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Spontaneous cortical granule release and alteration of zona pellucida properties during and after meiotic maturation of mouse oocytes.

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THE ANATOMICAL RECORD 237518426 (1993)
Spontaneous Cortical Granule Release and Alteration of
Zona Pellucida Properties During and After Meiotic Maturation
of Mouse Oocytes
Second Department of Anatomy, Toho University School of Medicine, Omori-Nishi, O h - k u ,
Tokyo 143 (A.O., K J . ) ; and Department of Obstetrics and Gynecology, Teikyo University
School of Medicine, University Hospital Mizonokuchi, Kanagawa 213 (T.N.), Japan
Exocytosis of cortical granules (CGs) and the concomitant
electron density changes of the zona pellucida (ZP)in the absence of sperm
penetration were investigated in mouse oocytes processed with tannic acid
containing fixation at various stages during and after maturation. After
fusion of the CG membrane with the plasma membrane, the CG contents
became very electron-dense, due to tannic acid. CG material is seen to be
made up of coarse granular structures which gradually change to fine
amorphous structures, which accumulate within the developing perivitelline space (PVS).
When the coarse CG material attaches to the ZP, small domains exhibiting higher electron density appeared, and the number of these domains
gradually increased. Release of CG was observed from metaphase I
through metaphase 11. In metaphase I to immediately after ovulation, the
higher electron density of ZP and CG release was restricted to the cortical
area overlying the meiotic spindle. Finally, the CG-free domain formed itself overlying the meiotic spindle as a result of CG release. However, in
oviductal ova, CG release additionally occurred in the hemisphere opposite
the spindle. At this stage the entire PVS was well developed and contained
numerous fine electron-dense materials. Moreover, the inner half of the ZP
increased in electron density as well. This change in electron density of the
ZP might be associated with released CG material.
These results suggest that the “partial cortical reaction” may play an
important role in conditioning the ZP prior to ZP reaction.
1993 Wiley-Liss, Inc.
Key words: Oocyte, Meiotic maturation, Cortical granule, Exocytosis,
Zona pellucida
In ovulated mature ova, exocytosis of cortical granules (CGs) takes place immediately after penetration
by sperm (Szollosi, 1967; Gulyas, 1980). The material
released from the CGs changes the properties of the
overlying zona pellucida (ZP).This change is called the
zona reaction (Braden et al., 1954). Changes in the ZP
andlor the ovum cell membrane are believed to play a
major role in the prevention of polyspermic fertilization (reviewed by Austin, 1961; Yanagimachi, 1977).
Recently, spontaneous exocytosis of the CGs has
been reported in immature ovarian oocytes after resumption of meiosis and in mature ova after ovulation
in several mammalian species, including the human
(Rousseau et al., 1977), mouse (Nicosia et al., 1977;
Okada et al., 1990; Ducibella et al., 1990), and hamster
(Okada et al., 1986). These studies also have revealed
that both maturing and mature ovulated ova possess a
CG-free or CG-poor domain in their cell cortices overlying the meiotic spindle (Nicosia et al., 1977; Gulyas,
1980). In the meta I1 of hamster ova, this domain is
formed as the result of CG release prior to fertilization
(Okada et al., 1986).
Several lines of evidence based on physiological and
biochemical studies indicate that the properties of ZP
are modified during oocyte maturation and sperm penetration of ZP (Iwamatsu and Chang, 1972; Oehninger
et al., 1991): the solubility of ZP by protease during in
vitro culture of follicular oocyte in serum-free medium
(Defelici and Shiracusa, 1982) and oviductal ova in
vivo (Aonuma et al., 1978), and the transition of ZP2 to
ZP2, in a serum-free medium (Ducibella et al., 1990).
Additionally, the only morphological changes to the ZP
are reported to be an increase in electron density of the
inner half of the ZP (Sathannanthan and Trounson,
Received December 19, 1991;accepted June 9,1993.
1982a; Familiari et al., 1992). However, it is still uncertain why andlor how the ZP is modified during maturation.
Standard electron microscopic fixation methods are
inadequate for staining difficult to observe glycoprotein structures (Sturgess et al., 1978) such as CG materials. On the other hand, ruthenium red (polycationic
inorganic dye) intensely stains CG released materials
(Gordon et al., 1975). However, ruthenium red does not
easily pass through the plasma membrane (Luft, 1971);
consequently, it is used primarily for staining of extracellular materials.
According to Maupin and Pollard (1983), tannic acid
staining in combination with fixation enhances electron density, both of intracellular structures (Anderson
et al., 1975; Mizuhira and Futaesaku, 1973) and extracellular structures of surface coats, etc. (Roubos and
van der Wal-Divendal, 1980).Tannic acid is considered
to serve as a mordanting agent of osmium-treated
structures. This preferential staining is particularly
evident in “exocytosing CG after fertilization” (Takeuchi and Takeuchi, 1985). Therefore, this staining technique facilitates the visualization of the premature CG
release process, both from qualitative and quantitative
In this study, we undertook to observe the behavior
of the CG in the ooplasm, as well as the releasing process of the CGs. We also investigated the interactions
of released CG materials and ZP, employing tannic
acid methods, in maturing oocytes and mature ova of
the mouse.
Collection of Oocytes and Ova
Mature female ICR strain mice, 8-15 weeks old,
were given an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (Teikoku Hormone
Mfg., Tokyo, Japan) followed 42-48 hr later by 5 IU
human chorionic gonadotropin (hCG; Teikoku Hormone Mfg., Tokyo, Japan). Four to 12 hr following hCG
injection, the ovaries were removed and placed in
dishes containing a modified Krebs-Ringer’s solution,
BWW medium (Biggers et al., 1971), containing 0.1%
bovine serum albumin (Fraction V, Sigma Chem., St.
Louis, MO). Maturing oocytes were recovered from the
ovaries by puncturing the antral follicles with a stainless steel needle under a dissecting microscope. Mature, ovulated ova were collected from 1) the ovarian
surface or bursae 13-14 hr after hCG injection when
ovulation was in progress and 2) the oviducts by cutting the wall of the ampulla with a pair of needles
between 14 and 17 hr after hCG injection.
Electron Microscopy
For thin sectioning, the oocytes and ova with cumulus cells were fixed for 1 hr a t 22°C with Karnovsky’s
paraformaldehyde-glutaraldehyde fixative containing
2% tannic acid (Takeuchi and Takeuchi, 1985). After
the specimens were washed with 0.1 M cacodylate
buffer (pH 7.2), they were left overnight a t 4°C in the
same buffer and then postfixed for 1 hr a t 4°C with
Dalton’s chrome-osmium fixative (pH 7.2). Fixed specimens were dehydrated in an ethanol series and embedded in Spurr’s resin. Thin sections were stained
with uranyl acetate and lead citrate.
Ovarian Oocytes at Metaphase I
All seven oocytes collected from the ovary 8-9 hr
after hCG injection exhibited a CG-free domain in their
cortices overlying the meiotic spindle and the beginning of a small PVS. Simultaneously, the CG area
could also be seen.
A CG-free domain, just above the chromosome (Ch)
area, and the initial PVS formation were observed (Fig.
l.A,B). The PVS formation area revealed a discontinuously narrow or absent PVS (8 hr after hCG injection)
(Fig. 1.B). The coarse granular materials attached to
the ZP exhibited higher electron density (Fig. lA, arrowheads). At the more developed stage (9 hr after hCG
injection), the cortical surface of the oocyte seemed to
form a valley-like structure with caverna (Fig. 2).
These areas of PVS contain high-electron-dense coarse
granular structures (arrows) and fine granular structures (Fig. 2). Additionally, the CG cavernous structure has high electron-dense remnants. These remnants were observed on the surface of the periphery of
the CG-free domain.
Release of dense material was not observed from the
processes of the corona cells and granulosa cells. The
process of CG release is shown in the following stages:
1. Approaching stage: Some CGs were observed migrating to the area immediately under the ooplasm
prior to membrane fusion (Fig. 3A, arrow, and B at
high magnification).
2. CG membrane fusion and opening stage: Just after membrane fusion occurred, the CG opens into the
PVS (arrowhead, Fig. 3 0 . Enhanced electron-dense
materials were observed from opened CGs by tannic
acid stain (Fig. 3C). Occasionally, the content of second
exocytosing granules (enhanced electron dense granule) was observed (Fig. 3C, arrow). Intraooplasmic intact CGs were only moderately stained with tannic acid
stain (Fig. 3D).
3. CG dispersing stage: The CGs were dispersed
within the PVS, and the cavernous structure was often
observed at the releasing area (Fig. 3D-GI. The high
electron density spot of the ZP was shown to be very
close to the released CG material (Fig. 3H, circled area,
and I). The material was not only found in the PVS but
also seemed to be produced by penetration of the CG
material into the overlying ZP (Fig. 31).
In the cortical area adjacent to the CG-free domain,
two types of CGs, differing in the electron density of
their contents, were observed (Fig. 35, arrow and arrowhead). In the hemisphere opposite the CG-free domain, on the other hand, there was no evidence of CG
release nor of CG material in the narrow PVS. Moreover, the narrow PVS was found to be clear (Fig. 3K).
cortical granule
first polar body
perivitelline space
meiotic spindle
zona pellucida
Fig. 1 (top). A, B Sections of ovarian oocytes at metaphase I, collected 8 hr after hCG injection. A: CG-free cortex near the chromosomes (Ch) and an adjacent cortical area with CGs (arrows), surrounded by the discontinuous perivitelline space (PVS) and overlying
ZP (ZP). Small electron dense regions (arrowheads) are observed in
the inner zone of the ZP. Scale bar: 1 pm. B: Part of a CG-free domain
over the chromosomes (Ch) under high magnification. Discontinuous
narrow perivitelline space or the complete absence of the perivitelline
space was observed. Scale bar: 0.5 pm.
Figs. 2.3.A-L Sections of ovarian oocytes at Meta I, collected 9 hr
after hCG injection.
Fig. 2 (bottom). The valley-like cortical surface is part of the CG-free
cortex. The released high-electron-dense CG material ( t ) contained
within the small PVS and ZP can be seen. Scale bar: 0.5 pm.
Fig. 3. A The CG domain and adjacent CG-free domain. CGs just
below the ooplasm are seen. Scale bar: 1 pm. B: One CG approaching
the ooplasm is seen under high magnification B. Scale bar: 1 pm. C:
Part of the CG-releasing domain and continuous PVS are seen. Soon
after CG membrane and ooplasm fusion, this membrane opened
mouth (V)and enhanced high-electron-dense material are observed
by tannic acid stain. Moreover, just below the released CG, the second
exocytosing granule is also observed ( t ). Scale bar: 0.5 pm. D Within
the caverna structure of releasing cortex. Granule-like high-electrondense materials (V)and another intact CG of moderately low electron
density (G+) are seen in the cortical surface of the oocyte. Scale bar: 0.5
pm. E-G: Released CG within PVS dispersed (AA),and the electron
high density of fine materials is seen. Scale bar: 0.5 pm.
Fig. 3. H, I: The recently released CG domain, the released CG
materials within PVS, and small high electron density of ZP and CG
free domain are observed. H: CG-free surface domain on left side and
just released CG materials within right side are visible. On the right
side of the ZP a small part showing increased electron density in the
circled area and a fine granule like structure are observed within the
PVS. Scale bar: 1 pm. I: Released CG material which is close to the
inner part of the ZP is seen. Under high magnification (in the circled
area H) only the small area of the internal ZP appears to have become
high in electron density (-1. Scale bar: 0.5 pm. J The part of CG
domain adjacent to the CG releasing domain is rich in CG. Here two
types of CG are seen, one of high electron density (A)and another one
of light electron density ( t ). Scale bar: 1 pm.
Fig. 3.K In the CG (arrow) domain and the overlying ZP in the hemisphere opposite the spindle no
high-electron-densematerial and no change of the inner part of the ZP are seen. Scale bar: 1 pm. L The
narrow PVS is clear; compare to the spindle pole of the CG-releasing domain (H,I). Under high magnification (L), no CG granule released materials or cortical caverna could be seen. Scale bar: 0.5 pm.
Even on higher magnification of the inner surface of
ZP, electron-dense structures were not observed (Fig.
Ovulated Ova at Metaphase II
Ovulated ova with the first polar body, collected from
the ovarian bursae 12-13 hr after hCG injection, exhibited a well developed CG-free cortex in the vicinity
of the meiotic spindle (Fig. 4A). In the cortex adjacent
to the CG-free domain, CG release was observed as a
burst of electron-dense CG material (Fig. 4A, arrowhead). The PVS was enlarged near the polar body and
fine and high electron-dense material accumulated in
it (Fig. 4A). A number of electron-dense spots appeared
within the ZP overlying the hemisphere containing the
spindle (Fig. 4A, B). The coarse granular materials released from the CGs were still observed frequently in
the PVS.
In the hemisphere opposite the spindle, neither CG
release nor electron-dense structural changes in the
PVS or ZP could be found a t this stage, and the PVS
formation was still small (not shown).
Unfertilized, Oviductal Ova Arrested at Metaphase II
In ova collected from the oviducts 17-19 hr after hCG
injection, the PVS was seen to be fully developed. At
this stage, CG release was finally observed from the
CG area in the hemisphere opposite the spindle. Fine,
amorphous materials were seen to accumulate within
the entire PVS. The inner half of the ZP, not only over
the CG-free domain (Fig. 5A) but also over other areas
of the ovum cortex, had a somewhat higher electron
density than the outer half (Fig. 5B, delimited by arrows). These observations are summarized in the diagram in Figure 6.
The fully mature (arrested a t metaphase 11) mouse
and hamster ova have a CG-free domain (Szollosi,
1967; Nicosia et al., 1977, Gulyas, 1980). In the hamster oocyte, the formation of this CG-free domain was
observed to be result of CG release (Okada et al., 1986).
In the mouse oocyte, use of a fluorescent microscopic
observation showed a decrease in the number of granules stained with Lens culinaris agglutinin during
maturation (Ducibella et al., 1990). However, CG release during meiotic development of the mouse oocyte
is still not clear. In the present study we found that
during maturation in the follicular and the post-ovulatory stage, the CG release is performed by a unique
mechanism, which occurs over a long period (6 hr or
more) in the absence of sperm stimulus.
After fertilization, it is well known that the ZP prevents polyspermic fertilization by releasing CG material. However, it is reported that artificial premature
CG release offers no prevention of sperm penetration,
Fig. 4. A, B Sections of just-ovulated ova at metaphase I1 with the
first polar body (FPB), collected 12-13 hr after hCG injection from
ovarian bursae or oviducts. Scale bar: 1 pm. A CG-free cortical domain and adjacent CG domain (upper right) near the meiotic spindle
(Sp). Electron-dense material (arrowhead) representing a burst of CG
material, is found on the surface of the CG domain. The PVS has
enlarged near the first polar body (FPB). The material in the PVS is
finer and denser than at metaphase I (cf. Fig. 1C). No CGs are found
within the first polar body. Note that a number of electron-dense spots
are distributed within the ZP. B: Part of the ZP in the same section as
that presented in Fig. 3A under higher magnification. The electrondense macula-like structures (spots) apparently represent densely
stained ZP components.
either ZP perletration or oocyte membrane fusion of
sperm (Wolf et al., 1979). At the Meta I stage, human
(Marrs et al., 1984;Trounson et al., 1982),mouse (Iwamatsu and Chang, 19721, and pig oocytes (Hunter,
1976) studies report that the ZP reveals polyspermic
penetration. Therefore, the effect of CG material on ZP
seems different between premature CG release and ZP
reaction. Why such a difference between polyspermic
and monospermic penetration of the ZP occurs is still
uncertain. The present study reveals two kinds of CGs,
in terms of the electron density of their contents (Fig.
35). Nicosia et al. (1977)and Ducibella et al. (1988)
have also demonstrated two kinds of CGs in the mouse
oocyte with different electron densities. The human,
monkey, hamster, and rabbit oocytes also have two
kinds of CGs (Gulyas, 1980).We speculate that allowing polyspermy during maturation of ova and the prevention of polyspermy after fertilization are due to differences in the materials contained in the two kinds of
CGs, as well as in the timing of their discharges. Further research is required to provide evidence for this
CG release after fertilization is known to alter the
ZP, which is called “ZP reaction” and “ZP hardening”
(reviewed by Austin, 1961;Yanngimachi, 1977).Prcvious morphological reports did not observe a direct correlation between CG materials and the ZP. Our present
study, using the tannic acid method, clearly showed
that the initial change of the high electron density of
ZP was observed on the Meta I spindle pole, very
closely located just above the CG-release material (Fig.
3A,B). Additionally, in more advanced stages, the high
electron density area of the ZP was always observed,
depending on the particular stage of meiosis, a t the site
where CG exocytosis was taking place simultaneously
(see diagram, Fig. 6).Finally, in oviductal ova, CG release occurred over almost the entire cell surface, and
the entire inner layer of the ZP had uniformly higher
electron density (Fig. 5A,B). A similar change in only
the inner part of the Z P has also been reported in unfertilized human ova (Sathananthan and Trounson,
1982a) and in mouse ova incubated in Con A (Longo,
After fertilization, it is known that the alteration of
ZP substance ZP2 to ZP2, (Bleil and Wassarman, 1981)
and an increased resistance to enzymatic digestion is
caused by the release of CG materials. The ZP hardening may result from cross-linking of tyrosine residues
Fig. 5. A, B: Sections of ovulated, unfertilized ova arrested a t metaphase 11, collected 19 hr after hCG injection (about 7 hr after ovulation). The further developed PVS contains fine amorphous materials.
Note that the inner half of the ZP (delimited by arrows) has higher
electron density than the outer half. Scale bar: 1 pm. A: CG-free
cortical domain above the meiotic spindle. The cell surface is comparatively smooth. B: Cortex of the hemisphere opposite from that of the
meiotic spindle. Numerous microvilli are densely distributed on the
cell surface.
Fig. 6. Correlation between cortical granule release (+) and conditioning of zona pellucida.
in the Z P substance induced by ovoperoxidase of CG 1982) and oviductal oocyte in vivo (Aonuma et al.,
origin (Schnell and Gulyas, 1980). On the other hand, 1978) but the reason for Z P hardening during maturaduring maturation a concomitant decrease in the num- tion is still not clear.
ber of CGs and conversion of ZP2 to ZP2, within serumThis study clearly shows a CG-free domain at Meta I
free medium have been observed in mouse oocytes us- owing to the CG release in mouse oocyte. The CG-free
ing electrophoretic methods (Ducibella et al., 1990). domain is formed again at Meta I1 by CG release. It
Also during maturation, a gradual increase in sponta- was reported that in the mature mouse oocyte, 40% of
neous Z P hardening was reported in the mouse oocyte the cortex is occupied by the CG-free domain (Ducibella
within serum-free medium (DeFelici and Siracusa, et al., 1988).Additionally, after the second CG-free do-
main formation at Meta 11, another CG release was
observed from the CG domain in the mature mouse
oocyte. Therefore, a number of CGs are released during
maturation. Such released CG material plays a positive role or roles in the modification of the overlying
structure, the ZP. The present observation shows CG
release and gradual enhanced electron density of the
inner layer of ZP which may have caused spontaneous
ZP hardening. It was reported that, in monospermic
fertilization of human oocytes, the effective block of
polyspermy seems to occur in the inner half of the Z P
(Sathananthan and Trounson, 1982b), since supplementary-reacted sperm were rarely found to penetrate
this region (Soupart and Morgenstern, 1973).
Therefore, the alteration of the inner layer of the Z P
may play an important role in maturation of ZP. Only
strong, active sperm may pass through the inner layer
of the ZP. After fertilization, ZP reaction immediately
occurs, with the ZP being previously conditioned by
partially premature CG release.
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