RESUMO
Mammalian spermatozoa must undergo biochemical and structural changes to acquire the capacity for fertilization, in a process known as capacitation. Activation of PKA enzymes is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation at the end of capacitation. Blockade of MRP4 inhibited induction of acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition generated an increase in Ca2+ levels mediated by PKA, and depletion of Ca2+ salts from the medium prevented the loss of motility and phosphotyrosine inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca2+ channel knockout mice. Taken together, these results suggest that cAMP efflux via MRP4 plays an essential role in mouse sperm capacitation.This article has an associated First Person interview with the first author of the paper.
Assuntos
AMP Cíclico/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Capacitação Espermática/fisiologia , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Propionatos/farmacologia , Quinolinas/farmacologia , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
The oviduct acts as a functional sperm reservoir in many mammalian species. Both binding and release of spermatozoa from the oviductal epithelium are mainly modulated by sperm capacitation. Several molecules from oviductal fluid are involved in the regulation of sperm function. Anandamide is a lipid mediator involved in reproductive physiology. Previously, we demonstrated that anandamide, through activation of the cannabinoid receptor type 1 (CB1), promotes sperm release from bovine oviductal epithelial cells, and through CB1 and the transient receptor potential vanilloid 1 (TRPV1), induces sperm capacitation. Herein we investigate co-activation between CB1 and TRPV1, and Ca(2+) influx as part of the mechanism of action of anandamide during sperm release from oviductal cells. Our results indicate that in the absence of Ca(2+) anandamide failed to release spermatozoa from oviductal epithelial cells. Additionally, sperm release promoted by cannabinoid and vanilloid agonists was abolished when the spermatozoa were preloaded with BAPTA-AM, a Ca(2+) chelator. We also determined Ca(2+) levels in spermatozoa preloaded with FURA2-AM co-cultured with oviductal cells and incubated with different cannabinoid and vanilloid agonists. The incubation with different agonists induced Ca(2+) influx, which was abolished by CB1 or TRPV1 antagonists. Our results also suggest that a phospholypase C (PLC) might mediate the activation of CB1 and TRPV1 in sperm release from the bovine oviduct. Therefore, our findings indicate that anandamide, through CB1 and TRPV1 activation, is involved in sperm release from the oviductal reservoir. An increase of sperm Ca(2+) levels and the PLC activation might be involved in anandamide signaling pathway.
Assuntos
Ácidos Araquidônicos/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Endocanabinoides/farmacologia , Oviductos/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Sinalização do Cálcio , Bovinos , Células Cultivadas , Técnicas de Cocultura , Feminino , Masculino , Oviductos/citologia , Capacitação EspermáticaRESUMO
BACKGROUND: Coronectomy is an alternative to complete removal of an impacted mandibular third molar. Most authors have recommended coronectomy to prevent damage to the inferior alveolar nerve during surgical extraction of lower third molars. The present study offers a systematic review and metaanalysis of the coronectomy technique. MATERIAL AND METHODS: A systematic review and meta-analysis was performed based on a PubMed and Cochrane databases search for articles published from 2014 and involving coronectomy of mandibular third molars located near the inferior alveolar nerve canal, with a minimum of 10 cases and a minimum follow-up period of 6 months. After application of the inclusion and exclusion criteria, a total of 12 articles were included in the study. RESULTS AND DISCUSSION: Coronectomy results in significantly lesser loss of sensitivity of the inferior alveolar nerve and prevents the occurrence of dry socket. No statistically significant differences were observed in the incidence of pain and infection between coronectomy and complete surgical extraction. After coronectomy, the remaining tooth fragment migrates an average of 2 mm within two years. CONCLUSIONS: Coronectomy is indicated when the mandibular third molar is in contact with the inferior alveolar nerve and complete removal of the tooth may cause nerve damage.
Assuntos
Dente Serotino , Extração Dentária , Dente Impactado , Humanos , Mandíbula , Nervo Mandibular , Coroa do Dente , Traumatismos do Nervo TrigêmeoRESUMO
STUDY QUESTION: Does fibronectin (Fn) stimulate the sperm capacitation process in humans? SUMMARY ANSWER: Fibronectin stimulates human sperm capacitation. WHAT IS KNOWN ALREADY: Capacitation is a process that occurs in the oviduct. It has been suggested that some molecules present in the oviductal fluid and cells as well as proteins present in the cumulus oophorus could be involved in the modulation of sperm function and their acquisition of fertilizing capacity. Fibronectin is a glycoprotein that is present in the fluid and the oviduct epithelium, and its receptor (alpha 5 beta 1 integrin) is present in human sperm. When alpha 5 beta 1 (α5ß1) integrin binds to fibronectin, intracellular signals similar to the process of sperm capacitation are activated. STUDY DESIGN, SIZE, DURATION: Human sperm were selected via a percoll gradient and were then incubated in non-capacitated medium (NCM) or reconstituted capacitated medium (RCM), in the presence or absence of fibronectin for different time periods. A total of 39 donors were used during the study, which lasted 3 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Freshly ejaculated sperm from healthy volunteers were obtained by masturbation. All semen samples were normal according to the World Health Organization parameters. Six approaches were used to determine the effects of fibronectin on sperm capacitation: chlortetracycline (CTC) assay, heterologous co-culture of human sperm with bovine oviductal epithelial cells (BOEC), measurement of cyclic (c) AMP levels, activity of protein kinase A (PKA), phosphorylation of proteins in tyrosine (Tyr) residues, and induction of acrosome reaction with progesterone. MAIN RESULTS AND THE ROLE OF CHANCE: When sperm were incubated in RCM in the presence of Fn, we observed differences with respect to sperm incubated in RCM without Fn (control): (i) a 10% increase in the percentage of sperm with the B pattern (capacitated sperm) of CTC fluorescence from the beginning of capacitation (P < 0.001); (ii) an effect on both the concentration of cAMP (P < 0.05) and PKA activity (P < 0.05) during early capacitation; (iii) an increase in the degree of phosphorylation of proteins on tyrosine residues after 60 min of capacitation (P < 0.01); (iv) an increase in the percentage of acrosome-reacted sperm in response to progesterone (P < 0.05); and (v) a decrease in the percentage of sperm attached to BOEC (P < 0.05). Moreover, we noted that the effect of Fn was specific and mediated by alpha 5 beta 1 integrin (P < 0.001). Fn by itself had no effect on sperm capacitation. LIMITATIONS, REASONS FOR CAUTION: This study was carried out with sperm from young adult men. Men with abnormal semen samples were excluded. The results cannot be directly extrapolated to other mammalian species. WIDER IMPLICATIONS OF THE FINDINGS: Currently, male subfertility has become a huge public health problem, which makes it imperative to develop new treatments. This is a novel discovery that extends our current knowledge concerning normal and pathological sperm physiology as well as events that regulate the process of fertilization. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from FONDECYT (1130341, E.S.D. and 1120056, P.M.) and FONCYT (PIP 2011-0496, S.P.-M). The authors have no conflicts of interest.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fibronectinas/fisiologia , Transdução de Sinais/fisiologia , Capacitação Espermática/fisiologia , Adulto , Humanos , Masculino , Adulto JovemRESUMO
With the progressive increase in the use of reproductive biotechnologies in the cattle industry, like artificial insemination and in vitro embryo production, the accurate determination of fertilizing competence of cryopreserved sperm samples is an essential issue. The routine methodology to assess bull sperm quality relies primarily on count, viability and motility of spermatozoa. However, these parameters do not tightly predict the reproductive success of samples. Therefore, identification of complementary markers of sperm functionality to strengthen the predictability of traditional spermogram is desirable to improve livestock reproduction practices. Previous results from our laboratory indicated that α5ß1 integrin plays a key role in bovine sperm function and mediates their interaction with the female reproductive tract. Thus, this study aimed to investigate whether the localization of α5ß1 held a correlation with fertilizing ability of bovine cryopreserved semen samples. Firstly, we assessed the quality of samples from six different bulls (A-F). We determined motility and viability of sperm samples after thawing and selection. Additionally, we measured the capacitation state of the samples by chlortetracycline (CTC) assay in the presence or absence of heparin, as an indicator of their responsiveness to a capacitating stimulus. Based on these assays, samples were classified being A the bull with the lowest quality and F the bull with the highest quality. Then, we studied the presence and localization of α5ß1 integrin. This protein showed a distribution pattern in the acrosomal (A), post-acrosomal (P) and acrosomal + post-acrosomal (A + P) regions with different localization percentages among the studied samples. Next, we determined the fertilizing ability of the samples in in vitro fertilization (IVF) assays and performed correlation analyses between IVF outcome and the routine spermogram parameters or α5ß1 integrin localization patterns. When the percentage of cells showing α5ß1 integrin was compared to fertilization rate, no correlation was observed. However, the presence of α5ß1 integrin in P and A + P regions (PA pattern), positively correlated with IVF rate (p < 0.05). These results suggest that while routine semen analyses failed to predict sperm reproductive competence, integrin localization in post-acrosomal region (PA pattern) showed a positive correlation with IVF outcome, thus posing an attractive marker to predict more accurately the reproductive performance of an individual.
Assuntos
Integrinas , Motilidade dos Espermatozoides , Animais , Bovinos , Criopreservação/veterinária , Feminino , Fertilidade , Masculino , EspermatozoidesRESUMO
Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 µg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 µM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5ß1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 µM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 µM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5ß1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.
Assuntos
Bovinos , Endocanabinoides/metabolismo , Fibronectinas/farmacologia , Preservação do Sêmen/veterinária , Capacitação Espermática/efeitos dos fármacos , Animais , Criopreservação/veterinária , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocanabinoides/genética , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Masculino , Óxido Nítrico , Motilidade dos EspermatozoidesRESUMO
N-acetylglucosaminidase (NAG) is particularly active in mammalian spermatozoa and appears to be involved in fertilization. Although it is assumed that this enzyme is acrosomal, previous results from our laboratory suggest the presence of NAG at the sperm plasma membrane level. The present study attempted to analyse the subcellular distribution of this enzyme in human spermatozoa. Sperm were incubated under different conditions and NAG activity measured in the soluble extracts and cell pellets using a specific fluorometric substrate. A significant proportion of NAG activity was released when sperm were incubated in culture medium, suggesting a weak association with the plasma membrane. This location was confirmed by western blot analysis of plasma membrane fractions and immunofluorescence on non-permeabilized sperm, which showed a positive signal mainly on the acrosomal domain. The distribution of NAG activity between plasma membrane and acrosome was analysed after cell disruption by freezing and thawing. Triton X-100 stimulated sperm and epididymal NAG activity but not the enzyme obtained from other sources. In addition, biotinylated human recombinant NAG was able to bind to human sperm. Finally, after sperm incubation under capacitating conditions, NAG total activity increased and the sperm enzyme lost its ability to be stimulated by Triton X-100. The possible connection of these results with sperm maturation, capacitation and NAG participation in primary binding to the zona pellucida, was discussed.
Assuntos
Acetilglucosaminidase/metabolismo , Espermatozoides/enzimologia , Acrossomo/enzimologia , Biotinilação , Western Blotting , Fracionamento Celular , Membrana Celular/enzimologia , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Epididimo/enzimologia , Imunofluorescência , Humanos , Masculino , Octoxinol/farmacologia , Ligação Proteica , Proteínas Recombinantes/metabolismo , Capacitação Espermática , Espermatozoides/efeitos dos fármacosRESUMO
We investigated the regulation of cyclooxygenase-2 (COX-2) by 17-beta-estradiol (E2) in the rat oviduct. We observed that COX-2 is expressed mainly in proestrous and estrous stages, periods under estrogenic influence. While exogenous administration of E2 (1 microg/rat) significantly increased COX-2 protein levels, progesterone did not modify it. COX-2 was mainly localized on oviductal epithelial cells from estrogenized rat. Induction of COX-2 expression by E2 was partially reverted by tamoxifen (1 mg/rat), an E2 receptor antagonist. Estradiol treatment also increased prostaglandins (PGs) synthesis: 6-keto-PGF(1alpha) (40%), a stable metabolite of prostacyclin (PGI2), PGF(2alpha) (40%) and PGE2 (50%). Tamoxifen completely suppressed this enhancement. In order to discriminate which isoform of COX was implicated in the stimulatory effect of E2 on PGs synthesis, oviducts were preincubated with meloxicam (Melo: 10(-9)M) or NS-398 (10(-7)M), two selective COX-2 inhibitors. Both Melo and NS-398 abolished the increase of PGs synthesis stimulated by E2. All together, these data indicate that E2 could upregulate COX-2 expression and activity in the rat oviduct and that the stimulatory effect of E2 may be receptor-mediated.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Estradiol/farmacologia , Oviductos/efeitos dos fármacos , Oviductos/enzimologia , Animais , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/análise , Inibidores de Ciclo-Oxigenase 2/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Ciclo Estral/metabolismo , Feminino , Imuno-Histoquímica , Meloxicam , Proteínas de Membrana/metabolismo , Nitrobenzenos/farmacologia , Oviductos/metabolismo , Gravidez , Progesterona/farmacologia , Prostaglandinas/biossíntese , Ratos , Ratos Wistar , Receptores de Estradiol/antagonistas & inibidores , Sulfonamidas/farmacologia , Tamoxifeno/farmacologia , Tiazinas/farmacologia , Tiazóis/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Many authors hypothesize that the epidermal growth factor (EGF) is involved in the onset of labor. Previous reports from our laboratory showed that intrauterine administration of EGF delays the beginning of labor. The aims of this study were: 1) to analyze the effect of intrauterine administration of 500 ng EGF on placental prostaglandins and nitric oxide, and 2) to characterize the expression of EGF receptors (EGF-R) in pregnant rat placentae. Saline solution (sham group) and 500 ng EGF (EGF-treated group) were administered via intrauterine injection on day 21 of gestation, and both groups of animals were sacrificed on day 22 (sham rats delivered on day 22). Results showed that EGF treatment: 1) inhibited the production of prostaglandin E (p<0.001) and F(2alpha) (p<0.01), 2) increased the synthesis of nitric oxide (p<0.001), and 3) reduced the expression of cyclooxygenase-II, the enzyme responsible for PG synthesis. Placentae were found to express EGF-R and its activated form, and the expressions of both forms were higher at mid and term pregnancy. Hence, EGF is a very interesting molecule for studying the regulation of placental prostaglandin and nitric oxide production related to the parturition process.
Assuntos
Dinoprosta/biossíntese , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Óxido Nítrico Sintase/metabolismo , Placenta/efeitos dos fármacos , Placenta/metabolismo , Prostaglandinas E/biossíntese , Animais , Western Blotting , Ciclo-Oxigenase 2/biossíntese , Receptores ErbB/biossíntese , Feminino , Isoenzimas , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/biossíntese , Placenta/enzimologia , Gravidez , RatosRESUMO
Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.
Assuntos
Óxido Nítrico Sintase/metabolismo , Espermatozoides/enzimologia , Animais , Anticorpos , Arginina/metabolismo , Radioisótopos de Carbono , Citrulina/biossíntese , Feminino , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Óxido Nítrico/biossínteseRESUMO
Previous studies in our laboratory indicated that immunization of male and female Wistar and Lewis rats with epididymal protein DE, resulted in the development of anti-DE antibodies in over 90% of the animals, with a significant and reversible reduction of fertility. In the present study, ELISA assays performed to analyze the evolution of the immune response indicated that antibody levels in the sera of immunized animals reached a maximum at 8 weeks after the initial injection and then gradually decreased, returning to control values by the end of the sixth month. Western blot experiments demonstrated that the immune sera specifically recognized DE in epididymal sperm extracts and epididymal cytosol, while no reaction was observed with different reproductive and essential organs. The immune sera were also capable of recognizing DE on the surface of both fresh and capacitated sperm as indicated by indirect immunofluorescence experiments. Finally, the exposure of sperm to immune sera prior to uterine insemination resulted in a significant (P < 0.05) reduction in the percentage of fertilized eggs compared to controls, with no effect on sperm motility and viability, nor on their ability to undergo capacitation. Together, these results support the participation of the raised antibodies as mediators of the antifertility effect and suggest a specific interference at the sperm-egg interaction level.
Assuntos
Anticoncepção Imunológica , Epididimo/imunologia , Isoanticorpos/farmacologia , Metaloproteínas/fisiologia , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Hormônios Testiculares/fisiologia , Animais , Western Blotting , Proteínas Secretadas pelo Epidídimo , Feminino , Soros Imunes , Imunização , Isoanticorpos/biossíntese , Isoanticorpos/imunologia , Masculino , Metaloproteínas/imunologia , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Aglutinação Espermática , Capacitação Espermática , Motilidade dos Espermatozoides , Hormônios Testiculares/imunologiaRESUMO
We examined the possible relationship between cytokines, nitric oxide and prostaglandins (PGs) in the estrogenized rat uterus. Results indicate that epidermal growth factor (EGF) enhances the synthesis of prostaglandins in estrogenized rat uteri and induces the augmentation of nitric oxide (NO) production in this tissue by stimulating iNOS. While the effect of EGF is abolished by L-NMMA, an NO antagonist, the NS-398, a cyclooxygenase-II (COX-II) inhibitor, prevents the augmentation of prostanoids induced by EGF. These results suggest that there is an interaction among EGF, NO and PGs and that in this interrelationship are involved COX-II and iNOS. This mechanism might be important during implantation and labor.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Estradiol/farmacologia , Óxido Nítrico/metabolismo , Prostaglandinas/biossíntese , Útero/metabolismo , Animais , Ácido Araquidônico/metabolismo , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Feminino , Técnicas In Vitro , Isoenzimas/análise , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitrobenzenos/farmacologia , Prostaglandina-Endoperóxido Sintases/análise , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Útero/efeitos dos fármacos , Útero/enzimologia , ômega-N-Metilarginina/farmacologiaRESUMO
We have studied the effect of nitric oxide (NO) on the production of arachidonic acid ([14C]-AA) metabolites in the rat oviduct. The basal synthesis of eicosanoids was measured by the conversion of ([14C]-AA) to the different radiolabeled products of cyclooxygenase (COX). The oviducts incubated for 1 h with the labeled substrate of COX were able to convert 3.3 +/- 0.3% of ([14C]-AA) to 6-ceto-PGF1alpha, 10.7 +/- 1.0% to PGF2alpha, 13.5 +/- 1.2% to PGE2 and 6.3 +/- 0.5% to TXB2. The tissues were incubated with different doses of two NO donors: SIN-1 and Spermine NONOate. The results indicated that SIN-1 produces a significant decrease (50%; P < 0.05) in all prostanoids evaluated in a dose-response fashion. The inhibitory effect was completely reversed by addition of 20 microg/ml of hemoglobin (Hb), a NO scavenger. The addition of Spermine NONOate to the incubation medium diminished significantly (65%) the synthesis of COX metabolites suggesting that NO acts by inhibiting COX activity in the rat oviduct. However, NOS inhibitors, N(G)-L-arginine-methyl-ester (L-NAME) nd N(G)-L-monomethyl-arginine (L-NMMA) had no effect on basal production of the prostanoids. These results indicate that in the rat oviduct the synthesis of COX metabolites is negatively regulated by nitric oxide.
Assuntos
Ácido Araquidônico/metabolismo , Tubas Uterinas/metabolismo , Molsidomina/análogos & derivados , Óxido Nítrico/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/biossíntese , Espermina/análogos & derivados , Animais , Radioisótopos de Carbono/metabolismo , Tubas Uterinas/efeitos dos fármacos , Feminino , Técnicas In Vitro , Molsidomina/farmacologia , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxidos de Nitrogênio , Ratos , Ratos Wistar , Espermina/farmacologiaRESUMO
In this paper, we evaluated the hypothesis that prostaglandin F2 alpha (PGF2 alpha) regulates NOS activity and we also investigated, by means of nitric oxide inhibitors (N-monomethyl-L-arginine monoacetate, L-NMMA) the role of endogenous NO on PGF2 alpha-induced contractions in rat oviduct. NOS activity was determined by measuring the conversion of 14[C]-L-arginine to 14[C]-L-citrulline on oviductal homogenates from estrogenized rats (1 microgram/rat). The presence of PGF2 alpha (10(-8) M) in the incubation medium produced an increase in NOS activity (p < or = 0.05). The effect of the prostanoid was blocked completely by the presence of two NOS inhibitors: N-nitro-L-arginine methyl ester (L-NAME, 0.6 mM) and aminoguanidine (Ag, 0.5 mM). These results suggested that PGF2 alpha could be modulating the Ca(2+)-independent NOS activity. We determined NOS activity using 1 mM EGTA, a chelator of Ca2+, in a free Ca2+ medium. These results indicated that PGF2 alpha produces an increase in Ca(2+)-independent NOS activity (p < or = 0.05). PGF2 alpha induces contraction of the oviductal smooth muscle in a concentration dependent manner. L-NMMA enhanced PGF2 alpha induced contraction of the oviduct, providing indirect evidence that there is a basal release of NO in the oviduct, which may reduce and/or modulate the contractile effects of PGF2 alpha.
Assuntos
Dinoprosta/farmacologia , Tubas Uterinas/fisiologia , Contração Muscular/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/fisiologia , Animais , Arginina/metabolismo , Cálcio/farmacologia , Quelantes , Citrulina/metabolismo , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Tubas Uterinas/enzimologia , Feminino , Guanidinas/farmacologia , Músculo Liso/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Nitric oxide (NO) is synthesized in a variety of tissues, including rat uterus, from L-arginine by NO synthase (NOS), of which there are three isoforms, namely neuronal, endothelial and inducible NOS (nNOS, eNOS and iNOS, respectively). Nitric oxide is an important regulator of the biology and physiology of the organs of the reproductive system, including the uterus. Some studies have shown increased variation in NO production and NOS expression during the oestrous cycle. However, the factors that regulate NO production in the uterus remain unclear. Therefore, in the present study, we investigated the effect of sex steroids on NOS expression and activity in the ovariectomized rat uterus. Ovariectomized rats received progesterone (4 mg per rat) or 17beta-oestradiol (1 microg per rat). All rats were killed 18 h after treatment. Both progesterone and oestradiol were able to augment NOS activity. The effect of oestradiol was abolished by pre-incubation with 500 micro M aminoguanidine, an iNOS inhibitor, or by coadministration of oestradiol with 3 mg kg(-1) dexamethasone, but the effect of progesterone was not affected by these treatments. Uterine nNOS, eNOS and iNOS protein levels were assessed using Western blots. Ovariectomized rat uteri expressed iNOS and eNOS. Progesterone increased the expression of eNOS and iNOS, whereas oestradiol increased iNOS expression only. These results suggest that oestradiol and progesterone are involved in the regulation of NOS expression and activity during pregnancy and implantation in the rat.
Assuntos
Estradiol/farmacologia , Hormônios Esteroides Gonadais/farmacologia , Óxido Nítrico Sintase/metabolismo , Progesterona/farmacologia , Útero/efeitos dos fármacos , Animais , Western Blotting , Cálcio , Inibidores Enzimáticos/farmacologia , Estradiol/fisiologia , Feminino , Hormônios Esteroides Gonadais/fisiologia , Guanidinas/farmacologia , Isoenzimas/análise , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/antagonistas & inibidores , Ovariectomia , Progesterona/fisiologia , Ratos , Ratos Wistar , Regulação para Cima , Útero/enzimologiaRESUMO
The effect of three nitric oxide (NO) synthase inhibitors, L-NG-nitro-arginine (NO2Arg), NG-Nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, on in vitro fertilization in the mouse was examined. Mouse epididymal spermatozoa were capacitated in a medium with or without NO synthase inhibitors. Oocytes were inseminated and the percentage of oocytes with two pronuclei was scored after an 8-h incubation. NO2Arg and L-NAME, but not aminoguanidine, inhibited fertilization. L-NAME inhibited fertilization in a dose-dependent manner, and its effects were stereospecific. The inhibitory effect was neutralized by L-arginine but not by D-arginine. Moreover, D-NAME did not inhibit fertilization. The results suggest that NO synthase activity (presumably of the constitutive type is necessary for spermatozoa to display their full fertilizing ability.
Assuntos
Inibidores Enzimáticos/farmacologia , Fertilização in vitro/efeitos dos fármacos , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Arginina/farmacologia , Guanidinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , NG-Nitroarginina Metil Éster/farmacologia , Nitroarginina/farmacologia , Capacitação EspermáticaRESUMO
In a recent work, we detected nitric oxide synthase (NO synthase) in the acrosome and tail of mouse and human spermatozoa by an immunofluorescence technique. Also, NO-synthase inhibitors added during sperm capacitation in vitro reduced the percentage of oocytes fertilized in vitro, suggesting a role for NO synthase in sperm function. Therefore, in the present study the effect of three NO-synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-D-arginine methyl ester (D-NAME) and L-NG-nitro-arginine (NO2-arg), and of a nitric oxide donor, spermine-NONOate, on the progesterone-induced acrosome reaction of mouse sperm was examined. NO-synthase inhibitors were added at 0, 60 or 90 min during capacitation; at 120 min, mouse epididymal spermatozoa were exposed to 15 microM progesterone for another 15 min. In another set of experiments, different concentrations of spermine-NONOate were added to capacitated spermatozoa for 15 min; in these experiments, progesterone was not included. NO2-arg and L-NAME blocked progesterone-induced exocytosis regardless of the time at which these inhibitors were added. Moreover, D-NAME did not inhibit exocytosis. In contrast, spermine-NONOate stimulated the acrosomal exocytosis in vitro directly. These results provide evidence that mouse sperm NO synthase participates in the progesterone-induced acrosome reaction in vitro and that nitric oxide induces this event.
Assuntos
Acrossomo/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Óxido Nítrico Sintase/fisiologia , Progesterona/farmacologia , Espermatozoides/efeitos dos fármacos , Animais , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , Espermatozoides/ultraestrutura , Estereoisomerismo , Estimulação QuímicaRESUMO
The localization of nitric oxide synthase was studied in mouse epididymal spermatozoa and freshly ejaculated human sperm. A rabbit antiserum against the neuronal isoform of the enzyme was used, and antibody binding was detected with a fluorescein isothiocyanate-conjugated polyclonal antibody specific for rabbit IgG. In mouse spermatozoa, the percentage of cells staining specifically ranged from 88% to 98%. Samples were examined after 0-, 90- and 150-min incubations in vitro. Three different patterns of staining were observed: (a) Pattern I, intense fluorescent staining localized in the acrosome and in a segment of the tail; (b) Pattern II, fluorescent staining localized only in the tail; and (c) Pattern III, faint fluorescent staining localized in the acrosomal cap and in the tail. The potential physiological significance of these patterns is discussed. Nitric oxide synthase was also localized in the acrosome of freshly ejaculated human sperm.
Assuntos
Óxido Nítrico Sintase/análise , Espermatozoides/enzimologia , Animais , Fluoresceína-5-Isotiocianato/química , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Soros Imunes/imunologia , Masculino , Camundongos , Óxido Nítrico Sintase/imunologia , Coelhos , Espermatozoides/imunologia , Fatores de TempoRESUMO
We do not know the normal relationship between altitude and PaCO2 in Mexico. We collected and analyzed the reports of reference values for gasometry in Mexico City (2240 m above sea level and a mean barometric pressure of 585 Torr) and other places in the country. The reports include arterial, capillary and expired gases in children and adults, with measurements done in resting and exercising subjects, breathing room air and 100% oxygen. In Mexico City we found 18 studies in normal subjects reporting a mean PaCO2 ranging from 25.5 to 38.4 Torr. Averaging arterial studies from children and adults, adjusting for the number of subjects studied, and discarding data with mean pH below 7.37 or above 7.43 (suggesting non steady state), 10 studies with a total of 581 subjects have the following average values (Torr, means and SD): PaCO2 = 31.1 +/- 2.6, PaO2 = 67.7 +/- 2.6, calculated PAO2 and P(A-a)O2 73.6 +/- 3.3 y 6.1 +/- 3.7 respectively. The PaCO2 found was much lower than that reported for native Peruvians in the Andes who have a mean PaCO2 of 37.8 Torr at an altitude of 2390 m, and a mean PaCO2 of 33.0 Torr only at 4860 m above the sea level. On the other hand, the average values in Mexico are similar to those found in North Americans who have a mean PACO2 of 33.1 Torr at 2131 m of altitude, a mean PACO2 of 30.7 at 2371 m and a mean PaCO2 of 31 Torr at 2238 m. Normal values for gasometry in Mexico are scarce and some of the existing ones are erroneous probably due to lack of adequate calibrations and to poor quality control. Values of PaCO2 in Mexico are more similar to those found in the USA than to those found in Peruvian natives.
Assuntos
Adaptação Fisiológica , Altitude , Dióxido de Carbono/análise , Adaptação Fisiológica/genética , Adulto , Ar/análise , Artérias , Pressão Atmosférica , Capilares , Criança , Humanos , México , Oxigênio/administração & dosagem , Pressão Parcial , Peru , Esforço Físico , Valores de Referência , Estados Unidos , População UrbanaRESUMO
INTRODUCTION: The decidual reaction and the formation of new vessels in the uterus are two crucial processes during embryo implantation. Previously, we observed that lysophosphatidic acid (LPA) increases cyclooxygenase-2 derived - prostaglandin E2 production during implantation in the rat uterus and that it augments the expression of decidualization (IGFBP-1) and vascularization (IL-10) markers. Both cyclooxygenase and nitric oxide synthase (NOS) are known enzymes involved in these processes. Thus, we became interested in studying which factors contribute to LPA receptor-specific role during the decidual and the vascular reaction at implantation. METHODS: We adopted a pharmacological approach in vitro incubating the uterus from rats on day 5 of gestation (day of implantation) with LPA, DGPP (a highly selective antagonist of LPA3, an LPA receptor) and cyclooxygenase and NOS selective and non-selective inhibitors. We determined NOS activity, prostaglandin E2 production and IGFBP-1 and IL-10 expression to evaluate decidualization and vascularization. RESULTS: We observed that LPA augmented the activity of the inducible NOS isoform through LPA1/LPA3. Inducible NOS activity participated in the induction of cyclooxygenase-2/prostaglandin E2 increase stimulated by LPA. Also, cyclooxygenase-2 derived prostaglandins mediated LPA-stimulatory action on NOS activity. Both cyclooxygenase-2 and inducible NOS mediated LPA effect on IGFBP-1 and IL-10 expression. CONCLUSIONS: These results suggest the participation of LPA/LPA3 in the production of crucial molecules involved in vascularization and decidualization, two main processes that prepare the uterine milieu for embryo invasion during implantation.