulation of the ratio of BAX/BCL2 mRNA expression and active caspase-3 concentration in ST cells (P < 0.05). Finally, GW9578 stimulated LE and ST cell proliferation, while rosiglitazone (PPARG agonist) increased the number of viable ST but not LE cells. In conclusion, this study demonstrated that conceptus products differentially modulate PPARs expression and activity in the porcine endometrium. Activation of PPARs may in turn affect nutrient transport, PG synthesis, angiogenesis, apoptosis, or cell proliferation in this tissue. Therefore, PPAR isoforms seem to play an important role in development and function of the porcine uterus.


Asunto(s)
Receptores Activados del Proliferador del Peroxisoma/fisiología , Compuestos de Quinolinio/metabolismo , Porcinos , Animales , Apoptosis , Proliferación Celular , Endometrio/metabolismo , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Receptores Activados del Proliferador del Peroxisoma/metabolismo , ARN Mensajero/metabolismo , Células del Estroma/metabolismo
6.
Biol Reprod ; 100(1): 162-174, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30481284

RESUMEN

The prostacyclin (prostaglandin I2) signaling system is an essential regulator of vascular homeostasis. Since the corpus luteum is a highly vascularized gland, prostacyclin seems to be crucial for luteal development and function. Although progress has been made in understanding the luteotropic action of prostacyclin in mammals, its role in the porcine corpus luteum remains to be determined. Therefore, studies were conducted to (1) determine profiles of prostacyclin synthase expression and prostacyclin metabolite concentration, as well as prostacyclin G-protein-coupled receptor expression in porcine luteal tissue on days 2 to 16 of the estrous cycle and days 10 to 30 of pregnancy using real-time PCR, western blot, or enzyme immunoassay; and (2) examine the effect of prostacyclin on progesterone synthesis in vitro. To accomplish the second aim, luteal cells were treated with prostacyclin analogs, iloprost and carbaprostacyclin, in the presence or absence of prostacyclin receptor antagonists. The mRNA expression of cytochrome P450 family 11 subfamily A member 1 and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 was analyzed using real-time PCR, while progesterone concentration in culture medium was assessed by radioimmunoassay.Dynamic changes of prostacyclin synthase and prostacyclin receptor expression were observed in porcine luteal tissue during the estrous cycle and early pregnancy. Moreover, prostacyclin stimulated progesterone production and this effect was abolished by the addition of prostacyclin receptor antagonists. Our findings provide strong evidence that prostacyclin and its signaling system are present in corpus luteum of the pig and may directly promote luteotropic activity through upregulation of progesterone synthesis.


Asunto(s)
Cuerpo Lúteo/metabolismo , Epoprostenol/biosíntesis , Células Lúteas/metabolismo , Receptores de Epoprostenol/genética , Animales , Células Cultivadas , Cuerpo Lúteo/citología , Femenino , Expresión Génica , Embarazo , Receptores de Epoprostenol/metabolismo , Porcinos
7.
Theriogenology ; 101: 53-61, 2017 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-28708516

RESUMEN

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are critical regulators of glucose homeostasis and lipid metabolism, and affect cell proliferation and differentiation. In the current study, we examined (1) the profiles of PPARA, PPARD, and PPARG mRNA expression and DNA binding activity in porcine conceptuses collected on Days 10-11 (spherical and tubular conceptuses), 11-12 (filamentous conceptuses), 13-14, and 15-16 (elongated conceptuses) of pregnancy, (2) the presence of PPARA, PPARD, and PPARG proteins in Days 10, 12, and 15 conceptuses. Moreover, we analyzed the abundance of retinoid X receptor (RXR; PPARs heterodimer partner) transcripts as well as the correlation between PPARs mRNA expression and the expression of genes important for and/or associated with elongation of porcine conceptuses: aromatase (CYP19A1), prostaglandin endoperoxide synthase 2 (PTGS2), glucose transporter 1 (SLC2A1), and interleukin 1B (IL1B). PPARA mRNA expression in conceptuses did not change during Days 10-14 of gestation, but was greater on Days 15-16 compared to Days 10-11 (P < 0.05). A considerable increase in PPARD and PPARG mRNA expression was observed in filamentous conceptuses from Days 11-12 compared to spherical and tubular conceptuses from Days 10-11 (P < 0.01), followed by a decrease on Days 13-14 and 15-16 (P < 0.05). PPARA, PPARD, and PPARG proteins were present in conceptus tissue demonstrating nuclear localization clearly visible on Days 12 and 15 of pregnancy. DNA binding activity of the PPARD isoform was greater in filamentous conceptuses from Days 11-12 than in spherical and tubular conceptuses from Days 10-11 (P < 0.01). Moreover, concentrations of active PPARD and PPARG proteins in nuclear fractions of conceptus tissue were greater on Days 11-12 compared to Days 13-14 and 15-16 of pregnancy (P < 0.05). RXRA, RXRD, and RXRG mRNA expression in conceptuses increased on Days 11-12 compared to Days 10-11 (P < 0.05). PPARD and PPARG mRNA expression showed strong positive correlations with PTGS2 mRNA expression (P < 0.0001). Additionally, PPARD gene expression correlated with SLC2A1 and IL1B mRNA expression (P < 0.01). Collectively, these results indicate that among all three PPARs expressed in peri-implantation porcine conceptuses, PPARD and PPARG may be involved in conceptus elongation before implantation.


Asunto(s)
Blastocisto/química , Receptores Activados del Proliferador del Peroxisoma/análisis , Isoformas de Proteínas/análisis , Sus scrofa/embriología , Animales , ADN/metabolismo , Implantación del Embrión , Femenino , Expresión Génica , PPAR alfa/genética , PPAR delta/genética , PPAR gamma/genética , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Embarazo
8.
Mol Reprod Dev ; 84(9): 842-855, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28628266

RESUMEN

Porcine conceptuses secrete pregnancy-recognition signals (estrogens, including estradiol-17ß) that inhibit luteolysis, thereby prolonging progesterone production by corpora lutea. The supportive mechanism by which the conceptus also inhibits luteolysis is by shifting endometrial prostaglandin (PG) synthesis to luteoprotective PGE2. Progesterone stimulates endometrial production of factors that are essential for conceptus development. Priming the uterus by progesterone and loss of progesterone receptors from the uterine epithelium by D1ay 10-12 after estrus are key for achieving endometrial receptivity for implantation. Conceptus implantation involves a series of events, many resembling the inflammatory reaction, that are greatly influenced by cytokines, growth factors, and prostaglandins. We herein present a novel, dual role for PGF2α in corpora lutea that depends on the acquisition of luteolytic sensitivity, based on the knowledge that PGF2α triggers pathways involved in luteolysis during the estrous cycle or/and may have an alternative function in maintaining progesterone synthesis during pregnancy. We also point out a new role for PGF2α that, together with PGE2, can act as embryonic signal mediators. PGF2α, which until recently was considered undesirable for promoting pregnancy, is now known to stimulate conceptus-maternal interactions and angiogenesis in the endometrium. This function is in line with other important prostaglandin functions, such as stimulating adhesion of trophoblasts (PGE2, PGI2) as well as endometrial vascular functions and trophoblast cell proliferation (PGI2). Finally, microRNAs have emerged as important post-transcriptional regulators of gene function, adding a new area of investigation that may enhance understanding of conceptus-endometrial interactions.


Asunto(s)
Embrión de Mamíferos/metabolismo , Endometrio/metabolismo , Estradiol/metabolismo , Intercambio Materno-Fetal/fisiología , Embarazo/fisiología , Prostaglandinas/metabolismo , Animales , Embrión de Mamíferos/citología , Femenino , Porcinos
9.
Anim Reprod Sci ; 174: 56-64, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27639502

RESUMEN

The aim of the study was to determine how supplementation with exogenous progesterone (P4) affects the expression of genes important for endometrial receptivity and conceptus development during the peri-implantation period in pigs. Gilts with PMSG/hCG-induced first estrus received daily injections of corn oil (CO; n=7) or P4 (n=7) on days 3 to 10 after insemination. The dose of P4 was 25mg/100kg BW on days 3 and 4 of gestation, then increased to 50mg/100kg BW on days 5 to 10. Blood samples were collected each day to monitor concentrations of circulating P4. Animals were slaughtered on day 12 of pregnancy to obtain endometrial tissue, conceptuses, and uterine luminal flushing (ULF). Gilts in the P4 group had consistently greater P4 concentrations in the blood compared with controls. Treatment of gilts with P4 increased uterine weight and enhanced total protein content and 6-keto PGF1α (a PGI2 metabolite) content in the uterine lumen. Moreover, injections of gilts with P4 increased the expression of prostaglandin endoperoxide synthase 2, microsomal PGE2 synthase and vascular endothelial growth factor A mRNA in the endometrium, but had no effect on gene expression in conceptuses. These results indicate that P4 supplementation stimulated the secretory activity of the endometrium and increased the expression of genes responsible for vascular function and luteotropic PGE2 synthesis, which are important components of successful pregnancy establishment in pigs. However, no effect of P4 treatment was detected in conceptuses.


Asunto(s)
Embrión de Mamíferos/efectos de los fármacos , Endometrio/efectos de los fármacos , Estro/efectos de los fármacos , Progesterona/farmacología , Porcinos/fisiología , Animales , Endometrio/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hormonas/metabolismo , Hormonas/farmacología , Embarazo , Progesterona/administración & dosificación
10.
Anim Reprod Sci ; 166: 28-35, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26781360

RESUMEN

The present study was designed to examine whether an estrus induction with gonadotropins could affect luteal P4 synthesis in early pregnant gilts. Sixteen prepubertal gilts received 750IU of PMSG and 500IU of hCG 72h later. Prepubertal gilts in the control group (n=17) were observed daily for estrus behavior. All gilts were inseminated in their first estrus. Corpora lutea (CLs) were collected on days 10, 12 and 15 of pregnancy and analyzed for (1) the mRNA and protein expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1), and 3ß-hydroxysteroid dehydrogenase (3ßHSD); (2) the tissue concentration of P4; and (3) the mRNA expression of luteinizing hormone receptor (LHR) and estrogen receptors (ESR1 and ESR2). Additionally, P4 concentration was analyzed in blood serum of all animals. PMSG/hCG injections to induce estrus decreased mRNA expression of StAR, CYP11A1 and 3ßHSD on day 10 and CYP11A1 on day 12 of pregnancy compared with the control group, while CYP11A1 and 3ßHSD proteins were down-regulated on day 10 in the hormonally-treated gilts. Concentrations of P4 in luteal tissue and blood serum were also lower in animals after gonadotropin-induced estrus. In contrast, LHR and ESR1 mRNA expression was greater in PMSG/hCG-treated than control gilts on day 15 of gestation. In conclusion, induction of estrus with a PMSG/hCG protocol in prepubertal gilts impaired expression of the luteal P4 synthesis system. Low P4 content may, in turn, induce local mechanisms involving LHR and ESR1 expression to support CL function.


Asunto(s)
Gonadotropina Coriónica/uso terapéutico , Cuerpo Lúteo/metabolismo , Sincronización del Estro , Gonadotropinas Equinas/uso terapéutico , Inducción de la Ovulación/métodos , Progesterona/biosíntesis , Porcinos , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Combinación de Medicamentos , Sincronización del Estro/fisiología , Femenino , Fase Luteínica/genética , Fase Luteínica/metabolismo , Inducción de la Ovulación/veterinaria , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Embarazo , Progesterona/metabolismo , Porcinos/genética , Porcinos/metabolismo
11.
Theriogenology ; 84(6): 969-82, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26139576

RESUMEN

The prostacyclin (PGI2) signaling pathway plays an important role during early pregnancy in rodents and ruminants. Abundant concentrations of PGI2 were also found in the endometrium and uterine lumen of gilts during the period of implantation. The present study was designed to examine (1) the expression of PGI2 receptor (PTGIR) messenger RNA (mRNA) and protein in the endometrium of cyclic and early-pregnant gilts; (2) possible regulation of endometrial PTGIR gene expression by conceptus products, estradiol and cytokines; (3) the effect of iloprost (a PGI2 analogue) on cAMP formation and the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF; isoform 164) mRNA in luminal epithelial (LE) and stromal (ST) cells. Increased PTGIR mRNA expression in the endometrium was detected on Days 11 to 12 and 18 to 20 of pregnancy compared with respective days of the estrous cycle (P < 0.05). Moreover, greater PTGIR protein level was observed in pregnant than in cyclic gilts on Days 11 to 12 after estrus (P < 0.05). Gilts with unilateral pregnancy revealed abundant PTGIR expression in the endometrium collected from the gravid uterine horn of Day-11 pregnant gilts compared with the respective horn of cyclic animals (P < 0.01). Both LE and ST cells of the endometrium possess PTGIR protein. Moreover, IL1ß, IFNγ, and conceptus-exposed medium, but not estradiol, stimulated PTGIR mRNA expression in LE and ST cells in vitro. Activation of PTGIR by incubation of LE and ST cells with iloprost resulted in greater cAMP generation (P < 0.01). Moreover, iloprost increased FGF-2 and VEGF164 mRNA expression in ST (P < 0.05), but not LE cells. In conclusion, this study revealed increased expression of PTGIR in the porcine endometrium during the peri-implantation period and reported a possible regulation of PTGIR abundance by conceptus-derived factors. Moreover, besides its important role in vascular system, PGI2 may promote the expression of proangiogenic genes in the uterine stroma.


Asunto(s)
Endometrio/metabolismo , Regulación de la Expresión Génica , Receptores de Epoprostenol/metabolismo , Porcinos/metabolismo , Animales , Células Epiteliales/metabolismo , Femenino , Embarazo , ARN Mensajero/metabolismo , Células del Estroma/metabolismo
12.
Theriogenology ; 82(6): 897-909, 2014 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-25123632

RESUMEN

The objective of the study was to investigate transcriptomic profile of pig endometrium on Days 12 and 16 of pregnancy in comparison with the respective days of the estrous cycle. Labeled complementary DNA was hybridized to Porcine Long Oligo microarray containing 13,297 oligonucleotide probes, which represented complementary DNA and expressed sequence tags. Statistical analysis revealed 110 differentially expressed genes (DEGs) on Day 12 of pregnancy and 179 DEGs on Day 16 of pregnancy. In silico analysis of gene function and functionality networks revealed links between genes implicated in cell death and survival, protein synthesis, lipid metabolism, cellular movement, tissue development, and cell-to-cell signaling. On Day 12 of pregnancy, estrogen, transforming growth factor (TGF) ß1, and fibroblast growth factor (FGF) 2, and on Day 16 of pregnancy, epidermal growth factor (EGF), insulin, interleukin 11 (IL-11), and FGF family members were indicated as possible upstream regulators of several DEGs. Obtained results showed changes in global endometrial gene expression at the time of maternal recognition of pregnancy and embryo implantation. Additionally, these data revealed signaling molecules, which together with E2, may evoke molecular changes in the uterus, leading to successful pregnancy establishment.


Asunto(s)
Endometrio/fisiología , Ciclo Estral/fisiología , Porcinos/fisiología , Transcriptoma/fisiología , Animales , Simulación por Computador , Estrógenos/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Embarazo , Progesterona/metabolismo
13.
Theriogenology ; 82(4): 546-56, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-24954635

RESUMEN

Prostacyclin (prostaglandin I2 [PGI2]) signaling system not only plays a pivotal role in vascular function in many species but is also important during early pregnancy in rodents and ruminants. Recently, abundant concentrations of PGI2 were found in the endometrium and uterine lumen of gilts at the time of implantation. In the present study, conceptuses collected on Days 10, 12, 14, 16, and 18 of pregnancy were examined for the expression of PGI2 receptors, PTGIR. Moreover, the effect of iloprost (a PGI2 analogue) on attachment, proliferation, and apoptosis in conceptus trophoblast (Tr) cells was investigated in vitro. Increased PTGIR mRNA expression was observed in Day 16 trophoblasts compared with Days 10, 12, and 14 conceptuses (P < 0.001) and Day 18 trophoblast tissue (P < 0.01). Embryos from Day 18 of gestation revealed greater PTGIR mRNA expression compared with Day 16 embryos (P < 0.01). In contrast to mRNA, PTGIR protein level in conceptus and trophoblast tissue was high on Days 12 and 14, followed by a decrease observed on Day 16. On Day 18 of pregnancy, PTGIR protein was detected in both trophoblast and embryonic tissue. Iloprost stimulated attachment and proliferation of Tr cells, but this effect was abolished by the addition of the PTGIR-specific antagonist, CAY10441, into culture medium. Addition of iloprost neither did affect the ratio of BAX/BCL-2 gene expression in cultured Tr cells nor did protect these cells from staurosporine-induced apoptosis. In summary, PTGIR is expressed in porcine conceptuses, and PGI2 acting through this receptor may promote the attachment and proliferation of Tr cells, thereby facilitating conceptus implantation.


Asunto(s)
Embrión de Mamíferos/metabolismo , Iloprost/farmacología , Receptores de Epoprostenol/metabolismo , Porcinos/metabolismo , Trofoblastos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Proliferación Celular/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Receptores de Epoprostenol/química , Alineación de Secuencia , Análisis de Secuencia de ADN , Trofoblastos/efectos de los fármacos
14.
J Reprod Dev ; 59(6): 512-9, 2013 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-24042214

RESUMEN

Transforming growth factor (TGF) ß and its receptors are expressed at the conceptus-maternal interface during early pregnancy in the pig. The present studies were conducted to examine: (1) the effect of conceptus products on TGFß1 mRNA expression and protein concentration in the porcine endometrium using in vivo and in vitro models, and (2) the effect of TGFß1 on proliferation of porcine trophoblast cells in vitro. During in vivo experiments, gilts with one surgically detached uterine horn were slaughtered on days 11 or 14 of the estrous cycle and pregnancy. For in vitro studies, endometrial explants and luminal epithelial (LE) cells co-cultured with stromal (ST) cells were treated with conceptus-exposed medium (CEM). Moreover, porcine trophoblast cells were treated with TGFß1, and the number of viable cells was measured. On day 11, the presence of conceptuses had no effect on TGFß1 mRNA expression, but decreased the TGFß1 protein concentration in the connected uterine horn compared with the detached uterine horn. In contrast to day 11, on day 14 after estrus, TGFß1 mRNA expression and protein content in the endometrium collected from the gravid uterine horn were greater when compared with the contralateral uterine horn. The treatment of endometrial slices with CEM resulted in greater TGFß1 mRNA expression and protein secretion. LE cells responded to CEM with an increased TGFß1 mRNA level. Moreover, TGFß1 stimulated the proliferation of day 14 trophoblast cells. In summary, porcine conceptuses may regulate TGFß1 synthesis in the endometrium at the time of implantation. TGFß1, in turn, may promote conceptus development by increasing the proliferation of trophoblast cells.


Asunto(s)
Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Endometrio/metabolismo , Mantenimiento del Embarazo , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba , Animales , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Cruzamientos Genéticos , Medios de Cultivo Condicionados/metabolismo , Técnicas de Cultivo de Embriones , Implantación del Embrión , Embrión de Mamíferos/citología , Endometrio/citología , Femenino , Inseminación Artificial/veterinaria , Embarazo , Células del Estroma/citología , Células del Estroma/metabolismo , Sus scrofa , Técnicas de Cultivo de Tejidos , Factor de Crecimiento Transformador beta1/genética , Trofoblastos/citología , Trofoblastos/metabolismo
15.
Theriogenology ; 76(9): 1684-94, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21890190

RESUMEN

The purpose of the study was to investigate an effect of estrus synchronization with prostaglandin (PG) F(2α) and PMSG/hCG on WNT4, WNT5A, WNT7A, ß-catenin (CTNNB1) and E-cadherin (CDH1) gene expression. The weight of the uterus, morphometrical parameters of the endometrium and the number of CL were recorded. The analysis of estradiol (E(2)), prostaglandin (PG) F(2α) and E(2) content in the uterine luminal flushings (ULFs) and progesterone (P(4)) level in the blood serum were conducted. RNA was isolated from endometrial, luteal and embryonic tissue of pregnant non-synchronized (Control; n = 15) and pregnant synchronized (PGF(2α)/PMSG/hCG; n = 15) pigs. Whereas there was no change in uterine weight, differences in height of endometrial surface and glandular epithelium were found. However, height of the endometrium, number of the glands and capillaries were unaffected. The total number of the CLs was higher (P < 0.05) in animals treated with PGF(2α)/PMSG/hCG. The amount of E(2) and P(4) was lower (P < 0.05, P < 0.001, respectively) in pregnant gilts administrated with PGF(2α)/PMSG/hCG. The concentration of PGF(2α) in ULFs was not affected by hormonal management, while PGE(2) was higher (P < 0.01) in hormonally in comparison to non-hormonally treated pigs. The content of WNT4 mRNA in conceptuses increased on particular Days studied in Control and PGF(2α)/PMSG/hCG administered animals. WNT7A and CTNNB1 were affected by PGF(2α)/PMSG/hCG treatment in both conceptuses (P < 0.001, P < 0.05) and endometrial tissue (P < 0.001, P < 0.01). The PGF(2α)/PMSG/hCG treatment resulted in elevated expression of WNT4 (P < 0.001) and CTNNB1 (P < 0.05) in luteal tissue in comparison to the Control gilts. Moreover, luteal amount of WNT5A mRNA was higher in PGF(2α)/PMSG/hCG animals in comparison to the Control group (P < 0.05). Presented data show that exogenous hormones administration can affect gene expression in the porcine reproductive tract and embryo.


Asunto(s)
Gonadotropina Coriónica/farmacología , Dinoprost/farmacología , Embrión de Mamíferos/efectos de los fármacos , Sincronización del Estro , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Gonadotropinas Equinas/farmacología , Porcinos/fisiología , Útero/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Animales , Cuerpo Lúteo/metabolismo , Embrión de Mamíferos/metabolismo , Endometrio/metabolismo , Estradiol/metabolismo , Femenino , Inseminación Artificial/veterinaria , Tamaño de los Órganos/efectos de los fármacos , Embarazo , Progesterona/sangre , Prostaglandinas/metabolismo , ARN Mensajero/metabolismo , Porcinos/genética , Porcinos/metabolismo , Útero/anatomía & histología , Proteínas Wnt/genética , Vía de Señalización Wnt/genética
16.
Mol Reprod Dev ; 78(8): 597-610, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21786362

RESUMEN

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis.


Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada/citología , Células Endoteliales/citología , Transfección/métodos , Venas Umbilicales/citología , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Transformada/metabolismo , Movimiento Celular/fisiología , Células Endoteliales/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Cariotipo , Microscopía , Reacción en Cadena de la Polimerasa , Virus 40 de los Simios , Porcinos , Venas Umbilicales/metabolismo , Factores de Crecimiento Endotelial Vascular/genética , Factores de Crecimiento Endotelial Vascular/metabolismo
17.
Theriogenology ; 76(4): 687-99, 2011 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-21652061

RESUMEN

WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. ß-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, ß-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17ß-estradiol (E(2)) and progesterone (P(4)) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, ß-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E(2) on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E(2) and P(4) level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, ß-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.


Asunto(s)
Cadherinas/biosíntesis , Implantación del Embrión/fisiología , Endometrio/fisiología , Regulación del Desarrollo de la Expresión Génica , Porcinos/fisiología , Proteínas Wnt/biosíntesis , beta Catenina/biosíntesis , Animales , Western Blotting/veterinaria , Cadherinas/genética , Implantación del Embrión/genética , Estradiol/farmacología , Femenino , Técnicas In Vitro , Microscopía Fluorescente/veterinaria , Embarazo , Progesterona/farmacología , ARN/química , ARN/genética , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Estadísticas no Paramétricas , Proteínas Wnt/genética , beta Catenina/genética
18.
Reprod Biol Endocrinol ; 9: 27, 2011 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-21349168

RESUMEN

BACKGROUND: The interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells. METHODS: The primary cultures of bovine luteal endothelial cells were immortalized by transfection with vector carrying the Simian virus 40 T-antigen (SV40 T-ag) sequence. Expression of SV40 T-ag gene in EnCL-1 cells was confirmed by RT-PCR and immunofluorescence staining showed the presence of endothelial cell markers: VE-cadherin and von Willebrand factor. EnCL-1 cells were stimulated by TNFalpha with IFNgamma (50 ng/ml each) for 24 h. Cell viability, mRNA expression (real time RT-PCR), protein expression (western blotting) for LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), PGE2 and PGF2alpha synthases and endothelin-1 (EDN-1), and levels of LTs (B4 and C4) and PGs (E2 and F2alpha) and EDN-1 in the medium (EIA) were evaluated. RESULTS: We received immortalized luteal endothelial cell line (EnCL-1). Cytokines did not change EnCL-1 cell viability but increased mRNA expression of LTC4S, LTA4H, PGE2 and PGF2alpha synthases and EDN-1. EDN-1/2/3, LTC4 and PGF2alpha synthases protein expression were elevated in the presence of TNFalpha/IFNgamma, and accompanied by increased EDN-1, LTC4 and PGF2alpha secretion. Cytokines had no effect on PGES and LTA4H protein expression, and PGE2 and LTB4 release. CONCLUSIONS: TNFalpha and IFNgamma modulate EnCL-1 cell function. Moreover, established EnCL-1 cell line appears to be a good model for investigating the molecular mechanisms related to cytokines action and aa metabolites production in cattle.


Asunto(s)
Ácido Araquidónico/metabolismo , Cuerpo Lúteo/fisiología , Citocinas/farmacología , Interferón gamma/farmacología , Células Lúteas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Bovinos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelina-1/biosíntesis , Epóxido Hidrolasas/biosíntesis , Femenino , Glutatión Transferasa/biosíntesis , Hidroxiprostaglandina Deshidrogenasas , Oxidorreductasas Intramoleculares/biosíntesis , Prostaglandina-E Sintasas , ARN Mensajero/metabolismo
19.
J Reprod Dev ; 56(6): 643-8, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20814169

RESUMEN

The homeobox A (HOXA) family of genes is responsible for segmental development of the female reproductive tract during embryogenesis. However, HOXA10 has been shown to be essential not only for uterus development, but also for implantation. Persistent expression and steroid-dependent regulation of this gene has been demonstrated in adult human, primate, murine and canine uteri. Moreover, HOXA10-dependent expression of prostaglandin H synthase-2 (PGHS-2), a key enzyme in prostaglandin production, has been previously detected. The role of the HOXA10 gene in the porcine uterus is not well established. Therefore, the present studies were undertaken to 1) examine the effect of E(2) and P(4) on HOXA10 mRNA and protein content in the endometrium collected on day 9 of the estrous cycle and 2) determine the PGHS-2 protein expression and PGE(2) and PGF(2α) secretion from endometrial tissue in response to steroid treatment. Endometrial explants collected from mature gilts on day 9 of the estrous cycle were incubated with E(2) (1-100 nM), P(4) (10-1000 nM) or E(2) (10 nM) and P(4) (100 nM) for 24 h. E(2) alone or E(2) in the presence of P(4) increased HOXA10 mRNA expression in the endometrium (P<0.05). The HOXA10 protein level was upregulated in response to E(2), P(4) and both steroids administered simultaneously (P<0.05). Moreover, E(2) and P(4) stimulated PGHS-2 protein expression in cultured endometrial explants. PGE(2), but not PGF(2α), secretion increased in the presence of E(2) (P<0.05). However, the release of both prostaglandins was decreased after treatment of endometrial explants with the highest dose of P(4) (P<0.01). These results demonstrate that E(2) and P(4) are important regulators of HOXA10 gene expression in the adult porcine endometrium during the mid-luteal phase of the estrous cycle. Additionally, the similar profiles of endometrial HOXA10 and PGHS-2 expression in the presence of E(2) and P(4) indicate that both genes are simultaneously regulated by steroids in the porcine uterus.


Asunto(s)
Endometrio/metabolismo , Estradiol/metabolismo , Estro/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Progesterona/metabolismo , Prostaglandinas/metabolismo , Animales , Ciclooxigenasa 2/metabolismo , Dinoprost/metabolismo , Dinoprostona/metabolismo , Femenino , Proteínas de Homeodominio/genética , Concentración Osmolar , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sus scrofa , Técnicas de Cultivo de Tejidos , Regulación hacia Arriba
20.
Theriogenology ; 74(7): 1207-20, 2010 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-20615530

RESUMEN

Seminal fluids introduced to the female reproductive tract at mating can affect subsequent events, such as ovulation, fertilization, conception, and pregnancy. Bioactive molecules present in seminal plasma can modify the cellular composition, structure, and function of local tissues and of tissues distal to the tract. The oviduct plays a decisive role in reproduction providing a beneficial milieu for gamete maturation, fertilization, and early embryonic development. Therefore we have investigated whether intrauterine infusion of seminal plasma can modulate prostaglandin (PG) synthesis in the porcine oviduct through regulation of gene and protein expression of enzymes of prostaglandin synthesis pathway. Among several enzymes involved in the prostaglandin synthesis pathway tested in the present study PGF(2α) synthase (PTGFS) and prostaglandin 9-ketoreductase (CBR1), which convert PGE(2) to PGF(2α), expression were significantly down-regulated in the oviducts on Day 1 after seminal plasma infusion into the uterine horns. The effects of the treatment were transient and by Day 5 levels of PTGFS and CBR1 were comparable in seminal plasma-treated and control animals. Additionally, increased PGE(2) to PGF(2α) and PGFM to PGF(2α) ratios in the oviductal tissues were indicated. Our results clearly demonstrate that seminal plasma affects prostaglandin synthesis in the porcine oviduct. Altered PTGFS and CBR1 expression in consequence changed PGE(2) to PGF(2α) and PGFM to PGF(2α) ratios in the porcine oviduct.


Asunto(s)
Oviductos/fisiología , Prostaglandinas/metabolismo , Semen/fisiología , Porcinos/fisiología , Animales , Femenino , Regulación de la Expresión Génica/fisiología , Masculino , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo
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