Molecular Human Reproduction vol.4 no.12 pp. 1122–1129, 1998
γ-tubulin during the differentiation of spermatozoa in various
mammals and man
J.-P.Fouquet1,4, M.L.Kann1, C.Combeau2 and R.Melki3
1Laboratoire de Biologie Cellulaire, Groupe Spermatogenèse et Maturation du Spermatozoı̈de, Université Paris V,
UFR Biomédicale, 45 rue des Saints-Pères, 75270 Paris cedex 06, 2Rhône Poulenc Rorer, Centre de recherche de Vitry,
13 Quai Jules Guesdes 94403 Vitry-sur-Seine, and 3Laboratoire d’Enzymologie et Biochimie Structurales, CNRS,
91198 Gif sur Yvette Cedex, France
4To
whom correspondence should be addressed
The distribution of γ-tubulin as a marker of microtubule organizing centres (MTOC) was studied during
spermiogenesis in rodents and in rabbit, monkey and man. A polyclonal antibody directed against human
γ-tubulin was used both for indirect immunofluorescence (IIF) and post-embedding immunogold procedures.
In all species, γ-tubulin was detected in the proximal and distal centrioles of round spermatids. In elongating
spermatids, γ-tubulin was predominantly found in the pericentriolar material (PCM) of both centrioles and
particularly around the adjunct of the proximal centriole. At this level, some labelling was also associated
with manchette microtubules, but other parts of the manchette and the nuclear ring were never labelled. We
propose a role for distal centriole γ-tubulin in axoneme nucleation and centriolar adjunct γ-tubulin in manchette
nucleation. The disappearance of γ-tubulin in mature spermatozoa indicates that sperm aster nucleation
should be dependent on oocyte γ-tubulin. Remnants of γ-tubulin in some human spermatozoa suggest that
paternal γ-tubulin also could contribute to sperm aster formation.
Key words: γ-tubulin/centrioles/MTOC/spermatid/spermatozoa
Introduction
In the majority of animal cells the microtubule cytoskeleton
is organized by the centrosome, a microtubule organizing centre
(MTOC) which consists of a pair of centrioles surrounded by
fibrous, and/or amorphous pericentriolar material (PCM). This
PCM is responsible for cytoplasmic microtubule nucleation.
In ciliated cells, the basal bodies found at the subapical cortex
are similar to centrioles. These MTOC nucleate cytoplasmic
microtubules, in addition to their role in the morphogenesis
of cilia, i.e. template elongation (Joshi, 1993, 1994). The
microtubule nucleating activity of MTOC(s) requires the presence of γ-tubulin which is mainly associated with PCM (Stearns
et al., 1991; Shu and Joshi, 1995; Zheng et al., 1995). Other
proteins could also be involved in microtubule nucleation
(Tassin et al., 1997). In mammals, gametes possess considerably modified MTOC. Oocytes are devoid of centrioles but still
contain PCM with microtubule nucleating activity (Wassarman
and Albertini, 1994). During sperm differentiation, the socalled distal centriole elongates to form axonemal microtubules
but ultimately disintegrates. In mature spermatozoa of human
beings and domestic mammals the proximal centriole is still
present (de Kretser and Kerr, 1994; Schatten, 1994) whereas
in rodents, as originally described for the rat (Woolley and
Fawcett, 1973), this centriole also disintegrates at the end of
spermiogenesis and/or during epididymal transit (de Kretser
and Kerr, 1994). In fertilized mouse oocytes, centrioles are
1122
detected for the first time at the 64-cell stage of development
thus indicating that centrioles and γ-tubulin rich MTOC are
maternally inherited (Gueth-Hallonet et al., 1993). This applies
to the hamster and probably other rodents (Hewitson et al.,
1997). On the other hand, in domestic mammals and in humans,
the proximal centriole of spermatozoa forms a spermaster in
the fertilized oocyte from which the first centriolar mitotic
spindle originates (Crozet, 1990; Sathananthan et al., 1991,
1996; Schatten, 1994; Navara et al., 1995). Thus in these
species, zygote centrioles are of paternal inheritance but their
microtubule nucleating ability is considered to be dependent
on the maternal γ-tubulin, already present in the oocyte before
fertilization and which should be attracted by the sperm
centriole (Navara et al., 1995). Although this sophisticated
model appears attractive, it has to be noted that the presence
and distribution of γ-tubulin during spermiogenesis has not
been investigated either in mice or in man or other mammals.
Another reason to study the transition of spermatid into
spermatozoa is that these cells (in addition to the conventional
MTOC, i.e. centrosome), develop a manchette, a unique set
of microtubules associated with a potential unique MTOC, the
nuclear ring (Brinkley, 1985).
We report here on the expression and localization of
γ-tubulin, in differentiating spermatids of various mammalian
species, using immunoelectron microscopy. In rodents, rabbit,
monkey and man the γ-tubulin was found to be associated
with centrioles and PCM of spermatids but not with the nuclear
© European Society of Human Reproduction and Embryology
γ-tubulin in mammalian spermatozoa
ring. In all species, mature spermatozoa appeared to be
essentially devoid of γ-tubulin.
Materials and methods
Animals
Testes and epididymides of sexually active mice, rats, hamsters,
guinea-pigs, rabbits and monkeys (Macaca fascicularis) were removed
under anaesthesia. Mouse and rat samples were used for both indirect
immunofluorescence (IIF) and immunoelectron miscroscopy (IEM).
Testes and epididymal spermatozoa of hamster, guinea pig and
monkey as well as testicular biopsies and ejaculated spermatozoa
from human donors were used only for IEM.
Other biological samples
HeLa cells were grown in Chamber Slides (Lab-tek) and used at
70% confluence for electrophoresis, immunoblotting and IIF. A
lymphoblastic KE 37 cell line was used for the isolation of centriole
pellets according to Bobinnec et al. (1998). In addition, lungs of
mouse and rat were used as selected material for the presence of
ciliated cells and basal bodies. Centrioles and basal bodies were
investigated as positive material to validate IEM detection of γ-tubulin.
Antibody directed to human γ-tubulin
Human γ-tubulin was expressed and purified as described in Melki
et al. (1993). Recombinant γ-tubulin, purified by sodium dodecyl
sulphate (SDS)–gel electrophoresis from inclusion bodies, was
injected into rabbits together with Freund’s adjuvant. Five booster
injections were given at intervals of 2 weeks.
The sensitivity of the antiserum was tested by the use of purified
recombinant γ-tubulin. The antibody thus generated was shown to
detect as much as 25 pg of γ-tubulin using immunoblots prepared
with known quantities of purified recombinant γ-tubulin probed by a
polyclonal anti-γ-tubulin antibody and detected by an enzyme-linked
chemiluminescence technique (ECL kit; Amersham, Amersham, UK).
The specificity of the antiserum was tested by the use of HeLa
cell extract. Cells were harvested and lysed by sonication and proteins
were separated by polyacrylamide gel electrophoresis (Laemmli,
1970) and transferred to nitrocellulose sheets. Immunodetection of
γ-tubulin was performed using the ECL technique (Amersham),
according to the manufacturer’s recommendations.
The antiserum immunoglobulin (Ig)G fraction was immunopurified
by the use of a Protein A Sepharose CL-4B (Pharmacia, Uppsala,
Sweden) column. The antiserum was incubated with the affinity
chromatography gel overnight at 4°C. The resin was then washed in
20 mM Na2HPO4, pH 7.0 and the IgG eluted in 0.1 M citric acid,
pH 3.0. After adjusting the pH at 7.5 by addition of Tris–HCl pH
9.5, the purified IgG(s) were divided into aliquots and stored at –20°C.
Indirect immunofluorescence
HeLa cells grown on slides and smears of isolated testicular cells
and epididymal spermatozoa from mouse and rat were isolated as
previously described (Fouquet et al., 1994; Kann et al., 1995). All
samples were prepared for IIF according to the simplest procedure.
Briefly, the cells were fixed-permeabilized either in ethanol or in
acetone (10 min at –20°C), incubated for 20 min at room temperature
in phosphate-buffered saline containing 0.2 % bovine serum albumin
(PBS–BSA) then successively incubated for 1 h with immunopurified
IgG directed to γ-tubulin then with anti-rabbit IgG fluorescein
isothiocyanate (FITC) conjugate (Amersham or Sigma/Aldrich Chimie
France), each diluted in PBS–BSA. Controls were performed by
omitting the primary antibody or by pre-adsorbing this antibody with
the immunogenic γ-tubulin at a molar ratio of 1:10. The preparations
were mounted in Aquamount (Lerner Laboratories, New York, NY,
USA) and examined on a Zeiss microscope equipped with epifluorescence optics.
Immunoelectron microscopy
As previously reported for tubulin isoforms detection in spermatozoa
(Prigent et al., 1996; Fouquet et al., 1997), all samples were
fixed for 1 h in 0.1 M cacodylate buffer, pH 7.3, containing 1%
glutaraldehyde and embedded in Lowicryl K4M. Thin sections
collected on uncoated nickel grids were incubated for 2 h with
immunopurified IgG directed to γ-tubulin then with the secondary
antibody conjugated to 10 nm gold particles (G–1021; Sigma/Aldrich
Chimie France). The antibodies were diluted in Tris-buffered saline
(TBS) pH 7.9 containing 0.2% bovine serum albumin (Fraction V,
IBF). Controls were as described above for IIF. The grids were
contrasted with saturated aqueous uranyl acetate before observation.
Results
Specificity of the antibody directed to γ-tubulin
The specificity of the polyclonal antibody to human γ-tubulin
was evaluated using both Western blot and IIF experiments.
Figure 1a, shows that the antibody directed to γ-tubulin
recognized a single band that has a molecular mass consistent
with that of γ-tubulin (47 kDa) in HeLa cell extracts. Since
γ-tubulin has been shown to be one conserved component of
the MTOC in eukaryotic cells we used our antibody to γtubulin to determine the localization of this protein in HeLa
cells by IIF. Immunostaining of HeLa cells with the immunopurified IgG revealed the centrosome in interphase (Figure 1b)
and the spindle poles in mitosis (not shown). No labelling was
observed (Figure 1c) when the immunopurified IgG(s) were
preincubated with recombinant γ-tubulin for 2 h at room
temperature. We conclude from these results that the polyclonal
antibody we generated is specific of γ-tubulin and that it does
not cross react with other HeLa cell proteins. The following
step was to test our IEM protocol using as models the two
major MTOC known to contain γ-tubulin i.e. centrosomes and
basal bodies. As shown in Figure 2a,c, γ-tubulin could be
detected both in PCM, centriolar triplets and/or centriolar
lumen at any level, proximal or distal, of isolated centrosomes.
The specificity of these labellings was confirmed by the fact
that they were not detected when either the gold secondary
antibody was used alone or the primary antibody was preincubated with the γ-tubulin immunogen (Figure 2b). Similarly the
observations of sections of lung ciliated cells showed a
γ-tubulin immunostaining at the periphery of the basal bodies
more specifically associated with basal feet (Figure 2d) which
are considered to be the actual MTOC (Sandoz et al., 1988).
IIF of γ-tubulin in rodent spermatids and spermatozoa
Smears of rat and mouse germ cells were used for this
investigation. In these conditions, small fluorescent dots, suggesting a centriole staining, were often difficult to recognize
in round cells the identification of which also was difficult.
By contrast, in elongating spermatids an unique staining of
the centriolar region was detected. In these cells, the manchette
and the nuclear ring were not stained (Figure 3a,a9). The
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J.-P.Fouquet et al.
Figure 2. Immunogold localization of γ-tubulin in (a), (b),
(c) isolated centrioles and (d) lung ciliated cells. (a) cross section
of the distal part of a centriole; (b) similar cross section to negative
control; (c) longitudinal section of a centriole; (d) labelling of basal
feet (〈〈) of basal bodies. Scale bars 5 0.1 µm.
Figure 1. HeLa Cells γ-tubulin. (a) Immunodetection by Western
blot: lane 1, Coomassie Brilliant Blue staining of an 8.5% sodium
dodecyl sulphate (SDS) polyacrylamide gel of whole extracts; lane
2, immunoblot with anti-γ-tubulin to a nitrocellulose membrane of a
lane identical to that shown in lane 1. Molecular weight markers
are indicated at the left. (b) Indirect immunofluorescence (IIF) of
cultured cells with anti-γ-tubulin. (c) IIF negative control with 49,6diamidino-2-phenylindole (DAPI) staining. Scale bars 5 10 µm.
centriolar labelling was still observed during most of the
maturation process of spermatids (Figure 3b,b9). However, in
older spermatids, i.e. testicular spermatozoa with a fully
differentiated midpiece the γ-tubulin signal was no longer
detected (Figure 3c,c9).
IEM of γ-tubulin in rodent spermatids and
spermatozoa
The distribution of γ-tubulin observed during spermiogenesis
was similar in all rodent species studied here: mouse, hamster,
rat and guinea-pig. Therefore the following description is
1124
illustrated as a composite plate (Figure 4), made of pictures
of spermatids from these different species.
In early round spermatids the γ-tubulin immunolabelling
was found in the centrosome, more specifically in the centrioles,
i.e. the microtubular triplets and/or the centriolar lumen, but
not in any PCM (Figure 4a). In late round spermatids when
the pair of centrioles was at the caudal pole of the nucleus
opposite to the acrosome, the distal centriole had already
developed a naked axoneme. At this stage, γ-tubulin was
detected in the PCM rather than in the distal centriole itself.
During the same period, the proximal centriole was developing
a mini-flagellum the so-called centriolar adjunct which showed
a very light γ-tubulin labelling. Then, dense material accumulated around the tip of the centriolar adjunct. This PCM-like
material simultaneously began to be labelled with the anti
γ-tubulin IgG (Figure 4b,c).
The elongation phase of spermatids is characterized by the
development of a transient microtubular manchette encasing
the caudal region of the nucleus. This cone-shaped sheath of
microtubules is oriented so that its extremity surrounds the
centriolar area whereas its base ends abruptly in the nuclear
ring at the limit of the acrosome near the equatorial region of
the nucleus. From the beginning to the end of the elongation
phase, γ-tubulin was not detected either in the nuclear ring or
in the major part of the manchette (Figure 4d). In contrast,
the γ-tubulin labelling was predominant in the PCM of both
centrioles and particularly around the centriolar adjunct of the
proximal centriole (Figure 4d–g). In the same region which is
γ-tubulin in mammalian spermatozoa
spermatids the PCM and the γ-tubulin associated labelling had
disappeared concomitant with the disintegration of the distal
and proximal centriole successively. Thus the neck region of
testicular and epididymal spermatozoa appeared devoid of
γ-tubulin whereas PCM-like bodies and γ-tubulin associated
labelling were found in the residual bodies (Figure 4i).
IEM of γ-tubulin in rabbit, monkey and human
spermatids and spermatozoa
The distribution of γ-tubulin was also studied during rabbit,
monkey and human spermiogenesis because in these species,
in contrast with rodents, mature spermatozoa still possess a
proximal centriole. In fact the observations described for
rodents also apply to the rabbit and primates. Therefore only
the most important features will be reported below. Thus, in
early round spermatids γ-tubulin was located in the centrioles
but not in any PCM (Figure 5a). From older round spermatids
to maturing spermatids the γ-tubulin was also detected in the
PCM of both centrioles (Figure 5b–h) but it was hardly
detected in the neck of testicular spermatozoa. In older round
spermatids, the changes of the proximal centriole were growth
of the centriolar adjunct without detectable γ-tubulin (Figure
5e) followed by setting of the PCM and associated γ-tubulin
around this centriolar adjunct (Figure 5g). In elongating and
maturing spermatids γ-tubulin was not detected in the nuclear
ring (data not shown). The manchette also appeared essentially
devoid of γ-tubulin except in its tip region around the pericentriolar area (Figure 5i). In late maturing spermatids, the PCM
was dispersed and the γ-tubulin associated labelling had
vanished. Moreover, in epididymal rabbit and monkey spermatozoa (not shown) or in human ejaculated spermatozoa
γ-tubulin was not detected in the remnants of the distal centriole
(Figure 5j,k). Trace amounts of γ-tubulin could be detected in
the proximal centriole of ~5% human ejaculated spermatozoa
(Figure 5j) and even in some centriolar adjunct-PCM remnants
but in ,1% of those cells (Figure 5k).
Discussion
Figure 3. Indirect immunofluorescence (IIF) of rat isolated germ
cells with anti γ-tubulin immunoglobulin (Ig)G. (a–c), fluorescence;
(a’–c’) phase contrast. (a) Elongating spermatid (→); (b) maturing
spermatids; (c) testicular spermatozoa with background staining of
the mitochondrial sheath. Scale bars 5 10 µm.
surrounded by the tip of the manchette some labelling was
found in association with microtubules (Figure 4g).
The maturation phase of spermatids begins with the backward movement of both the nuclear ring and the manchette.
At this stage, there was still intense γ-tubulin labelling in the
PCM of both centrioles which began to disperse. This was
particularly evident at the level of the centriolar adjunct
(Figure 4h). The dispersion of the PCM was confirmed during
the following steps of the maturation phase of spermatids, i.e.
the depolymerization of the manchette, the formation of the
mitochondrial helix of the middle piece and the regression of
the centriolar adjunct. At the end of the maturation phase of
Since the beginning of this decade tubulin has been shown to
be involved in microtubule nucleation (Joshi, 1994). The
distribution of this molecule, as a marker of MTOC, has been
investigated using immunoelectron microscopy. In permeabilized cells, before embedding (pre-embedding protocol),
γ-tubulin was found only in PCM (Stearns et al., 1991; Felix
et al., 1994). More recently, in isolated centrioles investigated
after embedding (post-embedding protocol) γ-tubulin was
detected both in the centrioles themselves and in their PCM
(Moudjou et al., 1996). Whether these different localizations
actually exist or are related to protocol differences, i.e. fact or
artefact, remains to be determined. In the present work, this
dual localization was confirmed not only in isolated centrioles
as reported above but also in situ using sections of intact cells
including spermatids and also in other germ cells (spermatogonia) and Sertoli somatic cells (data not shown). Moreover,
the γ-tubulin labelling was initially present only in centrioles
and then it became predominant in the PCM during spermiogenesis. These changes suggest a possible redistribution of
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J.-P.Fouquet et al.
Figure 4. γ-tubulin immunogold distribution in rodent spermatids. (a) Distal centriole in a mouse early round spermatid. (b) and (c)
Centriolar adjunct (*) in hamster late round spermatids. (d) Mouse elongating spermatid : nr 5 nuclear ring; m 5 manchette; centriolar
adjunct (*). (e) Centriolar adjunct (*) and distal centriole (→) in rat elongating spermatid. (f) Both centrioles in a mouse elongated
spermatid. (g) Cross section at the tip of the centriolar adjunct (*) in a mouse elongating spermatid : m 5 manchette. (h) Both centrioles,
pericentriolar material (PCM) and γ-tubulin dispersion in a mouse maturing spermatid. (i) γ-tubulin labelling in a residual body of an
hamster spermatozoon. Scale bars 5 0.1 µm.
γ-tubulin during spermatid differentiation as well as its elimination in residual bodies of mature spermatozoa at spermiation.
Such changes are reminiscent of those observed during the
cell cycle of HeLa cells (Moudjou et al., 1996). Thus, in spite
of the fact that centrioles are made of stable microtubules with
a predominance of both α acetylated and α-β glutamylated
tubulin isoforms (Fouquet et al., 1994; Bobinnec et al., 1998),
centrioles and their PCM are dynamic structures. The dual
distribution of the γ-tubulin could indicate two different abilities
of microtubule nucleation. PCM γ-tubulin is considered to be
involved in the nucleation of cytoplasmic microtubules (Joshi,
1126
1994). Centriolar γ-tubulin must be involved in the unknown
functions of the centriole; perhaps its nucleation/duplication,
or triplet stabilization (Moudjou et al., 1996) and even the
growth of an axoneme. Obviously there is a need to explore the
role of the γ-tubulin and other centriolar and/or pericentriolar
molecules working for these unique functions.
In this work, we were interested in determining the fate of
MTOC and their associated γ-tubulin in mammalian spermatids
during their differentiation into mature spermatozoa. When
young round spermatids arose from meiosis these haploid cells
contained a pair of centrioles with γ-tubulin similar to other
γ-tubulin in mammalian spermatozoa
Figure 5. γ-tubulin immunogold distribution during spermiogenesis in rabbit, monkey and man. (a) Centrioles in rabbit early round
spermatid. (b) Labelled pericentriolar material (PCM) of the distal centriole in rabbit late round spermatid. (c) remnants of labelled PCM in
rabbit late maturing spermatid. (d) and (e) Monkey late round spermatid : both centrioles in (d), centriolar adjunct (*) in (e). (f) and (g)
Human early elongating spermatids: labelled PCM around distal centriole (f) and centriolar adjunct (*) of proximal centriole (g). (h) and (i)
Human early maturing spermatids: centriolar adjunct (*), remnants of the manchette (m) and γ-tubulin associated labelling (i). (j) and (k)
Human ejaculated spermatozoa with remnants of centriolar adjunct (*). Scale bars 5 0.1 µm.
somatic cells. At the end of spermiogenesis, testicular spermatozoa are either devoid of both centrioles (as is the case in
rodents) or still contain the proximal centriole (as observed
in domestic mammals and primates). Whatever the species,
spermatozoa appeared practically devoid of γ-tubulin. Thus,
these results support the recently proposed hypothesis
(Schatten, 1994; Navara et al., 1995; Simerly et al., 1996)
that, in all studied mammals, the oocyte might be the only
source of γ-tubulin available for spindle microtubule nucleation
as the zygote begins to develop. The only apparent role of the
sperm centriole, when present, could be to attract the oocyte
γ-tubulin. This first step may be necessary for centriole
duplication and finally for sperm aster formation and restoration
of a centriolar spindle, as in somatic cells, from the first cell
cycle onwards. In this respect, it has been proposed that defects
in human sperm centrosomal constituents might cause novel
forms of male infertility (Sathananthan, 1991; Navara et al.,
1995; Simerly et al., 1996). However, in the mouse (GuethHallonet et al., 1993) and other rodents (Hewitson et al.,
1997), the restoration of centriolar organization of the zygote
remains unexplained since it occurs several cell cycles after
fertilization by a spermatozoon devoid of centriole. Finally,
even if there are two models of centriole inheritance, either
paternal or maternal, the microtubule nucleating ability of the
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J.-P.Fouquet et al.
zygote can be considered to be exclusively dependant on the
maternally inherited γ-tubulin. However, at least in man, a
blending of both maternal and paternal γ-tubulin in sperm
aster formation can be suspected, since a few spermatozoa
still contain γ-tubulin remnants.
Our data clearly demonstrate that mature mammalian
spermatozoa are devoid of γ-tubulin. In contrast, γ-tubulin was
present in the spermatid centrosome during most of spermiogenesis. Therefore, the question remains: what is the role
of this molecule in the spermatid differentiation? In round
spermatids, the most important event related to the microtubule
cytoskeleton is the growth of the naked flagellum arising from
the distal centriole. During this period the cell is acquiring its
specific polarity but there is no known network of cytoplasmic
microtubules and no detectable pericentriolar γ-tubulin. Rather,
the round spermatid is busy with tubulin dimer assembly into
the doublet microtubules of the axoneme. It is tempting to
speculate that the γ-tubulin detected at this stage in the distal
centriole could be involved in axoneme nucleation and/or
growth. In late round spermatids the proximal centriole
develops a centriolar adjunct which is quickly encircled with
a growing PCM and associated γ-tubulin at the beginning of
the elongation phase. This unique structure coincides with the
birth of the manchette. This cone-shaped sheath of microtubules
is considered to be involved in the shaping and elongation of
the spermatid nucleus (Fouquet and Kann, 1994). Up to now,
it has been proposed that manchette microtubules would
originate from the nuclear ring which would represent a
spermatid-specific MTOC (Brinkley, 1985). However, the
expected presence of γ-tubulin in the nuclear ring was not
verified. In contrast γ-tubulin was found exclusively near the
tip of the manchette in preferential association with the PCM
of the centriolar adjunct, i.e. at the opposite pole of the nuclear
ring. With the exception of some human spermatozoa, both
the centriolar adjunct, the PCM and the manchette disintegrate
concomitantly during the maturation phase of spermatids.
Taken together, these spatio–temporal relationships strongly
suggest that the centriolar adjunct might be the specific MTOC
of the spermatid manchette. In fact, this microtubule sheath
could be compared with a half-spindle originating at a modified
centrosome and ending abruptly at the nuclear ring. This, in
turn, could be compared with a giant kinetochore, a structure
known to capture spindle microtubules (Inoué and Salmon,
1995).
To conclude, we propose that in all mammals studied from
mice to men, spermatids contain γ-tubulin associated with a
single centrosome with a dual microtubule nucleating function:
the distal centriole might be the flagellar MTOC whereas the
PCM of the adjunct of the proximal centriole might be the
manchette MTOC.
Acknowledgements
We are indebted to Danielle Bligny for the excellent secretarial
assistance. The technical assistance of Annie Gonzales for tissue
embedding and sectioning and the photographic work of Eugenio
Prieto was appreciated. We thank Dr Mireille Kenigsberg for her help
in HeLa cells IIF. This work was supported by a grant from the
Association pour la Recherche sur le Cancer (to R.M.).
1128
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Received on May 15, 1998; accepted on September 15, 1998
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