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Gamete Biology |
a Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520
b Clinic of Sterility, Department of Obstetrics and Gynecology, University Hospital of Geneva, 1211 Geneva 14, Switzerland
c Department of Obstetrics and Gynecology, Reproductive Biology Unit, University of Stellenbosch and Tygerberg Hospital, Tygerberg, South Africa
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, fertilization, gamete biology, sperm, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The timing of protein phosphorylation in the various compartments of mouse sperm can be linked to the acquisition of specific sperm functions [6, 7]. Spermatozoa also show activity of specific metabolic pathways compartmentalized in different regions [8–10]. Travis et al. [6] proposed that ATP, specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum as opposed to ATP generated by mitochondria in the midpiece, is strictly required for protein tyrosine phosphorylation events that take place during mouse sperm capacitation. In agreement with the metabolic compartmentalization observed in the previous study, we have also shown in the mouse that glucose affects sperm-oocyte interaction and that its role is mediated through regulation of protein tyrosine phosphorylation in the fertilizing sperm [11–14]. In the presence of glucose, immunofluorescence assessed using the monoclonal anti-phosphotyrosine mouse antibody revealed that flagellum fluorescence appears during capacitation and the numbers of mouse sperm presenting principal piece and midpiece fluorescence becomes significantly higher over time. The increase in phosphorylation in the principal piece precedes that in the midpiece, and a fluorescent midpiece was never seen in sperm with an unstained principal piece. Upon binding to the zona pellucida, nearly all mouse sperm became progressively phosphorylated in both the principal piece and midpiece [7]. These results indicate that for both capacitation and fertilization to occur a precise sequence of phosphorylation is needed in the different compartments of the spermatozoon.
In the study by Carrera et al. [15], a small percentage of noncapacitated human sperm were positive for phosphotyrosine immunoreactivity, which was localized to the principal and neck region of the flagellum. Leclerc et al. [16] used indirect immunocytochemical techniques to determine that phosphotyrosine-containing proteins are mostly located in the principal piece of the flagellum. During capacitation in human sperm, at least 7 proteins are phosphorylated, as determined by the 32P metabolic labeling assay, and 14 proteins are autophosphorylated, as determined using the in vitro kinase assay. Of the 7–14 proteins, 2 proteins of 95 and 51 kDa were phosphorylated at tyrosine residues [17]. Emiliozzi and Fenichel [18] however showed that although protein tyrosine phosphorylation is associated with of human sperm in vitro it is not sufficient for completion of capacitation.
The changes occurring in human sperm protein tyrosine phosphorylation during the interaction between sperm and the zona pellucida have not been investigated. In particular, the compartmentalization of protein tyrosine phosphorylation has not been visualized during this process in human sperm. Our objective was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and during interaction between sperm and the zona pellucida and to ascertain whether the relative patterns of phosphorylation present on sperm could predict the fertilizing capacity of the spermatozoa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human tubal fluid (HTF) medium [19] was prepared and supplemented with 10 mg/ml BSA. IVF-20 or IVF-50 was purchased from Scandinavian IVF (Gothenburg, Sweden). Unless otherwise indicated, all chemicals were purchased from Sigma Pharmaceuticals (Buchs, Switzerland).
In Vitro Fertilization Patients
Forty-one couples coming for an in vitro fertilization (IVF) cycle in Geneva or New Haven clinics were included in this study. Eighty-three percent presented with female infertility (42% tubal, 7% ovulatory, 12% endometriosis, 12% age), and 17% presented with idiopathic infertility. The male patients displayed normal sperm counts and motility according to the 1999 World Health Organization (WHO) criteria [20]. Abnormal morphology was assessed using the WHO classification and was not lower than 15%. The male patients who did not meet these criteria or with a previous failed IVF treatment were not included in this study. The mean number (±SD) of inseminated oocytes was 9.5 ± 5.8.
Sperm
The semen samples of the 41 IVF male patients were used for IVF and for the localization of tyrosine phosphorylated proteins in sperm.
Semen were prepared in a 90%/45% gradient of PureSperm (Nidacon, Gothenburg, Sweden) in Geneva and in ISolate (Irvine Scientific, Santa Ana, CA) at the Yale Clinic. Both types of gradients are similar in composition and yielded suspensions with 80–100% mobile sperm. An initial 20 µl of the prepared sample was immediately fixed in formaldehyde (noncapacitated sperm). Of the remaining sperm, one part was diluted in IVF-20 medium for insemination for the routine IVF procedure and the other was diluted in HTF medium for phosphotyrosine residues detection. In preliminary experiments, phosphotyrosine localization was similar when sperm were capacitated in HTF or IVF-20 medium. Both sperm suspensions were maintained until 4 h after the initial preparation time at 37°C in 5% CO2 in air prior to fixation (capacitated sperm) or oocyte insemination. Samples suspected of containing insufficient sperm to inseminate were not used, and only postinsemination samples were used so patient treatment would not be jeopardized.
Human Zonae Pellucidae
Zonae pellucidae of prophase I human oocytes were provided by Dr. D. Franken. A limited number of prophase I oocytes were obtained from postmortem ovarian tissue. These oocytes were nonliving and had no developmental potential following storage in a Hepes buffer containing MgCl2 and polyvinylpyrrolidone at 4°C [21]. Oocytes were extensively washed in HTF medium for 24 h prior to use.
Sperm used for binding to human zonae pellucidae were obtained from four healthy donors. A donor of proven fertility was used for the first three experiments to eliminate patient variability. The experiment was then repeated with three different donors. Sperm were prepared as described for the IVF patients and were capacitated for 4 h in HTF medium. From this suspension, 50-µl sperm droplets were prepared (106 mobile sperm/ml) under oil for zona pellucida binding, and aliquots were fixed to determine the phosphorylation status of the capacitated sperm just before zona pellucida binding. The zonae pellucidae were then incubated for 3 h in the sperm droplets. At the end of incubation, sperm loosely attached to the zonae pellucidae were removed by minimal pipetting [22] and fixed in formaldehyde.
Indirect Immunofluorescence
Fixed sperm were washed in PBS, smeared on slides, and air dried. Sperm were permeabilized for 10 min in 0.2% Triton X-100 at room temperature. To block nonspecific sites, the slides were incubated for 1 h in 10% horse serum and washed in PBS. To demonstrate phosphotyrosine residues, the slides were then incubated in a monoclonal anti-phosphotyrosine mouse antibody (4G10; UBI, Luzern, Switzerland) diluted 1:100 for 1.5 h at room temperature in a humidified chamber, followed by incubation with a secondary mouse IgG antibody conjugated with tetramethyl rhodamine isothiocyanate (1:60). Negative controls were prepared by using an irrelevant mouse antibody (IgG2b) instead of the anti-phosphotyrosine antibody. A weak background fluorescence was observed in negative controls, and sperm presenting such background after reaction with the anti-phosphotyrosine antibody were considered negative.
Phosphotyrosine residues in the sperm bound to the zonae pellucidae were detected as in sperm deposited on slides except that labeling was performed in droplets of medium as previously described [7]. The zonae pellucidae were mounted on a slide just prior to observation. Sperm were then examined by the same observer with a Nikon fluorescence microscope using a 100x objective.
Statistics
Following arcsine transformation, the percentages of the different fluorescence patterns were compared using a paired Student t-test. The chi-square test was used to compare the frequencies of the different fluorescence patterns between capacitated and zona pellucida-bound sperm in individual experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the localization of tyrosine phosphorylated proteins in human sperm and possible changes during capacitation, phosphotyrosine residues were demonstrated by indirect immunofluorescence in noncapacitated and capacitated sperm. Only a small proportion of sperm contained tyrosine phosphorylated proteins; a large proportion was negative. Phosphotyrosine residues were localized to different compartments of spermatozoa: the principal piece, the whole acrosome, the equatorial segment (ES), and the neck region (Fig. 1). In individual sperm, tyrosine phosphorylation in the ES and the acrosome was not always associated with principal piece phosphorylation, but tyrosine phosphorylation in the neck and principal piece was always combined.
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Influence of Capacitation and Relationship to Fertilization Potential
To examine the influence of capacitation on the distribution of tyrosine phosphorylated proteins, sperm were incubated for 4 h, which is the reported time of appearance of tyrosine phosphorylation in capacitating human sperm [23]. The mean percentage of each tyrosine phosphorylation pattern was calculated for the noncapacitated and capacitated sperm of the 41 patients (Fig. 2). Phosphotyrosine residues limited to the principal piece was the most common pattern of phosphorylation and was observed in all but one of the samples assessed. The mean percentage of sperm presenting tyrosine phosphorylation in the principal piece increased significantly with capacitation but ranged from 0% to 50%, indicating variability among patients.
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Phosphorylation in the ES, whole acrosome, and neck was observed in 88%, 66%, and 68% of the patients, respectively. The mean percentages of sperm presenting these patterns were low, but when staining in these areas was associated with staining of the principal piece in individual sperm, the percentage of patients showing phosphorylation increased significantly with capacitation. Variability among patients was also evident: the percentages of capacitated sperm phosphorylated in the ES, whole acrosome, and neck were 0–66%, 0–12%, and 0–18%, respectively.
To investigate the relationship between tyrosine phosphorylation of sperm proteins and fertilization, the percentages of the different patterns of fluorescence were plotted according to the fertilization rates (Fig. 3). Sixteen patients achieved >60% fertilization rates and were considered high-fertilization patients. The high-fertilization patients displayed fluorescent principal pieces in 7–50% of their sperm (Fig. 3A). A significant correlation was found between the percentages of fluorescent principal piece and fertilization rates (r = 0.50, P = 0.045) in this group. The four patients (open circle) for whom <7% of sperm had a fluorescent principal piece achieved low fertilization rates. However, principal piece tyrosine phosphorylation above this threshold led to high fertilization rates in only 40% of the patients, suggesting that if required tyrosine phosphorylation in this compartment is not sufficient for successful fertilization.
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A number of high-fertilization patients lacked capacitated sperm with fluorescent acrosome (Fig. 3B), ES (Fig. 3C), or neck (Fig. 3D), indicating that tyrosine phosphorylation of proteins localized to these domains was not required for successful fertilization. High percentages of fluorescent acrosome and neck (open circles) were found only in the low-fertilization patients.
Figure 3 also shows that the three patients (open squares) who achieved 100% fertilization showed tyrosine phosphorylation in a high proportion of principal pieces (22%, 23%, and 30%) and a low proportion of acrosome (0.3%, 0.7%, and 2.3%), ES (0%, 2%, and 10%), and neck (3%, 3%, and 5%).
Localization of Phosphotyrosine Residues in Sperm Bound to Zonae Pellucidae
To determine whether protein tyrosine phosphorylation of sperm is modified upon binding to the oocytes and/or whether certain patterns of phosphorylation are beneficial for sperm binding to the oocyte, the phosphorylation status of sperm bound to human zonae pellucidae was determined (Fig. 4) and compared with that of capacitated sperm in suspension.
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Phosphotyrosine patterns were obtained in six zona-binding experiments (Fig. 5, a–f). Although some variation was evident in some of the individual patterns obtained in the different experiments, there was a consistent significant increase (P < 0.05) in the proportion of zona pellucida-bound sperm with phosphotyrosine residues localized to the neck and the principal piece together compared with the capacitated sperm in suspension. Although representing a lower proportion of sperm, the number of sperm showing a positive acrosome was also significantly higher in all but one experiment.
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The combined data for the experiments is shown in Table 1. The different proportion of the patterns in zona-bound sperm was not due to extended incubation because the phosphorylation patterns of sperm in suspension did not change by increasing the incubation time from 4 to 7 h.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A much smaller section of the population of spermatozoa assessed showed phosphorylation in the acrosome cap or ES. Because fertilization was not impaired in patients devoid of acrosome-positive sperm after capacitation, tyrosine phosphorylation in the acrosomal region during capacitation is probably not required for binding to the zona pellucida and fertilization. Under our experimental conditions, binding to the zona pellucida did promote a moderate increase in tyrosine phosphorylation in the acrosomal region in all but one donor tested. The appearance of a transient phosphorylation before our observation cannot be totally excluded and may explain why this pattern was limited and not observed in all the donors. A tyrosine phosphorylation step is necessary for the zona pellucida-induced acrosome reaction, and inhibition of tyrosine phosphorylation prevents the acrosome reaction [26, 27]. In addition, exposure to progesterone, which induces the acrosome reaction, promoted an increase in tyrosine phosphorylation in the head of a limited subpopulation of human sperm [28]. Burks et al. [29] proposed that a tyrosine kinase receptor localized to the surface of the acrosome in human sperm becomes tyrosine phosphorylated following binding to ZP3. In their study, they showed that a phosphoprotein recognized by monoclonal antibody 97.25 was localized on the sperm surface in the acrosomal region, but they did not present evidence that the phosphorylation status of this acrosomal protein changed according to capacitation or zona pellucida interaction. Although they showed that kinase activity of a putative sperm receptor, hu9 protein, was enhanced following zona pellucida interaction, they did not demonstrate unambiguously that the hu9 protein was the same as that localized to the acrosome by monoclonal antibody 97.25. Tyrosine phosphorylation in the ES may be linked to modifications of this region following the acrosome reaction [30, 31]. The specific step responsible for tyrosine phosphorylation in the ES during gamete interaction remains to be determined.
The main changes in phosphorylation observed following zona pellucida binding occurred in the neck region. This finding indicates that the zona pellucida promotes changes in protein tyrosine phosphorylation in bound sperm, but sperm phosphorylated during capacitation also may have a competitive advantage for binding to the zona pellucida. However, according to our data on the relationship between tyrosine phosphorylation in capacitated sperm and IVF, phosphorylation in the principal piece may be advantageous to fertilization and therefore to zona pellucida binding, whereas phosphorylation in the neck is not. The neck region is situated between the sperm head and the beginning of the midpiece [32]. It contains striated columns extending from the outer dense fibers of the midpiece to the capitulum close to site of attachment of the flagellum to the sperm head. In the human sperm, the centriole, which is embedded in the capitulum, is transmitted to the oocyte at fertilization and is required for sperm aster formation [33]. Tyrosine phosphorylation of proteins in the neck region following binding to the zona pellucida suggests that phosphorylation-dependent events are activated in the neck. An influence on centrosomal activity may be hypothesized. Phosphoproteins (recognized by the MPM-2 antibody) localized to the neck play a key role in regulating sperm aster formation at fertilization in different species, including humans [34–36].
The relevance of the different phosphorylation patterns observed before and after capacitation and upon zona pellucida binding provides evidence of the heterogeneity of the sperm population in an ejaculate and may give clues to the fertilizing potential of a patient's sperm. In this study however, we failed to observe a strict correlation when comparing sperm phosphorylation patterns with fertilization results. Successful fertilization depends on coordinated sequences of events between gametes until the sperm fuses with the oocyte. Both sperm and oocyte defects may be responsible for failed fertilization, and it is therefore difficult to identify which processes are relevant, particularly in humans where experimental fertilization cannot be performed for ethical reasons. Our data suggest that when a threshold number of spermatozoa showing principal piece tyrosine phosphorylation is not reached during capacitation, fertilization may be at risk. Principal piece phosphorylation above this threshold was not beneficial for every patient, suggesting that tyrosine phosphorylation is not sufficient for successful fertilization, and other gamete defects may be responsible for decreased fertilization. Tyrosine phosphorylation of proteins in the acrosome and the neck during capacitation was not needed for fertilization but may affect fertilization when present in too many sperm. However, a larger number of patients must be evaluated to confirm the relationship between phosphorylation patterns and fertilizing ability of sperm populations.
Evidence of anomalies in phosphorylation patterns during capacitation and an inability to achieve fertilization does however exist in the mouse. A delay in the appearance of tyrosine phosphorylation in sperm suspensions resulted in a failure of sperm penetration into oocytes [7]. This delay in phosphorylation and failure to achieve fertilization has been linked to the absence of glucose in culture medium [7, 11, 12]. In human sperm, there is also evidence that the absence of glucose can reduce fertilization results [37, 38]. This observation may be linked to the inability of human sperm to complete or undergo the correct sequence of phosphorylation events.
Different compartments of human spermatozoa undergo a specific sequence of phosphorylation both during capacitation and upon binding to the zona pellucida. Determination of how these events are controlled in the different compartments should provide an insight into basic mechanisms involving signalling within the various compartments. Determination of whether all spermatozoa are capable of undergoing this pattern of phosphorylation may provide insights into the heterogeneity of spermatozoa and assist us in understanding why some sperm samples are unable to achieve fertilization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Françoise Urner, Laboratoire des Gamètes, Clinique de Stérilité, Hôpital Cantonal, 30, Bd de la Cluse, 1211 Geneva 14, Switzerland. FAX: 41 22 38 24 385; francoise.urner{at}hcuge.ch
Received: 10 October 2002.
First decision: 23 October 2002.
Accepted: 1 November 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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