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Cell Motility and the Cytoskeleton 41:341–352 (1998)
Expression of Glycylated Tubulin During the
Differentiation of Spermatozoa in Mammals
Marie-Louise Kann,1* Yann Prigent,1 Nicolette Levilliers,2 Marie-Hélène Bré,2
and Jean-Pierre Fouquet1
1Laboratoire
de Biologie Cellulaire, Spermatogenèse et maturation du
spermatozoı̈de, Université Paris V, U.F.R. Biomédicale, Paris, France
2Laboratoire de Biologie Cellulaire 4, U.R.A. 1134 C.N.R.S.,
Université Paris-Sud, Orsay, France
Using quantitative immunogold analyses of tubulin isoforms we previously
demonstrated a unique differential expression of glutamylated tubulin in the
flagellum of mouse and man spermatozoa [Fouquet et al., 1997: Tissue Cell
29:573–583]. We have performed similar analyses for glycylated tubulin using two
monoclonal antibodies, TAP 952 and AXO 49, directed to mono- and polyglycylated tubulin respectively. Glycylated tubulin was not found in centrioles and
cytoplasmic microtubules (manchette) of germ cells. In mouse and man, axonemal
tubulin was first monoglycylated and uniformly distributed in all doublets at all
levels of the flagellum in elongating spermatids. In human mature spermatozoa
axonemal microtubules were enriched in monoglycylated tubulin from the base to
the tip of the flagellum. In mouse sperm flagellum a similar gradient of monoglycylated tubulin was also observed in addition to an opposite gradient of
polyglycylated tubulin. In both species, monoglycylated tubulin labeling predominated in doublets 3-8 whereas glutamylated tubulin labeling [Fouquet et al., 1997]
predominated in doublets 1-5-6. These differential labelings were suppressed after
motility inhibition of mouse spermatozoa by sodium azide treatment and in
non-motile human spermatozoa lacking dynein arms. The unique distribution of
these tubulin isoforms and the known inhibition of motility induced by their
specific antibodies are consistent with a complementary role of tubulin glycylation
and glutamylation in the regulation of flagellar beating in mammalian spermatozoa. Cell Motil. Cytoskeleton 41:341–352, 1998. r 1998 Wiley-Liss, Inc.
Key words: axoneme; microtubules; glycylated tubulin; spermatozoa; immunogold
INTRODUCTION
In the mammalian testis, germ cell microtubules are
made of three major tubulin isotypes: m␤ 3 and m␣ 3/7
[Lewis and Cowan, 1988] and pRD␣TT1 [Hecht et al.,
1988; Simerly et al., 1993]. These isotypes are either
expressed as unmodified polypeptides or as posttranslationally modified isoforms. For ␣ tubulin the
identified modifications are the N-terminal acetylation at
lys 40, the detyrosylation of C-terminal and excision of
the penultimate glutamyl residue after detyrosylation [for
review see MacRae, 1997; Ludueña, 1998]. In addition,
both ␣ and ␤ tubulin can be subjected to polyglutamylation and polyglycylation. The glutamylation consists of
r 1998 Wiley-Liss, Inc.
the C-terminal addition, of one to six glutamate units at
glu 445 for ␣ tubulin [Eddé et al., 1990] and at glu 435 for
␤ tubulin [Redeker et al., 1992; Rüdiger et al., 1992]. The
glycylation consists of the C-terminal addition of three to
34 glycine units as originally reported in Paramecium
cilia [Redeker et al., 1994] or one to 23 glycine units in
the bull sperm flagellum [Plessmann and Weber, 1997] at
glu 445 for ␣ tubulin and at glu 437 for ␤ tubulin. During
*Correspondence to: Dr M.L. Kann, Laboratoire de Biologie Cellulaire, Université Paris V, U.F.R. Biomédicale, 45 rue des Saints-Pères,
75270 Paris cedex 06, France.
Received 9 July 1998; accepted 8 September 1998
342
Kann et al.
spermiogenesis the spermatid build up two microtubular
structures: the manchette, a transient bundle of microtubules encasing the posterior region of the nucleus, and
axonemal microtubules of the flagellum. In the flagellum
all post-translational modifications described above have
been characterized whereas in the manchette the only
reported modification is the detyrosylation of ␣ tubulin
[Fouquet et al., 1994, 1997].
Recently it has been demonstrated that among a
panel of site directed antibodies to tubulin only those
directed to glutamylated and glycylated tubulin were
potent inhibitors of flagellar motility [Bré et al., 1996;
Gagnon et al., 1996], and only those directed to glutamylated tubulin evidenced a differential labeling of the
mammalian sperm flagellum using quantitative immunoelectron microscopy [Fouquet et al., 1996, 1997; Prigent
et al., 1996]. Both a proximo-distal decrease of the
labeling and its predominance in axonemal doublets
1-5-6 corresponding to the plane of the flagellar wave
were reported. Together, these results have suggested that
glutamylated tubulin could be involved in a functional
heterogeneity of peripheral doublets of the sperm flagellum, i.e., flagellar beating regulation.
Since polyglycylated tubulin is also expressed in
the mammalian sperm flagellum and since glycylation
and glutamylation occur on the same and/or adjacent
C-terminal glutamate residues of ␣ and ␤ tubulin [Bré et
al., 1996; Plessmann and Weber, 1997], it appears that the
glycylation of flagellar microtubules also might be involved in their functional heterogeneity. In the present
work the distribution of glycylated tubulin was studied in
the sperm flagellum of various mammals by immunoelectron microscopy using two monoclonal antibodies, AXO
49 and TAP 952, directed to glycylated tubulin [Bré et al.,
1996].
MATERIALS AND METHODS
Biological Samples
Testes and epididymides of sexually active mouse
(Swiss) were removed under anesthesia. Mouse motile
epididymal spermatozoa were collected in phosphate
buffered saline (PBS), pH 7.4 at 37°C within 15 min
[Fouquet et al., 1994]. In some experiments motility was
inhibited with sodium azide 0.1% before further investigation. Testicular biopsies and ejaculates from human
donors were also used. Ejaculated spermatozoa were
obtained after an abstinence period of 2–4 days from 10
fertile donors assessed as normal from their spermogram
and spermocytogram; sperm counts were ⬎20 ⫻ 106/ml,
⬎50% of spermatozoa were motile and ⬍50% were
morphologically abnormal. In addition, non-motile ejaculated spermatozoa, lacking dynein arms, from two nonfertile donors were also studied.
Testicular samples of other species including hamster, rat, rabbit and monkey (Maccaca fascicularis) as
previously prepared [Fouquet et al., 1994] were also
investigated.
Antibodies
Two monoclonal antibodies, TAP 952 and AXO 49,
raised against axonemal tubulin of Paramecium [Callen
et al., 1994] and directed to glycylated tubulin [Bré et al.,
1996] were used. TAP 952 mAb recognizes monoglycylated tubulin bearing single glycine unit on one or
several carboxyterminal glutamate residues. AXO 49
mAb recognizes polyglycine chains from three residues
upwards [Bré et al., 1998]. Neither TAP 952 mAb nor
AXO 49 mAb recognizes biglycylated peptides.
Indirect Immunofluorescence (IIF)
As in a previous study [Kann et al., 1995] smears of
mouse epididymal spermatozoa and mechanically isolated testicular germ cells were fixed-permeabilized in
acetone (⫺20°C) before immunostaining. In some cases,
germ cell samples diluted in phosphate buffer saline
(PBS) were frozen (1 h, ⫺20°C) before the preparation of
smears to enhance antigen accessibility. Immunostainings
were performed for 45 min at room temperature with TAP
952 or AXO 49 mAb, then with FITC conjugated
anti-mouse IgG (ICN) diluted in PBS–0.2% bovine
serum albumin (BSA). The preparations were mounted in
PBS-glycerol (10/90, v/v) before epifluorescence observations.
Immunoelectron Microscopy (IEM)
All biological samples were fixed for 1 h in 1%
glutaraldehyde–0.1 M cacodylate buffer pH 7.3 and
embedded in Lowicryl K4M. Thin sections collected on
uncoated nickel grids were incubated for 2 h in different
antibodies followed by incubation in goat antimouse
secondary antibodies conjugated either to 10 nm (Sigma,
Aldritchchimie, France) or 15 nm (Biocell, U.K.) gold
particles. All antibodies were diluted in Tris buffered
saline (TBS) pH 7.9 containing 0.2% bovine serum
albumin. The sections were contrasted with aqueous
saturated uranyl acetate before electron microscope observations.
Quantitative Immunogold Analysis
The number of gold particles was determined per
cross-section of mouse and human sperm flagellum. Five
regions were considered: part I, middle piece; part II,
proximal part of the principal piece with 9-6 outer dense
fibres (ODF); part III, principal piece with 4-2 ODF; part
IV, principal piece devoid of ODF; part V, terminal piece.
The results are presented as the mean ⫾ SEM for
peripheral doublets, central pair and total microtubules
(central pair plus peripheral doublets) of the axoneme in
Sperm Glycylated Tubulin
343
Fig. 1. IIF of mouse epididymal spermatozoa, after freezing pretreatment, with TAP 952 (a) and AXO 49
(b) mAb, and corresponding phase contrast (a8, b8). Bar, 10 µm. ⫻640.
50 cross-sections per region. Gold particle counts were
also performed on individual peripheral doublets to
search for a possible differential labeling. As in a previous
report [Fouquet et al., 1997], three groups of doublets
were compared: 1-5-6, 3-8 and 2-4-7-9.
One-way analysis of variance was used for the
statistical determination of significant differences in the
labeling patterns of various parts of the flagellum; the
Duncan multiple range test was used for specific comparisons. The Wilcoxon test was used for doublet comparisons in each region of the flagellum.
RESULTS
IIF of Glycylated Tubulin During Mammalian
Spermiogenesis
IIF was used to detect glycylated tubulin in spermatozoa and different germ cell types isolated from mouse
testes. The TAP 952 mAb did not label spermatogonia,
spermatocytes or round spermatids. A uniform labeling
was first detected in the flagellum of early elongating
spermatids. During the maturation phase of spermatids,
the labeling showed a greater intensity at the tip than at
the base of the flagellum, i.e., a proximo-distal increase of
labeling. In late spermatids with a well differentiated
middle piece, i.e., testicular spermatozoa, and in epididymal spermatozoa a similar labeling gradient was observed, but it was less intense than in previous steps of
differentiation. As in a previous work [Kann et al., 1995]
the less intense labeling observed in terminally differentiated cells than in younger cells was attributed to the
presence of compact periaxonemal sheaths which constitute physical barriers to the diffusion of antibodies despite
permeabilization treatments. Indeed, freezing of spermatozoa before the preparation of smears resulted in a
fluorescence intensity similar to that observed in maturing spermatids (Fig. 1a, a8).
The AXO 49 mAb did not label any germ cell types
except the flagellum of maturing spermatids, testicular
and epididymal spermatozoa. This labeling was also
enhanced after freezing pretreatment. A decreasing labeling was observed from the middle piece to the end of the
principal piece of the flagellum, i.e., a proximo-distal
decrease, with a predominant labeling of the flagellum tip
(Fig. 1b, b8).
In man, only ejaculated spermatozoa could be
studied. The labeling of the flagellum with the TAP 952
mAb showed a proximo-distal increase as described
344
Kann et al.
above for mouse spermatozoa. No labeling could be
detected with the AXO 49 mAb (data not shown).
IEM of Glycylated Tubulin During Mammalian
Spermiogenesis
In all studied species, mouse (Fig. 2a, b, c), hamster,
rat, rabbit, monkey and man, the TAP 952 mAb labeled
the whole axoneme with an increasing intensity from the
middle piece to the terminal piece (region I-V) of the
flagellum of epididymal and ejaculated spermatozoa.
During spermiogenesis the axoneme of round spermatids
was not yet labeled. The labeling appeared in the whole
axoneme of elongating spermatids with a uniform distribution and an increasing intensity. Gold particles were
first detected from the beginning of the elongation phase
in mouse spermatids but at later steps of spermiogenesis
(mid-elongation phase) in other species. The manchette
was never labeled (Fig. 2d). The proximo-distal differential labeling of the flagellum described above for mature
spermatozoa became detectable from the mid-maturation
phase of spermatids in all species.
In spermatozoa of rodents, as shown for the mouse
(Fig. 3) and the rabbit, the AXO 49 mAb produced a
decreasing labeling from the middle piece (region I) to
the end of the principal piece (region IV) of the flagellum
with a predominant labeling, however, in the terminal
piece (region V), including the junctional area of region
IV with V. In contrast, in primate spermatozoa, monkey
and man, no labeling could be detected at any level of the
flagellum. During spermiogenesis in rodents and rabbit,
the axoneme of round and elongating spermatids and the
manchette were not labeled. The labeling became detectable during the maturation phase of spermatids and
immediately exhibited the differential distribution described above for mature spermatozoa.
In summary, the results observed by IEM fit well
with those observed by IIF. In addition, it should be noted
that centrioles were never labeled either with TAP 952
(Fig. 2d) or with AXO 49 mAb (data not shown).
Quantitative Immunogold Analyses of Glycylated
Tubulin in Mammalian Spermatozoa
The distribution of gold particles was analysed at
the five levels of sperm flagellum in mouse and man to
determine more accurately the differential labeling patterns described above. Examples of pictures used in these
analyses are shown in Figure 2b, c and Figure 3 for mouse
spermatozoa and Figure 4 for human spermatozoa.
The results obtained with motile human spermatozoa (dynein-positive) labeled with TAP 952 mAb are
presented in Figure 5a. This graph shows that the end of
the principal piece and the terminal piece of the flagellum
(regions IV and V) are respectively twofold and 2.5-fold
more labeled than the middle piece (region I). From the
beginning of the principal piece (region II) the labeling
intensity was sufficient to perform a quantitative analysis
per doublet. This analysis (Fig. 5a8) shows in region II a
predominant labeling in doublets 3-8, whereas the labeling is twice less in doublets 1-5-6 and three to four times
less in doublets 2-4-7-9. In more distal regions of the
principal piece (regions III and IV) the labeling intensity
of doublets 3-8 is similar whereas that of other doublets
increases progressively but remains significantly less
(P ⬍ 0.001).
A similar study was also performed on non-motile
human spermatozoa devoid of dynein arms (dyneinnegative). At first sight these spermatozoa also exhibited
a proximo-distal increase of the labeling along the
flagellum. However, the predominant labeling of doublets
3-8 was not as evident as in motile (dynein-positive)
spermatozoa (compare panels a and b of Fig. 4). Gold
particle counts fully confirmed the simple observation. In
fact, the same proximo-distal labeling gradient was
demonstrated in both dynein-positive and dyneinnegative spermatozoa (compare panels a and b of Fig. 5).
However, the differential labeling of peripheral doublets
was dramatically reduced (compare panels a8 and b8 of
Fig. 5). More precisely at each level the doublets 3-8 and
2-4-7-9 were now uniformly labeled whereas the labeling
intensity was significantly less (P ⬍ 0.01) in doublets
1-5-6 (regions II and IV).
The expected differential labeling of mouse spermatozoa with TAP 952 mAb was also confirmed but the
profile was slightly different of that presented above for
human spermatozoa. Thus, the labeling increased slowly
from the region I (middle piece) to the mid principal piece
(region III) then increased sharply at the end of the
principal piece (region IV) and in the terminal piece
(region V) (Fig. 6a). In the mouse as in man, the
differential labeling of doublets was characterized by a
predominant labeling of doublets 3-8 in regions II, III,
whereas this labeling was almost uniform in region IV
(Fig. 6a8).
An analysis of TAP 952 mAb labeling was also
performed with sodium azide immobilized mouse spermatozoa. This treatment did not modify the profile of the
proximo-distal labeling gradient reported above (compare panels a and b of Fig. 6). However, in this case, the
predominant labeling of doublets 3-8 was observed only
in the region II (compare panels a8 and b8 of Fig. 6). On
the other hand the labeling of doublets 2-4-7-9 began
more intensely than did that of doublets 1-5-6, thus
corresponding to an inversion of the labeling intensity as
observed in motile spermatozoa.
The quantitative analysis with AXO 49 mAb was
performed only on mouse spermatozoa since as indicated
Sperm Glycylated Tubulin
Fig. 2. Monoglycylated tubulin immunogold labeling with the mAb TAP 952. a, b, c: Mouse epididymal
spermatozoa labeled with 15 nm gold particles. II, III, IV and V refer to successive regions of flagellum.
d: Elongated spermatid labeled with 10 nm gold particles. The axoneme is labeled but the centriole (arrow)
and manchette (m) are not. Bar, 0.25 µm. a, d, ⫻40,000; b, c, ⫻80,000.
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Kann et al.
Fig. 3. Polyglycylated tubulin immunogold labeling of mouse epididymal spermatozoa with the mAb
AXO 49 and 15 nm gold particles (a, b). I, II, III, IV and V as in Figure 2. Note the highest labeling at the
end of region IV and in region V (a). Bar, 0.25 µm. ⫻60,000.
above this mAb did not react with human spermatozoa.
The proximo-distal decrease of the labeling previously
described was confirmed. Thus (Fig. 7a), the middle piece
(region I) was seven times more labeled that the end of
the principal piece (region IV). However, the most
intense labeling was observed in the short terminal piece
(region V). Moreover, at each level, the labeling of each
doublet was similar (Fig. 7a8). After sodium azide
motility inhibition of mouse spermatozoa, the general
profile of AXO 49 labeling was not modified (compare
panels a and b of Fig. 7). Likewise, the uniform labeling
of doublets remained essentially unchanged (compare
panels a8 and b8 of Fig. 7).
DISCUSSION
The post-translational modifications (PTM) of tubulin isoforms and particularly polyglutamylation and
polyglycylation are universally expressed from ciliates to
mammals [for review see MacRae, 1997; Ludueña, 1998]
specifically in axonemes, i.e., in the most stable microtubules. Mammalian spermatids contain three microtubular
structures: the flagellar axoneme, itself arising from a
centriole, and the manchette, a transient bundle of
cytoplasmic microtubules involved in nuclear shaping
[for review see Fouquet and Kann, 1994]. As expected
flagella were labeled with antiglutamylated tubulin [Fou-
Sperm Glycylated Tubulin
347
Fig. 4. Monoglycylated tubulin immunogold labeling with the mAb TAP 952 and 15 nm gold particles.
Cross sections in the region II of flagellum in motile (dynein-positive) (a) and non-motile (dyneinnegative) (b) human spermatozoa. Bar, 0.25 µm. ⫻80,000.
quet et al., 1994, 1997] and antiglycylated tubulin antibodies (present results) whereas the manchette was not
labeled with the same antibodies. In spermatogenic cells
of all species studied here centrioles appeared glutamylated as previously reported [Fouquet et al., 1994, 1997]
and as also described for somatic cells [Bobinnec et al.,
1998] but they were not glycylated. Taken together these
differential expressions of tubulin isoforms in the centriole and axoneme suggest that centriole microtubules
might be less stable than axoneme microtubules. Indeed,
centrioles and their associated pericentriolar material are
dynamic structures during the cell cycle [Moudjou et al.,
1996; Bobinnec et al., 1998].
In the axoneme of mammalian sperm flagellum all
␣-tubulin isoforms and a majority of ␤-tubulin isoforms
are glutamylated [Fouquet et al., 1994]. Their labeling
assumes a decreasing proximo-distal gradient and a
prevalence in doublets 1-5-6 in rodents and man [Fouquet
et al., 1996; Prigent et al., 1996; Fouquet et al., 1997]. In
this work we also confirm the presence of glycylated
tubulin in mammalian spermatozoa [Bré et al., 1996;
Plessmann and Weber, 1997]. In addition our results
clearly demonstrate a differential expression of glycylation in particular peripheral doublets of the flagellum.
Results of IIF and IEM show that polyglycylation is
a late PTM of tubulin isoforms as compared to acetylation, detyrosylation and glutamylation during mammalian
spermatogenesis [Fouquet et al., 1997] and as already
observed in Drosophila [Bré et al., 1996]. Indeed, a
majority of PTM are observed from the round spermatid
stage in naked still growing axonemes whereas glycylation is only detectable in elongating spermatids with
axonemes of definitive length but which are building their
periaxonemal sheaths. In elongated spermatids, in mouse
as in man, the glycylated tubulin is, at first, detected with
the TAP 952 mAb. This allows the conclusion that
axonemal tubulin is, at first, monoglycylated and uniformly distributed in all doublets at all levels of axoneme.
During the following steps of spermatid differentiation,
i.e., maturing spermatids, the levels of glycylation are
different in the mouse and man.
In the mouse during the last steps of spermatid
differentiation into testicular spermatozoa, as well as in
epididymal spermatozoa, two opposite labeling gradients
were observed: a proximo-distal decreasing labeling with
AXO 49 mAb and a proximo-distal increasing labeling
with TAP 952 mAb. These gradients and antibody
specificity suggest that in proximal regions of the flagellum (I and II) microtubules carry a majority of polyglycylated chains with three or more glycine units whereas in
more distal region of the flagellum (III and IV) the
microtubules are essentially monoglycylated. In fact, the
labeling, both with TAP 952 and AXO 49 mAbs, indicates
the coexistence of both monoglycylated and polyglycylated chains in any region of the flagellum. In addition to
these linear labeling gradients, antibodies against glycylated tubulin revealed a differential labeling among
peripheral doublets. Thus, in regions II, III and probably I
of the flagellum, doublets 3-8 are richer in monoglycylated tubulin than doublets 1-5-6 and 2-4-7-9 respectively. In contrast, polyglycylated tubulin seems uniformly distributed in all microtubule doublets. On the
whole, these results suggest that in mouse sperm flagellum, axonemal microtubules have numerous glycylatable
sites bearing glycine chains of different length.
In man, the flagellum of spermatids and spermatozoa was never labeled with AXO 49 mAb. At first sight
this result indicates that axonemal tubulin is monoglycylated only as recognized by TAP 952 mAb, and predominantly in doublets 3-8. However, in human spermatozoa,
as in mouse, a proximo-distal increase of labeling was
observed in the whole flagellum. To explain this labeling
348
Kann et al.
Fig. 5. Quantitative analyses of monoglycylated immunogold labeling along dynein-positive (a) and
dynein-negative (b) human sperm flagellum with the mAb TAP 952. The mean number ⫾ SEM of gold
particles is indicated for the central pair, peripheral doublets and whole microtubules in cross sections of
the middle piece (I), principal piece (II, III, IV) and terminal piece (V). Right panels (a8, b8): the mean
number ⫾ SEM of gold particles per doublet in groups 2-4-7-9, 1-5-6 and 3-8 is indicated for the regions
II–IV of the flagellum.
Sperm Glycylated Tubulin
Fig. 6. Quantitative analyses of monoglycylated immunogold labeling along the flagellum of motile (a)
and non-motile (azide treated) (b) mouse epididymal spermatozoa. Legends as in Figure 5a, b. Right panels
(a8, b8): legends as in Figure 5a8, b8.
349
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Kann et al.
Fig. 7. Quantitative analyses of polyglycylated immunogold labeling along the flagellum of motile (a) and
non-motile (azide treated) (b) mouse epididymal spermatozoa. Legends as in Figure 5a, b. Right panels
(a8, b8): legends as in Figure 5a8, b8.
Sperm Glycylated Tubulin
gradient which appeared at the end of spermiogenesis two
interpretations can be proposed. The simplest one is that
the number of monoglycylated tubulin units increases
progressively from proximal to distal regions of the
flagellum. Another possibility is that a polyglycylation
limited to two glycyl units occurs in proximal regions of
the flagellum at least in some glycylatable sites. As a
consequence a decrease of TAP 952 mAb labeling can be
expected in corresponding regions, thus creating a labeling gradient with this mAb, but no reactivity with AXO
49 mAb, which do not recognize biglycylated chains [Bré
et al., 1998].
To conclude, in mouse as in man the monoglycylated tubulin predominates in distal regions of the
flagellum and in doublets 3-8 despite differences in
glycylation levels.
Since we have previously reported [Fouquet et al.,
1997] that glutamylated tubulin expression is modified in
abnormal or motility inhibited flagella we also investigated these models for glycylated tubulin expression. In
mouse, sodium azide inhibition of sperm motility did not
change the labeling of polyglycylated tubulin as revealed
with AXO 49 mAb. In contrast, with TAP 952 mAb, the
changes in monoglycylated tubulin labeling which occurred in various doublets tended to make uniform their
labeling intensity. Similar changes were also observed in
non-motile human spermatozoa lacking dynein arms.
Thus in both species the major change was the loss of the
predominant labeling in doublets 3-8. These two models
suggest a functional role for monoglycylated tubulin, but
not for polyglycylated tubulin, in doublets other than
1-5-6 and particularly in doublets 3-8 of the axoneme in
mammalian sperm flagellum. Indeed, the decrease of TAP
952 mAb labeling may be interpreted as an accessibility
decrease of this antibody for monoglycylated tubulin in
doublets 3-8, i.e., an increase of interactions of these
doublets with adjacent structures. These changes are just
opposite to those previously observed for glutamylated
tubulin labeling with the GT335 mAb [Fouquet et al.,
1997]. Thus, in motile spermatozoa, glutamylated tubulin
labeling is much more intense in doublets 1-5-6 than in
other doublets whereas in non-motile spermatozoa the
labeling is uniform in all doublets because it increases in
doublets other than 1-5-6, particularly in 3-8 doublets.
Therefore in the same sperm models the loss of flagellar
motility seems to be correlated with an unmasking of
glutamylated tubulin and a masking of glycylated tubulin
particularly in doublets 3-8 of the axoneme, i.e., a
decrease and an increase of interactions respectively with
adjacent molecules. This interpretation in terms of differential interactions of glycylated and glutamylated tubulin
with adjacent structures is supported by the fact that with
both tubulin isoforms [present results and Fouquet et al.,
1997] the highest labelings were observed in the terminal
351
piece of the flagellum which is devoid of periaxonemal
structures and dynein arms. Taking also into account that
among a panel of site-directed antibodies to C-terminal
tubulin epitopes only those directed to glutamylated
epitopes [Gagnon et al., 1996] and glycylated epitopes
[Bré et al., 1996] were able to inhibit sperm motility in
man and sea urchin it is tempting to speculate that
glutamylation and glycylation are two tubulin PTM
which might play a complementary role in the regulation
of flagellar beating. This hypothesis is consistent with the
plane of morphological symmetry and flagellar beating of
mammalian spermatozoa as defined by doublets 1-5-6
and the plane of fibrous sheath symmetry as defined by
doublets 3-8. Obviously, there is a need to determine
axonemal and/or periaxonemal molecules which might
specifically interact with glutamylated and/or glycylated
tubulin in the flagellum of mammalian spermatozoa. This
last point is important since the periaxonemal structures
are believed to be involved in the flagellar beating
regulation [Eddy and O’Brien, 1994].
To conclude, both glycylation and glutamylation
are the only PTM of tubulin which are differentially
expressed in the flagellum of mammalian spermatozoa
and for which a functional significance can be proposed.
ACKNOWLEDGMENTS
The technical assistance of A. Gonzales for tissue
embedding and sectioning and the photographic work of
E. Prieto are appreciated. We are indebted to Mrs. D.
Bligny for excellent secretariat assistance.
REFERENCES
Bobinnec, Y., Moudjou, M., Fouquet, J.P., Desbruyères, E., Eddé, B.,
and Bornens, M. (1998): Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells. Cell Motil.
Cytoskeleton 39:223–232.
Bré, M.H., Redeker, V., Quibell, M., Darmanaden-Delorme, J., Bressac, C., Cosson, J., Huitorel, P., Schmitter, J.M., Rossier, J.,
Johnson, T., Adoutte, A., and Levilliers, N. (1996): Axonemal
tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during
spermatozoan maturation and possible function in motility. J.
Cell Sci. 109:727–738.
Bré, M.H., Redeker, V., Vinh, J., Rossier, J., and Levilliers, N. (1998):
Tubulin polyglycylation: differential posttranslational modification of dynamic cytoplasmic and stable axonemal microtubules
in Paramecium. Mol. Biol. Cell., 9:2655–2665.
Callen, A.M., Adoutte, A., Andreu, J.M., Baroin-Tourancheau, A., Bré,
M.H., Ruiz, P.C., Clérot, J.C., Delgado, P., Fleury, A., JeanmaireWolf, R., Viklicky, V., Villalobo, E., and Levilliers, N. (1994):
Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium.
Biol. Cell 81:95–119.
Eddé, B., Rossier, J., Le Caer, J.P., Desbruyères, E., Gros, F., and
Denoulet, P. (1990): Posttranslational glutamylation of alphatubulin. Science 247:83–85.
352
Kann et al.
Eddy, E.M., and O’Brien, D.A. (1994): The spermatozoon. In Knobil,
E., and Neil, J.D. (eds): ‘‘The Physiology of Reproduction,’’
vol. 1. New York: Raven, pp. 29–77.
Fouquet, J.P., and Kann, M.L. (1994): The cytoskeleton of mammalian
spermatozoa. Biol. Cell 81:89–93.
Fouquet, J.P., Eddé, B., Kann, M.L., Wolff, A., Desbruyères, E., and
Denoulet, P. (1994): Differential distribution of glutamylated
tubulin during spermatogenesis in mammalian testis. Cell Motil.
Cytoskeleton 27:49–58.
Fouquet, J.P., Prigent, Y., and Kann, M.L. (1996): Comparative
immunogold analysis of tubulin isoforms in the mouse sperm
flagellum: unique distribution of glutamylated tubulin. Mol.
Reprod. Dev. 43:358–365.
Fouquet, J.P., Kann, M.L., Péchart, I., and Prigent, Y. (1997): Expression of tubulin isoforms during the differentiation of mammalian spermatozoa. Tissue Cell 29:573–583.
Gagnon, C., White, D., Cosson, J., Huitorel, P., Eddé, B., Desbruyères,
E., Paturle-Lafanechère, L., Multigner, L., Job, D., and Cibert,
C. (1996): The polyglutamylated lateral chain of alpha-tubulin
plays a key role in flagellar motility. J. Cell Sci. 109:1545–1553.
Hecht, N.B., Distel, R.J., Yelick, P.C., Tanhauser, S.M., Driscoll, C.E.,
Goldberg, E., and Tung, K.S.H. (1988): Localization of a highly
divergent mammalian testicular ␣-tubulin that is not detectable
in brain. Mol. Cell Biol. 8:996–1000.
Kann, M.L., Prigent, Y., and Fouquet, J.P. (1995): Differential distribution of glutamylated tubulin in the flagellum of mouse spermatozoa. Tissue Cell 27:323–329.
Lewis, S.A., and Cowan, N.J. (1988): Complex regulation and
functional versatility of mammalian ␣ and ␤-tubulin isotypes
during the differentiation of testis and muscle cells. J. Cell Biol.
106:2023–2033.
Ludueña, R.F. (1998): Multiple forms of tubulin: different gene
products and covalent modifications. Int. Rev. Cytol. 178:207–
275.
MacRae, T.H. (1997): Tubulin post-translational modifications, enzymes and their mechanisms of action. Eur. J. Biochem.
244:265–278.
Moudjou, M., Bordes, N., Paintrand, M., and Bornens, M. (1996):
␥-tubulin in mammalian cells: the centrosomal and the cytosolic
forms. J. Cell Sci. 109:875–887.
Plessmann, U., and Weber, K. (1997): Mammalian sperm tubulin: an
exceptionally large number of variants based on several posttranslational modifications. J. Protein Chem. 16:385–390.
Prigent, Y., Kann, M.L., Lach-Gar, H., Péchart, I., and Fouquet, J.P.
(1996): Glutamylated tubulin as a marker of microtubule
heterogeneity in the human sperm flagellum. Mol. Hum.
Reprod. 2:573–581.
Redeker, V., Melki, R., Promé, D., Le Caer, J.P., and Rossier, J.P.
(1992): Structure of tubulin c-terminal domain obtained by
subtilisin treatment. The major ␣ and ␤-tubulin isotypes from
pig brain are glutamylated. FEBS Lett. 313:185–192.
Redeker, V., Levilliers, N., Schmitter, J.M., Le Caer, J.P., Rossier, J.,
Adoutte, A., and Bré, M.H. (1994): Polyglycylation of tubulin: a
posttranslational modification of axonemal microtubules. Science 226:1688–1691.
Rüdiger, M., Plessmann, U., Klöppel, K.D., Wehland, J., and Weber, K.
(1992): Class II tubulin, the major brain ␤-tubulin isotype, is
polyglutamylated on glutamic acid residue 435. FEBS Lett.
308:101–105.
Simerly, C.R., Hecht, N.B., Goldberg, E., and Schatten, G. (1993):
Tracing the incorporation of the sperm tail in the mouse zygote
and early embryo using an anti-testicular ␣-tubulin antibody.
Dev. Biol. 158:536–548.
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