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Maturation antigen of the mouse sperm flagellumII. Origin from holocrine cells of the distal caput epididymis

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THE ANATOMICAL RECORD 217:146-152 (1987)
Maturation Antigen of the Mouse Sperm Flagellum:
II. Origin From Holocrine Cells of the Distal Caput
Department of Anatomy, School of Medicine, University of New Mexico,
Albuquerque, NM 87131
During epididymal transit, the mouse sperm flagellum acquires a
surface glycoprotein (SMA4)from epididymal fluid that functions as a sperm antiagglutinin. To determine the origin of this molecule, testes and epididymides of male
mice were sectioned for light microscopy and stained with wheat germ agglutinin
(WGA)-peroxidase, a probe that has been used previously to examine the biology of
SMA4. WGA reactivity was localized to the cytoplasm in a small population of cells
in the distal caput epididymis. Testis cells and principle cells of the caput were
nonreactive with WGA, while stereocilia were stained on principle cells in the corpus
and cauda. The WGA-positive cells in the distal caput were identified as holocrine
cells on the basis of morphology, distribution, and PAS+ reaction. At high magnification, intense WGA reactivity was due to the presence of numerous apical granules
in the cytoplasm. The location of the cells in distal caput coincided exactly with the
region of tubule in which sperm first acquired SMA4 on their flagellae. These data
suggest that holocrine cells near the junction of caput and corpus epididymis are the
source of the sperm antiagglutinin SMA4.
The mammalian epididymis produces a complex secretory product that bathes the sperm during passage
through the epididymis. In this environment sperm
undergo the final stages of development leading to fertility (Bedford, 1975; Orgebin-Crist et al., 1975). As
shown by autoradiography, principle cells of the epithelium are the source of proteins that interact with spermatozoa during this maturational period (Kopecny, 1971;
Kanka and Kopecny, 1977; Kopecny et al., 19841, although the functions and identities of the molecules are
largely unknown. Additionally, the method of release of
secretory products from the principle cells does not appear to be similar to that of typical secretory cells, since
they do not possess observable secretory granules (Flickinger, 1979,1981,1985).
Histochemical and electronmicroscopic evidence indicates that the epididymal epithelium is not homogeneous but varies in appearance throughout several regions
of the tube, and cells other than principle cells are present (Martan, 1969; Hoffer et al., 1972; Hamilton, 1975).
Among these subpopulations of cells, it has been proposed that “holocrine” cells may also have a secretory
function (Martan and Risley, 1963; Martan and Allen,
1964; Martan et al., 19641, but no specific epididymal
products have been traced to these cells. These cells are
present in specific regions of the epididymis in mouse
(Martan and Allen, 1964),rat (Martan and Risley, 1963;
Hoffer et al., 1972; Hamilton, 1975; Brown and Montesano, 1980; Sun and Flickinger, 1980), and human (Martan et al., 1964). Morphological evidence suggests that
they undergo a cyclic secretion process similar to that of
holocrine cells elsewhere (Martan and Risley, 1963).
0 1987 ALAN R. LISS, INC.
In previous studies using monoclonal antibodies, we
have shown that a 54-kd epididymal secretory product
is acquired by mouse sperm flagellae as they transit the
epididymis (Feuchter et al., 1981, 1986; Vernon et al.,
1982). This molecule, termed SMA4, is a specific receptor for wheat germ agglutinin (WGA), and its biology
can be studied by using WGA as a probe. In a separate
report we examine the functional significance of SMA4
as a sperm antiagglutinin (Feuchter et al., 1986). The
present study provides evidence that this sperm-binding
molecule, which covers the entire sperm flagellum, originates within holocrine cells of the distal caput epididymis in the mouse.
ICR retired breeder male mice (Harlan Labs) used for
these studies were housed in a controlled environment
on a 12 L:12 D (12 h r light, 12 h r dark) cycle and provided with Purina Lab Chow and water ad libitum.
Tissue Preparation
Animals were killed by cervical dislocation, and entire
reproductive tracts (testis, caput, corpus, and cauda epididymides, as well as ductus deferens) were excised.
After removal of excess fat, tissue was rinsed in modified
Tyrode’s solution (MTS) (2.7 mM KCI, 117 mM NaC1,0.5
mM MgClz x 6H20, 0.36 mM NaH2P04 x lHzO, 1.7
mM CaC12 x 2Hz0, 12 mM NaHC03, 0.25 mM Na Pyruvate, 5.5 mM glucose, and 3.3 mM HEPES, pH 7.4,
Received August 5,1986; accepted September 24, 1986.
room temperature) and fixed in 3% paraformaldehyde
for 24 h r (4°C). Samples were dehydrated in a series of
ethanols ranging from 70 to loo%, cleared in toluene,
and embedded in paraffin. Eight-micron-thick microtome sections of either whole epididymis or various portions of the reproductive tract were mounted with
albumin on acid-alcohol-cleaned glass slides. Frozen sections were prepared from udixed tissue that had been
immersed in liquid nitrogen-cooled isopentane and
mounted in Cryostat embedding medium. After airdrying, frozen sections were blocked and stained as described below.
Lectin-peroxidase Staining of Tissues
Staining was carried out a t room temperature by removing paraffin from slides with three changes of xylene and rehydrating in a n ethanol series of 100 to 70%,
followed by three rinses in phosphate-buffered saline
(PBS) (.E
M NaC1, 2.5 mM NaH2P04 x 1H20, and 7.5
mM Na2HP04 x 7H20, pH 7.4). Nonspecific protein
binding sites were blocked by incubation in 1% bovine
serum albumin (BSA) (Calbiochem) for 30 min. Slides
were then rinsed in PBS twice, followed by incubation
in 5 pg/ml biotinylated WGA (Vector) for 60 min to 24
hr. After two rinses in PBS, slides were incubated in a
mixture of avidin and biotinylated horseradish peroxidase (ABC Kit, V & P Scientific) for 1 hr. After three
rinses in PBS of 10 min each, slides were immersed in
substrate (.3 mg/ml diaminobenzidene [Sigma] and
.005% H202) for 10 min and rinsed in double-distilled
water for 10 min. WGA binding sites were localized by
a brown reaction product. In order to visualize the tissue
that was nonreactive with WGA, slides were immersed
in toluidine blue stain (30%ethanol and .05% toluidine
blue) for 5 min, followed by dehydration with 50-100%
ethanol and two changes of xylene. Slides were air-dried,
and coverslips were mounted with Permount for observation by bright-field microscopy. Specificity of WGA
binding was confirmed by incubation of slides in .1 M
N-acetyl-neuraminic acid, either simultaneously or subsequent to staining with WGA-biotin. Both procedures
resulted in no staining of the tissues by WGA.
The distribution of SMA4 within the male reproductive tract was examined by WGA-peroxidase staining of
Fig. 1. Paraffin section of mouse testis, stained with WGA-peroxidase
and counterstained with toluidine blue. Sperm were nonreactive with
WGA, and no WGA-positive cells were seen within the seminiferous
tubules. Therefore, tissue in this micrograph shows only toluidine blue
staining (dark circles at periphery of tubule are nuclei of spermatogonial cells). x200.
Fig. 2. Paraffin section of mouse caput epididymis, middle region,
stained with WGA-peroxidase and toluidine blue. Loosely packed sperm
in lumen (SP) were nonreactive with WGA and showed only light blue
staining. The columnar epithelium, consisting of principle cells, also
showed no WGA staining in either the surface stereocilia (arrows),
cytoplasm, or nuclei, which exhibited blue stain of varying density.
Fig. 3. Paraffin section of mouse corpus epididymis, stained with
WGA-peroxidase and toluidine blue. Densely packed sperm in the
lumen (SP) exhibited strong WGA reactivity on their flagellae, and
stereocilia (arrows) were heavily stained with brown reaction product
as well. Cytoplasm of epithelia1 cells appears dark blue in this micrograph owing to the thickness of section used to illustrate details of the
stereocilia. Nuclei were stained with toluidine blue. ~ 2 2 5 .
Fig. 4. Sketch of mouse testis and epididymis, illustrating region of
distal caput (shaded area) where epithelial cells were localized, which
stained strongly with WGA.
Fig. 5. Paraffin section of distal caput region indicated in Figure 4,
stained with WGA-peroxidase and toluidine blue. A subpopulation of
epithelial cells in this region exhibited heavy cytoplasmic staining
with WGA (arrows). There were about three to eight positive cells per
tubular profile, while the principle cells in this region were negative
and appear blue in the micrograph. Sperm throughout most of the
caput were nonreactive with WGA, but sperm in the distal parts
showed WGA reactivity. x 120.
Fig. 6. Higher magnification of cells shown in Figure 5. The WGApositive cells are similar to those described by others as holocrine or
apical cells, having a narrow or constricted base, a n expanded cell
apex toward the lumen, and an apically placed nucleus (large arrow,
upper right). In contrast, principle cells had a rectangular shape and
basally placed nuclei (small arrowheads). ~ 2 4 0 .
Fig. 7. Enlargement of the area inside rectangle in Figure 6. In this
micrograph, two darkly stained holocrine cells are flanked by columnar principle cells (with lightly stained nuclei). The apical nucleus
(NU) and constricted base of these cells were readily apparent, as well
as the granular nature of WGA-positive material within the cells (SG).
Granules averaged about 1 pm in diameter. ~ 6 0 0 .
paraffin sections. To rule out the possibility that fixation
and paraffin embedding produced staining artifacts, paraffin sections were compared to unfxed frozen sections
stained in a similar manner. No differences in staining
patterns were observed between the two methods. For
ease of handling, paraffin sections were used in most of
the studies. Regions of tissue with affinity for WGA
were characterized by a dark brown reaction product,
while areas that were nonreactive with WGA stained
blue, as indicated in the figure legends.
In sections of testis, no WGA-reactive cells were found
within the seminiferous tubules (Fig. 1).Sperm flagellae
were not reactive with WGA, nor were any cells in the
region of the rete testis (not shown).
Sperm flagellae acquire SMA4 during epididymal
transit, and this could be demonstrated by comparing
sections of caput, corpus, and cauda, or by using sagittal
sections of whole epididymis. Figure 2 illustrates a representative section through caput epididymis stained
with WGA-peroxidase and toluidine blue. Sperm in the
lumen (SP) were loosely packed and were not reactive
with WGA. Principle cells were likewise unreactive in
both their cytoplasm and stereocilia (arrows). Throughout the epididymis, nuclei stained with toluidine blue;
in thicker sections they appeared dark (Figs. 2,3), while
in thinner sections used for clarity at high magnification
they appeared lighter in color (Figs. 6, 7). In contrast,
sections through corpus epididymis were highly reactive
with WGA (Fig. 3). Sperm from this region (SP) were
densely packed and exhibited heavily stained flagellae.
Stereocilia of the principle cells were also WGA positive
(arrows). Cytoplasmic staining of the principle cells was
not detected.
The zone between unstained and stained sperm was
sharply demarcated near the caputlcorpus junction; a
zone of transition or zone of intermediate staining was
not seen between caput and corpus. In the region where
sperm first showed WGA staining, there were many
more sperm per luminal profile, and sperm were more
densely packed than in proximal parts of the caput.
Although the cytoplasm of most epithelial cells in the
epididymis was unreactive with WGA, a small population of WGA-positive cells was found in the distal segment of caput epididymis (Fig. 4). The subpopulation of
WGA-positive epithelial cells was confined solely to this
region and was not found elsewhere in the epididymal
ducts. The cells were sparsely distributed within the
epithelium, showing about three to eight cells per lumina1 profile (arrows, Fig. 5). The cells were found in
the same region where sperm first exhibited staining of
the flagellae, and where sperm first became noticeably
concentrated within the lumen.
At higher magnification (Fig. 6)the WGA-positive cells
exhibited a morphology that was similar to holocrine
cells described in the literature. They had a constricted
base, a n expanded apex toward the lumen, and apically
placed nuclei (large arrow, Fig. 6). In contrast, the principle cells in the rest of the epithelium were nonreactive
with WGA and had basally placed nuclei that were
lightly stained with toluidine blue (small arrowheads,
Fig. 6). Under oil immersion optics (Fig. 7) morphology
of the WGA-reactive material could be observed. Many
small, granular deposits were present throughout these
cells, with the majority of WGA-positive granules at the
cell apex. Granules varied in size, with a n average of
about 1pm diameter.
The epididymis produces many kinds of secretory
products that interact with spermatozoa during their
final maturation processes (Bedford, 1975; Hamilton,
1975). Secretory glycoproteins of the epididymis have
been examined in several mammalian species, and some
have been shown by indirect immunofluorescence to
originate from principle cells of the epididymal epithelium. For instance, bovine forward motility protein
(Acott and Hoskins, 19811, acrosome-stabilizing factor in
the rabbit (Thomas et al., 1984), and SSEA-1 antigen of
mouse sperm (Fox et al., 1982) appear to be produced by
principle cells. A protease inhibitor on mouse sperm
(Aarons et al., 1984; Poirier and Nicholson, 1984) and
three antigens on human sperm (Yan et al., 1984; Tezon
et al., 1985a,b)have also been traced to origins in the
principle cells. The rat has been most extensively studied, and several glycoproteins that are added to sperm
during epididymal transit have been discovered. These
include ol-lactalbumin-like proteins of low molecular
weight (Jones and Brown, 1982; Byers et al., 1984;
Klinefelter and Hamilton, 1985), a 37.5-kd protein (Faye
et al., 1980), specific epididymal proteins, or “SEP,”
(Garberi et al., 1979; Kohane et al., 1980), a 32-kd acrosomal protein (Jones et al., 1981; Wong and Tsang, 1982;
Zeheb and Orr, 1984), “HIS’ proteins of 100 and 66 kd
(Rifiin and Olson, 19851, a 50-kd molecule (Dravland
and Joshi, 19811, acidic epididymal glycoprotein, or
“AEG,” (Lea et al., 1976; Pholpramool et al., 19831,
sulfated “DAG’ protein (Sylvester et al., 1984), and several others (Brooks and Tiver, 1984). It is probable that
some of these apparently different proteins being investigated in the rat are similar, if not identical; all have
been localized to principle cells in various portions of
the epididymis by immunocytochemical techniques. The
functional significance of most of these molecules is unclear at the present time.
There are other cell types in the epithelium of epididymis that present a n appearance suggestive of secretory activity and that may be involved in sperm
maturation. The holocrine or apical cells have been considered secretory for over 20 years based on morphological evidence (Martan and Risley, 1963; Martan and
Allen, 1964; Martan et al., 1964),yet none of their secretory products have been identified. They have been described in mouse (Martan and Allen, 19641, rat (Martan
and Risley, 1963; Sun and Flickinger, 1980), hamster
(Flickinger et al., 19781, and human (Martan et al., 1964).
They are characterized by swollen cell apices with nuclei
placed above the basal row of principle cell nuclei. There
generally are few holocrine cells per luminal profile
(about three to eight). The cell types known collectively
as “holocrine,” “apical,” “basal,” and “clear” cells described by various authors may represent different
stages in the life cycle of holocrine secretory cells, and it
has been suggested that they may be a variation of
principle cells based on electronmicroscopic evidence
(Reid and Cleland, 1957; Sun and Flickinger, 1980). The
cells can be selectively stained using PAS or alcian blue
techniques, indicating a high glycoprotein content (Martan and Risley, 1963; Martan and Allen, 1964).They are
rich in mitochondria (Brown and Montesano, 1980) as
well as carbonic anhydrase (Cohen et al., 1976; AbouHaila and Fain-Maurel, 1985). Histochemical studies
show dehydrogenase, acid phosphatase, and Ca2+
ATPase activities higher than surrounding principle
cells (Abou-Haila and Fain-Maurel, 1985).
In the present study, we demonstrate the localization
of a sperm-binding epididymal secretion (SMA4) in holocrine cells of the distal caput epididymis. SMA4 is a
glycoprotein that coats the sperm flagellae during epididymal transit (Feuchter et al., 1981; Vernon et al.,
1982) and that is a specific receptor for wheat germ
agglutinin (F'euchter e t al., 1986). WGA has been useful
in examining the biology of this molecule, since it appears to be the only receptor for WGA on sperm surfaces.
On SDS-PAGE blots it stains a single, well defined 54kd band of glycoprotein, and the staining of blots, sperm
flagellae, and holocrine cells can be prevented by incubation with 0.1 M n-acetylneuraminic acid (Feuchter et
al., 1986).
WGA-reactive holocrine cells were found in the distal
caput, in a region initially identified using SMA4 antibodies (Vernon et al., 1982). The region is similar to
regions IV and V of the mouse epididymis described by
Abou-Haila (Abou-Haila and Fain-Maurel, 1984) and region B of Pavlok (Pavlok, 1974). Several secretory proteins have been traced to caput epididymis (Lea et al.,
1976; Sylvester et al., 19841, but none have been traced
to this particular region. The location of these cells coincides exactly with the region of the tubule where
sperm first exhibit staining of the flagellum, a n observation that supports the hypothesis that these cells secrete the tail-coating glycoprotein, SMA4.
In addition, sperm first become condensed and tightly
packed within the lumen immediately distal to the region of WGA-positive cells, a n observation that may be
of functional significance. The packing of sperm in the
lumen of corpus and cauda results from absorption of
much of the fluid by principle cells of the latter half of
the caput (Flickinger et al., 1978). In a separate study
we have shown that one of the functions of SMAC is to
prevent tail-to-tail agglutination of sperm (Feuchter et
al., 1986). Caput sperm, when diluted into saline, show
rapid tail-to-tail agglutination, whereas corpus and
cauda sperm, which possess SMA4 on their flagellae, do
not. When caput sperm are incubated with purified
SMA4, they become coated with it and do not agglutinate. The addition of this molecule to sperm flagellae
during epididymal transit prevents their agglutination
when packed together in the corpus and cauda. Addition
to the surface must necessarily precede condensation of
sperm into a tight mass, in order that they may untangle and become free-swimming at ejaculation. Therefore,
the location of these cells in the area immediately proximal to where sperm first stain for SMA4 and where
they first become tightly packed is appropriate for their
The WGA-reactive material in the cell cytoplasm appears as large granules, whose size (about 1 pm) and
distribution near the cell apex are reminiscent of typical
secretory granules. Electron microscopic studies have
determined that cells with the characteristics of holocrine cells contain numerous large vesicles with lightly
flocculent material near the cell apex (Flickinger et al.,
1978; Sun and Flickinger, 1980). Since their size and
location correspond to the WGA-positive granules
pointed out in this study, it is likely that these vesicles
contain the WGA-reactive material. Future electron microscopic studies should confirm this.
Principle cells of the epithelium did not stain with
WGA. Although principle cells have been confirmed to
be secretory by autoradiography (Flickinger, 1979,1981),
the mode of secretion from these cells is unknown, since
they have no recognizable secretory granules. It is possible that secretion of certain proteins occurs from numerous small coated vesicles or associated Golgi vesicles
of principle cells (Flickinger, 1985). Secretion products
that have been localized in principle cells of mouse and
other species are glycoprotein in nature, although they
do not appear to be as heavily glycosylated as SMA4
(Lea et al., 1976; Garberi et al., 1979; Jones et al., 1981;
Wong and Tsang, 1982; Zeheb and Orr, 1984). Thus,
their mechanisms of secretion may be different.
The principle cells distal to the region where SMA4 is
secreted by holocrine cells were stained on their stereocilia. Stereocilia are abundant on cells throughout the
epididymis and probably function in the absorption of
material from the epididymal lumen (Hamilton, 1975;
Turner, 1979,1984). The observation that sterocilia were
stained only distal to the point where sperm first acquire
WGA reactivity suggests that they may be coated with
SMA4 to prevent adherence or entanglement of sperm
during passage. They also may be involved in the recovery of excess SMA4 from epididymal fluid.
In our earlier studies, SMA4 was also localized to
large apical vesicles in a few of the principle cells (Vernon et al., 1982).This observation can be reconciled with
the present data by postulating that the principle cells
are involved in absorption of excess SMA4. Secretion
from principle cells does not involve typical large secretory granules but rather involves a small vesicle system
of coated vesicles and associated Golgi elements (Flickinger, 1985). Furthermore, there is evidence that secretory antigens from the proximal epididymis are absorbed
by principle cells distal to the secretion site (Faye et al.,
1980; Lea et al., 1976).
Biochemical studies on SMA4 confirm that it is a
heavily glycosylated molecule (Feuchter et al., 1986).
Prime receptors for WGA on glycoproteins are carbohydrate side chains with terminal or internally situated
N-acetyl-D-glucosamine residues; more recently it has
been shown that WGA also reacts specifically with Nacetyl-neuraminic acid (sialic acid) and has a lower aFinity for N-acetyl-D-galactosamine (Goldstein, 1980). We
have reported that the enzyme N-acetyl-D-glucosaminidase has no effect on the staining of sperm flagellae by
WGA, whereas N-acetyl-neuraminidase abolishes all
WGA reactivity (Feuchter et al., 1986). Therefore, terminal carbohydrate residues of SMA4 appear to be rich
in sialic acid.
Several species of spermatozoa have been reported to
increase in sialic acid content during epididymal transit
(Bedford, 1963; Bedford et al., 1973; Holt, 1980), although the functional consequences of sialic acid addition to the sperm are unknown. Addition of the
antiagglutinin SMA4 to mouse sperm is probably the
mechanism by which they increase in sialic acid content. Since the addition of sialic acid during epididymal
maturation appears to be a common phenomenon among
species, it may be a general mechanism by which sperm
agglutination is prevented. On other cell types in which
agglutination is undesirable, such as erythrocytes, high
surface sialic acid content is thought to play a role in
preventing agglutination.
Holocrine cells of the epididymis exhibit similarities
in morphology, distribution, and secretory product to
cells in other epithelia, such as goblet cells of the intestine. Both have a distinctive shape, a sparse arrangement within the epithelium, large secretory vesicles,
and a product rich in carbohydrate. The lubricating and
protective function of intestinal mucus can be compared
to the antiagglutination and surfactant functions of
SMA4. These cells may represent a general subset present in many epithelia that perform similar functions.
The discovery of a sperm-binding epididymal secretion
that originates from holocrine cells in the distal caput
raises questions about the functions of holocrine cells
elsewhere in the epididymis and about their relationship to sperm maturation. The observation that an epididymal secretion functions as an antiagglutinin
suggests that epididymal dysfunction may be one cause
of unexplained sperm agglutination, particularly in humans in which no antisperm antibodies are present.
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flagellum, holocrine, caputo, antigen, mouse, maturation, origin, epididymal, sperm, distal, cells
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