RESUMO
Mating shut down a 2-methoxyestradiol (2ME) nongenomic action necessary to accelerate egg transport in the rat oviduct. Herein, we investigated whether tumour necrosis factor-α (TNF-α) participates in this mating effect. In unmated and mated rats, we determined the concentration of TNF-α in the oviductal fluid and the level of the mRNA for Tnf-a (Tnf) and their receptors Tnfrsf1a and Tnfrsf1b in the oviduct tissues. The distribution of the TNFRSF1A and TNFRSF1B proteins in the oviduct of unmated and mated was also assessed. Finally, we examined whether 2ME accelerates oviductal egg transport in unmated rats that were previously treated with a rat recombinant TNF-α alone or concomitant with a selective inhibitor of the NF-κB activity. Mating increased TNF-α in the oviductal fluid, but Tnf transcript was not detected in the oviduct. The mRNA for TNF-α receptors as well as their distribution was not affected by mating, although they were mainly localized in the endosalpinx. Administration of TNF-α into the oviduct of unmated rats prevented the effect of 2ME on egg transport. However, the NF-κB activity inhibitor did not revert this effect of TNF-α. These results indicate that mating increased TNF-α in the oviductal fluid, although this not associated with changes in the expression and localization of TNF-α receptors in the oviductal cells. Furthermore, TNF-α mimicked the effect of mating on the 2ME-induced egg transport acceleration, independently of the activation of NF-κB in the oviduct. We concluded that TNF-α is the signal induced by mating to shut down a 2ME nongenomic action in the rat oviduct.
Assuntos
Estradiol/análogos & derivados , Tubas Uterinas/efeitos dos fármacos , Transporte do Óvulo/efeitos dos fármacos , Comportamento Sexual Animal/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , 2-Metoxiestradiol , Aceleração , Animais , Líquidos Corporais/química , Líquidos Corporais/metabolismo , Estradiol/farmacologia , Tubas Uterinas/metabolismo , Feminino , Genoma/efeitos dos fármacos , Transporte do Óvulo/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/fisiologiaRESUMO
Estradiol (E2) accelerates oviductal egg transport through nongenomic pathways involving oviductal protein phosphorylation in non-mated rats, and through genomic pathways in mated rats. Here we investigated the ability of cervico-vaginal stimulation (CVS) to switch the mode of action of E2 in the absence of other male-associated components. Pro-estrous rats were subjected to CVS with a glass rod and 12 hours later were injected subcutaneously with E2 and intrabursally with the RNA synthesis inhibitor Actinomycin D or the protein phosphorylation inhibitor H-89. The number of eggs in the oviduct, assessed 24 h later, showed that Actinomycin D, but not H-89 blocked the E2-induced egg transport acceleration. This clearly indicates that CVS alone, without other mating-associated signals, is able to shift E2 signaling from nongenomic to genomic pathways. Since mating and CVS activate a neuroendocrine reflex that causes iterative prolactin (PRL) surges, the involvement of PRL pathway in this phenomenon was evaluated. Prolactin receptor mRNA and protein expression in the rat oviduct was demonstrated by RT-PCR and Western blot, but their levels were not different on day 2 of the cycle (C2) or pregnancy (P2). Activated ST AT 5a/b (phosphorylated) was detected by Western blot on P2 in the ovary, but not in the oviduct, showing that mating does not stimulate this PRL signalling pathway in the oviduct. Other rats subjected to CVS in the evening of pro-estrus were treated with bromoergocriptine to suppress PRL surges. In these rats, H-89 did not block the E2-induced acceleration of egg transport suggesting that PRL surges are not essential to shift E2 signaling pathways in the oviduct. We conclude that CVS is one of the components of mating that shifts E2 signaling in the oviduct from nongenomic to genomic pathways, and this effect is independent of PRL surges elicited by mating.
Assuntos
Estradiol/farmacologia , Estrogênios/farmacologia , Tubas Uterinas/efeitos dos fármacos , Transporte do Óvulo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Dactinomicina/farmacologia , Estradiol/administração & dosagem , Estrogênios/administração & dosagem , Ciclo Estral , Tubas Uterinas/fisiologia , Feminino , Isoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologiaRESUMO
The oviducal transport of eggs to the uterus normally takes 72-96 h in the rat, but this is reduced to less than 20 h after a single injection of oestradiol (E2). This accelerated transport is associated with an increased frequency of pendular movements in the isthmic segment of the oviduct, with increased levels of the gap junction (GJ) component Connexin (Cx) 43, and is antagonised by progesterone (P). In the present study, we investigated the effect of these hormones on the instant and directional velocity of pendular movements and the role of the GJ and its Cx43 component in the kinetic response of the oviduct to E2 and P. Using microspheres as egg surrogates, microsphere instant velocity (MIV) was measured following treatment with E2, P or P + E2, which accelerate or delay egg transport. Microspheres were delivered into the oviduct of rats on Day 1 of pregnancy and their movement within the isthmic segment was recorded. Oestrogen increased MIV with faster movement towards the uterus. After P or P + E2, MIV was similar to that in the control group. Two GJ uncouplers, namely 18 alpha- and 18 beta-glycyrrhetinic acid, blocked the effect of E2 on MIV. Connexin 43 mRNA levels increased over that seen in control with all treatments. In conclusion, the effects of E2 on MIV resulted in faster movements that produced accelerated egg transport towards the uterus. Gap junctions are probably involved as smooth muscle synchronisers in this kinetic effect of E2, but the opposing effects of E2 and P are not exerted at the level of Cx43 transcription.
Assuntos
Estradiol/farmacologia , Tubas Uterinas/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Transporte do Óvulo/efeitos dos fármacos , Progesterona/farmacologia , Animais , Conexina 43/biossíntese , Conexina 43/genética , Tubas Uterinas/metabolismo , Feminino , Junções Comunicantes/metabolismo , Ácido Glicirretínico/farmacologia , Cinética , Masculino , Microesferas , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Desacopladores/farmacologiaRESUMO
Estradiol (E2) accelerates oviductal egg transport through nongenomic pathways involving oviductal protein phosphorylation in non-mated rats, and through genomic pathways in mated rats. Here we investigated the ability of cervico-vaginal stimulation (CVS) to switch the mode of action of E2 in the absence of other male-associated components. Pro-estrous rats were subjected to CVS with a glass rod and 12 hours later were injected subcutaneously with E2 and intrabursally with the RNA synthesis inhibitor Actinomycin D or the protein phosphorylation inhibitor H-89. The number of eggs in the oviduct, assessed 24 h later, showed that Actinomycin D, but not H-89 blocked the E2-induced egg transport acceleration. This clearly indicates that CVS alone, without other mating-associated signals, is able to shift E2 signaling from nongenomic to genomic pathways. Since mating and CVS activate a neuroendocrine reflex that causes iterative prolactin (PRL) surges, the involvement of PRL pathway in this phenomenon was evaluated. Prolactin receptor mRNA and protein expression in the rat oviduct was demonstrated by RT-PCR and Western blot, but their levels were not different on day 2 of the cycle (C2) or pregnancy (P2). Activated ST AT 5a/b (phosphorylated) was detected by Western blot on P2 in the ovary, but not in the oviduct, showing that mating does not stimulate this PRL signalling pathway in the oviduct. Other rats subjected to CVS in the evening of pro-estrus were treated with bromoergocriptine to suppress PRL surges. In these rats, H-89 did not block the E2-induced acceleration of egg transport suggesting that PRL surges are not essential to shift E2 signaling pathways in the oviduct. We conclude that CVS is one of the components of mating that shifts E2 signaling in the oviduct from nongenomic to genomic pathways, and this effect is independent of PRL surges elicited by mating.
Assuntos
Animais , Feminino , Ratos , Estradiol/farmacologia , Estrogênios/farmacologia , Tubas Uterinas/efeitos dos fármacos , Transporte do Óvulo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Dactinomicina/farmacologia , Ciclo Estral , Estradiol/administração & dosagem , Estrogênios/administração & dosagem , Tubas Uterinas/fisiologia , Isoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologiaRESUMO
Previously, we found that the dose of estradiol (E2) required to accelerate egg transport increases 5- to 10-fold, in mated compared to cyclic rats. Here we examined protein synthesis in the oviduct of mated and cyclic rats following a single injection of E2 known to accelerate oviductal egg transport or after concomitant treatment with progesterone (P4) known to block this acceleration. On Day 1 of the cycle or pregnancy, E2, P4, or E2 + P4 were injected s.c., and 4 h later oviducts were removed and incubated for 8 h in medium with 35S-methionine. Tissue proteins were separated by SDS-PAGE, and protein bands were quantitated by fluorography and densitometry. In mated rats, E2 and P4 increased different protein bands and P4 did not affect the fluorographic pattern induced by E2. In contrast with mated rats, none of these treatments changed the fluorographic pattern of the oviductal proteins in cyclic rats. Estradiol-induced egg transport acceleration was then compared under conditions in which oviductal protein synthesis was suppressed. Mated and cyclic rats treated with equipotent doses of E2 for accelerating egg transport also received actinomycin D (Act D) locally. Estradiol-induced oviductal egg loss was partially blocked by Act D in mated but had no effect in cyclic rats. We conclude that the oviduct of mated and cyclic rats differs in that only the former responds with increased protein synthesis to a pulse of exogenous E2 and P4 and requires an intact protein synthesis machinery in order to accelerate egg transport in response to E2.
Assuntos
Estradiol/farmacologia , Tubas Uterinas/efeitos dos fármacos , Transporte do Óvulo/efeitos dos fármacos , Biossíntese de Proteínas , Animais , Dactinomicina/farmacologia , Eletroforese em Gel de Poliacrilamida , Estradiol/administração & dosagem , Ciclo Estral , Tubas Uterinas/metabolismo , Feminino , Masculino , Progesterona/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
In order to explore nongenomic actions of estradiol (E2) and progesterone (P4) in the oviduct, we determined the effect of E2 and P4 on oviductal protein phosphorylation. Rats on Day 1 of the cycle (C1) or pregnancy (P1) were treated with E2, P4, or E2 + P4, and 0.5 h or 2.5 h later their oviducts were incubated in medium with 32P-orthophosphate for 2 h. Oviducts were homogenized and proteins were separated by SDS-PAGE. Following autoradiography, protein bands were quantitated by densitometry. The phosphorylation of some proteins was increased by hormonal treatments, exhibiting steroid specificity and different individual time courses. Possible mediation of the E2 effect by mRNA synthesis or protein kinases A (PK-A) or C (PK-C) was then examined. Rats on C1 treated with E2 also received an intrabursal (i.b.) injection of alpha-amanitin (Am), or the PK inhibitors H-89 or GF 109203X, and 0.5 h later their oviducts were incubated as above plus the corresponding inhibitors in the medium. Increased incorporation of 32P into total oviductal protein induced by E2 was unchanged by Am, whereas it was completely suppressed by PK inhibitors. Local administration of H-89 was utilized to determine whether or not E2-induced egg transport acceleration requires protein phosphorylation. Rats on C1 or P1 were treated with E2 s.c. and H-89 i.b. The number and distribution of eggs in the genital tract assessed 24 h later showed that H-89 blocked the E2-induced oviductal egg loss in cyclic rats and had no effect in mated rats. It is concluded that E2 and P4 change the pattern of oviductal protein phosphorylation. Estradiol increases oviductal protein phosphorylation in cyclic rats due to a nongenomic action mediated by PK-A and PK-C. In the absence of mating, this action is essential for its oviductal transport accelerating effect. Mating changes the mechanism of action of E2 in the oviduct by waiving this nongenomic action as a requirement for E2-induced embryo transport acceleration.
Assuntos
Tubas Uterinas/efeitos dos fármacos , Transporte do Óvulo/efeitos dos fármacos , Fosfoproteínas/metabolismo , Sulfonamidas , Amanitinas/farmacologia , Animais , Autorradiografia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Ciclo Estral , Tubas Uterinas/metabolismo , Feminino , Isoquinolinas/farmacologia , Cinética , Masculino , Radioisótopos de Fósforo , Fosforilação , Progesterona/farmacologia , Proteína Quinase C/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
The effect of the inhibition of nitric oxide synthase (NOS) on ovum transport and oviductal motility in rats was investigated. Three different NOS inhibitors were injected into the ovarian bursa at oestrus or day 3 of pregnancy. Oviducts and uteri were flushed 24 h later and the presence of ova was recorded. In oestrous and pregnant rats, treatment resulted in accelerated egg transport, as shown by a decrease in the number of ova present in the oviducts. In cyclic rats, intrabursal injection of 1 mg kg-1 of either N-monomethyl-L-arginine (L-NMMA) or N omega nitro-L-arginine methyl ester (L-NAME) elicited a 30% reduction in the number of ova present in the oviducts, whereas in pregnant animals, the same dose of L-NMMA produced a reduction of 40%. Simultaneous administration of the NO donor spermine NONOate (5 mg kg-1) completely reversed the effect of L-NMMA. Tubal motility was assessed by microsphere displacement analysis within the oviduct. Surrogate ova were transferred to the oviductal lumen at oestrus and 24 h later the effect of intraoviductal injection of 1 microgram L-NMMA or vehicle was assessed. The microspheres in the isthmus showed an oscillating motion, and periods in which movement was not detectable. However, L-NMMA treatment produced a 3.6-fold increase in the maximum instant velocities and a significant reduction in the resting periods of the microspheres compared with the control group (P < 0.001). These results provide evidence that NO inhibition increases tubal motility that results in accelerated ovum transport, and indicate that NO could act as a paracrine signal between different layers of the oviductal wall, providing a role for endogenous NO in regulation of tubal function.
Assuntos
Tubas Uterinas/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Transporte do Óvulo/efeitos dos fármacos , ômega-N-Metilarginina/farmacologia , Análise de Variância , Animais , Contagem de Células , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Microesferas , Músculo Liso/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Gravidez , Ratos , Ratos Wistar , Espermina/farmacologiaRESUMO
We have previously reported that a single injection of estradiol-17 beta (E2) given on day 3 of pregnancy (P3) is far more effective for accelerating oviductal transport in the rat, than treatment given on day 1 (P1). In order to quantify this change, dose-response curves were established for six different doses of E2 (range 0.031 to 1.00 micrograms per animal) given on P1, P2 or P3. In addition, a possible mechanism was explored by comparing the plasmatic and oviductal levels of E2 between 30 and 180 min following treatment with E2 on P1 or P3. As the interval from ovulation to treatment was increased, the transport of a larger number of embryos was accelerated and a smaller dose was required. The minimal effective dose decreased 30-fold from P1 to P3, the oviducts accumulated 20% to 90% more E2 on P3 than on P1, tissue levels were 6- to 48-fold higher than plasma levels and the latter did not differ between P1 and P3. It is concluded that the oviduct exhibits increased sensitivity and responsiveness to E2 on P3 and this is associated with greater accumulation of the hormone in the organ, not attributable to higher E2 plasma levels.
Assuntos
Estradiol/administração & dosagem , Transporte do Óvulo/efeitos dos fármacos , Prenhez/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Estradiol/sangue , Estradiol/farmacocinética , Feminino , Gravidez , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Distribuição TecidualRESUMO
We have previously reported that a single injection of estradiol-17 beta (E2) given on day 3 of pregnancy (P3) is far more effective for accelerating oviductal transport in the rat, than treatment given on day 1 (P1). In order to quantify this change, dose-response curves were established for six different doses of E2 (range 0.031 to 1.00 micrograms per animal) given on P1, P2 or P3. In addition, a possible mechanism was explored by comparing the plasmatic and oviductal levelsof E2 between 30 and 180 min following treatment with E2 on P1 or P3. As the interval from ovulation to treatment was increased, the transport of a larger number of embryos was accelerated and a smaller dose was required. The minimal effective dose decreased 30-fold from P1 to P3, the oviducts accumulated 20 percent to 90 percent more E2 on P3 than on P1, tissuelevels were 6- to 48-fold higher than plasma levels and the latter did not differ between P1 and P3. It is concluded that the oviduct exhibits increased sensitivity and responsiveness to E2 on P3 and this isassociated with greater accumulation of the hormone in the organ, not attributable to higher E2 plasma levels
Assuntos
Animais , Feminino , Ratos , Gravidez , Estradiol/administração & dosagem , Transporte do Óvulo/efeitos dos fármacos , Prenhez/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores de Tempo , Distribuição Tecidual , Relação Dose-Resposta a Droga , Estradiol/sangue , Estradiol/farmacocinéticaRESUMO
Administration of estradiol (E2) as a single subcutaneous injection, but not as a short intravenous infusion (less than 150 min), accelerates oviductal embryo transport in pregnant rats although the first mode determines lower E2 circulating levels. Since progesterone (P) can antagonize the effect of E2 on embryo transport we examined the circulating P levels under these two modes of E2 administration. Rats were treated on day 1 of pregnancy with 5 micrograms E2 given s.c. or i.v. (10 min infusion). Other groups were either hypophysectomized (HPX), adrenalectomized (ADX) or ovariectomized (OVX) prior to E2 treatment to prevent P rise, or were treated with E2 plus RU486 to block the action of P. Some groups were autopsied at short intervals following treatment to measure P levels and others 24 h later to assess the effect of treatments on embryo transport. P was increased several fold by i.v. infusions of E2 or vehicle alone in intact and OVX rats but not in HPX or ADX rats, whereas s.c. administration of E2 did not change P levels unless it was given concomitantly with i.v. infusion of vehicle. The short i.v. infusion of E2 accelerated embryo transport in HPX, ADX, or RU486 treated rats but not in intact rats. The s.c. injection of E2 accelerated embryo transport even when it was accompanied by an i.v. infusion of vehicle. The data does not exclude the participation of glucocorticoids in the above phenomena but agrees with the view that it is the transient increase in adrenal P secretion which blunts the oviductal response to a brief pulse of E2.