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Localization of Calmodulin in perinuclear structures of spermatids and spermatozoaA comparison of six mammalian species.

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THE ANATOMICAL RECORD 230:481-488 (1991)
Localization of Calmodulin in Perinuclear
Structures of Spermatids and Spermatozoa:
A Comparison of Six Mammalian Species
MARIE-LOUISE KANN, JACQUELINE FEINBERG, DOMINIQUE RAINTEAU,
JEAN-PIERRE DADOUNE, SERGE WEINMAN, AND JEAN-PIERRE FOUQUET
Groupe $Etude de la Formation et de la Maturation du Gam6te Mrile (M.L.K., J.P.D.,
J.P.F.) and Groupe $Etude des Mkcanismes de Regulation Intracellulaire (J.F., D.R.,
S.W.), UFR Biomkdicale des Saints-Peres, 75270 Paris Cedex 06, France
ABSTRACT
The distribution of Calmodulin was examined during spermiogenesis and sperm epididymal maturation in rabbit, hamster, mouse, rat, monkey,
and human. An affinity-purified antibody to Calmodulin was used to characterize
this protein in sperm extracts by immunoblot analysis. Post-embedding immunogold procedures were used to localize Calmodulin a t the ultrastructural level.
The pattern of Calmodulin distribution was similar in the six species studied. A
diffuse labeling was observed in round spermatids. Gold particles accumulated
first in the subacrosomal layer of elongating spermatids. The perinuclear ring was
also labeled. During the maturation phase of spermatids, Calmodulin labeling
extended to the postacrosomal sheath. Dramatic changes occurred at spermiation
so that in testicular sperm Calmodulin immunostaining was predominant in the
postacrosomal sheath. Some labeling was still detected in restricted areas of the
subacrosomal layer. This feature varied from species to species. Calmodulin location did not change during sperm epididymal maturation. A role for Calmodulin in
the control of manchette development and regulation of subacrosomal actin aggregation state during spermiogenesis is proposed. The unique location of Calmodulin
in the postacrosomal sheath of all species that have been studied in this work,
together with the known presence of calcium in this area suggest a pivotal role for
Calmodulin in sperm-egg fusion process.
Calmodulin (CaM) has been characterized in spermatozoa of different mammalian species. Moreover,
this Ca2+-regulatorprotein has been localized in various regions of sperm head and tail using indirect immunofluorescence (IIF). In the guinea-pig, rabbit
(Jones et al., 1980), bull (Feinberg et al., 19811, r a t
(Lagace et al., 19811, and hamster (Moore and Dedman,
1984) CaM was detected both in the acrosomal and
postacrosomal region as well as in the proximal andlor
distal part of the flagellum. More detailed descriptions
have also been reported using immunoelectron microscopy. In the guinea-pig, CaM was located in the cytoplasm around the acrosome but not inside (Yamamoto,
1985) and either along the coarse fibers (Gordon et al.,
1983) or in the axoneme and fibrous sheath of the tail
(Yamamoto, 1985). In boar sperm, CaM was found both
inside and outside of the acrosome, in the postacrosome, the neck, and the various structures of the flagellum middlepiece (Camatini et al., 1986). In bull and
ram sperm, CaM was mainly detected in the postacrosomal region and along the axoneme and fibrous
sheath of the tail principal piece (Weinman e t al.,
1986a,b).
The role of CaM in the regulation of sperm flagellar
motility has been extensively studied (for review, see
Tash, 1989). In addition, this protein might be involved
in other Ca2+-dependent mechanisms such as capaci0 1991 WILEY-LISS, INC
tation (Leclerc et al., 1990), acrosome reaction, and
sperm-egg fusion (Jones et al., 1980; Moore and Dedman, 1984; Weinman et al., 1986a,b; Camatini et al.,
1986; Aitken et al., 1988). To assume these various
functions CaM must be localized in specific subcellular
compartments. Indeed, the reported localizations using
IIF were not accurate enough to correlate structures
with functions. On the other hand, immunoelectron microscopic distribution of CaM varied in the four species
previously studied. Since it was not possible to determine whether these discrepancies were related to species differences, technical pitfalls, or antibody specificity, we decided to reinvestigate CaM distribution in
epididymal sperm of six species using the same immunoelectron microscopic method and the same antibody.
In addition, the distribution of this protein was also
examined during spermiogenesis because, except in
ram spermatids (Weinman et al., 1986b), a detailed
information was still lacking. A similar CaM localization was found in the perinuclear substance (PNS) of
spermatids in all species studied. Dramatic changes
occurred at spermiation so that this protein became
Received November 5, 1990; accepted February 5, 1991.
Address reprint requests to M.-L. Kann, Departement de Cytologie
et Histologie, 45 rue des Saints-Peres, F 75270 Paris Cedex 06,
France.
482
M.-L. KANN
essentially restricted to the postacrosomal area of
sperm head.
MATERIALS A N D METHODS
Testes and epididymides of adult hamster, mouse,
rat, rabbit, and monkey (Macaca fascicularis) were removed under anesthesia. Testicular biopsies and ejaculates from human donors were also used.
Preparation of Sperm Extracts
Epididymal or ejaculated sperm were collected in
phosphate buffered saline, pH 7.4 (PBS). They were
washed twice by centrifugation (2,0OOg, 5 min) in PBS
containing 4 mM ethylene diamine tetraacetic acid
(EDTA) and protease inhibitors: 1 pgiml leupeptin, 1
pgiml antipain, 1pgiml pepstatin A, 2 pgiml aprotinin,
and 0.5 mM phenylmethylsulfonyl fluoride (PMSF).
Pellets of intact sperm (108-109 spermatozoa) containing less than 0.1% contaminating cells were sonicated
for 2 min in 2.5 vol of 4 mM EDTA and 15 mM Tris-HC1
buffer, pH 6.8, containing the same antiproteolytic
agents as above. After another centrifugation (12,00Og,
5 min) the resulting pellets were resuspended in the
same solution also containing 1%sodium dodecyl-sulfate (SDS), boiled for 10 min, and pelleted for 5 min in
a microfuge. The supernatants were removed and frozen until electrophoresis.
Preparation of Calrnodulin and Antibody to Calmodulin
CaM from ram testis was purified to homogeneity
according to Autric et al. (1980). Performic acid-oxidized CaM was used to elicit antibodies in rabbit according to Van Eldik and Watterson (1981).The antibodies were affinity-purified as previously described
(Weinman e t al., 1986a).
lmmunoblot Analysis
The sperm extracts were processed for SDS-PAGE
and electrophoresed on a 15% polyacrylamide gel
(Laemmli, 1970) and transferred to nitrocellulose
sheets a s described by Towbin et al. (1979). CaM was
detected with the affinity-purified antibody according
to Van Eldik and Wolchok (1984).
Tissue Preparation for lmmunocytochemistry
Pieces of testes, epididymides, and pellets of human
ejaculated sperm were routinely fixed in 1%glutaraldehyde buffered with 0.1 M phosphate, pH 7.3. The
samples were embedded in Lowicryl K4M according to
Carlemalm et al. (1982) or Altman et al. (1984). In
addition, fixation in 0.5% glutaraldehyde plus 1%
paraformaldehyde, a s well as embedding in LR white
resin according to Timms (19861, were performed in
some cases. Thin sections were collected on uncoated
200 mesh nickel grids.
lrnrnunogold Procedure
It was carried out as previously described for actin
detection (Fouquet et al., 1989a; Kann and Fouquet,
1989). Briefly, the sections were treated with anti-CaM
antibody (20 pgiml) and then with goat antirabbit IgG
conjugated to 10-15 nm gold particles (Janssen) as secondary antibody. The grids were stained with uranyl
acetate solution before electron microscopic examination. Controls either omitting the anti-CaM antibody
17
kD
Fig. 1. Demonstration of Calmodulin in sperm extracts and characterization of affinity-purified antibodies to calmodulin. Purified ram
testis Calmodulin (lane a) was co-electrophoresed with extracts of rat
(lane b), hamster (lane c), mouse (lane d), rabbit (lane e), monkey
(lane f ) epididymal sperm, and human (lane g ) ejaculated sperm. The
proteins were transferred to a nitrocellulose membrane and immunodetected on the blot, as described in Materials and Methods.
or using this antibody preadsorbed with a 50-fold molar
excess of purified CaM were also performed to assess
the specificity of the immunostaining.
Staging and Cell Identification
The stages of the seminiferous epithelium cycle and
the steps of spermiogenesis were classified according to
Leblond and Clermont (1952) for the rat, Clermont
(1954) for the hamster, Oakberg (1956) for the mouse,
Clermont (1969) for the monkey, Ploen (1971) for the
rabbit, and Holstein (1976) for the human.
RESULTS
Identification of Calmodulin in Sperm Extracts and
Characterization of the Antibodies to Calrnodulin
As determined by immunoblot analysis, the IgGs
against Calmodulin recognized only one protein band
of 17 kDa in rat, hamster, mouse, rabbit, monkey, and
human sperm extracts (Fig. 1). This band comigrated
with purified ram testis Calmodulin.
lrnrnunogold Distribution of Calrnodulin
The preservation of CaM antigenicity was similar
with the different fixatives and embedding procedures
used (see Materials and Methods).
Rabbit
In round spermatids (steps A-D) immunolabeling
with the anti-CaM antibody showed a light and diffuse
distribution of gold particles over the cytoplasm, acrosome, subacrosomal layer and nucleus (Fig. 2). In
controls sections using the CaM-preadsorbed antibody
this light labeling was partially abolished in all cell
compartments, thus indicating both a low background
staining and no peculiar CaM location. During the
elongation phase (steps E-G), the subacrosomal layer
became regularly decorated with gold particles. A light
and specific CaM labeling was also observed a t the
level of the perinuclear ring (Fig. 3). Then, during the
CALMODULIN IN SPERMATIDS AND SPERMATOZOA
Figs. 2-6. CaM in rabbit spermatids and sperm (2-5 x 13,500;6 x
17,000).
Fig. 2. Step D round spermatid, no predominant labeling.
Flg. 3. Step F elongating spermatid, labeling of the subacrosomal
layer ( A ) and perinuclear ring (r).
most part of the maturation phase of spermatids (steps
H J ) , CaM labeling also extended to the postacrosomal
region so that the perinuclear substance (PNS) was
entirely labeled (Fig. 4).However, a t the time of spermiation (Fig. 51, CaM labeling in the subacrosomal
layer was restricted to local dilatations (bulges),
whereas the postacrosomal sheath remained strongly
labeled. CaM labeling was not modified during sperm
epididymal transit (Fig. 6).
483
Fig. 4. Step I maturing spermatid, the postacrosomal sheath (P) is
labeled in addition to the subacrosomal layer (A).
Figs. 5-6. Testicular sperm at spermiation and epididymal sperm,
respectively; labeling of the subacrosomal bulges (A) and postacrosoma1 sheath (PI.
Hamster
As in the rabbit, round spermatids (steps 1-7) were
diffusely labeled. A specific CaM labeling became
prominent in the subacrosomal layer and perinuclear
ring of elongating spermatids (steps 8-14) (Fig. 7).
During the maturation phase the postacrosomal sheath
was also labeled as soon as it began to develop (Fig. 8).
This was confirmed at later steps of spermiogenesis
484
M.-L.
KANN
Figs. 7-13. CaM in hamster spermatids and sperm ( x 13,500).
Fig. 7. Step 10 elongating spermatid, labeling of the subacrosomal
layer (A) and perinuclear ring (r).
Fig. 8. Step 15 spermatid at the beginning of the maturation phase,
the subacrosomal layer (A), postacrosomal sheath (P),and perinuclear
ring (r) are labeled.
Fig. 10. Testicular sperm a t spermiation, the subacrosomal ring (A),
and postacrosomal sheath (P) are labeled.
Figs. 11,12. Epididymal sperm with a labeling similar to that shown
in Figure 10, plus a labeling of the tip of the perforatorium (pe).
Fig. 13. In control sections, these specific labels are eliminated.
Some background gold particles are however still present in the acrosome.
Fig. 9. Step 16 maturing spermatids, the PNS is uniformely labeled.
(Figs. 9, 10). At spermiation, the subacrosomal layer
was no more uniformly labeled; gold particles were concentrated in the subacrosomal ring (Fig. 10) and in the
tip of the perforatorium. No further changes were observed during sperm epididymal transit (Figs. 11, 12).
In the various steps of spermiogenesis, some particles
were scattered throughout the acrosome. In testicular
and epididymal sperm, these particles were detected
mainly at the base of the acrosomal cap (Figs. 10, 11).
This label appeared not to be specific because i t was
also seen in control sections (Fig. 13).
Rat and mouse
The distribution of CaM during spermiogenesis in
these rodents was very similar to that observed in the
hamster. As shown in Figures 14-16 for the mouse and
Figures 17-19 for the rat, the perinuclear substance
was uniformly labeled in late spermatids. At spermiation, the labeling became predominant in the postacrosomal area although some gold particles were still
present in the perforatorium. CaM labeling in epididymal sperm was similar to that observed in testicular
sperm.
CALMODULIN IN SPERMATIDS AND SPERMATOZOA
Figs. 14-16. CaM in mouse spermatids and sperm x 13,500.
Fig. 14. Step 13 spermatid a t the beginning ofthe maturation phase,
labeling of the subacrosomal layer (AI, postacrosomal sheath (Pf, and
perinuclear ring (rl.
Flg. 15. Step 16 late spermatid. Partial labeling of the subacrosomal
layer (A], uniform labeling of the postacrosomal sheath (PI.
Fig. 16. Epididymal sperm. Predominant labeling of the postacroso-
ma1 sheath (P),scattered gold particles a t the tip of the perforatorium
(pel.
Monkey and human
As determined by immunogold procedure, the distribution of CaM during spermiogenesis in the monkey
485
Figs. 17-19. CaM labeling in rat spermatids ( x 13,500)
Fig. 17. Step 10 elongating spermatid showing gold particles in the
subacrosomal layer (A1 and perinculear ring (r).
Fig. 18. Step 16 maturing spermatid with CaM immunostaining
both in the subacrosomal layer ( A ) and postacrosomal sheath (PI.
Fig. 19. Step 19 spermatid (spermiation) with predominant labeling
of the postacrosomal sheath (PI. Note the light labeling of the perforatorium (pe).
(Figs. 20-23) and in human (Figs. 24-27) was quite
similar to that described in other species. A diffuse
labeling was present in round spermatids. In elongat-
486
M.-L. KANN
Figs. 20-23. CaM labeling in monkey spermatids and sperm
(20,000).
Fig. 20. Step 6 round spermatid. Some particles are already detected
in the subacrosomal layer (A).
Fig. 21. Step 13 early maturing spermatids, immunostaining of the
subacrosomal layer (A), postacrosomal sheath (PI, and perinuclear
ring (r).
Figs. 24-27. CaM in human spermatids and sperm (20,000).
Figs. 24,25. Early and late elongating spermatids. Labeling of the
subacrosomal layer (A1 and perinuclear ring (r).
Figs. 26,27. Ejaculated spermatozoa. Both the subacrosomal layer
and the postacrosomal sheath (P)are immunostained in this mature cell (Fig. 26);only the subacrosomal layer (A) is labeled in this
immature cell (Fig. 271.
(-1
Figs. 22,23. Epididymal sperm. Gold particles are found almost exclusively in the postacrosomal sheath (Fig. 22); no labeling in control
section using CaM preabsorbed antibody (Fig. 231.
ing spermatids, gold particles were concentrated in the
subacrosomal layer. The perinuclear ring was also labeled. Then, gold particles extended to the postacrosoma1 sheath in maturing spermatids. In monkey testicular and epididymal sperm, a dense CaM labeling was
restricted to the postacrosomal region (Fig. 22). In contrast, in human testicular and ejaculated sperm, such a
predominant labeling of the postacrosomal region was
seldom observed. In most sperm, the perinuclear substance remained entirely labeled (Fig. 261, although in
CALMODULIN IN SPERMATIDS A N D SPERMATOZOA
some immature cells the labeling did not extend to the
postacrosomal area (Fig. 27).
DISCUSSION
As visualized by immunogold labeling, a predominant CaM location in the post-acrosomal sheath of
sperm head was the most stricking feature in the six
species studied in this work. The presence of CaM in
this region has been already observed in sperm of the
guinea-pig, rabbit, and hamster using IIF (Jones e t al.,
1980; Moore and Dedman, 1984) and in that of the bull
and ram using immunoelectron microscopy (Weinman
et al., 1986a,b). Other CaM locations have been reported in the sperm head. Thus, the acrosomal region
was often labeled using fluorescent probes (Jones et al.,
1980; Feinberg e t al., 1981; Moore and Dedman, 1984).
In addition, CaM was detected between the plasma
membrane and the outer acrosomal membrane in the
guinea-pig (Yamamoto, 1985) and boar sperm (Camatini et al., 19861, as well as in the acrosome of boar
sperm (Camatini et al., 1986) using immunogold procedures. At first sight these different results seem difficult to reconcile. They may be due at least in part to
species differences. However it must be emphasized
that IIF and immunoelectron microscopy certainly
have different sensivity and accuracy. To give an example IIF did not allow to distinguish between an acrosomal or a subacrosomal CaM location. Thus various
acrosomal CaM locations previously reported in sperm
of different species could correspond to the presence of
CaM in limited areas of the subacrosomal layer as observed here.
In the present work, CaM was not detected in sperm
flagellum. In contrast, the presence of this protein was
previously reported in different regions and structures
of the sperm tail using IIF and immunoelectron microscopy (Jones et al., 1980; Moore and Dedman, 1984; Yamamoto, 1985; Weinman et al., 1986a,b; Camatini et
al., 1986).Perhaps the sensitivity of the postembedding
immunogold method used here was not sufficient to
detect CaM usually present in low concentration in
sperm tail (Feinberg et al., 1981). Up to now, there is
no agreement between the immunocytochemical results and those obtained using biochemical methods
(for review, see Tash, 1989). To conclude, the role of
CaM in flagellar motility has been demonstrated but
the problem of its location is not yet solved. As stated
above the discrepancies between the various CaM locations are not clearly explained and i t cannot be
claimed that CaM has been detected in all its locations
in the sperm of the six species studied here. Rather to
discuss extensively this point it seems more important
to emphasize the common CaM accumulation in the
postacrosomal region of sperm.
During spermiogenesis, CaM accumulated successively in the subacrosomal layer and in the postacrosoma1 sheath of spermatids. A CaM labeling of the perinuclear ring was also a common feature. The uniform
CaM distribution in the PNS changed to a predominant
location in the postacrosomal sheath of the testicular
sperm at spermiation. Thus, CaM location in testicular
sperm was identical to that found in epididymal sperm.
The only difference among species came from the presence of CaM in restricted areas of the subacrosomal
layer. Similar changes in CaM distribution during dif-
487
ferentiation and maturation of ram sperm have been
previously observed by Weinman et al. (1986b). In addition, these authors have suggested t h a t CaM left the
acrosome to reach the perinuclear substance. However,
such a transfer between these two compartments was
not seen in the present work.
CaM present in different regions of spermatids and
sperm may serve several functions. For the first time,
CaM was observed in the perinuclear ring. This structure has been considered as a special type of microtubule organizing center (Brinkley, 1985) from which the
manchette develops during the elongation phase. The
presence of CaM in this site suggests a role of this
protein as calcium mediator in microtubule assemblydisassembly, as proposed for the mitotic spindle (Welsh
e t al., 1979).
The PNS is considered the main cytoskeletal element
of the sperm head involved in the association of the
acrosome to the nucleus and in shape changes during
spermiogenesis (Longo e t al., 1987). Among various
proteins detected in the PNS of spermatids, F-actin is a
consistent component which would be involved in nuclear changes (Fouquet e t al., 1989b). The present
results showed that CaM also is a component of the
subacrosomal layer of elongating and maturing spermatids. In testicular sperm, subacrosomal F-actin was
depolymerised to G-actin (for review, see Fouquet et
al., 1990), whereas CaM was redistributed mainly in
the postacrosomal region. As reported during hamster
spermiogenesis (Gravis, 1979; Ruknudin e t al., 1988),
the distribution of calcium in the PNS followed a pattern similar to that described for CaM. Taken together,
these results suggest that during spermiogenesis the
aggregation state of subacrosomal actin might be controlled by CaM either by decreasing the level of free
Ca2 or by activating regulatory proteins of F-actin
(Means and Dedman, 1980). In sperm, a common role
for Ca2+-CaM and actin interactions cannot be proposed. In boar, CaM and G-actin were colocalized near
the acrosome equatorial segment (Camatini and
Casale, 1987). In rabbit, CaM location in the subacrosoma1 bulges and postacrosomal sheath coincided strictly
with the G-actin distribution described by Camatini et
al. (1987). In contrast, in the sperm head of hamster,
rat, monkey, human (Fouquet e t al., 1989a; Fouquet et
al., 19901, and in mouse and ram (unpublished data),
actin could not be visualized in the CaM-rich postacrosomal region.
In the sperm head, CaM was considered to play a role
during capacitation, acrosome reaction, and fertilization (Jones et al., 1980; Moore and Dedman, 1984;
Weinman et al., 1986a,b; Camatini et al., 1986; Aitken
et al., 1988; Leclerc et al., 1990). A universal role for
CaM still present in the subacrosomal layer appears
unlikely because its location varied from species to species. In contrast, the unique location of CaM in the
postacrosomal sheath suggests a fundamental function
for this protein. Sperm-egg fusion occurs close to the
postacrosomal region and the primary trigger for egg
activation is a detonation C a 2 + ,which might arise, at
least in part, from sperm (Yanagimachi, 1988). The
high level of Ca2' evidenced in the sperm postacrosoma1 region in different species [Fain-Maurel and
Dadoune, 1979; Gravis, 1979; Plummer and Watson,
1985; Ruknudin et al., 1988) and the presence of CaM,
+
488
M.-L. KANN
the ubiquitous Ca2 -modulator, reinforce this hypothesis. Work is now in progress to determine the fate of
CaM during the early events of fertilization and the
first results will be presented elsewhere (submitted for
publication).
+
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:
93-111.
Altman, L.G., B.G. Schneider, and D.S. Papermaster 1984 Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy. J . Histochem. Cytochem., 32t1217-1223.
Autric, F., C. Ferraz, M.C. Kilhoffer, J.C. Cavadore, and J.G. Demaille 1980 Large-scale purification and characterization of
calmodulin from ram testis: Its metal-ion-dependent conformers.
Biochim. Biophys. Acta, 631:139-147.
Brinkley, B.R. 1985 Microtubule organizing centers. Annu. Rev. Cell.
Biol., It145-172.
Camatini, M., G. Anelli, and A. Casale 1986 Immunocytochemical
localization of calmodulin in intact and acrosome-reacted boar
sperm. Eur. J. Cell Biol., 41t89-96.
Camatini, M., and A. Casale 1987 Actin and calmodulin coexist in the
equatorial segment of ejaculated boar sperm. Gamete Res., 17:
97-105.
Camatini, M., A. Casale, and M. Cifarelli 1987 Immunocytochemical
identification of actin in rabbit spermiogenesis and spermatozoa.
Eur. J . Cell Biol., 45t274-281.
Carlemalm, E., R.M. Garavito, and W. Villiger 1982 Resin development for electron microscopy and an analysis of embedding at low
temperature. J. Microsc., 126r123-143.
Clermont, Y. 1954 Cycle de l’epithelium seminal et mode de renouvellement des spermatozoides chez le hamster. Rev. Can. Biol.,
13t208-245.
Clermont, Y. 1969 Two classes of spermatogonial stem cells in the
monkey (Cercopithecus aethiops). Am. J. Anat., 126t57-72.
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., 3:l-11.
Feinberg, J.M.F., J.S. Weinman, S.J. Weinman, M.P. Walsh, M.C.
Harricane, J. Gabrion, and J.G. Demaille 1981 Immunocytochemical and biochemical evidence for the presence of calmodulin in bull sperm flagellum: Isolation and characterization of
sperm calmodulin. Biochim. Biophys. Acta, 673t303-311.
Fouquet, J.P., M.L. Kann, and J.P. Dadoune 1989a Immunogold distribution of actin during spermiogenesis in rat, hamster, monkey
and human. Anat. Rec., 223t35-42.
Fouquet, J.P., M.L. Kann, J.L. Courtens, and L. Ploen 198913 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, 22t291-300.
Gordon, M., E.G. Morris, and R.S. Young 1983 The localization of
Ca” -ATPase and Ca2+-binding proteins in the flagellum of
guinea pig sperm. Gamete Res., 8.49-55.
Gravis, C.J. 1979 Cytochemical localization of cations in the testis of
the Syrian hamster, utilizing potassium-pyroantimonate. Am. J.
Anat., 154t245-266.
Holstein, A.F. 1976 Ultrastructural observations in the differentiation of spermatids in man. Andrologia, 8t157-165.
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., 77.2772-2716.
Kann, M.-L., and J.-P. Fouquet 1989 Comparison of LR white resin,
Lowicryl K4M and epon postembedding procedures for immunogold staining of actin in the testis. Histochem., 91t221-226.
Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nature, 227t680-685.
Lagace, L., J.G. Chafouleas, R. Trejo, G. Delhumeau-Ongry, and J.R.
Dedman 1981 Colmodulin levels during rat testis development
and intracellular localization in isolated cells. Biol. Reprod., 22:
53A.
Leblond, C.P., and Y. Clermont 1952 Spermiogenesis of rat, mouse,
hamster and guinea pig as revealed by the “periodic acid fuchsin
sulfurous acid” technique. Am. J. Anat., 90:167-215.
Leclerc, P., M.A. Sirard, J.G. Chafouleas, and R.D. Lambert 1990
Decreased binding of cnlmodulin to bull sperm proteins during
heparin-induced capacitation. Biol. Reprod., 42t483-489.
Longo, F.J., G. Krohne, and W.W. Franke 1987 Basic proteins of the
perinuclear theca of mammalian spermatozoa and spermatids: A
novel class of cytoskeletal elements. J. Cell Biol., 105t1105-1120.
Means, A.R., and J.R. Dedman 1980 Calmodulin an intracellular calcium receptor. Nature, 285t73-77.
Moore, P.B., and J.R. Dedman 1984 Calmodulin, a calmodulin acceptor protein, and calcimedins: Unique antibody localizations in
hamster sperm. J . Cell Biochem., 25:99-107.
Oakberg, E.F. 1956 A description of spermiogenesis in the mouse and
its use in analysis of the cycle of the seminiferous efiithelium and
germ cell renewal. Am. J. Anat., 99t391-413.
Ploen, L. 1971 A scheme of rabbit spermateleosis based upon electron
microscopical observations. Z. Zellfosch, 115:553-566.
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.,
75t255-263.
Ruknudin, A., J.P. Dadoune, and LA. Silver 1988 Intracellular calcium in hamster spermatozoa in testis, epididymis and during the
acrosome reaction. Annu. Reprod. Sci., 16r145-153.
Tash, J.S. 1989 Protein phosphorylation: The second messenger signal
transducer of flagellar motility. Cell Mot. Cytoskeleton, 14t332339.
Timms, B.G. 1986 Postembedding immunogold labeling for electron
microscopy using “LR white” resin. Am. J. Anat., 175.267-275.
Towbin, H., T. Staehin, and J . Gordon 1979 Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some application. Proc. Natl. Acad. Sci. U.S.A., 76:
4350-4354.
Van Eldik, L.J., and D.M. Watterson 1981 Reproducible production of
antiserum against vertebrate calmodulin and determination of
the immunoreactive site. J. Biol. Chem., 256t4205-4210.
Van Eldik, L.J., and S.R. Wolchok 1984 Condition for reproducible
detection of calmodulin and S l O O in immunoblots. Biochem. Biophys. Res. Comm., 124t752-759.
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., 34:
1171-1179.
Weinman, S., C. Ores-Carton, F. Escaig, J. Feinberg, and S. Puszkin
198613 Calmodulin immunoelectron microscopy: redistribution
during ram spermatogenesis and epididymal maturation. 11. J.
Histochem. Cytochem., 34r1181-1193.
Welsh, M.J., J.R. Dedman, B.R. Brinkley, and A.R. Means 1979 Tw
bulin and calmodulin: Effects of microtubule and microfilament
inhibitors on localization in the mitotic apparatus. J. Cell Biol.,
81t624-634.
Yamamoto, N. 1985 Immunoelectron microscopic localization of calmodulin in guinea pig testis and spermatozoa. Acta Histochem.
Cytochem., 18t199-211.
Yanagimachi, R. 1988 Mammalian fertilization. In: Physiology of Reproduction. E. Knobil, J. Neil, eds. New York: Raven Press, Vol
11, pp. 135-185.
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species, structure, mammalia, perinuclear, spermatids, localization, six, comparison, spermatozoa, calmodulin
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