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The ultrastructure of the principal cells and intraepithelial leucocytes in the initial segment of the rat epididymis.

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The Ultrastructure of the Principal Cells and
Intraepithelial Leucocytes in the Initial
Segment of the R a t Epididymis '
ANITA P. HOFFER,2 DAVID W. HAMILTON3 AND DON W. FAWCETT
Department of Anatomy and Laboratories of Human Reproduction, and
Reproductive Biology, Harvard Medical School,
Boston, Massachusetts 021 15
ABSTRACT
The ultrastructure of the principal cells and intraepithelial leucocytes in the initial segment of the rat caput epididymidis was examined with the
electron microscope. Specializations of the principal cells associated with absorption include numerous endocytic invaginations of the cell surface, numerous
coated vesicles and multivesicular bodies in the apical cytoplasm. It was demonstrated that particulate tracers are taken into the cells and sequestered in secondary lysosomes and multivesicular bodies. Morphological features consistent with
secretory activity are also found in the principal cells and include numerous
cisternae of rough endoplasmic reticulum with a flocculent grey content and an
extremely well-developed Golgi apparatus. The speculation that the principal
cells are actively secretory despite the absence of secretory granules formed in
the Golgi and of a visible mechanism for release of the product at the cell surface
is discussed.
The "halo cells" in the epididymal epithelium were also examined and it is
shown that many of these cells are not typical migratory lymphocytes, Chief
among the differences are their granule-containing multivesicular bodies and
more abundant endoplasmic reticulum. Nonetheless, it is conceivable that the
halo cells are lymphocytes and that the conditions they encounter as they leave
the circulation and enter the epididymal epithelium may stimulate morphological changes. The possible immunological significance of these observations is
discussed.
There have been several descriptions of
the ultrastructure of the epididymal epithelium in recent years (Nicander, '65;
Friend and Farquhar, '67; Flickinger, '69;
Hamilton, '72a,b). The improved preservation and higher resolution achieved in
these studies have corrected a number of
persistent misconceptions of early light
microscopists who worked on the epididymis. Thus, irregular surface protrusions
previously interpreted as a manifestation
of apocrine secretion have been shown to
be artifacts of fixation and certain granules formerly regarded as secretory products have been shown to be lysosomes. The
structural differentiation of the cytoplasm
has proven to be far more elaborate than
previously supposed and it is quite evident
that this eoithelium is by no means the
metabolically inactive lining of an organ
for sperm storage. The functional correANAT. REC., 175: 169-202.
lates of the cytological features are largely
unknown, and, although considerable evidence is available on the morphology of
the absorptive process in principal cells
(Friend and Farquhar, '67; Nicander, '65;
Burgos, '64), the morphology of secretion of glycerophosphorylcholine (Dawson,
Mann and White, '57; Dawson and Rowlands, '59), carnitine (Marquis and Fritz,
'65), sialic acid (Rajalakshmi and Prasad,
'69) or steroids (Hamilton et al., '69; Hamilton, '72b) has not been elucidated.
In the present paper we report the morReceived July 17, '72.Accepted Oct. 4,'72.
1 Supported by a grant from The Population Council, by USPHS grant HO-04290 and by Contract NIH
69-2017from the National Institute of Child Health
and Human Development.
2 Recipient of a Pharmaceutical Manufacturers Association Foundation Award.
3 Recipient
of a , Research Career Development
Award from the Institute of Child Health and Human
Development, USPHS.
169
170
ANITA P. IIOFFER, DAVID W. HAMILTON AND DON W. FAWCETT
phology of the epithelium of a specific portion of the rat epididymis, the initial segment (Benoit, '26), that has been shown
to have interesting physiological properties
in many species (Crabo, '65; Setchell et al.,
'64; Levine and Marsh, '71) and which, in
the rat, is particularly susceptible to large
doses of the antifertility drug U-5897, an
alpha-chlorhydrin (Ericsson, '70; Ericsson
and Baker, '70). In a companion paper we
will report on the ultrastructural effects of
U-5897 on the initial segment epithelium.
It is our purpose here to examine in detail
certain cytological features of the epithelium and to offer suggestions as to their
possible functional significance.
MATERIALS AND METHODS
Six adult male rats were anesthetized
with ether or Nembutal and the epididymis
was fixed for electron microscopy by perfusion through the aorta (Vitale-Calpe,
Fawcett, and Dym, '73) with 5% glutaraldehyde in 0.16 M collidine buffer (pH 7.4).
Following perfusion with approximately
100 ml of the fixative, the initial segment
was dissected free from the rest of the
epididymis and immersed in collidine-buffered glutaraldehyde for an additional one
and one-half to two hours at 4°C. The
tissue was then rinsed in three changes of
0.2 M collidine buffer for one hour, postosmicated in 1.3% OsOl buffered with
0.067 M collidine, dehydrated in a series of
increasing concentrations of cold ethanol
and embedded in Epon. In one animal,
Thorotrast was injected into the lumen of
the rete testis prior to fixation. The incision
was sutured and the animal was allowed
to recover from anesthesia and move about
for approximately one hour. The initial segment was then fixed by immersion in 5%
glutaraldehyde in 0.16 M collidine buffer
for two hours, and processed as indicated
above. Sections showing pale gold interference colors were cut with a diamond
knife on a Porter-Blum MT-1 microtome
and stained with saturated aqueous uranyl
acetate (Watson, '58) followed by lead
citrate (Venable and Coggeshall, '65).
RESULTS
The histology of the initial segment of
the rat epididymis has been reported by
Reid and Cleland ('57), and recently gen-
eral features of its fine structure have been
described by Hamilton ('72b, in press).
There are four distinct cell types reported
to be present in the initial segment: principal cells, halo cells, apical cells, and basal
cells. Of these, the last two cell types have
a relatively simple morphology and our observations add nothing new to those
already reported by others. In the descriptions that follow, we will concentrate on
certain features of principal cells and on
new evidence bearing on the identification
of the halo cells (Reid and Cleland, '57) as
agranular leucocytes.
Principal cells
The tall columnar principal cells extend
the full thickness of the epithelium from
basement lamina to lumen and vary in
height from 30 to 60 p , They have a single
round or elliptical nucleus, a very prominent Golgi apparatus and a free surface
bearing long stereocilia (fig. 1). The finer
cytology of the principal cells in the initial
segment of the rat epididymis is similar in
many respects to that of the corresponding
cells in the vas deferens (Friend and
Farquhar, '67).
The stereocilia of the epididymal epithelium are often described as simply unusually long microvilli but, if one compares
them with the processes of the absorptive
cells of the intestine, there are noteworthy
differences. First, intestinal microvilli are
closely packed, highly ordered in their
arrangement, extremely uniform in diameter, and unbranched, Epididymal stereocilia are variable in diameter and may be
nearly as thick at their base as kinocilia
(figs. 2, 3). They are many microns in
length and have a tendency to branch
(Horstmann, '62; Nicander, '65). Although
examples of their bifurcation are not easily
found in thin sections, their branching
may be inferred from the fact that profiles
of stereocilia are rather sparse near the
cells, but more numerous farther into the
lumen. Second, the microvilli of the gut
contain a cytoplasmic matrix reinforced by
a longitudinal central core consisting of
two or three dozen discrete filaments.
These extend a short distance downward
into the apical cytoplasm where they mingle with a conspicuous transversely oriented mat of thinner filaments comprising
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
the terminal web. The stability of the brush
border and the precise parallel orientation
of its microvilli is thought to be due in part
to the anchoring of the core filaments in
the terminal web. No such high degree of
order or consistency of orientation is seen
in the stereocilia. Closely packed fine filaments completely fill the interior of stereocilia to the exclusion of all other formed
elements of the cytoplasmic matrix (fig.
3 ) . Although accurate measurement is difficult, these elements seem to be thinner
than those in the microvilli of the brush
border. The bundles of filaments often extend for several microns into the apical
cytoplasm. Since some stereocilia are erect
and perpendicular to the cell and others
are oblique to its surface, the bundles of
filaments that extend downward from
their bases converge or diverge at various
angles in the apical cytoplasm. The
straight course of the proximal portions of
the stereocilia suggests that the filaments
in their interior impart to them a certain
degree of stiffness, but a terminal web is
completely lacking in the principal cells of
the initial segment and this may permit
a greater degree of angular mobility and
variation in mobility of the stereocilia than
is seen in a typical brush border. The core
filaments of microvilli have been shown to
be composed of actin (Ishikawa et al., '69;
Tilney and Mooseker, '71) and hence are
potentially contractile. It remains to be determined whether the exceedingly thin filaments in stereocilia are of similar composition.
As in the principal cells of the vas deferens (Friend and Farquhar, '67), the
plasma membrane covering the stereocilia
and the remainder of the free surface of
principal cells in the initial segment is
somewhat thicker (100 A ) than that of
the lateral and basal cell surfaces and
much thicker than the membranes of the
endoplasmic reticulum and Golgi apparatus (fig. 3 ) . The free surface of the cell between the bases of the stereocilia is highly
irregular in contour and has numerous
shallow depressions and deeper pit-like invaginations. A local differentiation of the
membrane at these sites gives it a thickened or coated appearance. When Thorotrast is injected into the lumen of the rete
testis and the animal sacrificed 30 minutes
171
later, particles of the tracer are found in
these invaginations. Thorotrast is also observed in the coated vesicles and multivesicular bodies of the apical and supranuclear cytoplasm (figs. 4,5). Therefore, these
invaginations are evidently stages in the
formation of coated vesicles of the kind
that have been implicated in other cell
types in the pinocytotic uptake of proteinrich fluid (Roth and Porter, '64; Droller
and Roth, '66; Friend and Farquhar, '67).
Typical coated vesicles of various sizes that
have separated from the cell surface are
most plentiful in the apical cytoplasm, but
are present in smaller numbers in the
supranuclear Golgi region. The radial
striation of their limiting membrane appears to become indistinct or disappear altogether as they move downward into the
cytoplasm, but these pinocytotic vesicles
nevertheless remain easily distinguishable
from surrounding vesicular elements of the
reticulum by the thickness of their membrane, by the absence of a content of appreciable density, or, in injected specimens,
by their Thorotrast content.
One of the most conspicuous and characteristic features of the epididymal epithelium in general is the presence of nurnerous multivesicular bodies. In the present
material, multivesicular bodies are also
present and they exhibit many features in
common with the multivesicular bodies in
the vas deferens of the rat (Friend and
Farquhar, '67). In the epithelium of the
initial segment, most of these organelles
range from 0.1 to 4.0 /I in diameter with
a small number of vesicles (500 A ) randomly scattered in a matrix of very low
density (fig. 6 ) ; other less numerous multivesicular bodies have a denser matrix and
vesicles with a denser content (fig. 7). The
limiting membrane of these bodies is
thicker than that of the other cytoplasmic
membranes. In addition, segments of the
membrane bounding the multivesicular
bodies are thicker than other areas (fig. 6 )
and, at higher magnifications, exhibit a
faint radial striation suggesting that these
may be the sites of recent coalescence of
coated vesicles with these organelles. The
multivesicular bodies have been identified
as belonging to the intracellular digestive
apparatus by the demonstration of acid
phosphatase activity in their matrix and by
172
ANITA P. HOFFER, DAVID W. HAMILTON AND DON W. FAWCETT
the observation that peroxidase injected
into the lumen of the vas deferens is taken
up at the cell surface in coated vesicles and
later is found in the matrix of the multivesicular bodies (Friend and Farquhar,
'67). In the epithelium of the initial segment also, Thorotrast can be demonstrated
in the matrix of the multivesicular bodies
(fig. 4 ) . Membrane-bounded bodies with a
homogeneous dense content are also present in limited numbers. These are interpreted as primary lysosomes. They are very
abundant in some segments of the rabbit
epididymis (Nicander, 'SS), but are not a
conspicuous feature of the initial segment
in the rat.
The endoplasmic reticulum is extensively developed and shows a rather consistent pattern of regional distribution.
Parallel cisternae of typical rough surfaced
reticulum are concentrated in the basal
and paranuclear regions (fig. 8 ) . They may
also extend upward along the sides of the
cell nearly to the surface but are seldom
found more centrally situated in the apical
region (fig. 9). The bulk of the apical cytoplasm is filled with somewhat different appearing tubular elements of large caliber
that are usually described as smooth
reticulum although they do appear to have
widely scattered ribosomes adhering to
their membranes (fig. 9). These elements
have a thin limiting membrane ( 5 5 A )
and a content which is preserved as a very
fine-textured precipitate with a light gray
homogeneous or faintly mottled appearance. The prevailing orientation of the
tubules in the initial segment epithelium is
parallel to the cell axis. Near the luminal
surface, tubules gradually give way to vesicular profiles of irregular outline (fig. 3).
Single ribosomes and small polysomes in
small numbers are found scattered in the
cytoplasmic matrix between the closely
packed elements of the reticulum.
In many cell types and particularly in
epithelia, a thin ectoplasm rich in filaments can be identified beneath the plasmalemma. The terminal web of the intestinal absorptive cells already referred to is
an exaggerated example. In cells having a
well developed ectoplasm, the reticulum
and other organelles are excluded from this
layer and are never observed in close proximity to the plasma membrane. Epithelial
cells of the initial segment of the epididymis appear to have no ectoplasmic layer
(fig. 3 ) . Vesicular and tubular elements of
the reticulum are therefore found in very
close apposition to the apical plasmalemma. Although no images were seen that
are suggestive of coalescence and discharge of contents of the reticulum into
the lumen, this mode of secretion has been
claimed for other cell types (Ross and
Benditt, '65; Leduc, Avrameas and Bouteille, '68) and cannot be excluded here.
The Golgi complex of the principal cells
is extraordinaly extensive and appears to
consist of several stacks of cisternae, each
of which is as large as the entire Golgi
complex of many other cell types (fig.
10). In horizontal sections through the
supranuclear region, these several parallel
arrays of cisternae and their associated
vesicles occupy the greater part of the
cross-sectional area of the cell. Each assemblage of Golgi membranes consists of
8 to 14 parallel flattened saccules or cisternae. The two or three outermost cisternae - those on the forming face of the
Golgi stacks (Mollenhauer and Whaley,
'63) - are fenestrated and therefore appear discontinuous in section. When
viewed en face, they present a regular pattern of circular pores or fenestrae (fig. 7).
The periphery of the deeper lying cisternae
is also fenestrated but their central portions are unfenestrated, and the cisternae
are closely spaced with a very narrow
lumen which widens toward their periphery. Thus, in sections the profiles of the
cisternae are narrow, uninterrupted and
closely spaced in their mid-portions but are
expanded and discontinuous toward their
ends. The lumen of most of the Golgi saccules appears empty. There is no suggestion of a gradient of density in the content
of the flat saccules from one face of the
Golgi stack toward the other, such as one
observes in many glandular cells.
Both smooth-surfaced and coated vesicles are numerous in the vicinity of the
Golgi but there are no condensing vacuoles
or other evidence of concentration of a secretory product within this organelle or its
associated vesicles. The functional relation
of the large Golgi complex to the extensive
reticulum of these cells is an intriguing
problem.
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
173
The mitochondria of the principal cells
are very long and occasionally branched
(figs. 8, 9, 10). They have numerous foliate
or lamellar cristae generally oriented perpendicular to the long axis of the organelle.
The mitochondria1 matrix is of moderate
density and occasional matrix granules are
observed. Mitochondria may be found anywhere in the cytoplasm but tend to be concentrated along the lateral cell membranes.
Their orientation is generally parallel to the
longitudinal axis of the cells.
cells, including the amount and distribution of endoplasmic reticulum, ribosomes
and polyribosome rosettes, mitochondria
and multivesicular bodies. Additionally, the
multivesicular bodies of these cells often
contain dense granules surrounded by vesicles (figs. 14, 15, 16). On this evidence,
then, it seems clear that halo cells are
morphologically similar to circulating
leucocytes, but is not possible to identify
them as lymphocytes on the basis of morphological evidence alone.
Halo cells
The term "halo cells" was used by Reid
and Cleland ('57) to define a cell-type characterized by its small, dense nucleus surrounded by a small, very pale cytoplasm.
The cell-type was found at all levels of the
epididymal epithelium. These authors considered the cell to be a lymphocyte, but
could present no convincing evidence for
their hypothesis.
At the electron microscopical level these
cells have blunt, amoeboid processes which
extend between the principal cells (Hamilton, '72b). They do not have complex interdigitations of their surface membranes
with epithelial cells, and desmosomes or
other specialized attachment devices are
never found (figs. 11, 12). The nucleus is
often indented toward the cytocentrum and
contains abundant heterochromatin. Elements of rough endoplasmic reticulum are
infrequent, but free RNP particles and
occasional polyribosome rosettes are generally dispersed throughout the cytoplasm.
Large mitochondria and occasional multivesicular bodies are also present. A feature
often seen in these cells that is not common in lymphocytes from other species is
the presence of large dense granules in
the matrix of many of the multivesicular
bodies and more numerous elements of
endoplasmic reticulum (fig. 12).
In order to equate the halo cells with
non-granular leucocytes we prepared a
buffy coat of rat blood and examined it
with the electron microscope. Cells bearing
close resemblance to those described above
are commonly found in our buffy coat
preparations. These are illustrated in figures 13 through 16. As is clear, nuclear
and cytoplasmic characteristics of these
cells resemble very much those of halo
DISCUSSION
Secretion
The epididymal epithelium has been regarded by some as mainly absorptive in
function and by others as secretory. It has
become apparent, however, that conspicuous spherical inclusions formerly interpreted as secretory granules are in fact
lysosomes. Moreover, the prominent blobs
of apical cytoplasm seen projecting into the
lumen and often described as manifestations of apocrine secretion (Horstmann,
'61, '62; Holstein, '69) are not observed
when satisfactory fixation has been
achieved, and are now regarded as artifacts (Nicander, '65; Hamilton, '72b).
Nevertheless cytological studies reveal
such an extensive development of those
organelles commonly associated with synthesis of a product for export, that one can
scarcely escape the conclusion that the
cells must be secretory. Most other
epithelia with a large Golgi complex and
an extensive endoplasmic reticulum have
proved to be secretory and it has been possible to define a secretory pathway in
which a product synthesized on ribosomes
is segregated in the lumen of the reticulum; transported to the Golgi complex in
small intermediate vesicles; concentrated
there and formed into secretory granules
which discharge their content by coalescence of their limiting membrane with the
apical plasmalemma. This pattern of functional organization is so common in secretory cells and has been so thoroughly
studied in protein and glycoprotein secreting cells that we are unprepared for any
significant departure from it.
The principal cells of the initial segment
exhibit some but not all of the cytological
174
ANITA P. HOFFER, DAVID W. HAMILTON AND DON W. FAWCETT
characteristics of typical protein secreting
cells. They invariably show some degree of
distention of the sparsely granulated elements of the reticulum in the apical region, and these have a content of fine
textured gray material. This is presumed
to be a product of the cell. The missing
links are the absence of any evidence for
its transport to the large Golgi complex,
and the lack of any pictures suggesting a
mechanism for release of a secretory product from the cell. The presence of visible
product in the reticulum while the cisternae of the Golgi complex appear empty is
paradoxical in relation to the belief that
this organelle usually receives and concentrates secretory products. The paucity of
evidence in the epididymal epithelium for
intermediate vesicles passing from reticulum to Golgi is also inconsistent with current views on the origin and dynamic
state of the Golgi membranes inasmuch as
maintenance of the Golgi apparatus, at
least in some cells, is believed to depend
upon continual addition to its forming face
of new membrane brought to it in the form
of vesicles which bud off from agranular
regions of the reticulum (Zeigel and Dalton, '62; Jamieson and Palade, '67; Flickinger, '69). Smooth vesicles in moderate
numbers are associated with the Golgi in
the epididymal cells but if they originate
in the usual manner from the endoplasmic
reticulum, it is not at all clear how the
contents of the organelle formed by their
coalescence could have a content of lower
density than that of the reticulum. Although in other cell types one of the most
widely accepted functions of the Golgi
complex is concentration of secretory products, there is no evidence that the Golgi
subserves this function in the principal
cells of the epididymis, even though the
extensive development of the rough reticulum and the presence of cisternae distended with material strongly suggest that
the cell makes a product for export.
The only substances thus far identified
as possible products of secretion by epididyma1 epithelium are glycerophosphorylcholine (Dawson and Rowlands, '59) and
possibly dihydroepiandrosterone and testosterone (Hamilton, '72a), and there is no
reason to believe that the secretory path-
way for these would conform precisely to
that established for protein secretions. The
coexistence of an extensive endoplasmic
reticulum and a large Golgi that play no
obvious role in the synthetic and secretory
processes is not unique. Steroid secreting
endocrine glands, for example, have an
extensive smooth reticulum and a large
Golgi complex which enlarges in response
to trophic hormone stimulation (Fawcett,
Long and Jones, '69); yet there is no morphological evidence that i t functions to
concentrate or modify the secretory products. No morphological correlates of release of steroid from these cells have been
described.
Even among cell types known to secrete
proteins such as thyrotrophs, plasma cells
and fibroblasts, the Golgi complex may not
always be involved in packaging the product. For example, the ultrastructure of
TSH-secreting cells (thyrotrophs) in normal adult rats is similar to that of other
protein-secreting cells and includes a welldeveloped rough endoplasmic reticulum
and Golgi complex and numerous secretory granules. Following thyroidectomy,
however, thyrotrophs increase their production of thyroid-stimulating hormone
and yet their secretory granules disappear,
the Golgi decreases in size and the cisternae of rough endoplasmic reticulum become greatly distended with a faint grey
flocculent material (Farquhar, '7 1). It
seems, therefore, that in thyroidectomized
rats, TSH synthesis and secretion can
occur without visually demonstrable participation of the Golgi apparatus. Similarly,
the plamsa cell is very active in protein
synthesis and the cisternae of its extensive
reticulum become distended with accumulated immunoglobulin, but the large juxtanuclear Golgi characteristic of this cell
does not form secretory granules and has
no visually identifiable role in the secretory
process. Autoradiographic and biochemical
data showing that galactose is incorporated
in the Golgi while glucosamine seems to
be incorporated into nascent polypeptide
chains in the rough endoplasmic reticulum
(Zagury, Uhr, Jamieson and Palade, '70)
has been interpreted as evidence that the
Golgi complex is a way station in the intracellular transport of immunoglobulin
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
from the reticulum to its discharge at the
cell surface. Although Zagury et al. ('70),
favor the view that release of secretory
product in plasma cells is by fusion of
Golgi-derived vesicles with the plasmalemma, morphological evidence to support
this mechanism is lacking. It seems equally
plausible to us that in some cell types the
Golgi may be involved only in the elaboration of a carbohydrate component of the
product. The traffic in these instances may
conceivably be from the Golgi to the reticulum and might involve a mechanism of release that does not require pre-packaging
in Golgi-derived membranes. Moreover, the
studies of Avrameas and Leduc ('70), in
which antibody was localized immunohistochemically, show the antibody appearing
first in cisternae of rough reticulum adjacent to the nucleus and later in peripheral
cisternae, from which it appeared to be
released.
In the principal cells of the rat epididym i s , the absence of visible product in the
Golgi and presence of a content of appreciable density in the cisternae of the reticulum throughout the cell, suggests to us that
the Golgi may contribute a soluble carbohydrate moiety to a less soluble final product that accumulates in the reticulum.
There is little or no evidence that the
product is channeled through the Golgi
preliminary to its release. The close proximity of the tubules and cisternae of the
reticulum to the apical plasma membrane
without any intervening ectoplasmic layer
suggests the possibility that the product
may be released directly, but clear images
of such a process have not been obtained.
In this regard, it is interesting to point out
that an atypical form of endoplasmic reticulum is found in the apical cytoplasm of
the principal cells. The endoplasmic reticulum in the apical cytoplasm differs from
the granular reticulum of typical protein
synthesizing cells such as pancreatic acinar
cells or plasma cells in having many fewer
ribosomes. It differs from the agranular
reticulum of most steroid-secreting cells in
that ( 1 ) it is composed of vacuoles and
sinous tubules oriented predominantly in
the long axis of the cell ( a branching and
anastomosing reticulum is usually found in
steroidogenic cells); (2) the caliber of the
tubules is several times that of the ordinary
175
tubular elements of smooth endoplasmic
reticulum; (3) it has a conspicuous content of low density whereas the agranular
reticulum of steroid-secreting cells usually
appears empty in micrographs and ( 4 )
many of its elements exhibit an occasional
ribosome. The significance of this unusual
type of endoplasmic reticulum is not clear
but its form is not entirely unique. Endoplasmic reticulum of similar appearance is
found in the endometrial cup cells of the
horse (Hamilton, Allen and Moor, '73). In
this species, chorionic cells of the embryo
invade the uterine lining to form endometrial cups which synthesize and secrete
the complex glycoprotein, pregnant mare
serum gonadotrophin (PMSG). Like the
principal cells of rat initial segment, the
cup cells display numerous distended profiles of sparsely studded cisternae containing a faint grey flocculent material. The
Golgi does not play a visible role in processing of the secretory product and the mode
of release of the secretory material from
the cup cells has not been determined.
On the basis of the foregoing considerations, we would like to speculate that principal cells may be actively secretory despite
the absence of secretory vacuoles formed
in the Golgi and the lack of a visually identifiable mechanism for release of the product at the cell surface. Although conclusive
morphological evidence for such a mechanism is lacking, it is obvious that if communications between granular reticulum
and the extracellular spaces were intermittent, transient, and of small size, one
would very rarely observe them in electron
micrographs of thin sections. In cells of
vertebrates where the products are amorphous and rapidly carried away after release, actual examples of direct release
from the reticulum without channeling the
product through the Golgi are rare. However, in lower forms such as the zoospores
of xanthophycian algae, which have hispid
flagella, the highly characteristic hairs destined for assembly on the flagella are visible in peripheral cisternae of the reticulum
but not in the Golgi elements (Leedale et
al., '70). There can be little doubt that
these complex organelles are assembled
within the reticulum and released without
direct Golgi involvement. This example,
together with those of thyrotrophs, plasma
176
ANITA P. HOFFER, DAVID W. HAMILTON AND D 0 N . W . FAWCETT
cells and steroid secreting cells, lend indirect support to the speculation that the
principal cells are secretory, although they
do not prove it. Clearly further investigation is necessary to elucidate the less well
understood patterns of secretory product
discharge in this and other cell types.
Absorption
Let us consider now the possible absorptive functions of the initial segment epithelium. The literature contains numerous
observations which indicate that the caput
epididymidis is involved in absorption of
fluid and particulate material from the
lumen. In the mouse, the testis enlarges
after ligation of the efferent ducts but not
after ligation further down the epididymal
duct suggesting that fluid produced in the
testis is absorbed in the head of the epididymis (Young, '33). Spennatocrit values
in samples collected at various levels of
the epididymis indicate that almost 50%
(in rats) and 99% (in bulls and boars) of
the fluid leaving the testis is resorbed in
the head of the epididymis (Levine and
Marsh, '71; Crabo, '65). In rams 40 ml of
fluid per day enters one epididymis, yet
only 0.4 ml leaves the epididymis (Voglmayr et al., '66, '67; Waites and Setchell,
'69). Morphological studies with the light
microscope show that the head of the epididymis can also absorb particulate material
such as India ink or trypan blue from the
lumen in vivo (Gunn, '36; Mason and
Shaver, '52; Macmillan, '57; Grant, '58)
and in organ culture (Wagenseil, '28).
More recently ultrastructural studies have
extended these observations to show uptake of (1) colloidal mercuric sulphide by
the initial segment of the epididymis in
hamster (Burgos, '64); ( 2 ) peroxidase by
the head of the epididymis in hamster
(Sedar, '66) and (3) India ink by posterior
caput cells of the rabbit (Nicander, '65).
Although peroxidase uptake has been demonstrated in the vas deferens of the rat
(Friend and Farquhar, '67), absorption
studies with electron-dense tracers have
not been performed in the caput epididymidis of the rat.
It is puzzling that in spite of these
known functions, the ultrastructural specializations associated with absorption in
the initial segment epithelium of the rat
are quite unimpressive. They consist
mainly of the ( 1 ) presence of numerous
coated vesicles at the luminal surface, ( 2 )
the presence of numerous multivesicular
bodies in the apical cytoplasm, and ( 3 ) the
demonstration that particulate tracers are
in fact taken into the cells and are ultimately found in secondary lysosomes 6r in
the multivesicular bodies. The ultrastructure of the initial segment epithelium in
the rat is similar to that of absorbing cells
in hamster and rabbit caput (Burgos, '64;
Sedar, '66; Nicander, '65), yet none of
these epithelia exhibit other cytological specializations which one might expect of actively absorbing epithelia. The gallbladder
epithelium, for example, transports a large
volume of water from the free surface to
the base in the process of concentrating
the bile. The most significant specializations in this tissue are a juxtaluminal occluding junction and relatively loose association of the lateral surfaces of the cells
throughout the lower two-thirds of the epithelium. The lateral cell surfaces are
thrown into thin laminar or foliate processes that amplify the area of the membrane. During movement of water across
the epithelium, sodium ions are pumped
into the distensible intercellular cleft creating a standing gradient which acts osmotically to draw water into the space. This
then distends the intercellular space and
hydrostatic pressure drives isosmotic fluid
through the basal lamina. Although sodium reabsorption by active transport and
water uptake by osmosis also are believed
to occur in the initial segment and in the
head of the epididymis in rat (Levine and
Marsh, '71), the lateral cell surfaces in the
initial segment and head are relatively
straight and in close apposition. There is
no morphological indication, therefore,
that fluid reabsorption is accomplished by
pumping of solutes to create a standing
gradient in the intercellular clefts deep to
the occluding junctions in this tissue. Perhaps water transport in the epididymal
epithelium is accomplished in some unique
fashion which so far has escaped detection.
In some other absorbing epithelia, such
as the proximal convoluted tubule of the
nephron (Miller, '60; Strauss, '64), the
yolk sac (Dempsey and Wislocki, '56) or
the calf gut (Staley et al., '72), which are
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
concerned with absorption of protein rich
fluid, one finds an extensive system of apical canaliculi lined by a coated membrane.
These constitute a specialization for continuous pinocytosis with the ends of the canaliculi budding off vesicles that coalesce
with the numerous lysosomes in this region
of the cell. The lysosomal digestive system
clearly provides a mechanism for disposal
of the absorbed protein in such cells but
how the considerable volume of fluid imbibed at the same time is transported
across the base of the epithelium is unexplained. The epididymal epithelium more
closely resembles this category of absorptive cell than it does the gallbladder or
other epithelia transporting mainly water.
Although quantitatively much less efficient,
the formation of numerous coated vesicles
at the luminal surface of the epididymal
epithelial cells and their coalescence with
multivesicular bodies is qualitatively similar to the apical canaliculi and associated
lysosomes of epithelia absorbing or reabsorbing protein and carbohydrates. In
view of the low protein content of testicular
fluid, however, it is somewhat surprising
that the excurrent ducts appear to be specialized for absorption by bulk uptake of
fluid in pinocytotic vesicles rather than for
movement of water by creation of a standing gradient. The fact that this mechanism
for absorption is less highly developed in
epididymal epithelium than in those epithelia possessing apical canaliculi is probably related to the relatively small volume
of fluid that needs to be absorbed per unit
length of tubule. The statement that 90%
of the testicular fluid is reabsorbed in the
proximal part of the epididymis raises the
expectation of a highly specialized and
functionally efficient absorptive epithelium, but when one considers that the volume of fluid to be absorbed is only about
40 ml/day in rams (Voglmayr et al., '66,
'67; Waites and Setchell, '69), and this
function is spread along a convoluted
tubule several centimeters in length, it
should not be surprising that ultrastructural specializations for absorption are in
no sense comparable in degree to those of
the proximal renal tubule, which process
many liters of glomerular filtrate.
There is little precedent for the same
epithelium carrying out absorptive and
177
secretory functions without differentiation
into two distinct cell populations. Nevertheless, we have noted in the preceding
paragraphs that even though the absorptive function of the epithelium in the proximal epididymis is better documented, the
fine structural complexity of the principal
cells of the initial segment and particularly
their elaborate Golgi and extensive endoplasmic reticulum constitute strong morphological evidence for a high degree of
synthetic activity. Nor can we assign either
one of these functions to the basal cell
since (1) this cell type does not have the
cytoplasmic machinery for complex secretory or absorptive activity, (2) it is present
in too small numbers and ( 3 ) it is not in
contact with the lumen. What the products
of the synthetic and presumed secretory
activity of the principal cells are and what
their role is in sperm maturation remains
one of the principal challenges in male reproductive biology.
Agranular leucocytes ("halo cells")
In a period of great interest in the different populations of lymphocytes, it is rather
surprising that the halo cells first described
by Reid and Cleland ('57) have been
neglected. Some of the halo cells observed
in the present material are not typical migratory lymphocytes but no one has
troubled to compare their ultrastructure
with that of lymphocytes of the blood or intestinal epithelium heretofore. Accordingly
in the present study, a buffy coat was prepared from rat blood and examined in the
electron microscope. In addition to typical
granulocytes, monocytes and lymphocytes,
cells identical to the halo cell of the epididymal epithelium were found in the buffy
coat, thus indicating their origin from the
blood. However, the only basis for identifying these cells as tissue lymphocytes is
morphological similarity and their differences from typical circulating lymphocytes
(Zucker-Franklin, '69), are nearly as impressive as their similarities. Chief among
the differences are the granule-containing
multivesicular bodies and the more abundant endoplasmic reticulum of the atypical
cells. Although azurophilic granules are
occasionally found in normal lymphocytes
of peripheral blood (Bloom and Fawcett,
'68), electron micrographs indicate that
178
ANITA P. HOFFER, DAVID W. HAMILTON AND DON W. FAWCETT
these are pleomorphic lysosome-like dense
bodies (Brittinger, Hirschhorn, Douglas
and Weissman, '68) which have a different
appearance and are present in much
smaller numbers than the granule-containing multivesicular bodies of the epididymal
cells. On the other hand, recent studies
have shown that phytohemaglutanin and
pokeweed mitogen can stimulate the formation of acid hydrolase rich granules in
human lymphocytes cultured in vitro
(Hirschhorn, Hirschhorn and Weissman,
'67; Douglas, Hoffman, Borjeson and
Chessin, '67) and it is conceivable that the
conditions encountered by rat lymphocytes
as they leave the circulation and enter the
epididymal epithelium can stimulate the
formation of these unusual granules.
Nonetheless, the exact identity of these
cells cannot be established on the basis of
data presently available. If they are lymphocytes, one is left with the question of
their significance in this location. In the
gut with its large bacterial flora one can
see a clear advantage in having a reserve
of immuno-competent cells strategically located in the epithelium a s well as in the
lamina propria. In the epididymis, however, where the contents of the duct are
normally sterile, the advantages of a ready
reserve of lymphocytes is less obvious. It is
interesting to note in this connection that
migratory cells with lipid inclusions have
been observed in the prostate and seminal
vesicle epithelium of aging rats (Allison
and Cearley, '72) and these cells bear a
close resemblance to the agranular leukocytes in the epididymal epithelium. Allison
and Cearley report that the migratory cells
become more numerous in the seminal
vesicle and prostate of older rats and suggest that their presence reflects some sort
of an age-related immune response. In the
present study, we have seen these cells i n
the epididymal epithelium of young and
old male rats alike but we have not made
a careful count of their numbers in relation
to age. An alternate approach to investigation of the possible immunological role of
these cells might be to determine whether
they increase in animals immunized with
sperm antigens. In view of recent suggestions of autoimmune effects following
vasectomy (Shulman, '71 ; Alexander, '72),
this population of cells may deserve more
attention than it has received.
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PLATES
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamiltvn and Don W. Fawcett
EXPLANATION OF FIGURE
1
1 a2
Light micrograph showing the pseudostratified columnar epithelium of the initial segment of rat epididymis. Four cell types can be recognized: (1) principal cells, (2)
basal cells, ( 3 ) apical cells and ( 4 ) agranular leucocytes which correspond to the halo
cells described by earlier investigators. Toluidine blue. x 950.
PLATE 1
THE ULTRASTRUCTURE O F THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamilton and Don W. Fawcett
PLATE 2
EXPLANATION OF FIGURE
2
Electron micrograph of the apical cytoplasm of three principal cells. The free surface
between the stereocilia is irregular in contour due to numerous coated invaginations of
the plasma membrane (arrows). Coated vesicles (cv). multivesicular bodies (mvb) and
numerous profiles of endoplasmic reticulum are visible in the underlying cytoplasm.
x 11,300.
183
PLATE 3
EXPLANATION OF FIGURES
3
184
The apical cytoplasm of a principal cell is shown here at higher magnification. The closely packed fine filaments in the stereocilia extend
for several microns into the cytoplasm but a terminal web is not seen
in principal cells of the rat initial segment. The elements of the endoplasmic reticulum lie in close proximity to the apical plasmalemma.
A centriole is visible on the right. x 42,000.
PLATE 4
EXPLANATION O F FIGURES
4-5
186
Electron micrographs of the apical (fig. 4) and supranuclear (fig. 5)
cytoplasm of a principal cell 30 minutes after injection of Thorotrast
into the lumen of the rete testis. Particles of the tracer are visible
in coated vesicles and canaliculi beneath the cell surface and in
multivesicular bodies (MVB) but very little tracer is present in the
lumen i n this particular section. In many instances, large multivesicular bodies become so engorged with Thorotrast that their
vesicles are obscured. Figure 4, x 26,100; figure 5, x 26,650.
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P . Hoffer, David W. Hamilton and Don W. Fawcett
PLATE 4
PLATE 5
EXPLANATION OF FIGURES
6
Electron micrograph showing multivesicular bodies in the apical
cytoplasm of a principal cell. This type of rnultivesicular body has a
matrix of very low density and empty appearing vesicles. Dense
plaques are often found on the cytoplasmic surface of the limiting
membrane of these multivesicular bodies (arrows). x 35,200.
7 Electron micrograph showing a multivesicular body in the Golgi zone
of a principal cell. This type of multivesicular body has a denser
matrix and vesicles with a denser content than the multivesicular
body in figure 6. In the upper left hand corner of this micrograph, a
tangentially sectioned Golgi saccule exhibits a regular pattern of
circular pores of fenestrae. x 40,250.
188
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamilton and Don W. Fawcett
PLATE 5
PLATE 6
EXPLANATION OF FIGURE
8
190
The rough surfaced endoplasmic reticulum is extensively developed in
the basal cytoplasm of the principal cells. Cisternae are typically
oriented parallel to the basal and lateral surfaces of the cell. A portion of a basal cell can also be seen. x 16,200.
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamilton and Don W. Fawcett
PLATE 6
PLATE 7
EXPLANATION OF FIGURE
9 This electron micrograph shows the two different types of endoplasmic
reticulum found in the apical cytoplasm of principal cells in the
initial segment of rat epididymis. T h e bulk of the apical cytoplasm
is filled with large tubular elements of reticulum. These are usually
described as smooth reticulum even though they have widely scattered
ribosomes adhering to their membranes. Flattened profiles of typical
rough endoplasmic reticulum are found near the lateral plasmalemmata. x 60,000.
192
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamilton and Don W. Fawcett
PLATE 7
PLATE 8
EXPLANATION OF FIGURE
10
194
Electron micrograph showing the extremely well-developed Golgi complex in principal cells. Numerous parallel arrays of Golgi lamellae,
associated vesicular elements and multivesicular bodies (MVB ) can
be seen. x 26,000.
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamilton and Don W. Fawcett
PLATE
8
PLATE 9
EXPLANATION OF F I G U R E S
11
Electron micrograph showing an intraepithelial agranular leucocyte.
This cell type corresponds to the halo cell in earlier histological
descriptions of the epididymal epithelium. At the ultrastructural level,
blunt amoeboid processes, numerous free ribosomes and infrequent
elements of rough endoplasmic reticulum are characteristic features
of these cells. x 20,300.
12 Another intraepithelial leucocyte is shown in this electron micrograph.
A feature often seen in these cells, that is not common in lymphocytes of other species, is the presence of large dense granules in the
matrix of many multivesicular bodies (arrows). x 14,700.
196
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamilton and Don W. Fawcett
PLATE 9
PLATE 10
EXPLANATION O F FIGURES
Figures 13 through 16 are electron micrographs of agranular leucocytes
in a buffy coat preparation of rat blood. Large dense granules in the
multivesicular bodies and elements of endoplasmic reticulum are characteristically found in many of these cells but are not typical of lymphocytes
of other species.
13 Electron micrograph of an agranular leucocyte bearing a close resemblance to that shown in the initial segment epithelium in figure
11. The nucleus contains abundant heterochromatin. The cytoplasm
has free ribosomes but almost no vesicular elements or granular reticulum are found in this particular cell. x 15,750.
14 In this electron micrograph of an agranular leucocyte, cytoplasmic
membranes and dense cored granules are more numerous than in
figure 13. Although vesicles may not be observed in all of the dense
cored granules in a given section, they can be clearly seen in one of
the membrane-limited bodies in this section (arrow). x 17,200.
THE ULTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamilton and Don W. Fawcett
PLATE 10
PLATE 11
EXPLANATION OF FIGURES
15 The agranular leucocyte shown in this electron micrograph exhibits
a heterochromatic nucleus indented toward the cytocentrum, a centriole (CE), mitochondria and numerous small vesicles. A densecored granule similar to those seen in the other micrographs is present.
Other membrane-limited granules with a less dense content are also
present in the cytocentrum (arrowheads). x 16,600.
16 EIectron micrograph of another agranular leucocyte exhibiting numerous elements of endoplasmic reticulum and dense-cored granules.
Tiny vesicles c a n be found in each of the dense-cored granules.
X 16,000.
200
THE TJLTRASTRUCTURE OF THE RAT INITIAL SEGMENT
Anita P. Hoffer, David W. Hamilton and Don W. Fawcett
PLATE 11
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