II. Characterization and Development of the Regional- andCellular-Specific Abnormalities in the Epididymis of Micewith -Hexosaminidase A Deficiency
H. I. ADAMALI,* I. H. SOMANI,* J.-Q. HUANG,t D. MAHURAN4 R. A. GRAVEL,11 J. M. TRASLER,tII
AND L. HERMO*
From the *Departnzent of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada; the tMcGill
University-Montreal Childrens Hospital Research Institute, Montreal, Quebec, Canada; the Research Institute,
Hospital for Sick Children, Toronto, Ontario, Canada; the §Departments of Pediatrics, Biology, and Human
Genetics, McGill University, Montreal, Quebec, Canada; and the IlDepartments of Pediatrics, Pharmacology and
Therapeutics, and Human Genetics, McGill University, Montreal, Quebec, Canada.
ABSTRACT: 13-Hexosaminidase (Hex) is a lysosomal enzyme that ex-ists as two isoenzymes: Hex A (subunit structure af3) and Hex B (1313).Its presence in the testis and epididymis suggests important roles forHex and its substrates in male fertility and reproductive functions. Dis-ruption of the Hexa gene encoding the a-subunit of Hex has led to the
generation of a mildly affected mouse model of human Tay-Sachs dis-ease, allowing us the opportunity to analyze the effects of isolated HexA deficiency on epithelial cellular morphology of the male reproductivetract. At 5 weeks and at 3, 5, and 12 months, the testes, efferent ductsand epididymides of Hex A-deficient (Hexa -I-) and wild-type (Hexa+1+) mice were perfuse fixed and analyzed by routine light and electronmicroscopy as well as with immunocytochemistry employing antibodiesto lysosomal enzymes. In the testis, the seminiferous epithelium of Hexa
-I- mice appeared comparable to that of wild-typo mice in appearance
and topographical arrangement of its cell types at all ages examined.Also, no differences were noted for the efferent ducts. In contrast, therewere striking abnormalities in the epididymides of the mutant mice; how-
ever, the abnormalities were mainly restricted to the initial segment andintermediate zone. Principal cells of these regions at 5 weeks showed
a dramatic increase in the number of lysosomes as compared with
those from wild-type animals, and this progressed with increasing age.
Furthermore, unlike the few small lysosomes present in wild-type mice,those of Hexa -I- mice were at times enlarged and often filled the
supranuclear and basal regions of these cells. In the lightmicroscope,large, dense cellular aggregates were noted at the base of the epithe-
hum in the proximal initial segment that corresponded in the electronmicroscope to two different cell types, both of which increased in size
with age. One aggregate was considered to belong to narrow cells on
the basis of the presence of numerous cup-shaped vesicles character-istic of these cells; they appeared to be dislocated from the upper halfof the epithelium. In the distal initial segment and intermediate zone,
narrow cells were readily identified, but rather than being slender as inthe control animals, they were greatly enlarged and filled with pale ly-sosomes in mutant mice. The second type of cellular aggregate notedin the proximal initial segment corresponded to halo cells. They con-tained numerous small and large lysosomes and small, Golgi-related,dense, core granules characteristic of halo cells. On the basis of thelarge size of these cells, they appeared to be actively intemalizing sub-stances from the intercellular space. In contrast, principal and clear cells
of the caput, corpus, and cauda regions did not appear to show a sig-
nificant increase in number or size of lysosomes as compared withthose of wild-type animals. All structures identified as lysosomes in the
various cell types were immunoreactive for cathepsin D. The present
data thus reveal that isolated Hex A deficiency results in region- andcell-specific abnormalities in the epididymis but in no apparent abnor-malities in the testis or efferent ducts. Specific roles for Hex A thatcannot be compensated for by other isozymes of Hex appear to existwithin lysosomes of epithelial cells predominantly of the initial segmentand intermediate zone. Taken together, the results also suggest thatthe inability to degrade endocytosed substrates normally acted upon byHex A in lysosomes of principal and narrow cells leads to their accu-mulation, eventual fusion, and increased size.
(n = 3), and 12 months (n = 3) were utilized. These
animals were fixed by cardiac perfusion with 2.5% glu-
taraldehyde buffered in sodium cacodylate (0.1 M) con-
mining 0.05% calcium chloride (pH 7.4). The testes, ef-
ferent ducts, and epididymides were removed, cut into
small pieces, washed in 0.1 M cacodylate buffer, and then
postfixed in ferrocyanide-reduced osmium for 1 hour at
4#{176}C.Each epididymis was subdivided into its major re-
gions: the initial segment, intermediate zone, caput, cor-
pus, and cauda. Tissues were dehydrated in a graded se-
ries of ethanol and propylene oxide and then embedded
in Epon.
Light Microscopic Immunocytochemistry
Wild-type (n = 2) and Hexa -I- (n = 2) mice at 5 months ofage were anesthetized, and their testes and epididymides werefixed by immersion with Bouin’s fixative. After 2 hours of fix-
ation, the epididymides were removed and cut along their longaxis to include all regions. The tissue was then placed for severaldays in Bouin’s fixative and 70% ethanol. Subsequently, the tis-
sue was dehydrated in a series of graded ethanol solutions, fol-lowed by dioxane treatment and embedding in paraffin wax. Sec-tions 5 m thick were cut, mounted on glass slides, and treated
for light-microscope immunocytochemical analysis.A rabbit polyclonal anti-human Hex A antibody (Hou et a!,
1996; Hermo et al, 1997) that was raised against human placen-tal Hex and that recognizes both Hex A and Hex B was used at
Adamali et al Defects in the Epididymis in 3-Hexosaminidase A Deficiency 805
a dilution of 1:100 in 20 mM Tris-buffered saline (TBS), pH
7.4, containing 0.1% bovine serum albumin. A polyclonal anti-human cathepsin D antibody (Calbiochemical, La Jolla, Calif)
raised against human liver extract was used at a dilution of 1:50 in TBS as published previously (Igdoura et a!, 1995). Normal
rabbit serum was utilized as a control at a dilution of 1:50 and1:100 in TBS.
The paraffin sections were deparaffinized with xylene and hy-
drated in a series of graded ethanol solutions. Endogenous per-oxidase activity was inactivated in 70% ethanol containing 1%lithium carbonate. After hydration, the tissue sections were
washed in distilled water containing 300 mM glycine to block
free-aldehyde groups. Prior to immunostaining, the sections were
blocked for 15 minutes with 10% goat serum in TBS. Tissuesections were incubated at 37#{176}Cin a humidified chamber for 1hour with 50 l of the respective antibodies or rabbit serum.
Following several washes in TBS containing 0.1% Tween-20(TWBS), the sections were blocked with 10% goat serum for 15minutes in order to prevent nonspecific binding of the secondary
antibody.The secondary antibody incubation was performed at 37#{176}C
with goat anti-rabbit IgG conjugated to peroxidase (Sigma
Chemical Co., St. Louis, Mo) at a dilution of 1:250 in TBS. Allsections were washed and incubated for 10 minutes with per-
ide and 0.03% hydrogen peroxide in TBS containing 0.1 M im-
idazole at pH 7.6 (Straus, 1982). The sections were counter-
stained with 0.1% methylene blue and dehydrated in a gradedseries of ethanol solutions (for 2 minutes each) and xylene (for
2 minutes). Coverslips were mounted onto glass slides with Per-
mount.
Electron Microscopic Immunocytochemistry
Wild-type (n = 2) and Hexa -I- mice (n = 2) at 5 monthswere perfuse fixed, and pieces of the epididymis were embedded
in Lowicryl K4M as described previously (Oko, 1988; Hermo
et a!, 1997). Sections were labeled with anti-cathepsin D anti-body diluted at 1:20 in TBS as described previously (Igdoura eta!, 1995) and for anti-Hex A antibody as outlined in Adamali
et a! (1999). Normal rabbit serum was employed as a control at
a dilution of 1:20 in TBS.
Results
Light Microscopic Appearance of Epithelial Cells of theTestis and Epididymis in Hexa -I- Mice
For comparative purposes, controls for the testis, efferent
ducts, and epididymis can be seen in the accompanying
article on Hexb -I- mice (Adamali et al, 1999). In the
light microscope, the seminiferous epithelium of Hexa
-I- mice at 5 weeks (Fig. la) and at 3 (Fig. lb-d), 5,
and 12 months of age appeared comparable to those of
wild-type animals at the same age of development. Sertoli
cells were normal, and spermatogenesis revealed a full
complement of germ cells, with all stages of the cycle of
the seminiferous epithelium being observed (Fig. la-d).
Leydig cells, macrophages, and myoid cells showed no
deviations from controls (Fig. la-d). The efferent ducts
showing ciliated and nonciliated cells also appeared sim-
ilar to control animals at each age (not shown).
Morphological abnormalities were noted within the
proximal and distal regions of the initial segment of the
epididymis of Hexa -I- mice as compared with wild-
type mice. Principal cells of the proximal initial segment
of Hexa -I- mice at 5 weeks (Fig. 2a) showed few dense
lysosomes, whereas in older animals, their numbers in-
creased dramatically, such that their supranuclear and bas-
al regions were often filled with dense lysosomes (Fig.
2b-d). In the distal initial segment, at 5 weeks (Fig. 3a),
principal cells showed few pale lysosomes, which in-
creased in number by 3 months (Fig. 3b). At 5 months,
numerous pale and dense lysosomes were noted in these
cells, while at 12 months, principal cells were engorged
with dense lysosomes (Fig. 3c,d).
FIG. 1. Light micrographs of the seminiferous epithehium of the testis of (a) 5-week-old and (b-d) 3-month-old Hexa -I- mice at stages (b) V. (c)
VII, and (d) X-XI of the cycle. Sertohi cell nuclei (large arrowheads), spermatogonia (small arrows), zygotene (small arrowheads) and pachytenespermatocytes (double arrows), early spermatids (open arrows), and elongating spermatids (large arrows) are observed, and their topography is similar
to that seen in control animals of the same age. The processes of Sertohi cells at stage VII radiate towards the lumen and contain lysosomes (curvedarrows), as seen in control animals. Asterisk, lumen; L, Leydig cells; m, macrophage. a: 357x, b, d: 437x, c: 415x.
FIGS. 2, 3. FIG. 2. Light micrographs showing tubules of the proximal initial segment of the epididymis of (a) 5-week-old, (b) 3-month-old, (C) 5-
month-old, and (d) 12-month-old Hexa -I- mice. The epithehium is lined by columnar principal cells (P) that at 5 weeks (a) show few dense lysosomes
(arrowheads) but at later ages (b,c,d) show an increase in the number of lysosomes (arrowheads) supranuclearly and, at times, basally. Many basal
cells (small arrows) appear similar to control animals. Large, dense cellular aggregates (curved arrows) appear at the base of the epithehium by 3
months (b) and appear to increase in size and number with age, as seen in (c) and (d). Large arrows, capillaries; asterisks, lumen; IT, intertubular
space; open arrows, narrow cells. a: 338x, b, c: 234x, d: 305x. FIG. 3. Light micrographs showing tubules of the distal initial segment of the
epididymis of (a) 5-week-old, (b) 3-month-old, (c) 5-month-old, and (d) 12-month-old Hexa -I- mice. Principal cells (P) by 5 weeks (a) contain few
pale tysosomes (arrowheads), but by 3 months (b), such structures are abundant in their supranuclear region. At 5 months (c), numerous pale and
dense lysosomes (arrowheads) are evident in principal cells, whereas at 12 months (d), these cells become engorged with dense lysosomes (arrow-heads). Large, dense cellular aggregates (curved arrows) appear at the base of the epithehium by 5 months (c) and increase in size and number by
12 months (d). Narrow cells (large arrows) with an apically localized nucleus are filled with pale lysosomes at all ages. Many basal cells (small arrows)
appear similar to those of control animals. Asterisks, lumen; IT, intertubular space. a: 348x, b, c: 272x, d: 239x.
Journal of Andrology . November/December 1999
Adamali et al . Defects in the Epididymis in /3-Hexosaminidase A Deficiency 807
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Journal of Andrology . November/December 1999
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FIGS. 4-7. FIG. 4. Light micrograph showing tubules of the intermediate zone of the epididymis of a 3-month-old Hexa -I- mouse. Principal cells
(P) contain large numbers of pale lysosomes (arrowheads) in their basal and supranuclear regions. Large, pale aggregates (curved arrows) are present
at the base of the epithelium. Several basal cells (small arrows) comparable to those in control animals are noted, while narrow cells are filled With
pale lysosomes (open arrow). An apical cell (large arrow) contains dense supranuclear lysosomes. Asterisk, lumen. 325x. FIG. 5. Light micrograph
of tubules of the caput epididymidis of a 3-month-old Hexa -I- mouse. Principal cells (P) show clusters of pale lysosomes (arrowheads) supranuclearly;
the latter are not prominent in the basal region of these cells. Basal cells (small arrows) appear comparable to those of control animals. Asterisk,
lumen; IT, intertubular space. 319x. FIG. 6. Light micrograph of tubules of the corpus epididymidis of a 3-month-old Hexa -I- mouse. Principal cells
(P) contain several pale supranuclear lysosomes (arrowheads), with some appearing basally. Clear cells (large arrows) contain several large pale
lysosomes that appear comparable to those seen in control animals. Asterisk, lumen; IT, intertubular space; small arrows, basal cells. 319X. FIG.7. Light micrograph of tubules of the cauda epididymidis of a 3-month-old Hexa -I- mouse. Principal cells (P) show several pale lysosomes (largearrowheads) in their supranuclear and basal regions. Clear cells (large arrows) show both pale and dense lysosomes (small arrowheads) and are
comparable to those of control animals. Small arrows, basal cells; Asterisk, lumen; IT, intertubular space. 330X.
Adamali et al . Defects in the Epididymis in f3-Hexosaminidase A Deficiency 809
Narrow cells with an apically located nucleus were not-
ed in the proximal initial segment at 5 weeks of age in
Hexa -I- mice (Fig. 2a) and appeared comparable to
those of controls. However, they were not apparent in this
region at all other ages examined (Fig. 2b-d). In contrast,
narrow cells in the distal initial segment at all ages ex-
amined were characterized by an apically localized nu-
cleus, with pale lysosomes completely filling their entire
cytoplasm at 3 months of age and at all later ages (Fig.
3a-d). In both these regions, many basal cells appeared
similar to those in controls (Figs. 2 and 3). Large, dense
cellular aggregates were noted at the base of the epithe-
hum of the proximal initial segment by 3 months (Fig.
2b) and appeared to increase in size and number at later
ages (Fig. 2c,d). Some aggregates were also observed in
the distal initial segment at 5 (Fig. 3c) and 12 (Fig. 3d)
months of age.
In the intermediate zone, principal cells exhibited pale
lysosomes that progressively increased in number with
age as compared with controls and often filled their su-
pranuclear and basal regions (Fig. 4). Narrow cells pre-
sent in this region were completely filled with pale ly-
sosomes, while many basal cells appeared similar to those
in controls (Fig. 4). Large aggregates were noted at the
base of the epithelium, but unlike their counterparts in the
initial segment, they appeared pale and devoid of content
(Fig. 4).
In the caput (Fig. 5), corpus (Fig. 6), and cauda (Fig.
7) epididymidis of Hexa -I- mice, principal cells con-
tamed several lysosomes, often with a pale appearance;
they occupied the supranuclear region and only rarely the
basal region. Clear cells were readily identified by their
larger size and their frothy apical region and by the pres-
ence of both pale and dense lysosomes. Many basal cells
of these regions appeared similar to those in controls
(Figs. 5-7).
Light Microscopic Immunostaining of Epithelial Cells ofthe Proximal and Distal Initial Segment in Wild-Type andHexa -I- MiceIn the proximal initial segment of the epididymis of wild-
type mice at 3 and 5 months of age, lysosomes in prin-
cipal and narrow cells were intensely immunoreactive for
cathepsin D (Fig. 8a). In Hexa -I- mice, principal cells
showed a weak to moderate diffuse reaction, and while
narrow cells were not apparent, aggregates at the base of
the epithelium were intensely reactive and in some cases
extended as narrow strips towards the lumen (Fig. 8b).
With anti-Hex A antibody, principal cells of both wild-
type and Hexa -I- mice showed a weak reaction (Fig.
9a,b). Narrow cells in wild-type mice were intensely re-
active (Fig. 9a) as were aggregates at the base of the
epithelium in Hexa -I- mice, some of which occasion-
ally extended towards the lumen as narrow strips (Fig. 9b).
In the distal initial segment, with the anti-cathepsin D
antibody, principal cells at 3 and 5 months of age showed
a diffuse weak reaction in wild-type animals that was
more pronounced in Hexa -I- mice, while narrow cells
-4
FIGS. 8-11. FIG. 8. Light rnicrographs showing the proximal initialsegment of the epididymis of (a) 5-month-old wild-type and (b) Hexa -I- mice
stained with anti-cathepsin D antibody. In (a), principal cells (P) show several distinct reactive lysosomes (arrows) in their cytoplasm; however, in (b),the reaction is diffuse. Aggregates (curved arrows) at the base of the epithelium in (b) are intensely reactive and in some cases, the immunoperoxidase
reaction extends towards the lumen as narrow strips (double arrows). Asterisks, lumen. a: 315x, b: 282x. FIG. 9. Light micrographs showing the
proximal initial segment of the epididymis of (a) 5-month-old wild-type and (b) Hexa -I- mice stained with anti-Hex A antibody. Narrow cells
(arrowhead) in (a) show intense reactivity, whereas principal cells (P) show few reactive lysosomes in (a) and (b). Aggregates (curved arrows) at the
base of the epithelium in (b) are reactive, and in some cases, the immunoperoxidase reaction extends as narrow strips (double arrows) towards thelumen. a: 464x, b: 540x. FIG. 10. Light micrographs showing the distal initialsegment of the epididymis of 5-month-old (a) wild-type and (b) Hexa
-I- mice stained with anti-cathepsin 0 antibody. Narrow cells (arrowheads) are intensely reactive in (a) and, although greatly enlarged, are also
reactive in (b). Principal cells (P) show a weak reaction in (a) and a checkerboard-like staining pattern in (b). a: 228x, b: 437x. FIG. 11. Light
micrographs showing the distal initialsegment of the epididymis of 5-month-old (a) wild-type and (b) Hexa -I- mouse stained with anti-Hex A
antibody. Narrow cells (arrowheads) in (a) are intensely reactive, and although greatly enlarged in (b), show a reaction that is especially dense apically.
Principal cells (P) show reactivity in (a) and (b). Basal cells (arrows) are reactive in (a) and (b). Asterisk, lumen; IT, intertubular space. a: 242x, b:
250x.
FIG. 12. Electron micrographs of principal cells of the proximal initial segment of (a) 5-week-old, (b) 5-month-old, and (c) 12-month-old Hexa -I-mice. The supranuclear lysosomes (L) at all ages show a moderate or dense appearance and contain vesicles of different sizes and electron-dense
concentric membranous structures (arrowheads). The number of lysosomes in the supranuclear region increases with age and in some cells, they
accumulate basally and become very large (C); areas of fusion are apparent between lysosomes (large arrows). G, Golgi apparatus; N, nucleus; small
row cells in wild-type mice, while in Hexa -I- mice, the
reaction was only intense apically; principal cells showed
weak reactivity in both types of mice (Fig. lla,b). Ag-
gregates were immunoreactive for both Hex A and ca-
thepsin D, but these did not extend towards the lumen
(not shown). Use of normal rabbit serum failed to show
any reaction over sections of the epididymal tubules, with
images comparable to those previously published for
these antibodies (Igdoura et a!, 1995; Hermo et al, 1997).
Electron Microscopic Appearance of Epithelial Cells ofthe Testis, Efferent Ducts, and Epididymis of HexA -I-Mice
The testis of Hexa -I- mice in the electron microscope
revealed no morphological abnormalities in Sertoli, germ,
Leydig, and myoid cells or macrophages as compared
with wild-type animals at all ages examined. Noncihiated
and ciliated cells of the efferent ducts of Hexa -I- mice
were also comparable in appearance to those of wild-type
animals at each age examined (not shown).
In the proximal initial segment, principal cells of Hexa
-I- mice showed a progressive increase in lysosomes
with age, such that by 12 months, they often filled the
supranuclear and basal regions of these cells (Fig. 12a-
c). At all ages, lysosomes contained concentric membra-
nous structures, pale vesicles of different sizes, and lipid
inclusions, all embedded in a moderately or densely
stained matrix (Fig. 12a-c). By 12 months of age, some
of these lysosomes were extremely large, apparently re-
sulting from the fusion of one with another (Fig. l2c).
In the distal initial segment of Hexa -I- mice, prin-
cipal cells at 5 weeks showed few pale lysosomes (not
shown). At 3 months of age, the number of lysosomes
increased, and it continued to do so at 5 and 12 months
of age. While some principal cells at early ages presented
only pale lysosomes, others contained both pale and dense
lysosomes (Fig. l3a). However, by 12 months of age,
lysosomes were only of the dense variety (Fig. 13b).
These lysosomes contained membranous structures, yes-
ides, and lipid inclusions (Fig. l3a,b) and often filled the
supranuclear and basal regions of the cell.
In the intermediate zone at 5 weeks of age in Hexa -I-
mice, principal cells showed few pale !ysosomes (Fig.
l4a); however, their number progressively increased with
age (Fig. l4b), such that by 12 months of age, they often
filled the supranuclear and basal regions of these cells.
They were pale at all ages and contained few membra-
nous structures and vesicles (Fig. l4a,b). In wild-type an-
imals at all ages, principal cells of the proximal and distal
initial segment and intermediate zone showed only few
dense supranuclear lysosomes (Adamali et al, 1999). The
Golgi apparatus, secretory vesicles, and endoplasmic re-
ticulum in Hexa -I- mice of these three regions (Figs.
12b,c, 1 3a,b, and 14a,b) were comparable in appearance
to controls, as were coated pits and vesic!es and endo-
somes (not shown).
Narrow cells of the proximal initial segment in Hexa
-I- mice at 5 weeks of age appeared comparable to those
in controls of the same age, showing an occasional dense
lysosome, numerous apical cup-shaped vesicles, and a ta-
pering basal region. However, at later ages, narrow cells
were not conspicuous in this region. In the distal initial
segment and intermediate zone, narrow cells were readily
apparent, showing few pale lysosomes at 5 weeks (Fig.
l5a), whereas at all later ages, these cells became greatly
enlarged and filled with pale lysosomes, such that even
the basal region, normally tapered, was swollen and en-
gorged with lysosomes (Figs. l3b and 15b). Some of
these lysosomes were very large, apparently resulting
from the fusion of one with another (Fig. lsb). These
lysosomes contained few pale membranous structures and
vesicles. The apical region of these cells was filled with
cup-shaped vesicles, and a Go!gi apparatus and strands of
endoplasmic reticulum were also evident (Fig. l5a,b), all
of which were comparable in appearance to those seen in
wild-type animals.
In Hexa -I- mice at all ages, in the proximal and
distal initial segment and intermediate zone, many basal
cells showed few dense lysosomes, a Golgi apparatus, and
sparse endoplasmic reticulum (Fig. 16a), which was com-
parable to the case in controls. However, a few basal cells
-4
FIG. 14. Electron micrographs of the supranuclear region of principal cells of the intermediate zone of the epididymis of (a) 5-week-old and (b) 3-
month-old Hexa -I- mice. Few pale lysosomes (asterisks) are observed in (a); however, their number increases with age (b). Small vesicles and
few membranous structures (arrowheads) are seen in the lysosomes in (b). N, nucleus; G, Golgi apparatus; arrows, endoplasmic reticulum. a: 8,544x,b: 10,680x.
FIG. 15. Electron micrographs of narrow cells in the distal initial segment of the epididymis of (a) 5-week-old and (b) 12-month-old Hexa -I- mice.
In (a), few lysosomes (asterisks) are seen in the supranuclear region, whereas in (b), they fill the supranuclear and infranuclear regions. Fusion (large
arrowheads) between these structures is seen in (b). The contents of these structures include small vesicles and pale membranes (small arrowheads).
Numerous cup-shaped vesicles (arrows) are seen in the apical region of these cells. In (b), principal cells (P) show moderately dense lysosomes (L).
N, nucleus; G, Golgi apparatus. a: 12,240x, b: 7,429x.
Journal of Andrology . November/December 1999814
Adamali et al . Defects in the Epididymis in 3-Hexosaminidase A Deficiency
“c-. -
815
:1 ‘ -.
:.,
816 Journal of Andrology . November/December 1999
FIG. 16. Electron micrographs of basal cells of the proximal initial segment of the epididymis of a 3-month-old Hexa -I- mouse. The basal cell in
(a) is comparable to that seen in wild-type animals in that it shows few dense lysosomes (arrowheads), whereas in (b), the cell shows numerous palelysosomes (asterisks) along the slender process, extending towards the lumen. N. nucleus; G, Golgi apparatus; P. principal cells; Cap, capillary. a:
9,922x, b: 6,760x.
at ages 3 months and later showed an expanded apical
region containing numerous pale lysosomes (Fig. 16b); in
other respects, their features appeared similar to those of
wild-type mice. A few apical cells were noted in these
regions, presenting numerous small, dense lysosomes, as
shown in Adamali et a! (1999).
The use of anti-cathepsin D antibodies at the electron
microscope level revealed intense labeling over the nu-
merous pale, membrane-bound organelles located supra-
nuclearly and basally in principal (Fig 17a), narrow (Fig.
17b), and basal cells of Hexa -I- mice; gold particles
were present throughout the interior of these organelles,
confirming that they were lysosomal in nature. Only few
gold particles were evident, consistent with background
levels of labeling, when normal rabbit serum was em-
ployed (not shown).
The !arge, dense cellular aggregates noted in the light
microscope in the proximal initial segment were seen to
be of two types in the electron microscope. In one case,
the cytoplasm of such aggregates was filled with small
and large lysosomes containing an abundance of dense
concentric and crescentic membranous whorls of varying
sizes and few vesicles embedded in a moderately
dense matrix (Fig. 1 8a,b). Characteristically, these aggre-
gates presented a Golgi apparatus that was associated with
small, dense core granules, and a nucleus that was often
disfigured and indented by the impinging lysosomes (Fig.
18a). Such aggregates appeared to increase in size with
age (Fig. 18b). They did not show evidence of junctions
with adjacent epithelial cells, and at times, the intercel-
lular space surrounding them was slightly dilated and
contained membranous structures and vesicles (Fig. 1 8a).
Many of the features of these aggregates suggested that
they belonged to halo cells. In addition, small, normal-
looking halo cells of the type seen in wild-type animals,
in other words, cells with a pale cytoplasm with few
Adamali et al . Defects in the Epididymis in fJ-Hexosaminidase A Deficiency
p.:.
117
- , - 4’
:
FIG. 17. Electron micrographs of (a) the supranuclear region of a prin-
cipal cell and (b) infranuclear region of a narrow cell of the intermediatezone of a Hexa -I- mouse labeled with anti-cathepsin 0 antibody. Nu-
merous gold particles (arrowheads) are present over the supranuclear
(a) and infranuclear (b) pale structures, which indicate their lysosomal
nature (L). N, nucleus. 15,200x.
817
electron microscopy. Such aggregates were filled with
small and large lysosomes, but these contained numerous
vesicles of various sizes embedded in a moderately dense
matrix containing few membranous whorls (Fig. 19a,b).
The Golgi apparatus was evident, but small, Golgi-relat-
ed, dense core granules were not (Fig. l9a). Occasionally,
they made contact with the basement membrane, and des-
mosomes were shared with adjacent epithelial cells. The
nucleus was often indented and disfigured by the large
infringing lysosomes (Fig. l9b). In appropriate rare sec-
tions, these cells extended towards the lumen, where cup-
shaped vesicles were found apically, thus characterizing
them as narrow cells. Halo cells with a normal profile
were at times seen in the vicinity of these aggregates,
often sending processes around them (Fig. l9a). Use of
anti-cathepsin D antibodies revealed numerous gold par-
ticles over the small and large pale, membrane-bound or-
ganelles located within the aggregates, confirming their
lysosomal nature (Fig. 19c).
In the caput, corpus, and cauda epididymidis, principal
cells of 3-month-old Hexa -I- mice showed some mor-
phological abnormalities as compared with wild-type an-
imals of the same age. In wild-type mice, lysosomes were
more or less spherical in outline and contained small yes-
ides and whorls of electron-dense material, whereas in
Hexa -I- mice, some lysosomes were larger in size, ir-
regular in outline, and contained large vesicles (Figs. 20
and 21). However, in Hexa -I- mice, their number did
not appear to be significantly different from controls. Ly-
sosomes of clear cells of Hexa -I- mice at all ages also
did not appear to change significantly in number and were
comparable to those in wild-type animals.
small, dense core granules related to the Golgi apparatus,
were also seen in the epithelium (Fig. 19a), and such cells
were seen at times in large numbers in the intertubular
space.
A second type of large, dense cellular aggregate, pre-
sent in the proximal initial segment, was confirmed by
Discussion
Testes
In the light and electron microscopes, the testes of Hexa
-I- mice, examined at all ages, demonstrated no evi-
dence of morphological changes. The organelles of Sertoli
cells, including lysosomes, were comparable to those seen
in wild-type animals in terms of their appearance, number,
and topographical arrangement within the epithelium. All
the various types of germ cells were evident, including
all stages of the cycle of the seminiferous epithehium, as
described in normal mice (Oakberg, 1956). Sperm counts
were also comparable to those of wild-type animals, a!-
though litters sired by Hexa -I- males were smaller in
size (Trasler et al, 1998). In the rat, with the anti-Hex A
antibody, Hex was localized in lysosomes of Sertoli cells
at all stages of the cycle (Hermo et al, 1997), and in mice,
a pronounced reaction also was evident in germ cells,
particularly in spermatocytes (Adamali, unpublished data)
and in the acrosome of spermatozoa (Miller et a!, 1993;
Journal of Andrology . November/December 1999
‘-S’-P...’: ‘.. -
q.
818
FIG. 18. Electron micrographs of one type of large, dense cellular aggregate at the base of the epithelium of the proximal initial segment of the
epididymis of (a) 3-month-old and (b) 5-month-old Hexa -I- mice. In (a), the cellular aggregate is found in the intercellular space (asterisks) betweenprincipal cells (P). Lysosomes (L) of various sizes contain numerous, concentric, electron-dense membranous structures (arrowheads) and vesicles.Dense core granules (small arrows) characteristic of halo cells are observed next to the Golgi apparatus (G). In (b), the aggregate, also considered
to be a halo cell, is gigantic and spreads itself along the base of the epithelium between principal cells but does not contact the basement membrane.
It is filled with gigantic lysosomes (L) containing numerous concentric and crescentic membranous structures (arrowheads). The Golgi region shows
small, dense core granules. Large arrows, endoplasmic reticulum; Cap, capillary; N, nucleus, a: 9,680x, b: 7,757x.
Adamali et al . Defects in the Epididymis in fi-Hexosaminidase A Deficiency 819
Hall and Killian, 1987), suggesting a role for Hex in these
cells. Sertoli cells are endocytic cells involved in the up-
take of various substances from the lumen that eventually
appear within their lysosomes, where they are degraded
(Morales et al, 1985; Hermo et al, 1994). In addition,
lysosomes are involved in the degradation of residual
bodies phagocytosed by these cells (Morales and Cler-
mont, 1993). Germ cells are also endocytic cells and con-
tain lysosomes at certain steps of development (Hermo et
a!, 1994). The present data thus indicate that the absence
of the a-subunit does not have an effect on lysosomes,
despite the fact that Northern blot analysis revealed that
a-subunit mRNA levels were highest in the testis as com-
pared with levels in other tissues (Della Fazia et a!, 1994;
Wakamatsu et a!, 1994; Hermo et a!, 1997). The 13-subunit
also does not appear to be essential in the seminiferous
epithelium, as its absence, as noted in the accompanying
paper (Adamali et al, 1999), had no effect on the mor-
phological appearance of Sertoli or germ cells. Hence, at
present it is unclear how the deficiency of Hex is over-
come in these cell types or whether in fact substrates for
Hex are found in the testis.
Efferent Ducts
The nonciliated cells of the efferent ducts are active en-
docytic cells (Stoffel and Friess, 1994; Ihio and Hess,
1994) internalizing SGP- 1, SGP-2, and other substances;
Hex and cathepsins have been immunolocalized in their
lysosomes (Hermo et a!, 1992; 1994; 1997; Igdoura et a!,
1995). In the present study, these cells in the Hexa -I-
mice showed a normal appearance, a normal distribution,
and numbers of apical tubules, endosomes, and lysosomes
comparable to those in wild-type animals. In contrast, the
absence of Hex A and Hex B in the Hexb -I- mice, as
seen in Adamali et a! (1999), revealed a dramatic increase
in the number of lysosomes throughout the cytoplasm of
noncihiated cells. The difference between the two mouse
models confirms a requirement for Hex in the efferent
ducts but indicates that it would be fulfilled by either
enzyme, not requiring Hex A uniquely.
Epididymis
In the epididymis, differences noted between Hexa -l-
and wild-type mice were observed mainly within the ini-
tial segment and intermediate zone. Normally few in
number in wild-type mice, with age, lysosomes of prin-
cipal cells of the initial segment in Hexa -I- mice grad-
ually increased in number and size such that by 12
months, they often filled their supranuclear and basal re-
gions. The initial segment is a region of great importance
because in many species, including humans, it is the site
at which sperm begin to acquire their fertilizing capability
(Orgebin-Crist and Olson, 1984). In addition, it is char-
acterized by tall principal cells, which have also been not-
ed in the initial-like segment of the human epididymis
(Palacios et al, 1991, 1993; Yeung et al, 1991). In the rat,
these cells demonstrate fluid-phase and receptor-mediated
endocytosis and the synthesis and secretion of proteins
and are the major site of 5a-reductase synthesis (Djakiew
et al, 1984; Hermo et a!, 1991, 1994; Robaire and Viger,
1995). In the mouse, there is a dense vascularization of
fenestrated capillaries immediately beneath the basement
membrane of the epithelium and a high blood flow within
this region (Abe et a!, 1984), indicating that there must
be an active and rapid exchange of substances between
the epithelial cells and the blood.
The intermediate zone in the rat has been shown to be
a highly endocytic region in which principal cells possess
giant apical endosomes, suggesting an active role for
these cells in endocytosis (Hermo, 1995). The interme-
diate zone is also present in the mouse, as noted in the
present study. As principal cells of the initial segment and
intermediate zone are endocytic, it may be postulated that
many of the endocytosed substances end up in the lyso-
somes of these cells. Such substances may be derived as
direct secretory products from Sertohi cells as well as
-9
FIG. 19. Electron micrographs of a second type of large, dense cellular aggregate situated between principal cells (P) in the proximal initial segmentof the epididymis of a 12-month-old Hexa -I- mouse. In (a) and (b), the aggregate is filled with lysosomes (L) of various sizes, which show a plethoraof small vesicles and few irregular membranous structures (arrows) enclosing lipid inclusions (asterisks). A Golgi apparatus (G) is visible but does notshow dense core granules. The cytoplasm of the aggregate shows no sparsely granulated, dilated endoplasmic reticulum, unlike what is seen inadjacent principal cells (small arrows). Cellular aggregates of this type are considered to be narrow cells. In (a), a halo cell (I-I), comparable to thatseen in control animals, is noted next to a portion of the aggregate. In (b), the nucleus (N) is indented by impinging lysosomes. In (c), a paraformal-dehyde-fixed and Lowicryl-embedded aggregate present at the base of the proximal initial segment of the epididymis of a 12-month-old Hexa -I-mouse is seen labeled with anti-cathepsin 0 antibody. Gold particles (arrowheads) are present over the large pale stained structures of the aggregate,indicating their lysosomal nature (L). a: 7,271 x, b: 7,654x, c: 1O,413x.
FIGS. 20, 21. FIG. 20. Electron micrograph of the caput epididymidis of a wild-type 5-month-old mouse. The supranuclear region of principal cellsreveals several lysosomes (L) containing vesicles (small arrows) and an amorphous, electron-dense material (large arrows). Open arrow, endoplasmicreticulum; m, mitochondria. 27,284x. FIG. 21. Electron micrograph of the supranuclear region of a principal cell of the caput epididymidis of a 5-month-old Hexa -I- mouse. Lysosomes (L) contain vesicles, some of large size (asterisks), embedded in a moderately or densely stained matrix.Some lysosomes are small and dense (curved arrows), while others are large and have a spherical (arrows), or irregular (large arrowheads) outline.Open arrows, endoplasmic reticulum; m, mitochondria; N, nucleus. 37,453x.
820
-d
Journal of Andrology . November/December 1999
i; “.
Adamali et al . Defects in the Epididymis in ,3-Hexosaminidase A Deficiency 821
822 Journal of Andrology . November/December 1999
from the surface of spermatozoa as they traverse the lu-
men of the epididymal duct. Our present data suggest that
under normal conditions, substrates ending up within the
lysosomes of principal cells of these two regions are acted
upon by Hex A. Corroboration of this hypothesis stems
from evidence of immunolocalization of Hex in these
cells (Hermo et al, 1997) and from the fact that its ab-
sence in Hexa -I- mice results in an age-dependent in-
crease in the number and size of lysosomes. These data
thus suggest that Hex A plays an important role in the
lysosomes of principal cells of the initial segment and
intermediate zone that cannot be replaced by Hex B (Ada-
mali et al, 1999).
In the present study, some basal cells showed increased
numbers of lysosomes with a pale appearance in the ini-
tial segment and intermediate zone of Hexa -I- mice. In
wild-type mice, lysosomes were small, dense, and few in
number, and only some basal cells of these regions
showed reactivity for Hex (Hermo et al, 1997). This find-
ing may explain why only some basal cells appeared ab-
normal, whereas others appeared comparable to those of
controls. Presently, there is no evidence that basal cells
practice endocytosis; however, the present data strongly
suggest a role for endocytosis by some of these cells as
the number of lysosomes increase in Hexa -I- mice.
In the caput, corpus, and cauda regions of the epidid-
ymis in Hexa -I- mice, lysosomes in principal and clear
cells did not appear to be significantly increased as com-
pared with the major increases noted in Hexb -I- mice
(Adamali et a!, 1999). Northern blot analysis has revealed
that 13-subunit mRNA levels were higher in the corpus
region compared with levels in the initial segment (Her-
mo et al, 1997). Hex has also been immunolocalized with-
in lysosomes of both principal and clear cells of the caput,
corpus, and cauda, both of which show endocytic func-
tions in normal adult animals (Hermo et a!, 1988, 1994;
Vierula et a!, 1995). The absence of major lysosomal
changes in Hexa -I- mice in the caput, corpus, and cau-
da regions, in contrast to the dramatic changes observed
in the Hexb -I- mice, suggests a prominent role for Hex
that can be fulfilled by Hex A or Hex B in both of these
cell types of these regions.
Cellular AggregatesIn the proximal initial segment of Hexa -I- mice, two
types of dense cellular aggregates were noted with the
electron microscope at the base of the epithelium. Both
first appeared by 3 months and increased in size with age.
One of the aggregates contained numerous small and
large membrane-bound organelles filled with membra-
nous structures and identified as lysosomes by immuno-
cytochemistry. The aggregates characteristically present-
ed small, dense core granules adjacent to the Golgi ap-
paratus and showed no evidence of junctions with adja-
cent epithelial cells, nor did they adhere to the basement
membrane, all of which are features described for halo
cells (Dym and Romrell, 1975; Robaire and Hermo,
1988). Thus, our present data strongly suggest that these
aggregates in the epididymis of Hexa -I- mice are halo
cells actively involved in taking up substances from the
intercellular space, a role they must perform normally as
immunocompetent cells (Dym and Romrell, 1975). How-
ever, the accumulation of lysosomes in these cells and the
dramatic increase in their overall size suggest that these
halo cells may be affected by the absence of Hex A. Small
halo cells comparable to those in control animals were
also seen in the epithelium and intertubular space, where
they were often found in large numbers, indicating that
they may be actively recruited to the epithelium.
The second type of cellular aggregate seen in the prox-
imal initial segment of Hexa -I- mice appears to belong
to narrow cells, for several reasons. Firstly, narrow cells
normally present in the epithelium, where they contact
the lumen, were not readily apparent in this segment. Sec-
ondly, our immunocytochemical data revealed that nar-
row cells were immunoreactive for cathepsin D and Hex
A in wild-type mice, and in Hexa -I- mice, aggregates
were also reactive for these enzymes, with an occasional
one extending as a narrow strip towards the lumen. Third-
ly, some aggregates were immunoreactive for carbonic
anhydrase II (not shown), a marker exclusive for narrow
cells (Adamali and Hermo, 1996). Fourthly, the aggre-
gates showed no dense core granules near the Golgi ap-
paratus that excluded them as halo cells, nor did they
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and Sandhoff diseases and partial determination of accumulated gan-
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munolocalization of the 18 and 29 kilodalton secretory proteins in the
mouse epididymis: evidence for differential uptake by clear cells.Mi-
crosc Res Tech 1995:30:24-36.
Wakamatsu N, Benoit G. Lamhonwah A-M. Zhang Z-X. Trasler JM.
Triggs-Raine BL. Gravel RA. Structural organization, sequence, and
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osaminidase. Genomics l994;24:llO-119.
Yamanaka 5, Johnson MD, Grinberg A, Westphal H, Crawley JN,Taniike M. Suzuki K. Proia RL. Targeted disruption of the Hexagene results in mice with biochemical and pathological features
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