Effects of lactoferrin, a protein present in the female reproductive tract, on parameters of human sperm capacitation and gamete interaction.

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.

In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation.

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.

A protein isolated from human oviductal tissue in vitro secretion, identified as human lactoferrin, interacts with spermatozoa and oocytes and modulates gamete interaction.

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.

Proteins from human oviductal tissue-conditioned medium modulate sperm capacitation.

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.

[Is there an association between sperm normal morphology and their kinetic displacement?]. / Existe asociación entre la morfología normal del espermatozoide y su cinética de desplazamiento?

OBJECTIVE: To evaluate if there exist an association between the % of normal forms and the kinetic characteristics in human spermatozoa MATERIAL AND METHODS: A retrospective study was performed to analyze semen samples of 203 patients by Computer Assisted Semen Analysis. Sperm morphology was evaluated by the aid of a micrometric objective according to strict criteria. Only the patients presenting >20 x 106 sperm/ml and > 50% of progressive motility were included. Data from 168 patients were divided according to the % of normal forms in three groups a) <4% (n=22), b) between 4-13% (n=89) and c) >14% (n=57). Data collected among groups were compared. In order to select a motile sperm population 35 samples were treated by a discontinuous gradient and the % of normal forms as well as motility parameters evaluated before and after selection. RESULTS: The kinetic analysis showed that sperm concentration, the % of motile and rapid spermatozoa (>25 microm/s) as well as the average path velocity (VAP) and the lateral head displacement (LHD) were increased in association with the % of normal spermatozoa presenting the lowest values in the group <4% with respect to the > or = 14% group, (p<0.05). Linearity (LIN) remained constant among groups. Kinetic parameters and sperm morphology were significantly increased (p<0.0001) in the selected samples Results showed that the use of gradients even in teratozoospermic samples improves significantly the % of normal forms respect to baseline values (p<0.0002). CONCLUSIONS: Our results would support the hypothesis that morphologically better spermatozoa would be associated with those with better movement parameters measured in an objective way. In this manner we could suggest that in vivo the best spermatozoa would comprise one "elite" in the journey through the fertilization site.

¿ Existe asociación entre la morfología normal del espermatozoide y su cinética de desplazamiento? / Is there an association between sperm normal morphology and their kinetic displacement?

Objetivo: Evaluar si existe asociación entre el % de formas normales y la cinética de desplazamiento en espermatozoides humanos. Material y métodos: Se analizaron en forma retrospectiva los espermogramas de 203 pacientes evaluados mediante la ayuda de un sistema computarizado de análisis de movimiento. La evaluación de la morfología se realizó en forma micrométrica según el criterio estricto. Sólo se incluyeron los pacientes que presentaron >20 x 106 esp/ml y >50% de motilidad progresiva. Los datos de 168 pacientes fueron divididos según el % de formas normales iniciales en tres grupos a) =% (n=57) y se compararon los datos obtenidos. Con el objeto de seleccionar espermatozoides móviles, 35 muestras fueron tratadas con un gradiente discontinuo, evaluándose tanto el % de formas normales como los parámetros de movilidad basales y luego de la selección. Resultados: El análisis cinético mostró que tanto la concentración, el % de espermatozoides móviles y rápidos (>25 ìm/s), así como la velocidad media (VAP) y el desplazamiento de la cabeza (LHD) se vieron incrementados con la cantidad de espermatozoides morfológicamente normales presentando los valores más bajos en el grupo <4% respecto al grupo <=, (p<0,05). La LIN se mantuvo constante entre los grupos. En las muestras seleccionadas se incrementaron significativamente las variables cinéticas así como la morfología (p<0,0001). Los resultados muestran que aún en el caso de muestras teratozooespérmicas la utilización de gradientes mejora significativamente el % de formas normales respecto de los valores basales (p<0,0002). Conclusiones: Nuestros resultados apoyarían la hipótesis de que los espermatozoides morfológicamente mejores serían aquellos que presentan mejores características de movimiento medido en forma objetiva. De este modo podríamos suponer que in vivo, los mejores espermatozoides formarían parte de una 'élite' en la carrera hacia el sitio de fecundación

Objective: To evaluate if there exist an association between the % of normal forms and the kinetic characteristics in human spermatozoa Material and Methods: A retrospective study was performed to analyze semen samples of 203 patients by Computer Assisted Semen Analysis. Sperm morphology was evaluated by the aid of a micrometric objective according to strict criteria. Only the patients presenting >20 x 106 sperm/ml and > 50% of progressive motility were included. Data from 168 patients were divided according to the % of normal forms in three groups a) =% (n=57). Data collected among groups were compared. In order to select a motile sperm population 35 samples were treated by a discontinuous gradient and the % of normal forms as well as motility parameters evaluated before and after selection. Results: The kinetic analysis showed that sperm concentration, the % of motile and rapid spermatozoa (>25 ìm/s) as well as the average path velocity (VAP) and the lateral head displacement (LHD) were increased in association with the % of normal spermatozoa presenting the lowest values in the group =4% group, (p<0.05). Linearity (LIN) remained constant among groups. Kinetic parameters and sperm morphology were significantly increased (p<0.0001) in the selected samples Results showed that the use of gradients even in teratozoospermic samples improves significantly the % of normal forms respect to baseline values (p<0.0002). Conclusions: Our results would support the hypothesis that morphologically better spermatozoa would be associated with those with better movement parameters measured in an objective way. In this manner we could suggest that in vivo the best spermatozoa would comprise one 'elite' in the journey through the fertilization site

Modulation of human sperm function by follicular fluid.

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.

Relationship between antisperm antibodies, sperm movement, and semen quality.

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.

Does the hypoosmotic swelling test predict human sperm viability?

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.

Semen culture, leukocytospermia, and the presence of sperm antibodies in seminal hyperviscosity.

Seminal hyperviscosity is generally thought to reveal genitourinary infection. The aim of the present work was to study this hypothesis. A total of 65 semen samples were obtained from males presenting for infertility screening. The samples were evaluated according to WHO criteria and microbiologically investigated, including culturing for Mycoplasma hominis and Ureaplasma urealyticum, and microscopic observation of Chlamydia trachomatis by a direct fluorescence assay. Determination of local antisperm antibodies was performed. Semen was categorized according to consistency: normal (n = 31) and high (n = 34). No difference was recorded either in the number of positive cultures, or in the number of species found in each sample. The number of white blood cells and the percentage of antibody-bound sperm showed no difference in the groups under study. There was no association between seminal hyperviscosity, positivity in semen cultures, number of species isolated in semen cultures, leukospermia, or presence of sperm antibodies. Further studies should be performed considering the evolution of the genital-infected patients to clarify the etiology of the hyperviscosity.

Sperm motility and ATP content in seminal hyperviscosity.

Objective spermatic motility (Hamilton Thorne Research), the rapid progressive spermatozoa (grade A) recovery after swim-up, and the spermatozoa ATP content (bioluminescence) were studied in normoviscous and hyperviscous asthenospermic samples. The amplitude of lateral head displacement (ALH) was significantly lower in hyperviscous semen (normal: 4.6 +/- 0.7 microns [n = 20], high: 3.5 +/- 1.2 microns [n = 16]; p < .05). The grade A recovery percentage after swim-up was significantly higher in semens with high consistency (normal: 71.0 +/- 38.0 [n = 14], high: 181.3 +/- 108.9 [n = 6]; p < .05). The ATP content per living spermatozoa was in the normal consistency group 449.4 +/- 65.1 pmol per million living spermatozoa (n = 29) and in the high consistency batch 605.1 +/- 242.8 (n = 9), p < .05. In asthenospermia, the spermatozoa from hyperviscous samples have minor ALH values, better response to swim-up, and high ATP content than those from normoviscous ejaculates.

Mammalian sperm-egg fusion: the development of rat oolemma fusibility during oogenesis involves the appearance of binding sites for sperm protein "DE".

Rat epididymal protein DE mediates gamete fusion through complementary sites localized on the egg surface. To investigate whether these egg components are involved in the development of rat oolemma fusibility, both the presence of DE-binding components and the ability of the oolemma to fuse with sperm during oogenesis were examined. Localization of DE-complementary sites by indirect immunofluorescence revealed the absence of fluorescent labeling on growing oocytes with a diameter < 50 microns, and the presence of a uniform staining over the entire surface of germinal vesicle oocytes with a diameter > 50 microns. This localization of oolemma components changed progressively to a patchy distribution during maturation. Whereas sperm incorporation was observed only in maturing oocytes, the development of the Hoechst transfer technique to evaluate membrane fusion revealed that germinal vesicle oocytes with a diameter > 50 microns were already competent to fuse with sperm. The involvement of the DE-complementary sites in the oolemma fusibility of these oocytes was confirmed by the fact that the presence of DE during gamete coincubation significantly (p < 0.001) reduced the percentage of oocytes with fused sperm. Together, these observations indicate that the acquisition of fusibility by the rat oolemma occurs during the growth period and involves the appearance of DE-binding components on the oocyte surface. This study provides novel information on the molecular mechanism by which the mammalian egg plasma membrane becomes competent to fuse with sperm during oogenesis.

Sperm motility analysis using multi-exposure photography (MEP): validity of the method with normal and abnormal patterns.

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.