RESUMO
STUDY QUESTION: What are the levels of anandamide (N-arachidonoylethanolamide, AEA) in human seminal plasma and how are these related to abnormal spermatozoa? SUMMARY ANSWER: Seminal plasma AEA levels were lower in men with asthenozoospermia and oligoasthenoteratozoospermia compared with normozoospermic men. WHAT IS KNOWN ALREADY: AEA, a bioactive lipid, synthesized from membrane phospholipids may signal through cannabinoid receptors (CB1 and CB2) to regulate human sperm functions and male reproduction by modulating sperm motility, capacitation and the acrosome reaction in vitro. Local AEA levels are regulated by the synthetic and degradative enzymes, NAPE-PLD and FAAH, respectively. How the deregulation of this endogenous signalling pathway affects human sperm function(s) is not clear. STUDY DESIGN, SIZE AND DURATION: This was a cross-sectional study of 86 men presenting at an infertility clinic for semen analysis over a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: AEA was quantified, by ultra-high performance liquid chromatography-tandem mass spectrometry, in seminal plasma from 86 volunteers. Using qRT-PCR, CB1, CB2, NAPE-PLD and FAAH transcript levels were determined in spermatozoa from men with normozoospermia, asthenozoospermia, oligoasthenoteratozoospermia and teratozoospermia. Normal spermatozoa were exposed in vitro to methanadamide (meth-AEA) to determine its effect on sperm motility, viability and mitochondrial activity. MAIN RESULTS AND THE ROLE OF CHANCE: Seminal plasma AEA levels (mean ± SEM) were significantly lower in men with asthenozoospermia (0.080 ± 0.01 nM; P < 0.05) or oligoasthenoteratozoospermia (0.083 ± 0.01 nM; P < 0.05) compared with normozoospermic men (0.198 ± 0.03 nM). In addition, the levels of spermatozoal CB1 mRNA were significantly decreased in men with asthenozoospermia (P < 0.001) or oligoasthenoteratozoospermia (P < 0.001) compared with normozoospermic controls. Supra-physiological levels of meth-AEA decreased sperm motility and viability, probably through CB1-mediated inhibition of mitochondrial activity. LIMITATIONS, REASONS FOR CAUTION: The inhibitory effect of meth-AEA was only shown in vitro and may not reflect what happens in vivo. WIDER IMPLICATIONS OF THE FINDINGS: As the regulation of the endocannabinoid system appears to be necessary for the preservation of normal sperm function and male fertility, there may be implications for the adverse reproductive consequences of marijuana use. Exocannabinoids, such as Δ(9)-THC, are likely to compete with endocannabinoids at the cannabinoid receptors, upsetting the finely balanced endocannabinoid signalling system. The importance of the endocannabinoid system makes it an attractive target for pharmacological interventions to control male fertility. STUDY FUNDING/COMPETING INTEREST(S): This work was funded in part by miscellaneous educational funds from the University Hospitals of Leicester National Health Services Trust to support the Endocannabinoid Research Laboratory of University of Leicester. The authors declare no competing interests.
Assuntos
Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Infertilidade Masculina/patologia , Alcamidas Poli-Insaturadas/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Amidoidrolases/metabolismo , Ácidos Araquidônicos/metabolismo , Cromatografia Líquida de Alta Pressão , Estudos Transversais , Endocanabinoides/metabolismo , Citometria de Fluxo , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Fosfolipase D/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , RNA Mensageiro/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Sêmen/metabolismo , Espermatozoides/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
Quantitative real-time polymerase chain reaction (qRT-PCR) has been employed to study the gene expression profiles in human spermatozoa, but accurate analysis is dependent upon normalisation of data against an endogenous control. ß-Actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are the most commonly used reference genes for normalisation of gene expression in human spermatozoa, but the expression of these genes in many tissues has considerable variation under different physiological, pathological and experimental conditions which limits their effectiveness in normalisation. The expression stability of a panel of 12 reference genes was studied in normal and pathological human spermatozoa using geNorm and NormFinder software. Although there were some discrepancies in the ranking of reference gene stability, each software program ranked B2 M, ACTB, CYC1 and 18S RNA within the top 5 and recommended the combined use of at least two reference genes. Normalisation of qRT-PCR data for the cannabinoid receptor type 2 in spermatozoa using the different housekeeping genes demonstrated how, without validation, conflicting results are obtained. We recommend that the arbitrary use of reference genes should be avoided and the validation of reference gene stability should be undertaken prior to every study. For normalisation of CB2 expression, we would recommend using the geometric mean of B2 M and ACTB.
Assuntos
Perfilação da Expressão Gênica/normas , Infertilidade Masculina/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Espermatozoides/metabolismo , Humanos , Masculino , RNA Mensageiro/metabolismo , Receptor CB2 de Canabinoide/genética , Padrões de ReferênciaRESUMO
BACKGROUND: Marijuana, the most used recreational drug, has been shown to have adverse effects on human reproduction. Endogenous cannabinoids (also called endocannabinoids) bind to the same receptors as those of Δ(9)-tetrahydrocannabinol (THC), the psychoactive component of Cannabis sativa. The most extensively studied endocannabinoids are anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol. The endocannabinoids, their congeners and the cannabinoid receptors, together with the metabolic enzymes and putative transporters form the endocannabinoid system (ECS). In this review, we summarize current knowledge about the relationships of ECS, sex steroid hormones and cytokines in female fertility, and underline the importance of this endocannabinoid-hormone-cytokine network. METHODS: Pubmed and the Web of Science databases were searched for studies published since 1985, looking into the ECS, sex hormones, type-1/2 T-helper (Th1/Th2) cytokines, leukaemia inhibitory factor, leptin and reproduction. RESULTS: The ECS plays a pivotal role in human reproduction. The enzymes involved in the synthesis and degradation of endocannabinoids normalize levels of AEA for successful implantation. The AEA degrading enzyme (fatty acid amide hydrolase) activity as well as AEA content in blood may potentially be used for the monitoring of early pregnancies. Progesterone and oestrogen are involved in the maintenance of endocannabinoid levels. The ECS plays an important role in the immune regulation of human fertility. CONCLUSIONS: The available studies suggest that tight control of the endocannabinoid-hormone-cytokine network is required for successful implantation and early pregnancy maintenance. This hormone-cytokine network is a key element at the maternal-foetal interface, and any defect in such a network may result in foetal loss.
Assuntos
Moduladores de Receptores de Canabinoides/fisiologia , Citocinas/fisiologia , Endocanabinoides , Fertilidade/fisiologia , Hormônios Esteroides Gonadais/fisiologia , Moduladores de Receptores de Canabinoides/metabolismo , Feminino , Humanos , Transdução de SinaisRESUMO
The main endocannabinoids (EC) identified in mammalian tissues are N-arachidonoylethanolamide (AEA, anandamide), and 2-arachidonoylglycerol (2-AG). AEA levels are critical in pregnancy, especially during implantation, decidualization, and placental development. As 2-AG functions in pregnancy are still largely undefined, we hypothesized that it may also have a role during fetoplacental development. We showed that 2-AG is not only present in the rat mesometrial decidua and plasma during fetoplacental development, but that both 2-AG synthesizing (diacylglycerol lipase) and degradation (monoacylglycerol lipase) enzymes are expressed by decidual cells. While lower concentrations of 2-AG induced apoptosis of rat primary decidual cells, via the CB1 receptor, higher concentrations induced a dramatic effect on cell morphology, cell viability and lactate dehydrogenase release, triggered through a mechanism independent of CB1. This study provides evidences that 2-AG fluctuation in maternal tissues throughout normal pregnancy is primarily regulated by its metabolizing enzymes. Together, these data supports the hypothesis that a deregulation of the endocannabinoid system through aberrant cannabinoid signalling may impact normal uterine remodelling process and consequently normal pregnancy.
Assuntos
Ácidos Araquidônicos/metabolismo , Glicerídeos/metabolismo , Placentação/fisiologia , Útero/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Sobrevivência Celular , Células Cultivadas , Decídua/citologia , Decídua/metabolismo , Endocanabinoides , Feminino , Imuno-Histoquímica , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Masculino , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Placentação/genética , Reação em Cadeia da Polimerase , Gravidez , Ratos , Ratos WistarRESUMO
Decidualization is essential for a successful pregnancy and is a tightly regulated process influenced by the local microenvironment. Lipid-based mediators, such as the endocannabinoid anandamide, and other compounds that have cannabimimetic actions may act on the decidua during early pregnancy. In this study, the levels of N-arachidonoylethanolamine (anandamide) and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in rat plasma and maternal tissues between d 8 and 19 of pregnancy by ultraperformance liquid chromatography tandem mass spectrometry. The spatiotemporal expression of N-acylethanolamine metabolizing enzymes in implantation units were also determined by quantitative PCR, Western blot, and immunohistochemistry and shown to vary with gestation being mainly localized in decidual cells. The data also indicated that plasma and tissues levels of all three N-acylethanolamines fluctuate throughout pregnancy. Tissue levels of endocannabinoids did not correlate with plasma, suggesting that during pregnancy, maternal tissue levels of endocannabinoids are primarily regulated by in situ production and degradation to create endocannabinoid gradients conducive to successful pregnancy.
Assuntos
Enzimas/genética , Etanolaminas/sangue , Etanolaminas/metabolismo , Gravidez/sangue , Gravidez/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Moduladores de Receptores de Canabinoides/sangue , Moduladores de Receptores de Canabinoides/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Decídua/metabolismo , Decídua/fisiologia , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Enzimas/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , Fosfolipase D/genética , Fosfolipase D/metabolismo , Gravidez/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
OBJECTIVE: Plasma anandamide (AEA) levels have previously been shown to be elevated in labour and defective cannabinoid receptor type 1 signalling in mice has been shown to be associated with elevation of corticotrophin-releasing hormone and spontaneous onset of preterm labour. We measured plasma AEA levels in women undergoing induction of labour to define the changes during the transition from the nonlabouring to labouring state. DESIGN: A longitudinal observational study. SETTING: A large UK teaching hospital. POPULATION: Term pregnant women undergoing induction of labour. METHODS: Blood was collected from women before induction of labour and again when they were in active labour. Plasma AEA was extracted and measured using ultraperformance liquid chromatography-tandem mass spectrometry. MAIN OUTCOME MEASURES: The primary outcome variable was change in plasma AEA levels from the nonlabouring to the labouring state. The secondary outcome was induction-to-delivery interval. RESULTS: There was a 1.5-fold increase in mean plasma AEA levels from 1.20 +/- 0.57 nm in the nonlabouring state to 1.82 +/- 0.87 nm in the labouring state (P < 0.0001). Induction-to-delivery interval was predicted by both Bishop's score (P < 0.0001) and percentage change in plasma AEA levels (P < 0.0001). There was a negative correlation between the percentage change in plasma AEA level and the induction-to-delivery interval (r = - 0.28; P = 0.0481). This means that the greater the rise in the plasma AEA levels the shorter the duration of labour. CONCLUSIONS: Plasma AEA levels increase with active labour and the negative correlations between percentage change in plasma AEA levels and induction-to-delivery interval suggest that AEA is likely to be involved in the physiological mechanisms of labour. Whether this increase is essential for myometrial contraction is unclear and needs further investigation.
Assuntos
Ácidos Araquidônicos/sangue , Moduladores de Receptores de Canabinoides/sangue , Endocanabinoides , Trabalho de Parto/sangue , Alcamidas Poli-Insaturadas/sangue , Adolescente , Adulto , Cromatografia Líquida , Feminino , Humanos , Trabalho de Parto Induzido , Gravidez , Adulto JovemRESUMO
INTRODUCTION: Prostate cancer is an excellent target for chemoprevention strategies; given its late age of onset, any delay in carcinogenesis would lead to a reduction in its incidence. This article reviews all the completed and on-going phase III trials in prostate cancer chemoprevention. PATIENTS AND METHODS: All phase III trials of prostate cancer chemoprevention were identified within a Medline search using the keywords 'clinical trial, prostate cancer, chemoprevention'. RESULTS: In 2003, the Prostate Cancer Prevention Trial (PCPT) became the first phase III clinical trial of prostate cancer prevention. This landmark study was terminated early due to the 24.8% reduction of prostate cancer prevalence over a 7-year period in those men taking the 5alpha-reductase inhibitor, finasteride. This article reviews the PCPT and the interpretation of the excess high-grade prostate cancer (HGPC) cases in the finasteride group. The lack of relationship between cumulative dose and the HGPC cases, and the possible sampling error of biopsies due to gland volume reduction in the finasteride group refutes the suggestion that this is a genuine increase in HGPC cases. The other on-going phase III clinical trials of prostate cancer chemoprevention - the REDUCE study using dutasteride, and the SELECT study using vitamin E and selenium - are also reviewed. CONCLUSIONS: At present, finasteride remains the only intervention shown in long-term prospective phase III clinical trials to reduce the incidence of prostate cancer. Until we have the results of trials using alternative agents including the on-going REDUCE and SELECT trials, the advice given to men interested in prostate cancer prevention must include discussion of the results of the PCPT. The increased rate of HGPC in the finasteride group continues to generate debate; however, finasteride may still be suitable for prostate cancer prevention, particularly in men with lower urinary tract symptoms.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Próstata/prevenção & controle , Azasteroides/uso terapêutico , Ensaios Clínicos Fase III como Assunto , Dutasterida , Inibidores Enzimáticos/uso terapêutico , Finasterida/uso terapêutico , Previsões , Humanos , Masculino , Compostos de Selênio/uso terapêutico , Vitamina E/uso terapêuticoRESUMO
We have previously demonstrated the inhibitory effect of chlorophyllin, a green food additive, on the genotoxicities of various carcinogens in Drosophila. Recently, we reported that purpurin, a component of a red food additive produced from madder root (Rubia tinctorium), inhibits the bacterial mutagenicity of heterocyclic amines. In the present study, we examined antigenotoxic activities of various pigments that are either constituents of food or food additives, using Drosophila in vivo DNA repair assay. Third instar larvae of Drosophila were fed a mutagen with or without pigment. The resulting adult flies were monitored for their male (repair deficient)/female (repair proficient) ratios, which reflect the DNA damage. We tested a total of 20 pigments, which are mainly of plant origins, including flavonoids, carotenoids, anthocyanins, anthraquinones and beta-diketone (curcumin)-derivatives, against the genotoxicities of eight carcinogens; IQ, MeIQx, AFB1, NDMA, 2-AAF, DMBA, 4NQO, and MNU. Four anthraquinone pigments (alizarin, purpurin, lac color, and cochineal extract) showed significant antigenotoxic activities. Alizarin and purpurin suppressed the DNA damage induced by IQ, MeIQx, AFB1, NDMA, 2-AAF, DMBA, and MNU. Lac color and cochineal extract showed inhibition against IQ, MeIQx, AFB1, 2-AAF and DMBA. In these inhibitions, suppression of metabolic enzymes may be involved. Since purpurin and alizarin suppressed the activity of MNU, a direct alkylating agent, there may also be a mechanism distinct from enzyme inhibitions in these anthraquinone-mediated suppressions of DNA damage.
Assuntos
Antraquinonas/farmacologia , Antimutagênicos/farmacologia , Carcinógenos/toxicidade , Carmim/análogos & derivados , Dano ao DNA/efeitos dos fármacos , Corantes de Alimentos/farmacologia , Animais , Bioensaio , Carmim/farmacologia , Quimioprevenção , Relação Dose-Resposta a Droga , Drosophila , Feminino , Masculino , Testes de MutagenicidadeRESUMO
Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) is a naturally occurring anthraquinone pigment found in species of madder root. We have found that the presence of purpurin in bacterial mutagenicity assays is responsible for a marked inhibition of mutagenicity induced by food-derived heterocyclic amines. Purpurin was found to be a better inhibitor of Trp-P-2-dependent mutagenicity than either epigallocatechin gallate or chlorophyllin both of which are well-established anti-mutagenic components of diet. Inhibition of Trp-P-2(NHOH) mutagenicity by purpurin was dependent upon pH. It was a better inhibitor in neutral than acidic conditions. Purpurin was protective against the direct mutagen Trp-P-2(NHOH) in both the presence and the absence of hepatic S9 but required pre-incubation. Finally, purpurin was responsible for the inhibition of human CYP1A2 and human NADPH-cytochrome P450 reductase and a decrease in the bioactivation of Trp-P-2 by these enzymes when they were expressed in Salmonella typhimurium TA1538ARO. However, inhibition of Trp-P-2(NHOH)-dependent mutations suggests purpurin also has a direct effect on this mutagen in addition to inhibiting its formation by CYP1A2.