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THE ANATOMICAL RECORD 245157-64 (1996)
Cytoskeletal Differences Between Stereocilia of the Human Sperm
Passageway and Microvilli/Stereocilia in Other Locations
DIRK HOFER AND DETLEV DRENCKHAHN
Znstitute of Anatomy, Julius-Maximilians-University
Wurzburg,
0-97070 Wiinburg, Germany
ABSTRACT
Background: Stereocilia of the human ductus epididymidis
and ductus deferens display unique features in that they arise from an
apical cell protrusion (hillock)and contain thick stem portions which are
interconnected by cytoplasmic bridges. The molecular basis for this unique
fusion and branching pattern is hitherto unknown. These morphologic specialities led us to study the cytoskeleton of male spermway stereocilia with
respect to the major proteins that constitute the supportive cytoskeleton of
intestinal microvilli and inner ear stereocilia.
Methods: Samples of the human epididymidis and ductus deferens were
studied by immunoblotting and immunocytochemistry at the light and electron microscope levels.
Results: Spermway stereocilia are supported by an internal actin filament bundle crosslinked by fimbrin and associated with the membrane
linker molecule ezrin. The stem portions and hillock area are supplied with
the crossbridge forming molecule a-actinin. Spermway stereocilia differ
from brush border microvilli of the intestine, kidney, and ductuli efferentes
by the lack of the second bundling protein villin and the unusual expression of a-actinin in the stem region. They resemble inner ear stereocilia by
the presence of fimbrin and absence of villin, but differ from them by expression of ezrin and a-actinin. Thus, the main molecular difference between spermway stereocilia and stereocilidmicrovilli of other locations is
the presence of a-actinin in their stem portion and the hillock area.
Conclusions: Since a-actinin can form crossbridges between adjacent actin filaments (bundles)at longer distances than the other crosslinker of the
stereocilium core bundle, fimbrin, we assume that a-actinin is essential for
both the formation of the stem portions of spermway stereocilia and for the
generation of their striking branching pattern. A developmentally regulated temporal sequence of expression of fimbrin and a-actinin might control the unique architecture of spermway stereocilia.
0 1996 Wiley-Liss, Inc.
Key words: Stereocilia, Male Reproductive System, Cytoskeleton
The terminology of rod-shaped cell surface projections goes back to the end of the 19th century when
histologists grouped these extensions into two main
categories: i) motile hairs (kinocilia) and ii) non-motile
hairs (stereocilia). The term stereocilium (Gr. stereos =
stiff, Lat. cilium = hair, eyelash) comprised the rodshaped units of the intestinal and kidney tubular brush
border as well as the stereocilia of inner ear hair cells
(hair border) and the epithelium of the male sperm
passageway (Ductus epididymidis, Ductus deferens)
(Stohr, 1906). After introduction of the electron microscope the term “microvillus” was proposed to distinguish the short projections seen on many cell types
from the long stereocilia of the inner ear and male
sperm passageway (Fawcett and Porter, 1954).
Stereocilia and microvilli in several locations contain an internal supportive cytoskeleton composed of
0 1996 WILEY-LISS,
INC.
parallel-aligned actin filaments with uniform polarity
(Mooseker and Tilney, 1975; Flock and Cheung, 1977;
Matsudaira, 1991). In microvilli of the kidney and intestinal brush border actin filaments are bundled by
villin and fimbrin (Bretscher and Weber, 1980a,b).
This extensively crosslinked core bundle is associated
by an unknown mechanism with ezrin, a peripheral
membrane protein which is interposed between actin
filaments and proteins of the plasma membrane (Gould
et al., 1986; Bretscher, 1989; Luna, 1991). Myosin I is
another peripheral membrane protein involved in the
Received October 18, 1995; accepted December 15, 1995.
Address reprint requests to Prof. Dr. Drenckhahn, Institute of
Anatomy, Julius-Maximilians-University, Koellikerstrasse 6,
D-97070 Wiirzburg, Germany.
58
D. HOFER AND D.DRENCKHAHN
linkage of the actin filament core bundle to the microvillus membrane (Glenney et al., 1982; Matsudaira
and Burgess, 1979; Drenckhahn and Dermietzel, 1988;
Rodman et al., 1986). One difference between the actin
filament core bundles of inner ear stereocilia and brush
border microvilli is the absence of villin from stereocilia. The only crosslinker of actin filaments in stereocilia appears to be fimbrin (Flock et al., 1982; Drenckhahn et al., 1985, 1991). The cytoskeleton of spermway
stereocilia has not yet been analyzed.
The sperm passageway that connects the testes with
the urethra consists of ductuli efferentes, the ductus
epididymidis, and the ductus deferens. Ductuli efferentes (that are located in the head portion of the epididymis) are lined by ciliated and nonciliated cells. The
latter predominate and are distinguished by an apically located microvillus border that resembles structurally in many respects the brush border of enterocytes (Morita, 1966). The apical surface of the
remaining sperm passageway, i.e., the ductus epididymidis and ductus deferens is occupied with long, nonmotile microvillus-like projections, the height of which
decreases from 80 pm in the head region of the epididymal duct to 40 p,m in the epididymal tail and ductus
deferens (Horstmann, 1962; Holstein, 1969; Popovic et
al., 1973; Dym, 1988). The long and flexible spermway
stereocilia differ in their morphology from the stiff inner ear stereocilia and brush border microvilli in two
main aspects. First, the tuft of stereocilia arises from
an apical cell protrusion (further denoted as hillock)
that is absent from inner ear hair cells and from the
intestinal and kidney brush border region. Second, the
basal portions of spermway stereocilia frequently fuse
to form thicker stem portions several pm in length.
These stems of stereocilia may locally divide and fuse
again. The stems often contain two or more individual
actin filament bundles that are continuous with the
actin filament bundles of the two or more stereocilia
arising from each stem portion.
The molecular basis controlling this unique fusion
and branching pattern is hitherto unknown. To address
this question and to reveal possible differences of the
cytoskeleton between brush border microvilli, inner
ear stereocilia, and spermway stereocilia we attempted
to analyze the cytoskeleton of human epididymal stereocilia with respect to composition and localization of
the major cytoskeletal components of the intestinal
brush border and inner ear hair border, i.e., actin, villin, fimbrin, ezrin, and a-actinin. Myosin I was not included in this study because of the low degree of crossreactivity of antibodies against intestinal brush border
myosin I with the many isoforms of myosin I expressed
in different tissues (Goodson and Spudich, 1993).
buffered saline (PBS: 10 mM sodium phosphate, 140
mM NaC1; pH 7.4) warmed to 56°C or with 0.2 M glycine-HC1(pH 2.8) at 4°C (Drenckhahn and Franz, 1986;
Drenckhahn et al., 1993). In addition, we used a monoclonal mouse IgM antibody directed against a-actinin
(Sigma, Deisenhofen, Germany), a monoclonal mouse
IgM antibody against actin (Amersham, Braunschweig,
Germany), and a monoclonal mouse IgG antibody
against human villin (Camon, Immunotech, Marseille,
France). Tissue pieces of the human epididymis were
homogenized in 80°C sample buffer. Thirty minutes
later, insoluble material was pelleted (lO,OOOg, 10 min)
and the supernatant was subjected to sodium dodecyl
sulphate polyacrylamide (10%) gel electrophoresis
(SDS-PAGE)in the presence of 0.1 M dithiothreitol. The
separated proteins were electroblotted on nitrocellulose
membranes (Burnette, 1981) (Schleicher and Schull,
Darmstadt, Germany) which were then incubated with
antibodies. Bound immunoglobulins were visualized by
the peroxidase-antiperoxidase method (Drenckhahn et
al., 1983) using a-chloronaphthol (BioRad, Richmond,
VA) as chromogen.
lmmunostaining
Small tissue pieces of human epididymis, 1-5 mm3
in size, were excised. Tissue pieces were rapidly frozen
in melting isopentane cooled with liquid nitrogen. The
frozen tissue samples were carefully freeze-dried for 12
h at -40°C and
Torr and were then immersed for
48 h a t lop3Torr and 20°C with Epon containing 1.8%
(v/v) Epon accelerator. Afterwards, samples were
transferred to gelatin capsules and embedded in Epon
at 60°C for 24-48 h. Semithin sections (0.5-1 pm) in
thickness were mounted on glass coverslips and heated
to 80°C for 2 h. Removal of the resin was performed by
placing the coverslips for 5 min in a 1:l mixture of
methanol and toluene containing 10%sodium methoxide, which was prepared from metallic sodium as described by Major et al. (1961). The sections were then
rinsed with a 1:l mixture of methanol and toluene (5
rnin), acetone (2 x 5 rnin), distilled water (5 rnin), and
phosphate-buffered saline (PBS: 10 mM sodium phosphate, 140 mM NaC1, pH 7.4) for 5 min as described
elsewhere (Drenckhahn et al., 1983; Drenckhahn and
Franz, 1986). Antibody staining was done by overlaying each section for 2 h with 20 p1 of affinity-purified
antibodies (-1-10 pg/ml PBS). After several washes
with PBS, sections were incubated for 30 min at RT
with the secondary antibodies. As secondary antibodies
we used tetramethylrhodamine (TRITCI-labelled goat
anti-rabbit IgG and TRITC-labelled goat anti-mouse
IgG (Dianova, Hamburg, Germany; dilution 1 5 0 in
PBS). Afterwards the slides were washed again with
PBS (3 x 5 min) and mounted in 60%glycerol in PBS
MATERIALS AND METHODS
containing 1.5% n-propyl-gallate as antifading comAntibodies and lmmunoblotting
pound (Serva, Heidelberg, Germany). Negative conSpecificities of the polyclonal rabbit antibodies raised trols included omission of primary antibodies and, in
against chicken villin, fimbrin, and a-actinin have been case of the antibodies against villin, fimbrin, and actin
described elsewhere (Drenckhahn and Dermietzel, absorption with an excess of the respective antigens.
1988). The antibody against human placenta ezrin was
For double immunofluorescence, sections were incukindly provided by Dr. Bretscher (Ithaca, NY). The an- bated for 60 min at RT with a mixture of the polyclonal
tibodies against villin, fimbrin, and a-actinin were af- a-actinin antibody and the monoclonal actin IgM antifinity-purified using the respective antigens immobi- body. As secondary antibodies a mixture was used conlized by transfer to nitrocellulose. The bound taining fluorescein isothiocyanate (F1TC)-labelledgoat
immunoglobulins were eluted either with phosphate- anti-mouse IgM and Texas Red-labelled goat anti-rab-
59
SPERMWAY STEREOCILIA
Fig. 1 . Immunocytochemical localization of ezrin (a) and fimbrin (b,c ) in sections of the epididymal
duct and ductus deferens (c) (indirect immunofluorescence).Both antibodies show intense labelling of
apical stereocilia projecting into the ductal lumen. Staining is present along the entire length of the
stereocilia. L. lumen. Bar: 10 km.
bit IgG (the final dilution of both antibodies was 1 5 0 in
PBS). The secondary antibody mixture was applied for
1h at RT.
lmmunoelectron Microscopy
For immunoelectron microscopy tissue samples were
fixed overnight with a mixture of 0.1 glutaraldehyde
and 2% paraformaldehyde in PBS. After fixation, tissue pieces were treated for 15 min with 0.5 mg/ml sodium borohydride in PBS and embedded in the hydrophilic methacrylate resin LR-White (London Resin Co.,
Woking, UK).Ultrathin tissue sections were collected
on gold grids and processed for immunogold labelling
with affinity-purified anti-a-actinin exactly as described (Drenckhahn and Merte, 1987; Drenckhahn
and Dermietzel, 1988). Goat anti-rabbit IgG coupled to
colloidal gold particles (10 nM in diameter) were purchased from Janssen Pharmaceutica (Beerse, Belgium).
RESULTS
lmmunofluorescence
Sections of the tail and head portion of the epididyma1 duct incubated with polyclonal antibodies to fimbrin, ezrin, and actin resulted in a bright staining of
the long wavy bundles of stereocilia that project from
the apical surface of the columnar principal cells (Figs.
la,b, 2a). Immunofluorescence was observed along the
entire length of the stereocilia. Identical results were
obtained with stereocilia of the ductus deferens (Fig.
lc). No immunostaining specific for villin was observed
in stereocilia using both poly- and mono-clonal antibodies directed against villin. In this respect, sperm-
way stereocilia differ from brush border microvilli of
enterocytes and kidney tubules but resemble inner ear
stereocilia that do also not contain villin.
However, in contrast to inner ear stereocilia and
brush border microvilli stereocilia of the epididymis
reacted with two different antibodies specific for a-actinin. In sections double-labelled with antibodies to
a-actinin and actin it became evident that the a-actinin label was restricted to the basal portions (stem regions) of the stereocilia and absent from their remaining mid and distal portions (Fig. 2a,b). Anti-a-actinin
and anti-actin reacted also strongly with the hillock
area of the apical cell pole. A dotted staining pattern
along the lateral cell borders is most likely due to labelling of the zonula adhaerens and type I1 desmosomes (adhaerens plaques) (Drenckhahn and Franz,
1986). In sections of ductuli efferentes antibodies to
actin, fimbrin, ezrin, and villin labelled the rather
short microvilli of the nonciliated epithelial cells. a-Actinin-like immunoreactivity was confined to the zonula
adhaerens and the lateral cell border but was absent
from the microvillus border of ductuli efferentes (Fig.
3a,b,c,d). Thus, the microvillus border of ductuli efferentes resembles by these criteria the intestinal and
kidney brush borders.
lmmunoelectron Microscopy
Immunofluorescence microscopy did not allow to resolve the subcellular location of a-actinin in the spermway stereocilia. In particular, we wanted to know
whether a-actinin extends into the stereocilia or
whether it is restricted to the cone-shaped hillock area
that contains the rootlets of stereocilia. By immu-
60
D. HOFER AND D. DRENCKHAHN
Fig. 2. Luminal border of the epididymal duct double-immuno-labelled with a monoclonal antibody against actin (a),and a polyclonal
antibody against a-actinin (b).Whereas the actin antibody shows
intense labelling of the entire length of stereocilia (indicated by arrowhead), the a-actinin immunostaining is restricted to the hillock
area and proximal portion of the stereocilia. Note also staining of the
area of the zonulae adhaerentes (small arrows) and numerous small
dots along the basolateral cell margins probably representing type I1
desmosomes (puncta adhaerentia). Bar: 10 pm.
nogold labelling of ultrathin sections of epididymal tissue we confirmed our impression that the immunolabel
specific for a-actinin is present in both the hillock and
the stem portions of stereocilia (Fig. 4).The label was
strongest in the hillock area and in the stem region
with little label extending into stereocilia beyond their
branching points. Within the remaining mid and apical
portion of stereocilia the immunogold label was negligible. Significant immunogold labelling was also seen
in association with the zonula adhaerens (Fig. 4) and
type I1 desmosomes (not shown).
crease the membrane surface area available for integral membrane proteins that participate in resorption
(e.g., kidney and intestinal brush borders, spermway
stereocilia), secretion [microvilli of gastric parietal
cells (Ito, 198713, or chemosensation [microvilli of receptor cells in taste buds and the vomeronasal organ
(Vaccarezza et al., 1981; Roper, 198911. Other types of
microvilli/stereocilia provide lever-like processes involved in mechanosensation [e.g., stereocilia of inner
ear hair cells (Hudspeth, 1989) and microvilli of skin
Merkel cells (Gould et al., 198511.
According to these different functions, microvilli/stereocilia display pronounced morphological differences
ranging from the uniformly sized and regularly arranged microvilli of the intestinal and kidney brush
border to the thick and long stereocilia of the inner ear
hair cells with their organpipe-like arrangement and,
finally, to the long flexible stereocilia of the male
sperm passageway that display an unusual branching
pattern in their basal portions.
These morphologic differences indicate differences in
the organization of the scaffolding cytoskeleton that is
generally believed to control stiffness and size of microvilli/stereocilia.
In the present study we investigated the microvillil
stereocilia lining the male sperm passageway with respect to the major proteins that constitute the support-
lrnrnunoblotting
In immunoblots fimbrin (68 kD band) and a-actinin
(100 kD band) were detected in both the tail and head
portions of the epididymis (Fig. 5 ) . In contrast poly- and
mono-clonal antibodies to villin did not detect any polypeptide band in the tail of the epididymis. However,
both antibodies against villin detected a distinct villin
band (95 kD band, comigrating with intestinal villin)
in the head of the epididymis that is a mixed tissue
containing both ductuli efferentes and the ductus epididymidis.
DISCUSSION
Microvilli and stereocilia are rod-shaped cellular
projections serving various functions such as to in-
SPERMWAY STEREOCILIA
61
Fig. 3. Localization of ezrin (a), fimbrin (b), a-actinin (c), and villin (d) in cross sections of ductuli
efferentes from the head of the epididymis. Immunoreactivityis confined to the short apical microvilli in
a, b and d. Note absence of a-actinin staining in apical microvilli but immunolabelling of zonulae
adhaerentes (arrows) and type I1 desmosomes. L, lumen. Bar: 10 pm.
ive cytoskeleton of microvilli/stereocilia a t other fimbrin but not villin (Flock et al., 1982; Drenckhahn
locations. The main outcome can be summarized as fol- et al., 1985, 1991). Thus, spermway stereocilia provide
lowed (see also Table 1): First of all we observed that another example of a microvillus type lacking one of
the cytoskeleton of the microvilli associated with the the actin bundling proteins of the kidney and intestinal
luminal surface of nonciliated epithelial cells of ductuli brush border.
Beside this similarity to inner ear stereocilia, spermefferentes resembles strongly the cytoskeleton of brush
border microvilli in other locations in that these mi- way stereocilia differ from their counterparts in the
crovilli contain an actin core supplemented with the inner ear in two aspects. First, spermway stereocilia
two actin-bundling proteins villin and fimbrin and contain ezrin, which we did not observe in inner ear
with the plasmalemmal linker molecule, ezrin. In this stereocilia (unpublished observations). In this respect
respect the microvillus border of nonciliated epithelial spermway stereocilia are related to brush border micells of ductuli efferentes can be considered as a further crovilli. Although ezrin has been recently shown in fiexample of a typical brush border.
broblasts (Tsukita et al., 1994) to link actin filaments
In contrast, stereocilia of the ductus deferens and to the cell surface glycoprotein CD 44, the binding site
ductus epididymidis displayed some conspicuous differ- of ezrin at the microvillus membrane is still unknown.
ences with respect to the composition of the cytoskele- Since ezrin is typically found in cell surface projections
ton. Similarities shared by all three types of microvilli/ involved in resorptive processes as for example in mistereocilia exist in the actin filament core bundle crovilli of the intestinal and kidney brush border
crosslinked by one actin filament bundling protein, (Bretscher, 1983; Gould et al., 1986) and in the amninamely, fibrin. Unlike brush border microvilli, sperm- otic epithelium (Wolf et al., 19911, it is likely that ezrin
way stereocilia do not contain the second bundling pro- links membrane-bound enzymes or transporters to the
tein, villin. In this respect spermway stereocilia resem- actin core bundle.
ble inner ear stereocilia that do also contain only
Spermway stereocilia differ from all other microvilli/
62
D. HOFER AND D. DRENCKHAHN
M,x103
1
2
3
4
5 6
10068 -
5
Fig. 5. Immunoblot analysis of the tail (lanes 2,4,6) and head portion
(lanes 1,3,5) of the human epididymis incubated with antibodies to
fimbrin (lanes 1,2),a-actinin (3,4), and villin (5,6).Note that fimbrin
and a-actinin are expressed in both the tail and head portion whereas
villin is only expressed in the head but absent from the tail.
tuft-like arrangement of the stereocilia including the
formation of the apical hillock from which the stereocilia arise. It has been recently shown that the actin
binding sites for a-actinin and fimbrin overlap (Holtzman et al., 1994; Honts et al., 1994; McGough et al.,
1994), suggesting that a-actinin and fimbrin might
compete for the same binding site in the stem portions
and rootlets of spermway stereocilia. Taking into account that fimbrin forms crosslinks of about 15 nm in
Fig. 4. Electron micrograph of the apical portion of the epithelium of width, whereas a-actinin can form crossbridges of up to
the epididymal duct (head portion) labelled with an affinity-purified 100 nm in size, it is possible that a-actinin occupies
antibody against a-actinin and 10 nm gold particles coated with antiare separabbit IgG. Beside the zonula adhaerens (ZA) the a-actinin label is binding sites where adjacent
also present in both the coneshaped hillock area (H) and the stem rated by a distance greater than the length of the fimregions (*) of stereocilia. Note negligible immunolabel in remaining brin molecule. At such places the long flexible a-actinin
portions of stereocilia (arrows). Bar: 1 km.
molecule could serve to hold adjacent core bundles together, thereby creating the thicker stem portions (Fig.
6).
~I
stereocilia so far investigated in that they contain in
their basal portions a-actinin. a-Actinin is a widespread
actin filament crosslinking protein, found in the muscular Z-lines, densities of smooth muscle, densities of
stress fibres, adhaerens type intercellular junctions,
and focal cell-to-substrate contacts (Critchley, 1993).
The function of a-actinin is to crosslink actin filaments
of both opposite and uniform polarity (Meyer and Aebi,
1990). In addition, a-actinin can bind actin filaments to
integral membrane proteins of the plasma membrane,
such as the p,-subunit of integrins (Otey et al., 1990).
Since in spermway stereocilia a-actinin was confined to
the branching stem portions of stereocilia in the apical
hillock, it appears reasonable to assume that a-actinin
is involved in this unique anastomosing morphology in
that it forms crosslinks between the core bundles of
adjacent stereocilia. These crosslinks might be important for generation of the thicker stem portions and for
stabilization of the cytoplasmic adhesive bridges that
exist between neighbouring stereocilia. Such crosslinks
could play an important role in the formation of the
Once separated into individual stereocilia enveloped
by the plasma membrane, a-actinin would lose its bivalent (cross-linking) binding sites and detach from the
tightly packed core bundles crosslinked by fimbrin.
Such a mechanism could explain why a-actinin is more
or less restricted to the basal portion (stem portion) of
stereocilia and is absent from the distal two thirds of
their length. However, such a mechanism could not
explain why the stem portions branch.
One way of how this branching pattern might be
controlled would be by a temporally separated initiation of the expression of fimbrin and a-actinin during
development. If fimbrin is expressed at high levels
early during stereociliogenesis, individual stereocilia
might grow out lacking stem portions and cytoplasmic
bridges. The expression of a-actinin might be low at
this early stage. As soon as a-actinin becomes expressed a t higher levels, the rootlet portions of stereocilia would become crosslinked and, during further
growth of stereocilia, the rootlets might become incorporated into the hillock and stem region.
63
SPERMWAY STEREOCILIA
TABLE 1. Components of the actin filament cytoskeleton of microvillVstereociliain different locations
Intestine,
enterocvtesl
Actin
Villin
Fimbrin
Myosin-I
Ezrin
a-Actinin
Microvilli
Retina,
Kidney, proximal
tubule'
Miiller cells3
+
+
+
+
+-
+
+
+
+
+-
+
+
+
-
-
Ductuli
efferentes
+
+
+
+
+-
stereocilia
Ductus epididymidis
ductus deferens
+
+
nd5
+
-
+
Inner
ear4
+-
+
26
-
'Drenckhahn and Dermietzel, 1988.
*Rodman et al., 1986.
3Hofer and Drenckhahn, 1993.
4Drenckhahn et al., 1982; 1991 and additional personal data.
'nd not detected.
'Not detected in chicken (Drenckhahn et al., 1991), but present in stereocilia of frog saccular macula (Gillespie et al., 1993).
Stereocilia
Actin filaments
bundled by fimbrin
Stems with
actin bundles
interconnected
by a-actinin (-)
Hillock --.
Zonula
occludens
Zonula
adhaerens
ype II - Desmosome
Fig. 6. Summarizing diagram illustrating the location and possible
role of a-actinin in generation and/or maintenance of the unique architecture of spermway stereocilia.
In order to test this hypothesis, we have begun to
study the development of spermway stereocilia in laboratory animals.
ACKNOWLEDGMENTS
We thank Martina Zink for skillful technical assistance. This work was supported by grants of the
Deutsche Forschungsgemeinschaft.
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