RESUMO
The present research used immunohistochemistry to analyse the detection and localisation of nitric oxide synthase (NOS) isoforms in the ductuli efferentes and epididymis of prepubertal and adult alpaca. In the ductuli efferentes and epididymis of prepubertal and adult animals, nNOS and eNOS were similarly expressed in epithelial lining cells, conversely differences were observed in the immunopresence of iNOS. Our data provide evidence that NOS isoforms may have roles in reproductive functions and in the developmental processes of the excurrent duct system in the alpaca.
RESUMO
The aim of the present study was to evaluate: (1) the presence of nerve growth factor (NGF), neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR) in the rabbit uterus; and (2) the in vitro effects of NGF on PGF2α and PGE2 synthesis and on the PGE2-9-ketoreductase (PGE2-9-K) activity by the rabbit uterus. Nerve growth factor, NTRK1, and NGFR were immunolocalized in the luminal and glandular epithelium and stroma cells of the endometrium. reverse transcriptase polymerase chain reaction indicated the presence of messenger RNA for NGF, NTRK1, and NGFR in the uterus. Nerve growth factor increased (P < 0.01) in vitro secretions of PGF2α and PGE2 but coincubation with either NTRK1 or oxide nitric synthase (NOS) inhibitors reduced (P < 0.01) PGF2α production and blocked (P < 0.01) PGE2 secretion. Prostaglandins releases were lower (P < 0.01) than control when uterine samples were treated with NGF plus cyclooxygenase inhibitor. However, addition of NGFR inhibitor reduced (P < 0.01) PGF2α secretion less efficiently than NTRK1 or NOS inhibitors but had no effect on PGE2 yield. Nerve growth factor increased (P < 0.01) the activity of PGE2-9-K, whereas coincubation with NTRK1 or NOS inhibitors abolished (P < 0.01) this increase in PGE2-9-K activity. However, cotreatment with either cyclooxygenase or NGFR inhibitors had no effect on PGE2-9-K activity. This is the first study to document the distribution of NGF/NTRK1 and NGFR systems and their effects on prostaglandin synthesis in the rabbit uterus. NGF/NTRK1 increases PGF2α and PGE2 productions by upregulating NOS and PGE2-9-K activities, whereas NGF/NGFR augments only PGF2α secretion, through an intracellular mechanism that is still unknown.
Assuntos
Expressão Gênica , Fator de Crescimento Neural/genética , Prostaglandinas/biossíntese , Coelhos/metabolismo , Receptores de Fator de Crescimento Neural/genética , Útero/metabolismo , Animais , Dinoprosta/biossíntese , Feminino , Hidroxiprostaglandina Desidrogenases/metabolismo , Imuno-Histoquímica , Fator de Crescimento Neural/análise , Fator de Crescimento Neural/farmacologia , RNA Mensageiro/análise , Receptor trkA/análise , Receptor trkA/genética , Receptor trkA/fisiologia , Receptores de Fator de Crescimento Neural/análise , Receptores de Fator de Crescimento Neural/fisiologia , Útero/químicaRESUMO
The aim of this study was to elucidate the possible direct regulatory role of the endocannabinoids in the modulation of LH secretion in rabbits, a reflex ovulator species. The cannabinoid receptor type 1 (CB1) was characterized by RT-PCR techniques in the anterior pituitary of intact and ovariectomized does treated with GnRH and primed with estrogen and CB1 antagonist, rimonabant. Cannabinoid receptor type 1 immune reaction was evidenced by immunohistochemistry in the cytoplasm of approximately 10% of the pituitary cells with a density of 8.5 ± 1.9 (per 0.01 mm(2)), both periodic acid-Schiff positive (30%) and negative (70%). All CB1-immunoreactive cells were also immune reactive for estrogen receptor type 1. Ovariectomy, either alone or combined with estrogen priming, did not modify the relative abundances of pituitary CB1 mRNA, but decreased (P < 0.01) the expression of estrogen receptor type 1 mRNA. Treatment with CB1 antagonist (rimonabant) inhibited (P < 0.01) LH secretory capacity by the pituitary after GnRH injection, and estrogen priming had no effect. The present findings indicate that the endocannabinoid system is a potential candidate for the regulation of the hypothalamic-pituitary-ovarian axis in reflex ovulatory species.
Assuntos
Hormônio Luteinizante/metabolismo , Hipófise/fisiologia , Coelhos/fisiologia , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/fisiologia , Animais , Antagonistas de Receptores de Canabinoides/farmacologia , Receptor alfa de Estrogênio/análise , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Expressão Gênica , Hormônio Liberador de Gonadotropina/farmacologia , Masculino , Ovariectomia , Piperidinas/farmacologia , Hipófise/química , Hipófise/efeitos dos fármacos , Pirazóis/farmacologia , RNA Mensageiro/análise , Receptor CB1 de Canabinoide/análise , RimonabantoRESUMO
Immunopresence, gene expression, and enzymatic activity of cyclooxygenase 1 (COX1), COX2, PGE2-9-ketoreductase (PGE2-9-K), endothelial (eNOS), and inducible nitric oxide synthases (iNOS), and hormone in vitro production were examined in early, mid, late, and regressive buffalo corpora lutea (CL). COX1 immunosignals were detected in the cytoplasm of small luteal cells, COX2 in large luteal cells, and PGE2-9-K in all luteal cells. COX2 and PGE2-9-K immunosignals were greater in late CL. Immunopresence of both NOS types were evidenced in the nuclei and cytoplasm of all luteal cells, as well as in the nuclei of endothelial cells, during all stages studied. The eNOS and iNOS immunosignals increased during the early stage. COX1 transcripts were lower in late and regressive CL, COX2 in late, PGE2-9-K higher in regressive, and iNOS higher in early and lower in regressive CL. COX1 enzymatic activity was lower in regressive CL, COX2 increased in mid and late stages, and PGE2-9-K was higher in late CL. Endothelial NOS activity was higher during mid and late stages and lower in regressive, whereas iNOS was greater in late and lower in early. Progesterone in vitro release was higher in mid and lower in late phase, while PGF2α synthesis was higher in late CL and lower in regressive, and PGE2 was higher during regressive stage. These results support the idea that COX, NOS, and PGE2-9-K regulate buffalo CL life span. In particular, regressive CL seems involved in the development of the contralateral early CL, through the production of the luteotrophic PGE2.
Assuntos
Búfalos/fisiologia , Corpo Lúteo/enzimologia , Diestro , Expressão Gênica , Hidroxiprostaglandina Desidrogenases/análise , Óxido Nítrico Sintase/análise , Prostaglandina-Endoperóxido Sintases/análise , Animais , Núcleo Celular/química , Citoplasma/química , Feminino , Perfilação da Expressão Gênica , Hidroxiprostaglandina Desidrogenases/genética , Imuno-Histoquímica , Microscopia , Óxido Nítrico Sintase/genética , Prostaglandina-Endoperóxido Sintases/genéticaRESUMO
The main objective of this study was to examine the modulatory in vitro effects of gonadotropin-releasing hormone (GnRH) on isolated Leydig cells of adult alpaca (Lama pacos) testis. We first evaluated the presence of GnRH receptor (GnRHR) and cyclooxygenase (COX) 1 and COX2 in alpaca testis. We then studied the in vitro effects of buserelin (GnRH analogue), antide (GnRH antagonist), and buserelin plus antide or inhibitor of phospholipase C (compound 48/80) and COXs (acetylsalicylic acid) on the production of testosterone, PGE(2), and PGF(2α) and on the enzymatic activities of COX1 and COX2. Immunoreactivity for GnRHR was detected in the cytoplasm of Leydig cells and in the acrosomal region of spermatids. COX1 and COX2 immunosignals were noted in the cytoplasm of spermatogonia, spermatocytes, spermatids, Leydig cells, and Sertoli cells. Western blot analysis confirmed the GnRHR and COX1 presence in alpaca testis. The in vitro experiments showed that buserelin alone increased (P < 0.01) and antide and buserelin plus acetylsalicylic acid decreased (P < 0.01) testosterone and PGF(2α) production and COX1 activity, whereas antide and compound 48/80 counteracted buserelin effects. Prostaglandin E(2) production and COX2 activity were not affected by buserelin or antide. These data suggest that GnRH directly up-regulates testosterone production in Leydig cells of adult alpaca testis with a postreceptorial mechanism that involves PLC, COX1, and PGF(2α).
Assuntos
Camelídeos Americanos/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/biossíntese , Testosterona/biossíntese , Animais , Busserrelina/farmacologia , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/análise , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/biossíntese , Dinoprostona/biossíntese , Hormônio Liberador de Gonadotropina/análogos & derivados , Células Intersticiais do Testículo/química , Células Intersticiais do Testículo/metabolismo , Masculino , Oligopeptídeos/farmacologia , Prostaglandina-Endoperóxido Sintases/análise , Receptores LHRH/análise , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
The dynamic expression for estrogen receptor subtype-1 (ESR1), interleukin-1beta (IL1B), and apoptosis-associated genes, as well as nitric oxide synthase activity, were examined in corpora lutea (CL) of rabbits after prostaglandin F(2alpha) (PGF(2alpha)) administration on either day 4 or day 9 of pseudopregnancy. By reverse transcriptase polymerase chain reaction, the steady-state level of ESR1 transcript was lower (P < 0.01) and that of anti-apoptotic B-cell CLL/lymphoma 2 (BCL2) -like 1 (BCL2L1) was greater in day 4 (P < 0.01) than in day 9 CL. Western blot analysis revealed that BCL2-associated X protein (BAX) abundance was greater in day 4 (P < 0.01) than in day 9 CL, whereas BCL2L1 protein was undetectable at both luteal stages. After PGF(2alpha), ESR1 transcript decreased (P < 0.01) in day 9 CL, whereas IL1B mRNA showed a transitory increase (P < 0.01) at both stages. The pro-apoptotic tumor protein p53 (TP53) gene had diminished (P < 0.01) on day 4 and on day 9 after a transitory increase (P < 0.01), whereas the BAX/BCL2L1 expression ratio increased (P < 0.01) in day 9 CL 24 h after treatment. Following PGF(2alpha), TP53 protein increased (P < 0.01) at both luteal stages, and BAX decreased (P < 0.01) in day 4 CL but increased (P < 0.01) 24 h later in day 9 CL; BCL2L1 became detectable 6 h later in day 4 CL. Nitric oxide synthase activity temporarily increased (P < 0.01) following PGF(2alpha). These findings suggest that PGF(2alpha) regulates luteolysis by ESR1 mRNA down-regulation and modulation of pro- and anti-apoptotic pathways in CL that have acquired a luteolytic capacity.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Dinoprosta/fisiologia , Receptor alfa de Estrogênio/metabolismo , Interleucina-1/metabolismo , Luteólise/metabolismo , Pseudogravidez/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Corpo Lúteo/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Regulação da Expressão Gênica/fisiologia , Interleucina-1/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , RNA Mensageiro/análise , Coelhos , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2alpha synthesis following PGF2alpha treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P < 0.01) 4- to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P < 0.01) COX-2 and PGE2-9-K basal activities, and PGF2alpha synthesis rate, but higher (P < 0.01) PGE2 production. Independent of luteal stage, PGF2alpha treatment did not affect COX-1 activity. In day-4 CL, PGF2alpha induced an increase (P < 0.01) in both COX-2 activity and PGF2alpha synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2alpha up-regulated (P < 0.01) both COX-2 and PGE-9-K activities, and PGF2alpha production, but decreased (P < 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2alpha challenge and were more marked in day-9 CL. Our data suggest that PGF2alpha directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2alpha synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2alpha in rabbits.
Assuntos
Corpo Lúteo/metabolismo , Dinoprosta/farmacologia , Dinoprostona/biossíntese , Luteólise , Pseudogravidez/metabolismo , Regulação para Cima , Animais , Corpo Lúteo/efeitos dos fármacos , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 2/biossíntese , Dinoprosta/biossíntese , Dinoprostona/análise , Feminino , Expressão Gênica/efeitos dos fármacos , Hidroxiprostaglandina Desidrogenases/análise , Hidroxiprostaglandina Desidrogenases/metabolismo , Progesterona/análise , RNA Mensageiro/análise , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
In this study, we have examined the presence and the distribution of receptors for leptin (Ob-R) in the oviduct of rabbits, and the effects of leptin on the release of prostaglandin (PG) F2alpha and PGE2 and on the activity of nitric oxide (NO) synthase (NOS) by oviducts cultured in vitro. Rabbits were killed during the follicular phase and the oviducts were incubated in vitro with leptin, PGF2alpha, PGE2, NO donor and inhibitors of NOS and cyclo-oxigenase (COX). Using immunohistochemistry, Ob-R-like positive reaction was observed only in the cytoplasm of secretory cells, having stronger intensity in the infundibulum and ampulla tracts than in the isthmus. Both leptin and NO donor inhibited PGE2 release, whereas they enhanced PGF2alpha release; NOS inhibitor alone or with leptin increased PGE2 and decreased PGF2alpha production; NOS activity was enhanced by leptin, while PGs did not affect this enzyme. This study suggests that the oviduct could be a potential target for endocrine regulation by leptin, whose circulating levels may act as a metabolic signal modulating oviductal PG release through mediation of the NOS/NO system.
Assuntos
Leptina/farmacologia , Óxido Nítrico Sintase/metabolismo , Oviductos/metabolismo , Prostaglandinas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Aspirina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Feminino , Imuno-Histoquímica/métodos , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroprussiato/farmacologia , Técnicas de Cultura de Órgãos , Oviductos/química , Coelhos , Radioimunoensaio , Receptores de Superfície Celular/análise , Receptores para LeptinaRESUMO
Total activity of nitric oxide synthase (NOS) and the gene expression of both endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms in corpora lutea of pseudopregnant rabbits were examined during prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis. Corpora lutea were collected at 0, 6, 12, 24 and 48 h after an injection of PGF(2alpha) at day 9 of pseudopregnancy. At 12 h after PGF(2alpha) administration, luteal mRNA encoding eNOS decreased (PAssuntos
Corpo Lúteo/metabolismo
, Dinoprosta/farmacologia
, Luteólise/efeitos dos fármacos
, Óxido Nítrico Sintase/metabolismo
, Óxido Nítrico/fisiologia
, Animais
, Western Blotting/métodos
, Corpo Lúteo/química
, Inibidores Enzimáticos/farmacologia
, Feminino
, NG-Nitroarginina Metil Éster/farmacologia
, Óxido Nítrico Sintase/antagonistas & inibidores
, Óxido Nítrico Sintase/genética
, Óxido Nítrico Sintase Tipo II
, Óxido Nítrico Sintase Tipo III
, Progesterona/análise
, Progesterona/sangue
, Pseudogravidez/metabolismo
, RNA Mensageiro/análise
, Coelhos
, Reação em Cadeia da Polimerase Via Transcriptase Reversa
RESUMO
Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P=0.05) by day 13. By contrast, luteal eNOS proteins increased 2-fold (P=0.05) from the early- to late-luteal phase. Independently of CL age, iNOS mRNA was very poorly expressed while protein levels gradually declined from the early- to late-luteal stage. Intense eNOS-like immunoreactivity was detected in large luteal cells, while iNOS staining was targeted to a few, isolated cells, probably macrophages. Basal NOS activity was greater in day 4 CL than in both day 9 and day 13 CL. These data are the first to characterize in rabbit CL the temporal expression patterns of NOS isoforms across different luteal stages of pseudopregnancy and, collectively, suggest the existence of an expressional control for this constitutive isoform, which might have a physiological role in regulating CL function during development.
Assuntos
Corpo Lúteo/enzimologia , Óxido Nítrico Sintase/análise , Pseudogravidez/enzimologia , Análise de Variância , Animais , Western Blotting/métodos , Feminino , Imuno-Histoquímica/métodos , Modelos Animais , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Progesterona/sangue , RNA Mensageiro/análise , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Angiotensin converting enzyme (ACE) was demonstrated to modulate the production of 17beta-estradiol, progesterone and prostaglandin E2 (PGE2) in frog ovary of Rana esculenta. However, the activity was not mediated by angiotensin II (Ang II). In an attempt to identify the peptide involved in the pathway modulated by ACE, bradykinin, another physiological substrate of ACE, was chosen and incubated in the presence of the membrane suspension purified from the frog ovary homogenate. The hydrolytic products were analysed by reverse-phase high-pressure liquid chromatography (HPLC) analysis and the results showed that bradykinin was metabolized by membrane suspension. The presence of the protease inhibitors in the incubation mixture indicated ACE and neutral endopeptidase as being responsible for the bradykinin hydrolysis. Frog ovary was incubated in vitro in the presence of bradykinin (10 microM), bradykinin receptor antagonist NPC 567 (1 mg mL-1), bradykinin fragment (1-7) (10 microM), ACE (2.5 mU mL-1), captopril (0.1 mM) and lisinopril (0.1 mM). The results showed no modulating activity by bradykinin on ovarian 17beta-estradiol and PGE2 production, thus demonstrating that it was not involved in the ACE-modulated pathway.
Assuntos
Bradicinina/metabolismo , Ovário/metabolismo , Peptidil Dipeptidase A/metabolismo , Prostaglandinas/biossíntese , Rana esculenta/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Bradicinina/farmacologia , Antagonistas dos Receptores da Bradicinina , Extratos Celulares/farmacologia , Cromatografia Líquida de Alta Pressão , Dinoprostona/biossíntese , Estradiol/biossíntese , Feminino , Hidrólise/efeitos dos fármacos , Ovário/citologia , Ovário/efeitos dos fármacos , Peptidil Dipeptidase A/isolamento & purificação , Peptidil Dipeptidase A/farmacologia , Receptores da Bradicinina/metabolismo , Fatores de TempoRESUMO
Studies were conducted to characterize receptors for prostaglandin (PG) F(2alpha) (PGF(2alpha)) and PGE(2), and the signalling pathways regulating total nitric oxide synthase activity and progesterone production in rabbit corpora lutea (CL) of different luteal stages. CL were obtained at days 4, 9 and 13 of pseudopregnancy and cultured in vitro for 2 h with PGF(2alpha) or PGE(2) and with activators and inhibitors of G protein (Gp), phospholipase C (PLC), protein kinase C (PKC), adenylate cyclase (AC) and protein kinase A (PKA). High affinity PGF(2alpha) receptor (K(d)=1.9+/-0.6 nM mean+/-s.e.m. ) concentrations increased (P< or =0.01) four- to five-fold from early to mid- and late-luteal phases (50.6+/-8.5, 188.3+/-36.1 and 231.4+/-38.8 fmol/mg protein respectively). By contrast, PGE(2) receptor (K(d)=1.6+/-0.5 nM) concentrations decreased (P< or =0.01) from day 4 to day 9 and 13 (27.5+/-7.7, 12.4+/-2.4 and 16.5+/-3.0 fmol/mg protein respectively). The Gp-dependent AC/PKA pathway was triggered only on day 4 CL, mimicking the PGE(2) treatment and increasing progesterone production. In both day 9 and day 13 CL, the Gp-activated PLC/PKC pathway evoked a luteolytic effect similar to that induced by PGF(2alpha). The time-dependent selective resistance to PGF(2alpha) and PGE(2) by rabbit CL is mediated by factors other than a lack of luteal receptor-ligand interactions.
Assuntos
Corpo Lúteo/metabolismo , Dinoprosta/farmacologia , Proteínas de Ligação ao GTP/farmacologia , Fase Luteal/metabolismo , Receptores de Prostaglandina/metabolismo , Transdução de Sinais/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenilil Ciclases/farmacologia , Análise de Variância , Animais , Colforsina/farmacologia , Técnicas de Cultura , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/farmacologia , Imipramina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Progesterona/análise , Progesterona/biossíntese , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Pseudogravidez , Coelhos , Receptores de Prostaglandina E/metabolismo , Estatísticas não Paramétricas , Estaurosporina/farmacologia , Fosfolipases Tipo C/farmacologiaRESUMO
The possible role of Substance P (SP) was studied in the modulation of basal and gonadotopin-releasing hormone (GnRH)-induced gonadotropin secretion in the urodele crested newt, Triturus carnifex. During prereproduction, reproduction (noncourtship and courtship), refractory, recovery and aestivation, male and female pituitaries were incubated with medium-alone, GnRH, SP, GnRH receptor antagonist (antide), and SP receptor antagonist (L-703606). Since antisera raised against gonadotropins are not available for this species, we measured these hormones indirectly through their effects on the secretion of testicular androgens and ovarian progesterone from gonads superfused with the preincubated pituitaries. Pituitaries of both sexes preincubated with medium-alone, GnRH, GnRH plus L-703606, and GnRH plus SP plus L-703606 increased steroid secretion during prereproduction, noncourtship, courtship, and recovery; the increase induced by the pituitaries incubated with medium-alone was lower during prereproduction, noncourtship, and recovery. Pituitaries preincubated with SP, GnRH plus SP, GnRH plus SP plus antide, and SP plus antide did not change basal steroid secretion in any of the reproductive phases considered. Antide, L-703606, GnRH plus antide, GnRH plus SP plus antide plus L-703606, SP plus L-703606, and antide plus L-703606 experimental groups showed the same results as those with medium-alone. These results suggest that SP downregulates gonadotropin release in both Triturus carnifex sexes. In addition, an antagonist role, through receptor-independent mechanisms, exists between GnRH (upregulation) and SP (downregulation) in the modulation of pituitary.
Assuntos
Hormônio Liberador de Gonadotropina/farmacologia , Gonadotropinas/metabolismo , Hipófise/metabolismo , Substância P/farmacologia , Triturus/fisiologia , Androgênios/metabolismo , Animais , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Estivação/fisiologia , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Antagonistas de Hormônios/farmacologia , Técnicas In Vitro , Masculino , Antagonistas dos Receptores de Neurocinina-1 , Oligopeptídeos/farmacologia , Progesterona/metabolismo , Quinuclidinas/farmacologia , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismoRESUMO
By examining in vitro the effects of prostaglandin E-2 (PGE-2) and prostaglandin F-2alpha (PGF-2alpha) induced in the corpora lutea (CL) of pseudopregnant rabbits, we have demonstrated that these prostaglandins modulate luteal nitric oxide synthase (NOS) activity and progesterone production differently, depending on the age of the CL. On CL obtained on day 4 of pseudopregnancy (day-4), PGE-2 was found to depress NOS total activity to 13% of control and to significantly increase basal progesterone secretion by 61%, while PGF-2alpha had no effect. On day-9 CL, PGE-2 was ineffective, but PGF-2alpha caused a 2.5-fold increase of NOS activity and a marked decrease in progesterone production. Using specific inhibitors, we found that the regulatory actions of PGE-2 in vitro are mediated via the adenyl cyclase/protein kinase A (PKA) second messenger system, while the PGF-2alpha-induced luteolytic effects on day-9 CL depend upon activation of the phospholipase C/protein kinase C (PKC) system. The different responsiveness of day-4 and day-9 CL to PGE-2 and PGF-2alpha could depend on receptor availability for these two prostaglandins, even if other cellular mechanisms cannot be excluded. The present study supports a functional role for NOS in regulating the steroidogenic capacity of rabbit CL, and reveals a novel interaction between a stimulatory G-protein-coupled receptor and PKC/PKA-mediated signal transduction modulating NOS activity.
Assuntos
Adenilil Ciclases/metabolismo , Óxido Nítrico Sintase/metabolismo , Progesterona/metabolismo , Prostaglandinas/farmacologia , Pseudogravidez/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Células Cultivadas , Corpo Lúteo/metabolismo , Dinoprosta/farmacologia , Dinoprostona/farmacologia , Feminino , Fase Luteal/fisiologia , CoelhosRESUMO
The aim of the present research was to study the role of angiotensin-converting enzyme (ACE) and ANG II in amphibian (Rana esculenta) testicular steroidogenesis and prostaglandin production. Hormonal effects of ACE, ACE inhibitors, synthetic bullfrog ANG I, and [Val(5)]ANG II were determined in frog testis of prereproductive period. Production of 17beta-estradiol, progesterone, androgens, and PGE(2) and PGF(2alpha) was determined by incubating frog testes with ACE (2.5 mU/ml), captopril (0.1 mM), lisinopril (0.1 mM), [Val(5)]ANG II (1 microM), and synthetic bullfrog ANG I (1 microM). The analysis of the data showed an independent modulation of 17beta-estradiol and androgen production by ACE and ANG II. The ACE pathway caused a decrease of 17beta-estradiol production and an increase of androgen production in frog testes; on the other hand, the ANG II pathway increased 17beta-estradiol production and decreased androgen production. The determination of testicular aromatase activity showed a positive regulation by ANG II and a negative regulation by ACE. As for prostaglandin production, only ANG II influenced PGF(2alpha). These results suggest a new physiological role of ACE and ANG II in modulating steroidogenesis and prostaglandin production.
Assuntos
Angiotensina II/fisiologia , Aromatase/metabolismo , Peptidil Dipeptidase A/fisiologia , Testículo/enzimologia , Androgênios/biossíntese , Angiotensina II/farmacologia , Animais , Dinoprosta/biossíntese , Estradiol/biossíntese , Feminino , Técnicas In Vitro , Cinética , Masculino , Oligopeptídeos/metabolismo , Ovário/enzimologia , Peptidil Dipeptidase A/farmacologia , Rana esculenta , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
The role of nitric oxide (NO) synthase, prostaglandin E2-9-ketoreductase, and aromatase brain activities in regulating frog amplexus was assessed in the water frog (Rana esculenta). Plasma concentrations of testosterone were higher, and concentrations of 17beta-oestradiol lower, in amplexing males than in unamplexing males; while concentrations of testosterone and PGE2 were lower, and those of 17beta-oestradiol and PGF2alpha higher, in amplexing females compared to unamplexing females. Hormone release rescued from frog brains in vitro mirrored plasma hormone measures. Brain aromatase activity was lower in amplexing males; NO synthase was lower and PGE2-9-ketoreductase and aromatase were higher in amplexing females. In male brains, PGE2-9-ketoreductase inhibitor decreased PGF2alpha release and increased that of PGE2; aromatase inhibitor decreased 17beta-oestradiol and increased testosterone release. In female brains, NO donor and PGE2-9-ketoreductase inhibitor increased testosterone and PGE2 release and decreased that of 17beta-oestradiol and PGF2alpha; NO synthase inhibitor decreased testosterone release and PGE2 and increased 17beta-oestradiol and PGF2alpha release; PGF2alpha decreased testosterone release and increased 17beta-oestradiol release; aromatase inhibitor decreased 17beta-oestradiol release and increased testosterone release. In female brains, NO donor and PGE2-9-ketoreductase inhibitor decreased PGE2-9-ketoreductase and aromatase activities; PGF2alpha increased aromatase activity; NO synthase inhibitor increased PGE2-9-ketoreductase and aromatase activity. The data suggest that, in amplexing female brains, external and/or internal stimuli inhibit NO synthase, decreasing NO and activating PGE2-9-ketoreductase; in turn, PGF2alpha increases aromatase activity and 17beta-oestradiol release; while, in amplexing male brains, stimuli inhibit aromatase activity, thereby increasing testosterone production.
Assuntos
Aromatase/metabolismo , Encéfalo/enzimologia , Hidroxiprostaglandina Desidrogenases/metabolismo , Óxido Nítrico Sintase/metabolismo , Rana esculenta/fisiologia , Comportamento Sexual Animal/fisiologia , Animais , Inibidores da Aromatase , Encéfalo/fisiologia , Dinoprosta/sangue , Dinoprostona/sangue , Inibidores Enzimáticos/farmacologia , Estradiol/sangue , Feminino , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Masculino , Óxido Nítrico Sintase/antagonistas & inibidores , Testosterona/sangueRESUMO
The objective of this study was to test the possible nitric oxide (NO) involvement in pituitary gonadotropin secretion in the male crested newt, Triturus carnifex. Pituitaries were incubated in vitro with medium alone, GnRH, NO donor (NOd, sodium nitroprusside), NO synthase inhibitor (NOSi, Nomega-nitro-L-arginine methyl ester), cGMP analogue (cGMPa, 8-bromo-cGMP), soluble guanylate cyclase inhibitor (sGCi, cystamine), GnRH plus NOSi, GnRH plus sGCi, and NOd plus sGCi during the annual reproductive cycle: pre-reproduction, reproduction (noncourtship and courtship), and the refractory, recovery, and estivation periods. To determine pituitary gonadotropin secretion indirectly, newt testes were superfused in vitro with preincubated pituitaries, and androgen release was determined. NO synthase (NOS) activity and cGMP levels were assessed in the preincubated pituitaries. Medium alone- and GnRH-preincubated pituitary increased androgen secretion during pre-reproduction, noncourtship, courtship, and recovery; the GnRH-induced increase was higher than the medium alone-induced increase during pre-reproduction, noncourtship, and recovery. NOd and cGMPa increased androgens in all reproductive phases considered except courtship; the NOd- and cGMP-induced increase was higher than the medium alone-induced increase during pre-reproduction, noncourtship, and recovery. NOS activity was highest during courtship and lowest during the refractory and estivation periods. GnRH increased NOS activity during pre-reproduction, noncourtship, and recovery. Cyclic GMP levels were highest during courtship and lowest during the refractory period and estivation. GnRH increased cGMP levels during pre-reproduction, noncourtship, and recovery, while NOd did so during all reproductive phases considered. These results suggest that basal and GnRH-induced gonadotropin secretion are up-regulated by NO in the pituitary gland of the male Triturus carnifex.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Óxido Nítrico/farmacologia , Hipófise/metabolismo , Reprodução/fisiologia , Androgênios/metabolismo , Animais , GMP Cíclico/metabolismo , Técnicas In Vitro , Masculino , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/metabolismo , Nitroprussiato/farmacologia , Perfusão , Hipófise/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/metabolismo , TriturusRESUMO
We examined the presence and the regulation of nitric oxide (NO) synthase (NOS) using in vitro cultured corpora lutea (CL) obtained from rabbits at days 4 and 9 of pseudopregnancy. The role of NO and NOS on steroidogenesis was also investigated using the same CL preparations after short-term incubations (30 min and 2 h) with the NO donor, sodium nitroprusside (NP), the NOS inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME) and prostaglandin (PG) F-2alpha. The basal NOS activity was greater in CL at day 4 than at day 9, and was also differently modulated by PGF-2alpha, depending on the age of the CL. The addition of PGF-2alpha to day 4 CL had no effect, but PGF-2alpha on day 9 caused a threefold increase in NOS activity. NP caused a two- to fivefold decrease in release of progesterone from CL of both ages, and this inhibitory effect on steroidogenesis was reversed by l-NAME. All treatments failed to modify basal androgens and 17beta-oestradiol was not detectable in either control or treated CL. These results suggest that NO is effectively involved in the regulation process of steroidogenesis, independently of 17beta-oestradiol. PGF-2alpha had no effect on day 4, but induced luteolysis on day 9, by reducing progesterone (P=0. 01) to about 18% of control. The luteolytic action of PGF-2alpha was completely reversed by co-incubation with l-NAME, thus supporting the hypothesis that luteolysis is mediated by NO. The addition of NP or l-NAME did not modify the in vitro release of PGF-2alpha. We hypothesised that PGF-2alpha upregulates NOS activity and, consequently, the production of NO, which acutely inhibits progesterone release from day 9 CL of pseudopregnant rabbits.
Assuntos
Corpo Lúteo/metabolismo , Óxido Nítrico Sintase/metabolismo , Progesterona/biossíntese , Análise de Variância , Animais , Corpo Lúteo/efeitos dos fármacos , Técnicas de Cultura , Dinoprosta/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroprussiato/farmacologia , Pseudogravidez , Coelhos , Fatores de TempoRESUMO
The responsiveness of rabbit corpus luteum to 200 micrograms of the prostaglandin F2 alpha (PGF2 alpha) analogue, alfaprostol, between Days 3 and 9 of pseudopregnancy was assessed by evaluating the decline in plasma progesterone after treatment with PGF2 alpha in 81 New Zealand White (NZW) rabbits. On Days 3-5, functional luteolysis was not observed. On Days 6, 7, and 8 of pseudopregnancy, the number of rabbits responsive to PGF2 alpha, rose from 38% to 71% and 83%, respectively. In the other cases, the effect of the PGF2 alpha analogue was transient as CL recovered in the following 2 or 3 days. By contrast, on Day 9 luteolysis was effective and persistent in all the animals. In rabbits treated on Day 9, progesterone decreased gradually from 10.6 +/- 0.7 within the first 6 h, but fell to 3.6 +/- 1.5 ng/mL (p < 0.01) 12 h after PGF2 alpha and to 0.2 +/- 0.1 ng/mL (p < 0.01) 24 h later.
Assuntos
Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/fisiologia , Progesterona/sangue , Prostaglandinas F/farmacologia , Pseudogravidez/tratamento farmacológico , Animais , Dinoprosta/análogos & derivados , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Gonadotropinas Equinas/farmacologia , Pseudogravidez/induzido quimicamente , CoelhosRESUMO
We studied the possible role of nitric oxide (NO) in GnRH-induced gonadotropin secretion in the female water frog, Rana esculenta. During pre-reproduction, pre-ovulation, ovulation, post-ovulation, refractory, recovery and hibernation, pituitaries were incubated with medium-alone, GnRH, NO donor (NOd), NO synthase inhibitor (NOSi), cyclic GMP analogue (cGMPa), soluble guanylate cyclase inhibitor (sGCi), GnRH plus NOSi, GnRH plus sGCi, and NOd plus sGCi. Because antisera raised against gonadotropins are not available for this species, we measured these hormones indirectly through their effects on ovarian progesterone secretion. The ovaries were superfused with the pituitaries pre-incubated as reported above. In addition, NOS activity and cGMP levels were determined in the pre-incubated pituitaries. Those pre-incubated with medium-alone and with GnRH increased progesterone secretion during pre-reproduction, pre-ovulation, ovulation and recovery; the increase induced by GnRH was higher than that induced by medium-alone during pre-reproduction, pre-ovulation and recovery. NOd and cGMPa increased progesterone in all considered reproductive phases except ovulation; the increase induced by NOd and cGMP was higher than that induced by medium-alone during pre-reproduction, pre-ovulation and recovery. NOS activity was highest during ovulation and lowest during post-ovulation, refractory and hibernation. GnRH increased NOS activity during pre-reproduction, pre-ovulation and recovery. Cyclic GMP levels were highest during ovulation and lowest during post-ovulation, refractory and hibernation. GnRH increased cGMP levels during pre-reproduction, pre-ovulation and recovery, NOd during all considered reproductive phases. These results suggest that NO mediates basal and GnRH-induced gonadotropin secretion in female Rana esculenta.