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.