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Myosin VI (MVI) is a versatile actin-based motor protein that has been implicated in a variety of different cellular processes, including endo- and exocytic vesicle trafficking, Golgi morphology, and actin structure stabilization. A role for MVI in crucial actin-based processes involved in sperm maturation was demonstrated in Drosophila. Because of the prominence and importance of actin structures in mammalian spermiogenesis, we investigated whether MVI was associated with actin-mediated maturation events in mammals. Both immunofluorescence and ultrastructural analyses using immunogold labeling showed that MVI was strongly linked with key structures involved in sperm development and maturation. During the early stage of spermiogenesis, MVI is associated with the Golgi and with coated and uncoated vesicles, which fuse to form the acrosome. Later, as the acrosome spreads to form a cap covering the sperm nucleus, MVI is localized to the acroplaxome, an actin-rich structure that anchors the acrosome to the nucleus. Finally, during the elongation/maturation phase, MVI is associated with the actin-rich structures involved in nuclear shaping: the acroplaxome, manchette, and Sertoli cell actin hoops. Since this is the first report of MVI expression and localization during mouse spermiogenesis and MVI partners in developing sperm have not yet been identified, we discuss some probable roles for MVI in this process. During early stages, MVI is hypothesized to play a role in Golgi morphology and function as well as in actin dynamics regulation important for attachment of developing acrosome to the nuclear envelope. Next, the protein might also play anchoring roles to help generate forces needed for spermatid head elongation. Moreover, association of MVI with actin that accumulates in the Sertoli cell ectoplasmic specialization and other actin structures in surrounding cells suggests additional MVI functions in spermatid movement across the seminiferous epithelium and in sperm release.
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Histochem Cell Biol (2017) 148:445–462
DOI 10.1007/s00418-017-1579-z
Expression and localization of myosin VI in developing mouse
Przemysław Zakrzewski1 · Robert Lenartowski2 · Maria Jolanta Re˛dowicz3 ·
Kathryn G. Miller4 · Marta Lenartowska1
Accepted: 4 May 2017 / Published online: 12 May 2017
© The Author(s) 2017. This article is an open access publication
and Sertoli cell actin hoops. Since this is the first report of
MVI expression and localization during mouse spermio-
genesis and MVI partners in developing sperm have not yet
been identified, we discuss some probable roles for MVI
in this process. During early stages, MVI is hypothesized
to play a role in Golgi morphology and function as well
as in actin dynamics regulation important for attachment
of developing acrosome to the nuclear envelope. Next,
the protein might also play anchoring roles to help gener-
ate forces needed for spermatid head elongation. Moreo-
ver, association of MVI with actin that accumulates in
the Sertoli cell ectoplasmic specialization and other actin
structures in surrounding cells suggests additional MVI
functions in spermatid movement across the seminiferous
epithelium and in sperm release.
Keywords Actin · Immunocytochemistry · Myosin VI
splice variants · Spermiogenesis · Ultrastructure
ABP/s Actin-binding/regulating protein(s)
AF/s Actin filament(s)
F-actin Filamentous actin
JLA20 MAb Monoclonal antibody against actin
LI Large insert in MVI tail region
MVa Myosin Va
MVI Myosin VI
MVI PAb Polyclonal antibody against MVI
NoI MVI Isoform with no inserts in the tail domain
SI Small insert in MVI tail region
sv/sv mice Snell’s waltzer mutants that lack MVI
TGN trans-Golgi network
Abstract Myosin VI (MVI) is a versatile actin-based
motor protein that has been implicated in a variety of dif-
ferent cellular processes, including endo- and exocytic
vesicle trafficking, Golgi morphology, and actin structure
stabilization. A role for MVI in crucial actin-based pro-
cesses involved in sperm maturation was demonstrated in
Drosophila. Because of the prominence and importance of
actin structures in mammalian spermiogenesis, we inves-
tigated whether MVI was associated with actin-mediated
maturation events in mammals. Both immunofluorescence
and ultrastructural analyses using immunogold labeling
showed that MVI was strongly linked with key structures
involved in sperm development and maturation. During the
early stage of spermiogenesis, MVI is associated with the
Golgi and with coated and uncoated vesicles, which fuse to
form the acrosome. Later, as the acrosome spreads to form
a cap covering the sperm nucleus, MVI is localized to the
acroplaxome, an actin-rich structure that anchors the acro-
some to the nucleus. Finally, during the elongation/matura-
tion phase, MVI is associated with the actin-rich structures
involved in nuclear shaping: the acroplaxome, manchette,
* Przemysław Zakrzewski
1 Laboratory of Developmental Biology, Faculty of Biology
and Environmental Protection, Nicolaus Copernicus
University in Torun´, Torun´, Poland
2 Laboratory of Isotope and Instrumental Analysis, Faculty
of Biology and Environmental Protection, Nicolaus
Copernicus University in Torun´, Torun´, Poland
3 Laboratory of Molecular Basis of Cell Motility, Department
of Biochemistry, Nencki Institute of Experimental Biology,
Polish Academy of Sciences, Warsaw, Poland
4 Department of Biology, Washington University in St. Louis,
St. Louis, MO, USA
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446 Histochem Cell Biol (2017) 148:445–462
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Spermiogenesis is a complex developmental process that
entails extensive morphological and biochemical alterna-
tions resulting in formation of fully differentiated male
gametes—spermatozoa. Two key events of spermiogenesis
are acrosome biogenesis and nuclear shaping, accompanied
by sperm tail formation. This process in mammals is typi-
cally divided into three main phases, during which round
spermatids transform into elongated mature sperm (Fig. 1):
the Golgi, acrosome cap/elongation, and maturation phases
(see review by Toshimori 2009). During the Golgi stage,
Golgi-derived proacrosomal vesicles form the acrosome
adjacent to the anterior pole of the spermatid nucleus. These
granules tether, dock, and fuse along the acroplaxome, a
cytoskeletal plate stabilized by the keratin 5/Sak57-con-
taining marginal ring that anchors the developing acrosome
to the nuclear envelope (Kierszenbaum et al. 2003b, 2011
and see review by Kierszenbaum and Tres 2004). The pair
of centrioles migrates distally and initiates formation of
the flagellum. A cloud-like structure, called the chromatoid
body, establishes contact with intranuclear material through
the pore complexes at the caudal pole of the spermatid
nucleus. Next, the giant acrosomal vesicle spreads over the
spermatid nucleus to form a distinct cap, while the Golgi
complex migrates toward the posterior pole of the sperm
nucleus. Soon after acrosome biogenesis starts, a transient
cytoskeletal structure—the manchette—develops caudally
to the acrosome–acroplaxome around the nucleus and the
spermatid initiates elongation, starting the acrosome/elon-
gation subphase. Golgi-derived non-acrosomal vesicles
are mobilized and transported along the manchette to the
centrosome region and developing flagellum (see reviews
by Kierszenbaum 2002; Kierszenbaum and Tres 2004). As
the elongation progresses, the acrosome contents gradually
condense, the cap continues to cover the spermatid nucleus,
and distal centriole produces an axoneme. During the last
step of spermiogenesis, the spermatid nucleus is remodeled
by chromatin condensation, the manchette disappears upon
completion of the sperm head elongation, and mitochondria
are packed into the midpiece of elongating tail (see review
by Toshimori 2009). Immediately prior to spermiation,
excess cytoplasm is eliminated from the future sperm as the
residual body, which is phagocytosed by the surrounding
Sertoli cell.
The actin cytoskeleton, including a number of actin-
binding/regulating proteins (ABPs), has been implicated
in various aspects of mammalian spermiogenesis. First,
filamentous actin (F-actin) has been identified as a cen-
tral component of several unique cytoskeletal structures
assembled during spermatid differentiation including the
acrosome–acroplaxome complex, the manchette, and the
apical ectoplasmic specialization of the Sertoli cell adja-
cent to developing spermatid (Kierszenbaum et al. 2003b
and see reviews by Kierszenbaum et al. 2011; Sun et al.
2011; Qian et al. 2014). Second, two different molecular
motor systems operate to mobilize vesicle cargos required
for acrosome biogenesis and tail development. Besides
microtubules (see reviews by Berruti and Paiardi 2011;
Lehti and Sironen 2016), an actin-related pathway using
the MVa/Rab27a/b complex is involved in Golgi-derived
vesicle transport along the acroplaxome and the manchette
(Kierszenbaum et al. 2003a, 2004; Hayasaka et al. 2008).
In addition, MVa-decorated vesicles surround a portion of
the chromatoid body, suggesting a possible role of actin fil-
aments in the disposal of nuclear material generated during
spermiogenesis (Kierszebaum et al. 2003a and see review
by Kierszenbaum and Tres 2004). Third, the acrosome–
acroplaxome–manchette complex contains ABPs such as
cortactin and profilin-3, which are thought to modulate
Fig. 1 Schematic representation of the main stages of spermiogen-
esis in mouse: the Golgi, acrosomal, and maturation phases. ag acro-
somal granule, am acrosome membrane, av acrosome vesicle, ax
acroplaxome, c centriole, cy cytoplasm, dp dense plaque, es apical
ectoplasmic specialization, g Golgi apparatus, iam inner acrosomal
membrane, if intermediate filaments, m mitochondria, mp midpiece, n
spermatid nucleus, nm nuclear envelope, oam outer acrosomal mem-
brane, pp principal piece
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447Histochem Cell Biol (2017) 148:445–462
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actin dynamics during acrosomogenesis and head shaping
(Obermann et al. 2005; Kierszenbaum et al. 2008; Behnen
et al. 2009). Finally, the apical ectoplasmic specialization
associated with the tubulobulbar complexes at the concave
side of the elongating spermatid head contains actin fila-
ments. These actin structures form a stack of hoops stabi-
lized by espin and adhesion protein complexes (Kierszen-
baum et al. 2003b and see reviews by Kierszenbaum and
Tres 2004; Kierszenbaum et al. 2007; Xiao and Yang 2007).
Spatiotemporal expression of testis-specific actin assembly/
disassembly regulators modulates adhesion of spermatids
to the Sertoli cells during their movement across the semi-
niferous epithelium, and then allows the release of mature
sperm at spermiation. Although F-actin structures seem to
play important roles during the key events of spermiogene-
sis in mammals, the molecular basis of their regulation and
roles in the processes is still poorly understood.
During Drosophila spermatogenesis, some processes
similar to those described for mammalian spermatogenesis
occur, and the actin cytoskeleton plays several important
roles. Stable actin structures, called actin cones, mediate
spermatid individualization during the final step of Dros-
ophila spermiogenesis when 64 syncytial spermatids are
reorganized into individual mature sperms (Noguchi and
Miller 2003; Noguchi et al. 2006). As these cones move
along the axonemes from the spermatid nuclei to the end
of the tails, cytoplasm is removed from maturing sperma-
tids and the cyst membrane is remodeled into individual
sperm membranes. Actin cones are composed of two struc-
tural domains, a front meshwork that excludes the cyto-
plasmic contents and a tail of parallel bundles driving the
cone movement (Noguchi et al. 2006, 2008). We have pre-
viously found that localization of MVI to the cones’ fronts
is required for their proper formation and function during
spermatid individualization (Noguchi et al. 2006; Isaji et al.
2011; Lenartowska et al. 2012). In MVI mutants, actin
cone organization is disrupted, leading to cessation of the
individualization process and male infertility. In addition,
when MVI is absent or mislocalized, distribution of other
ABPs is abnormal. Some components usually localized to
the front of cones are spread throughout the cones, suggest-
ing that MVI might function by anchoring specific cargos
in the front meshwork (Rogat and Miller 2002; Noguchi
et al. 2008; Isaji et al. 2011).
MVI is the only known pointed-end-directed actin-based
motor (see review by Buss and Kendrick-Jones 2008). Sim-
ilar to other myosins, MVI has an N-terminal motor domain
(containing an ATP-binding pocket and actin-binding inter-
face), a neck or “lever arm” region (binding two calmo-
dulin or calmodulin-like light chains), and a tail with the
C-terminal cargo-binding domain. MVI also contains a two
unique inserts in the head/neck region, including a 22-aa
Insert2, responsible for minus end-directed movement
along actin. Moreover, four alternative MVI splice variants
have been identified in mammals, containing a large insert,
a small insert, both inserts, or no insert within the C-ter-
minal tail. These isoforms are differentially expressed in
different tissues/cell types and are associated with specific
subcellular compartments and functions. MVI has been
implicated in several processes through functional studies
in flies, worms, and mammals, including clathrin-mediated
endocytosis, Golgi organization and secretion, basolateral
targeting and sorting, cell adhesion and epithelial integrity,
cell migration, actin dynamics, cytokinesis, transcription
(see review by Buss and Kendrick-Jones 2008), and myo-
genesis (Karolczak et al. 2013, 2015). In these seemingly
different cellular processes, MVI may function as a cargo
transporter or as a protein anchor involved in actin organi-
zation/dynamics in specialized cells.
Mutation in the MVI gene in Snell’s waltzer mice (sv/sv
mutants) leads to deafness as a result of neurosensory epi-
thelia degeneration in the inner ear (Avraham et al. 1995;
Self et al. 1999). These mice display also several other
defects in different cell types such as aberrations in Golgi
morphology, reduced secretion, defective endocytosis, and
impaired morphology of brush border enterocytes and hip-
pocampal neurons. In addition, profound fibrosis and both
cardiac and pulmonary vascular endothelial defects were
also observed (Hegan et al. 2012, 2015 and references
therein). Although sv/sv males exhibit somewhat reduced
fertility (Avraham et al. 1995 and our unpublished obser-
vations), no studies have been published that address the
possible role of MVI in mouse spermatogenesis. However,
given that: (1) dynamic actin structures modulated by spe-
cific ABPs determine the success of spermiogenesis in both
invertebrate and mammals, and (2) MVI is a key element
of these functional actin-related protein complexes during
spermatid maturation in Drosophila, we hypothesized that
MVI may be involved in mammalian spermatid maturation.
To test this hypothesis, we examined the MVI expression
and localization using immunocytochemical approaches
complemented with ultrastructural analysis of mouse tes-
tes. To the best of our knowledge, this is the first detailed
study of MVI during spermatid development in mammals.
Materials and methods
Wild-type adult male mice were used in the study. All
animal work, until the mouse tissues were harvested, was
performed at the Nencki Institute of Experimental Biology
(Warsaw, Poland). Animal housing and killing procedures
were performed in compliance with the European Commu-
nities Council directives adopted by the Polish Parliament
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448 Histochem Cell Biol (2017) 148:445–462
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(Act of 15 January 2015 on the use of animals in scientific
investigations). All conducted experiments were repeated a
minimum of three times with similar results.
MVI splice variant analysis by RT‑PCR
To determine the MVI isoform(s) expressed in mouse tes-
tes, organs were dissected from adult males and total RNA
was extracted with TRI Reagent® (Sigma-Aldrich) accord-
ing to the manufacturer’s protocol. First-strand cDNA
synthesis was performed with 1 μg of total RNA, dART
reverse transcriptase, and an oligo(dT)20 primer following
the manufacturer’s instruction (EURx). Nested PCR was
done to identify the splicing isoforms of the mouse MVI.
A 2 μl of first-strand cDNA was used as template for PCR
amplification with OptiTaq DNA polymerase and outer
gene-specific primers (forward 5-GATGAGGCACAG
TA-3). A 2-μl aliquot of the first PCR mixture served as
the template in a second PCR using the inner gene-specific
primers (forward 5-ATGAGGCACAGGGTGACAT-3 and
cycles were as follows: 95 °C for 2 min followed by 35
cycles of 95 °C for 30 s, 57 °C for 30 s, 72 °C for 30 s,
followed by a final extension step of 72 °C for 10 min. The
PCR products were visually inspected on a 2% agarose gel
in TBE buffer.
To verify the specificity of the commercial primary anti-
bodies in mouse used during the subsequent immunolocali-
zation studies, testes, liver, kidneys, heart, lungs, and brain
dissected from the male adult mice were homogenized
in liquid nitrogen, and soluble proteins were extracted in
100 mM Tris–HCl (pH 7.5), 5 mM EDTA, 5 mM EGTA,
10% sucrose, and Complete Protease Inhibitor Cocktail
(Roche) according to the manufacturer’s protocol. The
homogenates were centrifuged at 16,000g for 30 min at
4°C and concentrations of the supernatants were meas-
ured with the Bio-Rad DC Protein Assay according to the
manufacturer’s instructions. Equal amounts of proteins
were separated by electrophoresis on a 7.5% SDS-PAGE
gels and then the proteins were semi-dry transferred to
Amersham PVDF Hybond-P membrane (GE Healthcare).
Blocked blots were probed with a rabbit polyclonal anti-
body against MVI at 1:50 dilution (MVI PAb, Proteus) or a
mouse monoclonal anti-actin antibody (JLA20 MAb, Cal-
biochem) at 1:5000 dilution, washed, and probed with the
corresponding anti-rabbit IgG or anti-mouse IgG/IgM sec-
ondary antibodies, conjugated with horseradish peroxidase
(HRP, Sigma-Aldrich and Merck, respectively). Signals
were detected with the Amersham ECL Advance Western
Blotting Detection Kit according to the manufacturer’s
guidelines (GE Healthcare).
Immunofluorescence studies
Dissected testes were fixed with 4% (v/v) formaldehyde
and 0.25% (v/v) glutaraldehyde in 0.1 M phosphate-buff-
ered saline (PBS, pH 7.4) for 2 h at room temperature
(slight vacuum infiltration). Pre-fixed testes were then cut
into small pieces and further fixation was proceeded over-
night at 4 °C. Fixed samples were washed with PBS, dehy-
drated in a graded series of increasing ethanol concentra-
tions, and embedded in LR Gold resin (Sigma-Aldrich)
according to the standard protocol. Samples were then
sectioned with a diamond knife into semithin sections
(cross sections of seminiferous tubules) and transferred
onto microscope slides covered with Biobond (BB Interna-
tional). For preliminary analysis, sections were stained with
0.1% toluidine blue according to the standard protocol and
observed under the light microscope. For immunolocaliza-
tion (single labeling technique), sections were blocked with
1% (MVI localization) or 3% (actin localization) bovine
serum albumin (BSA, Sigma-Aldrich) for 2 h and then
incubated with the primary MVI PAb or the JLA20 MAb
overnight at 4 °C, at dilutions 1:50 or 1:500, respectively.
Signals were detected using the corresponding anti-rabbit
IgG Cy3® (Sigma-Aldrich) or anti-mouse IgG/IgM Alexa
Fluor 488® secondary antibodies (ThermoFisher). In the
final step, DNA was stained with 2 μg/ml 4,6-diamidino-
2-phenylindole (DAPI, Fluka). Specimens were covered
with MobiGLOW mounting medium (MoBiTec) to prolong
the fluorescence. Negative controls were processed in the
same way except that no primary antibodies were added.
Images were acquired using an Olympus BX50 fluores-
cence microscope, Olympus Xc50 digital color camera, and
cellB software (Olympus Soft Imaging Solutions gmbH).
Immunogold electron microscopy
For detailed ultrastructural analysis, dissected testes were
fixed in 2% (v/v) glutaraldehyde in 0.1 M PBS (pH 7.4)
for 2 h at room temperature (slight vacuum infiltration).
Pre-fixed testes were then cut into small pieces and further
fixation was proceeded overnight at 4 °C. Next, the sam-
ples were post-fixed with 1% (v/v) osmium tetroxide (Pol-
ysciences) in PBS for 2 h at 4 °C, dehydrated in ethanol,
and embedded in Spurr resin (Sigma-Aldrich) according
to the standard protocol. Ultrathin sections (cross sections
of seminiferous tubules) were collected on copper grids,
post-stained with 5% uranyl acetate and 0.4% lead citrate
solutions, and examined on a Joel EM 1010 transmission
electron microscope.
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449Histochem Cell Biol (2017) 148:445–462
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For post-embedding immunogold MVI localization in
developing spermatids, samples were prepared according
to the same protocol as for previous immunolocalization.
Ultrathin cross sections of seminiferous tubules were cut,
collected on nickel grids, and incubated with blocking solu-
tion containing 1% BSA in 0.1 M PBS (pH 7.4) for 5 min
at room temperature. Next, sections were incubated in
1:100 dilution of the MVI PAb in PBS supplemented with
0.1% BSA for 1.5 h, followed by incubation with a gold-
conjugated anti-rabbit IgG 15-nm secondary antibody (BB
International) at dilution 1:100 in PBS with 0.1% BSA for
45 min. Both incubations were proceeded at room tem-
perature. In the negative control, the primary antibody was
omitted. Finally, the sections were post-stained with 2.5%
uranyl acetate and 0.4% lead citrate solutions and exam-
ined by transmission electron microscopy as above.
MVI expression in mouse testis
Four MVI posttranscriptional splice variants (Fig. 2a) can
be expressed in mammals due to the presence of two inserts
[long (LI) and short (SI)] in the C-terminal globular tail:
MVI with LI only, with SI only, with both long and short
(LI + SI) or with no insert (NoI). Given that these isoforms
are differentially expressed in various tissues/cell types
where they have diverse localization and function, we first
examined which of the MVI splice variants were expressed
in mouse testes. To establish this, RT-PCR was performed.
Bands corresponding to SI and NoI MVI tail isoforms were
detected. Thus, these two isoforms are the primary iso-
forms expressed in mouse testis (Fig. 2a, last lane).
We next performed immunofluorescence studies of MVI
and actin distributions during mouse spermatogenesis.
Because we used commercial primary antibodies, west-
ern blot analysis was performed to confirm the specificity
of MVI PAb and JLA20 MAb in mouse testes (Fig. 2b, c).
Our western blots showed that both antibodies recognized
the appropriately sized target proteins in different mouse
tissues, including testes (Fig. 2b, c, lane 1).
Immunofluorescence localizations of MVI (Figs. 3, 4,
red) and actin (Figs. 3, 4, green) were performed to exam-
ine distributions of these proteins in the seminiferous epi-
thelium. As shown in the toluidine-stained semithin cross
sections, seminiferous tubules contain differentiating
generative line cells including spermatogonia, spermato-
cytes, and spermatids, associated with somatic Sertoli cells
(Fig. 3a–g). Successive developmental phases were vis-
ible, including the Golgi phase (Fig. 3b), the acrosome/cap
phase (Fig. 3c), the acrosome/elongation phase (Fig. 3d),
and finally the maturation phase (Fig. 3e, f). MVI was
preferentially localized to the acrosomes that spread over
the spermatid nuclei at the acrosome stage (Fig. 3h, dou-
ble arrows) and to the elongated spermatid heads (Fig. 3h,
arrows). In the developing spermatid acrosomes during the
acrosomal and maturation phases, MVI and actin are both
present (compare Fig. 3h, i, double arrows and arrows),
suggesting that MVI is associated with actin-based pro-
cesses involved in sperm development and maturation.
Actin staining was also found within the cytoplasm of the
seminiferous epithelium cells (Fig. 3i) and accumulated in
the basal ectoplasmic specializations (Fig. 3i, stars) and the
basement membrane of the testis (Fig. 3i, dotted line). No
labeling was observed when the primary antibodies were
omitted (data not shown).
Because the most intensive MVI PAb and JLA20 MAb
immunoreactivities were observed in developing sperma-
tids during the transformation into mature spermatozoa,
further detailed analysis of MVI and actin distributions was
performed during this prolonged cell differentiation stage.
Observations focused on the Golgi phase, the acrosome
cap/elongation phase, and the maturation phase (Fig. 4).
During the early stage of the acrosome biogenesis, the
strongest immunofluorescence signals for MVI and actin
were associated with the nascent acrosome vesicle (Fig. 4a,
b, arrows). However, localization patterns of these two
proteins in developing acrosome were somewhat differ-
ent. While MVI was found in the acrosomal sac with the
exception of the hydrolytic enzyme-rich interior (Fig. 4a,
arrowheads), actin staining was strictly limited to the acro-
some–acroplaxome complex linking developing acrosome
with the spermatid nucleus (Fig. 4b, arrowhead). Both pro-
teins were also localized in some regular spots detectable
within the cytoplasm of round spermatids and adjacent to
Fig. 2 Verification of MVI splice variants expressed in mouse testes
and specificity of used commercial antibodies in these organs. a RT-
PCR products obtained with primers designed to produce MVI frag-
ments containing either a large insert (LI), a small insert (SI), both
inserts (LI + SI) or no insert (NoI) from control plasmids (first four
lanes) and mouse testis (last lane). b, c Immunoblotting of crude
protein extracts from different mouse tissues with MVI PAb (b) and
anti-actin JL20 MAb antibodies (c). Lane 1 testis, 2 liver, 3 kidney, 4
heart, 5 lung, 6 brain
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450 Histochem Cell Biol (2017) 148:445–462
1 3
Fig. 3 Toluidine blue staining
(ag) and immunofluorescence
labeling of MVI (h) and actin
(i) of mouse seminiferous
tubules during spermatogenis.
aSpT spermatid at the acrosome
phase, BV blood vessel, gSpT
spermatid at the Golgi phase,
Lc Leydig cell, mSpT sperma-
tid at the maturation phase, Sc
Sertoli cell, SE seminiferous
epithelium, SpC spermatocyte,
SpG spermatogonium, SpZ
spermatozoa, STL seminifer-
ous tubule lumen. Arrows or
double arrows show MVI (red)
and actin (green) staining in
spermatids at maturation or
acrosome phase, respectively;
stars in i show actin localization
in basal ectoplasmic specializa-
tion; dashed lines basement
membrane. Nuclei are stained
with DAPI (blue). Bars 50 μm
(a), 20 μm (gi), 5 μm (b‑f)
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451Histochem Cell Biol (2017) 148:445–462
1 3
Fig. 4 Immunofluorescence
localization of MVI (red) and
actin (green) in mouse develop-
ing spermatids during the Golgi
phase (gSpT), the acrosomal
phase (aSpT), and the matura-
tion phase (mSpT) as well as in
spermatozoa (SpZ). All other
indications are explained in the
text (see “Results”). Nuclei are
stained with DAPI (blue) or
outlined with dashed line. Bars
5 μm
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452 Histochem Cell Biol (2017) 148:445–462
1 3
the spermatid nuclei (Fig. 4a, b, stars). These MVI/actin-
enriched spots may correspond with the chromatoid bodies.
When the giant acrosome vesicle spread over the nucleus,
MVI and actin were associated with the acrosome (Fig. 4c,
d, arrows). However, the actin labeling was distinctly
stronger in the acrosome–acroplaxome complex and at the
top of the acrosome vesicle (compare Fig. 4c, d, arrow-
heads, and double arrowhead). Similar patterns of MVI
and actin distributions were observed during the acrosome/
elongation and early/late maturation phases (Fig. 4e–h).
When the spermatid nuclei started elongation, MVI was
detectable mainly at the tip region of the acrosome (Fig. 4e,
e, arrows) while actin was clearly visible along the acro-
some–acroplaxome complex (Fig. 4f,f’ arrows), including
the top of the acrosome vesicle (Fig. 4f’, arrowhead) and
the acroplaxome marginal ring region (Figs. 4f, arrow-
head). As spermatid elongation progressed, both proteins
were distributed along the acrosome–acroplaxome complex
(Fig. 4e–h), including the post-acrosomal region (Fig. 4e,
arrowhead, and g–h). In addition, actin staining was
detected inside the nuclei during spermatid elongation and
maturation (Fig. 4f, f, h, h). Finally, just before spermia-
tion, MVI and actin were accumulated around the elon-
gated sperm heads (Fig. 4i, j), particularly in the regions
adjacent to the apical ectoplasmic specializations (Fig. 4i,
j, stars). MVI was also detected inside the sperm nuclei
(Fig. 4i) and in the midpiece of the sperm tail (Fig. 4i,
arrowheads) along with actin (Fig. 4j, sperm nuclei and
mid piece pointed by arrowheads).
Ultrastructural analysis of MVI localization in mouse
We next sought to determine distribution of MVI in devel-
oping spermatids at ultrastructural level using immuno-
electron microscopy with the MVI PAb and a gold-
conjugated secondary antibody. Since the fixation for
immunocytochemistry usually does not preserve some
organelles and cell structures/membranes well enough
for ultrastructural localization, our immunogold experi-
ments were completed using conventionally fixed ultrathin
sections. With this combined strategy, we were able to
distinguish MVI localization in defined sub-domains
of cell compartments (such as the trans-Golgi network,
TGN) or highly specialized cell structures (such as the
Golgi phase
During the Golgi phase, the giant acrosomal vesicle,
which contains electron-dense acrosomal granule mate-
rial, is formed by numerous Golgi-derived vesicles
(Fig. 5a–d), including both uncoated (Fig. 5b–d, arrows)
and clathrin-coated (5b, c, double arrows) vesicles. These
vesicles dock and fuse along the actin-containing plaque
acroplaxome that links developing acrosome to the sper-
matid nuclear envelope (Fig. 5b–d, stars). As shown in
Fig. 5e–g, gold particles representing MVI location were
observed in the Golgi region adjacent to the acrosome-
nuclear pole of round spermatids, including the TGN
enriched with proacrosomal vesicles (Fig. 5e–g, circles).
Immunogold labeling showed MVI localization along the
outer acrosome membrane (Fig. 5e–g, arrows) and in the
inner acrosome membrane–acroplaxome interface (Fig. 5e,
g, stars), including developing acrosome–acroplaxome
leading edges (Fig. 5f, h, stars). Some gold traces were also
found on the surface of the acrosomal granule (Fig. 5e, g,
arrowheads) and occasionally within the acrosome vesicle
space (Fig. 5f, white arrow). We also detected MVI associ-
ated with clathrin-coated vesicles near the acrosomal outer
membrane (Fig. 5h, double arrow). During the Golgi phase,
the electron-dense granulo-filamentous chromatoid body
was clearly visible in the spermatid cytoplasm (Fig. 5i). As
acrosome biogenesis progressed and Golgi-derived vesi-
cles continued to fuse with developing acrosome (Fig. 5j,
arrows), the chromatoid body migrated to the caudal cyto-
plasmic region of the round spermatid and established
contact with the nuclear envelope (Fig. 5k, square brack-
ets). At this stage, MVI was still apparent in the sperma-
tid cytoplasm around the acrosome (Fig. 5l, circles) and in
the acroplaxome (Fig. 5l, star). However, some gold traces
were also detected inside the acrosome vesicle; they were
localized near the acrosome membrane (Fig. 5l, arrows)
or on the periphery of the acrosomal granule (Fig. 5l,
arrowheads). In addition, a strong MVI immunoreactiv-
ity was associated with electron-dense fibrillar material of
the chromatoid body near the spermatid nucleus, as well
as adjacent to the spermatid nucleus (Fig. 5m, n, respec-
tively). Numerous gold traces were also found within the
chromatin (Fig. 5n, arrows), while only few were localized
in the posterior region of the spermatid cytoplasm (Fig. 5m,
Acrosome cap/elongation phase
During the acrosome phase, the acrosome vesicle spreads
over the spermatid nucleus to form a distinct cap and the
spermatid initiates its elongation (Fig. 6). At the early cap
subphase, the outer acrosomal membrane was strongly
pleated, demonstrating that Golgi-derived vesicles still
undergo fusion during this period (Fig. 6a, b, arrows). We
could also discern the inner acrosomal membrane-associ-
ated plaque at the leading edge of the acrosome–acroplax-
ome complex (Fig. 6a, c, boxed regions). As before, MVI
was present in the TGN (Fig. 6d, circles), enriched with
the clathrin-coated vesicles that undergo fusion with the
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453Histochem Cell Biol (2017) 148:445–462
1 3
Fig. 5 Ultrastructural analysis (ad, ik) and immunogold localiza-
tion (eh, ln) of MVI in developing mouse spermatids during the
Golgi phase. ag acrosomal granule, av acrosome vesicle, cb chro-
matoid body, cy cytoplasm, g Golgi complex, gSpT spermatid at the
Golgi phase, m mitochondria, n nucleus. Square brackets in k show
the contact region between the nucleus and the chromatoid body.
Dotted lines mark a boundary between the spermatid cytoplasm, the
acrosomal vesicle and the nucleus (h) or between the spermatid cyto-
plasm and the nucleus (n). All other indications are explained in the
text (see “Results”). Bars 1 μm (a, i), 500 nm (bg, in), 250 nm (h)
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454 Histochem Cell Biol (2017) 148:445–462
1 3
acrosome vesicle (Fig. 6d, arrows). MVI was also localized
in the outer acrosomal membrane (Fig. 6d, e arrowheads).
In the acroplaxome marginal ring zone, gold traces were
found in the interspace between the outer and inner acroso-
mal membranes (Fig. 6e, f arrows) and in the acroplaxome
(Fig. 6e, f stars).
As the acrosome phase progressed, distinct struc-
tural elements of the spermatid anterior pole were
distinguishable (Fig. 6g), such as the spermatid mem-
brane, the acrosome cap, the acrosome outer and inner
membranes, and the acroplaxome (Fig. 6g, arrow). At
higher magnification, we could also discern the acro-
somal electron-dense plaque in the acroplaxome mar-
ginal ring (Fig. 6h, i, arrows). The Sertoli cell membrane
(Fig. 6g) together with adjacent ectoplasmic F-actin bun-
dles (Fig. 6g, i, asterisks) and the endoplasmic reticulum
Fig. 6 Ultrastructural analysis (ac, gi) and immunogold localiza-
tion (df, jm) of MVI in developing mouse spermatids during the
acrosome phase. apx acroplaxome, aSpT spermatid at the acrosome
phase, av acrosome vesicle, cy cytoplasm, er endoplasmic reticulum,
g Golgi complex, iam inner acrosome membrane, m mitochondria, n
nucleus, oam outer acrosome membrane, Sc Sertoli cell, Scm Sertoli
cell membrane, SpTm spermatid membrane. Boxed regions in a and c
include the acrosome–acroplaxome marginal ring regions. All other
indications are explained in the text (see “Results”). Bars 1 μm (a),
500 nm (b, d, jl), 250 nm (gi, m), 200 nm (c, e, f)
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455Histochem Cell Biol (2017) 148:445–462
1 3
cisternae (Fig. 6i, er ) were also clearly visible along the
spreading acrosome. During this subphase, a pattern simi-
lar to the previously described MVI localization was
observed, with immunogold staining present in the outer/
inner acrosome membranes (Fig. 6j–m, black arrows) and
in the acroplaxome (Fig. 6j–m, white arrows), including its
marginal ring (Fig. 6k, double arrow). We also found MVI
in the F-actin-containing apical ectoplasmic specializa-
tions (Fig. 6j, k arrowheads) and the Sertoli cell cytoplasm
(Fig. 6j, circles). Interestingly, association of MVI with
actin filaments in the apical ectoplasmic specializations
was not always apparent (compare Fig. 6j, k). This associa-
tion became more intense as the spermatid matured (see the
next results). Gold traces were still present within the sper-
matid chromatin (Fig. 6k, m, circles).
Maturation phase
During the maturation phase, nuclear shaping based on
cooperative exogenous and endogenous clutching forces
of the Sertoli cell apical ectoplasmic specializations and
the acrosome–acroplaxome–manchette complex coincides
with spermatid chromatin condensation, sperm tail forma-
tion, and residual cytoplasm removal from the future sperm
(Fig. 7a). The apical pole of the elongating and condens-
ing spermatid nucleus (Fig. 7a–k) is capped by the homog-
enous acrosomal vesicle (Fig. 7a, boxed region, and b, g,
k) tightly juxtaposed to the acroplaxome and to the F-actin
hoops (marked by asterisks) of the apical ectoplasmic
specialization in the peri-acrosomal (Fig. 7d, h), the sub-
acrosomal (Fig. 7c, e, j), and the post-acrosomal (Fig. 7f,
i) regions. Highly specialized structures such as the acro-
plaxome marginal ring (Fig. 7b, k, arrowheads and 7f, i,
arrows), the manchette (Fig. 7b, i, k), the manchette perinu-
clear ring (Fig. 7b, f, i, k) as well as developing sperm tail
(Fig. 7g) are clearly visible during the maturation phase.
Cross sections of the flagellum show mitochondria gath-
ered around the axoneme (Fig. 7l) to form the mitochon-
drial sheath in the sperm midpiece (Fig. 7m). In contrast,
the principal piece of the sperm tail lacks mitochondria.
However, fibrous material is present surrounding this tail
region (Fig. 7n, arrows).
As spermatid shaping progressed and the manchette
developed just below the marginal ring of the acroplax-
ome, the MVI localization pattern remained similar to pre-
vious stages, predominantly detected in the F-actin hoops
(marked by asterisks) surrounding the apical pole of the
elongating nuclei (Fig. 8a–d, g, h, l, black arrows), the
outer/inner acrosomal membranes (Fig. 8a–d, f, g, l, black
arrowheads), and the acroplaxome interface (Fig. 8a–d,
g–i, white arrows), including its marginal ring (Fig. 8d–f,
j, k, white arrowheads). Some gold traces were also local-
ized throughout the manchette flanking the elongating
spermatid nucleus (Fig. 8e, f, circles) and in the perinu-
clear ring (Fig. 8f, j, double arrows). Interestingly, sperma-
tid chromatin exhibited intense MVI immunoreactivity as
condensation progressed (Fig. 8d, circles, and g–l). MVI
was also found in the endoplasmic reticulum adjacent to
the F-actin hoops within the Sertoli cytoplasm (Fig. 8b,
white stars) and in developing flagellum (Fig. 8m–o). As
expected, the axoneme was devoid of immunolabeling, but
some gold traces were associated with the mitochondrial
sheath in the midpiece of the flagellum (Fig. 8m–o, arrows)
or localized at the axoneme periphery near the outer dense
fibers (Fig. 8m–o, black arrowheads), where they occasion-
ally form concentrated patches (Fig. 8m, double arrow).
It was also noticeable that during the tail formation,
MVI was detected in the tail residual cytoplasm (Fig. 8n,
white arrowheads). MVI was absent in both the principal
(Fig. 8p) and the end (Fig. 8p, arrow) pieces of the sperm
tail. Control sections in which no MVI PAb was used were
devoid of labeling (data not shown).
Taken together, our immunocytochemical studies con-
firm MVI localization in highly specialized F-actin-con-
taining structures present during mouse spermiogenesis,
including the acrosome–acroplaxome–manchette complex
and the apical ectoplasmic specializations surrounding the
head region of the elongating spermatids. MVI was also
prominently associated with the Golgi (including TGN),
migrating chromatoid body, the nucleus, and mitochon-
drial sheath of the flagellum midpiece during the sperm tail
development. A schematic summary of the obtained results
is shown in Fig. 9.
Here, we present the first evidence that the unconventional
actin-based motor protein, MVI, may be involved in some
key events of spermiogenesis in mammals including: (1)
acrosome biogenesis, (2) spermatid elongation, and (3)
spermiation. We have also found that MVI-SI and MVI-
NoI splice variants are the predominant isoforms expressed
in mouse testes. Our findings are consistent with an earlier
report showing that these MVI isoforms are expressed in rat
testes (Buss et al. 2001). MVI immunolocalization shown
here confirm that these isoforms are present in developing
mouse spermatids and the Sertoli cells adjacent to them.
Thus, we conclude that MVI with no LI splice variants are
preferentially expressed in the seminiferous epithelia dur-
ing spermiogenesis in mammals.
MVI’s possible roles in acrosome biogenesis in mouse
Immunofluorescence and immunogold studies demon-
strated the association of MVI with the anterior pole of
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456 Histochem Cell Biol (2017) 148:445–462
1 3
developing spermatids, including the Golgi stacks and
uncoated/clathrin-coated vesicles as well as the nascent,
and then maturing acrosome–acroplaxome complex. These
findings suggest that MVI may be involved in acrosomo-
genesis in two different ways. First, MVI association with
vesicles is consistent with the idea that MVI plays a trans-
port role in proacrosomal vesicle trafficking. Second, MVI
association with the acrosome–acroplaxome complex sug-
gests an anchoring role during the acrosome development.
Previous evidence indicates that proacrosomal vesi-
cles utilize two different routes to reach their docking
sites in the acroplaxome: a microtubule route, involving
kinesin KIFC1 (Yang and Sperry 2003) and an F-actin
route, involving MVa and Rab27a/b small GTPases that are
known to play a general role in exocytosis (Kierszenbaum
et al. 2003a, 2004). Moreover, it is believed that acrosome
biogenesis involves both the anterograde vesicular transport
from the TGN and the retrograde transport based on endo-
cytic pathway (Ramalho-Santos et al. 2001 and see review
by Berruti and Paiardi 2011). One of the best candidates to
facilitate transport of vesicles derived from the Golgi and
from the plasma membrane is MVI, which moves toward
the minus end of actin filaments and is involved in both
clathrin-mediated endocytosis and secretion (see review
Fig. 7 Ultrastructural analysis of developing mouse spermatids dur-
ing the maturation phase. apx acroplaxome, av acrosome vesicle,
ax axoneme, c centriole, dmp developing midpiece of the sperm
tail, er endoplasmic reticulum, m mitochondria, mp midpiece of the
sperm tail, mSpT spermatid at the maturation phase, mt manchette, n
nucleus, pp principal piece of the sperm tail, pr perinuclear ring of the
manchette, Sc Sertoli cell. Boxed region in a includes the spermatid
head with spreading acrosome. All other indications are explained in
the text (see “Results”). Bars 2 μm (a), 1 μm (b, c), 500 nm (gh,
ln), 200 nm (df, ik)
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457Histochem Cell Biol (2017) 148:445–462
1 3
by Buss and Kendrick-Jones 2008). Interestingly, MVI-SI
and MVI-NoI splice variants, the isoforms we detected in
mouse testis, have previously been shown to play a role in
transport of clathrin-coated as well as uncoated vesicles in
different mammalian cell lines (Aschenbrenner et al. 2003;
Dance et al. 2004; Naccache et al. 2006; Au et al. 2007;
Chibalina et al. 2007; Puri 2009; Majewski et al. 2010;
Bond et al. 2012; Tumbarello et al. 2012; Tomatis et al.
2013). Given that plus ends of actin filaments typically
are positioned towards cell membranes (Cramer 1999),
MVI acting as a minus-end-directed motor would move
vesicles away from the Golgi surface to the center of the
cell. MVI direct binding to multiple proteins involved in
different steps along endocytic pathway (Chibalina et al.
2007; Spudich et al. 2007; Tumbarello et al. 2012 and see
review by Tumbarello et al. 2013) and in Golgi complex
Fig. 8 Immunogold localization of MVI in developing mouse sper-
matids during the maturation phase. apx acroplaxome, av acrosome
vesicle, ax axoneme, dmp developing midpiece of the sperm tail, ep
end piece of the sperm tail, er endoplasmic reticulum, m mitochon-
dria, mp midpiece of the sperm tail, mSpT spermatid at the maturation
phase, mr marginal ring of the acroplaxome, mt manchette, n nucleus,
pp principal piece of the sperm tail, pr perinuclear ring of the man-
chette, Sc Sertoli cell. All other indications are explained in the text
(see “Results”). Bars 250 nm
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458 Histochem Cell Biol (2017) 148:445–462
1 3
(Sahlender et al. 2005) is consistent with this hypothesis.
Additionally, a central role of MVI in exocytosis, includ-
ing the Golgi ribbon formation and sorting of proteins into
different cargo vesicles has been suggested. Specifically,
MVI concentration in the TGN is thought to facilitate post-
Golgi secretion, including vesicle formation and budding,
and then fusion of secretory vesicles with the plasma mem-
brane during the final stage of exocytosis (Buss et al. 1998;
Warner et al. 2003; Au et al. 2007; Majewski et al. 2010;
Bond et al. 2011; Tomatis et al. 2013). Thus, association of
MVI with the Golgi apparatus, coated, and uncoated ves-
icles argue that MVI may be involved in the anterograde
and/or the retrograde vesicular transport pathways during
acrosome biogenesis in mouse.
Other hypotheses for MVI’s role are possible given pre-
vious work that support MVI ability to anchor various car-
gos to actin. For example, MVI-SI isoform controls neuro-
exocytosis in PC12 cells by tethering secretory granules to
the actin network near the target plasma membrane (Toma-
tis et al. 2013). Given that MVa has been also implicated in
neurosecretion (Tomatis et al. 2013 and references therein),
these authors suggest synergistic roles for these two motor
proteins, with MVI acting in recruitment and retention of
secretory granules to the cortical actin network and MVa
involved in their active transport toward the plasma mem-
brane along actin filaments. Our results coupled with the
previously documented role of MVa in acrosome formation
could suggest a similar cooperation of these two molecular
motors during acrosomogenesis in mouse.
In support of an anchoring role for MVI during acro-
some development, this protein is strongly associated
with the acrosome–acroplaxome complex, which is
important for anchoring the acrosome to the nucleus.
MVI is specifically located in developing acrosome–acro-
plaxome complex and its leading edges, including the
outer/inner acrosomal membranes, the space between the
acrosome membrane and the internal granule, the acro-
plaxome, and the acroplaxome marginal link. Such local-
izations suggest that MVI could help to mediate anchor-
ing of the acrosome vesicle to the spermatid nucleus
via the acroplaxome. The molecular basis of acrosome
biogenesis, and particularly its attachment and spread
over the nucleus, is poorly understood and to date only
one protein, a transmembrane protein Dpy19l2, has been
suggested to link the acrosome to the nuclear envelope
(Pierre et al. 2012). However, the acroplaxome contains
F-actin and actin-associated proteins such as cortac-
tin and profilin-3 (Obermann et al. 2005; Kierszenbaum
et al. 2008; Behnen et al. 2009), and potentially other
ABPs (including motor proteins) which might modu-
late actin dynamics during acrosomogenesis and may be
responsible for linking developing acrosome to the sper-
matid nucleus. A role for MVI in this linking is consistent
with its crucial role in cell membrane tethering to cortical
F-actin during development of the cochlear hair cells in
the inner ear and the brush border cells in mammals (see
reviews by Frank et al. 2004; Crawley et al. 2014).
We confirmed that actin accumulates in the acrosome–
acroplaxome complex from the Golgi stage to the matura-
tion stage. In contrast, MVI was initially localized in the
nascent acrosomal sac, including its outer membrane, the
inner membrane–acroplaxome site, and the interspace
between the acrosomal membrane and the granule inside.
MVI may be accumulated on the acrosomal membrane as a
Fig. 9 Schematic representa-
tion of MVI distribution (black
dots) during mouse spermiogen-
esis. aSpT round spermatid at
the acrosome phase, gSpT round
spermatid at the Golgi phase,
mSpT elongated spermatid at
the maturation phase
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459Histochem Cell Biol (2017) 148:445–462
1 3
result of participating in transport of Golgi vesicles, before
gaining access to the acroplaxome. At this time, we have no
hypotheses about a potential role of MVI inside the acro-
some vesicle.
An additional anchoring or structural role of MVI has
been suggested in Golgi apparatus organization. In fibro-
blasts from sv/sv mice or cells depleted of MVI or its bind-
ing partner, optineurin, Golgi complexes were significantly
altered in morphology and reduced in size compared to the
wild-type cells (Warner et al. 2003; Sahlender et al. 2005;
Majewski et al. 2011; Karolczak et al. 2015). In developing
mouse spermatids at the Golgi stage, we found MVI asso-
ciated with not only the TGN, but distributed throughout
the Golgi apparatus from the cis to the trans-faces. This
observation is consistent with potential structural role for
Golgi-localized MVI during mouse spermiogenesis. How-
ever, we have not been able to confirm so far that optineu-
rin is a MVI binding partner in developing mouse sperma-
tids (data not shown).
MVI may play a role in nuclear shaping during the
maturation stage
The association of MVI with the actin structures that
mediate sperm nuclear shaping suggests an important
role during the elongation stage. Nuclear shaping is a
critical event during sperm development involving the
condensation of the future sperm nucleus from a spheri-
cal configuration into an elongated structure. Two struc-
tural components of maturing spermatids are critical in
this process—the acroplaxome marginal ring and the
manchette, both containing actin filaments. These struc-
tures together with F-actin hoops, the main cytoskeletal
element of the Sertoli cell apical ectoplasmic specializa-
tions, seem to stabilize the spermatid head as it is under-
going elongation and to mediate the sperm–Sertoli cell
association during spermiation (see reviews by Kiersze-
nbaum and Tres 2004; Kierszenbaum et al. 2011; Sun
et al. 2011; Qian et al. 2014; Xiao et al. 2014). The acro-
plaxome marginal ring, the manchette, and actin dynam-
ics within them seem to be important in modulation of
clutching forces that mediate spermatid head elongation.
On the other hand, the Sertoli cell actin hoops surround-
ing the elongating spermatid head also are important
for nuclear shaping. Numerous testis-specific ABPs that
regulate actin assembly/disassembly have been identified
in the acroplaxome–manchette complex and the apical
tubulobulbar–Sertoli cell ectoplasmic specialization com-
plexes in mammals, including profilin-3, gelsolin, Eps8,
Arp2/3, drebrin E, Rai14, and palladin (Braun et al. 2002;
Guttman et al. 2002; Lie et al. 2009, 2010; Li et al. 2011;
Qian et al. 2013a, b). The components and mechanism
of action of these actin-regulating protein complexes
are poorly understood. We postulate that a key element
of these protein complexes may be MVI. This protein is
continuously associated with the acroplaxome–manchette
complex from the early acrosome stage to the late mat-
uration stage, and with the actin hoops surrounding the
elongating spermatid/sperm head. Phosphorylated cortac-
tin is also present in both the acroplaxome and the Sertoli
cell hoops whereas non-phosphorylated cortactin pre-
dominates in the manchette (Kierszenbaum et al. 2008).
Phosphorylated cortactin interacts with the actin nuclea-
tor, Arp2/3 complex, that allows actin remodeling from
bundled to branched configuration (Weaver et al. 2001;
Young et al. 2012 and see reviews by Cheng and Mruk
2011; Qian et al. 2014). Our previous studies showed that
during Drosophila spermatid individualization MVI colo-
calizes with cortactin and Arp2/3 in the front meshwork
of actin cones, and that lack of functional MVI results in
impaired distribution of these ABPs (Rogat and Miller
2002; Noguchi et al. 2008; Isaji et al. 2011). One model
for the MVI role in actin cone structure stabilization is
anchoring specific cargos to the front meshwork. We pos-
tulate that MVI may play a similar role in developing
mouse spermatids by anchoring actin regulators that are
responsible for mediating the actin dynamics required for
forces involved in nuclear shaping.
Possible additional roles for MVI during sperm
MVI is associated with the chromatoid body at the Golgi
stage, a cloud-like structure packed with RNA and many
of RNA-binding and RNA-processing proteins (see
review by Meikar et al. 2011). It also progressively accu-
mulates in the spermatid nucleus from the Golgi stage
to the maturation stage. In addition, MVI was present in
condensing spermatid nucleus. Previous work showed
that MVI colocalizes with RNA polymerase II and newly
synthesized mRNA transcripts (Jung et al. 2006; Vreugde
et al. 2006) and may play a role in nucleo-cytoplasmic
trafficking (Majewski et al. 2010). Thus, additional roles
for MVI are possible in nuclear processes. In addition,
association with the mitochondrial sheath may suggest a
role in formation of this structure. Much less is known
about binding proteins that might mediate MVI associa-
tion with and specific functional roles for MVI in such
structures and compartments. Finally, actin structures
are important for movement of spermatids across the
seminiferous epithelium and for spermiation, and MVI
association with those structures may also be important.
Additional studies are required to define the processes
in which MVI plays a role and its precise function(s)
throughout sperm differentiation.
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460 Histochem Cell Biol (2017) 148:445–462
1 3
Based on the strong and prominent association of MVI
with a number of structures important for key steps of sper-
miogenesis, we suggest dual roles for MVI in this process.
First, MVI may play a transport role during formation the
Golgi–acrosome–acroplaxome–manchette complex. Sec-
ond, MVI may play an anchoring role in maintaining the
morphology of the Golgi complex and may help tether actin
regulators to several unique F-actin-containing structures
involved in acrosomal attachment to the nucleus, acroso-
mal cap formation, and nuclear shaping: the acroplaxome,
the manchette and the apical ectoplasmic specializations of
Sertoli cells. In addition, MVI along with other ABPs may
play additional roles in a variety of other processes since
MVI was also observed to be associated with chromatoid
bodies, mitochondrial sheath of the tail midpiece, and the
condensing nucleus. Future studies using MVI mutant
males and identification of the testis-specific MVI binding
partner/s are needed to verify these hypotheses.
Acknowledgements We would like to thank Vira Chumak and
Małgorzata Suszek from the Laboratory of Molecular Basis of Cell
Motility of the Nencki Institute for their invaluable help with animals
and the PCR technique. This project was supported by statutory funds
from Ministry of Science and Higher Education (PL) for (1) Nicolaus
Copernicus University in Torun´, including a grant for young scientist
(to PZ) and (2) the Nencki Institute.
Author contributions Conceived and designed the experiments: PZ,
ML, and MJR. Performed the experiments: PZ and RL. Analyzed
data: PZ, ML, RL, MJR, and KGM. Wrote the paper: PZ, ML, and
Compliance with ethical standards
Conflict of interest The authors declare that no competing interests
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://crea-, which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
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... Myosin VIIa (MYO7a) has been implicated in the adhesion and transport of developing spermatids across the rat seminiferous epithelium and its depletion perturbs the spatiotemporal expression of different ABPs involved in spermiogenesis (Velichkova et al. 2002;Wen et al. 2019). Finally, gene expression profiling in rodent tissues has revealed that transcripts of the minus-directed myosin VI (MYO6) are present in mouse testes (Avraham et al. 1995) and that two different MYO6 isoforms are expressed in mouse and rat testes (Buss et al. 2001;Zakrzewski et al. 2017). ...
... MYO6 is broadly expressed in different animal tissues including the testes in humans, rodents, worms and Drosophila (Kelleher et al. 2000;Kellerman and Miller 1992;Hasson and Mooseker 1994;Avraham et al. 1995Avraham et al. , 1997. Moreover, PCR analysis demonstrated that two MYO6 isoforms (the SI and NI) are expressed in rodent testes (Buss et al. 2001;Zakrzewski et al. 2017) and are associated with several key actin-rich structures throughout sperm development and maturation in mice (Figs. 5 and 6); (Zakrzewski et al. 2017(Zakrzewski et al. , 2020a. During the Golgi phase, MYO6 is present at/around the Golgi complex adjacent to the acrosome-nuclear pole, including the trans-Golgi network and uncoated and as well as coated vesicles and at the inner acrosome membrane-acroplaxome interface (Fig. 5a-d); (Zakrzewski et al. 2017(Zakrzewski et al. , 2020a. ...
... MYO6 is broadly expressed in different animal tissues including the testes in humans, rodents, worms and Drosophila (Kelleher et al. 2000;Kellerman and Miller 1992;Hasson and Mooseker 1994;Avraham et al. 1995Avraham et al. , 1997. Moreover, PCR analysis demonstrated that two MYO6 isoforms (the SI and NI) are expressed in rodent testes (Buss et al. 2001;Zakrzewski et al. 2017) and are associated with several key actin-rich structures throughout sperm development and maturation in mice (Figs. 5 and 6); (Zakrzewski et al. 2017(Zakrzewski et al. , 2020a. During the Golgi phase, MYO6 is present at/around the Golgi complex adjacent to the acrosome-nuclear pole, including the trans-Golgi network and uncoated and as well as coated vesicles and at the inner acrosome membrane-acroplaxome interface (Fig. 5a-d); (Zakrzewski et al. 2017(Zakrzewski et al. , 2020a. ...
Full-text available
Spermiogenesis is the final stage of spermatogenesis, a differentiation process during which unpolarized spermatids undergo excessive remodeling that results in the formation of sperm. The actin cytoskeleton and associated actin-binding proteins play crucial roles during this process regulating organelle or vesicle delivery/segregation and forming unique testicular structures involved in spermatid remodeling. In addition, several myosin motor proteins including MYO6 generate force and movement during sperm differentiation. MYO6 is highly unusual as it moves towards the minus end of actin filaments in the opposite direction to other myosin motors. This specialized feature of MYO6 may explain the many proposed functions of this myosin in a wide array of cellular processes in animal cells, including endocytosis, secretion, stabilization of the Golgi complex, and regulation of actin dynamics. These diverse roles of MYO6 are mediated by a range of specialized cargo-adaptor proteins that link this myosin to distinct cellular compartments and processes. During sperm development in a number of different organisms, MYO6 carries out pivotal functions. In Drosophila, the MYO6 ortholog regulates actin reorganization during spermatid individualization and male KO flies are sterile. In C. elegans, the MYO6 ortholog mediates asymmetric segregation of cytosolic material and spermatid budding through cytokinesis, whereas in mice, this myosin regulates assembly of highly specialized actin-rich structures and formation of membrane compartments to allow the formation of fully differentiated sperm. In this review, we will present an overview and compare the diverse function of MYO6 in the specialized adaptations of spermiogenesis in flies, worms, and mammals.
... In addition to myosin Va and VIIa, myosin VI (MYO6) is also expressed in mouse testes, where it is present in actin-rich structures during acrosome biogenesis such as the Golgi complex and the acrosome-acroplaxome complex [ [12]; summarized in Figure 1]. MYO6 is expressed in most cell types and tissues, and since it is the only pointed-end-directed actin-based motor, it participates in several cellular processes including endocytosis, Golgi organization and function, basolateral targeting and sorting in the secretory pathway, epithelial integrity, and cell adhesion and migration [13][14][15]. ...
... Our previous results have shown that in wild-type mice, MYO6 is present at actin-rich structures involved in acrosome formation, such as the Golgi complex and the acrosome-acroplaxome complex ( Figure 1) [12]. We therefore analyzed here whether this motor protein has a role in acrosomogenesis by testing whether MYO6 is important for maintaining the morphology of the testis-specific structures during acrosome biogenesis. ...
... MYO6 together with its binding partner TOM1/L2 are localized at the Golgi complex and developing acrosome Acrosome biogenesis involves the delivery of proacrosomal vesicles from either the Golgi complex or from late endosomes/lysosomes to the nascent acrosome [28]. We previously demonstrated that MYO6 localizes to the Golgi stacks during the acrosomogenesis in wildtype mouse spermatids [12]. Indeed, our present immunofluorescent analysis shows that MYO6 is associated with the Golgi complex including the cis-Golgi domain (Figure 4A.a-a') and the region corresponding to the trans-Golgi network (Figure 4A.a-a") in sv/+ spermatids, whereas no signal was observed in sv/sv cells confirming the specificity of our MYO6 antibodies ( Figure 4A.b-b"). ...
Full-text available
During spermiogenesis in mammals actin filaments and a variety of actin-binding proteins are involved in the formation and function of highly specialized testis-specific structures. Actin-based motor proteins, such as myosin Va and VIIa, play a key role in this complex process of spermatid transformation into mature sperm. We have previously demonstrated that myosin VI (MYO6) is also expressed in mouse testes. It is present in actin-rich structures important for spermatid development, including one of the earliest events in spermiogenesis-acrosome formation. Here, we demonstrate using immunofluorescence, cytochemical and ultrastructural approaches that MYO6 is involved in maintaining the structural integrity of these specialized actin-rich structures during acrosome biogenesis in mouse. We show that MYO6 together with its binding partner TOM1/L2 is present at/around the spermatid Golgi complex and the nascent acrosome. Depletion of MYO6 in Snell's waltzer mice causes structural disruptions of the Golgi complex and affects the acrosomal granule positioning within the developing acrosome. In summary, our results suggest that MYO6 plays an anchoring role during the acrosome biogenesis mainly by tethering of different cargo/membranes to highly specialized actin-related structures..
... In somatic cells, unconventional myosins function in organelle and vesicle transport and mechanotethering (Bar-Shira Maymon et al., 2000;Dolezel et al., 2008), cell migration and mitosis (Buss et al., 2004;Chibalina et al., 2009 anticipated that myosins will take part in a similar range of functions. Specifically, at least two myosin motor proteins play roles in haploid germ cell development (as detailed within this review) across several species: myosin Va in mice (Kierszenbaum et al., 2004), Drosophila melanogaster (Mermall et al., 2005), rats (Kierszenbaum et al., 2003b) and crabs (Sun et al., 2010;Ma et al., 2017) and myosin VI in mice (Zakrzewski et al., 2017), Drosophila (Rogat and Miller, 2002) and Caenorhabditis elegans (Hu et al., 2019). ...
... While largely unexplored in spermatids, it is anticipated that similar motor-based mechanisms regulate Golgi organisation and vesicle formation. The kinesin KIF1C, myosins VI and Va and the myosin Va adapter RAB27a/b have all been localised to the spermatid Golgi (Yang and Sperry, 2003;Kierszenbaum et al., 2004;Zakrzewski et al., 2017). As detailed below, however, these motor proteins also decorate the surface of Golgi-derived pro-acrosomal vesicles, and the expression of myosin Va and RAB27a/b within the Golgi appears to be restricted to the region wherein pro-acrosomal vesicles accumulate. ...
... Actin has also been detected at the manchette (Kierszenbaum et al., 2003b), but its role in protein/vesicle transport is poorly understood. Myosin Va-decorated vesicles have been detected in association with manchette MTs in the rat (Kierszenbaum et al., 2003b), and myosin VI is localised to the manchette in the mouse (Zakrzewski et al., 2017), implying actin-myosin networks function in the transport of vesicle and/or protein complexes along the manchette. ...
Background: The precise movement of proteins and vesicles is an essential ability for all eukaryotic cells. Nowhere is this more evident than during the remarkable transformation that occurs in spermiogenesis-the transformation of haploid round spermatids into sperm. These transformations are critically dependent upon both the microtubule and the actin cytoskeleton, and defects in these processes are thought to underpin a significant percentage of human male infertility. Objective and rationale: This review is aimed at summarising and synthesising the current state of knowledge around protein/vesicle transport during haploid male germ cell development and identifying knowledge gaps and challenges for future research. To achieve this, we summarise the key discoveries related to protein transport using the mouse as a model system. Where relevant, we anchored these insights to knowledge in the field of human spermiogenesis and the causality of human male infertility. Search methods: Relevant studies published in English were identified using PubMed using a range of search terms related to the core focus of the review-protein/vesicle transport, intra-flagellar transport, intra-manchette transport, Golgi, acrosome, manchette, axoneme, outer dense fibres and fibrous sheath. Searches were not restricted to a particular time frame or species although the emphasis within the review is on mammalian spermiogenesis. Outcomes: Spermiogenesis is the final phase of sperm development. It results in the transformation of a round cell into a highly polarised sperm with the capacity for fertility. It is critically dependent on the cytoskeleton and its ability to transport protein complexes and vesicles over long distances and often between distinct cytoplasmic compartments. The development of the acrosome covering the sperm head, the sperm tail within the ciliary lobe, the manchette and its role in sperm head shaping and protein transport into the tail, and the assembly of mitochondria into the mid-piece of sperm, may all be viewed as a series of overlapping and interconnected train tracks. Defects in this redistribution network lead to male infertility characterised by abnormal sperm morphology (teratozoospermia) and/or abnormal sperm motility (asthenozoospermia) and are likely to be causal of, or contribute to, a significant percentage of human male infertility. Wider implications: A greater understanding of the mechanisms of protein transport in spermiogenesis offers the potential to precisely diagnose cases of male infertility and to forecast implications for children conceived using gametes containing these mutations. The manipulation of these processes will offer opportunities for male-based contraceptive development. Further, as increasingly evidenced in the literature, we believe that the continuous and spatiotemporally restrained nature of spermiogenesis provides an outstanding model system to identify, and de-code, cytoskeletal elements and transport mechanisms of relevance to multiple tissues.
... These findings allow us to hypothesize that UBE2J1 and RNF133 are the first ER-localized E2 and E3 transmembrane proteins, respectively, that function in ERAD during spermiogenesis and for the formation of normal spermatozoa. During spermiogenesis, spermatozoa are dramatically remodeled architecturally into a morphology required for proper fertilization [16]. This includes mitochondrial rearrangement around the flagella at the midpiece of the tail, and proteins, organelles, and bulk cytoplasm that are no longer needed are discarded through the extrusion of cytoplasmic droplets, which are eventually removed from the sperm head and neck region [17,18]. ...
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Background Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. Results We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 ( Rnf133 ); RING finger protein 148 ( Rnf148 ); RING finger protein 151 ( Rnf151 ); and Zinc finger SWIM-type containing 2 ( Zswim2 )]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148 , Rnf151 , and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. Conclusions Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.
... In early spermiogenesis, the Golgi apparatus secretes numerous small pro-acrosomal vesicles or pro-acrosomal granules that gradually migrate into the apical cytoplasm (Berruti and Paiardi 2011). Motor proteins such as kinesin (KIFC1, kinesin-7, KIF3A, and KIF3B) and myosin (myosin Va and myosin VI) have been reported to function in vesicle trafficking from the Golgi to the acrosome during acrosome biogenesis (Yang and Sperry 2003;Kierszenbaum et al. 2003;Zhao et al. 2017;Zakrzewski et al. 2017;She et al. 2020). Relevant evidence indicates that cytoplasmic dynein is involved in the vesicle transport of the Golgi apparatus (Papoulas et al. 2005;Horgan et al. 2010a, b), we speculated that dynein may function in acrosome biogenesis through vesicle transport, so we performed immunofluorescence analysis of Pt-DHC and the Golgi marker GM130. ...
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The mechanism of acrosome formation in the crab sperm is a hot topic in crustacean reproduction research. Dynein is a motor protein that performs microtubule-dependent retrograde transport and plays an essential role in spermatogenesis. However, whether cytoplasmic dynein participates in acrosome formation in the crab sperm remains poorly understood. In this study, we cloned the cytoplasmic dynein intermediate chain gene (Pt-DIC) from Portunus trituberculatus testis. Pt-DIC is composed of a p150glued-binding domain, a dynein light chain (DLC)-binding domain, and a dynein heavy chain (DHC)-binding domain. The Pt-DIC gene is widely expressed in different tissues, showing the highest expression in the testis, and it is expressed in different stages of spermatid development, indicating important functions in spermatogenesis. We further observed the colocalization of Pt-DIC and Pt-DHC, Pt-DHC and tubulin, and Pt-DHC and GM130, and the results indicated that cytoplasmic dynein may participate in nuclear shaping and acrosome formation via vesicle transport. In addition, we examined the colocalization of Pt-DHC and a mitochondrion (MT) tracker and that of Pt-DHC and prohibitin (PHB). The results indicated that cytoplasmic dynein participated in mitochondrial transport and mitochondrial degradation. Taken together, these results support the hypothesis that cytoplasmic dynein participates in acrosome formation, nuclear shaping, and mitochondrial transport during spermiogenesis in P. trituberculatus. This study will provide valuable guidance for the artificial fertilization and reproduction of P. trituberculatus.
... Several myosin actin-based motor proteins, including MYO6, have been linked to the Golgi complex and depletion of MYO6 results in changes in size of the Golgi complex and reductions in post-Golgi membrane trafficking in several cell types [54][55][56]. Recent study has further shown that MYO6 together with its binding partner TOM1/L2 is expressed in the mouse testes, where it is presented in actin-rich structures involved in acrosome biogenesis such as the Golgi complex throughout the Golgi stack from the CGN to the TGN [57]. Lacking of MYO6 in mice causes structural disruptions of the Golgi complex during early acrosome biogenesis. ...
Sexual reproduction requires the fusion of two gametes in a multistep and multifactorial process termed fertilization. One of the main steps that ensures successful fertilization is acrosome reaction. The acrosome, a special kind of organelle with a cap-like structure that covers the anterior portion of sperm head, plays a key role in the process. Acrosome biogenesis begins with the initial stage of spermatid development, and it is typically divided into four successive phases: the Golgi phase, cap phase, acrosome phase, and maturation phase. The run smoothly of above processes needs an active and specific coordination between the all kinds of organelles (endoplasmic reticulum, trans-golgi network and nucleus) and cytoplasmic structures (acroplaxome and manchette). During the past two decades, an increasingly genes have been discovered to be involved in modulating acrosome formation. Most of these proteins interact with each other and show a complicated molecular regulatory mechanism to facilitate the occurrence of this event. This Review focuses on the progresses of studying acrosome biogenesis using gene-manipulated mice and highlights an emerging molecular basis of mammalian acrosome formation.
... The granule enlarges with Golgi-derived glycoprotein-rich contents and gradually flattens and spreads over the nucleus to form a cap structure, while the Golgi apparatus migrates toward the posterior pole of the nucleus. As the spermatid elongates, the acrosome contents condense and the cap continues to spread over the nucleus [192]. ...
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Infertility is considered a global public health issue since it affects more than 50 million couples worldwide. Current assisted reproductive technologies (ARTs) have minimal requirements for gametes that are competent for fertilisation and subsequent embryo development. In cases where genetic abnormalities lead to arrested gametogenesis and the production of immature, defective or degraded gametes, treatment is not usually possible. Identifying the molecular causes of these types of infertility is crucial for developing new strategies to treat affected couples. Moreover, these patients represent a unique opportunity to discover new actors of oogenesis and spermatogenesis and to decipher the molecular pathways involved in the production of competent gametes.Genetic analysis of cohorts of infertile patients with shared ancestry can allow the identification of inherited genetic variants as possible causal factors. Using whole exome sequencing, we identified a homozygous pathogenic variant of the gene PATL2 in a cohort of patients with a phenotype of arrested oogenesis due to oocyte meiotic deficiency (OMD). OMD is a rare pathology characterised by the recurrent ovulation of immature oocytes. PATL2 encodes an oocyte ribonucleoprotein whose amphibian orthologue had been shown to be involved in oocyte translational control and whose function in mammals was poorly characterised. We also identified a pathogenic variant of the gene SPINK2 in a familial case of azoospermia. SPINK2 encodes a serine protease inhibitor essential for the neutralisation of acrosin activity during sperm acrosome formation.We showed, through generation of Patl2 and Spink2 knockout (KO) mice and Patl2 tagged mice (the latter using CRISPR-Cas9), that both corresponding proteins play essential respective roles in gametogenesis. We demonstrated that Patl2 is strongly expressed in growing mouse oocytes and that its absence leads to the dysregulation of numerous transcripts necessary for oocyte growth, meiotic maturation and preimplantation embryo development. This was accompanied by a phenotype of subfertility in KO females in natural mating, a large proportion of ovulated oocytes lacking a polar body (immature) and/or displaying spindle assembly defects in immunostaining, and high rate of oocytes with an aberrant response to fertilisation in IVF experiments. In Spink2 KO mice, we demonstrated that absence of Spink2 protein, which is located in the acrosome of maturing and mature spermatozoa, leads to arrested spermiogenesis and azoospermia due to autophagy at the round-spermatid stage. This is plausibly due to aberrant acrosin activity in the absence of its inhibitor, corroborated by fragmentation of the Golgi and absence of the acrosome in immunostaining.We have thus characterised two genetic subtypes of human infertility associated with mutation of these two genes. In doing so, we have furthered our understanding of the respective roles of these crucial actors of mammalian gametogenesis, potentially paving the way for improvement of current ARTs and development of new, personalised therapies.
... Furthermore, spermiogenesis can be divided into the following main phases: Golgi, acrosome cap/elongation, and maturation phases [7]. The Golgi apparatus produces proacrosomal vesicles during the Golgi phase, which coalesce and result in the acrosomal vesicle adjacent to the nuclear membrane on the opposite side of the tail. ...
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Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 knockout (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying a potential target for non-hormonal contraception.
... As one of the important cytoskeleton, the microfilament network not only provides structural support for cell morphology and movement, but also participates in transporting intracellular molecules myosin (Zakrzewski et al., 2017;Li et al., 2017). The disruption of actin-based cytoskeleton during spermatogenesis may affect male fertility (Johnson, 2015;Li et al., 2016a). ...
Spermatogenesis is a highly complex physiological process which contains spermatogonia proliferation, spermatocyte meiosis and spermatid morphogenesis. In the past decade, actin binding proteins and signaling pathways which are critical for regulating the actin cytoskeleton in testis had been found. In this review, we summarized 5 actin-binding proteins that have been proven to play important roles in the seminiferous epithelium. Lack of them perturbs spermatids polarity and the transport of spermatids. The loss of Arp2/3 complex, Formin1, Eps8, Palladin and Plastin3 cause sperm release failure suggesting their irreplaceable role in spermatogenesis. Actin regulation relies on multiple signal pathways. The PI3K/Akt signaling pathway positively regulate the mTOR pathway to promote actin reorganization in seminiferous epithelium. Conversely, TSC1/TSC2 complex, the upstream of mTOR, is activated by the LKB1/AMPK pathway to inhibit cell proliferation, differentiation and migration. The increasing researches focus on the function of actin binding proteins (ABPs), however, their collaborative regulation of actin patterns and potential regulatory signaling networks remains unclear. We reviewed ABPs that play important roles in mammalian spermatogenesis and signal pathways involved in the regulation of microfilaments. We suggest that more relevant studies should be performed in the future.
Background: The importance of phosphorylation in sperm during spermatogenesis has not been pursued extensively. Testis-specific serine kinase 3 (Tssk3) is a conserved gene, but TSSK3 kinase functions and phosphorylation substrates of TSSK3 are not known. Objective: The goals of our studies were to understand the mechanism of action of TSSK3. Materials and methods: We analyzed the localization of TSSK3 in sperm, used CRISPR/Cas9 to generate Tssk3 knockout (KO) mice in which nearly all of the Tssk3 open reading frame was deleted (ensuring it is a null mutation), analyzed the fertility of Tssk3 KO mice by breeding mice for 4 months, and conducted phosphoproteomics analysis of male testicular germ cells. Results: TSSK3 is expressed in elongating sperm and localizes to the sperm tail. To define the essential roles of TSSK3 in vivo, heterozygous (HET) or homozygous KO male mice were mated with wild-type females, and fertility was assessed over 4 months; Tssk3 KO males are sterile, whereas HET males produced normal litter sizes. The absence of TSSK3 results in disorganization of all stages of testicular seminiferous epithelium and significantly increased vacuolization of germ cells, leading to dramatically reduced sperm counts and abnormal sperm morphology; despite these histologic changes, Tssk3 null mice have normal testis size. To elucidate the mechanisms causing the KO phenotype, we conducted phosphoproteomics using purified germ cells from Tssk3 HET and KO testes. We found that proteins implicated in male infertility, such as GAPDHS, ACTL7A, ACTL9, and REEP6, showed significantly reduced phosphorylation in KO testes compared to HET testes, despite unaltered total protein levels. Conclusions: We demonstrated that TSSK3 is essential for male fertility and crucial for phosphorylation of multiple infertility-related proteins. These studies and the pathways in which TSSK3 functions have implications for human male infertility and nonhormonal contraception.
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The last phase of spermatogenesis involves spermatid elongation (spermiogenesis), where the nucleus is remodeled by chromatin condensation, the excess cytoplasm is removed, and the acrosome and sperm tail are formed. Protein transport during spermatid elongation is required for correct formation of the sperm tail and acrosome and shaping of the head. Two microtubular based protein delivery platforms transport proteins to the developing head and tail; the manchette and the sperm tail axoneme. The manchette is a transient skirt-like structure surrounding the elongating spermatid head and is only present during spermatid elongation. In this review we consider current understanding of the assembly, disassembly and function of the manchette and the roles of these processes in spermatid head shaping and sperm tail formation. Recent studies have shown that at least some of the structural proteins of the sperm tail are transported through the intra manchette transport (IMT) to the basal body at the base of the developing sperm tail and through the intra flagellar transport (IFT) to the construction site in the flagellum. This review focuses on the microtubule based mechanisms involved and the consequences of their disruption in spermatid elongation.
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The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873-885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.
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Epithelial cells from diverse tissues, including the enterocytes that line the intestinal tract, remodel their apical surface during differentiation to form a brush border: an array of actin-supported membrane protrusions known as microvilli that increases the functional capacity of the tissue. Although our understanding of how epithelial cells assemble, stabilize, and organize apical microvilli is still developing, investigations of the biochemical and physical underpinnings of these processes suggest that cells coordinate cytoskeletal remodeling, membrane-cytoskeleton cross-linking, and extracellular adhesion to shape the apical brush border domain. © 2014 Crawley et al.
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Transport of germ cells across the seminiferous epithelium is crucial to spermatogenesis. Its disruption causes infertility. Signaling molecules, such as focal adhesion kinase, c-Yes, c-Src, and intercellular adhesion molecules 1 and 2, are involved in these events by regulating actin-based cytoskeleton via their action on actin-regulating proteins, endocytic vesicle-mediated protein trafficking, and adhesion protein complexes. We critically evaluate these findings and provide a hypothetical framework that regulates these events.
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The coordinated trafficking and tethering of membrane cargo within cells relies on the function of distinct cytoskeletal motors that are targeted to specific subcellular compartments through interactions with protein adaptors and phospholipids. The unique actin motor myosin VI functions at distinct steps during clathrin-mediated endocytosis and the early endocytic pathway - both of which are involved in cargo trafficking and sorting - through interactions with Dab2, GIPC, Tom1 and LMTK2. This multifunctional ability of myosin VI can be attributed to its cargo-binding tail region that contains two protein-protein interaction interfaces, a ubiquitin-binding motif and a phospholipid binding domain. In addition, myosin VI has been shown to be a regulator of the autophagy pathway, because of its ability to link the endocytic and autophagic pathways through interactions with the ESCRT-0 protein Tom1 and the autophagy adaptor proteins T6BP, NDP52 and optineurin. This function has been attributed to facilitating autophagosome maturation and subsequent fusion with the lysosome. Therefore, in this Commentary, we discuss the relationship between myosin VI and the different myosin VI adaptor proteins, particularly with regards to the spatial and temporal regulation that is required for the sorting of cargo at the early endosome, and their impact on autophagy.
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Rai14 (retinoic acid induced protein 14) is an actin binding protein first identified in the liver, highly expressed in the placenta, the testis, and the eye. In the course of studying actin binding proteins that regulate the organization of actin filament bundles in the ectoplasmic specialization (ES), a testis-specific actin-rich adherens junction (AJ) type, Rai14 was shown to be one of the regulatory proteins at the ES. In the rat testis, Rai14 was found to be expressed by Sertoli and germ cells, structurally associated with actin and an actin cross-linking protein palladin. Its expression was the highest at the ES in the seminiferous epithelium of adult rat testes, most notably at the apical ES at the Sertoli-spermatid interface, and expressed stage-specifically during the epithelial cycle in stage VII-VIII tubules. However, Rai14 was also found at the basal ES near the basement membrane, associated with the blood-testis barrier (BTB) in stage VIII-IX tubules. A knockdown of Rai14 in Sertoli cells cultured in vitro by RNAi was found to perturb the Sertoli cell tight junction-permeability function in vitro, mediated by a disruption of F-actin, which in turn led to protein mis-localization at the Sertoli cell BTB. When Rai14 in the testis in vivo was knockdown by RNAi, defects in spermatid polarity and adhesion, as well as spermatid transport were noted mediated via changes in F-actin organization and mis-localization of proteins at the apical ES. In short, Rai14 is involved in the re-organization of actin filaments in Sertoli cells during the epithelial cycle, participating in conferring spermatid polarity and cell adhesion in the testis.
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Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.
In mice and humans, loss of myosin VI (Myo6) function results in deafness, and certain Myo6 mutations also result in cardiomyopathies in humans. The current studies have utilized the Snell's waltzer (sv) mouse (a functional null mutation for Myo6) to determine if this mouse also exhibits cardiac defects and thus used to determine the cellular and molecular basis for Myo6-associated heart disease. Myo6 is expressed in mouse heart where it is predominantly expressed in vascular endothelial cells (VECs) based on co-localization with the VEC cell marker CD31. Sv/sv heart mass is significantly greater than that of sv/+ littermates, a result of left ventricle hypertrophy. The left ventricle of the sv/sv exhibits extensive fibrosis, both interstitial and perivascular, based on histologic staining, and immunolocalization of several markers for fibrosis including fibronectin, collagen IV, and the fibroblast marker vimentin. Myo6 is also expressed in lung VECs but not in VECs of intestine, kidney or liver. Sv/sv lungs exhibit increased peri-aveolar fibrosis and enlarged air sacs. Electron microscopy of sv/sv cardiac and lung VECs revealed abnormal ultrastructure, including luminal protrusions and increased numbers of cytoplasmic vesicles. Previous studies have shown that loss of function of either Myo6 or its adaptor binding partner synectin/GIPC results in impaired arterial development due to defects in VEGF signaling. However, examination of synectin/GIPC -/- heart revealed no fibrosis or significantly altered VEC ultrastructure, suggesting that the cardiac and lung defects observed in the sv/sv mouse are not due to Myo6 function in arterial development. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.
In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES.