RESUMO
Aluteal cycles were induced in the mare to evaluate the effects of progesterone deprivation on the gene expression of embryos and endometrium collected eight days after ovulation. We hypothesized that the transcript expression would be altered during induced aluteal (AL) cycles (low progesterone <1â¯ng/mL) when compared with control cycles during diestrus (high progesterone; > 4â¯ng/mL) for 1) the embryonic expression of progesterone-mediated transcripts and those related to normal embryo growth and development and 2) the endometrial expression of progesterone-mediated transcripts and those related to prostaglandin synthesis and normal pregnancy establishment. Seven cyclic mares with a median age of 6.5 years (range 3-16) were utilized in a crossover design. Mares in estrus were artificially inseminated to a fertile stallion and randomly assigned to control or AL groups. Mares received either saline solution (control mares) or PGF2α (AL mares), twice daily on days 0, 1, and 2 and once daily on days 3 and 4. Serial blood samples were collected daily from day 0 (ovulation) until the day of embryo collection and endometrial biopsy on day 8. Mares were monitored until they returned to estrus, and artificially inseminated. Mares were switched to the opposite treatment group only after a successful embryo collection occurred during the previous cycle and only cycles that produced embryos were used for analyses. The study design resulted in paired samples from each mare for analyses. No significant rise in progesterone was observed in the AL group with mean concentrations of plasma progesterone remaining <1.0â¯ng/mL from ovulation until embryo collection on day 8. This is in sharp contrast to the control (luteal) cycle where a post-ovulatory rise in plasma progesterone was observed. Real-time RT-PCR was utilized to evaluate the expression of ESR1, PGR, CYP19A1, P19, SLC35A1, OCD, APOB, AQP3, NEU2 transcripts in the embryos and PTGS2, P19, ESR1, HK2, sPLA2, PGR, CTGF, IFNE, FGF9, SLC36A2 expression in the endometrium. Four transcripts showed increased expressed in embryos developed during AL cycles ESR1, P19, APOB and PGR (pâ¯<â¯0.05). Four transcripts showed increased expressed in endometrium developed during AL cycles sPLA2, PGR, ESR1, FGF9 (pâ¯<â¯0.05) and four transcripts showed decreased expression P19, CTGF, IFNE, HK2 (pâ¯<â¯0.05). Additionally, staining differences were present in endometrial staining for both ERα and PR receptor during AL cycles compared with control cycles. Embryos and endometrium developed in a progesterone-deprived environment during induced aluteal cycles demonstrated altered transcript expression. These results indicate that adequate progesterone levels may be a key mediator of the appropriate embryo-maternal environment during early preimplantation embryo development.
Assuntos
Embrião de Mamíferos/metabolismo , Endométrio/metabolismo , Cavalos/embriologia , Animais , Desenvolvimento Embrionário , Ciclo Estral , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cavalos/metabolismo , Inseminação Artificial/veterinária , Progesterona/fisiologiaRESUMO
The present experiment was divided into three studies to investigate the effect of age and endometrial degeneration on uterine blood flow of mares. In study 1, the influence of semen infusion and conception was evaluated using noninseminated (non-AI), inseminated nonpregnant (AI-NP), and inseminated pregnant (AI-P) mares (n = 7 mares/group). In study 2, the effect of age was investigated using young (≤6 years), adult (from 8 to 12 years), and old (≥15 years) pregnant mares (n = 7 mares/group). In study 3, uterine blood flow was also characterized in pregnant mares (n = 7 mares/group) with minimal, moderate, or severe endometrial degenerative changes (GI, GII, and GIII, respectively). Uterine vascular perfusion and pulsatility index from mesometrium attachment arteries were recorded daily from Day 0 (day of ovulation) to Day 12 and between Days 0 and 20 in nonpregnant and pregnant mares. Analysis according to the position of the embryo (uterine horn with embryo vs. opposite horn) was performed from Day 12 until Day 20. In study 1, increased uterine vascular perfusion and decreased pulsatility index were detected (P < 0.001) between Days 3 and 5 in non-AI and AI-P mares, whereas the uterine vascular perfusion of AI-NP mares did not change (P > 0.05) throughout the experiment. In study 2, the vascular perfusion of the uterine horn with embryo was higher (P < 0.001) than in the opposite uterine horn from Day 12 until Day 20 in both young and adult mares. With exception of Day 15, both uterine horns of old mares showed similar (P > 0.1) vascular perfusion from Day 12. In study 3, vascular perfusion of both uterine horns was lower (P < 0.001) in GIII mares than in GI and GII mares. After Day 15, the scores for uterine vascular perfusion were consistently greater (P < 0.001) in the uterine horn with embryo than in the opposite horn in GI mares. Results showed, for the first time, the reduced blood flow of the gravid uterus during early gestation in older mares and in mares with significant endometrial degeneration.
Assuntos
Envelhecimento/fisiologia , Endométrio/fisiologia , Idade Gestacional , Cavalos/fisiologia , Útero/irrigação sanguínea , Animais , Velocidade do Fluxo Sanguíneo , Feminino , Inseminação Artificial/veterinária , Gravidez , Fluxo Pulsátil , Estudos Retrospectivos , Ultrassonografia , Útero/diagnóstico por imagemRESUMO
Glycogen storage disease type Ia (GSD-Ia) stems from glucose-6-phosphatase (G6Pase) deficiency and causes hypoglycemia, hepatomegaly, hypercholesterolemia and lactic acidemia. Three dogs with GSD-Ia were initially treated with a helper-dependent adenovirus encoding a human G6Pase transgene (HDAd-cG6Pase serotype 5) on postnatal day 3. Unlike untreated dogs with GSD-Ia, all three dogs initially maintained normal blood glucose levels. After 6-22 months, vector-treated dogs developed hypoglycemia, anorexia and lethargy, suggesting that the HDAd-cG6Pase serotype 5 vector had lost efficacy. Liver biopsies collected at this time revealed significantly elevated hepatic G6Pase activity and reduced glycogen content, when compared with affected dogs treated only by frequent feeding. Subsequently, the HDAd-cG6Pase serotype 2 vector was administered to two dogs, and hypoglycemia was reversed; however, renal dysfunction and recurrent hypoglycemia complicated their management. Administration of a serotype 2 HDAd vector prolonged survival in one GSD-Ia dog to 12 months of age and 36 months of age in the other, but the persistence of long-term complications limited HDAd vectors in the canine model for GSD-Ia.
Assuntos
Doenças do Cão/terapia , Terapia Genética/métodos , Glucose-6-Fosfatase/genética , Doença de Depósito de Glicogênio Tipo I/terapia , Adenoviridae/genética , Animais , Peso Corporal , Cães , Terapia Genética/efeitos adversos , Vetores Genéticos , Doença de Depósito de Glicogênio Tipo I/veterinária , Hipoglicemia/complicações , Hipoglicemia/prevenção & controleRESUMO
In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.
Assuntos
Gonadotropina Coriônica/administração & dosagem , Líquido Folicular/química , Cavalos/metabolismo , Óxido Nítrico/análise , Animais , Inibidores Enzimáticos/farmacologia , Estradiol/análise , Feminino , Guanidinas/farmacologia , Cinética , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ovulação , Progesterona/análiseRESUMO
Oxytocin is released in response to teasing during both estrus and diestrus in mares, and at least during estrus, teasing results in an increase in electromyographic activity in the uterus. Exogenous oxytocin causes an increase in intrauterine pressure and prior studies have shown that this response is correlated to the day of the estrous cycle. To determine if teasing causes an increase in intrauterine pressure and if this response varies by day of the cycle, intrauterine pressure was measured while mares were teased with a stallion 2 days before ovulation, on the day ovulation was detected and 2 days after ovulation. A significant increase in intrauterine pressure was observed in response to teasing both 2 days before ovulation and on the day of ovulation, when plasma concentrations of progesterone were low. No significant increase in intrauterine pressure was observed in response to teasing 2 days after ovulation when progesterone concentrations were elevated. Management practices that include teasing or stallion exposure may be beneficial in stimulating uterine clearance mechanisms in mares during the preovulatory period.
Assuntos
Ciclo Estral/fisiologia , Cavalos/fisiologia , Comportamento Sexual , Útero/fisiologia , Animais , Eletromiografia , Estradiol/sangue , Feminino , Masculino , Ovulação , Pressão , Progesterona/sangue , Fatores de Tempo , Contração UterinaRESUMO
Recent studies suggest that nitric oxide (NO) may have a role in regulating ovarian physiology. To investigate the role of NO during ovulation in mares, inhibitors of the nitric oxide synthase (NOS) were administered to estrous mares. Forty cycling mares (20 horses and 20 pony mares) were allotted to one of the three treatment groups. Once a follicle was at least 27 mm in diameter, but smaller than 35 mm, mares were given one of the following treatments: saline solution 0.9% (n = 20, w/v, i.v., every 12 h), Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME; n = 10, 148 micromol/kg, i.v., every 12 h), or aminoguanidine hemisulfate (AG; n = 10, 406 micromol/kg, i.v., every 12 h). When a follicle >30 mm was present on one of the ovaries, ovulation was induced with hCG (2,500 IU, i.v.). The median time of ovulation (+/-6 h) after hCG administration for the treatment groups was 42, 84 and 54 h for mares treated with saline solution, L-NAME and AG, respectively. There was no significant difference between the groups treated with AG or L-NAME (P = 0.06); however, these groups were different from the control group (P < 0.05). The delayed ovulation caused by the administration of NOS inhibitors suggests a role for NO in follicular growth and ovulation in horses.
Assuntos
Gonadotropina Coriônica/farmacologia , Inibidores Enzimáticos/farmacologia , Cavalos/fisiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ovulação/efeitos dos fármacos , Animais , Estro , Feminino , Guanidinas/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Folículo Ovariano/anatomia & histologia , Progesterona/sangueRESUMO
The effect of phorbol 12-myristate-13-acetate (PMA), a tumor-promoting phorbol ester, on the synthesis of proteoglycans of endothelial cells in culture was investigated. This phorbol activates protein kinase C (PKC) when added to cells in culture. PKC, in turn, modulates the activity of growth factors. Using [35S]-sulfate or [3H]-glucosamine to label the proteoglycans we have observed a 4-24-fold increase of the heparan sulfate (HS) synthesis in a dose-dependent manner (0-100 ng/ml). Chondroitin sulfate (CS) synthesis was not affected by PMA. The effect of PMA could be completely abolished by a calcium ionophore (A23187). By the use of synchronized cells and PMA pulses at different periods of the cell cycle, as well as [3H]-thymidine incorporation, we were able to show that the enhancement of heparan sulfate synthesis is most prominent during G1. Our data suggest that the release of HS to the medium could be one of the responses of the cell to a mitogenic stimulus.
Assuntos
Fase G1/efeitos dos fármacos , Heparitina Sulfato/biossíntese , Proteoglicanas/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Animais , Calcimicina/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Glicosaminoglicanos/biossíntese , Proteoglicanas de Heparan Sulfato , Coelhos , Fatores de TempoRESUMO
The effect of phorbol 12-myristate-13-acetate (PMA), a tumor-promoting phorbol ester, on the synthesis of proteoglycans of endothelial cells in culture was investigated. This phorbol activates protein kinase C (PKC) when added to cells in culture. PKC, in turn, modulates the activity of growth factors. Using [35S]-sulfate or [3H]-glucosamine to label the proteglycans we have observed a 4-24-fold increase of the heparan sulfate (HS) synthesis in a dose-dependent manner (0-100 ng/ml). Chondroitin sulfate (CS) synthesis was not affected by PMA. The effect of PMA could be completely abolished by a calcium ionophore (A23187). By the use of synchronized cells and PMA pulses at different periods of the cell cycle, as well as [3H]-thymidine incorporation, we were able to show that the enhancement of heparan sulfate synthesis is most prominent during G1. Our data suggest that the release of HS to the medium could be one of the responses of the cell to a mitogenic stimulus