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Gamete Biology |
a Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, gamete biology, kinases, signal transduction, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanisms of capacitation remain poorly understood, particularly in the pig; most studies have been conducted on other mammals. Cholesterol efflux from the head plasma membrane is believed to initiate a signal transduction pathway [12, 13], and capacitation is strongly associated with reduced membrane cholesterol and, consequently, a lower cholesterol:phospholipid (C:P) ratio [10]. Cholesterol efflux has been demonstrated in numerous studies but with only human or rodent sperm [14–17]. During capacitation in vitro, cholesterol efflux is mediated by BSA, which is thus considered to be necessary for capacitation in many species [18]. BSA is less critical for capacitation of pig sperm in vitro [19, 20].
In many species, calcium influx to sperm also occurs during capacitation [21–25] and may activate one or more enzymatic systems or pathways. For example, adenylate cyclase increases during capacitation [26, 27] and in response to calcium [28–30]. Bicarbonate also activates sperm adenylate cyclase [31–33] and rapidly fluidizes pig sperm plasma membranes [4]. Thus, both calcium and bicarbonate support capacitation, and their roles are probably interrelated, as indicated by observations that bicarbonate stimulates calcium uptake in porcine sperm [25] and is necessary for calcium influx into mouse sperm [34].
As in many mammals, sperm capacitation in pigs requires Ca2+ [19, 24] and bicarbonate [35–39]. The sequence of events for pig sperm capacitation is similar inother ways to that in other species. Tyrosine phosphorylation of mouse, human, bull, and hamster sperm proteins is associated with capacitation [40–42]; tyrosine phosphorylation of sperm proteins also occurs during capacitation in the pig [43, 44]. We have recently demonstrated that tyrosine phosphorylation of an Mr 32 000 pig sperm protein (p32) and the activation of an Mr 32 000 tyrosine kinase (TK-32) occur concomitant with capacitation [45].
The primary objective of the present study was to evaluate the importance of medium components on capacitation of porcine sperm, tyrosine phosphorylation of sperm proteins (particularly p32), and activity of TK-32. The secondary objective was to use these findings in combination with immunoprecipitation and inhibitor experiments to determine whether p32 and TK-32 are the same molecule. The results presented here suggest that capacitation of pig sperm is mediated by a signal transduction pathway that is slightly different than that of other species and that p32 and TK-32 are different proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Molecular weight standards were obtained from Amersham International (Oakville, ON, Canada). Acrylamide N,N'-methylene bisacrylamide, ammonium persulfate, and tris (hydroxymethyl) aminoethane (Tris) were from BioRad Laboratories (Mississauga, ON, Canada). Monoclonal mouse anti-phosphotyrosine antibody (clone 4G10) was purchased from Upstate Biotechnology (Lake Placid, NY), peroxidase-conjugated goat anti-mouse antibody was from BioRad, and anti-mouse IgG agarose conjugates were from Santa Cruz Biotechnology (Santa Cruz, CA). Other chemical products were from Sigma Chemical Company (St. Louis, MO).
Culture Media
The principal culture media used were based on Krebs Ringer bicarbonate [46] as described by Tardif et al. [45]. Capacitating medium (CM) was composed of 4.8 mM KCl, 1.2 mM KH2PO4, 95 mM NaCl, 5.56 mM glucose, 25 mM NaHCO3, 2 mM CaCl2, 0.6% BSA, and 1 mM pyruvate (pH 7.4). The noncapacitating medium (NCM) was similar to CM but without calcium, bicarbonate, and BSA (2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 137 mM NaCl, 5.55 mM glucose, and 1 mM pyruvate, pH 7.4).
Sperm Preparation and Treatments
Semen was collected from fertile boars by the gloved-hand method [47]. The sperm-rich fraction was diluted with Beltsville Thawing Solution [48] at the Centre d'Insémination Porcine du Québec (St-Lambert, QC, Canada) and transported to the laboratory at 16°C within 20 min. The diluted semen was divided into two portions, centrifuged once (10 min, 22°C, 270 x g), and diluted (4 x 107 sperm/ml). The first portion was diluted in CM to induce capacitation, and the second portion was resuspended in NCM as a noncapacitating negative control. Sperm were then incubated at 39°C in a 5% CO2 humidified atmosphere for up to 4.5 h.
Evaluation of Sperm Capacitation
Sperm capacitation was determined by the ability of the sperm to undergo the A23187-induced AR as described previously [49]. Calcium (2 mM) was added to calcium-depleted medium (NCM) before induction with A23187. Acrosome-reacted sperm were identified using fluorescein-labeled Pisum sativum agglutinin as previously described [49].
Isolation of Pig Sperm Proteins
Sperm proteins were isolated as described previously [45]. During incubation in either NCM or CM, sperm aliquots were taken (1–5 x 106 sperm) at different times. Sodium orthovanadate (0.2 mM) was added, and the aliquots were centrifuged briefly to isolate a sperm pellet (2–5 min, 13 000 x g). The pellet was resuspended in sample buffer [50] without ß-mercaptoethanol and boiled for 2–5 min. The sperm solution was centrifuged (2–3 min, 13 000 x g), and the resulting supernatant was boiled in sample buffer with ß-mercaptoethanol (5%) for 2–5 min. The sperm protein sample was then subjected to SDS-PAGE.
SDS-PAGE and Western Blotting
Sperm proteins were separated by SDS-PAGE on 12% polyacrylamide gels and transferred electrophorectically [45, 51] (4–8 mA h-1 [cm2]-1) to polyvinyldienne fluoride (PVDF) membranes. Nonspecific protein-binding sites on the membrane were blocked with 5% dry nonfat milk in Tris-buffered saline (TBS; 25 mM Tris-HCl pH 7.4, 150 mM NaCl). The PVDF membrane was incubated with anti-phosphotyrosine antibodies for 1 h at 1:20 000 in TTBS (TBS + 0.5% Tween 20). After washing (10 min, three times), the blot was incubated with peroxidase-conjugated goat anti-mouse antibodies at 1:20 000 in TTBS for 45 min and washed again. Labeled proteins were revealed using enhanced chemiluminescence detection with the ECL kit (Amersham) according to the manufacturer's instructions.
Enzyme Renaturation after SDS-PAGE
Sperm proteins prepared as described and separated according to their molecular weights by SDS-PAGE with 100 µg/ml poly glu-tyr (4:1) copolymerized in the gel matrix. Sperm protein kinase renaturation was performed as previously described [45]. SDS was removed at room temperature (RT) from gels by one wash overnight in buffer A (20% propan-2-ol, 50 mM imidazole, 28 mM iminodiacetic acid) followed by another wash for 60 min (buffer A containing 10 mM ß-mercaptoethanol; RT). To unfold the proteins, gels were placed in plastic bags for 90 min (buffer A containing 8 M guanidine-HCl, 10 mM ß-mercaptoethanol). Gels then were incubated four times (and buffer refreshed) at 4°C with gentle agitation in buffer A (containing 10% sucrose, 0.04% Tween 20, 10 mM ß-mercaptoethanol); the first and second incubations were for 90 min, the third was overnight, and the last was for 60 min. Gels were equilibrated in buffer B (10 mM Hepes-NaOH pH 7.4, 20 mM MgCl2, 5 mM MnCl2, 0.2 mM Na3VO4, 10 mM ß-mercaptoethanol) for 60 min at RT. Subsequently, kinase renaturation, activation, and substrate phosphorylation proceeded in plastic bags on a rotative agitator in buffer B including 100 µCi ATP-
-32P. Gels were successively washed with 5% trichloroacetic acid, 1% pyrophosphate, and 10% phosphate (monobasic). When the matrices were considered to be nonradioactive as evaluated by Geiger counting (i.e., in the corner of the gel away from the proteins), the gels were dehydrated in 10% methanol (1 h, RT) and dried. Gels were then exposed on phosphorscreens, and substrate phosphorylation was quantified by phosphorimagery (PhosphorImager; Molecular Dynamics, Sunnyvale, CA).
Immunoprecipitation
Sperm were solubilized (45 min, 40 x 106 cells) in 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 50 mM Tris-HCl pH 7.4, 0.2 mM Na3VO4, 2 mM EGTA, 2 mM EDTA, 0.1 mg/ml leupeptin, 0.05 mg/ml aprotinin, 0.02 mg/ml pepstatin, and 1 mM PMSF. Anti-phosphotyrosine antibodies (5–10 µg) were added to the sperm proteins and gently agitated at 4°C overnight. The immunocomplexes were sequestered by adding 100 µl of agarose bead slurry and gently agitating at 4°C for 2 h. The agarose beads containing the immunocomplexes were collected by pulsed centrifugation (5 sec, 13 000 x g), and the supernatant was reserved for acetone precipitation of the proteins not immunoprecipitated by the anti-phosphotyrosine antibody. The agarose beads were washed three times with PBS, resuspended in sample buffer, and boiled. Western blotting and enzyme renaturation of the anti-phosphotyrosine immunoprecipitates and supernatant proteins were performed as described above.
Statistical Analyses
Differences in the percentages of capacitated and viable sperm due to treatment (NCM vs. CM) or time were determined by ANOVA using general linear model procedures [52]. A Fisher protected least significant difference test was conducted when the main effect was significant (P < 0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein tyrosine phosphorylation patterns were unchanged by the addition of BSA or HCO3- to NCM separately (Fig. 1, A and B, left). However, a faint band corresponding to p32 was occasionally apparent from sperm incubated in calcium + NCM after 3 h (Fig. 1C, left). The capacitation state was unaffected by the inclusion of each these compounds in NCM (P = 0.76; Table 1).
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When sperm were incubated in CM devoid of either BSA or HCO3-, no effect on protein tyrosine phosphorylation was observed over time (Fig. 1, A and B, right). Elimination of either BSA or bicarbonate from CM did not change the appearance of any phosphotyrosine-containing proteins, including p32. No change in the percentage of capacitated sperm was observed when BSA was absent in the CM (Table 1); however, the removal of bicarbonate inhibited capacitation (Table 1; P < 0.001). Figure 1C shows that when the sperm were incubated in CM depleted of calcium, the appearance of the tyrosine phosphoprotein, p32, decreases markedly. Capacitation was also inhibited when the sperm were incubated in CM without calcium for 3 h (Table 1; P < 0.001).
Effect of Bicarbonate and Calcium on Protein Tyrosine Kinase (TK-32) Activity
The tyrosine kinase activity of TK-32, an Mr 32 000 enzyme, is stimulated in capacitated sperm, and we previously speculated that TK-32 is the same molecule as p32 (a tyrosine kinase substrate) [45]. As evaluated by phosphoimagery, TK-32 activity was inhibited when the sperm were incubated for 3 h in CM depleted of either bicarbonate or calcium (Fig. 2; P < 0.001). The activity of TK-32 was similar whether the CM was depleted of bicarbonate or calcium (P = 0.86).
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p32 and TK-32 Are Not the Same Protein
Tyrosine kinase activity was detected only in the supernatant of the tyrosine phosphotyrosine immunoprecipitation experiment (Fig. 3). The identities of neither TK-32 nor p32 are known, although we have speculated that they may be the same protein [45]. However, contrary to this hypothesis, p32 (from the immunoprecipitate) possesses no tyrosine kinase activity, although p32 was still detected in the supernatant after immunoprecipitation because all sperm antigens cannot be precipitated (data not shown). The fact that the anti-phosphotyrosine antibody did not immunoprecipiate TK-32 (Fig. 3, lane 2) strongly suggests that p32 and TK-32 are not the same protein.
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In agreement with the immunoprecipitation results, the appearance of p32 and TK-32 activity differed in response to the tyrosine kinase inhibitor bistyrphostin (Fig. 4). The appearance of p32 was not reduced at all by the inclusion of bistyrphostin during incubation for 3 h (Fig. 4A). In contrast, the activity of TK-32 was eliminated (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BSA Is Not Essential
Both capacitation and the appearance of p32 were unchanged by the exclusion of BSA from CM. These data support previous suggestions that BSA is not essential for capacitation of porcine sperm [19, 20]. Albumin mediates the loss of cholesterol from sperm membranes, thus reducing the C:P ratio [14, 16, 54]. However, the C:P ratio of the plasma membrane of boar sperm is already considerably lower than that of other species even before capacitation; it is only 0.2 in boar sperm [55] compared with 0.4 in bovine sperm [56] and 0.99 in human sperm [57]. Thus, because the C:P ratio of boar sperm plasma membrane is relatively low, albumin-mediated cholesterol efflux may not be necessary during capacitation, as indicated by the apparent unimportance of BSA to capacitation and p32 appearance. For this reason, the effect of BSA on the activity of the TK-32 enzyme was not evaluated.
Importance of Bicarbonate and Calcium
In contrast, bicarbonate was necessary for capacitation of pig sperm and maximal TK-32 activity but not for the appearance of p32. The importance of calcium for boar sperm is similar to that for hamster sperm; exclusion of bicarbonate from the medium did not block the appearance of phosphotyrosine-containing proteins associated with capacitation in hamster sperm [58]. However, in the mouse, bicarbonate is required for both protein tyrosine phosphorylation and capacitation [40]. In human sperm, bicarbonate is also required for capacitation and is associated with the redox status linked to tyrosine phosphoprotein appearance [59].
Bicarbonate rapidly destabilizes boar sperm plasma membranes and leads to increased calcium flux into both the head and tail [25]. The plasma membranes from pig sperm were also fluidized by elevating cAMP using the phosphodiesterase inhibitor isobutylmethylxanthine [4, 39]. Bicarbonate itself also stimulates adenylate cyclase activity and transiently increases sperm cAMP levels [31] that are thought to favor protein tyrosine phosphorylation during capacitation [31, 60]. Kalab and coworkers [43] noted that pig sperm contain endogenous phosphodiesterase inhibitors. During capacitation, these natural inhibitors may permit a sufficiently high level of cAMP and membrane destabilization to facilitate calcium influx. The subsequent action of calcium would then cause tyrosine phosphorylation and the appearance of p32. In human sperm, protein tyrosine phosphorylation is also associated with capacitation, and as observed here, tyrosine phosphorylation alone was not sufficient for the completion of capacitation [61]. The appearance of the tyrosine phosphoprotein p32 is not itself indicative of full capacitation of pig sperm. Conversely, TK-32 activation may be more representative of the capacitation state; the absence of bicarbonate inhibited both its kinase activity and capacitation. Supplementing NCM with bicarbonate alone did not induce capacitation or p32 appearance, suggesting that bicarbonate acts in concert with calcium (Table 1 and Fig. 1).
The appearance of p32 in CM without bicarbonate is therefore due to calcium action on tyrosine kinase(s) and/or phosphatases(s) that are as yet not identified. Capacitation, the appearance of p32, and TK-32 activity were all inhibited when the sperm were incubated in CM without calcium. Furthermore, the emergence of p32 and TK-32 activation are both calcium dependent and associated with capacitation. Nevertheless, in the CM depleted of bicarbonate, the calcium alone was not able to induce capacitation, underlining the necessity of bicarbonate even in the presence of calcium. Therefore, despite its importance in the signaling events related to capacitation of porcine sperm, calcium alone is insufficient.
These results suggest that the roles of bicarbonate and calcium are difficult to separate and there is probably an interaction between them. Figure 1 shows that NCM with calcium does not support the appearance of p32, even though its presence was expected based on the findings with CM ± calcium, suggesting that calcium alone is not sufficient for p32 appearance. The appearance of p32 must be associated with some interaction between calcium and bicarbonate or even BSA. In the case of CM without bicarbonate, the calcium presumably initiates the pathway leading to the appearance of p32. However, the sperm remain unable to undergo capacitation, probably because the requisite architectural modifications to the plasma membrane cannot occur in the absence of bicarbonate [39].
p32 and TK-32 Are Not the Same Protein
Moreover, the differing effects of bicarbonate and calcium on the in-gel tyrosine kinase renaturation experiments indicate that p32 is not the same protein as TK-32. The TK-32 activity is appreciably diminished in the absence of either bicarbonate or calcium in the CM (Fig. 2), whereas only calcium depletion affected p32 appearance (Fig. 1). Also, immunoprecipitation with anti-phosphotyrosine antibodies to remove p32 did not eliminate TK-32 activity from the supernatant (Fig. 3). TK-32 is not tyrosine phosphorylated because TK-32 activity was not present in the anti-phosphotyrosine antibody immunoprecipitates. This experiment indicates that these two proteins of Mr 32 000 (p32 and TK-32) are different and that TK-32 itself is not tyrosine phosphorylated. These results are supported by experimentation with a tyrosine kinase inhibitor, bistyrphostin (Fig. 4), which did not alter p32 appearance but abolished TK-32 activity. However, definitive differentiation of these two proteins will only be achieved after proteomic identification and immunoprecipation using antibodies specific to one or both. Although TK-32 has not been identified, its activity, which is clearly maximized by the simultaneous presence of bicarbonate and calcium (both of which are required for capacitation), probably is involved in the pathway leading to complete capacitation.
Speculative Mechanism of Capacitation
In this study, we observed two levels of regulation during capacitation of pig sperm at different stages: 1) the appearance of p32, a tyrosine kinase substrate, and 2) the activity of TK-32, a putative tyrosine kinase. The appearance of p32 is calcium dependent, and p32 is downregulated without this ion. However, p32 appears even in the absence of bicarbonate in calcium-containing medium. Calcium, therefore, is able to stimulate the formation of p32, but without bicarbonate the degree of membrane destabilization is not adequate for the completion of capacitation. Possible explanations for these findings are that the calcium acts to activate a tyrosine kinase or to block a tyrosine phosphatase. Either way, the outcome would be the enhancement of p32. Alternatively, calcium may interact with an effector enzyme such as PLC or PK-C, which could stimulate a specific tyrosine kinase whose activity would yield p32. Leclerc et al. [62] showed that calmodulin was modulated during capacitation of bull sperm. Calmodulin-binding proteins have recently been localized on the bovine sperm subacrosomal and postacrosomal regions during capacitation in the presence of calcium [63]. Capacitation of pig sperm may be accomplished via a tyrosine kinase using this amplification system. Calcium has a high affinity for calmodulin, and their association changes the conformation of calcium and induces binding to an effector, such as a kinase or a cAMP-phosphodiesterase [64]. Although the activity of sperm TK-32 increases in a manner dependent on the presence of bicarbonate and calcium, the details of its regulation are unknown. However, identification of TK-32 will facilitate the elucidation of this mechanism.
Although enhanced tyrosine phosphorylation of sperm proteins due to calcium during capacitation has been shown in the mouse [40], extracellular calcium inhibits protein tyrosine phosphorylation in human sperm [65, 66]. Such dephosphorylation may be regulated through the activation of a phosphatase, calcineurin [66]. Such phosphatases may be less sensitive to calcium in mouse and pig sperm than in human sperm.
Our data demonstrate that in pig sperm, bicarbonate and calcium play crucial roles in the capacitation process. The tyrosine phosphoprotein p32 appears during capacitation in a calcium-dependent manner, but its presence is not a prerequisite for capacitation of pig sperm, although capacitation always occurs when this protein is present. However, the presence of BSA does not alter capacitation or the appearance of p32. We also demonstrated that both bicarbonate and calcium stimulate the tyrosine kinase activity of TK-32. Although it is generally accepted that protein tyrosine phosphorylation accompanies capacitation of mammalian sperm, certain aspects of this process are species specific, and the specific tyrosine phosphoproteins and the regulation of their appearance differ considerably among species. Further studies to identify both p32 and TK-32 and to elucidate their roles in capacitation are currently underway in our laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Janice L. Bailey, Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Pavillon Paul-Comtois, Université Laval, Sainte-Foy, QC, Canada G1K 7P4. FAX: 481 656 3766; janice.bailey{at}crbr.ulaval.ca
Received: 1 March 2002.
First decision: 31 March 2002.
Accepted: 5 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P. Mol Reprod Dev 1999 54:292-302[CrossRef][Medline]This article has been cited by other articles:
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  C. Campagna, C. Guillemette, P. Ayotte, and J. L. Bailey Effects of an Environmentally Relevant Organochlorine Mixture and a Metabolized Extract of This Mixture on Porcine Sperm Parameters In Vitro J Androl, May 1, 2009; 30(3): 317 - 324. [Abstract] [Full Text] [PDF]
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 S. Aquila, V. Rago, C. Guido, I. Casaburi, S. Zupo, and A. Carpino Leptin and leptin receptor in pig spermatozoa: evidence of their involvement in sperm capacitation and survival Reproduction, July 1, 2008; 136(1): 23 - 32. [Abstract] [Full Text] [PDF]
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M. L. Vadnais and K. P. Roberts Effects of Seminal Plasma on Cooling-Induced Capacitative Changes in Boar Sperm J Androl, May 1, 2007; 28(3): 416 - 422. [Abstract] [Full Text] [PDF]
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 |
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 M.-F. Lusignan, A. Bergeron, M.-H. Crete, C. Lazure, and P. Manjunath Induction of Epididymal Boar Sperm Capacitation by pB1 and BSP-A1/-A2 Proteins, Members of the BSP Protein Family Biol Reprod, March 1, 2007; 76(3): 424 - 432. [Abstract] [Full Text] [PDF]
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P. Grasa, J. A. Cebrian-Perez, and T. Muino-Blanco Signal transduction mechanisms involved in in vitro ram sperm capacitation. Reproduction, November 1, 2006; 132(5): 721 - 732. [Abstract] [Full Text] [PDF]
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C. Dube, P. Leclerc, T. Baba, C. Reyes-Moreno, and J. L. Bailey The Proacrosin Binding Protein, sp32, Is Tyrosine Phosphorylated During Capacitation of Pig Sperm J Androl, July 1, 2005; 26(4): 519 - 528. [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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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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H. Harayama Viability and Protein Phosphorylation Patterns of Boar Spermatozoa Agglutinated by Treatment With a Cell-Permeable Cyclic Adenosine 3',5'-Monophosphate Analog J Androl, November 1, 2003; 24(6): 831 - 842. [Abstract] [Full Text] [PDF]
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J. C. Kirkman-Brown Editorial Commentary J Androl, September 1, 2003; 24(5): 734 - 735. [Full Text] [PDF]
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