RESUMO
Mobile phone usage has become an integral part of our lives. However, the effects of the radiofrequency electromagnetic radiation (RF-EMR) emitted by these devices on biological systems and specifically the reproductive systems are currently under active debate. A fundamental hindrance to the current debate is that there is no clear mechanism of how such non-ionising radiation influences biological systems. Therefore, we explored the documented impacts of RF-EMR on the male reproductive system and considered any common observations that could provide insights on a potential mechanism. Among a total of 27 studies investigating the effects of RF-EMR on the male reproductive system, negative consequences of exposure were reported in 21. Within these 21 studies, 11 of the 15 that investigated sperm motility reported significant declines, 7 of 7 that measured the production of reactive oxygen species (ROS) documented elevated levels and 4 of 5 studies that probed for DNA damage highlighted increased damage due to RF-EMR exposure. Associated with this, RF-EMR treatment reduced the antioxidant levels in 6 of 6 studies that discussed this phenomenon, whereas consequences of RF-EMR were successfully ameliorated with the supplementation of antioxidants in all 3 studies that carried out these experiments. In light of this, we envisage a two-step mechanism whereby RF-EMR is able to induce mitochondrial dysfunction leading to elevated ROS production. A continued focus on research, which aims to shed light on the biological effects of RF-EMR will allow us to test and assess this proposed mechanism in a variety of cell types.
Assuntos
Radiação Eletromagnética , Estresse Oxidativo/efeitos da radiação , Espermatozoides/fisiologia , Animais , Telefone Celular , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/efeitos da radiaçãoRESUMO
STUDY QUESTION: What are the mechanisms by which the preparation of spermatozoa on discontinuous density gradients leads to an increase in oxidative DNA damage? SUMMARY ANSWER: The colloidal silicon solutions that are commonly used to prepare human spermatozoa for assisted reproduction technology (ART) purposes contain metals in concentrations that promote free radical-mediated DNA damage. WHAT IS KNOWN ALREADY: Sporadic reports have already appeared indicating that the use of colloidal silicon-based discontinuous density gradients for sperm preparation is occasionally associated with the induction of oxidative DNA damage. The cause of this damage is however unknown. STUDY DESIGN, SIZE, DURATION: This study comprised a series of experiments designed to: (i) confirm the induction of oxidative DNA damage in spermatozoa prepared on commercially available colloidal silicon gradients, (ii) compare the levels of damage observed with alterative sperm preparation techniques including an electrophoretic approach and (iii) determine the cause of the oxidative DNA damage and develop strategies for its prevention. The semen samples employed for this analysis involved a cohort of >50 unselected donors and at least three independent samples were used for each component of the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computer-aided sperm analysis to measure sperm movement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media. MAIN RESULTS AND THE ROLE OF CHANCE: Oxidative DNA damage is induced in spermatozoa prepared on PureSperm(®) discontinuous colloidal silicon gradients (P < 0.001 versus repeated centrifugation) because this medium contains metals, particularly Fe, Al and Cu, which are known to promote free radical generation in the immediate vicinity of DNA. This damage can be significantly accentuated by reducing agents, such as ascorbate (P < 0.001) and inhibited by selective chelation (P < 0.001). This problem is not confined to PureSperm(®); analysis of additional commercial sperm preparation media revealed that metal contamination is a relatively constant feature of such products. LIMITATIONS, REASONS FOR CAUTION: While the presence of metals, particularly transition metals, may exacerbate the levels of oxidative DNA damage seen in human spermatozoa, the significance of such damage has not yet been tested in suitably powered clinical trials. WIDER IMPLICATIONS OF THE FINDINGS: The results explain why the preparation of spermatozoa on discontinuous colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Australian Health and Medical Research Council. One of the authors (R.J.A.) has had a consultantship with a biotechnology company, NuSep, interested in the development of electrophoretic methods of sperm preparation. He has no current financial interest in this area. None of the other authors have a conflict of interest to declare.
Assuntos
Dano ao DNA , Silício/farmacologia , Espermatozoides/efeitos dos fármacos , Centrifugação/efeitos adversos , Estudos de Coortes , Coloides/química , Citometria de Fluxo , Humanos , Peroxidação de Lipídeos , Masculino , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Manejo de Espécimes/efeitos adversos , Espermatozoides/citologia , Elementos de Transição/análise , Elementos de Transição/farmacologiaRESUMO
This article considers the origins of DNA damage in human spermatozoa, the methods that are available to monitor this aspect of semen quality and the clinical significance of such measurements. DNA damage in spermatozoa appears to be largely oxidative in nature, inversely correlated with levels of nuclear protamination and frequently associated with the activation of a truncated apoptotic pathway. DNA base adducts formed as a result of oxidative attack are released from the spermatozoa into the extracellular space through the action of a glycosylase, OGG1. This creates an abasic site, which is not resolved until fertilization because spermatozoa do not possess the molecular machinery needed to continue the base excision repair pathway. The abasic sites so generated in human spermatozoa are readily detected by SCSA or the Comet assay; however, no signal is detectable with TUNEL. This is because spermatozoa lack the enzyme (APE1) needed to create the free 3' hydroxyl groups required by this detection system. Nevertheless, spermatozoa do eventually become TUNEL positive as they enter the perimortem. The American Society of Reproductive Medicine Practice Committee has suggested that DNA damage in spermatozoa should not be assessed because the correlation with pregnancy is inconsistent across independent studies. However, this is a straw man argument. The reason why such assays should be undertaken is not just that they reflect the underlying quality of spermatogenesis but, more importantly, that the DNA damage they reveal may have detrimental effects on the developmental normality of the embryo and the health of possible future children.
Assuntos
Dano ao DNA/genética , Análise do Sêmen , Espermatogênese/genética , Espermatozoides/anormalidades , Apoptose/genética , Feminino , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/genética , Masculino , Oxirredução , Estresse Oxidativo , GravidezRESUMO
The discovery of a truncated base excision repair pathway in human spermatozoa mediated by OGG1 has raised questions regarding the effect of mutations in critical DNA repair genes on the integrity of the paternal genome. The senescence-accelerated mouse prone 8 (SAMP8) is a mouse model containing a suite of naturally occurring mutations resulting in an accelerated senescence phenotype largely mediated by oxidative stress, which is further enhanced by a mutation in the Ogg1 gene, greatly reducing the ability of the enzyme to excise 8-hydroxy,2'-deoxyguanosine (8OHdG) adducts. An analysis of the reproductive phenotype of the SAMP8 males revealed a high level of DNA damage in caudal epididymal spermatozoa as measured by the alkaline Comet assay. Furthermore, these lesions were confirmed to be oxidative in nature, as demonstrated by significant increases in 8OHdG adduct formation in the SAMP8 testicular tissue (P<0.05) as well as in mature spermatozoa (P<0.001) relative to a control strain (SAMR1). Despite this high level of oxidative DNA damage in spermatozoa, reactive oxygen species generation was not elevated and motility of spermatozoa was found to be similar to that for the control strain with the exception of progressive motility, which exhibited a slight but significant decline with advancing age (P<0.05). When challenged with Fenton reagents (H2O2 and Fe2+), the SAMP8 spermatozoa demonstrated a highly increased susceptibility to formation of 8OHdG adducts compared with the controls (P<0.001). These data highlight the role of oxidative stress and OGG1-dependent base excision repair mechanisms in defining the genetic integrity of mammalian spermatozoa.
Assuntos
Envelhecimento/fisiologia , DNA Glicosilases/fisiologia , Reparo do DNA , Modelos Animais , Estresse Oxidativo/fisiologia , Espermatozoides/metabolismo , Animais , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Fertilidade/fisiologia , Masculino , Camundongos , Fenótipo , Espermatogênese/fisiologiaRESUMO
Our ability to diagnose and treat male infertility is gradually improving in concert with advances in our understanding of the molecular mechanisms underpinning defective sperm function. In this context, one of the factors to emerge as a major causative agent in male infertility is oxidative stress. Spermatozoa are particularly susceptible to such stress because they are exceptionally rich in vulnerable substrates such as polyunsaturated fatty acids, proteins and DNA. The lack of sperm cytoplasm also provides these cells with little capacity to protect themselves from oxidative attack or to effect any repair, should damage occur. Similarly, sperm chromatin is in a quasi-crystalline state and has very little capacity to respond to any DNA damage induced by oxidative attack. When the latter does occur, it appears to be initiated by reactive oxygen species (ROS) generated by the sperm mitochondria. These free radicals attack the lipids present in the sperm mitochondria generating electrophilic aldehydes, which bind to components of the mitochondrial electron transport chain stimulating yet more ROS production. The oxidative stress created via this self-propagating mechanism initiates an apoptotic cascade as a result of which the spermatozoa loose their capacity for fertilization and suffer damage to their DNA. Phosphatidylserine externalization is a late event in sperm apoptosis and may facilitate the silent phagocytosis of moribund cells in the female reproductive tract, that is, the phagocytosis of senescent spermatozoa without the accompanying generation of an inflammatory response. Encouragingly, the involvement of oxidative stress in the aetiology of male infertility has opened up new opportunities for therapeutic interventions involving the judicious administration of nucleophiles and other forms of antioxidants.
Assuntos
Apoptose/fisiologia , Dano ao DNA/fisiologia , Estresse Oxidativo/fisiologia , Espermatozoides/fisiologia , Animais , Infertilidade Masculina , MasculinoRESUMO
The purpose of this study was to evaluate the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay as a method for assessing DNA damage in human spermatozoa. The conventional assay was shown to be insensitive and unresponsive to the DNA fragmentation induced in human and mouse spermatozoa on exposure to Fenton reagents (H2O2 and Fe(2+) ). However, both time- and dose-dependent responses could be readily detected if the chromatin was exposed to 2 mm dithiothreitol (DTT) for 45 min prior to fixation. This modified version of the assay significantly enhanced the TUNEL signals generated by subpopulations of spermatozoa isolated on discontinuous Percoll gradients as well as the responses triggered by reagents (arachidonic acid and menadione) that are known to stimulate superoxide anion production by human spermatozoa. DTT exposure also improved the signals detected with chromomycin A3 (CMA3), a probe designed to determine the efficacy of chromatin protamination, and enhanced the correlation observed between this criterion of sperm quality and the TUNEL assay. Finally, the output of the TUNEL assay was found to be highly correlated with sperm vitality. The TUNEL methodology was therefore further refined to incorporate a vital stain that covalently bound to intracellular amine groups in non-viable cells. This tag remained associated with the spermatozoa during fixation and processing for the TUNEL assay so that ultimately, both DNA integrity and vitality could be simultaneously assessed in the same flow cytometry assay. The methods described in this article are simple and robust and should facilitate research into the causes of DNA damage in human spermatozoa.
Assuntos
Dano ao DNA , DNA/análise , Marcação In Situ das Extremidades Cortadas/métodos , Espermatozoides , Animais , Sobrevivência Celular , Cromatina , Cromomicina A3 , Ditiotreitol , Citometria de Fluxo , Humanos , Peróxido de Hidrogênio/farmacologia , Ferro/farmacologia , Masculino , Camundongos , Sensibilidade e Especificidade , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , Coloração e RotulagemRESUMO
DNA damage in the male germ line has been linked with a variety of adverse clinical outcomes including impaired fertility, an increased incidence of miscarriage and an enhanced risk of disease in the offspring. The origins of this DNA damage could, in principle, involve: (i) abortive apoptosis initiated post meiotically when the ability to drive this process to completion is in decline (ii) unresolved strand breaks created during spermiogenesis to relieve the torsional stresses associated with chromatin remodelling and (iii) oxidative stress. In this article, we present a two-step hypothesis for the origins of DNA damage in human spermatozoa that highlights the significance of oxidative stress acting on vulnerable, poorly protaminated cells generated as a result of defective spermiogenesis. We further propose that these defective cells are characterized by several hallmarks of 'dysmaturity' including the retention of excess residual cytoplasm, persistent nuclear histones, poor zona binding and disrupted chaperone content. The oxidative stress experienced by these cells may originate from infiltrating leukocytes or, possibly, the entry of spermatozoa into an apoptosis-like cascade characterized by the mitochondrial generation of reactive oxygen species. This oxidative stress may be exacerbated by a decline in local antioxidant protection, particularly during epididymal maturation. Finally, if oxidative stress is a major cause of sperm DNA damage then antioxidants should have an important therapeutic role to play in the clinical management of male infertility. Carefully controlled studies are now needed to critically examine this possibility.
Assuntos
Dano ao DNA/fisiologia , Espermatozoides/metabolismo , Cromatina/metabolismo , Dano ao DNA/genética , Humanos , Masculino , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismoRESUMO
We report that the naphthalimide analogue 2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (NAP-6) is a highly potent and selective breast cancer targeting molecule. These effects are mediated via the aryl hydrocarbon receptor (AHR) pathway and the subsequent induction of CYP1 metabolising monooxygenases in breast cancer cell line models. Indeed the triple negative breast cancer cell line MDA-MB-468 with a GI50 value of 100 nM is greater than 500-fold more sensitive to NAP-6 compared with other tumour derived cell models. Within 1 h exposure of these cells to NAP-6, CYP1A1 expression increases 25-fold, rising to 250-fold by 24 h. A smaller concurrent increase in CYP1A2 and CYP1B1 is also observed. Within 24 h these cells present with DNA damage as evident by enhanced H2AXγ expression, cell cycle checkpoint activation via increased CHK2 expression, S-phase cell cycle arrest and cell death. Specific small molecule inhibitors of the AHR and CYP1 family ameliorate these events. A positive luciferase reporter assay for NAP-6 induced XRE binding further confirms the role of the AHR in this phenomenon. Non-sensitive cell lines fail to show these biological effects. For the first time we identify 2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione as a new AHR ligand that selectively targets breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Naftalimidas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Dano ao DNA , Indução Enzimática/efeitos dos fármacos , Feminino , Células HT29 , Humanos , Células MCF-7 , Estrutura Molecular , Naftalimidas/química , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: Whereas studies have revealed that the cryopreservation of human semen increases sperm DNA fragmentation, the mechanisms involved in this type of cryo-injury are largely unknown. Elucidation of these mechanisms may provide insight into preventing such injury. METHODS: We obtained 60 semen samples from 60 men and conducted experiments to determine the cause of cryopreservation-induced DNA fragmentation using 8-oxo-7,8-dihydro-2'deoxyguanosine (8OHdG) as a biomarker of oxidative stress, percentage caspase positive cells as an indicator of apoptosis, the potential antioxidant genistein and the caspase inhibitor Z-VAD(OMe)-FMK. RESULTS: Cryopreservation led to a significant increase in percentage DNA fragmentation, percentage 8OHdG and percentage caspase positive cells (P < 0.001). Percentage DNA fragmentation was positively correlated with percentage 8OHdG before (r = 0.756, P < 0.001) and after cryopreservation (r = 0.528, P = 0.017). The addition of 50 and 100 microM genistein to the cryoprotectant had a significant protective effect on sperm DNA (P < 0.001) although the caspase inhibitor demonstrated no difference to the control. CONCLUSIONS: Human sperm DNA fragmentation is associated with an increase in oxidative stress during cryopreservation, rather than the activation of caspases and apoptosis. The estrogenic compound genistein may be useful in reducing this effect but larger trials are needed to confirm this.
Assuntos
Apoptose , Criopreservação , Dano ao DNA , Estresse Oxidativo , Preservação do Sêmen/efeitos adversos , Espermatozoides/fisiologia , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Caspases/metabolismo , Dano ao DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Desoxiguanosina/análogos & derivados , Genisteína/farmacologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Our understanding of epididymal physiology and function has been transformed over the three decades in which the International Meeting Series on the Epididymis has been hosted. This transformation has occurred along many fronts, but among the most significant advances has been the unexpected discovery of the diversity of small non-protein-coding RNAs (sRNAs) expressed in the epididymal epithelium and differentially accumulated in the luminal population of spermatozoa. OBJECTIVES: Here we survey recent literature pertaining to profiling the sRNA landscape of the mammalian epididymis with the goal of demonstrating the contribution that these key regulatory elements, and their associated pathways, make to epididymal physiology and sperm maturation. RESULTS AND DISCUSSION: High throughput sequencing strategies have fueled an unprecedented advance in our understanding of RNA biology. In the last decade, such high throughput profiling tools have been increasingly applied to study the mammalian epididymis, presaging the discovery of diverse classes of sRNA expressed along the length of the tract. Among the best studied sRNA classes are the microRNAs (miRNA), a sRNA species shown to act in concert with endocrine signals to fine-tune the segmental patterning of epididymal gene expression. In addition to performing this homeostatic role, epithelial cell-derived sRNAs also selectively accumulate into the epididymosomes and spermatozoa that occupy the duct lumen. This exciting discovery alludes to a novel form of intracellular communication that contributes to the establishment of the sperm epigenome and its modification under conditions of paternal stress. CONCLUSION: Compelling literature has identified sRNAs as a crucial regulatory tier that allows the epididymis to fulfill its combined roles of sperm transport, maturation, and storage. Continued research in this emerging field will contribute to our growing understanding of the etiology of male factor infertility and potentially allow for the future design of rational therapeutic options for these individuals.
Assuntos
Epididimo/metabolismo , MicroRNAs/genética , RNA Interferente Pequeno/genética , Maturação do Esperma/genética , Espermatozoides/metabolismo , Animais , Microambiente Celular/fisiologia , Epididimo/citologia , Epitélio/metabolismo , Humanos , Masculino , Espermatozoides/citologiaRESUMO
A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.
Assuntos
Adutos de DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Estrogênios/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Ensaio Cometa , Reagentes de Ligações Cruzadas , Etídio/análogos & derivados , Etídio/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Oxirredução , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
The need to protect human spermatozoa from oxidative stress during assisted reproductive technology, has prompted a detailed analysis of the impacts of phenolic compounds on the functional integrity of these cells. Investigation of 16 individual compounds revealed a surprising variety of negative effects including: (i) a loss of mitochondrial membrane potential (Δψm) via mechanisms that were not related to opening of the permeability transition pore but associated with a reduction in thiol expression, (ii) a decline in intracellular reduced glutathione, (iii) the stimulation of pro-oxidant activity including the induction of ROS generation from mitochondrial and non-mitochondrial sources, (iv) stimulation of lipid peroxidation, (v) the generation of oxidative DNA damage, and (vi) impaired sperm motility. For most of the polyphenolic compounds examined, the loss of motility was gradual and highly correlated with the induction of lipid peroxidation (r=0.889). The exception was gossypol, which induced a rapid loss of motility due to its inherent alkylating activity; one consequence of which was a marked reduction in carboxymethyl lysine expression on the sperm tail; a post-translational modification that is known to play a key role in the regulation of sperm movement. The only polyphenols that did not appear to have adverse effects on spermatozoa were resveratrol, genistein and THP at doses below 100µM. These compounds could, therefore, have some therapeutic potential in a clinical setting.
Assuntos
Dano ao DNA , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Polifenóis/efeitos adversos , Espermatozoides/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , Masculino , Potencial da Membrana Mitocondrial/genética , Estresse Oxidativo/genética , Polifenóis/administração & dosagem , Polifenóis/química , Técnicas de Reprodução Assistida , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Espermatozoides/patologiaRESUMO
Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5-diaminofluorescein diacetate and 2',7'-dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2-hydroxyestradiol, menadione, 4-hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol-peroxidase and, to a lesser extent, 2',7'-dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.
Assuntos
Citometria de Fluxo/métodos , Medições Luminescentes/métodos , Espécies Reativas de Oxigênio/metabolismo , Espectrofotometria/métodos , Espermatozoides/metabolismo , Aldeídos/análise , Aldeídos/química , Aldeídos/metabolismo , Ácido Araquidônico/análise , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Estradiol/análogos & derivados , Estradiol/química , Estrogênios de Catecol/metabolismo , Etídio/análogos & derivados , Etídio/química , Fluoresceínas/química , Humanos , Leucócitos/metabolismo , Luminescência , Luminol , Masculino , Mitocôndrias/metabolismo , Estresse Oxidativo , Fenantridinas/química , Sensibilidade e Especificidade , Vitamina K 3/análise , Vitamina K 3/química , Vitamina K 3/metabolismoRESUMO
Dasyurids are a diverse group of Australian native carnivores and insectivores that contains several threatened species. Despite successful cryopreservation of sperm from several marsupials, only 3% postthaw motility is reported for dasyurid marsupials. This study examined sperm preservation in the fat-tailed dunnart (Sminthopsis crassicaudata), an experimental model, with supplementary observations on the eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In S. crassicaudata, a toxicity trial demonstrated that incubation with up to 40% glycerol did not reduce sperm viability, suggesting that glycerol is not toxic to dasyurids. On the basis of this finding, S. crassicaudata, D. viverrinus, and D. hallucatus sperm were extended to a final concentration of 20% or 40% glycerol in Tris-citrate fructose and frozen in liquid nitrogen vapor. Postthaw sperm from all three species were nonmotile, and vital staining (SYBR14 and propidium iodide) indicated that sperm were nonviable. However, there was no evidence suggesting disruption of normal gross morphology or loss of acrosomal integrity when assessed by Bryan's staining. After freeze drying, Bryan's staining indicated that approximately 80% of S. crassicaudata sperm had normal acrosomes and no head loss. Despite being nonviable, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling showed that S. crassicaudata sperm frozen in 40% glycerol or freeze-dried had no DNA damage compared with fresh controls. This study has described a method for preservation of the dasyurid sperm nuclei, but continued studies are required to achieve viable motile sperm and establish tools for the long-term storage of dasyurid sperm.