TISSUE & CELL, 1990 22 (3) 291-300 0 1990 Longman GroupUK Ltd
JEAN-PIERRE
FOUQUET,
MARIE-LOUISE KANN AND JEAN-PIERRE DADOUNE
IMMUNOELECTRON MICROSCOPIC DISTRIBUTION OF ACTIN IN HAMSTER SPERMATIDS AND EPIDIDYMAL, CAPACITATED AND ACROSOME-REACTED SPERMATOZOA Keywords:
Actin, immunocytochemistry.
sperm&ids,
spermatozoa
ABSTRACT. The distribution of actin in hamster sperm cells was studied during spermiogenesis, epididymal transit, in vitro capacitation and acrosome reaction by immunogold procedures using a polyclonal and two monoclonal antiactin antibodies. A predominant actin labeling (F-actin) was detected in the subacrosomal space of spermatids. Actin labeling was also observed under the plasma membrane of intercellular bridges and along the outer acrosomal membrane. In late spermatids there was both F-actin depolymerization and a loss of actin immunolabeling, thus suggesting a dispersion of G-actin monomers. No obvious labeling was evidenced in residual bodies. This pattern was observed with the three antiactin probes. In contrast, an actin labeling reappeared over the fibrous sheath of the flagellum in epididymal spermatozoa but only when the polyclonal antibody was used. Only one single actin reactive band was detected by immunoblotting of sperm extracts. Since the sperm tails were NBD phallacidin negative they were considered to contain either G-actin or actin oligomers rather than bundles of actin filaments. It is suggested that G-actin originating in the head of late spermatids was redistributed to the flagellum of epidymal spermatozoa. No further changes were noted after capacitation and acrosome reaction thus indicating no apparent effect on actin polymerization and distribution.
ration in the ground squirrel (Vogl et al., 1986) and the rat (Russell et al., 1986). In addition, subacrosomal actin has also been detected by immunogold staining of the spermatids in the boar (Camatini et al., 1986), the rat, hamster, monkey and human (Fouquet et al., 1989 a) and the rabbit (Camatini et al., 1987; Fouquet et al., 1989 b). The disappearance of NBD phallacidin as well as myosin decoration in late spermatids indicates a depolymerization of subacrosomal Factin to G-actin. In these cells as well as in epididymal spermatozoa actin also becomes undetectable in the subacrosomal layer by immunocytochemistry. However spermatozoa of various species still contain monomeric actin (Ochs and Wolf, 1985; Welch and O’Rand, 1985; Flaherty et al., 1986; Camatini et al., 1986) whose distribution varies from
Introduction During mammalian spermiogenesis filamentous actin accumulates in the subacrosoma1 space of spermatids. This has been demonstrated, using the fluorescent probe NBD phallacidin, in the rabbit (Welch and O’Rand, 1985), the ground squirrel (Vogl et al., 1986), the rat (Masri et al., 1987) and the guinea-pig (Halenda et al., 1987) as well as by electron microscopy using myosin decoGroupe d’Etude de la Formation et de la Maturation du Gamete Mile. Dkpartement de Cytologic et Histologie, UFR Biomtdicale des Saints-Pkres, 45 rue des SaintsPbres - F 75270 Paris Cedex 06, France. Address for correspondence: J. P. Fouquet, Dtpartement de Cytologic et Histologie, 45 rue des Saints-Peres - F 75270 Paris Cedex 06, France. Received Revised
18 December 16 January
1989
1990. 291
FOUQUET ET AL
species to species as shown using either immunofluorescence (for review see Flaherty et al., 1986) or immunoelectron microscopy (Camatini et al., 1986; Lora-Lamia et al., 1986; Camatini et al., 1987; Casale et al., 1988; Flaherty et al., 1988). In view of these results the fate of actin in late spermatids then during sperm epididymal transit remains unknown, Subacrosomal F-actin has been considered to have a structural function being involved in acrosome shaping and/or capping (Welch and O’Rand, 1985; Russell et al., 1986; Vogl et al., 1986; Fouquet et al., 1989a). In contrast , a capping function for the nuclear membrane has been proposed from the observations of abnormal spermatids in the experimentally cryptorchid rabbit (Fouquet et al., 1989b). The diversity of actin location in sperm cells seems to preclude a universal function for this protein (Flaherty et al., 1988). However, sperm actin may function either to anchor the plasma membrane to underlying structures or to affect the mobility of plasma membrane proteins (Flaherty et al., 1988). In this respect it has been suggested that actin polymerization could be involved in plasma membrane changes during capacitation of boar sperm (Saxena et al., 1986). In this study, the distribution of actin in hamster sperm cells was reinvestigated during spermiogenesis and epididymal transit as well as after in vitro capacitation and acrosome reaction using immunogold electron microscopy with three different antiactin antibodies. NBD phallacidin staining was used for F-actin detection whereas immunoblotting was performed for actin characterization. The results suggest an actin redistribution from the head of late spermatids to the flagellum of epididymal spermatozoa but further changes were not evidenced in capacitated and acrosome reacted sperm cells. Material and Methods Animals.
Twelve mature 2-5 months old male hamsters were anesthetized with 6% chloral and bled: then their testes and/or epididymides were removed for further preparations. Four adults 2-3 months old female hamsters were induced to superovulate by successive treatment with 25 U.I. PMSG and 25 U.I. HCG. These females were killed 16
hours later and the oocytes were collected from the oviduct (Yanagimachi, 1982). Preparation of epididymal, capacitated and acrosome reacted spermatozoa
Cauda epididymides were cleared of adherent fat and blood vessels to avoid contamination of sperm suspensions with erythrocytes. Then, the distal part of the tubules were carefully dissected and spermatozoa were allowed to swim in the appropriate medium. Except for capacitation, epididymal spermatozoa were collected in phosphate buffered saline (PBS: 20mM sodium phosphate, 150 mM NaCl, pH 7.4). Epididymal spermatozoa were capacitated in a modified Tyrode’s medium (m. TAPL 1) containing pyruvate, lactate, taurine, epinephrine and BSA (Yanagimachi, 1982) for 3h. After washing by centrifugation (1OOOg 5 min) spermatozoa were added to oocytes to obtain acrosome reacted spermatozoa during in vitro fertilization for 2-4 h. The oocytes had been previously freed from the cumulus by a 5 minutes treatment with 0.1% hyaluronidase in m. TALP 1 (Yanagimachi, 1982). Immunogold localization of actin
Pieces of testes, epididymal, capacitated and acrosome reacted spermatozoa linked to oocytes were fixed in 1% glutaraldehyde buffered with 0.1 M phosphate pH 7.3 for 1 h at room temperature. Then, free aldehyde groups were blocked by washing in the same buffer containing 0.1 M glycine overnight at 4°C. Embedding was performed in lowicryl K 4M (Balzers) according to Altman et al. (1984). Thin sections were collected on nickel grids and the immunogold procedure for actin detection was carried out as previously described (Fouquet et al., 1989; Kann and Fouquet, 1989). Three antiactin antibodies were used; an affinity purified rabbit polyclonal IgG (Gounon and Karsenti, 1981) at a final concentration of l&1.5 kg/ml, a mouse monoclonal IgG designated C, (Lessard, 1988) at a concentration of 2&40 &ml and a mouse monoclonal IgM (code 350 Amersham) at a concentration of IO-20 l.rg/ml. These primary antibodies were immunolocalized with gold particles coated with secondary antibodies (Janssen) diluted 1:15, respectively: GAR IgG-G 15; GAM IgGG 10 and GAM IgM-G 10. Positive controls of immunoreactivity were verified from the
ACTIN IN HAMSTER
SPERM CELLS
labeling of Sertoli cells and myoid cells whereas for negative controls the antiactin antibodies were omitted or preadsorbed with chicken gizzard actin (Sigma). The sections were stained with uranyl acetate before electron microscope examination. The steps of hamster spermiogenesis were classified according to Clermont (1954). NBD phallacidin staining ofJilamentol,ts actin
Suspensions of epididymal, capacitated and acrosome-reacted spermatozoa linked to oocytes were, washed twice in PBS, attached to coverslips and fixed in 3.7% formaldehyde in PBS for ltX1.5 min at room temperature. The cells were labeled with NBD phallacidin (Molecular Probes Inc., Junctions City) 0.3>0.16bM in PBS using the protocol recommended by the supplier. Cryostat sections of testes and epididymides also were used as positive control. Preparation of epididymal sperm extracts
293
After electrophoresis, the proteins were transferred to nitrocellulose sheets according to Towbin et al. (1979). These sheets were rinsed in PBS and air dried. For immunostaining, the blots were first incubated l-2 h at room temperature in PBS containing 0.3% gelatin (Sigma, type II) and 0.1% Tween 20 to block nonspecific binding of antibodies. Then the blots were incubated overnight at 4°C in the blocking mixture also containing the primary antiactin antibody, either l-l.5 p,g/ml rabbit polyclonal IgG or 2-4 pgi ml mouse monoclonal IgG or 5-lOpg/ml mouse monoclonal IgM. After washes in the blocking solution the nitrocellulose papers were incubated for 2 h at room temperature either in a 1: 1000 dilution of goat antirabbit IgG (Paesel) or in 1:400 dilution of goat antimouse Ig s (Dako) peroxidase conjugates. Then, the strips were rinsed and stained with 4 Chloro-l-naphtol (0.5 mgiml) in PBS containing 0.02% hydrogen peroxide.
Results The epididymal sperm suspensions in PBS Immunogold staining of actin were washed by centrifugation (ZOOOg, 5min) and the pellets were resuspended in During spermiogenesis a similar actin distriPBS containing 4 mM Ethylene diamine bution was observed with the polyclonal and tetraacetic acid (EDTA), 0.1% sodium azide two monoclonal antiactin antibodies. In round spermatids from step 4-5, actin labeland a protease inhibitor cocktail (PIC; 1 kg/ ing was detected in the subacrosomal layer ml leupeptine, antipai’n and pepstatin A, between the nuclear envelope and the inner 2ug/ml aprotinin and 1 mM phenyl methyl sulfonyl fluoride (PMSF), Sigma Chemical acrosomal membrane (Fig. 1). No particular labeling could be observed in other cellular Co.). Sperm counts with an hemocytometer indicated 1.5-2. 10s spermatozoa per sample regions except along the plasma membrane with less than 10m3blood cells and no other at the level of intercellular bridges (Fig. 2). contaminating cells. After a second centrifuIn elongating spermatids (steps 8-13) the gation (12,000 g, 5 min) the pellets were subacrosomal layer and its actin labeling resuspended in Tris buffer saline (TBS; extended simultaneously (Fig. 3, 4). In 20mM Tris-HCl buffer, 150 m NaCl, addition, discrete alignments of gold particles pH6.8) also containing 4mM EDTA, 0.1% were found occasionally in those areas of the sodium azide, 1% sodium dodecyl-sulfate plasma membrane connected with the outer (SDS) and the PIC mixture, boiled for 10 min acrosomal membrane just beneath the heaand pelleted for 5min in a microfuge. The vily stained Sertoli ectoplasmic specializasupernatants were removed and mixed with tions (Fig. 3). In these two locations the label 2 x sample buffer of Laemmli (1970) boiled was not detected in control sections using for 5min and frozen until electrophoresis. actin preadsorbed antiactin probes. At the The pellets were fixed in glutaraldehyde and beginning of the maturation phase (steps embedded in lowicryl as described above to 14-15) actin labeling of the subacrosomal check both ultrastructural alterations and the layer also extended under the postacrosomal loss of reactivity to antiactin antibodies. lamina up to halfway (Fig. 5). Actin immunostaining decreased gradually in step 16 SDS-PAGE and immunoblotting spermatids during their migration toward the The sperm extracts were analysed by lumen of the seminiferous tubules. No speciSDS-PAGE on 12% gels (Laemmli, 1970). fic label could be detected in the head of step
ACTIN
IN
HAMSTER
SPERM
795
CELLS
NBD phallacidin staining
17 spermatids as well as in the residual bodies at the time of spermiation (Fig. 6). Though the neck and tail of spermatids remained unlabeled at any phase of spermiogenesis a light label of the principal piece of the flagellum was observed in some step 17 spermatids but only with the polyclonal antiactin antibody. In epididymal spermatozoa actin was detected in the principal piece of the flagellum, except in the distal part devoid of outer dense fibers. This label occurs in clusters between the fibrous sheath and the plasma membrane as visualized with the polyclonal antiactin antibody (Figs 7, 8). In contrast, only a faint staining was detected with the C, monoclonal antiactin antibody (Fig. 9) and no staining with the Amersham antibody. Actin was not detected in other parts of epididymal sperm including cytoplasmic droplet and no changes were observed after capacitation (Figs 10-12). Likewise in the acrosome - reacted spermatozoa found in the perivitelline space of oocytes actin labeling remained restricted to the principal piece of the flagellum (Figs 13-15). This actin labeling in epididymal, capacitated and acrosome reacted spermatozoa was abolished when the first antibody was omitted or preadsorbed with actin as shown for capacitated spermatozoa (Fig. 12) but some background staining was associated with the sperm heads (Figs 13-14).
Figs l-6. either
Immunogold
C, monoclonal
monoclonal Figs
antiactin
1-2.
Step
and intercellular Fig.
3. Early
Sertoli portion
Fig.
Fig.
arrows).
step 8 spermatid.
Note
specializations plasma
~27,ooO actin
hamster
spermiogenesis
labeling
actin
membrane
antiactin
as visualized
(Fig.
using
3) or Amersham
except Fig. 6, ~20,000. of the subacrosomal
The chromatoid
(arrow)
and immunoblotting
To ascertain the presence of actin in epididyma1 spermatozoa, SDS protein extracts were subjected to unidimensional SDS-PAGE and immunoblotting. The two monoclonal antibodies as well as the polyclonal antiactin antibody both recognized actin form chicken gizzard and one single reactive band of 43 Kd in sperm extracts (Fig. 17). In the extracted spermatozoa as expected (O’Brien and BelIve, 1980) microtubules and the membranes except the outer mitochondrial membrane were solubilized, correlatively the immunocytochemical signal for actin was no
labeling
of the subacrosomal
and the gold particles (between
layer
body (cb) is unlabeled,
small
layer
alignment
arrows).
Some
(arrowhead)
A (acrosome). (arrowhead),
close to a discrete background
staining
(A).
13 spermatid.
specialization
5. Step
showing
(ICB,
SDS-PAGE
1, 2. 5, 6) or polyclonal
4) antibodies.
in the acrosome
Fig. 4. Step
of actin during
(Figs
5 spermatid
of spermatid
ectoplasmic
previous
(Fig.
bridge
ectoplasmic
is observed
distribution ant&tin
Since it is established that spermatozoa contain G-actin, the NBD phallacidin staining was used to detect a possible polymerization to F-actin after capacitation and acrosome reaction. As expected, testicular spermatozoa observed at the time of spermiation and epididymal spermatozoa were NBD phallacidin negative and no difference was noted in capacitated and acrosome - reacted spermatozoa. In testes sections used as positive controls myoid cells and Sertoli cells were heavily labeled. The intensity of the fluorescence in the Sertoli ectoplasmic specializations surrounding most spermatids made difficult the detection of the faint signal in the subacrosomal areas.
Actin
labeling
of the subacrosomal
layer
(arrowhead)
and Sertoli
(arrow).
15 spermatid.
Actin
labeling
of the postacrosomal
lamina
(PL)
in addition
to
locations. 6. Actin
17 spermatids
labeling contrary
is undetected to the Sertoli
in the head.
ectoplasmic
neck
(N)
specializations
and residual (arrow).
body
(rb)
of step
FOUQUET ET AL
more detected in the principal piece of the flagellum (Fig. 16). Discussion
Using polyclonal IgG (Gounon and Karsenti, 1981) and monoclonal IgM (Amersham U.K.) antiactin antibodies, both of which recognizing F and G - actin in muscle and non-muscle cells (Chailley et al., 1986; Casale et al., 1988; Flaherty et al., 1988) it was previously shown that actin (F-actin) is a component of the subacrosomal layer of hamster spermatids during the greater part of spermiogenesis (Fouquet et al., 1989a). These results were confirmed here using a third antibody, a monoclonal antiactin, which reacts with all six known vertebrate actin isoforms (Lessard, 1988). It has been suggested that subacrosomal actin may interact with inner acrosomal membrane for acrosome capping or shaping (Welch and O’Rand, 1985; Russell et al., 1986). However a study of abnormal acrosomes and nuclei in spermatids of experimentally cryptorchid rabbit showed that subacrosomal actin is in fact a component of the so-called perinuclear substance (PNS) (Fouquet et al., 1989b). This PNS and its actin labeling developed normally even if partially covered by a small and discontinuous acrosome. Furthermore the
PNS actin remained adherent to the nuclear membrane rather than the overlying inner acrosomal membrane when the subacrosoma1 space was dilated. These observations suggested actin involvement in nuclear changes during spermiogenesis. In addition to the predominant location of actin in the subacrosomal space this protein was also detected in two other sites. First at the level of intercellular bridges where it appears to maintain these structures as reported in rat spermatids (Russell et al., 1987). Second between the plasma membrane and the outer acrosomal membrane in elongating and maturing spermatids, where a light but specific label was observed. A similar light staining was also described in boar spermatids by Camatini et al. 1986) who suggested that actin could be involved in plasma membrane-outer acrosomal membrane interactions. On the other hand, no actin was detected in the chromatoid body in contrast to rat spermatids (Walt and Armbruster, 1984). Likewise, no actin labeling could be observed in the tail and the neck of spermatids at any step of spermiogenesis. The lack of NBD phallacidin staining of testicular spermatozoa indicated a depolymerization of subacrosomal actin as reported in other species (Vogl et al., 1986; Halenda et al., 1987; Masri et al., 1987). At the same
Figs 7-8. In epididymal spermatozoa actin labeling, using polyclonal antiactin, occurs in small clusters (rosettes) over the fibrous sheath of the tail principal piece: ribs (arrow) and longitudinal columns (arrowhead). Fig. 7: longitudinal axial section between two longitudinal grazzingsections (wide arrows). x27,lMO. Fig. 8. transverse section. ~36,ooO. Fig 9. In epdidymal sperm actin labeling, using the C4 monoclonal anti&in, low signal only on the principal piece of the flagellum. ~27,000.
gives a very
Figs l&12. Actin immunostaining of capacitated spermatozoa using polyclonal antiactin. Fig. 10. Head and middle piece showing only a low background staining. ~18,000. Fig. 11. Decoration of the fibrous sheath of the tail principal piece. ~27,000. Fig. 12. Control using actin preadsorbed antibody. x27,ooO. Figs 1>15. Actin immunolabeling of acrosome-reacted spermatozoa, in the perivitelline space of oocyte, using polyclonal antiactin antibody. Note actin labeling in the cortex (c) of the oocyte (Fig. 13). In the sperm head and middle piece of the flagellum there is some background staining (Figs 1%14, x 18,COO)whereas the specific labeling of the principal piece is unchanged (Fig. 15, x27,ooO). Fig. 16. Actin immunostaining, with the polyclonal antibody, of epididymal sperm pelleted after protein extraction with SDS: no actin labeling neither in the principal piece (arrows) nor in other cell part (X 18,000).
0
9
.
FOUQUET
298
b’
c
c’
Fig. 17. lmmunoblots stained with the polyclonal antiactin antibody (a, a’), monoclonal antiactin from Amersham (b, b’) and C, monoclonal antiactin (c, c’), a and b, c chicken gizzard actin 1.5 and 2.5 pg respectively a’, b’. c’ extracts of 5106 hamster spermatozoa.
time there was also a loss of actin immunolabeling in the head of late spermatids. This may be explained by G-actin dispersion after F-actin depolymerization so that the local concentration for this protein would become too low to give an immunocytochemical signal. Also G-actin could be degraded or shed in the residual bodies. Although no peculiar actin labeling was noted in these cytoplasmic remnants of testicular spermatozoa some actin loss could be evidenced by immunoblotting on purified fractions. On the other hand, the immunoreactive epitopes might no longer be available after steric changes of the actin molecules due to interaction with other proteins. In fact, a light labeling reappeared in the principal piece of the flagellum in some testicular spermatozoa which increased in intensity and was present in all epididymal spermatozoa as evidence with polyclonal antiactin antibody. This label could indicate either the presence of actin or a cross reactive protein in the principal piece of the flagellum. However a single actin band was detected in immunoblots of protein extracts from epididymal sperm virtually uncontamined by other cell types. Therefore this suggests there was, at least in part, some transfer of G-actin monomeres from the head to the tail of sper-
ET AL
matozoa during spermiation and epididymal transit. Since the epididymal sperm tails were not stained with NBD phallacidin certainly they do not contain large bundles of actin filaments. However, if actin oligomers are present between the fibrous sheath and the plasma membrane their size and number may be too low to be detected with this fluorescent F-actin probe. On the other hand actin immunostaining in form of gold-clusters might evidence G-actin storage as already reported (Kordeli et al., 1987). The characterization of peculiar actin-binding proteins at this site could help to clarify this point. Flaherty et al, (1988) reported on the presence of actin in the neck and concave margin of the hamster sperm head using the Amersham antiactin antibody. In the present study actin was not detected in these regions neither with the same antibody nor with two other antiactin antibodies. However these authors used immunogold preembedding procedures in contrast to the postembedding procedures used here. Similar discrepancies in sperm actin distribution possibly due either to methodology or antibody specificity have been frequently reported as for example in the boar (Flaherty et al., 1986; Camatini et al., 1986; Lora-Lamia et al., 1986; Casale et al., 1988) and the human (Clarke et al., 1982; Virtanen et al., 1984; Flaherty et al., 1988). As regards the possible role of actin which has been detected in the principal piece of the flagellum, an actin-mediated anchorage of the plasma membrane to the fibrous sheath may be suggested. This assumption of actin interactions with various membranes or structures is defended for any actin location in spermatozoa of various species (Flaherty et al., 1988) but such a function remains to be explored at first the presence of various actin-binding proteins. After in vitro capacitation of boar spermatozoa the NBD phallacidin staining detected in most regions of the cells, suggested an actin polymerization involved in migration of plasma membrane proteins (Saxena et al., 1986). In contrast, neither NBD phallacidin staining nor immunogold changes were observed in capacitated and acrosomereacted hamster spermatozoa. Therefore in hamster spermatozoa, capacitation and acrosome-reaction do not seem to have any effect on actin polymerization and distribution
ACTIN
IN HAMSTER
SPERM
CELLS
In conclusion, this study suggests that a depolymerization of the subacrosomal Factin in late spermatids gives rise to G-actin monomeres which then can be transferred to other regions of the spermatozoa during spermiation and/or epididymal transit. The final distribution of actin should be dependent upon its affinity for various proteins encountered during the transfer. These changes also include a possible masking of actin which would prevent its immunocytochemical
detection. Similar changes probably occur in the spermatozoa of other species thus resulting in various actin locations as has been reported. Acknowledgements
The authors are indebted to Dr Pierre Gounon for the gift of the polyclonal antiactin antibody and to Dr James L. Lessard for the gift of the C, monoclonal antibody.
References Altman, L. G., Schneider, B. G. and Papermaster. D. S.. 1984. Rapid embedding of tissues in Lowicryl K 4M forimmunoelectron microscopy. J. Hisrochem. Cytochem., 32,1217-1223. Camatini, M., Anelli, G. and Casale, A. 1986. Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy. Eur. J. Cell Bio[., 42,311-318. Camatini, M., Casale, A. and Cifarelli, M. 1987. immunocytochemical identification of actin in rabbit spermiogenesis and spermatozoa. Eur. J. Cell Biol., 45,27~281. Casale, A., Camatini, M., Skalli, 0. and Gabbiani, G. 1988. Characterization of actin isoforms in ejaculated boar spermatozoa. Gamete Res., 20,13%144. Chailley, B., Bork, K., Gounon, P. and Sandoz. D. 1986. Immunological detection of actin in isolated cilia from quail oviduct. Biol. Cell., 58.43-52. Clarke, G. N., Clarke, F. M. and Wilson, S. 1982. Actin in human spermatozoa. Biol. Reprod., 26,319-327. Clermont, Y. 1954. Cycle de I’epithelium seminal et mode de renouvellement des spermatozoldes chez le hamster. Rev. Can. Biol., 13,20&245. FIaherty, S. P.. Winfrey, V. P. and Olson, G. E. 1986. Localization of actin in mammalian spermatozoa: a comparison of eight species. Anar. Rec., 216,504515. Flaherty, S. P., Winfrey, V. P. and Olson, G. E. 1988. Localization of actin in human, bull, rabbit and hamster sperm by immunoelectron microscopy. Anat. Rec., 221,599-610. Fouquet, .I. P., Kann, M. L. and Dadoune, .I. P. 1989a. Immunogold distribution of actin during spermiogenesis in the rat, hamster, monkey, and human. Anat. Rec., 223,35-42. Fouquet, J. P., Kann, M. L., Courtens, J. L. and Ploen, L. 1989b. Immunogold distribution of actin during spermiogenesis in the normal rabbit and after experimental cryptorchidism. Gamete Ref., 24,281-290. Gounon, P. and Karsenti., E. 1981. Involvement of contractile proteins in the changes in consistency of oocyte nucleoplasm of the newt Pleurodeles waltlii. .I. CeN Biol., 88,41&421. Halenda, R. M., Primakoff, P. and Myles, D. G. 1987. Actin filaments. localized to the region of the developing acrosome during early stages, are lost during later stages of guinea pig spermiogenesis. Biol. Reprod., 36.491-499. Kann, M. L. and Fouquet, J. P. 1989. Comparison of LR white resin, Lowicryl K4M and epon post-embedding procedures for immunogold staining of actin in the testis. Himchemistry. 91.221-226. Kordeli, E., Cartaud, J., Nghiem, H. 0. and Changeux, J. P. 1987. In situ localization of soluble and tilamentous actin in Torpedo marmorata electrocyte. Biol. Cell., 59. 61-68. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage Tq. Nature. 227,680-685. Lessard, J. L. 1988. Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Ceil Motil. Cytoskel., 10,34%362. Lora-Lamia, C., Castellani-Ceresa, L.. Andreetta, F., Cotelli, F. and Brivio, M. 1986. Localization and distribution of actin in mammalian sperm heads. J. Ultrastruct. Mol. Struct. Rex., 96, 12-21, Masri, B. A., Russell, L. D. and Vogl, A. W. 1987. Distribution of actin m spermatids and adjacent Sertoli cell regionsof the rat. Anat. Rec., 218,2&26. O’Brien, D. A. and Bellve, A. R. 1980. Protein constituents of the mouse spermatozoon. Develop. Biol., 75,38&404. Ochs, D. and Wolf, D. P. 1985. Actin in ejaculated human sperm cells. Biol. Reprod., 33, 1223-1226. Russell, L. D., Weber, J. E. and Vogl, A. W. (1986). Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis. Tissue & Ceil, IS, 887-898. Russell, L. D., Wogl, A. W. and Weber, J. E. 1987. Actin localization in male germ cell intercellular bridges in the rat and ground squirrel and disruption of bridges by cytochalasin D. Am. J. Amt., 180.25-40.
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ET AL
Saxena, N., Peterson, R. N., Saxena. N. K. and Russell. L. D. 1986. Microfilaments appear in boar spermatozoa during capacitation in vitro. J. Exp. Zool., 239,42%427. Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocelhdose sheets: Procedure and some applications. Proc. Nad. Acad. Sci., USA, 76.435&4354. Virtanen, I., Badley, R. A., Paasivuo, R. and Lehto, V. P. 1984. Distinct cytoskeletal domains revealed in sperm cells. J. CellBid., 99, 108%1091. Vogl, A. W. Grove. B. D. and Lew, G. J. 1986. Distribution of actin in Sertoli cell ectoplasmic specializations and associated spermatids in the ground squirrel testis. Anal. Rec., 215,331-341. Walt, H. and Armbruster, B. L. 1984. Actin and RNA arc components of the chromatoid bodies in spermatids of the rat. Cell Tissue Res., 236,487-490. Welch, J. E. and O’Rand, M. G. 1985. Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit. Dev. Bid., 109,411-417. Yanagimachi, R. 1982. Requirement of extracellular calcium ions for various stages of fertilization and fertilizationrelated phenomena in the hamster. Gamete Res.. 5,323-344.