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Regular Article |
a Clinic of Sterility, Department of Obstetrics and Gynecology, University Hospital of Geneva, 1211 Geneva 14, Switzerland
b Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520-8063
ABSTRACT
A key intracellular event during capacitation is protein tyrosine phosphorylation, but its involvement during sperm interaction with the oocyte has not been investigated. Glucose is necessary to achieve fertilization and thus may have an influence on sperm protein tyrosine phosphorylation. The objectives of this study were to 1) visualize protein tyrosine phosphorylation patterns in sperm during capacitation and interaction with the oocyte and 2) determine the influence of glucose. Protein tyrosine phosphorylation was investigated by Western analysis and immunofluorescence. Protein tyrosine phosphorylation was increased during capacitation, and immunofluorescence revealed that zona binding and gamete fusion were correlated with an increase in tyrosine phosphorylation of proteins in the midpiece. During capacitation, the absence of glucose led to a delay in the appearance of protein tyrosine phosphorylation. Following binding to the zona pellucida and the oolemma, tyrosine phosphorylation in the flagellum was also delayed in the absence of glucose and resulted in a significant inhibition of the midpiece phosphorylation. The correlation between successful gamete fusion and the tyrosine phosphorylation of midpiece proteins suggests that the effect of glucose on sperm-oocyte interaction is mediated through regulation of protein tyrosine phosphorylation in a specific area of the fertilizing sperm.
fertilization, gamete biology, ovum, sperm, sperm capacitation
INTRODUCTION
Mammalian sperm are required to undergo a process known as capacitation before they can undertake the fertilization process [1, 2]. These processes can also be supported in vitro; however, the role of various components of the culture medium is only now being better identified at the cellular and molecular level [3–5]. One of the key intracellular events during capacitation is protein tyrosine phosphorylation, which can be drastically affected by HCO3-, Ca2+, and albumin levels in medium [3, 4]. Other important components in medium that can influence mammalian sperm activity include the energy substrates glucose, pyruvate, and lactate.
Glucose is important for fertilization to occur in the mouse [6, 7], rat [8], and human [9], and both zona pellucida penetration and sperm-oocyte fusion are prevented in the absence of glucose in the mouse [10, 11]. Sperm rather than the oocyte need to metabolize glucose to achieve fertilization, probably through the pentose phosphate pathway (PPP) because exogenous NADPH, which is a metabolic product of the PPP, is able to substitute for glucose in sperm-oocyte fusion [12]. However, specific functions, which are regulated by glucose in mouse sperm, have yet to be determined. The importance of glucose in mammalian spermatozoa is attracting more interest, and the characterization of several enzymes involved in glucose metabolism are showing unique idiosyncrasies in structure and positioning. For example, a spermatogenic cell-specific glyceraldehyde 3-phosphate dehydrogenase, has been localized to the principal piece [13–15], and a germ cell-specific type 1 hexokinase has been associated with head membranes, the mitochondria, and the fibrous sheath [16].
The observation that NADPH can modulate protein tyrosine phosphorylation in human sperm [17, 18] suggests that glucose metabolized by the PPP may participate in the regulation of protein tyrosine phosphorylation. In bovine sperm, glucose inhibits heparin-induced protein tyrosine phosphorylation associated with capacitation, although its mechanism of action has not been demonstrated [19]. In addition, tyrosine phosphorylation of the protein SNAP-25, which is involved in insulin secretion, is increased by glucose in an insulinoma cell line [20].
The changes occurring in protein tyrosine phosphorylation in sperm during its interaction with the oocyte have not been investigated. In particular, the compartmentalization of protein tyrosine phosphorylation has not been visualized in sperm during this process. The objectives of this study were 1) to characterize the role of glucose in protein tyrosine phosphorylation patterns in mouse sperm, 2) to investigate and visualize protein tyrosine phosphorylation patterns in sperm during capacitation and interaction with the oocyte, and 3) to determine whether glucose can modulate protein tyrosine phosphorylation in sperm during these different steps.
MATERIALS AND METHODS
Chemicals and Culture Media
The basic culture medium used in all experiments was M16 [21] containing 23.3 mM lactate, 0.33 mM pyruvate, and 5.56 mM glucose and supplemented with 15 mg/ml type V BSA. We have previously shown that this medium supports sperm capacitation and fertilization in the mouse [10]. Unless otherwise indicated, all chemicals were obtained from Sigma (Buchs, Switzerland). ß-NADPH was stored at -20°C and dissolved in glucose-free culture medium just prior to use.
Gametes
Spermatozoa were obtained from the cauda epididymidis of 10- to 16-wk-old OF1 male mice (RCC, Fullinsdorf, Switzerland). Spermatozoa were released into 200 µl of M16 and were either incubated further as concentrated suspensions (30–50 x 106 sperm/ml) or diluted fivefold in M16 (5–10 x 106 sperm/ml). To study sperm in suspension, sperm were incubated for different time periods before extraction or fixation. To study sperm interacting with oocytes, sperm were incubated as diluted suspensions for 1.5 h or 3 h prior to an additional dilution (0.05–0.1 x 106 motile sperm/ml) and insemination of zona-intact or zona-free oocytes, respectively.
Oocytes were obtained from 4- to 5-wk-old B6D2F1 mice (RCC, Fullinsdorf, Switzerland) and stimulated with an i.p. injection of 5 IU eCG (Folligon, Veterinaria, Switzerland) followed 48 h later by 5 IU hCG (Choluron, Zurich, Switzerland). Fourteen to 16 h after administration of hCG, oocytes surrounded by their cumulus cells were collected. Cumulus cells were removed with 0.2 mg/ml hyaluronidase. To prepare zona-free oocytes, zonae were mechanically removed as previously described [11].
Zona-intact or zona-free oocytes were incubated in 50 µl of sperm suspension for 15 min and transferred into fresh medium without sperm. Loosely attached sperm were removed from zona-free oocytes by using a 100-µm pipette according to the method of Wolf and Hamada [22] and from zona-intact oocytes by using a 150-µm pipette according to the method of Inoue and Wolf [23]. The oocytes were fixed at different time periods after sperm-oocyte coincubation. All the incubations were performed at 37°C in 5% CO2 in air.
Western Analysis
Sperm were washed twice in PBS, and proteins were solubilized in Laemmli's buffer without mercaptoethanol (1 x 106 spermatozoa/20 µl) according to the method of Aitken et al. [17]. The sperm extracts were centrifuged, and the supernatants were stored at -20°C until use. Prior to electrophoresis, proteins were denatured by heating the samples for 5 min to 100°C in Laemmli's buffer containing 5% mercaptoethanol. The same amount of protein, equivalent to 0.5 x 106 spermatozoa, was loaded into each well of a 10% SDS-polyacrylamide gel, and electrophoresis was performed using a minigel system (Bio-Rad, Zurich, Switzerland) prior to transfer to a nitrocellulose membrane with the Bio-Rad mini transfer system. The membranes were stained with Ponceau red to confirm that each lane contained the same amount of protein. Phosphotyrosine was immunodetected by first incubating the membranes with a monoclonal anti-phosphotyrosine mouse antibody (4G10; UBI, Luzern, Switzerland) diluted 1:1000 for 1 h at room temperature. An anti-mouse IgG antibody conjugated with peroxidase was used as secondary antibody, and detection was performed by the enhanced chemiluminescence (ECL) technique using ECL reagents (Amersham, Dübendorf, Switzerland).
Indirect Immunofluorescence
Sperm in suspension and zona-intact and zona-free oocytes were fixed in 3.7% formaldehyde, washed in PBS, and permeabilized for 10 min in 0.2% Triton X-100 at room temperature. To block nonspecific sites, samples were incubated for 1 h in 10% horse serum and washed in PBS. Sperm were then incubated with a monoclonal antiphosphotyrosine mouse antibody (4G10, 1:100) for 1.5 h at room temperature, washed, and incubated with a secondary anti-mouse IgG antibody conjugated with tetramethylrhodamine isothiocyanate (1:60) for 1 h. Zona-free oocytes were counterstained with Hoechst to visualize sperm chromatin decondensation in the oocytes. Negative controls were prepared by preincubating the first antibody with o-phosphotyrosine or by omitting the first antibody. Sperm in suspension or bound to the oocytes were then observed with a Nikon fluorescence microscope using a 100x objective.
Statistics
The percentages of the different fluorescence patterns were compared, after arcsine transformation, by using the Student t-test or an analysis of variance followed by the Scheffé test for multiple comparisons when two or more than two groups were to be compared, respectively.
RESULTS
Protein Tyrosine Phosphorylation of Sperm in Suspension
The effect of glucose on tyrosine phosphorylation of sperm proteins was first investigated by using Western analysis. When epididymal sperm were incubated as dilute suspensions in the presence of 5.5 mM glucose, a set of proteins (
45–100 kDa) became tyrosine phosphorylated after 90 min (Fig. 1A), whereas incubation of concentrated suspensions did not result in any increase in protein tyrosine phosphorylation (Fig. 1B). Upon dilution of sperm suspensions, previously incubated for 3 h in glucose-containing medium, protein tyrosine phosphorylation increased only when glucose was still present in the culture medium (Fig. 1C). However, longer incubation (3 h) of diluted suspensions in the absence of glucose resulted in a delayed increase in protein tyrosine phosphorylation as compared with the normal appearance of phosphotyrosine in the presence of glucose (Fig. 2). The 116-kDa protein, which has been described as a special type of hexokinase [24], appeared similarly phosphorylated in both concentrated and diluted suspensions and did not appear to be affected by the absence of glucose.
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We have previously shown that glucose is required for sperm to fuse with the oocyte plasma membrane [11]. To determine whether the delayed increase in phosphorylation would allow sperm to enter oocytes despite the absence of glucose, sperm were incubated as diluted suspensions for 3 h with or without glucose before inseminating zona-free oocytes. This delayed increase in phosphorylation did not compensate for the absence of glucose; 85.7% (30/35) and 8.6% (3/35) of zona-free oocytes were penetrated by sperm capacitated with and without glucose, respectively.
To demonstrate its specific effect on tyrosine phosphorylation, glucose was added for a brief period of time to sperm previously incubated under conditions preventing tyrosine phosphorylation, i.e., concentrated suspensions followed by dilution in glucose-free medium. As shown in Figure 3, a rapid increase in tyrosine phosphorylation was observed upon the addition of 5.5 mM glucose. Because the metabolic product of the PPP, NADPH, is able to substitute for glucose in sperm-oocyte fusion [12] we added 5 mM NADPH instead of glucose. An increase in tyrosine phosphorylation was observed as early as 5 min after NADPH exposure (Fig. 3).
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Indirect immunofluorescence was then performed to examine localization of phosphotyrosine residues in sperm incubated as diluted suspensions in the presence or absence of glucose. Representative patterns of fluorescence are shown in Figure 4. In the absence of primary antibody or following its absorption to o-phosphotyrosine, a weak background of fluorescence was observed (data not shown), and sperm presenting such background fluorescence after reaction with the anti-phosphotyrosine antibody were considered negative. As summarized in Figure 5, upon release from the epididymis, most of the sperm were negative and only a small percentage of sperm displayed fluorescence in the acrosomal region over the anterior head, usually associated with an unstained flagellum. The number of acrosome-positive sperm did not increase significantly during capacitation, in both the presence and the absence of glucose. In the presence of glucose, flagellum fluorescence appeared at 30 min of incubation and the numbers of sperm presenting principal piece (with or without positive midpiece) and midpiece fluorescence became significantly higher at 60 and 90 min, respectively. The increase in phosphorylation in the principal piece appeared to precede that in the midpiece, and a fluorescent midpiece was never seen in sperm with an unstained principal piece. In the absence of glucose, the appearance of flagellum fluorescence was delayed to 60 min and the increase in the number of sperm with principal piece fluorescence (with or without positive midpiece) became significant only after 90 min of incubation. At the end of incubation, flagellum fluorescence represented the principal pattern of fluorescence and a large proportion of sperm (>50%) were still negative in the presence and the absence of glucose.
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Protein Tyrosine Phosphorylation of Sperm Bound to Zona-Intact Oocytes
The effect of glucose on phosphotyrosine residue localization in sperm bound to the zona pellucida was investigated by immunofluorescence before and after the reported time of acrosome reaction [25]. The acrosome reaction does not occur before 20–25 min after insemination, and 3 h are necessary for the majority of bound sperm to acrosome react [25]. Sperm and oocytes were coincubated in the presence or absence of glucose for 15 min, and the oocytes were either immediately fixed (time = 0) or incubated for 3 h in fresh medium without spermatozoa. The mean number (±SEM) of bound sperm was not significantly different at t = 0 (15.0 ± 5.5 vs. 10.8 ± 3.0) and sperm detachment occurred significantly in the presence (6.3 ± 2.1) but not in the absence (9.1 ± 4.5) of glucose following 3 h of incubation. As shown in Figure 6A, most of the bound sperm displayed principal piece fluorescence, but spreading of the fluorescence to the midpiece increased significantly with time, so the majority of the bound sperm displayed both midpiece and principal piece fluorescence (Fig. 7, A and B) after 3 h of incubation. In the absence of glucose, the proportion of sperm presenting midpiece fluorescence increased significantly with time, but the onset of this phenomenon was considerably delayed and remained significantly lower when compared with that of sperm from glucose-containing medium. Sperm exhibiting acrosomal fluorescence, associated with flagellum fluorescence, were occasionally seen at both time points and in both media, but their numbers were not significantly different.
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Protein Tyrosine Phosphorylation During Binding and Fusion of Sperm with Zona-Free Oocytes
The effect of glucose on phosphotyrosine residue localization in sperm bound to the oolemma or penetrating into the oocytes was then examined. Sperm and zona-free oocytes were coincubated for 15 min, and the oocytes were either immediately fixed (time = 0) or incubated for a further 20, 30, and 60 min in fresh medium. The mean number (±SEM) of sperm bound to the oolemma was similar in glucose-free (0, 30 min: 5.6 ± 1.7, 7.2 ± 2.0) and glucose-containing medium (0, 20, 30, 60 min: 7.8 ± 2.3, 5.3 ± 1.2, 5.8 ± 1.2, 4.5 ± 0.8), and most of the oocytes were fertilized in the presence of glucose. As shown in Figure 6B, the proportion of bound sperm presenting midpiece fluorescence increased significantly at 30 min only in the presence of glucose. By omitting glucose, a significantly higher proportion of bound sperm were negative and the number of sperm presenting principal piece fluorescence (with or without positive midpiece) and midpiece fluorescence remained significantly lower as compared with sperm from glucose-containing medium. As observed in sperm attached to the zona pellucida, sperm exhibiting acrosomal fluorescence were occasionally seen.
As reported previously, sperm penetration into zona-free oocytes occurred only in the presence of glucose (Fig. 6C). Because sperm chromatin decondensation in the oocyte was not consistently observed before 15–20 min after sperm-oocyte coincubation, we examined the penetrated sperm after 20 min. The fertilization index, i.e., the number of decondensed sperm per oocyte [26], increased between 20 and 60 min from 0.68 to 1.06, indicating that the majority of sperm had penetrated the oocyte by 20 min but that additional sperm may enter the oocyte later. Most of the penetrated sperm displayed a positive flagellum at 20 and 30 min, half of them with whole flagellum fluorescence (Fig. 7, C–E), whereas in others fluorescence was limited to the principal piece. At 60 min, the number of negative sperm increased significantly.
DISCUSSION
Glucose supports sperm-oocyte fusion in the mouse [11], but the target function of glucose has not been clearly identified. Because tyrosine phosphorylation of sperm proteins during capacitation is required for subsequent fertilization [3, 4] and the glucose metabolic product NADPH is able to promote tyrosine phosphorylation in human sperm [17, 18], we hypothesized that one role of glucose is to modulate protein tyrosine phosphorylation during fertilization. In this study, we examined the protein tyrosine phosphorylation in sperm during capacitation and interaction with the oocyte and determined the influence of glucose on this process.
Protein Tyrosine Phosphorylation in Sperm During Capacitation and Interaction with the Oocyte
In the present study, we confirmed the observations of Visconti et al. [3] that phosphorylation of a set of proteins (45–100 kDa) on tyrosine residues increased with capacitation in mouse sperm. In contrast with our immunofluorescence results, Leyton and Saling [27] did not report tyrosine phosphorylation localized to the flagellum but only to the acrosomal region of a small proportion (<5%) of mouse spermatozoa, which increased with capacitation. However, protein tyrosine phosphorylation has been localized to the flagellum in human [28–31] and hamster [32] sperm.
The identity of the tyrosine phosphorylated proteins we observed by immunofluorescence was not determined in this study, but their localization corresponds to that of the germ cell-specific hexokinase associated with the plasma membrane of the head, the mitochondria of the midpiece, and the fibrous sheath of the principal piece [16, 33]. However, the invariable tyrosine phosphorylation of the germ cell-specific hexokinase (116 kDa) during capacitation, when examined by Western analysis, is not consistent with the proposal that the phosphoprotein detected by immunofluorescence is hexokinase, unless changes in phosphorylation of this protein may be quantitatively too small to be detected by Western analysis. Alternatively, the phosphorylated proteins found in the flagellum may represent the 45- to 100-kDa proteins that have been shown by Western analysis to be increased with capacitation [3].
As observed in human sperm [29], a large population of mouse sperm (50%) did not display tyrosine phosphorylation at the end of the capacitation period, raising the question of the identity and role of the phosphorylated sperm. Suspensions include different subpopulations of spermatozoa, and the specific phosphorylation pattern expressed by the fertilizing sperm remains to be clearly determined. Analysis of tyrosine phosphorylation of sperm bound to zona-intact oocytes revealed that tyrosine phosphorylation was not required for sperm to attach to the zona pellucida. Although we cannot conclude that phosphorylation in the principal piece occurred in suspension or upon initial interaction with the zona-pellucida, tyrosine phosphorylation clearly increased in the midpiece after binding to the zona pellucida. Phosphorylation of proteins of the flagellum may be linked to functions involved in sperm penetration of the zona pellucida, such as hyperactivated motility. The observation that tyrosine phosphorylation of flagellum proteins is associated with hyperactivated motility in human [34] and hamster [32] sperm is consistent with this hypothesis. Using solubilized zona pellucida, Leyton and Saling [27] observed an increase in tyrosine phosphorylation of an acrosomal 95-kDa (nonreducing conditions) protein that appears to be required for the acrosome reaction in mouse sperm [35, 36]. Although we failed to detect an increase in the number of sperm with acrosomal phosphorylation, the possibility that a transient increase in phosphorylation in the acrosomal region occurred between sperm attachment and the 3-h observation time point cannot be excluded.
Binding of sperm to the oolemma induced modulation of protein tyrosine phosphorylation similar to that observed after zona pellucida binding. Phosphorylation of the midpiece may be a prerequisite for bound sperm to enter the oocyte. The observation of penetrated sperm displaying protein tyrosine phosphorylation limited to the principal piece may be explained by a process of dephosphorylation of the flagellum following fusion, culminating in the dephosphorylation of the whole flagellum. Although the significance of this dephosphorylation is unclear, it may be correlated with the plasma membrane block to polyspermy, which is believed to operate approximately 40 min after sperm penetration in the mouse [37].
Similar modulation of phosphorylation when sperm were interacting with the zona pellucida or the oolemma was surprising. In contrast to what happens during sperm penetration through the zona pellucida, hyperactivated motility has not been reported to be necessary for bound sperm to fuse with the oolemma [38, 39]. The possibility that the tyrosine phosphorylated proteins may be linked to another sperm function important for both zona pellucida and oocyte penetration may be envisaged and would imply that a common pathway may be activated in sperm interacting with the zona pellucida and the oolemma.
Influence of Glucose on Protein Tyrosine Phosphorylation
In the mouse, glucose is not essential for sperm capacitation [7, 26] but is required for sperm to penetrate the zona pellucida [10] and the oocyte itself [11]. Therefore, glucose can influence sperm function during various phases of gamete interaction.
In the absence of glucose, the appearance of tyrosine phosphorylation in sperm suspensions was delayed and sperm did not successfully penetrate oocytes, indicating that 1) tyrosine phosphorylation of sperm proteins prior to gamete interaction is not sufficient to allow sperm to fuse with the oocyte and to complete capacitation [40, 41], although phosphorylation has been correlated with capacitation [3], and 2) the influence of glucose on phosphorylation of sperm proteins during gamete interaction may be relevant to successful fertilization.
When sperm were bound to the zona pellucida or to the oolemma, glucose was required for a rapid increase in tyrosine phosphorylation spreading over the entire flagellum, including the midpiece. Under our experimental conditions, only one or two sperm out of approximately five bound sperm were able to penetrate zona-free oocytes by 30 min, before the oolemma block to polyspermy is functional [37]. Thus, the remaining bound sperm apparently failed to accomplish a series of events required for sperm fusion with the oocyte. The existence of subpopulations of sperm that have an altered response to glucose, preventing or delaying midpiece phosporylation, could explain their inability to penetrate the oocyte.
Redkar and Olds-Clarke [26] reported that prolonged sperm-oocyte incubation in the absence of glucose (3–5 h) resulted in sperm penetration, although at a lower rate, suggesting that tyrosine phosphorylation in the midpiece may increase over longer time periods, subsequently allowing sperm penetration despite the absence of glucose.
Because glucose must be metabolized to allow sperm to fuse with the oocytes [11], products of glucose metabolism may play a significant role in regulating tyrosine phosphorylation. Glucose is metabolized through glycolysis and the PPP in mouse sperm, but glycolysis may not be essential for sperm-oocyte fusion [12]. In addition, sperm is not deprived of ATP in the absence of glucose because ATP is generated by mitochondrial respiration using pyruvate and lactate [42], which are both components of routine fertilization culture medium. In contrast, NADPH, which increased protein tyrosine phosphorylation in mouse sperm during capacitation in this study, may rely exclusively on glucose metabolism; the main source of NADPH appears to be the PPP in human sperm [43]. In the absence of glucose, extramitochondrial NADH could partially substitute for NADPH in NADPH-dependent protein tyrosine phosphorylation [18, 44] and may explain the cause for a delay in this process.
Visconti and Kopf [45] proposed a model for the control of protein tyrosine phosphorylation in sperm during capacitation. They proposed that removal of cholesterol from the sperm plasma membrane would induce a sequence of events including an increase in bicarbonate uptake, activation of adenylate cyclase, activation of protein kinase A, and an increase in protein tyrosine phosphorylation. In addition, Aitken et al. [46] reported regulation of intracellular cAMP by reactive oxygen species (ROS) in human sperm. Although the relationship between glucose and this sequence remains to be investigated, NADPH could be involved in ROS generation by activating NADPH oxidase [44]. The absence of tyrosine phosphorylation when the sperm suspensions were concentrated may also be related to ROS metabolism. The presence of antioxidant enzymes originating from the epididymis in concentrated sperm suspensions may result in limited production of H2O2 and therefore limited tyrosine phosphorylation [47].
The correlation between successful gamete fusion and the tyrosine phosphorylation of proteins in the sperm midpiece strongly suggests that effect of glucose on sperm-oocyte interaction is mediated through regulation of protein tyrosine phosphorylation in the fertilizing sperm. Further investigations are needed to ascertain the role of the glucose metabolic pathways and any secondary effects of glucose on specific sperm functions. Characterization of the timing of protein phosphorylation in the various compartments of sperm and its link to the acquisition of specific sperm functions will also inform us as to the sequence of molecular events needed by an individual sperm to fertilize the oocyte.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of Caroline Schaetti, Ingrid Wagner, and Nicole Jaquenoud and the continued support of Prof. A. Campana.
FOOTNOTES
First decision: 6 September 2000.
1 Supported by the Fonds National Suisse de la Recherche Scientifique (32-55693.98). 
2 Correspondence: Françoise Urner, Laboratoire des Gamètes, Clinique de Stérilité, Hôpital cantonal, 30 Bd. de la Cluse, 1211 Geneva 14, Switzerland. FAX: 41 22 382 43 85; francoise.urner{at}hcuge.ch
Accepted: December 12, 2000.
Received: August 3, 2000.
REFERENCES
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|
|
|
|
 |
|
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|
|
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|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
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|
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|
|
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|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
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A. J. Travis, L. Tutuncu, C. J. Jorgez, T. S. Ord, B. H. Jones, G. S. Kopf, and C. J. Williams Requirements for Glucose Beyond Sperm Capacitation During In Vitro Fertilization in the Mouse Biol Reprod, July 1, 2004; 71(1): 139 - 145. [Abstract] [Full Text] [PDF]
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S. Shadan, P. S. James, E. A. Howes, and R. Jones Cholesterol Efflux Alters Lipid Raft Stability and Distribution During Capacitation of Boar Spermatozoa Biol Reprod, July 1, 2004; 71(1): 253 - 265. [Abstract] [Full Text] [PDF]
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S. Mededovic and L. R Fraser Angiotensin II stimulates cAMP production and protein tyrosine phosphorylation in mouse spermatozoa Reproduction, May 1, 2004; 127(5): 601 - 612. [Abstract] [Full Text] [PDF]
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V. G. Da Ros, M. J. Munuce, D. J. Cohen, C. I. Marin-Briggiler, D. Busso, P. E. Visconti, and P. S. Cuasnicu Bicarbonate Is Required for Migration of Sperm Epididymal Protein DE (CRISP-1) to the Equatorial Segment and Expression of Rat Sperm Fusion Ability Biol Reprod, May 1, 2004; 70(5): 1325 - 1332. [Abstract] [Full Text] [PDF]
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K. Mitra and S. Shivaji Novel Tyrosine-Phosphorylated Post-Pyruvate Metabolic Enzyme, Dihydrolipoamide Dehydrogenase, Involved in Capacitation of Hamster Spermatozoa Biol Reprod, April 1, 2004; 70(4): 887 - 899. [Abstract] [Full Text] [PDF]
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M. A. Baker, L. Hetherington, H. Ecroyd, S. D. Roman, and R. J. Aitken Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation J. Cell Sci., January 15, 2004; 117(2): 211 - 222. [Abstract] [Full Text] [PDF]
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H. Ecroyd, R. C. Jones, and R. J. Aitken Tyrosine Phosphorylation of HSP-90 During Mammalian Sperm Capacitation Biol Reprod, December 1, 2003; 69(6): 1801 - 1807. [Abstract] [Full Text] [PDF]
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H. W. Ecroyd, R. C. Jones, and R. J. Aitken Endogenous Redox Activity in Mouse Spermatozoa and Its Role in Regulating the Tyrosine Phosphorylation Events Associated with Sperm Capacitation Biol Reprod, July 1, 2003; 69(1): 347 - 354. [Abstract] [Full Text] [PDF]
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D. Sakkas, G. Leppens-Luisier, H. Lucas, D. Chardonnens, A. Campana, D.R. Franken, and F. Urner Localization of Tyrosine Phosphorylated Proteins in Human Sperm and Relation to Capacitation and Zona Pellucida Binding Biol Reprod, April 1, 2003; 68(4): 1463 - 1469. [Abstract] [Full Text] [PDF]
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