A Histochemical Study of the Cytoplasmic Inclusions of the
Epithelial Cells in the Epididymis of the Mouse
By A. C. CHRISTIE
(From the Cytological Laboratory, Dept. of Zoology, Oxford; present address, Royal Hospital
for Women, Paddington, Sydney, Australia)
SUMMARY
I . A histochemical study has been made of the cytoplasmic inclusions of the epithelial cells lining the storage section of the canal in the head of the epididymis of the
mouse.
2. Spherical bodies, usually situated in the vicinity of the nucleus and here referred
to as juxta-nuclear bodies, are shown to consist of cerebroside with some phospholipid.
The juxta-nuclear bodies have been regarded by other authors as derived from the
nucleolus, but there is no histochemical similarity.
3. A large supra-nuclear body exists, consisting of a matrix of protein and carbohydrate with enmeshed spheres. The latter do not colour with any dye or histochemical
reagent used in this investigation. They are partially or completely surrounded by
rims or crescents consisting wholly or mainly of lipid.
4. Droplets and much smaller granules are present in the region between the supranuclear body and the free border of the cell. The contents of the former show no
histochemical evidence of the presence of organic matter, while the latter consist of
protein and carbohydrate.
INTRODUCTION
T
HE epithelial cells of the head of the epididymis of the mouse have
recently been subjected to careful study by the electron-microscope
(Dalton and Felix, 1954). The present investigation was undertaken in the
belief that such studies might lead to error unless controlled by very full use
of the light-microscope on the same material. In particular, the work with the
electron-microscope has been done mainly with a single fixative, osmium
tetroxide, which is capable of misleading through the deposition of osmium
dioxide, which scatters electrons; and in its ordinary usage the electronmicroscope gives no chemical information. Some of the objects in the cytoplasm of the epithelial cells of the head of the epididymis of the mouse are
quite large enough for morphological and chemical studies with the lightmicroscope.
Good morphological descriptions of the cell in question have been published
by Nassonov (1924), Ludford (1925), and Benoit (1926).
In the present studies their findings have been carefully checked. It was
felt that there was a considerable gap in knowledge, partly because there has
been too much reliance in the past on the study of deposited particles of
osmium dioxide or of silver, and partly because scarcely any histochemical
findings have been reported.
As Benoit (1926) showed, the head of the epididymis of the mouse contains
tubules of two kinds: (1) the initial segment, in which spermatozoa are not
stored, and (2) the storage region. Everything in the present paper refers to
the latter region except where the contrary is distinctly stated.
[Quarterly Journal of Microscopical Science, Vol. 96, part 2, pp. 161-168, 1955.]
2421.2
M
162
Christie—A Study of the Cytoplasmic Inclusions of the
In the present paper the end of the cell next the lumen of the tubule will be
called the apical or upper end; the other end, the basal or lower.
MATERIAL AND METHODS
Pieces of fresh epididymis of the common house-mouse were placed in a
number of fixatives (referred to later) within i to 2 minutes of killing the
animal. After fixation overnight and thorough washing in running water next
day, both paraffin and gelatine sections were prepared and examined unstained
or stained by various techniques. In addition to routine histological staining
methods (Ehrlich's haematoxylin and eosin, Heidenhain's iron haematoxylin,
toluidine blue, phloxine-tartrazine (Lendrum, 1947)), Golgi techniques (of
Kolatchev, Mann-Kopsch, and Aoyama), and Metzner's modification of
Altmann's stain for mitochondria, the following histochemical techniques
were applied to appropriately fixed and embedded tissue:
(1) Sudan black B on gelatine and paraffin sections of tissue fixed in various
mixtures referred to below. Also, Baker's (1949) method, with formaldehydefixed, postchromed, gelatine-embedded sections.
(2) Sakaguchi's (1925) test for arginine, according to the method adapted
to histochemical use by Baker (1947a).
(3) Baker's (1946) acid haematein and pyridine extraction test for phospholipids.
(4) The coupled tetrazonium reaction of Danielli (1947, 1950), the technical details being those described by Pearse (1953).
(5) The Feulgen reaction (hydrolysis for 15 minutes at 6o° C ) . The tissue
was fixed in Altmann's or Mann's fluid or in Heidenhain's mercuric-saline.
(6) The periodic acid / Schiff reaction of McManus (1946) and Hotchkiss
(1948). The former's \°/0 watery solution of periodic acid was used for
5 minutes. The additional techniques of acetylation and of subsequent saponification in weak alkali, before the reaction, were also employed (McManus and
Cason, 1950).
(7) Examination for glycogen. Paraffin sections of tissue fixed in Rossman's
fixative were treated by the periodic acid / Schiff technique, some being previously treated with salivary amylase and commercial diastase for 1-2 hours
at 370 C. After treatment with the enzymes the slides were covered with
celloidin before proceeding further (Lillie and Greco, 1947).
(8) The lead tetra-acetate / Schiff reaction (Hashim and Acra, 1953), combined with the acetylation technique of McManus and Cason (1950).
(9) The Schiff reaction after sodium bismuthate oxidation. Rigby's (1950)
solution was employed, viz:
Sodium bismuthate
Glacial acetic acid
Distilled water .
.
.
.
.
.
.
.
.
.
.
.
.
.
2 gm.
. 1 0 ml.
. 1 0 ml.
Sections of Champy-fixed tissue were brought to water and placed in this
Epithelial Cells in the Epididymis of the Mouse
163
cavity of tubule-
free border-
"secretion" droplet"secretion" granule—
sphere in supra nuclear body
lipoidal coat of
sphere
10 ji
uxta-nuclear
juxta
body
d
nucleus
nucleolus
mitochondrion
Fie. 1. Diagram of the epithelial cell that lines the storage section of the head of the
epididymis of the mouse.
164
Christie—A Study of the Cytoplasmic Inclusions of the
solution for half an hour with constant agitation. After thorough washing they
were subsequently treated with Schiff s reagent.
RESULTS •
Juxta-nuclear bodies
In the region of the nucleus, and generally above it but occasionally below,
spheres or spheroids are observed. They vary somewhat in size. Most of them
are about iju. or less in diameter, but occasionally they almost attain 2[M,
especially when situated basally. Sometimes the larger ones are irregular in
shape, having a crenated periphery spotted with one or more small deposits of
reduced osmium. This appearance is seen in Mann-Kopsch and Kolatchev
preparations, and also after fixation in osmium tetroxide alone.
They were observed by Benoit (1921), Nassonov (1924), and Ludford
(1925). Benoit considered them to be lipid in nature and all three workers
believed that they arise from the nueleolus. Ludford referred to them as 'complex granules', and stated that they occur not only in and above the region of
the cytoplasm occupied by the Golgi element but also between the latter and
the nucleus. In my material they are mostly adjacent to the nucleus and mainly
above it; hence the name I have given them.
They are present in paraffin and gelatine sections of tissue fixed in Altmann's
and Champy's fluids, 2% osmium tetroxide, formaldehyde-calcium, cold
acetone, and Mann's fluid. They are clearly seen in unstained sections of
tissue fixed in the first three fixatives just mentioned, being slightly darkened
by the first two and considerably so by the third. Before applying many of the
histochemical techniques it was found necessary to treat sections with periodic
acid for 5 minutes to bleach them; this completely decolorizes the bodies,
no matter how dark previous fixation in osmium-containing fixatives has
rendered them. Champy's and Altmann's fixatives give the best morphological
detail, and wherever possible sections thus fixed have been employed.
After appropriate fixation they can be coloured even in paraffin sections
with Sudan black B or its acetylated derivative. The sudanophilia is alcohollabile.
With Baker's (1946) acid haematein test, a thin rim of positively-reacting
phospholipid is present round the periphery of each body. In life the phospholipid is presumably distributed throughout the bodies, mixed with the other
constituent. After pyridine extraction the test is negative.
The bodies are not fixed by hot acetone.
After oxidation by periodic acid (McManus, 1946; Hotchkiss, 1948;
Shimizu and Kumamoto, 1952; Glegg and others, 1952; Lhotka, 1952;
Hashim and Acra, 1953), or sodium bismuthate (Rigby, 1950), the juxtanuclear bodies give a strongly positive reaction with Schiff's reagent. Without
preliminary oxidation there is no reaction.
The juxta-nuclear bodies are not stained by the coupled tetrazonium reaction. The Sakaguchi test is also negative.
Epithelial Cells in the Epididymis of the Mouse
165
These results, taken together, indicate that the juxta-nuclear bodies consist
mostly of cerebroside, with some phospholipid.
The bodies are visible and present a black rim in 'Golgi' preparations made
by the Mann-Kopsch and Kolatchev methods, but they are not seen in
Aoyama preparations.
Histochemically, the juxta-nuclear bodies contrast strongly with nucleolar material, as the following table shows.
TABLE I
Test
Baker's acid haematein test:
(a) first part
(6) second part .
Feulgen reaction* .
Reduction of 2% osmium
tetroxide employed as
fixative
Sudan black .
Schiff reaction after oxidation with:
periodic acid
lead tetra-acetate .
sodium bismuthate
Coupled tetrazonium reaction
.
.
.
.
Mann-Kopsch technique .
Kolatchev's technique
Nucleolar material
Juxta-nuclear bodies
Moderately strong positive reaction
Very strong positive
Positive (partially)
Positive rim only
Slight
Not coloured
Strong
Strongly coloured
Negative
Positive
);
it
Positive
Visible but colourless
Not present
Negative (completely)
Negative
Black
.,
• Performed on paraffin sections of tissue fixed in Altmann's, Zenker's, and Mann's fluids.
Supra-nuclear body
Between the nucleus and the free border of the cell there is a specialized area
of the cytoplasm. This area, which is usually roughly spherical, is about 6/* in
diameter. Sometimes it is more elongated in the long axis of the cell, up to
10 fi. In this area there are numerous spherical or spheroid bodies of various
sizes up to about i-$fi in diameter. These spheres are often arranged in rows,
generally slightly wavy. The wavy rows are usually arranged in the long axis
of the cell. Sometimes the row is shaped like a horseshoe, with the convex
side of the curve pointing towards the nucleus.
None of the staining or histochemical techniques used coloured these
spheres, and there is therefore no positive information about their chemical
composition in life.
Each sphere is surrounded by a complete or incomplete investment of
material that reacts positively to Sudan black. When the investment is incomplete it appears in optical section as a crescent. The black colour is quickly
removed by 70% alcohol. The investment therefore contains or consists
of lipid. Among all the other histochemical tests used, only the periodic
acid / Schiff method gives a positive reaction at or near the surfaces of the
spheres.
166
Christie—A Study of the Cytoplasmic Inclusions of the
The whole region in which the spheres lie reacts to dyes and histochemical
tests somewhat differently from the ground cytoplasm of the rest of the cell.
With basic dyes (toluidine blue and Nile blue) this region stains less strongly
than the general cytoplasm; it colours also by the periodic acid / Schiff
method, but less intensely than the rims round the spheres. The coupled
tetrazonium reaction is positive, resembling that in the general ground cytoplasm. It appears that there is a diffuse substance between the spheres. This
substance contains protein and probably carbohydrate (but apparently not
glycogen).
When the tissue is fixed in Mann's or Champy's fluid and post-osmicated,
much osmium is precipitated in this specialized area of the cell. It is seen in
the form of strands, which appear to correspond to the rows of granules. When
the sections are treated for 10-15 seconds with \°/0 aqueous periodic acid
solution, part of the black material is oxidized and thus removed, and the
appearance then approaches more closely to that given by Sudan black, for
rims and crescents are seen. It would appear that the osmium is most strongly
deposited on or in the lipid rims of the spheres, but also extends between
these in a diffuse manner so as to join them. The substance of the spheres
themselves does not reduce osmium in the techniques.
The general appearance given by the silver technique of Aoyama is in
general similar to that given by the osmium methods, but the fixation is not so
good.
An interesting figure is produced if the tissue is fixed in 1% cadmium
chloride, embedded in gelatine, and coloured with Sudan black. The lipid
rims of the spheres have now run together to form a tubular structure colouring with Sudan black; the centre of the tube is uncoloured.
'Secretion' products
In the upper part of the cell, between the zone just described and the free
border of the cell, spheres are frequently seen, larger than those in the supranuclear body. These spheres have been regarded by Fuchs (1902) and Ludford (1925) as secretory products. They have generally been regarded as
derived from the spheres in the supra-nuclear zone (see Ludford, 1925;
Bowen, 1926). For a discussion as to whether they are in fact secretory
products or absorbed material, see Wagenseil (1928). None of the stains or
histochemical tests used in the present investigation coloured these bodies.
They are presumably aqueous, and contain little or no organic matter.
In the same region of the cell as the large spheres just described, small
granules are often demonstrable. They are too small for accurate measurement, but may be about \ to |/u. in diameter. They withstand various fixatives
(Rossman, Champy, Altmann) and are seen in both gelatine and paraffin
sections. The only histochemical tests that showed them were the coupled
tetrazonium, periodic acid / Schiff, and lead tetra-acetate / Schiff tests. The
coupled tetrazonium test showed them slightly darker than the ground cytoplasm. The Schiff reactions were strongly positive, even after saponification
Epithelial Cells in the Epididymis of the Mouse
167
of acetylated sections. Digestion with saliva or commercial diastase for 1 hour
at 370 C. did not prevent the reaction. The granules presumably contain
protein and a carbohydrate other than glycogen. These bodies appear to
correspond with the 'secretory granules' of Ludford (1925), previously seen
by Fuchs (1902). Nicander has recently (1954) described glycogen granules
in this region of the corresponding cell of the dog.
Mitochondria
The mitochondria are mostly rod-shaped and slightly wavy. They are well
seen in Altmann-Metzner preparations, and also, from their phospholipid
content, after acid haematein. Where the mitochondria happen to be closely
aggregated together, they are seen to give a positive reaction with the coupled
tetrazonium test. They are most numerous in the apical part of the cell,
between the supra-nuclear body and the nucleus, and below the nucleus;
that is to say, they occur where not excluded by other cytoplasmic inclusions.
DISCUSSION
There is histochemical evidence indicating that the predominant epithelial
cell in the head of the epididymis of the mouse contains spheres near the
nucleus, consisting mainly of cerebroside but also in part of phospholipid.
Bodies of similar nature have previously been reported in the nerve-cells of the
thoracic ganglia of the locust (Shafiq and Casselman, 1954) and the sympathetic nerve-cell of the rabbit (Baker and Casselman, 1955). There is no histochemical support for the contention that they are nucleolar extrusion-products.
It is possible that spheres ('lipochondria') consisting of cerebrosides and
phospholipid may be widely distributed cytoplasmic inclusions of diverse
cells. It would appear profitable to search for them elsewhere.
Between the nucleus and the free border of the cell there is a supra-nuclear
body consisting of a diffuse matrix containing carbohydrate and protein, interspersed with (and partly encompassed by) spheres that are unstained by any
staining method or histochemical technique used in the present investigation.
These spheres are usually surrounded by rims or capped by crescents of lipid
material reacting positively to the periodic acid / Schiff technique, and therefore presumably containing cerebroside. In post-osmication techniques,
osmium is deposited on the lipid rims or crescents, and to a less extent on the
diffuse matrix.
Between the supra-nuclear body and the free border of the cell, droplets
and granules are present, the former containing material that gives no histochemical reactions for organic material, and the latter, much smaller in size,
consisting of both protein and carbohydrate. These have been considered to
be two types of secretion product by Fuchs (1902) and Ludford (1925), but
others, notably Wagenseil (1928), have considered the possibility that the
droplets may be absorption products.
The author is grateful to Dr. J. R. Baker for suggesting the subject of this
168
Christie—Cytoplasmic Inclusions of the Epididymis
communication, and acknowledges with pleasure much helpful advice and
discussion.
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