Adriana Caille

Glucose-regulated protein 78 (Grp78/BiP) is secreted by human oviduct epithelial cells and the recombinant protein modulates sperm-zona pellucida binding.

OBJECTIVE To determine the secretion of Grp78 by human oviduct epithelial cells, its association to spermatozoa, and its involvement in gamete interaction. DESIGN Prospective study. SETTING Basic research laboratory. SUBJECT(S) Semen samples obtained from normozoospermic volunteers. Tubal tissue provided by patients undergoing hysterectomies. Oocytes collected from women undergoing IVF-ET. INTERVENTION(S) Analysis of Grp78 expression and secretion by oviductal tissue. Gamete incubation with recombinant Grp78 (rec-Grp78). MAIN OUTCOME MEASURE(S) Assessment of protein expression and secretion by immunohistochemistry and Western immunoblotting, respectively. Evaluation of rec-Grp78 binding to human spermatozoa by immunocytochemistry, and analysis of its effect upon gamete interaction using the hemizona assay. RESULT(S) Grp78 was found in the surface of oviduct epithelial cells. Soluble Grp78 was detected in oviductal fluids from women in the periovulatory period and in oviductal tissue conditioned medium. Rec-Grp78 was able to bind to the sperm acrosomal cap, and its presence during gamete interaction led to a decrease in the number of spermatozoa bound to the zona pellucida (ZP). When calcium ions from the incubation medium were replaced by strontium, rec-Grp78 enhanced sperm-ZP interaction. CONCLUSION(S) Grp78 is expressed and secreted by oviduct epithelial cells. The protein would bind to the gametes and may modulate their interaction in a calcium-dependent manner.

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.

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/Hams 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.

Comparative concentrations of steroid hormones and proteins in human peri-ovulatory peritoneal and follicular fluids

Despite the fact that both peritoneal (PF) and follicular (FF) fluids have a common ovarian origin, FF is a natural inducer of sperm acrosome reaction (AR) while PF is not. To better understand these effects, concentrations of oestradiol, progesterone and proteins in peri-ovulatory PF and FF were determined and compared. PF was aspirated by laparoscopy at the peri-ovulatory stage from women with unexplained infertility. FF was collected from patients undergoing IVF and pooled. PF and FF were tested for the presence of antisperm antibodies. Oestradiol and progesterone were measured by enzyme immunoassay, and total protein concentration was determined and analysed. The AR was determined in spermatozoa that were exposed to PF alone, progesterone-supplemented PF, progesterone, control medium, or ethanol. No antisperm antibodies were found in any fluid tested. Oestradiol and progesterone and concentrations in PF were significantly lower than in FF. Protein concentration was also significantly lower in PF than in FF, but no differences were observed between the electrophoretic patterns. When capacitated spermatozoa were exposed to progesterone-supplemented PF there was a significant increase in the percentage of AR with respect to those in PF, control medium or ethanol. These results suggest that the lack of AR-stimulating activity of PF was related to its lower progesterone concentration compared with FF.

Modulation of human sperm function by follicular fluid

Summary.  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 α‐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 α‐d‐mannose receptors.

d-Mannose-binding sites are putative sperm determinants of human oocyte recognition and fertilization

The aim of the present study was to further evaluate the participation of D-mannose in the process of human sperm-egg interaction. Zona pellucida binding competitive assays in the presence of D-mannose were carried out using discarded oocytes from IVF. Spermatozoa were capacitated and D-mannose-binding site (MBS) expression, sperm viability and follicular fluidinduced acrosome reaction (AR) were evaluated. MBS were visualized using a fluorescein-neoglycoprotein probe. The capacity of free D-mannose and mannosylated albumin to induce the AR was also tested. MBS and the IVF outcome were also analysed. The involvement of D-mannose in sperm binding to the zona pellucida was verified by the inhibitory effect produced when the sugar was present during binding assays. MBS expression increased during capacitation, in parallel with the ability to undergo the induced AR. Mannosylated albumin, but not the free sugar, induced the AR. In acrosome-reacted spermatozoa, the MBS was located at the plasma membrane, as shown by confocal analysis. No significant difference in the increase in MBS expression was observed among the different IVF groups of patients. The data show that D-mannose is involved in the sperm-zona pellucida interaction, and that the expression of MBS on the sperm surface occurs during the acquisition of in-vitro sperm fertilizing ability.

Effects of ulipristal acetate on sperm DNA fragmentation during in vitro incubation

Abstract Objective Ulipristal acetate (UPA) acts as an emergency contraceptive by inhibiting ovulation. This study explores possible additional effects on the fragmentation of sperm DNA during in vitro incubation. Methods Motile spermatozoa from healthy donors were selected by swim-up and incubated under capacitating conditions in control medium or with UPA (1, 10, 100, 1,000 or 10,000 ng/ml). In some experiments, 200 μM of H2O2 were added to induce oxidative stress. The sperm chromatin dispersion test was performed to analyse DNA integrity (400 cells; 1000×). Lipid peroxidation (thiobarbituric acid assay), induced-acrosome reaction (AR) and sperm vitality (Eosin Y) were also evaluated in spermatozoa exposed to UPA and/or H2O2. Results During sperm incubation, the percentage of fragmented DNA increased significantly, from 15.0 ± 1.3 to 41.0 ± 4.5% (p < 0.001). In the presence of UPA, DNA fragmentation decreased significantly (p < 0.05), in a dose-dependent manner. At 100 and 1000 ng/ml, UPA also counteracted the effect of H2O2 and prevented DNA fragmentation. No effect on sperm vitality, lipid peroxidation or induced-AR was found with any treatment. Conclusions During in vitro sperm capacitation DNA fragmentation increased but the latter was counteracted in the presence of UPA, which possibly acted as a scavenger of reactive oxygen species produced by spermatozoa.

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.

Peritoneal fluid modifies the response of human spermatozoa to follicular fluid

The aim of this study was to elucidate the mechanism involved in the acrosome reaction (AR) induced by follicular fluid (FF) in spermatozoa previously exposed to peritoneal fluid (PF). The influence of progesterone was also investigated. Semen samples were from 18 normozoospermic donors. PF samples were from 13 women with unexplained infertility and from a woman treated with synthetic progestagen. FF samples were collected from six women undergoing IVF/embryo transfer and pooled. Motile spermatozoa were capacitated overnight and a kinetic and inhibition study on the FF-induced AR was performed. Spermatozoa pretreated with PF were challenged with either FF or progesterone. The ability of progesterone- and progestagen-supplemented PF to induce AR was analysed. Enzyme-digested PF was also tested. Pre-incubation with PF for 60 min completely prevented the FF-induced AR; spermatozoa treated with PF were unable to respond to FF or progesterone and this effect was not reversible. Progesterone- and progestagen-supplemented PF stimulated the AR relative to controls. Enzyme-digested PF did not have an inhibitory capacity. These data strongly suggest that there are one or more inhibitory proteins in PF that interact with spermatozoa so as to prevent access of progesterone to its receptor and thus inhibit the occurrence of the AR. The oviduct, or Fallopian tube, provides a place for spermatozoa and egg transport and storage, fertilization and early embryo development. If ovulation has not occurred, spermatozoa may reside in the oviduct for several hours or even a few days, awaiting oocyte arrival. It is assumed that fluids present in the female genital tract may have a role in synchronizing the timing required to guarantee the success of fertilization. We previously observed that the peritoneal fluid that bathes the peritoneal cavity is a suitable medium for sperm survival and we also reported that this fluid could stabilize spermatozoa. In this study we show further evidence that the exposure to peritoneal fluid modifies the response of spermatozoa to oocyte signals.

Sperm peritoneal recovery in infertile couples with male factors

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.