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THE ANATOMICAL RECORD 255:227–239 (1999)
Ultrastructural and
Ultracytochemical Features of
Secretory Granules in the Ampullary
Epithelium of the Hamster Oviduct
MAJID EL-MESTRAH AND FREDERICK W.K. KAN*
Department of Anatomy and Cell Biology, School of Medicine, Faculty of Health
Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6
ABSTRACT
The epithelium of mammalian oviducts consists mainly of ciliated and
non-ciliated secretory cells. In some mammals, secretory products originating from oviductal secretory cells have been shown to bind to the surface of,
or accumulate within, ovulated eggs and/or developing embryos. These
findings suggest that the secretions of the oviductal epithelial cells may play
an important role in reproductive and developmental events that occur in
the oviduct.
In the present study, ultrastructural and cytochemical features of
secretory cells in the hamster ampullary epithelium were shown by routine
electron microscopy, lectin-gold cytochemistry and both conventional freezefracture and rapid-freezing techniques with special reference to the organizational aspects of their secretory granules. The use of ferrocyanide-reduced
osmium tetroxide as a post-fixative in the Epon embedment of ampullary
tissue samples also proved to be advantageous especially in revealing the
carbohydrate contents of certain cellular compartments. The most conspicuous characteristic of the secretory cells, based on their staining property,
was the presence of two types of secretory granules: those with a homogeneous electron-dense matrix and those with an electron-lucent matrix.
Under favorable conditions, distinct features of the organizational arrangement of a crystalline lattice inside the secretory granules were also revealed.
This well organized crystalline lattice shown in sections of Epon-embedded
oviductal tissue was confirmed by examination of replicas of freeze-fractured
oviducts prepared by the rapid-freezing technique. We also demonstrated with
high resolution lectin-gold cytochemistry the intracellular distribution of lectinbinding glycoconjugates in the secretory cells of the hamster oviductal
ampulla often in a linear array following the crystalline lattice.
The results obtained in this study, taken together, provide insight into a
possible link of the internal topographical features of oviductal secretory
granules along with the cytochemical properties of their contents to the
anticipated regulatory mechanism underlying their process of secretions.
Anat Rec 255:227–239, 1999. r 1999 Wiley-Liss, Inc.
Key words: oviductal glycoconjugates; oviductal ampullary epithelium; lectin-gold cytochemistry; oviductal secretion
The mammalian oviduct is more than a simple tubular
conduit for the transport of gametes and developing embryos. The oviductal ampulla is particularly important due
to its unique role as the site of egg fertilization. In its
functional state, the oviduct is also an active secretory
r 1999 WILEY-LISS, INC.
Grant sponsor: Medical research Council of Canada.
*Correspondence to: Dr. Frederick W.K. Kan, Department of
Anatomy and Cell Biology, School of Medicine, Faculty of Health
Sciences, Queen’s University, Kingston, Ontario, Canada K7L
3N6. E-mail: KANFWK@POST.QUEENSU.CA
Received 3 November 1998; Accepted 25 February 1999
228
EL-MESTRAH AND KAN
organ that maintains and modulates a dynamic fluid-filled
milieu in which many crucial developmental events occur.
Oviductal fluid, which is composed predominantly of
plasma derivatives (Leese, 1988) and contains secretory
products that originate from the oviductal epithelium
(Stone et al., 1980), provides the necessary environment
for the maturation of gametes, fertilization, and early
embryo development.
The epithelium of the oviduct is simple columnar and
consists of two main types of cells, ciliated and secretory.
The non-ciliated secretory cells synthesize and secrete
non-serum macromolecules or glycoproteins that are dissolved in the oviductal fluid (Oliphant, 1986). In the
mammalian oviduct, glycoconjugates play an important
role in mediating the interaction between male and female
gametes. In some mammals, secretory products originating from the oviductal epithelium have been shown to bind
to the surface of, or accumulate within, ovulated eggs
and/or developing embryos (Kapur and Johnson, 1986,
1988; Léveillé et al., 1987; Kan et al., 1989; Abe and
Oikawa, 1990; Boice et al., 1990; Gandolfi et al., 1991; Abe
et al., 1992). Glycoconjugates can also act as hormone
receptors and are essential for the successful implantation
of blastocysts in the uterus (Friedricsson, 1969; Mastroianni, 1969; Menghi et al., 1985, 1986).
In an earlier study, Kan et al. (1988) combined immunocytochemistry and the use of a specific monoclonal antibody against a 200-kDa oviductal glycoprotein to localize
the antigenic sites exclusively to the zona pellucida of
postovulatory oocytes isolated from the ampullary region
of golden hamsters. Biochemical studies with monoclonal
antibodies performed in the hamster demonstrated that
glycoproteins of oviductal origin are detected in the ZP of
postovulatory oocytes (Robitaille et al., 1988). Moreover,
Araki and coworkers (1987) showed, in the hamster, a
monoclonal antibody (AZPO-8) reacting with the zona
pellucida of the oviductal egg but not with that of the
ovarian egg. Furthermore, other reports have also indicated that embryonic development in vitro is enhanced by
co-culture with oviductal epithelial cells or conditioned
medium (for a review, see Bavister, 1988). These findings,
taken together, suggest particular roles for the oviductal
secretory cells and their secretions in reproductive and
developmental events that occur in the oviduct.
The ultrastructural morphology of the non-ciliated cells
has been the subject of several investigations (for a review,
see Ellington, 1991). Abe and Oikawa (1991) described the
ultrastructural features of secretory cells in the golden
hamster oviductal epithelium. However, their observations were limited to the examination of regional differences in the morphological features of oviductal secretory
cells. Ferrocyanide-reduced osmium tetroxide is known to
be a fixative which can enhance the staining of carbohydrate contents of certain cellular compartments (Karnovsky, 1971). Particularly, the use of ferrocyanidereduced osmium tetroxide has been shown by other investigators to allow a selective staining of the sarcotubular
system, glycogen, glycocalyx as well as of unsaturated
fatty acids of membranes (White et al., 1979; Aguas, 1982).
The objective of the present investigation was to examine some of the ultrastructural and cytochemical features
of secretory cells with special reference to the secretory
granules in the ampulla of golden hamster. In this study,
Epon-embedded tissue sections and freeze-fractured
samples of rapidly frozen oviducts revealed the internal
topography of secretory granules in the non-ciliated secretory cells. These data correlated with results obtained with
lectin cytochemistry to show the distribution of different
carbohydrate residues in relation to the internal organization of the secretory granules. The use of ferrocyanidereduced osmium tetroxide allowed a better distinction
between two different types of secretory granules based on
the staining property of their carbohydrate contents. On
the basis of these observations, we suggest that the unique
internal topography of both types of secretory granules,
added to their differential staining and cytochemical properties, may reflect a difference in the secretory products of
ampullary secretory cells as well as a possible link of such
topography to the anticipated mechanism regulating their
secretions.
MATERIALS AND METHODS
Preparation of Oviductal Tissue
Eighteen sexually mature (8 to 12 weeks) female golden
hamsters (Mesocricetus auratus) (Charles River, StConstant, Quebec, Canada) were housed over hardwood
bedding (Beta chip; Northeastern Products Corp., Warrensburg, NY). The hamsters were maintained on a 12-hr
light/dark cycle, and were freely provided with food (Purina Rodent Chow; Ralston Purina International, Strathray, Ontario, Canada) and drinking water. They were
acclimatized to laboratory conditions for at least 7 days
before experimental procedures were initiated. To obtain
oviductal tissue, six of the animals designated for cytochemical study were killed by cervical dislocation. Their
ventral abdominal wall was immediately cut open and the
oviducts were excised, washed briefly with phosphatebuffered saline (PBS), pH 7.4, and examined under a
dissecting microscope to localize the ampullary region of
the oviducts. The ampullary portions were then fixed for 2
hr at 4°C by immersion in 2.5% glutaraldehyde in 0.1 M
cacodylate buffer (pH 7.4). For post-embedding labeling,
the ampullary tissue samples were trimmed into small
cubes and then dehydrated in a series of graded methanol
solutions, infiltrated, and embedded in Lowicryl K4M
according to routine procedure.
The ampullary tissue samples of another six animals,
designated for morphological study only, were post-fixed
with 1% osmium tetroxide in nanopure water for 2 hr at
4°C following glutaraldehyde fixation by immersion as
detailed above. Some of the tissue samples were treated
with the ferrocyanide-reduced osmium tetroxide method
(Karnovsky, 1971). Post-fixation with the latter method
was carried out at 4°C for 2 hr. In either case, between the
fixation and post-fixation, tissue samples were washed
three times in 0.1 M cacodylate buffer, trimmed into small
cubes, and after post-fixation they were dehydrated in a
series of graded ethanol solutions, infiltrated, and embedded in Epon 812. Sections of Epon- and Lowicryl-embedded tissue samples measuring 1 µm in width were first
examined by light microscopy in order to locate the area of
interest. Lowicryl K4M thin sections of pale gold interference color were then cut with glass knives on a LKB
ultramicrotome and mounted on 200 mesh nickel grids
having a formvar-carbon-coated film. Epon sections were
cut with a diamond knife and were mounted on copper
grids having a formvar-carbon-coated film.
SECRETORY GRANULES IN OVIDUCTAL AMPULLA
Cytochemical Labeling
For lectin cytochemistry, colloidal gold was used as a
marker. Cytochemical labeling was performed by the
one-step and two-step postembedding methods as described by Benhamou (1986). The one-step method (direct
labeling) was performed with HPL and RCA I lectins, and
the two-step method (indirect labeling) with WGA and LFA
lectins.
Preparation of Colloidal Gold, Lectin-Gold and
Glycoprotein-Gold Complexes
Colloidal gold particles of 15 nm diameter were prepared
by the sodium citrate method as described by Frens (1973).
The lectins used were Helix pomatia lectin (HPL), specific
for N-acetyl- D-galactosamine (Gal NAc) (Hammarstrom
and Kabat, 1969, 1971); wheat germ agglutinin (WGA),
specific for N-acetylglucosamine (GlcNAc) (Nagata and
Burger, 1974)/sialic acid (Peters et al., 1979); Limax flavus
agglutinin (LFA), specific for sialic acid (Miller et al.,
1982); and Ricinus communis agglutinin I (RCA I), specific
for D-galactose (Olsnes et al., 1974; Irimura et al., 1975).
Direct Helix pomatia lectin-colloidal gold (HPL-CG) complex and direct Ricinus communis agglutinin I-colloidal
gold (RCA I-CG) complex were prepared as described by
Roth (1983). Ovomucoid-gold complex and fetuin-gold complex were prepared as described by Geoghegan and Ackerman (1977) and Roth et al. (1984), respectively.
Ovomucoid, fetuin, HPL, WGA, and RCA I were purchased from Sigma Chemical Co. (St. Louis, MO) and LFA
from Terochem Lab Ltd. (Mississauga, ON).
Cytochemical Controls
The labeling specificity of both HPL-CG and RCA I-CG
was assessed by incubating the tissue sections at room
temperature in presence of their blocking sugars, GalNAc
(0.2 M) and D-galactose (0.2 M), respectively. For LFA, the
labeling specificity was assessed as follows: 1) sections
were incubated at room temperature with LFA in presence
of its blocking sugar sialic acid (0.2 M) followed by incubation with fetuin-gold complex; 2) sections were digested
with neuraminidase for 2 hr at 37°C prior to labeling with
LFA followed by incubation with fetuin-gold complex; 3)
sections were incubated at room temperature with fetuingold complex alone. For WGA, the following controls were
used: 1) sections were incubated at room temperature with
WGA in presence of its blocking sugars GlcNAc (0.1 M) and
sialic acid (0.1 M) followed by incubation with ovomucoidgold complex; 2) sections were incubated at room temperature with ovomucoid-gold complex alone; 3) sections were
incubated at room temperature with WGA in presence of
its blocking sugar Glc NAc (0.2 M) followed by incubation
with ovomucoid-gold complex.
Freeze-Fracture Procedure
Four mature female hamsters were used for routine
freeze-fracture studies. For freeze-fracture, the animals
were sacrificed by cervical dislocation. The oviducts were
excised as described above and fixed immediately by
immersion in 2.5% glutaraldehyde in 0.1 M cacodylate
buffer. While in fixative, the ampullary region was identified and isolated under a dissecting microscope. The
ampullary tissue samples were further fixed for 2 hr at
229
4°C, washed with 0.1 M cacodylate buffer and then infiltrated with 25% glycerol in 0.15 cacodylate for at least 1 hr
at 4°C. Blocks of tissue samples were mounted on Balzers
type gold disks and frozen in liquid nitrogen cooled Freon
22. Freeze-fracture was carried out in a Balzers unit at
⫺130°C under a vacuum of 2 ⫻ 10⫺6 Torr without etching.
This was followed by shadowing with platinum (Pt) at a
fixed angle of 45° and coating with carbon (C) at a 90°
angle. The thickness of the replica was approximately 2
nm Pt and 25 nm C as determined by a Balzers crystal
thin-film monitor.
In addition to routine freeze-fracture study, two other
animals were sacrificed in the same manner and the
ampullary portions of the oviducts were isolated and
mounted immediately on Balzers gold disks without fixation and cryoprotection. The freshly isolated, unfixed
tissue samples were rapidly frozen at ⫺170°C in a rapidfreezing device (Life Cell). The rapid-frozen samples were
then freeze-fractured in a Balzers unit at ⫺130°C and
etched for 3 min at ⫺100°C before replication was made as
described above. Platinum-carbon replicated specimens
prepared by both routine freeze-fracture method and
rapid-freezing technique were digested in sodium hypochlorite to remove the tissue debris. The replicas were then
washed three times in bidistilled water and mounted on
300 mesh copper grids before being examined on an
electron microscope.
RESULTS
Ultrastructural Morphology of the Ampullary
Epithelium of the Hamster Oviduct
Electron microscope examination of the ampullary epithelium showed the presence of two main types of cells,
ciliated cells and non-ciliated secretory cells. The ciliated
cells possessed long cilia interspersed between microvilli
at their apical cell surface (Fig. 1). Mitochondria and
membrane-bound lysosome-like vesicles were frequently
seen in the supranuclear region. Secretory cells could be
identified by the absence of cilia at their cell surface (Fig.
1). The most conspicuous characteristic of the secretory
cells, however, was the presence of secretory granules of
variable sizes and distinct staining properties (Figs. 1 and
2). The intracellular location of granules was typically
restricted to the supranuclear and apical cytoplasm although a few isolated granules could be observed elsewhere in the cytoplasm, including the basolateral compartment. Immature secretory granules, with their contents
showing a granular-like appearance in cross section, were
commonly located in the vicinity of the Golgi zone (Fig. 2).
Many coated vesicles were also seen associated with the
Golgi saccules (Fig. 2). Occasionally, well-preserved and
favorably cut sections of the ovoid and, presumably, more
mature secretory granules located at the apical region of
secretory cells revealed their highly organized contents.
The latter appeared in the form of parallel, lamella-like
structures with one of their two ends radiating from a
round dense core located at one pole of the granule and
with their other ends converging to meet at the opposite
pole (Fig. 3a). In secretory granules with a different plane
of cut, cross-sectioned profiles and oblique view of the
lamella-like structures constituting the contents of the
granules were also revealed (Fig. 3b).
The internal organization of the secretory granules were
further examined using platinum/carbon coated replicas
230
EL-MESTRAH AND KAN
Figs. 1, 2. Electron photomicrographs of thin-sections of Eponembedded hamster oviductal ampulla. Ciliated cells (CC) are characterized by the presence of cilia (Ci) shown in cross-section at the apical cell
surface with mitochondria (M) distributed in clusters in the apical region of
the cell. Secretory cells (SC) are characterized by the presence of
numerous secretory granules (SG) at their apical pole (Fig. 1) and their
cytoplasm appears to be more compact and darkly stained. Secretory
cells also possess a well-developed Golgi apparatus (Gol) made up of six
to eight saccules frequently seen to be associated with secretory granules
(SG) (Fig. 2). Note the presence of the many coated vesicles that are
associated with the Golgi saccules (arrowheads) (Fig. 2). Note also the
slight difference in the staining property of secretory granules (SG1 and
SG2) in Figure 2.
Nu, nucleus; Lu, lumen. Fig. 1, ⫻16,000; Fig. 2, ⫻25,500.
SECRETORY GRANULES IN OVIDUCTAL AMPULLA
231
Fig. 3. a: A favorably cut section of a secretory granule with its internal distinct lamella-like structures in
beaded-chain appearance (arrowheads) are seen radiating from a small and round dense core (arrow) at one
pole of the secretory granule. b: A secretory granule sectioned in a different plane showing cross-sectioned
profiles (asterisk) of the lamellae. a, ⫻16,900; b, ⫻18,200.
prepared by both routine freeze-fracture and rapidfreezing techniques. In replicas prepared from frozen
samples of glutaraldehyde-fixed and cryoprotected oviductal tissue, many cross-fractured profiles of secretory
granules were seen (Fig. 4). The contents of crossfractured granules had a relatively smooth appearance
with a very fine granular texture (Fig. 4a). Parallel arrays
of lamella-like profiles corresponding to those seen in Epon
sections were not seen in routine freeze-fracture preparations. However, when unfixed and rapidly frozen samples
were fractured and etched followed by shadowing with
platinum and carbon, the resulting replicas revealed both
longitudinally and cross-fractured profiles of lamella-like
lattice in the secretory granules corresponding to similar
structures observed in Epon sections (Figs. 4b and c).
The post-fixation of tissue samples with ferrocyanidereduced osmium tetroxide provided the opportunity for a
better demonstration of the morphology of the secretory
cells. The darkly stained cytoplasm of the secretory cells
was typical when compared to that of the ciliated cells (Fig.
5). The secretory granules were usually of two types: 1)
those with a homogeneous electron-dense matrix and 2)
those with an electron-lucent matrix. The heavily stained
secretory granules were easily distinguishable from those
displaying a lesser degree of opacity. Both types of granules appeared to occur within the same cell type (i.e. in
secretory cells) and possessed a relatively small dense core
(Fig. 6a). In both cases, the dense core was seen to be
preferentially located at one pole of the secretory granule
and appeared to be in contact with the granule membrane.
Some of these granules appeared to have coalesced into a
large expanse (Fig. 6b). The release of granule content to
the ampullary lumen at the apical surface of the nonciliated cells and cytoplasmic protrusions were frequently
observed. The glycocalyx covering the microvilli was heavily
stained due to its glycoprotein content, whereas the cilia
possessed no such appearance. Electron microscopic observation of the ampullary secretory cells revealed, in the
cytoplasm, the presence of a well developed Golgi complex
with elaborate organization (Fig. 7). Electron-lucent secretory granules were always found in the trans-face of the
Golgi complex whereas densely stained granules were
found in both the trans- and cis-face of the Golgi apparatus. Some palely stained secretory granules displayed
cross-sectioned profiles of the lamella-like structures previously seen in Epon-embedded oviduct tissue without prior
treatment with ferrocyanide-reduced osmium tetroxide
(Fig. 7a). In addition, many small vesicles of uniform size
were also found in the vicinity of the Golgi apparatus (Fig.
7a). The small round vesicles were located between cisternae of endoplasmic reticulum and the cis-face of the Golgi
apparatus, between the ends of stacks of Golgi saccules
and also concentrated in an area close to the trans-face of
the Golgi complex. Most of the cisternae of endoplasmic
reticulum adjacent to the cis-face of the Golgi complex
appeared to be of the smooth type. However, individual
endoplasmic reticulum cisternae studded with ribosomes
on one surface were also seen in the neighbourhood of the
cis-face of the Golgi complex. Occasionally, a row of these
small, uniform-sized vesicles were found to lie adjacent to
the first cis Golgi saccule (Fig. 7a). In some Golgi stacks, a
gradient of staining intensity was seen in the saccules with
a progressive increase in staining intensity from the
cis-face towards the trans-face (Fig. 7b).
Fig. 4. Electron photomicrographs of freeze-fractured oviductal ampulla prepared by both conventional freeze-fracture (a) and rapid-freezing
techniques (b,c). a: Cross-fractured secretory granules (SG) in secretory
cells of oviductal ampulla prepared by the routine freeze-fracture method
show their contents with a relatively smooth texture (asterisk). However,
when rapidly frozen ampullary samples were fractured and etched, the
resulting replicas revealed both longitudinally (arrowheads) and cross-
fractured (asterisk) profiles of the lamella-like lattice occupying the matrix
of the secretory granules (SG) (b,c); Such lamella-like profiles correspond
to similar structures seen in Epon sections. Note that the presence of both
longitudinally and cross-fractured profiles can be seen within the same
secretory granule (SG) as a result of the fracturing process (c). a,
⫻26,000; b, ⫻29,900; c, ⫻27,200.
Fig. 5. Electron photomicrograph of Epon-embedded ampulla previously post-fixed with ferrocyanide-reduced osmium tetroxide. The columnar-shaped ciliated cells (CC) have a palely stained cytoplasm with the
nucleus (Nu) located basally. However, secretory cells (SC) characterized
by the presence of microvilli (Mi) at their apical surface and the assembly
of numerous secretory granules (SG) at their apical pole are seen
protruding into the ampullary lumen (Lu). Note the presence of two types
of granules in the secretory cells (SC); the electron-dense secretory
granules (SG) are much more abundant than those which are electronlucent (asterisk). Ci, cilia; M, mitochondria. ⫻12,500.
Fig. 6. Electron micrographs showing the secretory granules of the
ampullary secretory cells at high magnifications (a). The two types of
secretory granules (SG) are easily identifiable due to their differential
staining; a dense core (arrowheads) is located at one pole of each of both
types of secretory granules. b: A secretory granule (SG) is seen releasing
its content through the free edge of the apical cell surface (arrowheads)
into the ampullary lumen (Lu), whereas other secretory granules appear
to coalesce to form a larger expanse (asterisk) . a, ⫻25,000; b, ⫻30,000.
SECRETORY GRANULES IN OVIDUCTAL AMPULLA
Fig. 7. Electron photomicrographs showing the Golgi region of secretory cells in the oviductal ampulla previously treated with ferrocyanidereduced osmium tetroxide prior to Epon embedment. a: A well-developed
Golgi apparatus (Gol) is seen embracing both electron-dense (asterisk)
and electron-lucent (SG) secretory granules. Their contents reveal
cross-sectioned profiles (thick arrows) of beaded chains of lamella-like
structures previously seen cut longitudinally in Figure 3a. Note the
235
presence of several darkly stained granules located near both the cis-face
and trans-face of the Golgi complex, and the presence of a row of small
uniform-sized vesicles adjacent to the cis-face of the Golgi stack (arrowheads). b: A Golgi apparatus (Gol) showing a gradient of staining intensity
increasing progressively from the cis- to the trans-face; note also the
presence of a fenestrated saccule (arrowheads) at the trans-face of the
Golgi apparatus (Gol). a, ⫻42,500; b, ⫻43,000.
236
EL-MESTRAH AND KAN
Cytochemical Localization of Lectin-Binding
Glycoconjugates in the Hamster Oviductal
Ampulla
In the present study, we also examined the distribution
of lectin-binding glycoconjugates in relation to the contents of the granule in Lowicryl-embedded tissue sections
of the ampullary epithelium. For this purpose, four different lectins were used. Quantitative results of cytochemical
labeling with various lectins have been detailed in a
previous study (El-Mestrah and Kan, 1999).
Since the cytochemical features of secretory granules
are of major interest in this study, special attention was
directed toward the distribution of gold particles in the
secretory granules of oviductal ampulla with the possible
correlation of such distribution to their internal morphology. Although the use of Lowicryl K4M hydrophilic resin
for lectin cytochemistry compromised the ultrastructure of
the cells, tissue sections incubated with each of HPL-CG
(Figs. 8a and b), RCA I-CG (not shown), LFA (Fig. 8c), and
WGA (not shown) presented similar patterns of labeling by
gold particles. The distribution of gold particles observed
over the secretory granules of non-ciliated secretory cells is
best illustrated by electron micrographs taken from ampullary sections incubated with HPL-CG (Figs. 8a and b) and
LFA (Fig. 8c). The labeling of lectin-binding glycoconjugates in some secretory granules appeared to be aligned
with the lamella-like lattice occupying the matrix of the
granules (Figs. 8b and c) with no sign of labeling over the
dense core (Fig. 8a). Occasionally, labeling by gold particles could be seen associated with the secretory products
in the process of their release into the oviductal lumen
(Fig. 8b). On the other hand, ciliated cells which are
characterized by the presence of long cilia interspersed
between microvilli at their apical cell surface, showed no
indication of any labeling in their cytoplasm and its
associated organelles except for a weak labeling over the
ciliary extensions in the ampullary lumen (not shown).
Control incubations of HPL, RCA I, and LFA (not shown)
showed negative reaction to the corresponding lectin-gold
complex demonstrating the specificity of the labelings.
Tissue sections incubated with WGA in presence of its
blocking sugars GlcNAc and sialic acid or with ovomucoidgold complex alone were also negative (not shown).
DISCUSSION
The mammalian oviduct is a secretory organ which
provides the environment necessary for the maturation
and transport of gametes and embryo development. In
particular, the oviductal ampulla has a fundamental importance owing to the fact that it is the site where fertilization
takes place. The ultrastructural morphology of the oviduct
is closely related to its functions. In the present study, we
examined the ultrastructure and cytochemical properties
of secretory cells of the ampullary epithelium in the golden
hamster using various techniques in electron microscopy.
One distinct feature of the oviductal ampulla observed
in this study was the ultrastructural appearance of secretory granules. The morphological features of secretory
granules of oviductal secretory cells have been investigated in several mammalian species including the human.
It has been suggested that two types of secretory granules,
namely, electron-dense and electron-lucent granules, are
present in the oviductal secretory cells of the mouse
(Komatsu and Fujita, 1978), golden hamster (Abe and
Oikawa, 1991), rabbit (Brower and Anderson, 1969; Jansen
and Bajpai, 1982), cow (Abe et al., 1993), monkey (Odor et
al., 1983), and human (Björkman and Fredricsson, 1962;
Clyman, 1966), while Willemse and Van Vorstenbosch
(1975) demonstrated the presence of four types of secretory
granules in the sheep oviduct. In a previous study performed in the hamster oviduct, Abe et al. (1991) demonstrated the presence of regional differences in the ultrastructural features of secretory cells and their related
secretory granules. These various studies, taken together,
have shown that there are marked differences among
species as well as regional differences in the morphological
features of secretory granules.
In the present study, favorable Epon sections of the
secretory granules revealed their distinctive internal organization whereby well organized lamella-like structures
appeared to be radiating from a dense core located at one
end of the granule; this finding may suggest a possible role
for the dense core in the secretory mechanism of the
secretory granule. However, the unknown nature and the
functional role of the dense core as well as that of the
lamella-like structures need to be further investigated.
The dense core found located at one pole of some secretory
granules was not present in all secretory granules. This is
possibly due to the plane of cut. However, in the golden
hamster, dense cores were rarely seen in the immature
secretory granules, but they appeared during the maturation process of secretory granules (Abe and Oikawa, 1989).
Similar phenomena were observed in the sheep (Willemse
and Van Vorstenbosch, 1975). These findings suggest that
the presence of dense cores is a characteristic of mature
secretory granules in the oviduct. Furthermore, some
secretory granules which were located in the vicinity of the
Golgi apparatus, and are therefore considered immature,
showed cross-sectioned profiles of lamellar-like structures
similar to those of the mature ones, suggesting that these
immature granules might have attained their actual internal topography early on during the process of maturation.
Although two types of secretory granules appeared to
occur within the non-ciliated secretory cells, a distinction
between the two types was made more evident with the
use of ferrocyanide-reduced osmium tetroxide by which
the secretory granules were shown to be differentially
stained. This is suggestive of the presence of a heterogeneous population of secretory granules in the hamster
oviductal ampulla and that the secretory granules might
contain different secretory contents.
Although the presence of similar lamella-like lattice in
the oviduct secretory granules has been reported in sevFig. 8. Electron photomicrographs of the ampullary epithelium labeled
with HPL-gold (a,b) and with LFA and fetuin-gold complexes (c). a: A high
concentration of gold particles is localized over the secretory granules
(SG), Golgi apparatus (Gol) and microvilli (Mi) of a secretory cell. Note
that the dense core (thick arrows) occupying one pole of the secretory
granules (SG) is devoid of any labeling. b: Colloidal gold particles are
uniformly distributed throughout the secretory granules (SG) which
appear to have coalesced. The labeling by gold particles is shown here to
be aligned with the lamella-like profiles (arrowheads) occupying the
matrix of the secretory granules (SG) during the secretory process. c: A
region of a secretory cell from a tissue section incubated with LFA and
fetuin-gold complex showing secretory granules (SG) heavily labeled with
gold particles. In this micrograph, gold particles can be seen superimposed over the lamella-like structures (arrowheads) of the secretory
granules (SG) . Lu, lumen; Nu, nucleus. a, ⫻34,000; b, ⫻47,000; c,
⫻17,000.
SECRETORY GRANULES IN OVIDUCTAL AMPULLA
Figure 8.
237
238
EL-MESTRAH AND KAN
eral other animal species (Nayak and Ellington, 1977;
Odor et al., 1983), results obtained in our study have
provided, perhaps, the best view of the arrangement of
such lamella-like structures in the granules at the ultrastructural level. Using the rapid-freezing and freeze-etch
techniques on freshly isolated, unfixed oviductal tissue, we
have been able to confirm the distinct entity of these
lamella-like structures shown in thin sections of Eponembedded oviducts.
In the present study, as shown by results obtained with
both the routine osmium tetroxide post-fixation Eponembedded specimens and tissue treated with ferrocyanidereduced osmium tetroxide, the Golgi apparatus in the
non-ciliated cells of the hamster oviduct is a highly elaborate organelle typical of secretory cells. The small round
vesicles that were observed in between the endoplasmic
reticulum cisternae and the first cis Golgi saccules are
likely transport vesicles shuttling newly synthesized proteins from the endoplasmic reticulum cisternae to the
forming (cis) face of the Golgi complex. The progressive
increase in staining intensity of the Golgi saccules from
the cis-face to the trans-face (Fig. 7b), as shown by
ferrocyanide-reduced osmium tetroxide impregnation, reflects an increase in the carbohydrate moieties associated
with newly synthesized macromolecules in the secretory
cells during their processing through the Golgi stacks. It is
interesting to note that small coated vesicles were being
budded off the edges of the Golgi saccules and that some of
these coated vesicles could be seen to deliver their content
to immature secretory granules in formation (Fig. 2). It
appears that some of the coated vesicles that pinched off
from the ends of the Golgi saccules were being diverted to a
specific destination (formation of secretory granules) other
than the plasma membrane in the hamster oviductal
secretory cells.
The high resolution cytochemistry has allowed for the
localization of lectin-binding glycoconjugates in intracellular organelles involved in their biosynthesis and secretion.
The high density of labeling for HPL-, WGA-, LFA-, and to
a lesser extent RCA I-binding glycoconjugates over the
Golgi apparatus and secretory granules indicates an active
synthesis and secretion of the products by the ampullary
non-ciliated secretory cells.
In a previous study performed in the hamster, we
demonstrated by lectin-gold cytochemistry that HPLbinding glycoconjugates are absent from the zona pellucida (ZP) of ovarian oocytes but are synthesized and
secreted by the non-ciliated secretory cells in the oviduct
and later transferred to the zona pellucida of ovulated eggs
(Kan et al., 1990). Subsequently, we demonstrated, with
the high resolution lectin-gold approach and quantitative
analysis, changes of glycoconjugates in the hamster ZP
during oocyte growth and development in the ovary and
oviduct (Roux and Kan, 1991). Similarly, previous studies
from other laboratories demonstrated the distribution of
lectin-binding glycoconjugates in relation to the estrous
cycle (Vrcic, 1993; Raychoudhury et al., 1993; Wu et al.,
1993; Menghi et al., 1985). Together, these results suggest
that the oviductal epithelium is a bona fide site for the
synthesis and secretion of glycoconjugates that may be
involved in many developmental events occurring in the
oviduct.
One of the aims of our study was to draw a correlation
between the distribution of lectin-binding glycoconjugates
in the secretory granules and their internal structure. In
the present study, cytochemical labeling with the various
lectins (in particular, HPL and LFA) showed that the gold
particles were directly located over the crystalline lattice
in the granule matrix. The organization of lamella-like
structures within the secretory granules may serve as an
anchor for glycoconjugates prior to their release into the
ampullary lumen, or the glycoconjugate macromolecules
themselves may be anchored to each other in a highlyorganized manner. The absence of labeling over the dense
core of secretory granules requires further investigation in
order to unravel its chemical composition and its possible
role in the secretory process.
In summary, we have revisited the ultrastructure and
cytochemical properties of secretory granules in the ampullary region of the golden hamster using a combination of
routine electron microscopy, freeze-fracture techniques
and lectin-gold cytochemistry. We have provided new
ultrastructural information on the internal organization of
the ampullary secretory granules and the structural entity
of the crystalline lamella-like lattice decorated by lectinbinding glycoconjugates as demonstrated in the present
study. With the application of the ferrocyanide-reduced
osmium tetroxide staining procedure, we have been able to
elucidate the heterogeneity of secretory contents based on
the differential staining properties of the secretory granules. The ampullary region of the female reproductive
tract is a strategic site in the complex process of fertilization since it is in the lumen of the ampulla where the
sperm fertilizes the egg. The ampulla plays a vital role in
this process by contributing its secretion to the luminal
environment. Therefore, the revelation of the internal
topographical features of oviductal secretory granules, at
both morphological and cytochemical levels, allowed us to
raise some valid speculations on the possible correlation of
such internal organization to the regulatory mechanism
underlying their secretory process. In the future, it would
be necessary to unravel the precise role of the dense core in
the secretory mechanism as well as the function of the
highly organized secretory contents within the membrane
bound granules.
ACKNOWLEDGMENTS
The authors wish to thank Mr. Bob Temkin and Ms.
Verna Norkum for reproduction of the original photomicrographs.
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