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High-resolution localization of hyaluronic acid in the golden hamster oocyte-cumulus complex by use of a hyaluronidase-gold complex.

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THE ANATOMICAL RECORD 228:370-382 (1990)
High-Resolution Localization of Hyaluronic Acid in
the Golden Hamster Oocyte-Cumulus Complex by
Use of a Hyaluronidase-GoldComplex
FREDERICK W.K. KAN
Department of Anatomy, Faculty of Medicine, University of Montreal, Montreal, Quebec,
Canada H3C 357
ABSTRACT
The distribution of hyaluronic acid in the oocyte-cumulus complexes collected from the oviduct ampulla of superovulated hamsters was revealed
by use of hyaluronidase coupled to colloidal gold. On thin sections of Lowicrylembedded oocyte-cumulus complexes, gold particles were associated specifically
with interconnecting fibrillar materials that make up the cumulus matrix. Inside
the cumulus cells, gold particles were found over the cisternal membrane of the
rough endoplasmic reticulum, in the contents of lysosomes and multivesicular
bodies, and over Golgi vesicles of some cumulus cells. A high concentration of gold
labeling was observed over the peripheral condensed chromatin and perinucleolar
components in the nucleus. The cell surface of the cumulus cells also appeared to
be labeled. Gold particles, however, were absent over the mitochondria and lipid
vacuoles. In the oocytes, labeling was found t o be associated mainly with rough
endoplasmic reticulum and arrays of lamellar structures; cortical granules, mitochondria, and coated vesicles were essentially devoid of gold particles. Gold particles were also seen along the plasma membrane of the oocytes and within the
perivitelline space. The zona pellucida was not labeled by hyaluronidase-gold.
Different control experiments confirmed the specificity of the labeling. Digestion of
thin sections with hyaluronidase prior to incubation with hyaluronidase-gold abolished the initial reaction, whereas treatment of thin sections with chondroitinase
did not prevent labeling of oocyte-cumulus complexes by hyaluronidase-gold. Although the function of hyaluronic acid in the oocyte-cumulus complex a t the time
of ovulation and fertilization is not known, the high concentration of this particular compound in the cumulus matrix and the cumulus cells and its specific locations in the perivitelline space and in the superovulated oocytes implicate the
significance of its presence and warrant future investigations.
In order to fertilize the egg, mammalian spermatozoa
have to penetrate through two extracellular egg investments, the cumulus matrix and the zona pellucida.
Although numerous studies have been done on the
morphological, biochemical, and immunochemical properties of the zona pellucida (see reviews by Yanagimachi, 1981; Dunbar, 1983a,b; Dunbar and Wolgemuth, 1984; Wassarman et al., 19861, those of the
cumulus matrix have been less characterized. Moreover, the distribution of hyaluronic acid within the egg
investments and the precise intracellular location of
this macromolecular component in the oocyte-cumulus
complex are not known. Biochemically, hyaluronic acid
has been shown to be the major component of the bovine cumulus matrix (Ball et al., 1982). In cattle and
mice, the synthesis and deposition of hyaluronic acid in
the oocyte-cumulus complex appear to be stimulated by
follicle-stimulating hormone (Eppig, 1979; Ball et al.,
1982), the action of which is believed to be mediated by
cyclic adenosine monophosphate (AMP) (Eppig, 1979;
Ball et al., 1982). Ultrastructurally, a recent report on
the golden hamster cumulus matrix shows that this
0 1990 WILEY-LISS, INC
egg investment consists of a network of fibrillar units
with a considerable amount of interconnection (Yudin
et al., 1988). Cytochemically, however, the distribution
of hyaluronic acid in the oocyte-cumulus complex has
not been thoroughly described, due mainly to the unavailability of a suitable marker for its detection a t the
electron microscopic level. Previous studies on the localization of hyaluronic acid have relied on cationic
dyes, such as Alcian blue, in conjunction with enzymatic digestion of tissue sections (Fisher and Solursh,
1977; Derby, 1978; Derby and Pintar, 1978; Markwald
et al., 1978; Pintar, 1978; Singley and Solursh, 1980;
Delgado and Zoller, 1987). However, these histochemical staining techniques are used mainly at the light
microscopic level. Although ruthenium red has been
utilized for the ultrastructural detection of hyaluronic
Received July 11, 1989; accepted March 30, 1990.
Address reprint requests to Dr. F.W.K. Kan, Department of Anatomy, University of Montreal, C.P. 6128, Succ. A, Montreal, Quebec,
Canada H3C 357.
371
LOCALIZATION OF HYALURONIC ACID
acid in the hamster oocyte-cumulus complex (Talbot,
1984), results reported in this study are scanty. Attempts have been made to employ the hyaluronic acid
binding region and monoclonal antibodies against this
component in cartilage proteoglycans as probes for the
ultrastructural localization of hyaluronic acid in brain
tissue sections (Ripellino et al., 1985, 1988, 1989).
These attempts have yielded results of specificity. Radioautography has also been used to examine the preovulatory synthesis of proteoglycans by human oocytes
and cumulus cells and their secretion into the extracellular matrices of the oocyte-cumulus complex (Tesarik and Kopecny, 1986). This approach, too, suffers
the limitation of resolution and cannot specifically
identify the labeled macromolecular component to be
hyaluronic acid. Recently, hyaluronidase (HUD) adsorbed to colloidal gold particles has been used with
success to detect hexuronic acid-containing macromolecules on thin sections of a variety of rat tissues (Londono and Bendayan, 1988). This HUD-gold method,
with minor modification, was used in the present study
for the high-resolution localization of hyaluronic acid
in the superovulated hamster oocyte-cumulus complex.
I report here the specific association of hyaluronic
acid with interconnecting fibrillar materials of the cumulus matrix and the subcellular distribution of these
macromolecular component in the cumulus cells and
oocytes. In addition, the failure of labeling of the zona
pellucida by HUD-gold confirms previous histochemical (Delgado and Zoller, 1987) and biochemical (Newport and Carrol, 1976; Dunbar et al., 1980) findings of
absence of hyaluronic acid in this particular egg investment.
MATERIALS AND METHODS
Collection of Oocytes
Sexually mature female golden hamsters (Mesocricetus auratus, 8 weeks old) were obtained from Charles
Rivers Laboratories (St-Constant, Quebec, Canada).
They were stimulated to superovulate by a n intraperitoneal (i.p.) injection of 25 IU pregnant mare’s serum
gonadotropin (PMSG, Equinex; Ayerst, Montreal, Quebec, Canada) followed, 48 hours later by a n i.p. injection of 25 IU human chorionic gonadotropin (hCG,
APL; Ayerst). The animals were killed by cervical dislocation 17 hours after injection with hCG. Their ventral abdominal wall was immediately cut open, and the
oviducts were removed and placed in the well of a porcelain dish containing Dulbecco’s phosphate-buffered
saline (PBS-D, pH 7.4, Gibco Inc., Burlington, Ontario,
Canada). To collect oocyte-cumulus complexes, the ampullary portion of the oviduct was identified and torn
open with fine steel tweezers under a dissecting microscope. The oocyte-cumulus complexes collected were
fixed for 2 hours at room temperature by immersion in
2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH
7.4). At the end of the fixation, the specimens were
washed three times in 0.1 M cacodylate buffer and kept
overnight at 4°C in the same buffer.
For preparation of thin sections, the oocyte-cumulus
complexes were dehydrated in a series of graded methanols, infiltrated, and embedded in Lowicryl K4M according to routine procedure. Superovulated oocytes
were first located in 1 pm-thick sections of Lowicrylembedded specimens by light microscopy. Thin sections
(pale gold interference color) were then cut with glass
knives on a LKB ultramicrotome and mounted on 200
mesh nickel grids having a carbon-coated parlodion
film and processed for HUD-gold labeling.
Preparation of Colloidal Gold and Hyaluronidase
VI-Gold Complex
Colloidal gold particles of 15 nm diameter were prepared by the sodium citrate method as described by
Frens (1973). Preparation of the HUD-gold complex
was performed essentially as recently reported (Londono and Bendayan, 1988). The major difference was
that HUD type VI (from bovine testes, EC 3.2.1.35,
purchased from Sigma Chemical Co., St. Louis, MO)
was used in the present study instead of HUD type
IV-S as previously used by the above authors. Preliminary experiments indicated that a higher labeling intensity was obtained when HUD VI was used in place
of type IV-S in preparation of the gold probe. In brief,
for preparation of HUD VI-gold, 50 pg of HUD VI were
dissolved in 0.1 ml of twice-distilled water, to which 10
ml of colloidal gold sol previously adjusted to pH 7.5
was added, and the two solutions were gently mixed.
Then 0.1 ml of 1% polyethylene glycol (MW 20,000)
was added and mixed well with the HUD VI-gold complex. The complex was centrifuged for 30 minutes at
26,500 rpm. The resulting red sediment was resuspended in 1ml of 0.05 M acetate buffer, pH 5, containing 0.02% polyethylene glycol. The HUD VI-gold complex was stored at 4°C in a capped, round-bottom
polypropylene tube until use. Freshly prepared HUD
VI-gold complex was normally used for incubation of
sections within 3 days after preparation.
Cytochemical Labeling
Thin sections of Lowicryl-embedded oocyte-cumulus
complexes were incubated for 5 minutes on a drop of
0.05 M acetate buffer, pH 5, followed by 30 minutes
incubation at room temperature on a drop of the HUD
VI-gold complex diluted 1:lO with the same buffer. After labeling with the HUD VI-gold complex, the sections were washed with acetate buffer, rinsed with
twice-distilled water, and dried on a filter paper. The
sections were stained with uranyl acetate and lead citrate for examination in a Philips 300 electron microscope operated at 80 KV.
Cytochernical Controls
The HUD VI-colloidal gold complex is a one-step
post-embedding enzyme-gold labeling technique. Although the exact mechanism of the action of the enzyme-gold technique remains to be elucidated, it appears that upon incubation of the tissue section with a
specific enzyme-gold complex, the enzyme molecules at
the surface of the gold particle interact with their specific substrate molecules (in this case the hyaluronic
acid) exposed at the surface of the section and that the
biological activity of many enzyme-gold complexes previously studied was found to be retained (for reviews
see Bendayan, 1984,1989). As for any other cytochemical technique, the specificity of the HUD VI-gold labeling was assessed through several control experiments listed below:
1. Lowicryl-embedded thin sections of oocytecumulus complexes were digested with the free enzyme
372
F.W.K. KAN
(0.5 mg/ml of HUD VI) in the presence of protease inhibitors (Talbot, 1984) for 6 hours a t 37°C prior to labeling with the HUD VI-gold complex. In this control
experiment, the nonlabeled enzyme (HUD VI) should
extract the substrate molecules (hyaluronic acid) so
that the subsequent enzyme-gold complex should yield
little or no specific labeling. Protease inhibitors were
added to prevent any activity that might occur due to
possible protease contamination of the HUD VI.
2. Thin sections of oocyte-cumulus complexes were
incubated with the HUD VI-gold complex, to which the
specific substrate (1 mg/ml of hyaluronic acid, from bovine vitreous humor, Sigma Chemical Co.) was added.
The free substrate should compete with the hyaluronic
acid exposed on the surface of the section for binding t o
the HUD VI-gold complex.
3. Thin sections were incubated with HUD VI-gold in
presence of excess amount of the free enzyme (1 mg/ml
of HUD VI). In this case, the free enzyme should compete with the HUD VI-gold complex for binding to the
hyaluronic acid that is exposed on the tissue section.
4. Because testicular hyaluronidase degrades not
only hyaluronic acid but also chondroitin and chondroitin sulfates as well (Leppi and Stoward, 1965; Zergibe, 1962), in order to ensure that HUD VI-gold complex detects mainly hyaluronic acid and not other
glycosaminoglycans in the oocyte-cumulus complex,
some thin sections were digested with chondroitinase
ABC (1 mg/ml or 0.55 unit/ml, Sigma Chemical Co.) for
5 hours a t 37°C prior to labeling with HUD VI-gold
complex as described above.
with the cumulus matrix and was unlabeled by HUD
VI-gold, indicating the absence of hyaluronic acid in
this particular egg investment (Fig. 3). The junction
between the inner limit of the cumulus matrix and the
outer surface of the zona pellucida is illustrated a t high
magnification in Figure 3, in which the heavily labeled
cumulus matrix is shown in contrast to the zona pellucida, which was almost completely devoid of labeling
by gold particles. The inner border of the unlabeled
zona pellucida is sharply interrupted by the resurgence
of gold labeling in the perivitelline space, which is a
discrete extracellular compartment located between
the inner surface of the zona pellucida and the plasma
membrane of the oocyte (Fig. 3, lower inset). Gold labeling in the perivitelline space appears to be associated with cloudy materials that possess a higher staining density than that of the zona pellucida.
Distribution of Hyaluronic Acid in the Cumulus Cells
Cumulus cells in the cumulus matrix were found in
clusters but more often in solitude. Occasionally, erythrocytes were found in apposition to the cumulus cells.
HUD VI-gold labeling of thin sections of Lowicryl-embedded oocyte-cumulus complex revealed intense labeling of the cell surface of the cumulus cells (Figs. 4).
Inside the cumulus cells, particles associated with ribosome-bound membrane of the rough endoplasmic reticulum (rER) were found (Fig. 4).Gold particles were
predominant over the cisternal membrane of the rER,
with occasional particles inside the lumen of the cisternae. Colloidal gold labeling was also observed over
lysosome-like structures, multivesicular bodies, and
RESULTS
Golgi apparatus (Fig. 5). Mitochondria and lipid vacuLocalization of Hyaluronic Acid in the Cumulus Matrix
oles in the cumulus cells were not labeled by HUD
Lowicryl-embedded thin sections of oocyte-cumulus VI-gold (Figs. 1 , 2 , 4 , 5 ) .An unusually high concentracomplex, after labeling with HUD VI-gold, revealed tion of gold labeling was found in nuclear compartintense labeling of the cumulus matrix by gold parti- ments, in particular over the condensed peripheral
cles (Fig. 1). The opacity of the gold particles facilitates chromatin (heterochromatin) and condensed perinucledelineation of the inner (Fig. 1) and outer (Fig. 2) olar chromatin (Figs. 1,2,4, 5). The diffused chromatin
boundaries of this extracellular matrix. At low magni- (euchromatin) and the fibrillar region of the nucleolus
fication, its structural organization resembles that of did not appear to be labeled.
an “expanded spider-web’’made up of a network of interconnections of fibrillar materials woven into a Distribution of Hyaluronic Acid in the Superovulated Oocyte
Thin sections of Lowicryl-embedded oocyte-cumulus
sphere in which cumulus cells are suspended (not
shown). The intense labeling of the fibrillar materials complex showed that the oocyte is surrounded by an
by gold particles reflects the presence of a high concen- uniform thickness of zona pellucida, unlabeled by HUD
tration of hyaluronic acid in the cumulus matrix. Indi- VI-gold, which in turn is enclosed by the heavily lavidual or clusters of cumulus cells are frequently seen beled cumulus matrix (Fig. 1). Externally, in addition
a t the outer boundary of the cumulus matrix, which is to the labeling of the perivitelline space by HUD VIwell delineated by gold particles. Occasionally a cumu- gold as described earlier, gold particles were seen aslus cell is seen pressing against the matrix, creating an sociated with microvilli and along the plasma memoutward protrusion of the fibrillar network (Fig. 2). In brane (Fig. 3). Inside the oocyte, many cortical
this part of the cumulus matrix, the stretched outer granules were found immediately below the plasma
limit outlined by gold particles remained continuous membrane. These granules were not labeled (Fig. 3).
and intact, showing no sign of disruption despite the Mitochondria, frequently appearing in clusters, were
force exerted by the cumulus cell. This stretchable fea- also devoid of HUD VI-gold labeling (Fig. 3). The most
ture of the cumulus matrix indicates the fibroelastic numerous and evident organelle found in the oocyte
property of the interwoven strands, which appear to be proper were parallel arrays or swirls of cytoplasmic
capable of sustaining weights and pressure exerted by lamellae (Figs. 1,6).Transverse section of these lamellar structures showed their concentric orientation
the cells within the cumulus matrix.
Labeling of the cumulus matrix by HUD VI-gold around clusters of mitochondria (Figs. 1 , 3 , 6 ) ,whereas
stopped abruptly a t the inner boundary of the cumulus tangential section showed their appearance in crystalmatrix, which bordered along the outer surface of the line pattern (Fig. 6b). These cytoplasmic lamellae were
zona pellucida (Figs. 1,3). The matrix of the zona pel- strongly labeled by HUD VI-gold (Fig. 6). Gold partilucida exhibited a more compact texture as compared cles, however, appeared not to be directly over these
LOCALIZATION OF HYALURONIC ACID
Fig. 1. Electron photomicrograph of an ultra-thin Lowicryl section
of superovulated hamster oocyte stained for hyaluronic acid by use of
the hyaluronidase VI-gold complex. The cumulus matrix (CM) delineated by arrowheads a t the left of CM is intensely labeled with gold
particles. The zona pellucida (ZP) does not appear to be labeled. Immediately outside the oocyte, labeling is found over the microvilli
373
(arrows) and along the plasma membrane. Labeling is also localized to
the perivitelline space (PV). Inside the oocyte, gold particles are associated mainly with the lamellar structures (smaller arrows),
whereas cortical granules (c) and mitochondria (m) are unlabeled. CC,
cumulus cell nucleus; Li, lipid droplets. X 8,000.
374
F.W.K. KAN
Fig. 2.A cumulus cell (CC) is seen protruding outward from the cumulus network creating a bulge in
the cumulus matrix (CM). X 9,500.
particular structures; rather, they labeled fluffy materials t h a t are associated with the borders of the cytoplasmic lamellae (Fig. 6). This is evident in tangential
section of lamellae in oocytes in which gold particles
were not found over the crystalline lattice but were
localized to the borders of the lamellae (Fig. 6b). Isolated cisternae of rER in the oocyte were also labeled
(results not shown).
Controls
The specificity of the postembedding labeling over
the cumulus matrix, in cumulus cells and oocytes, were
confirmed by several controls. Addition of a n excess of
the free enzyme (HUD VI, 1 mg/ml) to the HUD VIgold complex or addition of the specific substrate (hyaluronic acid, 1 mg/ml) prevented labeling of the
oocyte-cumulus complex by gold particles (Figs. 7a,b).
Predigestion of the Lowicryl sections of oocyte-cumulus
complexes with free HUD VI (1mg/ml) in the presence
of protease inhibitors (6 hours at 37°C) greatly reduced
the labeling previously seen over the cumulus matrix
(Fig. 7c).
Treatment of thin sections of oocyte-cumulus complexes with chondroitinase ABC did not prevent label-
375
LOCALIZATION OF HYALURONIC ACID
Fig. 3.High magnification of the oocyte-cumulus complex showing
the transition from the cumulus matrix to the oocyte proper. The
cumulus matrix (CM) is heavily labeled with gold particles. The labeling stops abruptly a t the outer surface of the zona pellucida (ZP),
which, characterized by its compact feature, is not labeled by gold
particles. The labeling reappears in the perivitelline space (PV) and is
seen over the microvilli (arrowheads) and on the plasma membrane of
the oocyte. Cortical granules (arrows) and mitochondria (m) are not
labeled. However, intense labeling by gold particles is associated with
the cytoplasmic lamellae. x 13,200. Upper inset: The intensely labeled cumulus matrix (CM) is shown here in contrast to the unlabeled
zona pellucida (ZP). x 54,000. Lower inset: High magnification of a
superovulated hamster oocyte showing a region of the perivitelline
space (PV) labeled strongly by HUD VI-gold complex. The zona pellucida (ZP), cortical granules (arrowheads), mitochondria (m) and the
contents of two coated vesicles (arrows) are devoid of labeling.
x 21,300.
ing of the cumulus matrix, cumulus cells, or oocytes by
HUD VI-gold (Fig. 8). However, inside certain cumulus
cells, there appeared to be a lessened accumulation of
gold particles over the organelles. These results indicated that the reaction was due mainly to hyaluronic
acid, and not to chondroitin or chondroitin sulfates,
although a relatively small amount of the latter cornponents may also be present.
DISCUSSION
In this study, HUD VI coupled to gold particles was
used for high-resolution mapping of hyaluronic acid in
the golden hamster oocyte-cumulus complex. Four major findings resulted from this study: The first is that a
high concentration of hyaluronic acid was detected in
the cumulus matrix by use of the HUD VI-gold com-
376
F.W.K. KAN
Fig. 4. Electron micrograph of a cumulus cell. Gold particles are
localized over the plasma membrane (arrows). Inside the cell, labeling
is seen along the cisternal membrane of the rough endoplasmic reticulum or in association with ribosomes (arrowheads). In the nucleus
(N), labeling is preferentially associated with the peripheral chromatin and patches of condensed chromatin throughout the nucleoplasm.
The perinucleolar condensed chromatin is also strongly labeled. Nu,
nucleolus; CM, cumulus matrix. x 19,200.
plex. Colloidal gold labeling reveals the structural cumulus matrix. Recent experiments performed in our
organization of the cumulus matrix in the form of a laboratory with Limus flavus agglutinin (specific for
network of interconnecting fibrillar structures, as de- sialic acid), Ricinus communis I (specific for D-galacscribed recently (Yudin et al., 1988). Similar to what tose), and wheat-germ agglutinin (specific for sialic
has been described in the cumulus matrix of the mouse acid/N-acetyl-D-glucosamine) also showed that these
(Eppig, 1979) and in cattle (Ball, 1982), hyaluronic acid lectins failed to label the hamster cumulus matrix
is probably also the major component of glycosamino- (Roux and Kan, 1990). Collectively, the present findglycans in the golden hamster cumulus matrix. This is ings as well as those from our previous studies indicate
evidenced by the absence or great reduction of gold that the hamster cumulus matrix is poor in glycoprolabeling in the cumulus matrix in all control experi- teins in general but contains copious hyaluronic acid.
ments and by the fact that pretreatment of oocyte-cuAlthough biochemical studies have shown that mammulus complexes with chondroitinase ABC did not pre- malian oocyte-cumulus complexes are capable of synvent their labeling by HUD VI-gold. Previous studies thesizing hyaluronic acid in vitro (Eppig, 1979; Ball,
in our laboratory using a monoclonal antibody against 1982) and a recent radioautographic study has sugan oviductal glycoprotein of high molecular weight gested that the cumulus cells and the oocyte in human
(Kan et al., 1988,1989a) and Helix pomatia lectin (spe- are the cellular sources that supply glycosaminoglycific for N-acetyl-D-galactosamine residues) (Kan et cans to the cumulus matrix (Tesarik and Kopecny,
al., 1989b) showed, respectively, the absence of the gly- 1986), the exact pathway of synthesis and secretion of
coprotein and the corresponding sugar residues in the hyaluronic acid and other glycosaminoglycans is not
LOCALIZATION O F HYALURONIC ACID
377
Fig. 5. Portion of a cumulus cell showing the vicinity of a Golgi
complex (Gol). Multivesicular body (mvb) and lysosome-like structures (arrowheads) are strongly reactive to hyaluronidase VI-gold labeling. The Golgi apparatus (Gol) and its peripheral vesicles also
show strong reactions. Gold particles in the nucleus (N) are localized
over peripheral heterochromatin and condensed chromatin. m, mitochondria. X 29,000.
Fig. 6. Transverse (a) and tangential (b) sections of lamellae in
cytoplasm of a superovulated hamster oocyte. a: Shows the concentric
orientation of the lamellae labeled by gold particles on their sides; b
The peripheral labeling pattern is evident in tangentially cut section,
which shows the interior crystalline lattice (asterisk) unlabeled by
hyaluronidase VI-gold. Gold particles are localized to the peripheries
of the lamellae (arrowheads). x 19,000.
clear. Recently, a study with the HUD-gold technique
(London0 and Bendayan, 1988) has localized the labeling to the rER membrane, plasma membrane, nuclei,
and basement membrane components in the pancreas,
duodenum, and kidney. The Golgi apparatus and its
associated vesicles of certain cell types were also reported to be labeled. Similarly, in the present study,
hyaluronic acid was also localized to the aforementioned structures and compartments. Presumably, the
synthesis of hyaluronic acid in the cumulus cells takes
place in the rER membrane with completion of its elongation in the Golgi apparatus. There is no firm evidence to date that hyaluronic acid is covalently linked
to protein (Chakrabarti and Park, 1980; Roden, 1980);
therefore, in the present study, it could not be deter-
mined whether the labeling detected on the cell surface
represents membrane proteins that carry hyaluronic
acid or if it is due merely to the general electrostatic
binding nature of glycosaminoglycans (Lindahl and
Hook, 1978). In this study, hyaluronic acid was detected in lysosome-like structures and multivesiclular
bodies, with more intense labeling over the latter. Internalization of cell-associated proteoglycans by receptor-mediated endocytosis has been reported (Fransson,
1987). Internalized proteoglycans are believed to appear first in endosomes (Hassel et al., 1986), which
then divert the products to lysosomes, the Golgi apparatus, or the nucleus (Farquhar, 1985; Ishihara et al.,
1987). The products may be recycled back to the cell
surface via the Golgi complex (Fransson, 1987). It ap-
378
F.W.K. KAN
Fig. 7. Control Lowicryl sections. a: Incubation of thin section with
hyaluronidase VI-gold in the presence of excess enzyme (hyaluronidase, 1 mgiml). The labelings previously seen over the cumulus matrix (CM), cytoplasmic and nuclear components of the cumulus cell
(CC), as well as the oocyte are abolished. P V perivitelline space.
x 9,500. b: The lamellae (arrows), the perivitelline space (PV),.and
the microvilli (arrowheads) are completely devoid of gold particles
when the specific substrate (hyaluronic acid, 1 mgiml) was added to
the incubation medium. x 17,500. c: Digestion of the thin section of
oocyte-cumulus complex with free hyaluronidase VI (0.5 mgiml, 6
hours, 37°C) in the presence of protease inhibitors prior to labeling
with hyaluronidase VI-gold also substantially reduces the labeling
previously seen over the cumulus matrix (CM). ZP, zona pellucida.
X 17,500.
pears that hyaluronic acid associated with the plasma
membrane of the cumulus cells could also be eventually internalized and degraded through the lysosomal
system in a manner similar to that described above. In
this case, free hyaluronic acid may be shed into the
extracellular space and incorporated into the cumulus
matrix or taken up by receptor-mediated endocytosis
(for a review, see Fransson, 1987). The receptor for hy-
LOCALIZATION OF HYALURONIC ACID
379
Fig. 8. Prior treatment of the oocyte-cumulus complexes with chondroitinase ABC does not affect
labeling of the cumulus matrix (CM) by hyaluronidase VI-gold. a: Cytoplasmic lamellae (arrows) are
intensely labeled; b: Condensed chromatin in the nucleus of a cumulus cell also are intensely labeled. ZP,
zona pellucida. (a) x 12,000, (b) x 9,500.
aluronate has been recently identified in adult Syrian
hamsters (Tarone et al., 1984; Underhill et al., 1987)
and localized to the basolateral surfaces of bronchial
and bronchiolar epithelium as well as to the surfaces of
pulmonary macrophages (Green et al., 1988). Alternatively, the high concentration of gold particles detected
in the multivesicular bodies of the cumulus cells could
be the result of post-translation regulation of produc-
380
F.W.K. KAN
tion and secretion of hyaluronic acid by intracellular
degradative mechanisms as suggested by several authors (Smith, 1979; Bienkowski, 1983; Nanci et al.,
1987).
Another interesting finding was the detection of hyaluronic acid in the nucleus, an observation corroborated with results obtained in other tissues using a
similar HUD-gold complex (London0 and Bendayan,
1988). Several biochemical studies have indicated the
involvement of glycosaminoglycans in nuclear functions (Stein et al., 1975; Bhavanandan and Davidson,
1975; for a review, see Lindahl and Hook, 1987). In
particular, heparin has been shown to induce dispersion of chromatin structure (Arnold et al., 1972; Cook
and Aikawa, 1973; Smith and Cook, 1977) and to stimulate the DNA template characteristic of chromatin
and nuclei during DNA synthesis (Kraemer and Coffey, 1970). Although hyaluronic acid has been identified as the major component of nuclear glycosaminoglycans in isolated rat liver nuclei (Furukawa and
Terayama, 1977), to date its function in the nucleus is
still unclear. Most recently, immunocytochemical studies using antibodies to protein epitopes also demonstrated the presence of hyaluronic acid in the cytoplasm and nuclei of certain brain cells during brain
development (Ripellino et al., 1989). The localization of
hyaluronic acid to the peripheral condensed chromatin
and to the condensed perinucleolar chromatin in the
cumulus cells suggests that this macromolecular component could be involved in transcriptional activities.
The exact role(s) of hyaluronic acid in the cumulus cells
remains to be elucidated.
The third major finding was the localization of hyaluronic acid associated with the superovulated oocyte.
As mentioned earlier, the zona pellucida was not labeled by HUD VI-gold, whereas labeling was detected
in the perivitelline space. A previous report on hamster
oocyte-cumulus complex using ruthenium red for staining hyaluronic acid claimed to have detected reaction
products in the upper third of the zona pellucida (Talbot, 1984). However, this staining property is likely
due to extensions of the cumulus matrix in the upper
porous region of the zona pellucida rather than to
staining of the zona pellucida itself. Such occurrence is
also found in the present study (shown in Fig. l),in
which labeled filamentous extensions of the cumulus
matrix are seen infiltrating the upper region of the
zona pellucida. Morphologically,the perivitelline space
of the superovulated oocytes is occupied by filamentous
materials, which are highly compact and appear to be
different from those of the zona pellucida. This marked
difference is best illustrated in Figure 7a,b. Previous
studies in our laboratory using a monoclonal antibody
(Kan et al., 1988,1989a) and lectins (Kan et al., 1989b;
Roux and Kan, 1990) demonstrated that the zona pellucida of the superovulated hamster oocytes is rich in
glycoproteins, which, however, are absent in the perivitelline space. In the present study, the detection of
hyaluronic acid in the latter suggests that in the superovulated oocyte there exists different structural
components that are unique to the zona pellucida and
the perivitelline space. Glycosaminoglycans, such as
hyaluronic acid, heparin, and chondroitin sulfate, are
known to stimulate acrosome reaction in the hamster
(Meizel and Turner, 1985) and rabbit (Lenz et al.,
1983). It has been suggested that one or more glycosaminoglycans that are present in the perivitelline
space may be involved in acting synergistically with
the zona to induce the acrosome reaction (Gwatkin,
1989).The cytochemical detection of hyaluronic acid in
the perivitelline space in the present study seems to fit
well into this scheme.
Another noteworthy finding is the labeling of cytoplasmic lamellae by HUD VI-gold complex inside the
oocyte. These lamellar structures were first described
by Weakley (1966) in the hamster. Study of the fixation
properties of these lamellar structures (Weakley, 1967)
and freeze-fracture studies carried out on hamster
oocytes (Suzuki and Yanagimachi, 1983; Koehler et al.,
1985)indicated that the lamellae are largely protein in
nature. Although the cytoplasmic lamellae are believed to be nutritive to embryo development (Nilsson,
1980), the exact chemical composition and functional
significance of these structures remains obscure. In the
present study, labeling by HUD VI-gold was observed
a t the peripheries of the lamellae. This is evidenced on
tangential section (Fig. 6b), which shows the absence of
gold labeling over the crystalline lattice of the tangentially cut lamellae. Due to the polyanionic nature of
glycosaminoglycans (Chakrabarti and Park, 1980), the
binding of hyaluronic acid to the lamellae is presumably a result of electrostatic interaction. Future ultrastructural studies aided by cytochemical probes will
likely shed light on the chemical composition and function of the cytoplasmic lamellae in relation to their
association with hyaluronic acid.
Mammalian sperm cells have to traverse the cumulus matrix and the zona pellucida before fertilization
can occur. The presence of copious hyaluronic acid in
the cumulus matrix, its absence in the zona pellucida,
and its reappearance in the perivitelline space collectively point to the unique nature of the existence of this
macromolecular component in the extracellular compartments within the oocyte-cumulus matrix. The localization of hyaluronic acid in different intracellular
compartments of the cumulus cells reveals the possible
intracellular traffic of this macromolecular component.
The detection of hyaluronic acid along the peripheries
of cytoplasmic lamellae has added new information to
these prominent but less well-characterized structures.
Based on these studies, I suggest that the hyaluronic
acid associated with the cumulus matrix originates
from the cumulus cells and that the hyaluronic acid
found in the perivitelline space comes from the oocyte.
Although the significance of this distribution and the
exact function of hyaluronic acid remain to be unraveled, it is hoped that the findings of this study will pave
the way for future investigations into the role of hyaluronic acid in sperm-cumulus-oocyte interaction,
which is so critical to successful fertilization.
ACKNOWLEDGMENTS
The author thanks Dr. Gilles Bleau for comments on
the manuscript, Ms. Christiane Rondeau, and Cecile
Venne for their expert technical assistance and Mr.
Jean Leveille for photographic work. Appreciation is
also extended to Johanne Chainey for clerical assistance. This investigation was supported by the Fonds
de la Recherche en Sante du Quebec (87 118) and
LOCALIZATION OF HYALURONIC ACID
CAFIR of Universite de Montreal. The author is a
scholar from the Medical Research Council of Canada.
381
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