RESUMO
In a recent study, lactoferrin (LF) was detected in human oviductal secretion. The protein was able to bind to oocytes and sperm, and modulated gamete interaction. The aim of the present study was to investigate the effect of LF on parameters related to human sperm capacitation and sperm-zona pellucida interaction. Semen samples were obtained from healthy normozoospermic donors (n = 7). Human follicular fluids and oocytes were collected from patients undergoing in vitro fertilization. Motile sperm obtained by swim-up were incubated for 6 or 22 h under capacitating conditions with LF (0-100 µg/mL). After incubations, viability, motility, presence of α-d-mannose receptors (using a fluorescent probe on mannose coupled to bovine serum albumin), spontaneous and induced acrosome reaction (assessed with Pisum sativum agglutinin conjugated to fluorescein isothiocyanate), and tyrosine phosphorylation of sperm proteins were evaluated. Sperm-zona pellucida interaction in the presence of LF was investigated using the hemizone assay. The presence of LF did not affect sperm viability or motility, but caused a dose-dependent significant decrease in sperm α-d-mannose-binding sites, and the effect was already significant with the lowest concentration of the protein used after 22 h incubation. Dose-dependent significant increases in both induced acrosome reaction and tyrosine phosphorylation of sperm proteins were observed in the presence of LF. The present data indicate that LF modulates parameters of sperm function. The inhibition of gamete interaction by LF could be partially explained by the decrease in sperm d-mannose-binding sites. The presence of the LF promoted sperm capacitation in vitro.
Assuntos
Lactoferrina/farmacologia , Oócitos/efeitos dos fármacos , Capacitação Espermática/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Feminino , Humanos , Lactoferrina/metabolismo , Lectinas Tipo C/efeitos dos fármacos , Lectinas Tipo C/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/efeitos dos fármacos , Lectinas de Ligação a Manose/metabolismo , Oócitos/metabolismo , Fosforilação , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Fatores de Tempo , Tirosina , Adulto JovemRESUMO
The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham's F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen-free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.
Assuntos
Fragmentação do DNA , Espécies Reativas de Oxigênio/análise , Espermatozoides/metabolismo , Reação Acrossômica , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos , Masculino , Oxirredução , Técnicas de Reprodução Assistida/efeitos adversos , Capacitação Espermática , Espermatozoides/química , Espermatozoides/fisiologiaRESUMO
STUDY QUESTION: Is lactoferrin (LF) (detected in oviductal secretion) able to bind to oocytes and sperm and modulate gamete interaction? SUMMARY ANSWER: LF binds to zona pellucida (ZP) and spermatozoa (depending upon the capacitation stage and acrosome status) and inhibits gamete interaction in vitro. WHAT IS KNOWN ALREADY: Proteins from human oviductal tissue secretion modulate gamete interaction and parameters of sperm function in vitro and some of them bind to sperm, but they remain to be isolated and identified. STUDY DESIGN, SIZE, DURATION: Proteins were isolated from human oviductal tissue secretion using their sperm membrane binding ability. One of the isolated proteins was identified as human LF and immunolocalized in tubal tissues. LF expression was analyzed in native oviductal fluid and oviduct epithelial cells (at different phases of the menstrual cycle: proliferative, periovulatory and secretory). In addition, the LF binding sites on spermatozoa (at different capacitation and acrosome reaction stages) and on ZP and the dose-dependent effect of LF on gamete interaction were investigated. All experiments were performed at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tubal tissues obtained from premenopausal patients (scheduled for hysterectomy, n = 23) were cultured in DMEM/Ham's F12 medium and conditioned media (CM) were collected. Motile spermatozoa were obtained by swim-up from normozoospermic semen samples from healthy donors (n = 4). An affinity chromatography with sperm membrane extracts was used to isolate proteins from CM. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophresis and further identified by nano liquid chromatography tandem mass spectrometry peptide sequencing. The presence of LF in oviductal tissue was investigated by immunohistochemistry and immunofluorescence and was detected in native oviductal fluid and oviduct epithelial cells homogenates by western blot. LF binding sites on gametes were investigated by incubating gametes with the protein coupled to fluorescein isothiocyanate (FITC). The acrosome reaction was assessed with Pisum sativum agglutinin conjugated with rhodamine. The effect of increasing concentrations of LF (0.1-100 µg/ml) on gamete interaction was evaluated by a sperm-ZP binding assay, using human oocytes donated by women undergoing IVF procedures. MAIN RESULTS AND THE ROLE OF CHANCE: A protein isolated by the affinity column was identified as human LF. LF was immunolocalized in human oviductal tissue and detected in oviductal fluid and oviduct epithelial cell homogenates. In the latter case, LF expression was highest at the periovulatory phase of the menstrual cycle (P < 0.01). Different LF binding patterns were observed on spermatozoa depending upon capacitation stage and if the acrosome reaction had occurred. Unstained sperm were most prevalent before capacitation, but after incubation for 6 h under capacitating conditions and in acrosome-reacted sperm LF binding was observed, mainly localized in the equatorial segment and post-acrosomal region of the sperm head. LF binding studies on ZP showed homogenous staining. LF caused a dose-dependent significant inhibition of sperm-ZP interaction, and the effect was already significant (P < 0.01) with the lowest LF concentration used. LIMITATIONS, REASONS FOR CAUTION: This study has investigated the effect of LF only on human gamete interaction in vitro and thus has some limitations. Further investigations of the potential mechanisms involved in LF action both on gamete function in vitro and in vivo in animal models are needed to confirm the role of this protein in the reproductive process. WIDER IMPLICATIONS OF THE FINDINGS: The present data indicate that human oviductal LF expression is cycle dependent and inhibited gamete interaction in vitro. No previous data were available about potential direct effects of LF on gamete interaction. It could be thought that the protein is involved in the regulation of the reproductive process, perhaps contributing to prevent polyspermy. Thus, further research is needed to clarify the potential role of LF in the regulation of the fertilization process. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from FONCYT (PICT 01095, S.A.G., M.J.M) and SECyT UNR (PIDBIO238, S.A.G). The authors have no conflict of interest to declare.
Assuntos
Tubas Uterinas/metabolismo , Lactoferrina/metabolismo , Oócitos/metabolismo , Espermatozoides/metabolismo , Reação Acrossômica , Adulto , Sítios de Ligação , Membrana Celular/metabolismo , Feminino , Fertilização , Fluoresceína-5-Isotiocianato/farmacologia , Humanos , Técnicas In Vitro , Masculino , Ligação Proteica , Interações Espermatozoide-Óvulo , Zona Pelúcida/metabolismoRESUMO
BACKGROUND: Spermatozoa acquire the ability to fertilize an oocyte when they become capacitated. Capacitation takes place when sperm pass through the female reproductive tract, interacting with female fluids. Both tyrosine phosphorylation of sperm proteins and the ability to respond to acrosome reaction (AR) inducers have been associated with sperm capacitation. Recent data indicate that conditioned media (CM) from human oviductal tissue culture decrease sperm affinity for the zona pellucida in vitro. Since capacitation enables the sperm-oocyte interaction, the aim of the present study was to investigate the effect of CM on events related to sperm capacitation and to assess whether these effects were permanent. METHODS: Oviductal tissue was obtained from premenopausal patients (scheduled for hysterectomies because of uterine fibromyoma). The tissues were cultured as explants and CM were collected. Explant viability was assessed as tissue DNA integrity. Normozoospermic semen samples were obtained from healthy donors. Motile spermatozoa were incubated under capacitating conditions with or without increasing protein concentrations of CM for 6 or 22 h. Human follicular fluid-induced AR was detected by the Pisum sativum technique. Tyrosine phosphorylated proteins were detected with a monoclonal anti-phosphotyrosine antibody. RESULTS: The incubation of spermatozoa in the presence of increasing concentrations of conditioned medium (CM) proteins caused a dose-dependent decrease in both tyrosine phosphorylation of sperm proteins and in the level of AR induction. When CM was removed from the sperm incubation media, the effects were reversed. Heat-inactivated CM did not affect either tyrosine phosphorylation or the induction of AR. CONCLUSIONS: The present data suggest that proteins secreted from human oviductal tissue are able to inhibit events associated with sperm capacitation in vitro.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Tubas Uterinas/metabolismo , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/metabolismo , Análise de Variância , Sobrevivência Celular , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Dano ao DNA , Feminino , Líquido Folicular/metabolismo , Humanos , Masculino , Fosforilação , Proteínas/metabolismo , Proteínas/farmacologia , Motilidade dos Espermatozoides , Espermatozoides/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Human follicular fluid (hFF), present in the ampullary environment, can reduce the number of sperm bound to the zona pellucida. The aim of the present study was to investigate the effects of follicular fluid on sperm function. The presence of 50% v/v follicular fluid resulted in a significant reduction in the number of bound spermatozoa with respect to control medium (12.7 +/- 5.5 sp HZ(-1) versus 24.6 +/- 5.7 sp HZ(-1), P = 0.03) as measured by the hemizona binding assay. This reduction in zona binding capacity was not associated with a loss of sperm viability, motility or a premature acrosomal reaction. When capacitated spermatozoa were previously exposed 1 h to follicular fluid, a significant reduction in the number of alpha-d-mannose binding sites on sperm head was detected (23.7 +/- 3.1% versus 15.5 +/- 2.4%, P < 0.05). In addition, sperm fertilizing capacity (assessed as the acrosome reaction to ionophore challenge score) in the presence of follicular fluid was also diminished (38.0 +/- 4.8% versus 22.6 +/- 4.9%, P < 0.01). No modification in the pattern of protein tyrosine phosphorylation which occurs during capacitation was observed in the presence of the fluid. Taken together, the results indicate that the decrease in sperm zona-binding capacity observed in the presence of hFF was related to a lower number of sperm containing alpha-d-mannose receptors.
Assuntos
Líquido Folicular/fisiologia , Interações Espermatozoide-Óvulo , Espermatozoides/fisiologia , Reação Acrossômica , Feminino , Humanos , Lectinas Tipo C/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Espermatozoides/metabolismoRESUMO
The presence of antisperm antibodies might impair fertilization by affecting sperm function in different ways. The aim of this study was to analyze the incidence of antisperm antibodies and evaluate its impact on sperm movement, in normozoospermic and abnormal semen samples. Samples from 144 patients were classified as normozoospermic or abnormal, according to WHO guidelines and Kruger's strict criteria. Motilility was assessed by a computer-assisted system by placing a drop of semen in a Makler chamber. Antisperm antibodies were detected using the Mar Test and Immunobead tests to identify immunoglobulin location and isotype. Results showed no association between the presence of antisperm antibodies and semen quality. Antibody incidence was 15 and 9.5% for normozoospermic and abnormal semen samples, respectively. Motion analysis showed that, although sperm linearity was significantly reduced in abnormal patients with antibodies, the existence of an immune response did not affect the sperm pathway in most cases. This study suggests that sperm motion analysis would not be of great assistance in the screening of an immunological male fertility factor.
Assuntos
Autoanticorpos/análise , Infertilidade Masculina/imunologia , Sêmen/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/imunologia , Adulto , Diagnóstico por Computador , Humanos , Infertilidade Masculina/diagnóstico , Masculino , Estudos de Amostragem , Sêmen/citologia , Sensibilidade e EspecificidadeRESUMO
Human sperm viability is essential for successful fertilization. Eosin Y is the usually accepted method for sperm viability assessment, though the hypoosmotic swelling test has been proposed for the selection of viable spermatozoa in procedures such as intracytoplasmic sperm injection. The present study was designed to determine the value of hypoosmotic swelling test in the prediction of sperm viability. For this purpose, hypoosmotic swelling and eosin Y were performed in parallel and in combination, on both fresh and freeze-thawed semen. Rates for eosin Y were significantly higher than for the hypoosmotic swelling test in fresh semen, with a weak, though significant correlation between the two tests (r = 0.47, p < 0.05). When both tests were performed in succession (hypoosmotic swelling test followed by eosin Y), 14.6% of swollen sperm incorporated the dye. Following exposure to hypoosmotic conditions, sperm viability decreased by 35%. When sperm were killed by freezing, hypoosmotic swelling test rates were higher than eosin Y. Results indicate that these two tests cannot be used interchangeably, since 15% of the swollen sperm apparently died, suggesting that plasma membrane integrity is lost before the capacity to maintain osmotic equilibrium.
Assuntos
Espermatozoides/fisiologia , Congelamento , Humanos , Masculino , Pressão OsmóticaRESUMO
Spermatozoon motility is an important semen parameter that can be correlated with fertilizing capacity. It can be evaluated by subjective or objective methods such as the multiple exposure photography technique (MEP). This technique was: (1) validated in terms of accuracy and sensitivity, and (2) used to investigate the possibility of differentiating patient subgroups on the basis of sperm motility. The method was shown to be accurate. The within and between assay variation coefficients were less than 6% and 17% respectively. Maximal sensitivity required 100 spermatozoa, since coefficients of variation were high when counting smaller numbers. A significant difference between fertile, normozoospermic and asthenozoospermic groups on the basis of the population pattern was found in a retrospective study. Moreover, the asthenozoospermic group could be divided into three subgroups, depending on whether one, two, or three motility variables were altered.
Assuntos
Infertilidade Masculina/diagnóstico , Fotografação/métodos , Motilidade dos Espermatozoides , Humanos , Infertilidade Masculina/classificação , Infertilidade Masculina/fisiopatologia , Masculino , Fotografação/normas , Estudos RetrospectivosRESUMO
To validate an in-vitro bioassay for assessing chromatin stability of human sperm, 38 semen samples from infertile men were studied using sodium dodecyl sulphate, an anionic detergent which disorganizes only the cytoplasmic membrane. Assay sensitivity was 50 sperm, whilst the within- and between-assay variation, and the between-observer variation were found to be within the accepted range for this type of bioassay. The influence of different in-vitro treatments currently used in some clinical assisted fertilization programmes was evaluated: a destabilizing effect occurred in Grade I (stable) and Grade III (swollen) sperm. In the former, all treatments reduced stable sperm; in the latter, a significant (P less than 0.001) increase in swollen sperm was shown with two methods that used Ham's F-10 as culture medium. Different chromatin patterns found in the treated sperm suggest the possibility that the recovered samples could be modified compared to their status at the time of isolation.
Assuntos
Cromatina/metabolismo , Capacitação Espermática , Espermatozoides/metabolismo , Bioensaio , Humanos , Infertilidade Masculina/patologia , Masculino , Dodecilsulfato de Sódio , Manejo de Espécimes , Espermatozoides/citologiaRESUMO
Several studies (1-3) dealing with the presence of spermatozoa in preovulatory peritoneal liquid have shown a low correlation with the situation prevailing in the uterine cervix. Although all previous studies have worked with normospermic populations, we aimed at answering whether alterations of seminal variables are associated with alterations in transportation along the feminine tract. Different sperm migrations (in concentration and motility) were found, depending on the number of seminal variables altered.