THE ANATOMICAL RECORD 231:316-323 (1991)
Sperm Actin and Calmodulin During Fertilization in the Hamster: An Immune Electron Microscopic Study JEAN-PIERRE FOUQUET, BENITO FRAILE, AND MARIE-LOUISE KANN Groupe dEtude de la Formation et de la Maturation du Gam&teMcile, Laboratoire d'Histologie, UFR BiomCdicale, 45 rue des Saints-Peres, Paris Cedex 06, France
ABSTRACT
The distribution of actin and CaM in hamster spermatozoa was examined during the early events of fertilization using postembedding immunogold procedures. Actin was immunolocalized with a polyclonal antibody and two monoclonal antibodies. CaM was immunodetected with a polyclonal antibody. In epididymal sperm, actin labeling was found solely in the principal piece of the flagellum. CaM labeling was observed in the postacrosomal lamina, subacrosomal ring, and tip of the perforatorium. These distributions were not modified after capacitation and acrosome reaction. During the successive steps of sperm-egg fusion actin remained undetected in the sperm head whereas its location did not change in the flagellum. CaM distribution remained unmodified until the sperm head begins to decondense. At later stages of sperm head decondensation the postacrosomal lamina and its CaM labeling disappeared, whereas gold particles were still detected in the subacrosomal layer. The predominant location of actin into the egg cortex, particularly the microvillus-free area was confirmed. Except for the CaM labeling of the meiotic spindle, no special CaM location could be found throughout the egg. Thus, in hamster, a role for sperm actin in sperm-egg fusion appears unlikely. In contrast the CaM present in the Ca2+-rich postacrosomal lamina could be involved in the regulation of egg activation.
Actin has been extensively studied in mammalian male germ cells. In mouse testis the synthesis of p and y cytoplasmic isoforms is a continuous process during spermatogenesis. In addition, a smooth-muscle y actin is specifically synthesized in spermatids (Kim et al., 1989). In all species studied so far, actin (F-actin) accumulates in the subacrosomal layer of spermatids (for references, see Fouquet et al., 1990). This cytoskeletal protein might be involved in the shaping and/or capping of the nucleus (Fouquet et al., 1989) rather than the overlying acrosome a s originally proposed (Welch and O'Rand, 1985). In most species sperm cells also contain actin (in form of monomers and/or oligomers), but in contrast to spermatids the diversity of actin locations seems to preclude a universal function for this protein (Flaherty et al., 1988). At least in boar sperm, a role for actin in plasma membrane changes during sperm epididymal transit, capacitation, and acrosome reaction was previously suspected (Saxena et al., 1986; Camatini e t al., 1986; Peterson et al., 1990). However, in the hamster, it has been suggested that after depolymerisation the subacrosomal actin would be redistributed to the sperm flagellum during spermiation and epididymal transit, whereas no other changes could be detected after capacitation and acrosome reaction (Fouquet et al., 1990). Calmodulin (CaM) is present in high levels in spermatozoa of all species examined. The role of this calcium-binding protein in the regulation of sperm flagellar motility is well documented (Tash, 1989).Moreover, CaM might be involved in other Ca2+-dependentmechanisms, including capacitation (Leclerc et al., 1990), 0 1991 WILEY-LISS, INC
acrosome reaction, and sperm-egg fusion (Jones et al. 1980; Moore and Dedman, 1984; Weinman et al., 1986 a,b; Aitken et al., 1988). In addition CaM-actin interactions with sperm plasma membrane proteins have been suggested in the boar (Camatini and Casale, 1987). However, as for actin location, no generalized pattern for sperm CaM distribution can be evidenced from the reported localizations (Jones et al., 1980; Moore and Dedman, 1984; Yamamoto, 1985; Weinman et al., 1986a,b; Camatini et al., 1986; Camatini and Casale, 1987). Therefore it remains difficult to correlate sperm structures with the functional significance of CaM locations. In a recent study of CaM distribution during spermiogenesis and sperm epididymal transit (Kann et al., 1991), a n accumulation of CaM in the postacrosomal region of sperm head was demonstrated in the hamster and five other species, thus reinforcing the idea of a role for sperm CaM during fertilization. In the present work the distribution of actin and CaM in hamster spermatozoa was studied by immunoelectron microscopy during the early events of fertilization to shed some light on the function of these two proteins in terms of sperm-egg interaction.
Received March 6, 1991; accepted May 13, 1991. Address reprint requests to Jean-Pierre Fouquet, Laboratoire d'Histologie, UFR Biomedicale, 45 rue des Saints-Peres, F 75270 Paris Cedex 06, France. Dr B. Fraile is now at Departamento de Biologia Celular y Genetica, Universidad de Alcala de Henares, Madrid, Espana.
ACTIN AND CALMODULIN DURING SPERM-EGG FUSION
MATERIALS AND METHODS In Vitro Fertilization
In three different experiments, six adult 2-3-monthold female hamsters were induced to superovulate by successive treatment with 25 UI PMSG and 25 UI HCG. These females were anesthetized with 6% chloral and killed 16 hours later. The oocytes were collected from the oviduct in a modified Tyrode’s medium mTALPl (Yanagimachi, 1982). The oocytes were freed from the cumulus by a 5 minute treatment with 0.1% hyaluronidase (bovine testis type IV, Sigma Chemical Co., St. Louis, MO) in the same medium, washed and transferred into 0.1% trypsin (bovine pancreas type I, Sigma Chemical Co., St. Louis, MO) for 30-60 seconds to remove the zona pellucida and then washed twice more. For each experiment one mature 2-5-month-old male hamster was used to collect epididymal spermatozoa, which were capacitated in m-TALP1 during 3 hours a t a concentration of lo6 cells per ml. Aliquots (200 ~ 1of) the capacitated sperm were placed in Petri dishes under mineral oil. Ten zona-free oocytes were added to each sperm suspensions and incubated a t 37°C under 6% COz in air (Yanagimachi, 1982). lmmunoelectron Microscopy
From 10 up to 180 minutes after insemination the eggs and associated sperm were fixed for 30 minutes in phosphate-buffered 1% glutaraldehyde solution and processed for postembedding immunogold procedures as previously described (Kann and Fouquet, 1989). Briefly, Lowicryl K,M embedded eggs were serially sectioned in alternating series of 0.5 pm semithin sections with ultrathin sections. The latter were used to detect actin and CaM in acrosome-reacted spermatozoa and in those incorporated into the eggs. Samples of epididymal and capacitated sperm were similarly prepared for immunoelectron microscopy. The following antibodies were used: a n affinity-purified polyclonal antiactin (Gounon and Karsenti, 1981) a t a final concentration of 10 p.g/ml, a mouse monoclonal antiactin IgM (code 350 Amersham, Arlington Heights, IL), and a mouse monoclonal antiactin IgG designated C4 (Lessard, 1988) at a final concentration of 20 pg/ml; and a n affinity-purified rabbit polyclonal antiCaM (Weinman et al., 1986a) at a final concentration of 20 kg/ml. I t has been previously shown that the three antiactin probes recognized only actin in hamster sperm extracts (Fouquet et al., 1990). Likewise the antiCaM antibody recognized only CaM on western blots (Kann e t al., 1991). These primary antibodies were immunolocalized with the appropriate 10-15 nm gold-labeled secondary antibodies (Janssen or Sigma). Controls either omitting each of these primary antibodies or using these antibodies preadsorbed with a 50-fold molar excess of corresponding purified antigens overnight at 4°C were also performed to assess the specificity of the immunostainings. The sections were examined with a Philips 201 electron microscope at 80 kV. RESULTS Actin and CaM in Epididymal, Capacitated, and Acrosome-Reacted Spermatozoa
As already reported (Fouquet et al., 1990), the same actin localization was found in these three types of
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spermatozoa. Actin was detected in the principal piece of the flagellum, between the fibrous sheath and the plasma membrane, when the polyclonal antiactin was used (Fig. 1).No other regions of the head and tail were labeled using either this antibody or the two monoclonal antiactin antibodies. Likewise no difference in CaM distribution was noted between epididymal (Fig. 21, capacitated, and acrosome-reacted spermatozoa (Fig. 7). This protein was located mainly along the postacrosomal lamina and in two restricted areas of the subacrosomal layer namely the subacrosomal ring and the tip of perforatorium (Figs. 2 and 7). Actin and CaM of the Spermatozoa During Their Incorporation Into the Eggs
The distribution of these two proteins was first scrutinized as soon as the spermatozoa reached the surface of the eggs, when numerous egg microvilli made contact and began to fuse with the sperm heads (10-15 minutes). Actin remained undetected with the polyclonal and the two monoclonal antiactin antibodies in the various regions of the sperm head (Figs. 3-5). In contrast, the three antibodies detected the presence of actin in the cortex of the eggs, the protein being particularly prominent in the microvilli. At the same time the distribution of CaM in the sperm head of the fertilizing spermatozoa (Fig. 6) was strictly identical to th a t described previously for epididymal, capacitated, and acrosome-reacted sperm. The cytoplasm of the eggs was also diffusely labeled and no special CaM labeling could be noted in the actin-rich cortex and microvilli. At later stages (15-30 minutes), when the postacrosomal region of spermatozoa was incorporated into the egg cytoplasm, actin labeling tended to be more intense in this area. Actin was not detected in the perinuclear substance (PNS) of sperm heads whatever the antibody used (Figs. 9-11). However, the granular material which appeared under the swelling postacrosoma1 lamina was labeled with the C, monoclonal antiactin (Fig. 11) but not with the two other antibodies (Figs. 9-10]. In the adjacent sections of the same cells the CaM labeling was observed in association with the sperm head structures already mentioned: the intact perforatorium and subacrosomal ring as well as the swelling postacrosomal lamina (Figs. 7, 8). The observation of numerous serial sections showed that the postacrosomal lamina locally began to disintegrate. The resulting fragments were still labeled with the antibody to CaM (data not shown). In more advanced steps of sperm head incorporation (30-60 minutes), during chromatin decondensation, no further changes were observed a s regards actin labeling. Except for some gold particles in the perinuclear granular material the actin labeling around the sperm head structures could be attributed only to the cortex of the egg (Figs. 12, 13, 14). In the sperm tail actin remained located between the fibrous sheath and the plasma membrane of the principal piece (Fig. 16). In the postacrosomal lamina CaM labeling gradually disappeared concomitantly with the dispersion of this material whereas in the subacrosomal ring and perforatorium this labeling was still detected (Fig. 15). Two to three hours after insemination it was verified that total nuclear decondensation and pronuclei formation normally took place in these eggs but the ultimate
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Fig. 1. Acrosome-reacted spermatozoa in the vicinity of an egg. Actin labeling of the principal piece of the flagellum (-) with the polyclonal antibody: gold particles over the fibrous sheath (left section), under the plasma membrane (right section). A low background staining occurs over the mitochondria and around the axoneme. x 18,000. Fig. 2. Epididymal spermatozoa. CaM labeling in its postacrosomal sheath (+) and the subacrosomal ring (b). x 18,000.
fate of actin and CaM labeling originating in sperm structures (head and tail) was not determined. Regarding these proteins in the eggs no special changes were
Figs. 3-6. Successive sections of the same spermatozoon in contact with egg microvilli 15 minutes after insemination: Figure 3, actin detection with the polyclonal antibody. x 18,000; Figures 4-5, actin detection with the IgM (Amersham) and the IgG (C,) monoclonal antiactin, respectively. x 25,000, gold particles are only present in the cortex of the egg; Figure 6 , CaM labeling is identical to that observed in epididymal sperm (compare with Fig. 2). x 25,000.
noted at any steps. However, as expected, it was observed that the cortex overlying the meiotic spindle, the so-called microvillus-free area (Longo, 1988) was
ACTIN AND CALMODULIN DURING SPERM-EGG FUSION
Figs. 7-1 1. Sperm incorporation into a n egg 30 minutes after insemination: Figures 7, 8, 9, 11, successive sections of the same spermatozoon. Figure 7, CaM labeling of the postacrosomal sheath (-+) and perforatorium (pe) of an acrosome-reacted spermatozoon. x 15,000. Figures 7, 8, labeling of the subacrosomal ring 0) and
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swelling postacrosomal lamina (+) of the fused sperm (Fig. 8. x 36,000). Figures 9, 10, 11, actin labeling with the monoclonal IgM (9) the polyclonal IgG (10)and the monoclonal IgG (11). Except for some gold particles in the granular material (g, 11) actin is detected only into the egg. x 36,000.
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Figs. 12-16. Sperm-egg fusion 1 hour after insemination: Figures 12-15 successive sections. x 27,000. Figures 12-14, actin immunostaining with: the monoclonal IgM (Fig. 12), the polyclonal IgG (Fig. 13),and the monoclonal IgG (Fig. 14). Gold particles are present in the granular material (g) next to the decondensing sperm nucleus
(Fig. 14) in addition to the cortex of the egg. Figure 15, CaM labeling Figure 16, actin labeling still detected in the subacrosomal ring 0). with the polyclonal IgG of the principal piece of the flagellum (-1. x 18,000.
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Figs. 17-18. Cortex of an egg in the region of the meiotic spindle, 30 minutes after insemination. x 10,800. Figure 17, actin labeling with the monoclonal IgM. Figure 18, CaM labeling. The microvillus-
free area (+) is stained for actin but not for CaM whereas the meiotic microtubules (mt) are stained for CaM but not for actin.
intensely stained with antiactin antibodies (Fig. 17), as was the cortex of the polar bodies (data not shown) but the meiotic spindle area was not particularly labeled. In contrast, no CaM labeling was observed in the cortex over the meiotic spindle whereas a labeling of the microtubules near the poles of the spindle was clearly evidenced (Fig. 18).
some actin (G-actin) may be still present but masked in hamster sperm head. If this is true one can postulate that this protein could be detected again during the morphological changes associated to the early events of sperm-egg fusion. As previously reported, actin remained undetected in the sperm head after capacitation and acrosome reaction using three antiactin probes (Fouquet e t al., 1990). Likewise, actin was unDISCUSSION detected in sperm head structures during sperm incorThe distribution of actin in the mammalian egg and poration into the egg except in the granular material its role during fertilization has been studied under nor- which appeared during postacrosomal lamina and numal conditions and in the presence of drugs disrupting clear decondensation. The origin of actin in this matemicrofilaments in various species (see Longo, 1988 and rial could not be clearly established but its detection at Le Guen et al., 1989 for references). This cytoskeletal advanced stages of sperm-egg cytoplasmic fusion sugprotein would be involved in fertilization cone develop- gests it is egg actin. Thus, during the early events of ment, meiotic spindle anchorage to the egg surface, and fertilization actin was never detected in sperm head polar body abstriction but not in sperm-egg fusion. and its location in the sperm tail was not modified. However, preincubation of guinea-pig and human Therefore, a role for sperm actin in sperm-egg fusion sperm with cytochalasin-D has been reported to reduce appears unlikely. Sperm calcium and CaM are considered to be inzona-free hamster egg penetration (Rogers et al., 1989). These results remain difficult to explain since actin volved in various Ca2' -dependent mechanisms: capacwas not detected in guinea-pig spermatozoa, whereas itation, acrosome reaction, and sperm-egg fusion in adonly monomeric actin was evidenced in spermatozoa of dition to flagellar motility (see Introduction for man and other species (for references, see Fouquet et references). Thus a n uptake of calcium has been demal., 1990). Therefore the role of actin, particularly that onstrated during capacitation of sperm in the mouse originating from sperm cells remains uncertain during and some other species (Ruknudin and Silver, 1990). In sperm-egg fusion. Actin could not be detected in the bull sperm, a decrease in the binding of CaM to CaMhead of epididymal hamster sperm. It has been sug- binding proteins has been observed (Leclerc et al., gested that this protein would be redistributed from 1990). Moreover, changes in CaM compartmentalizathe head to the principal piece of the flagellum at sper- tion after capacitation and acrosome reaction of miation (Fouquet et al., 1990). Although undetected guinea-pig spermatozoa have been also reported (Trejo
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and Mujica, 1990). However, in the present work, the distribution of CaM which was observed in the head of hamster testicular spermatozoa remained unchanged during their epididymal transit a s well a s after capacitation and acrosome reaction. Therefore these events do not seem to have any effect on CaM location in hamster spermatozoa. The primary trigger for egg activation is a detonation Ca2+ believed to originate principally in a Ca2+ release from intracellular stores of the egg (Miyazaki, 1989). It has also been suggested that sperm calcium could be involved in this process (Yanagimachi, 1988). The predominant location of calcium in the postacrosomal region of spermatozoa in the hamster (Gravis, 1979; Ruknudin et al., 1988), a s well as in ram (Plummer and Watson, 1985) and human (Fain-Maurel and Dadoune, 1979) support this hypothesis. Moreover, the postacrosomal region is rich in CaM in these species and others (Weinman et al. 1986a,b; Kann et al. 1991). The localization both of calcium and CaM in the postacrosomal lamina suggests that this structure is a reservoir of calcium which could be used to trigger egg activation a s already discussed by others (Jones et al., 1980; Weinman et al., 1986b). The stability of CaM anchorage to the postacrosomal lamina during the early events of sperm-egg fusion favors this hypothesis but we do not yet know if there is a release of calcium at this level. On the other hand sperm cells contain other Ca2 -binding proteins which could be also involved in this process (Feinberg et al., in press). The predominant location of actin in the cortex of the egg particularly around the sperm head and its accumulation above the meiotic spindle agrees with previous results (see Longo, 1988; Le Guen et al., 1989). CaM labeling was diffusely distributed throughout the cytoplasm of the egg without special location except in the meiotic spindle. The labeling of the microtubules near the poles of the metaphase I1 spindle is in agreement with the reported immunofluorescent locations in mitotic cells where CaM might mediate calcium effects on microtubule assembly-disassembly (Welsh et al., 1979). No colocalization of actin and CaM was observed neither in the eggs nor in the spermatozoa which could suggest a n interaction between the two proteins in these cells. As stated above the granular material present at the vicinity of the decondensing sperm head was labeled at least with one of the antiactin probes used here. The origin both of this material and actin is unknown. A similar material also occurs during human sperm interaction with zona-free hamster eggs and i t was proposed i t would originate from the postacrosomal lamina splitting (Courtot and Lin-Tong, 1988). This appears unlikely in the hamster sperm-egg model because the granular material was already present and not labeled by CaM antibodies while a t the same time the postacrosomal lamina and its CaM labeling were essentially unchanged. In conclusion, actin in hamster sperm head, if any, does not seem involved in the early events of sperm-egg fusion. In contrast, CaM located in the postacrosomal lamina is a good candidate as a reservoir of calcium which would trigger egg activation a t the beginning of fertilization. +
ACKNOWLEDGMENTS
We thank Ms. Annie Gonzales for expert technical assistance. We also thank, for the gift of antibodies, Dr. Pierre Gounon for the polyclonal antiactin, Dr. James L. Lessard for the C, monoclonal antiactin, and Dr. Serge Weinman for the polyclonal anticalmodulin. LITERATURE CITED Aitken, R.J., J.S. Clarkson, M.J. Hulme, and C.J. Henderson 1988 Analysis of calmodulin acceptor proteins and the influence of calmodulin antagonists on human spermatozoa. Gamete Res., 21: 93-1 11. Camatini, M., G. Anelli, and A. Casale 1986 Immunocytochemical localization of calmodulin in intact and acrosome-reacted boar sperm. Eur. J . Cell Biol., 4lr89-96. Camatini, M., and A. Casale 1987 Actin and calmodulin coexist in the equatorial segment of ejaculated boar sperm. Gamete Res., 17: 97-105. Courtot, A.M. and W. Lin-Tong 1988 Initial stages of sperm-egg interaction in a heterospecific system: Fate of the post-acrosomal sheath and appearance of a particular material within the oocyte. Hum. Reprod., 3551-655. Fain-Maurel, M.A., and J.P. Dadoune 1979 Scanning-electron-microscopy-X-ray microanalysis and distribution of elements within the head of human spermatozoa. Arch. Androl., 3t1-11. Feinberg, J., D. Rainteau, M.A. Kaetzel, J.L. Dacheux, J.R. Dedman, and S. Weinman 1991 Differential localization of annexins in ram germ cells: A biochemical and immunocytochemical study. J . Histochem. Cytochem., in press. Flaherty, S.P., V.P. Winfrey, and G.E. Olson 1988 Localization of actin in human, bull, rabbit and hamster sperm by immunoelectron microscopy. Anat. Rec. 221.599-610. Fouquet, J.P., M.L. Kann, J.L. Courtens, and L. Ploen 1989 Immunogold distribution of actin during spermiogenesis in the normal rabbit and after experimental cryptorchidism. Gamete Res., 24: 281-290. Fouquet, J.P., M.L. Kann, and J.P. Dadoune 1990 Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa. Tissue Cell, 22:291-300. Gounon, P., and E. Karsenti 1981 Involvement of contractile proteins in the changes in consistency of oocyte nucleoplasm of the newt Pleurodeles waltlii. J. Cell Biol., 88t410-421. Gravis, C.J. 1979 Cytochemical localization of cations in the testis of the Syrian hamster, utilizing potassium-pyroantimonate. Am. J. Anat., 154t245-266. Jones, H.P., R.W. Lenz, B.A. Palevitz. and M.J. Cormier 1980 Calmodulin localization in mammalian spermatozoa. Proc. Natl. Acad. Sci. U.S.A., 77:2772-2776. Kann, M.L., and J.P. Fouquet 1989 Comparison of LR white resin, Lowicryl K4M and epon postembedding procedure or immunogold staining of actin in the testis. Histochemistry, 91t221-226. Kann, M.L., J . Feinberg, D. Rainteau, J.P. Dadoune, S. Weinman, and J.P. Fouquet 1991 Localization of calmodulin in perinuclear structures of spermatids and spermatozoa: A comparison of six mammalian species. Anat. Rec., in press. Kim, E., S.H. Waters, L.E. Hake, and N.B. Hecht 1989 Identification and developmental expression of a smooth-muscle y-actin in postmeiotic male germ cells of mice. Mol. Cell Biol., 9r1875-1881. Leclerc, P., M.A. Sirard, J.G. Chafouleas, and R.D. Lambert 1990 Decreased binding of calmodulin to bull sperm proteins during heparin-induced capacitation. Biol. Reprod., 42:483-489. Le Guen, P., N. Crozet, D. Huneau, and L. Gall 1989 Distribution and role of microfilaments during early events of sheep fertilization. Gamete Res., 22,411-425. Lessard, J.L. 1988 Two monoclonal antibodies to actin: One muscle selective and one generally reactive. Cell Motil. Cytoskeleton, lOt349-362. Longo, F.J. 1988 Reorganization of the egg surface at fertilization. Int. Rev. Cytol., 113t233-269. Miyazaki, S. 1989 Signal transduction of sperm-egg interaction causing periodic calcium transients in hamster eggs. In: Mechanisms of Egg Activation. Nucitelli, R., G. Cherr, and W.H. Clark, eds. Plenum Publishing Corp., New York, pp. 231-246. Moore, P.B., and J.R. Dedman 1984 Calmodulin and calmodulin acceptor protein and calcimedins: Unique antibody localizations in hamster sperm. J. Cell Biochem., 25t99-107. Peterson, R.N., J.J. Bozzola, W.P. Hunt, and A. Darabi 1990 Charac-
ACTIN AND CALMODULIN DURING SPERM-EGG FUSION terization of membrane-associated actin in boar spermatozoa. J. Exp. Zool., 253:202-214. Plummer, J.M., and P.F. Watson 1985 Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique. J. Reprod. Fertil., 75.255-263. Rogers, B.Y., C. Bastias, R.L. Coulson, and L.D. Russell 1989 Cytochalasin D inhibits penetration of hamster eggs by guinea pig and human spermatozoa. J. Androl., 10t275-281. Ruknudin, A., J.P. Dadoune, and I.A. Silver 1988 Intracellular calcium in hamster spermatozoa in testis, epididymis and during acrosome reaction. Anim. Reprod. Sci., 16t145-153. Ruknudin, A,, and I.A. Silver 1990 Ca2' uptake during capacitation of mouse s ermatozoa and the effect of an anion transport inhibitor on Cap+ uptake. Molec. Reprod. Dev., 26:63-68. Saxena, N., R.N. Peterson N.K. Saxena, and L.D. Russell 1986 Microfilaments appear in boar spermatozoa during capacitation in vitro. J. Exp. Zool., 239t423-427. Tash, J.S. 1989 Protein phosphorylation: The second messenger signal transducer of flagellar motility. Cell Motil. Cytoskeleton, 14: 332-339. Trejo, R., and A. Mujica 1990 Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa. Molec. Reprod. Dev., 26t366-376.
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Weinman, S., C. Ores-Carton, D. Rainteau, and S. Puszkin 1986a Immunoelectron microscopic localization of calmodulin and phospholipase A, in spermatozoa. I. J . Histochem. Cytochem.,34tll711179. Weinman, S., C. Ores-Carton, F. Escaig, J. Feinberg, and S. Puszkin 19861, Calmodulin immunoelectron microscopy: Redistribution during ram spermatogenesis. 11.J. Histochem. Cytochem.34tl1811193. Welch, J.E., and M.G. ORand 1985 Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit. Dev. Biol., 109t411-417. Welsh, M.J., J.R. Dedman, B.R. Brinkley, and A.R. Means 1979 Tubulin and calmodulin: Effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus. J . Cell Biol., 81:624 -634. Yamamoto, N. 1985 Immunoelectron microscopic localization of calmodulin in guinea pig testis and spermatozoa. Acta Histochem. Cytochem., 18.199-21 1. Yanagimachi, R. 1982 Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster. Gamete Res., 5:323-344. Yanagimachi, R. 1988 Mammalian fertilization. In: Physiology of Reproduction. Knobil, E. and J. Neil, eds. Raven Press, New York, Vol. 2, Chap. 5, pp. 135-185.
Sperm Actin and Calmodulin During Fertilization in the Hamster: An Immune Electron Microscopic Study JEAN-PIERRE FOUQUET, BENITO FRAILE, AND MARIE-LOUISE KANN Groupe dEtude de la Formation et de la Maturation du Gam&teMcile, Laboratoire d'Histologie, UFR BiomCdicale, 45 rue des Saints-Peres, Paris Cedex 06, France
ABSTRACT
The distribution of actin and CaM in hamster spermatozoa was examined during the early events of fertilization using postembedding immunogold procedures. Actin was immunolocalized with a polyclonal antibody and two monoclonal antibodies. CaM was immunodetected with a polyclonal antibody. In epididymal sperm, actin labeling was found solely in the principal piece of the flagellum. CaM labeling was observed in the postacrosomal lamina, subacrosomal ring, and tip of the perforatorium. These distributions were not modified after capacitation and acrosome reaction. During the successive steps of sperm-egg fusion actin remained undetected in the sperm head whereas its location did not change in the flagellum. CaM distribution remained unmodified until the sperm head begins to decondense. At later stages of sperm head decondensation the postacrosomal lamina and its CaM labeling disappeared, whereas gold particles were still detected in the subacrosomal layer. The predominant location of actin into the egg cortex, particularly the microvillus-free area was confirmed. Except for the CaM labeling of the meiotic spindle, no special CaM location could be found throughout the egg. Thus, in hamster, a role for sperm actin in sperm-egg fusion appears unlikely. In contrast the CaM present in the Ca2+-rich postacrosomal lamina could be involved in the regulation of egg activation.
Actin has been extensively studied in mammalian male germ cells. In mouse testis the synthesis of p and y cytoplasmic isoforms is a continuous process during spermatogenesis. In addition, a smooth-muscle y actin is specifically synthesized in spermatids (Kim et al., 1989). In all species studied so far, actin (F-actin) accumulates in the subacrosomal layer of spermatids (for references, see Fouquet et al., 1990). This cytoskeletal protein might be involved in the shaping and/or capping of the nucleus (Fouquet et al., 1989) rather than the overlying acrosome a s originally proposed (Welch and O'Rand, 1985). In most species sperm cells also contain actin (in form of monomers and/or oligomers), but in contrast to spermatids the diversity of actin locations seems to preclude a universal function for this protein (Flaherty et al., 1988). At least in boar sperm, a role for actin in plasma membrane changes during sperm epididymal transit, capacitation, and acrosome reaction was previously suspected (Saxena et al., 1986; Camatini e t al., 1986; Peterson et al., 1990). However, in the hamster, it has been suggested that after depolymerisation the subacrosomal actin would be redistributed to the sperm flagellum during spermiation and epididymal transit, whereas no other changes could be detected after capacitation and acrosome reaction (Fouquet et al., 1990). Calmodulin (CaM) is present in high levels in spermatozoa of all species examined. The role of this calcium-binding protein in the regulation of sperm flagellar motility is well documented (Tash, 1989).Moreover, CaM might be involved in other Ca2+-dependentmechanisms, including capacitation (Leclerc et al., 1990), 0 1991 WILEY-LISS, INC
acrosome reaction, and sperm-egg fusion (Jones et al. 1980; Moore and Dedman, 1984; Weinman et al., 1986 a,b; Aitken et al., 1988). In addition CaM-actin interactions with sperm plasma membrane proteins have been suggested in the boar (Camatini and Casale, 1987). However, as for actin location, no generalized pattern for sperm CaM distribution can be evidenced from the reported localizations (Jones et al., 1980; Moore and Dedman, 1984; Yamamoto, 1985; Weinman et al., 1986a,b; Camatini et al., 1986; Camatini and Casale, 1987). Therefore it remains difficult to correlate sperm structures with the functional significance of CaM locations. In a recent study of CaM distribution during spermiogenesis and sperm epididymal transit (Kann et al., 1991), a n accumulation of CaM in the postacrosomal region of sperm head was demonstrated in the hamster and five other species, thus reinforcing the idea of a role for sperm CaM during fertilization. In the present work the distribution of actin and CaM in hamster spermatozoa was studied by immunoelectron microscopy during the early events of fertilization to shed some light on the function of these two proteins in terms of sperm-egg interaction.
Received March 6, 1991; accepted May 13, 1991. Address reprint requests to Jean-Pierre Fouquet, Laboratoire d'Histologie, UFR Biomedicale, 45 rue des Saints-Peres, F 75270 Paris Cedex 06, France. Dr B. Fraile is now at Departamento de Biologia Celular y Genetica, Universidad de Alcala de Henares, Madrid, Espana.
ACTIN AND CALMODULIN DURING SPERM-EGG FUSION
MATERIALS AND METHODS In Vitro Fertilization
In three different experiments, six adult 2-3-monthold female hamsters were induced to superovulate by successive treatment with 25 UI PMSG and 25 UI HCG. These females were anesthetized with 6% chloral and killed 16 hours later. The oocytes were collected from the oviduct in a modified Tyrode’s medium mTALPl (Yanagimachi, 1982). The oocytes were freed from the cumulus by a 5 minute treatment with 0.1% hyaluronidase (bovine testis type IV, Sigma Chemical Co., St. Louis, MO) in the same medium, washed and transferred into 0.1% trypsin (bovine pancreas type I, Sigma Chemical Co., St. Louis, MO) for 30-60 seconds to remove the zona pellucida and then washed twice more. For each experiment one mature 2-5-month-old male hamster was used to collect epididymal spermatozoa, which were capacitated in m-TALP1 during 3 hours a t a concentration of lo6 cells per ml. Aliquots (200 ~ 1of) the capacitated sperm were placed in Petri dishes under mineral oil. Ten zona-free oocytes were added to each sperm suspensions and incubated a t 37°C under 6% COz in air (Yanagimachi, 1982). lmmunoelectron Microscopy
From 10 up to 180 minutes after insemination the eggs and associated sperm were fixed for 30 minutes in phosphate-buffered 1% glutaraldehyde solution and processed for postembedding immunogold procedures as previously described (Kann and Fouquet, 1989). Briefly, Lowicryl K,M embedded eggs were serially sectioned in alternating series of 0.5 pm semithin sections with ultrathin sections. The latter were used to detect actin and CaM in acrosome-reacted spermatozoa and in those incorporated into the eggs. Samples of epididymal and capacitated sperm were similarly prepared for immunoelectron microscopy. The following antibodies were used: a n affinity-purified polyclonal antiactin (Gounon and Karsenti, 1981) a t a final concentration of 10 p.g/ml, a mouse monoclonal antiactin IgM (code 350 Amersham, Arlington Heights, IL), and a mouse monoclonal antiactin IgG designated C4 (Lessard, 1988) at a final concentration of 20 pg/ml; and a n affinity-purified rabbit polyclonal antiCaM (Weinman et al., 1986a) at a final concentration of 20 kg/ml. I t has been previously shown that the three antiactin probes recognized only actin in hamster sperm extracts (Fouquet et al., 1990). Likewise the antiCaM antibody recognized only CaM on western blots (Kann e t al., 1991). These primary antibodies were immunolocalized with the appropriate 10-15 nm gold-labeled secondary antibodies (Janssen or Sigma). Controls either omitting each of these primary antibodies or using these antibodies preadsorbed with a 50-fold molar excess of corresponding purified antigens overnight at 4°C were also performed to assess the specificity of the immunostainings. The sections were examined with a Philips 201 electron microscope at 80 kV. RESULTS Actin and CaM in Epididymal, Capacitated, and Acrosome-Reacted Spermatozoa
As already reported (Fouquet et al., 1990), the same actin localization was found in these three types of
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spermatozoa. Actin was detected in the principal piece of the flagellum, between the fibrous sheath and the plasma membrane, when the polyclonal antiactin was used (Fig. 1).No other regions of the head and tail were labeled using either this antibody or the two monoclonal antiactin antibodies. Likewise no difference in CaM distribution was noted between epididymal (Fig. 21, capacitated, and acrosome-reacted spermatozoa (Fig. 7). This protein was located mainly along the postacrosomal lamina and in two restricted areas of the subacrosomal layer namely the subacrosomal ring and the tip of perforatorium (Figs. 2 and 7). Actin and CaM of the Spermatozoa During Their Incorporation Into the Eggs
The distribution of these two proteins was first scrutinized as soon as the spermatozoa reached the surface of the eggs, when numerous egg microvilli made contact and began to fuse with the sperm heads (10-15 minutes). Actin remained undetected with the polyclonal and the two monoclonal antiactin antibodies in the various regions of the sperm head (Figs. 3-5). In contrast, the three antibodies detected the presence of actin in the cortex of the eggs, the protein being particularly prominent in the microvilli. At the same time the distribution of CaM in the sperm head of the fertilizing spermatozoa (Fig. 6) was strictly identical to th a t described previously for epididymal, capacitated, and acrosome-reacted sperm. The cytoplasm of the eggs was also diffusely labeled and no special CaM labeling could be noted in the actin-rich cortex and microvilli. At later stages (15-30 minutes), when the postacrosomal region of spermatozoa was incorporated into the egg cytoplasm, actin labeling tended to be more intense in this area. Actin was not detected in the perinuclear substance (PNS) of sperm heads whatever the antibody used (Figs. 9-11). However, the granular material which appeared under the swelling postacrosoma1 lamina was labeled with the C, monoclonal antiactin (Fig. 11) but not with the two other antibodies (Figs. 9-10]. In the adjacent sections of the same cells the CaM labeling was observed in association with the sperm head structures already mentioned: the intact perforatorium and subacrosomal ring as well as the swelling postacrosomal lamina (Figs. 7, 8). The observation of numerous serial sections showed that the postacrosomal lamina locally began to disintegrate. The resulting fragments were still labeled with the antibody to CaM (data not shown). In more advanced steps of sperm head incorporation (30-60 minutes), during chromatin decondensation, no further changes were observed a s regards actin labeling. Except for some gold particles in the perinuclear granular material the actin labeling around the sperm head structures could be attributed only to the cortex of the egg (Figs. 12, 13, 14). In the sperm tail actin remained located between the fibrous sheath and the plasma membrane of the principal piece (Fig. 16). In the postacrosomal lamina CaM labeling gradually disappeared concomitantly with the dispersion of this material whereas in the subacrosomal ring and perforatorium this labeling was still detected (Fig. 15). Two to three hours after insemination it was verified that total nuclear decondensation and pronuclei formation normally took place in these eggs but the ultimate
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Fig. 1. Acrosome-reacted spermatozoa in the vicinity of an egg. Actin labeling of the principal piece of the flagellum (-) with the polyclonal antibody: gold particles over the fibrous sheath (left section), under the plasma membrane (right section). A low background staining occurs over the mitochondria and around the axoneme. x 18,000. Fig. 2. Epididymal spermatozoa. CaM labeling in its postacrosomal sheath (+) and the subacrosomal ring (b). x 18,000.
fate of actin and CaM labeling originating in sperm structures (head and tail) was not determined. Regarding these proteins in the eggs no special changes were
Figs. 3-6. Successive sections of the same spermatozoon in contact with egg microvilli 15 minutes after insemination: Figure 3, actin detection with the polyclonal antibody. x 18,000; Figures 4-5, actin detection with the IgM (Amersham) and the IgG (C,) monoclonal antiactin, respectively. x 25,000, gold particles are only present in the cortex of the egg; Figure 6 , CaM labeling is identical to that observed in epididymal sperm (compare with Fig. 2). x 25,000.
noted at any steps. However, as expected, it was observed that the cortex overlying the meiotic spindle, the so-called microvillus-free area (Longo, 1988) was
ACTIN AND CALMODULIN DURING SPERM-EGG FUSION
Figs. 7-1 1. Sperm incorporation into a n egg 30 minutes after insemination: Figures 7, 8, 9, 11, successive sections of the same spermatozoon. Figure 7, CaM labeling of the postacrosomal sheath (-+) and perforatorium (pe) of an acrosome-reacted spermatozoon. x 15,000. Figures 7, 8, labeling of the subacrosomal ring 0) and
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swelling postacrosomal lamina (+) of the fused sperm (Fig. 8. x 36,000). Figures 9, 10, 11, actin labeling with the monoclonal IgM (9) the polyclonal IgG (10)and the monoclonal IgG (11). Except for some gold particles in the granular material (g, 11) actin is detected only into the egg. x 36,000.
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Figs. 12-16. Sperm-egg fusion 1 hour after insemination: Figures 12-15 successive sections. x 27,000. Figures 12-14, actin immunostaining with: the monoclonal IgM (Fig. 12), the polyclonal IgG (Fig. 13),and the monoclonal IgG (Fig. 14). Gold particles are present in the granular material (g) next to the decondensing sperm nucleus
(Fig. 14) in addition to the cortex of the egg. Figure 15, CaM labeling Figure 16, actin labeling still detected in the subacrosomal ring 0). with the polyclonal IgG of the principal piece of the flagellum (-1. x 18,000.
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Figs. 17-18. Cortex of an egg in the region of the meiotic spindle, 30 minutes after insemination. x 10,800. Figure 17, actin labeling with the monoclonal IgM. Figure 18, CaM labeling. The microvillus-
free area (+) is stained for actin but not for CaM whereas the meiotic microtubules (mt) are stained for CaM but not for actin.
intensely stained with antiactin antibodies (Fig. 17), as was the cortex of the polar bodies (data not shown) but the meiotic spindle area was not particularly labeled. In contrast, no CaM labeling was observed in the cortex over the meiotic spindle whereas a labeling of the microtubules near the poles of the spindle was clearly evidenced (Fig. 18).
some actin (G-actin) may be still present but masked in hamster sperm head. If this is true one can postulate that this protein could be detected again during the morphological changes associated to the early events of sperm-egg fusion. As previously reported, actin remained undetected in the sperm head after capacitation and acrosome reaction using three antiactin probes (Fouquet e t al., 1990). Likewise, actin was unDISCUSSION detected in sperm head structures during sperm incorThe distribution of actin in the mammalian egg and poration into the egg except in the granular material its role during fertilization has been studied under nor- which appeared during postacrosomal lamina and numal conditions and in the presence of drugs disrupting clear decondensation. The origin of actin in this matemicrofilaments in various species (see Longo, 1988 and rial could not be clearly established but its detection at Le Guen et al., 1989 for references). This cytoskeletal advanced stages of sperm-egg cytoplasmic fusion sugprotein would be involved in fertilization cone develop- gests it is egg actin. Thus, during the early events of ment, meiotic spindle anchorage to the egg surface, and fertilization actin was never detected in sperm head polar body abstriction but not in sperm-egg fusion. and its location in the sperm tail was not modified. However, preincubation of guinea-pig and human Therefore, a role for sperm actin in sperm-egg fusion sperm with cytochalasin-D has been reported to reduce appears unlikely. Sperm calcium and CaM are considered to be inzona-free hamster egg penetration (Rogers et al., 1989). These results remain difficult to explain since actin volved in various Ca2' -dependent mechanisms: capacwas not detected in guinea-pig spermatozoa, whereas itation, acrosome reaction, and sperm-egg fusion in adonly monomeric actin was evidenced in spermatozoa of dition to flagellar motility (see Introduction for man and other species (for references, see Fouquet et references). Thus a n uptake of calcium has been demal., 1990). Therefore the role of actin, particularly that onstrated during capacitation of sperm in the mouse originating from sperm cells remains uncertain during and some other species (Ruknudin and Silver, 1990). In sperm-egg fusion. Actin could not be detected in the bull sperm, a decrease in the binding of CaM to CaMhead of epididymal hamster sperm. It has been sug- binding proteins has been observed (Leclerc et al., gested that this protein would be redistributed from 1990). Moreover, changes in CaM compartmentalizathe head to the principal piece of the flagellum at sper- tion after capacitation and acrosome reaction of miation (Fouquet et al., 1990). Although undetected guinea-pig spermatozoa have been also reported (Trejo
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and Mujica, 1990). However, in the present work, the distribution of CaM which was observed in the head of hamster testicular spermatozoa remained unchanged during their epididymal transit a s well a s after capacitation and acrosome reaction. Therefore these events do not seem to have any effect on CaM location in hamster spermatozoa. The primary trigger for egg activation is a detonation Ca2+ believed to originate principally in a Ca2+ release from intracellular stores of the egg (Miyazaki, 1989). It has also been suggested that sperm calcium could be involved in this process (Yanagimachi, 1988). The predominant location of calcium in the postacrosomal region of spermatozoa in the hamster (Gravis, 1979; Ruknudin et al., 1988), a s well as in ram (Plummer and Watson, 1985) and human (Fain-Maurel and Dadoune, 1979) support this hypothesis. Moreover, the postacrosomal region is rich in CaM in these species and others (Weinman et al. 1986a,b; Kann et al. 1991). The localization both of calcium and CaM in the postacrosomal lamina suggests that this structure is a reservoir of calcium which could be used to trigger egg activation a s already discussed by others (Jones et al., 1980; Weinman et al., 1986b). The stability of CaM anchorage to the postacrosomal lamina during the early events of sperm-egg fusion favors this hypothesis but we do not yet know if there is a release of calcium at this level. On the other hand sperm cells contain other Ca2 -binding proteins which could be also involved in this process (Feinberg et al., in press). The predominant location of actin in the cortex of the egg particularly around the sperm head and its accumulation above the meiotic spindle agrees with previous results (see Longo, 1988; Le Guen et al., 1989). CaM labeling was diffusely distributed throughout the cytoplasm of the egg without special location except in the meiotic spindle. The labeling of the microtubules near the poles of the metaphase I1 spindle is in agreement with the reported immunofluorescent locations in mitotic cells where CaM might mediate calcium effects on microtubule assembly-disassembly (Welsh et al., 1979). No colocalization of actin and CaM was observed neither in the eggs nor in the spermatozoa which could suggest a n interaction between the two proteins in these cells. As stated above the granular material present at the vicinity of the decondensing sperm head was labeled at least with one of the antiactin probes used here. The origin both of this material and actin is unknown. A similar material also occurs during human sperm interaction with zona-free hamster eggs and i t was proposed i t would originate from the postacrosomal lamina splitting (Courtot and Lin-Tong, 1988). This appears unlikely in the hamster sperm-egg model because the granular material was already present and not labeled by CaM antibodies while a t the same time the postacrosomal lamina and its CaM labeling were essentially unchanged. In conclusion, actin in hamster sperm head, if any, does not seem involved in the early events of sperm-egg fusion. In contrast, CaM located in the postacrosomal lamina is a good candidate as a reservoir of calcium which would trigger egg activation a t the beginning of fertilization. +
ACKNOWLEDGMENTS
We thank Ms. Annie Gonzales for expert technical assistance. We also thank, for the gift of antibodies, Dr. Pierre Gounon for the polyclonal antiactin, Dr. James L. Lessard for the C, monoclonal antiactin, and Dr. Serge Weinman for the polyclonal anticalmodulin. LITERATURE CITED Aitken, R.J., J.S. Clarkson, M.J. Hulme, and C.J. Henderson 1988 Analysis of calmodulin acceptor proteins and the influence of calmodulin antagonists on human spermatozoa. Gamete Res., 21: 93-1 11. Camatini, M., G. Anelli, and A. Casale 1986 Immunocytochemical localization of calmodulin in intact and acrosome-reacted boar sperm. Eur. J . Cell Biol., 4lr89-96. Camatini, M., and A. Casale 1987 Actin and calmodulin coexist in the equatorial segment of ejaculated boar sperm. Gamete Res., 17: 97-105. Courtot, A.M. and W. Lin-Tong 1988 Initial stages of sperm-egg interaction in a heterospecific system: Fate of the post-acrosomal sheath and appearance of a particular material within the oocyte. Hum. Reprod., 3551-655. Fain-Maurel, M.A., and J.P. Dadoune 1979 Scanning-electron-microscopy-X-ray microanalysis and distribution of elements within the head of human spermatozoa. Arch. Androl., 3t1-11. Feinberg, J., D. Rainteau, M.A. Kaetzel, J.L. Dacheux, J.R. Dedman, and S. Weinman 1991 Differential localization of annexins in ram germ cells: A biochemical and immunocytochemical study. J . Histochem. Cytochem., in press. Flaherty, S.P., V.P. Winfrey, and G.E. Olson 1988 Localization of actin in human, bull, rabbit and hamster sperm by immunoelectron microscopy. Anat. Rec. 221.599-610. Fouquet, J.P., M.L. Kann, J.L. Courtens, and L. Ploen 1989 Immunogold distribution of actin during spermiogenesis in the normal rabbit and after experimental cryptorchidism. Gamete Res., 24: 281-290. Fouquet, J.P., M.L. Kann, and J.P. Dadoune 1990 Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa. Tissue Cell, 22:291-300. Gounon, P., and E. Karsenti 1981 Involvement of contractile proteins in the changes in consistency of oocyte nucleoplasm of the newt Pleurodeles waltlii. J. Cell Biol., 88t410-421. Gravis, C.J. 1979 Cytochemical localization of cations in the testis of the Syrian hamster, utilizing potassium-pyroantimonate. Am. J. Anat., 154t245-266. Jones, H.P., R.W. Lenz, B.A. Palevitz. and M.J. Cormier 1980 Calmodulin localization in mammalian spermatozoa. Proc. Natl. Acad. Sci. U.S.A., 77:2772-2776. Kann, M.L., and J.P. Fouquet 1989 Comparison of LR white resin, Lowicryl K4M and epon postembedding procedure or immunogold staining of actin in the testis. Histochemistry, 91t221-226. Kann, M.L., J . Feinberg, D. Rainteau, J.P. Dadoune, S. Weinman, and J.P. Fouquet 1991 Localization of calmodulin in perinuclear structures of spermatids and spermatozoa: A comparison of six mammalian species. Anat. Rec., in press. Kim, E., S.H. Waters, L.E. Hake, and N.B. Hecht 1989 Identification and developmental expression of a smooth-muscle y-actin in postmeiotic male germ cells of mice. Mol. Cell Biol., 9r1875-1881. Leclerc, P., M.A. Sirard, J.G. Chafouleas, and R.D. Lambert 1990 Decreased binding of calmodulin to bull sperm proteins during heparin-induced capacitation. Biol. Reprod., 42:483-489. Le Guen, P., N. Crozet, D. Huneau, and L. Gall 1989 Distribution and role of microfilaments during early events of sheep fertilization. Gamete Res., 22,411-425. Lessard, J.L. 1988 Two monoclonal antibodies to actin: One muscle selective and one generally reactive. Cell Motil. Cytoskeleton, lOt349-362. Longo, F.J. 1988 Reorganization of the egg surface at fertilization. Int. Rev. Cytol., 113t233-269. Miyazaki, S. 1989 Signal transduction of sperm-egg interaction causing periodic calcium transients in hamster eggs. In: Mechanisms of Egg Activation. Nucitelli, R., G. Cherr, and W.H. Clark, eds. Plenum Publishing Corp., New York, pp. 231-246. Moore, P.B., and J.R. Dedman 1984 Calmodulin and calmodulin acceptor protein and calcimedins: Unique antibody localizations in hamster sperm. J. Cell Biochem., 25t99-107. Peterson, R.N., J.J. Bozzola, W.P. Hunt, and A. Darabi 1990 Charac-
ACTIN AND CALMODULIN DURING SPERM-EGG FUSION terization of membrane-associated actin in boar spermatozoa. J. Exp. Zool., 253:202-214. Plummer, J.M., and P.F. Watson 1985 Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique. J. Reprod. Fertil., 75.255-263. Rogers, B.Y., C. Bastias, R.L. Coulson, and L.D. Russell 1989 Cytochalasin D inhibits penetration of hamster eggs by guinea pig and human spermatozoa. J. Androl., 10t275-281. Ruknudin, A., J.P. Dadoune, and I.A. Silver 1988 Intracellular calcium in hamster spermatozoa in testis, epididymis and during acrosome reaction. Anim. Reprod. Sci., 16t145-153. Ruknudin, A,, and I.A. Silver 1990 Ca2' uptake during capacitation of mouse s ermatozoa and the effect of an anion transport inhibitor on Cap+ uptake. Molec. Reprod. Dev., 26:63-68. Saxena, N., R.N. Peterson N.K. Saxena, and L.D. Russell 1986 Microfilaments appear in boar spermatozoa during capacitation in vitro. J. Exp. Zool., 239t423-427. Tash, J.S. 1989 Protein phosphorylation: The second messenger signal transducer of flagellar motility. Cell Motil. Cytoskeleton, 14: 332-339. Trejo, R., and A. Mujica 1990 Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa. Molec. Reprod. Dev., 26t366-376.
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Weinman, S., C. Ores-Carton, D. Rainteau, and S. Puszkin 1986a Immunoelectron microscopic localization of calmodulin and phospholipase A, in spermatozoa. I. J . Histochem. Cytochem.,34tll711179. Weinman, S., C. Ores-Carton, F. Escaig, J. Feinberg, and S. Puszkin 19861, Calmodulin immunoelectron microscopy: Redistribution during ram spermatogenesis. 11.J. Histochem. Cytochem.34tl1811193. Welch, J.E., and M.G. ORand 1985 Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit. Dev. Biol., 109t411-417. Welsh, M.J., J.R. Dedman, B.R. Brinkley, and A.R. Means 1979 Tubulin and calmodulin: Effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus. J . Cell Biol., 81:624 -634. Yamamoto, N. 1985 Immunoelectron microscopic localization of calmodulin in guinea pig testis and spermatozoa. Acta Histochem. Cytochem., 18.199-21 1. Yanagimachi, R. 1982 Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster. Gamete Res., 5:323-344. Yanagimachi, R. 1988 Mammalian fertilization. In: Physiology of Reproduction. Knobil, E. and J. Neil, eds. Raven Press, New York, Vol. 2, Chap. 5, pp. 135-185.
