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Subcellular distribution of the nonspecific esterase in the mouse epididymis with special reference to regional differences.

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THE ANATOMICAL RECORD 214:148-153 (1986)
Subcellular Distribution of the Nonspecific Esterase
in the Mouse Epididymis With Special Reference to
Regional Differences
Laboratoire de Biologie cellulaire, Universitd Rend Descartes, 75270 Paris Cedex 06, France
The subcellular distribution of esterases was studied in mouse epididymis by using 5-bromo-indoxyl-acetate as a substrate. In all the cells of the duct,
a low level of esterase activity was detected except in one of the five segments of the
head-segment Tv; in one of the three types of apical cells-the “prominent cells”;
and in the “clear cells” scattered in the middle and distal parts. In these cells, the
intensity of the reaction was high.
The reaction product was consistently found in the endoplasmic reticulum and
was more abundant in cells showing a high level of activity than in others. In cells
with low esterase activity, the reaction was mainly restricted to this organelle. In
highly active cells, the spectrum of subcellular locations was selectively enlarged
and esterase was demonstrated in almost all cell compartments, including the cell
membrane, nuclear envelope, mitochondria, lytic structures, and, more rarely in the
Golgi apparatus or microvilli. These locations were dependent on cell type. A weak
enzyme activity also appeared on mature spermatozoa.
Numerous studies have demonstrated the structural
complexity of the epididymis in various mammals: the
rat (Reid and Cleland, 1957),the rabbit (Nicander, 19571,
man (Holstein, 1969), the macaque (Ramos and Dym,
1977), the guinea pig (Hoffer and Greenberg, 1978), the
hamster (Flickinger et al., 19781, and the mouse (Soranzo et al., 1982).
A previous study of postnatal differentiation of enzyme activities in mouse epididymis has shown that an
esterase activity was present in the epithelium at birth,
and persisted along the undifferentiated duct at the
same intensity during the first 2 weeks and then varied,
leading to the regional differentiation of the adult state
(Abou-Ha‘ilaand Fain-Maurel, 1986). This suggests that
these later variations could be related to a differentiated
pattern of gene activation associated with the maturation of the different cell types and the functional zonation of the duct. Thus, esterase appears to be a valid
marker enzyme for cellular activity.
In the postpubertal mouse epididymis, esterase activity detected histochemically differed across cell types
(Allen and Slater, 1958; Kirkeby and Blecher, 1981) and
even in histologically identical cells (Abou-Ha‘ila and
Fain-Maurel, 1984). These variations are probably the
expression of different functional states. They may correspond to quantitative changes, or to a different subcellular distribution in cell compartments as shown in other
organs (Deimling and Bocking, 1976). For example, in
the interstitial cells or prepubertal mouse testis, esterase is exclusively associated with lipid droplets whereas
in the Leydig cells of testosterone-producing adult testis,
the esterase reaction is predominantly in mitochondria
and at cellular membranes.
0 1986 ALAN R. LISS, INC.
The aim of the present investigation was to determine
the subcellular distribution of esterase along the mouse
epididymis by comparing cells with a low or a high level
of enzyme activity. This study may provide insight into
regional and cellular differences in the function of the
epididymis. Furthermore subcellular location of esterase has not previously been reported in this organ.
This study was conducted on six male mice (Swiss OF
1) aged about 3 months. The basic procedure was repeated three times on two animals. The male mice were
anesthetized with chloral hydrate and intracardially
perfused with fixative (3%paraformaldehyde and 1.5%
glutaraldehyde in 20 mM collidine-S buffer pH 7.4) for 5
minutes. For each animal, the two epididymides were
removed, subdivided into ten parts (five for the head,
three for the corpus, two for the cauda) under a binocular microscope while in the fixative solution, fragmented, fixed for 2 hours (4OC), and washed in several
changes of collidine-S buffer. The samples, chopped into
minced slices, were rinsed twice in 10 mM Tris-HC1
kuffer (pH 6.5) and incubated (37OC, 70 or 90 minutes)
iil Holt’s esterase medium (1958) containing 5-bromoincloxyl-acetate (Sigma) as a substrate and buffered with
Tris-HC1 to pH 6.5. Controls made on each of the ten
parts of one animal, included incubation of tissues in
medium lacking the substrate, and secondly in the complete medium after preincubation for 30 minutes in
M eserine, a n inhibitor of cholinesterases. After incubaReceived April 2, 1985; accepted September 23, 1985.
tion, the tissues were washed in Tris-HC1 buffer and
then rinsed with several changes of collidine-S bufYer.
The samples were postfixed in buffered osmium tetroxide (1hour a t 4OC), dehydrated, and embedded in Epon.
For each animal, semithin sections (1 pm) were used to
localize the different segments for electron microscopy.
For each part, several ultrathin sections were examined
in a Philips EM 300 without grid staining.
In the mouse, as in other mammals, the epididymis is
made up of three portions: the proximal (caput), middle
(corpus), and distal (cauda) parts. The proximal part was
subdivided into five segments (1,-V) characterized by the
cytological and histochemical features of the principal
cells. In this part, scattered between the principal cells,
which contained a basal nucleus, “cells with apical nuclei” were found (Abou-Hai’la and Fain-Maurel, 1984).
In the middle and distal parts “clear cells” have been
cytologically (Soranzo et al., 1982) and histochemically
(Abou-Hai’laet al., 1985) described.
The ultracytochemical study of esterase activity in the
mouse epididymis led to the following results:
1) In all the principal cells of the epididymis, a low
level of esterase activity was detected (Fig. l),except in
segment IV of the proximal part, where the amount of
reaction product was abundant (Fig. 2). However, in this
segment, groups of cells having a stronger reaction than
in certain neighbouring cells were seen.
2) In all the principal cells of the duct, a reaction
product was found in the vesicular or saccular endoplasmic reticulum (ER). The reaction product was more
abundant in cells showing a high level of activity than
in others. In cells with low activity, the reaction was
mainly restricted to this organelle (Fig. 1). In highly
active cells, the spectrum of subcellular locations was
found to be selectively enlarged and esterase was shown
to be present in almost all the cell compartments: the
nuclear envelope, mitochondria, Golgi apparatus, cellular membrane (Fig. a), cytoplasmic structures resembling lysosomes or residual bodies (Fig. 31, and microvilli
(Fig. 4). The nuclear envelope and mitochondria presented low activity in segment 11, high activity in segment IV,and a variable pattern in the middle and distal
parts. The reaction deposit was observed in the Golgi
apparatus essentially in segment IV.It appeared at the
cellular membrane and in the lytic inclusions in segment IV in the middle and distal parts. The microvilli
showed a heterogeneously distributed reaction in the
middle and distal parts.
3) Among the three types of “cells with apical nuclei”
described in the five segments of the proximal part (narrow cells in segment I, prominent cells in segment 11,
mitochondria-goblet cells in segments 111-V) (Abou-Haila
and Fain-Maurel, 1984), only the “prominent cells” of
segment I1 presented a high esterase activity in the ER,
nuclear envelope, lytic inclusions, as well as in the mitochondria and basal membrane (Fig. 5a, b). In segment
IV,the level of activity was lower in the “mitochondriagoblet cells” with apical nuclei than in the adjacent
principal cells, but remained pronounced in the nuclear
envelope and lytic inclusions (Fig. 6).
The “clear cells,” scattered in the medial and distal
parts, were characterized by their high pattern of enzyme activity, which was located in the ER, the nuclear
envelope, and the mitochondria (Fig. 7). The lytic inclusions which were numerous in the perinuclear area of
this cell type showed a marked activity on their membrane or in their contents (Figs. 7,8).
The basal cells generally presented a n undetectable
level of esterase activity.
4) In the lumen of the middle and distal parts, the
spermatozoa showed a low pattern of enzyme activity on
the mitochondria and the plasma membrane lining the
acrosome and principal piece (Fig. 9).
5) In the case of incubation in the substrate-free medium, the deposition of reaction product did not occur in
the cells, thus confirming the specificity of the reaction
(Fig. 10). In the case of incubation in the standard medium containing eserine, the enzymatic activity of the
epididymis remained unaffected (Fig. 11).
A preliminary electrophoretical study employing several inhibitors has shown that carboxylic ester hydrolases were the major group of esterases in the mouse
epididymis (unpublished data). Thus the substrate chosen for the ultrastructural demonstration of this enzyme
was 5-bromo-indoxyl-acetate,one of the more selective
substrates used to study the specific hydrolysis patterns
of carboxylic esters (Deimling and Bocking, 1976). Apparently incubation duration (70 or 90 minutes) did not
influence the enzyme distribution in ultrathin sections.
Fixation with aldehydes is the usual procedure of
keeping soluble esterases in the sections. In some cases,
the demonstration of the presence of esterases is feasible
only after prior fixation (Niemi and Kormano, 1965). It
is known that in sections fixed with glutaraldehyde,
activity is weaker than after formaldehyde treatment,
but the distribution of reaction product is comparable in
the two cases (Bocking et al., 1973; Kirkeby and Moe,
1984). In the present study, the fixative used for ultracytochemistry contained paraformaldehyde (3%) and
glutaraldehyde (1.5%)in a n attempt to obtain a satisfactory preservation of cell structures and soluble enzymes.
The distribution and variations of esterases on ultrathin
sections corresponded to the histochemical data obtained on cryostat section (Abou-Hai’laand Fain-Maurel,
This study particularly demonstrated a high level of
esterase activity in the principal cells of segment IV,
which, however, showed the same ultrastructural features as the cells of segment V. It also showed some
differences in the location of reaction deposit across the
five segments of the proximal part. Thus, it can be postulated that each segment plays a specific role. The
variations in the level of activity in the histologically
identical principal cells of segment IV are probably the
expression of different functional states, as shown in
other tissues by biochemical and cytochemical determinations (Feustel et al., 1970; Bocking and Deimling,
The main reaction, with the substrate used, is found
in the ER in all cell types despite the regional differences described for the mouse epididymis (Soranzo et al.,
1982; Abou-Hai’la and Fain-Maurel, 1984). This preferential location was found in many tissues and has been
revealed with different substrates (cf. Deimling and
Bocking, 1976). In the mouse epididymis, no difference
was seen in staining patterns of the ER, which can
Fig. 1. Ultracytochemical demonstration of the very low level of
nonspecific esterase activity in the principal cells of segment I11 of the
proximal part. ~ 6 , 9 0 0 .
Fig. 2. Ultracytochemical demonstration of the high level of nonspecific esterase activity in the principal cells of segment Iv of the proxima1 part. Note the reaction deposits on cell membrane, ER, nuclear
envelope, mitochondria (el, and Golgi apparatus (b).~ 6 , 9 0 0 .
Fig. 3. Nonspecific esterase in lytic inclusions of the principal cells
of the distal cauda. x 13,600.
Fig. 4. Nonspecific esterase on microvilli of the principal cells of the
distal part. x7,800.
Fig. 5. Ultracytochemical demonstration of nonspecific esterase in
segment I1 with a high level of activity in the apical (a) and basal (b)
parts of “prominent cells.” Note the distribution of reaction product in
the nuclear envelope, mitochondria, and cell membrane. a, X6,900; b,
Fig. 6. Demonstration of a higher esterase activity in a principal cell
(m) than in a “mitochondria goblet-cell’’(b)in segment IV. X7,800.
Fig. 7. Demonstration of a higher level of activity i n the “clear cell”
than in the two adjacent principal cells (proximal caudal. Note the
location of reaction deposit around the lytic inclusions, on ER-rich
cytoplasm, mitochondria, and plasma membrane. x 7,800.
Fig. 8. High magnification of a “clear cell” basal part evidencing the
location of reaction product in the RER saccular caveolae (a),perinuclear space (b),and on the membrane surrounding the lytic inclusions.
Fig. 9. Location in the lumen of the epididymal medial part of the
reaction product on the spermatozoa mitochondria1 sheath (*) and the
plasma membrane of the principal piece. X7,800.
Flg. 10. Principal cells in proximal corpus after incubation of tissue
in medium lacking the substrate. ~ 7 , 8 0 0 .
Fig. 11. The basal part of a “clear cell” in the distal corpus after
treatmentwith 10-4Meserine.Notethedistributionofenzymaticactivity
on the nuclear envelope, cytoplasm, mitochondria, residual bodies, and
plasma membrane. ~ 6 . 9 0 0 .
appear (Soranzo et al., 1982) either as vesicular, with
rarely studded ribosomes in segments I and 11, or a s
saccular, with numerous ribosomes in the other parts.
On the contrary, the nuclear envelope which is a part of
the ER presented a variable enzymatic reaction, depending on the regional differences of the canal and the
various cell types. It will, therefore, be of interest to
know whether these two subcellular components contain identical esterases. The location of esterase on the
membrane or in the matricial substrate of the mitochondria and the lytic inclusions (lysosomes, residual bodies)
could be correlated with their functional state. However,
no activity was seen in the multivesicular bodies which
now are considered as secondary lysosomes.
Because of their esterase activities, as is the case for
other enzymes (acid phosphatase, 6-glucuronidase) (unpublished data), the “prominent cells” seemed to play
the same function as “clear cells,” which are implicated
in reabsorption (Moore and Bedford, 1979). Even if their
supranuclear cytoplasmic content was less rich in multivesicular bodies than “clear cells,” they showed a n
active endocytosis process. This suggests that reabsorption occurs in the two cell types but that the ingested
substances can differ.
The presence of esterase activity, observed on cell microvilli and spermatozoa plasmalemma in the middle
and distal parts of mouse epididymis, could reflect the
selective adsorption on spermatozoa of a cell product
released in the lumen. No reaction deposit was observed
on acrosomes containing the “corona penetrating enzyme” (CPE), which nevertheless is believed to be a n
esterase (Bradford et al., 1976a,b).
Other inhibitors and methods are required to further
specify individual esterases inside the cell. A preliminary study of electrophoretic banding patterns has already shown the presence of several specific esterases
and isoenzymes, some of which were regulated by androgens, in tissues, luminal fluids, or spermatozoa of the
different parts of the mouse epididymis (unpublished
The authors wish to thank Mrs. Georgette Hedouin
for her skillful technical assistance, Mrs. Myriam Largeau in typing the manuscript, and Constance Greenbaum for stylistic corrections.
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subcellular, distributions, nonspecific, regional, differences, mouse, esterase, references, epididymal, special
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