BIOLOGY OF REPRODUCTION 69, 373–378 (2003)
Published online before print 2 April 2003.
DOI 10.1095/biolreprod.103.015719
M i n i r ev i ew
New Insights into the Assembly of the Periaxonemal Structures
in Mammalian Spermatozoa
Denise Escalier1
Histologie Fonctionnelle et Moléculaire, Université Paris 5, 75270 Paris Cedex 6, France
ABSTRACT
flagellar growth that impairs the normal distribution of periaxonemal structures, and 4) aberrant organization of the
periaxonemal structures with seemingly normal axonemal
structure. This latter category has been described in humans
[7, 8], and it was recently observed in mice that were deficient in the ubiquitin-conjugating enzyme UBE2B [9].
That this enzyme should be involved in the organization of
the periaxonemal structures was unexpected, raising the
question regarding possible involvement of the proteasomeubiquitin pathway in sperm tail morphogenesis, as has already been suggested [10, 11].
The present review will consider whether the known
roles of the ubiquitin-proteasome pathways in other cells
support this notion. We will explore potential targets of
UBE2B regarding components of the mammalian flagella
that have been identified. We will also discuss data in the
literature concerning the possible role of axonemal differentiations in the spatial distribution of the periaxonemal
structures. Finally, Ube2b-null mice will be compared to a
known human sperm pathology, suggesting that Ube2b-null
mice could be a mouse model for human sperm tail pathology.
Disruption of Ube2b in the mouse has revealed that the regular and symmetric organization of the fibrous sheath of the
sperm flagella is dependent on expression of the ubiquitin-conjugating enzyme UBE2B. These data could cast light on how a
component of the ubiquitin-proteasome pathway participates in
the assembly of flagellar periaxonemal structures. Data in the
literature support the notion of involvement of ubiquitin-proteasome pathways in the assembly of cytoskeletal components in
somatic cells. This review attempts to integrate recent knowledge regarding flagellar components that could be related to
proteasome components and, therefore, could be targets of
UBE2B in the spermatid. An attempt is made to characterize the
human flagellar anomalies of infertile patients, which are the
closest to those of Ube2b-deficient mice. These new insights regarding the assembly of mammalian sperm flagella provide a
basis for studying the ontogenesis of flagellar accessory structures and suggest leads for medical and genetic investigations.
gamete biology, gametogenesis, sperm, spermatogenesis, testis
INTRODUCTION
Our understanding of the morphogenesis of the sperm
tail comes chiefly from mutants of Trypanosoma sp., Drosophila sp., and mammals [1–4]. In mammals, the sperm
flagellum contains highly organized periaxonemal structures, suggesting the involvement of complex cellular pathways that regulate their assembly. The periaxonemal structures that surround the axoneme consist of nine outer dense
fibers (ODFs) and the fibrous sheath (FS) [5]. Each ODF
is strongly fastened to an axonemal doublet. Three types of
ODFs can be distinguished according to length [6]. The FS
is made up of two longitudinal columns (LCs) associated
with axonemal doublets 3 and 8 and a series of ribs connected to the LCs.
Anomalies of the flagella can be classified as 1) axonemal anomalies only, 2) anomalies of the assembly of both
the axoneme and periaxonemal structures, 3) a defect of
ABNORMAL FLAGELLAR MORPHOGENESIS
BY DISRUPTION OF Ube2b
Ube2b-null male mice were found to be sterile. Their
spermatozoa presented with a misshapen sperm head, irregular flagellar diameter, and impaired motility [12]. An increase in the apoptosis of Ube2b-null spermatocytes was
associated with damaged synaptonemal complex structures
and a higher cross-over rate [13]. Ube2b encodes the ubiquitin-conjugating enzyme E2, also known as UBE2B. The
aberrant morphology of the sperm head seems to indicate
that the major impairment of spermatogenesis in Ube2bnull mice occurs during condensation of the spermatid’s
nucleus [14]. Surprisingly, Ube2b-null sperm flagella also
bore a spectacular flagellar phenotype characterized by an
aberrant disposition of the periaxonemal structures (Fig. 1)
[9]. Thus, UBE2B-deficient mice appeared as the first
mouse model to present well-defined anomalies in the spatial distribution of the periaxonemal structures. This strongly supported the concept established by Kierszenbaum [15]
regarding possible involvement of the ubiquitin-proteasome
pathway in flagellar morphogenesis in mammals. Other investigations demonstrated the existence of multiple ubiquitin-proteasome-dependent pathways, raising the question
of the pathway through which UBE2B plays a role in organization of the periaxonemal structures.
1
Correspondence: Denise Escalier, Histologie Fonctionnelle et Moléculaire, Université Paris 5, 45, rue des Saints Pères, 75270 Paris Cedex 6,
France. FAX: 33 (0)1 42 86 20 84;
e-mail: [email protected]
Received: 23 January 2003.
First decision: 17 February 2003.
Accepted: 28 March 2003.
Q 2003 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
373
374
ESCALIER
FIG. 1. Transverse sections of flagella from wild-type (a–c) and Ube2b2/2
mice (d–f). Testes were fixed in glutaraldehyde and embedded in epon as
previously described [9]. a) LC-As normally are present on doublets 3 and
8 in newly formed flagellum. b) Higher-magnification view showing an LCA. c) LCs in mature spermatids are assembled on LC-As and thus bound to
doublets 3 and 8. d–f) Predominant anomalies in Ube2b2/2 flagella. d) One
LC-A is on doublet 2 instead of doublet 3 in a young spermatid. e and f)
Both LC-A and LC are on doublet 2 instead of doublet 3 (e) or doublet 8
(f) in mature spermatids. Bars 5 0.1 mm.
The ‘‘classical’’ biochemical steps of the ubiquitin-proteasome pathway are now well identified [16]. Ubiquitin is
covalently attached to abnormal and short-lived proteins,
which are destined to ATP-dependent proteolysis in eukaryotic cells. In brief, ubiquitin is first activated by the
ubiquitin-activating enzyme E1. Ubiquitin-activating enzyme E2 (ubiquitin carrier protein or ubiquitin-conjugating
enzyme) transfers the activated ubiquitin to the substrate
that is bound to E3, a member of the ubiquitin protein ligase family. The 26S proteasome can recognize and break
down nonubiquitinated proteins as well, and multiple ubiquitin- or proteasome-dependent pathways exist [17]. Additionally, ubiquitin pathways are involved in many different
cell processes [18] and also participate in nonproteolytic
functions, such as routing of cellular proteins to their subcellular compartments [16]. Functional interactions of components of the ubiquitin-proteasome pathway with each of
the components of the cytoskeleton (actin filaments, tubulin, and intermediate filaments) have been found [19]. For
example, an actin-ubiquitin conjugate is necessary for the
assembly of myofibrils of flight muscles [20].
In addition, it would be of interest to investigate the
ultrastructure and kinetics of the spermatozoa of mice that
are deficient in ubiquitin ligase E6-AP because of the functional disability of their sperm to penetrate the egg, even
though the spermatozoa are capable of progression [21].
PERIAXONEMAL COMPONENTS ARE TRANSPORTED
FROM THE CELL BODY TO THE FLAGELLUM
Proteins of the periaxonemal structures in the mammal
are first synthesized in the cytoplasm, as has been demonstrated for the AKAP82 FS protein. This protein is synthesized as a precursor (pro-AKAP82) in the cell body, transported down the axoneme to its site of assembly in the FS,
and then proteolytically clipped to form mature AKAP82
(known in the new nomenclature as AKAP4) [22]. Intraflagellar transport (IFT) is essential for the development and
maintenance of cilia and flagella. Among the factors found
in spermatids and possibly involved in the transport of
mammalian flagellar proteins are the motor protein
KIFC5A (kinesin) [23] and Tg737-Polaris [24]. Indeed, IFT
involves kinesin, which transports rafts of proteins for axoneme assembly in an anterograde movement, and Tg737Polaris is involved in ciliogenesis and is a component of
the macromolecular protein complex IFT [25, 26]. Tg737null mice have both defective ciliogenesis and abortive flagella [11]. In addition, components of the 26S proteasome,
including the ATPase TBP-1, have been found in the manchette and the sperm tail [10, 15, 27–29]. A member of the
highly evolutionarily conserved TBP-1-like subfamily of
putative ATPases, TBP-1 has high homology with several
ATPase-like subunits of the human 26S proteasome [30].
Therefore, TBP-1 shares regions of identity, including ATPbinding sites, with several subunits of the 26S proteasome,
which is involved in the ATP-dependent degradation of
ubiquitin-conjugated proteins [28]. Considering that an important element in the function of the 26S proteasome is
the ubiquitin pathway by which proteins are tagged for degradation, it was suggested that the 26S proteasome could
degrade proteins that are discarded during the structural
tuning of the sperm tail and could have a role in the remodeling of tail components [10, 27]. A model has been
proposed and fully illustrated [11, 15].
ONTOGENESIS OF AXONEMAL-ASSOCIATED
STRUCTURES
Analysis of the assembly of the periaxonemal structures
is another approach to determine the origin of LC anomalies in Ube2b-null mice. The LCs normally are assembled
symmetrically along axonemal doublets 3 and 8. Before
LCs are assembled, ‘‘LC-anchorage structures’’ (LC-As;
also known as small ridges or stud-like projections) emerge
from the outer wall of doublets 3 and 8 (Fig. 1, a and b)
[31]. The molecular nature of the LC-As is not known. The
LC precursor framework or anlagen is assembled on these
crescent structures [32]. Therefore, it is through the LC-As
that the LCs are connected to the axoneme (Fig. 1, a–c).
In Ube2b-null flagella, LC-As emerge on improper axonemal doublets (Fig. 1d). Consequently, LCs are badly positioned (Fig. 1, e and f). This assembly aberration in Ube2bnull mice suggests that the LC-As determine the future position of the LCs and that UBE2B could be specialized to
the correct positioning of LC-As and, consequently, of the
periaxonemal structures in the mammalian flagellum. Moreover, the LC-As normally appear on doublets 3 and 8 in
the flagella of mice that are deficient for the major FS protein AKAP4 [33], suggesting that malpositioning of LC-As
in Ube2b-null mice is not the consequence of an anomaly
of the FS proteins.
Figure 2 depicts the possible role of UBE2B in the proper assembly of LC-As and LCs in the flagellum of murine
spermatozoa. The improper positioning of LC-As in
Ube2b-null mice raises the question of the factors that determine on which doublets the LC-As appear, especially
whether particular tubulins or microtubule-associated proteins (MAPs) or posttranslational modifications of these
components determine the binding of LC-As on defined
axonemal doublets. In Chlamydomonas sp., the presence of
a particular tubulin protofilament in the doublets is also
suggested for the localization of the dynein-arm docking
complex (a small protrusion necessary for binding of the
outer dynein arms) [34].
AXONEMAL POSTTRANSLATIONAL MODIFICATIONS
The microtubules that make up the flagellar axoneme
display constitutional differences, which are revealed by
375
A MOUSE MODEL OF SPERM TAIL ASSEMBLY
FIG. 2. UBE2B and mammalian flagella
morphogenesis. Possible involvement of
UBE2B in the assembly of flagellar cytoskeletal structures, as suggested by the
murine Ube2b2/2 phenotype, is shown.
The yellow arrows represent the stages of
the assembly of flagellar structures. First,
the distal centriole gives rise to the axoneme (green), and then the structure of the
LC-As (nature as yet unknown) appears on
doublets 3 and 8 (red), on which the LCs
of the FS are finally assembled (gray). The
primary defect observed in Ube2b2/2 mice
seems to affect LC-As structures (red).
differential distribution of the tubulin epitopes along the
flagellar axoneme. This suggests either a difference in the
types of tubulins or differences in tubulin posttranslational
modifications [35]. In addition, the distribution of glutamylated tubulin in the axonemal doublets seems to be related to the length of the ODFs [36, 37]. Polyglutamylation
of tubulin controls the binding of different MAPs, which
leads to a selective recruitment of MAPs in the microtubule
wall [38]. The importance of posttranslational modifications
of tubulins in sperm tail morphogenesis has been reviewed
[39].
In Chlamydomonas reinhardtii, a-tubulin is synthesized
as a precursor, which is modified by acetylation in the flagellum and during its assembly [40]. A link between acetylation and ubiquitination has been suggested by the finding
of cytoplasmic deacetylase in the murine testis that, first,
binds ubiquitin through its C-terminal region that contains
a zinc-finger domain and, second, binds to proteins whose
interaction is mediated by the ubiquitin signaling pathway
[41]. Acetylation of a-tubulin occurs in the flagella of Trypanosoma brucei, which contain an extra-axonemal structure, the paraflagellar rod (PFR) [42]. Trypanosoma sp. tubulin acetylation occurs in the flagellum during the assembly of axonemal microtubules (MTs), and this acetylation
is stable. On the other hand, polymerizing flagellar MTs in
Trypanosoma sp. have a high proportion of tyrosinated atubulin, which is subsequently brought back to a basal level
in flagella containing PFRs [42]. All these data show that
posttranslational modifications of a-tubulin can occur after
the assembly of the axoneme and can be concomitant to
the assembly of extra-axonemal structures. It would be interesting to know whether the ubiquitin pathway plays a
role in the acetylation of flagellar tubulin and whether
anomalies of a-tubulin acetylation could have a domino
effect, causing anomalies in a subset of other proteins and
leading to an altered organization of axonemal-associated
structures.
Posttranslational modifications of tubulins thus define a
difference in the longitudinal constitution of doublets that
could be a key process for the correct assembly of periaxonemal structures. Moreover, the assembly of mammalian
axonemal doublets appears to be controlled by different elements, as illustrated by human phenotypes that exhibit a
lack of specific doublets [43]. This raises the question of
whether the spatial distribution of doublets is altered in
Ube2b-null mice, because the constitution of the doublets
is different. Human flagellar phenotypes that share ectopic
LCs, but on different doublets (see paragraph before conclusion), also raise this question.
POSSIBLE UBE2B TARGETS DURING FLAGELLAR
MORPHOGENESIS
The PFR proteins of Trypanosoma sp. have to be imported from the cytoplasm into the flagellum, and the two
major components of the PFR possess a segment that is
necessary for this flagellar import or binding [44]. This segment contains a sequence that is similar to a sequence in
376
ESCALIER
FIG. 3. Periaxonemal structure organization in Ube2b-deficient mice as compared
to that seen in infertile men with ‘‘aberrant
distribution of the periaxonemal structures.’’ The axoneme is shown here so that
only the microtubules are visible. In both
Ube2b-null mice and infertile patients,
LCs were not found in the plane of the
central microtubules, and/or one LC was
absent. Wild-type mice and Csnk2a2
knock-out mice (that present with anomalies of sperm morphology) served as controls. a) Normal localization of the two
LCs on doublets 3 and 8. b and c) Abnormal localization of one LC. d) Both LCs
are abnormally positioned. e) Absence of
one of the LCs.
axonemal dynein from Chlamydomonas sp., suggesting that
a common signal for flagellar targeting may be conserved
across these species [45]. The flagellar protein targeting in
T. brucei also depends on a tripeptide motif, which is present on both a PRF protein and an actin-related protein [46].
In mammals, many genes whose products are constituents of the FS have been identified. Among them are structural proteins [47], but only Sptrx-1 has been found to be
restricted to LCs [48]. In addition, the FS contains several
anchoring proteins for the subcellular localization of protein
kinase A (AKAPs) [49]. The AKAPs function as scaffolding proteins for signal-transducing molecules [50]. Phosphorylation of AKAP targets the protein to structures. The
AKAPs have domains for targeting to various subcellular
compartments, including the actin cytoskeleton [49]. It is
worth noting that actin is associated to the FS [51].
The flagellar protein OIP1 has recently been identified
as a ring-finger protein [52]. Ring fingers are candidates for
E3 ubiquitin-protein ligases. They mediate binding activity
toward E2 ubiquitin-conjugating enzymes and display E2dependent ubiquitination activity in vitro. OIP1 binds to the
ODF1 protein and is present in the sperm tail. Further studies should allow its structural localization in the flagellum
to be pinpointed [52]. Dorfin is another ring-finger protein
with E3-like ubiquitin activity that is present in the centrosome region of rodent spermatids [11, 53].
The centrosomal g-tubulin (centriolar and pericentriolar)
is in dynamic association with factors of the ubiquitin pathway (ubiquitin-specific processing proteases, or UBPs)
[54], and it has been proposed that the centrosome gathers
components of a proteolytic machinery [53]. It could be
interesting to investigate whether UBE2B plays a part in
control of the metabolism of the centrosome during axonemal assembly.
The FS contains the small GTPase Rho-binding protein,
rhophilin. It was suggested that rhophilin may act as a regulator of FS assembly and/or function [55]. In somatic cells,
Rho induces actin polymerization and regulates cell motility and cytokinesis through reorganization of the actin cytoskeleton [55]. Moreover, Rho is known to detyrosinate
MTs, leading to their stabilization [56]. Interestingly, a high
proportion of detyrosinated tubulins is found in the MT of
the sperm tail axoneme [57].
Consequently, some proteins localized in the FS could
have a metabolic relationship with MT components and
with the E2 ubiquitin-conjugating enzyme. Further studies
should define whether they participate in periaxonemal
structure assembly and/or sperm motility.
Ube2b2/2 FLAGELLA ARE STRIKINGLY SIMILAR TO THE
HUMAN FLAGELLAR PHENOTYPE
Ube2b-null mice are important as an animal model, because their flagellar phenotype is strikingly similar to the
known human phenotype of ‘‘aberrant distribution of the
periaxonemal structures’’ [7]. Figure 3 presents the distribution of LCs in Ube2b2/2 mice in comparison to that previously reported in humans [7, 8]. The morphological pattern of Ube2b2/2 flagella is remarkably reminiscent of this
human flagellar phenotype. Previous analysis of this human
sperm phenotype has led to the hypothesis that LC-As assembly could be the primary structural defect and that the
distribution of tubulins in the axoneme could be affected
[7].
It is worth noting that the assembly of the axonemal
substructures is not affected in the flagella of either Ube2bnull mice or these infertile patients. This is a rare situation
in humans, because most patients with periaxonemal anomalies have anomalies of the axoneme [43]. Interestingly,
malposition of the LCs is found in human spermatozoa that
lack the two dynein arms [43]. The association of a lack
of MT-associated structures and malpositioning of LCs
strengthens the concept of intrinsic MT defects.
In humans, aberrant distribution of the periaxonemal
structures leads to severe sperm motility defects, with abnormal flagellar curvature or impaired progressivity [7, 8].
Cases of brothers who share the same phenotype (unpublished data) suggest a genetic origin for the anomaly. Despite numerous murine models with spermiogenetic defects
[58, 59], it was not possible to assign a candidate gene for
A MOUSE MODEL OF SPERM TAIL ASSEMBLY
this particular human phenotype, because a similar anomaly
has not been reported in any other species. Patients with
ectopic LCs could present a mutation in the human Ube2b
homologue (on chromosome 5q23-q31) [60] that could theoretically be investigated. It is worth noting, however, that
the human flagellar phenotype of ‘‘aberrant distribution of
the periaxonemal structures’’ is not totally homogeneous.
For example, LCs can be seen predominantly on doublets
3 and 4 or on doublets 3 and 9. In other cases, LCs are
bound to different doublets from the proximal region to the
distal region, and some patients present a predominance of
only one LC (unpublished data). Moreover, defects in factors interacting with UBE2B could theoretically lead to a
Ube2b2/2-like flagellar phenotype. In further investigations,
it should be necessary to examine a large number of patients, and care should be taken to precisely note the exact
description of their sperm tail anomalies.
CONCLUSION
Recently, many biochemical components have been
identified in the flagellum of spermatids and spermatozoa
in mammals. They open future prospects for determining
the genetic pathways controlling flagellar morphogenesis.
The recent findings of flagellar anomalies in Ube2b-deficient mice, associated with a better understanding of flagellar elements, could help to identify candidate flagellar
proteins as targets of UBE2B. Additionally, Ube2b-null
mice come as an unexpected and precious murine model
for a similar human sperm pathology.
ACKNOWLEDGMENTS
I would like to thank Dr. H. Roest, Prof. J.H. Hoeijmakers, Prof. J.A.
Groodegoed, and Dr. W.M. Baarends (University of Rotterdam, The Netherlands) for providing Ube2b-null mice and fruitful discussions. I also
wish to thank anonymous reviewers for their useful critical comments. I
am grateful to Dr. X. Xu (McLaughlin Research Institute, Great Falls, MT)
for providing Csnk2a2-null mice and to Dr. E. Broneer for English translation. I apologize to those investigators whose works were not referenced
because of the brevity of this review.
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