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RESEARCH |
Laboratory of Animal Reproduction, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan and 1 Department of Molecular and Life Sciences, University of Abertay Dundee, Dundee DD1 1HG, UK
Correspondence should be addressed to Koji Ashizawa; Email: ashizawa{at}cc.miyazaki-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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In fowl spermatozoa, the addition of extracellular Ca2+ is found to be an absolute requirement for acrosomal exocytosis at 40 °C, the normal body temperature of these birds. When incubated with preparations of the homogenized inner perivitelline layers (IPVLs), which may be considered to be analogous to the mammalian zona pellucida (Waclawek et al. 1998), fowl spermatozoa were unable to undergo the acrosome reaction. However, the addition of Ca2+ to the salt solution resulted in a significant increase in acrosomal exocytosis (Robertson 1999). Additionally, unlike mammalian spermatozoa, fowl sperm motility is reversibly inhibited as the temperature is raised from 30 °C to 40 °C. Motility is restored by decreasing the temperature or by the addition of Ca2+ at 40 °C (Munro 1938, Ashizawa & Nishiyama 1978, Ashizawa & Wishart 1987, 1992, Ashizawa et al. 1989a, 1994a, Wishart & Ashizawa 1987). Therefore, Ca2+ seems to play a key role for the stimulation of the acrosome reaction and the motility of fowl spermatozoa at the avian body temperature.
Ca2+-dependent cysteine protease (calpain), an enzyme responsible for degradation of axonal and muscle cytoskeletal elements, has been isolated and characterized from fowl tissues such as skeletal muscle (Kawashima et al. 1984, Wolfe et al. 1989, Johari et al. 1993, Birkhold & Sams 1994, Sorimachi et al. 1995), brain, sciatic nerve and gastrocnemius muscle (el-Fawal et al. 1990). In most mammalian tissues and cells, there are more than two forms of calpain – namely calpain 1 and calpain 2 – which have identical substrate specificities, but require low and high Ca2+ concentrations respectively, for their activation (Murachi 1989, Croall & DeMartino 1991). The small GTPase Rho also plays pivotal roles in the Ca2+ sensitization of smooth muscle contraction (Hirata et al. 1992, Gong et al. 1996, Otto et al. 1996) and a recent study demonstrates that Rho-kinase (Rho-associated kinase), one of target proteins of Rho, modulates smooth muscle contraction in a Ca2+-dependent manner (Kureishi et al. 1997). Rho and Rho-kinase activities have also been identified in fowl smooth muscle (Feng et al. 1999, Anabuki et al. 2000, Stevenson et al. 2004).
Although there is no previous evidence for the existence of these proteins in fowl spermatozoa, both calpain (Schollmeyer 1986, Rojas et al. 1999, Rojas & Moretti-Rojas 2000, Yudin et al. 2000, Aoyama et al. 2001) and Rho (Castellano et al. 1997) have been found in mammalian and sea urchin spermatozoa respectively: calpain appears to be involved in the regulation of the acrosome reaction (Schollmeyer 1986, Yudin et al. 2000, Aoyama et al. 2001) and the cell fusion process that takes place during penetration of the oocyte (Rojas et al. 1999, Rojas & Moretti-Rojas 2000). The presence of Rho in the acrosomal region, the middle piece of the head and in the flagellum of sea urchin spermatozoa has been demonstrated by both immunofluorescence and immunogold staining and, based on this cellular localization, it has been assumed that Rho may participate in regulating motility and the actin polymerization that accompanies the acrosome reaction (Castellano et al. 1997). If so, Rho-kinase should also participate in the regulatory mechanisms of the acrosome reaction and motility of spermatozoa.
However, no information is available concerning the effects of calpain and Rho-kinase on the regulation of the acrosome reaction and motility of avian spermatozoa, although potent and selective inhibitors of calpain and Rho-kinase, namely PD 150606 (Wang et al. 1996) and Y-27632 (Uehata et al. 1997) respectively, have been developed. In the following experiment, therefore, attempts were made to investigate the effects of PD 150606 and Y-27632 on the acrosome reaction and motility of fowl spermatozoa.
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Materials and Methods |
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Semen was collected by the method of Bogdonoff and Shaffner (1954). Samples of semen pooled from four to six males were diluted approximately 10-fold in 150 mmol NaCl/l with 20 mmol TES (N-Tris-(hydroxymethyl)-methyl-2-aminoethanesulfonic acid)/l at pH 7.4 and centrifuged at 700 g for 13 min at room temperature (20–25 °C). The washed spermatozoa were reconstituted in the same buffer to give a final concentration of approximately 6 x 108 cells/ml.
Chemicals
Y-27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide), a cell-permeable selective inhibitor of Rho-kinase, a generous gift from Yoshitomi Pharmaceutical Industries, Ltd (Osaka, Japan), was dissolved in distilled water as a stock solution (100 mmol/l) and stored at –30 °C until use. PD 150606 (3-(4-lodophe-nyl)-2-mercapto-(Z)-2-propenoic acid), a cell-permeable selective calpain inhibitor, obtained from Calbiochem-Novabiochem Co. (La Jolla, CA, USA), was dissolved in DMSO as a stock solution (10 mmol/l) and stored at –30 °C until use. Calyculin A, a specific inhibitor of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). ATP, bovine serum albumin, desiccated firefly tails, fluorescence isothiocynate (FITC)-conjugated peanut agglutinin (PNA) and TES were obtained from Sigma. Tween 20 was purchased from MP Biomedicals, Inc. (Aurora, OH, USA). Bicinchoninic acid (BCA) protein assay regent was from Pierce Chemical Co. (Rockford, IL, USA). SDS-PAGE molecular weight standards were purchased from Amersham. Other chemicals were of reagent grade from Nacalai Tesque, Inc. (Kyoto, Japan).
Antibodies
A rabbit polyclonal antibody raised against a synthetic peptide based on the amino-terminal end of domain-I in the large subunit of calpain 12 (molecular weight, 80 kDa) was purchased from Triple Point Biologics, Inc. (Forest Grove, OR, USA). Horseradish peroxidase-conjugated anti-rabbit immunoglobulins donkey serum was obtained from Amersham.
Analysis of the acrosome reaction of spermatozoa
IPVLs were separated from laid fowl eggs (Robertson et al. 1997) and were homogenized using a Teflon glass homogenizer on ice. The protein concentrations of IPVL homogenates were adjusted to 75 µg/ml with TES/NaCl buffer (pH 7.4), using bovine serum albumin as a standard. Fowl spermatozoa, at concentrations adjusted to 1.2 x 107 cells/ml were incubated, with or without IPVL, for 30 min at 40 °C. The dose–response of the acrosome reaction was measured in the presence of various concentrations of PD 150606 or Y-27632 and the effects of the addition of CaCl2 after the addition of PD 150606 or Y-27632 were also examined. The inhibition constant (Ki) values of PD 150606 for calpains are around 0.2–0.4 µmol/l (Wang et al. 1996) and the Ki value of Y-27632 for Rho-kinase is 0.14 µmol/l (Uehata et al. 1997). Ordinarily, 10- to 100-fold higher concentrations are required for the whole cells. Therefore, both inhibitors were used in the range of 1–100 µmol/l in this study.
Acrosome-reacted spermatozoa were identified using a fluorescent microscope at x 1000 magnification and FITC-conjugated PNA – which binds to acrosome-reacted, but not acrosome-intact, spermatozoa – as described by Horrocks et al.(2000). The percentages of acrosome reaction were calculated from a total of approximately 100 spermatozoa distributed uniformly among three or more fields.
Analysis of motility of intact and demembranated spermatozoa
Sperm samples were pre-incubated aerobically in a water bath at 30 or 40 °C for 10 min. After the pre-incubation, the dose–response and time course of motility of intact spermatozoa were measured at 30 or 40 °C after addition of PD 150606 or Y-27632. The effects of the addition of CaCl2 or calyculin A, after the addition of PD 150606 or Y-27632 were also examined at 30 and 40 °C. The diluent for the incubation and measurement of sperm motility was TES/NaCl buffer without IPVL, as described above.
Demembranation and reactivation of spermatozoa were performed at 30 and 40 °C according to the method described previously (Ashizawa et al. 1989b). The extraction medium used consisted of 0.1% (v/v) TritonX-100, 200 mmol sucrose/l, 25 mmol potassium glutamate/l, 1 mmol MgSO4/l, 1 mmol dithiothreitol (DTT)/l and 20 mmol Tris–HCl buffer/l (pH 7.9). The reactivation medium consisted of 0.5 mmol ATP/l, 200 mmol sucrose/l, 25 mmol potassium glutamate/l, 1.5 mmol MgSO4/l, 1 mmol DTT/l and 20 mmol Tris–HCl buffer/l (pH 7.9). To examine the effects of PD 150606 or Y-27632, various concentrations of PD 150606 or Y-27632 were added to the reactivation medium. Addition of EGTA, CaCl2 or calyculin A to inhibitor-treated spermatozoa was also performed.
The suspension of intact or demembranated spermatozoa was placed into a microscope slide chamber (UR-157 type; Sekisui Chemical Co., Ltd, Tokyo, Japan) on a thermostatically controlled warm plate and the motility of spermatozoa was recorded by videomicroscopy (magnification on the 12 inch black and white monitor was approximately x 600) at 30 or 40 °C (Katz & Overstreet 1981). Measurements were made on a total of 200–300 spermatozoa, distributed uniformly among three or more fields, to determine the percentage of motile spermatozoa.
Analysis of ATP concentrations of intact spermatozoa
The ATP content of spermatozoa in the absence of IPVLs was assayed in boiled sperm extracts by firefly bioluminescence (Wishart 1982). The numbers of spermatozoa were estimated by the method of Wishart and Ross (1985), using a double-beam spectrophotometer (Shimadzu, Model UV-150-02, Kyoto, Japan). The concentration of ATP was expressed in terms of nanomoles ATP/109 spermatozoa.
Western immunoblot analysis of calpain
Spermatozoa that had been washed as described above, and with concentrations adjusted to 4 x 108 cells/ml, were mixed with equal volumes of concentrated ( x 2) Laemmli (1970) sample buffer and were boiled for 5 min. Samples containing approximately 15 µg protein were loaded onto 7.5% SDS-polyacrylamide slab gel, and subjected to electrophoresis. Western blotting was performed according to the protocol of Towbin et al.(1979), with some modifications. Briefly, proteins were transferred electrophoretically to a polyvinylidene difluoride membrane sheet (BioRad). After transfer, non-specific sites on the membranes were blocked by incubating them overnight at 4 °C in 0.1% Tween 20 in Tris-buffered saline (TTBS) containing 5% skimmed milk powder. The blots were then incubated for 1.5 h at room temperature (20–25 °C) with the antibody to calpain 12 (1:1000 dilution with TTBS containing 5% skimmed milk powder). For negative control, the blots were incubated in TTBS containing 5% skimmed milk powder alone. The blots were further incubated for 1 h at room temperature (20–25 °C) with anti-rabbit immunoglobulins donkey serum conjugated with horseradish peroxidase (1:2000 dilution with TTBS containing 5% skimmed milk powder). After each incubation, the membranes were rinsed extensively in TTBS. Finally, blots were developed with the Amersham enhanced chemiluminescence (ECL) detection kit for 5 min. Immunocomplexes were detected with Amersham photoimager system (Tyhoon 9410) exposures for around 15–20 min.
Statistical analysis
The percentages of acrosome reaction and motility were transformed using arc sine transformation. All data were subjected to statistical analysis by Duncan’s multiple-range tests (Duncan 1955).
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Results |
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, various concentrations of PD 150606 or Y-27632 alone were added at the start of incubation and spermatozoa were incubated for 30 min at 40 °C. In contrast, in Fig. 1b and d, 15 min after the start of the 30 min incubation, CaCl2 was added in the PD 150606- or Y-27632-treated spermatozoa, to stimulate the acrosome reaction. During the incubation of spermatozoa for 30 min at 40 °C, the acrosome reaction of spermatozoa with or without IPVL was almost negligible in the absence of Ca2+ in TES/NaCl buffer (Fig. 1a and c). When 2 mmol CaCl2/l was added, the acrosome reaction was stimulated in the presence, but not in the absence of IPVL. However, no inhibition or stimulation of acrosome reaction was observed in the presence of PD 150606 or Y-27632 within the range of 1–100 µmol/l (Fig. 1b and d).
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The time course of motility in the presence of CaCl2 or calyculin A, an inhibitor of PP1 and PP2A, after the addition of PD 150606 or Y-27632 at 30 and 40 °C is shown in Fig. 2a–d. Even the presence of CaCl2 after the addition of PD 150606 could not prevent the inhibition of motility of intact spermatozoa at 30 °C, although no inhibition of motility of spermatozoa was observed after the addition of Y-27632 (Fig. 2a). At 40 °C, the motility of intact spermatozoa was negligible, and the addition of PD 150606 or Y-27632 retained the immotility, but the motility was immediately restored by the subsequent addition of 2 mmol CaCl2/l in the control (no addition of inhibitor) and Y-27632-treated spermatozoa. In contrast, restoration of Ca2+-supplemented sperm motility was not observed in the presence of PD 150606 (Fig. 2b). In the absence of inhibitors (control), the addition of calyculin A did not appreciably affect the intact sperm motility at 30 °C (Fig. 2c), but permitted restoration of motility of at 40 °C (Fig. 2d). Similar effects were observed with the addition of Y-27632 (Fig. 2c and d). In contrast, the presence of calyculin A after the addition of PD 150606 could not prevent the inhibition of motility of intact spermatozoa at 30 and 40 °C (Fig. 2c and d).
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The effect of more prolonged exposure is shown in Fig. 3a and b. At 30 °C, the motility was inhibited immediately after the addition of 2 mmol EGTA/l. Then, motility was restored by the subsequent addition of 2 mmol CaCl2/l without the addition of inhibitors (control). As well as control spermatozoa, restoration of motility by Ca2+ was observed in the presence of either PD 150606 or Y-27632 (Fig. 3a). The presence of calyculin A permitted reactivation of demembranated spermatozoa at 40 °C, and this effect was maintained after addition of PD 150606 or Y-27632 as well as in the control (no addition) (Fig. 3b).
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). Additionally, at 40 °C, the ATP concentrations of spermatozoa after the addition of PD 150606 or Y-27632 were almost the same values compared with those of untreated spermatozoa (control). However, the ATP concentrations of spermatozoa decreased in the presence of Ca2+ or calyculin A alone and Ca2+ or calyculin A with Y-27632, presumably due to the restoration of motility (Fig. 4b).
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Discussion |
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The regulatory serine/threonine protein phosphatases, such as myosin light chain phosphatase, are classified into four main enzymes: type 1 (PP1), type 2A (PP2A), type 2B (PP2B) and type 2C (PP2C); myosin light chain phosphatase activity in smooth muscle is classified as PP1 (Cohen 1989). On the other hand, PP1 appears to be dominant in the temperature-dependent inhibition of flagellar movement of fowl spermatozoa at body temperatures of 40 °C, since the motility of demembranated fowl spermatozoa at 40 °C was stimulated by the addition of calyculin A or okadaic acid (specific inhibitors of PP1 and PP2A), and inhibitors 1 and 2 (small heat-stable proteins which inhibit PP1 activity) (Ashizawa et al. 1994b). In addition, the motility of demembranated fowl spermatozoa at 30 °C decreased markedly following the addition of recombinant PP1 supplemented with Mn2+ (Ashizawa et al. 1997). These results, together with the Rho-kinase involvement in smooth muscle contraction by phosphorylating PP1 (Velasco et al. 2002), invoke the following hypothesis: if Rho-kinase is involved in the reversible temperature-dependent immobilization of fowl spermatozoa, then the addition of Y-27632 would not permit the restoration of motility at 40 °C, because there would be no inhibition of PP1 activity. In the present study, however, no inhibition of motility of spermatozoa by the presence of Y-27632 was observed after the addition of stimulators, such as CaCl2 or calyculin A at 40 °C (Fig. 2b and d
). Furthermore, the vigorous motility of spermatozoa at 30 °C was not inhibited following the addition of Y-27632 (Fig. 2a and c). Therefore, Rho-kinase cannot be demonstrated to be involved in the regulation of motility of fowl spermatozoa in vitro.
It has been suggested that the acrosome reaction of spermatozoa is caused by the increase of intracellular Ca2+ concentration and Ca2+ sensitization (for review see Fraser 1995, Benoff 1998, Baldi et al. 2000, Guraya 2000, Topfer-Petersen et al. 2000). As mentioned earlier, Y-27632 appears to inhibit the Ca2+ sensitization mechanism in smooth muscle (Uehata et al. 1997). Thus, if Rho-kinase is involved in the acrosome reaction of spermatozoa, the addition of Y-27632 may inhibit the induction of the acrosome reaction. However, during incubation for 30 min at 40 °C, the presence of Y-27632 within the range 1–100 µmol/l could not inhibit the CaCl2/IPVL-induced acrosome reaction (Fig. 1d). Therefore, as with motility, Rho-kinase does not appear to be involved in the regulation of the acrosome reaction of fowl spermatozoa in vitro or Rho-kinase may not exist in fowl spermatozoa.
Calpain, a Ca2+-dependent cysteine protease, has been found in mammalian spermatozoa (Schollmeyer 1986, Rojas et al. 1999, Rojas & Moretti-Rojas 2000, Yudin et al. 2000, Aoyama et al. 2001), and this protease appears to be involved in the regulation of sperm motility (Aoyama et al. 2001, Ozaki et al. 2001), the acrosome reaction (Schollmeyer 1986, Yudin et al. 2000, Aoyama et al. 2001) and the cell fusion process that takes place during penetration of the oocyte (Rojas et al. 1999, Rojas & Moretti-Rojas 2000).
In the study reported here, immunoblot analysis of fowl sperm proteins showed that a protein of approximately 80 kDa was recognized by polyclonal antibodies raised against a synthetic peptide based on the amino-terminal end of domain-I in the large subunit of calpain 12 (Fig. 5), suggesting that calpain 12 may be present in fowl spermatozoa. The present study also demonstrated that unlike in mammalian spermatozoa, calpain appears not to be involved in the regulation of the acrosome reaction of fowl spermatozoa, since the presence of PD 150606 before the addition of CaCl2 did not inhibit the acrosome reaction within the range of 1–100 µmol/l during incubation for 30 min at 40 °C (Fig. 1b). However, as for mammalian spermatozoa, the flagellar movement of fowl spermatozoa may be controlled by calpain, since even in the presence of CaCl2 or calyculin A, the motility of intact spermatozoa at 40 °C remained inhibited following the addition of PD 150606 (Fig. 2b and d). Under all of these circumstances, sperm ATP concentrations were not reduced by the addition of PD 150606 (Fig. 4). Thus, it appears that the addition of PD 150606 was not simply affecting membrane damage or inhibiting energy production in these spermatozoa, but may be acting on some part of the regulatory cascade of flagellar movement. Furthermore, it seems that calpain might be present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components, since the motility of demembranated spermatozoa was not affected by the addition of the same concentrations of PD 150606 (Fig. 3). This suggestion was consistent with the observations that calpain 2 seems to be associated with porcine sperm membranes (Schollmeyer 1986) and both calpains 1 and 2 were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque spermatozoa (Yudin et al. 2000).
In conclusion, Rho-kinase could not be shown to be involved in the regulation of the acrosome reaction or motility of fowl spermatozoa, but calpain appears to be involved in the calcium-initiated cascade that regulates flagellar movement of fowl spermatozoa, but not in that which induces the acrosome reaction.
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Footnotes |
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), 100 µmol Y-27632/l and 2 mmol CaCl2/l (
) or 2 mmol CaCl2/l alone (control, •) at (a) 30 °C and (b) 40 °C; 100 µmol PD 150606/l and 100 nmol calyculin A/l (
