RESUMEN
The aim of this study was to evaluate the role of prorenin/(pro)renin receptor activation on luteal progesterone (P4) secretion. Our hypothesis was that the nonproteolytic activation of (pro)renin receptor [P(RR)] is part of the regulatory mechanism responsible for corpus luteum (CL) function. In the first three experiments, prorenin was found to stimulate the production of P4, which is not inhibited by an angiotensin receptor antagonist (saralasin), but rather by a renin/prorenin inhibitor (aliskiren), a MAPK1/3 inhibitor (PD325901) or an EGFR inhibitor (AG1478), which are evidence of nonproteolytic activation of prorenin. Moreover, prorenin induced phosphorylation of MAPK1/3 in luteal cells. Following these in vitro experiments, a sequence of in vivo experiments was performed demonstrating that the intrafollicular injection of aliskiren in preovulatory follicles impaired P4 secretion in cows that ovulated. Furthermore, all profibrotic genes studied were present in the CL and TGFB1 and FN1 mRNA were upregulated from day 5-10 post-ovulation. During luteolysis, REN was downregulated at 48 h, whereas TGFB1 and SERPINE1 were dramatically upregulated in luteal tissue at 12 h after PGF. In summary, these data are evidence that nonproteolytic activation of (P)RR is involved in luteal function.
Asunto(s)
Células Lúteas , Renina , Animales , Bovinos , Cuerpo Lúteo/fisiología , Dinoprost/farmacología , Femenino , Luteólisis , Progesterona/farmacología , Renina/genéticaRESUMEN
The objective of this study was to investigate the effects of inhibiting the epidermal growth factor receptor (EGFR) pathway on meiosis blockage and resumption, mRNA expression of genes involved in oocyte maturation and cumulus expansion, and embryo development. Bovine cumulus-oocyte complexes (COCs) were cultured for 15 h in the presence of the EGFR inhibitor (AG1478) and follicular hemisections (FHS). Most of the oocytes (89.3%) remained at the germinal vesicle (GV) stage when cultured in the presence of FHS and 5 µM AG1478. The inhibitory effect was reversible as most oocytes (83.8%) completed meiosis after additional 20 h maturation. Embryo development to the blastocyst stage was similar (P > 0.05) between FHS and 5 µM AG1478 treated (39.3%) and control (41.1%) groups. In cumulus cells, mRNA abundance of early growth response protein 1 (EGR1), tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) genes, and phosphorylated extracellular regulated kinase (p-ERK1/2) protein were lower in COCs treated with AG1478 plus FHS compared with FHS alone (P < 0.05). In granulosa cells of FHS, AG1478 treatment reduced transcript levels of PGR and ADAMTS1 (P < 0.05). The inhibitory effect of AG1478 on meiotic progression was not reverted by treatment with angiotensin II (ANG II) or prostaglandins (PGF2α or PGE2). This study demonstrates that inhibition of EGFR in the presence of FHS is a reliable approach to promote reversible arrest of bovine oocytes at the GV stage.
Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/efectos de los fármacos , Folículo Ovárico , Quinazolinas/farmacología , Tirfostinos/farmacología , Angiotensina II/farmacología , Animales , Bovinos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Células del Cúmulo , Dinoprostona/farmacología , Técnicas de Cultivo de Embriones/métodos , Técnicas de Cultivo de Embriones/veterinaria , Femenino , Hormona Folículo Estimulante/farmacología , Células de la Granulosa , Técnicas de Maduración In Vitro de los Oocitos/métodos , Meiosis/efectos de los fármacos , Oocitos/fisiología , Quinazolinas/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Tirfostinos/administración & dosificaciónRESUMEN
The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (P<0.05). To determine the involvement of angiotensin II in prorenin-induced meiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200µM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (P<0.05). Only the oocytes' cyclic adenosine monophosphate levels seemed to be regulated by prorenin and/or forskolin treatment after incubation for 6h. To the best of our knowledge, this is the first study to identify the (pro)renin receptor in ovarian cells and to demonstrate the independent role of prorenin in the resumption of oocyte meiosis in cattle.
Asunto(s)
Cuerpo Lúteo/trasplante , Meiosis/fisiología , Ovario/fisiología , Receptores de Superficie Celular/metabolismo , Renina/metabolismo , Reproducción/fisiología , Amidas/farmacología , Angiotensina II/metabolismo , Animales , Bovinos , Células Cultivadas , Colforsina/farmacología , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/fisiología , Células del Cúmulo/citología , Células del Cúmulo/efectos de los fármacos , Células del Cúmulo/fisiología , Femenino , Fumaratos/farmacología , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/fisiología , Humanos , Meiosis/efectos de los fármacos , Nucleótidos Cíclicos/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Folículo Ovárico/citología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Ovario/citología , Ovario/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Renina/antagonistas & inhibidores , Renina/genética , Reproducción/efectos de los fármacos , Saralasina/farmacología , Células Tecales/citología , Células Tecales/efectos de los fármacos , Células Tecales/fisiología , Receptor de ProreninaRESUMEN
Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n=29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n=9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1+TAI, n=9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI+NC-1, n=11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Student's t-test. At 35 days of gestation the pregnancy rates in the group NC-1+TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P<0.05). At 60 days, the pregnancy rates in the NC-1+TAI group (0/4, 0%) was lower compared to TAI+NC-1 (5/7, 71.4%) and control (6/8, 75.0%) groups (P<0.05). Animals from the group NC-1+TAI were re-inseminated 60 days after the first TAI. After pregnancy losses throughout the study, 5 animals (TAI), 3 animals (NC-1+TAI) and 5 animals (TAI+NC-1) maintained pregnancy until 170 days of gestation. TaqMan RT-PCR demonstrated the presence of N. caninum DNA in the medulla and right posterior cortex in 3 out of 5 fetuses from the TAI+NC-1 group. We concluded that heifers infected after TAI had a higher incidence of the parasite at the fetus CNS. Identification of N. caninum by TaqMan RT-PCR would assist in the investigation of infection and in the evaluation of vaccines or therapeutic drugs to control neosporosis in cattle.