MICROSCOPY RESEARCH AND TECHNIQUE 61:349 –357 (2003)
Perivitelline Space: Does It Play a Role in Blocking
Polyspermy in Mammals?
P. TALBOT*
AND
PRAMILA DANDEKAR
Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521
KEY WORDS
fertilization; oocytes; cortical granules; extracellular matrix
ABSTRACT
The perivitelline space of mammalian oocytes changes in size and composition
during preimplantation development. Often overlooked in the past, this space contains a hyaluronan-rich extracellular matrix prior to fertilization and a cortical granule envelope following release
of the cortical granules at fertilization. The hyaluronan-containing matrix of unfertilized oocytes is
well developed in some species such as opossums and humans but is scant in rodents including the
hamster and mouse. The significance of the hyaluronan-rich matrix, which attaches to the plasma
membrane of the oocytes, is not fully understood. However, hyaluronan, which can inhibit membrane fusion, is present in the perivitelline space (PVS) of unfertilized oocytes and must be
negotiated by the fertilizing sperm. Following fertilization, the cortical granule envelope forms as
the cortical granules disperse, thereby causing the PVS to increase significantly in size. Calcium is
important in the dispersion of the cortical granules following exocytosis. Once formed, the cortical
granule envelope in some species is about the same thickness as the zona pellucida, but it is not
readily visualized unless it is stained with fluorescent probes or examined ultrastructurally after
following stabilization with ruthenium red. The envelope contains proteins that remain in the PVS
until the time of blastocyst hatching. Although little work has been done on the functions of the
cortical granule envelope, several studies are consistent with the idea that it plays a role in blocking
polyspermy. While nicotine increases polyspermy in sea urchins, its effects on polyspermy in
human smokers have not been characterized, but could be addressed in human in vitro fertilization
labs. Microsc. Res. Tech. 61:349 –357, 2003. © 2003 Wiley-Liss, Inc.
WHAT IS THE PERIVITELLINE SPACE?
The perivitelline space (PVS) is a “space” between
the surface of the oocyte or more specifically the oolemma and the zona pellucida, an extracellular matrix
synthesized by the oocyte. The term space is a misnomer as it implies something that is empty and perhaps
uninteresting. In fact, the PVS has contents that
change during development and that appear to play
various roles before, during, and after fertilization. The
purpose of this review will be to discuss what is known
about the PVS of mammals before and after fertilization and to examine the evidence that the contents of
the PVS play a role in blocking polyspermy.
SIZE OF THE PVS
The PVS varies considerably in size at different
times in development. The PVS around germinal vesicle containing oocytes is relatively small and difficult to
visualize with light microscopy (Fig. 1A). After extrusion of the first polar body, the PVS becomes asymmetrical and enlarged around the first polar body, and it
retains this appearance at the time of fertilization (Fig.
1B). After fertilization, the PVS remains asymmetrical
and large around the polar bodies (Fig. 1C). As cleavage occurs, the PVS takes on a different shape as it
follows the contours of the blastomeres (not shown).
FORMATION OF THE PVS
The PVS forms during oogenesis. Resting oocytes
(10 –15 ␮m) lack a zona pellucida. When oocytes in
©
2003 WILEY-LISS, INC.
follicles begin to grow, the proteins ZP1, ZP2, and ZP3
are synthesized by the oocytes, secreted, and assembled extracellularly to form the zona pellucida (Flechon
et al., 1984; Liang and Dean, 1993; Shimizu et al.,
1983). Although the developing zona is initially made
up of discontinuous clumps of zona matrix, these
clumps eventually coalesce forming a complete encasement around the oocyte (Zuccotti et al., 1991). Formation of the complete zona defines the area known as the
PVS. The PVS exists as long as the zona exists, which
is until the time of blastocyst hatching in the uterus
just prior to implantation (Yanagimachi, 1994).
CONTENTS OF THE PVS BEFORE
FERTILIZATION
The PVS surrounding follicular oocytes is small (Fig.
1A). The zona pellucida has been shown to be quite
permeable, even to relatively large molecules (Hastings
et al., 1972; Sellens and Jenkinson, 1975) and virus
particles (Gwatkin, 1967). The PVS around follicular
oocytes would thus contain follicular fluid that permeates through the zona from the fluid accumulated in
*Correspondence to: P. Talbot, Department of Cell Biology and Neuroscience,
University of California, Riverside, CA 92521. E-mail: Talbot@citrus.ucr.edu
Received 20 August 2002; accepted in revised form 20 October 2002.
Grant sponsor: Tobacco Related Disease Research Program of California;
Grant sponsor: NIH.
DOI 10.1002/jemt.10348
Published online in Wiley InterScience (www.interscience.wiley.com).
350
P. TALBOT AND P. DANDEKAR
the surrounding antrum of the follicle. This fluid is
very similar to serum (Collins et al., 1997). It is not
known if the zona filters the follicular fluid allowing
only certain components to pass into the PVS.
Prior to ovulation and in response to luteinizing hormone, the cumulus cells undergo expansion by secreting and assembling an extracellular matrix that is rich
in hyaluronic acid (Phillips and Dekel, 1982; Zhuo and
Kimata, 2001). While this matrix has been reported to
contain a number of different proteins and glycosaminoglycans (Zhuo and Kimata, 2001), hyaluronan covalently bonded to inter-alpha trypsin inhibitor appears to be the major component of the matrix (Chen et
al., 1994, 1996; Hess et al., 1999). CD-44, a hyaluronan
receptor has recently been reported on cumulus cells
(Campbell et al., 1995; Kimura et al., 2002; Ohta et al.,
1999) and by binding hyaluronan may hold the assembled matrix within the oocyte cumulus complex. The
secretion of this matrix causes the cumulus cells to
expand apart from each other. The cumulus matrix of
unfertilized mammalian oocytes is not readily seen
using light microscopy unless methods to stain it are
employed. When oocyte cumulus complexes are fixed
for microscopy in the presence of ruthenium red, the
ECM between the cumulus cells is composed of electron
dense granules and filaments (Talbot, 1984; Talbot and
DiCarlantonio, 1984a). The granules are sensitive to
trypsin while the filaments are digested by hyaluronidase (Talbot and DiCarlantonio, 1984b). Thin projections of the cumulus cells adjacent to the zona pellucida
pass through the zona and attach via gap junctions to
the surface of the oocyte (Anderson and Albertini,
1976). These projections apparently also secrete the
same extracellular matrix that is present between cumulus cells into the PVS (Fig. 2A–C). Similar granules
and filaments are present in the PVS of unfertilized
mammalian oocytes after cumulus cell expansion, and
both filaments and granules appear to attach to the
oolemma (Dandekar et al., 1992, 1995; Dandekar and
Talbot, 1992). Hyaluronan has also been localized ultrastructurally within the PVS and on the surface of
the hamster oolemma using hyaluronidase conjugated
to gold (Kan, 1990), consistent with the data obtained
using ruthenium red. This granular filamentous matrix has been demonstrated in the PVS of rodents such
as the hamster and mouse where it is relatively sparse
(Dandekar and Talbot, 1992), in humans (Fig. 2A) and
pigs where it is more abundant (Dandekar et al., 1992;
Talbot, 1985), and in marsupials (Fig. 2B) and rabbits
(Fig. 2C) where it is particularly well developed (Breed
and Leigh, 1988; Dandekar et al., 1995; Talbot and
DiCarlantonio, 1984b). Therefore, unfertilized oocytes
of eutherian mammals and marsupials are characterized by a relatively small PVS that contains hyaluronan and probably the other proteins found in the matrix between cumulus cells.
The content of the PVS is modified after passage of
the oocyte cumulus complex into the oviduct where the
Fig. 1. Light micrographs showing the PVS (arrow) around a germinal vesicle stage oocyte (A), mature unfertilized oocyte (B), and a
recently fertilized oocyte (C). The space is initially small, but becomes
asymmetric and increases around the polar body after its extrusion.
POLYSPERMY AND THE PVS
351
thesize and secrete oviduct secretory glycoprotein
(OGP) in their non-ciliated cells (Buhi, 2002). This
protein has been referred to by various names including OSP, EAP, EGP, oviductin, MUC-9, and GP215,
and it is synthesized only by the oviduct (Buhi, 2002).
A number of studies have shown that OGP passes into
the zona pellucida and PVS and that it accumulates in
both of these regions prior to fertilization (Buhi et al.,
2000). It is interesting that passage of OGP through
the zona may be mainly one-directional. In mice, OGP
(GP215) appears to be selectively sequestered in the
PVS (Kapur and Johnson, 1986). For example, nonimmune secondary antibodies used as controls gain
access to the PVS but are readily removed by brief
washes in antibody free medium (Kapur and Johnson,
1986). Moreover, mouse serum albumin can readily
penetrate the zona and is retained by the zona after
removal from the oocyte, but albumin is not found
in the PVS of zona intact oocytes, even though albumin (⬃66 kDa) is a much smaller protein than OGP
(⬃215 kDa) (Kapur and Johnson, 1986). OGP, however, once in the PVS is retained there and can be
demonstrated using immunocytochemical methods.
These data suggest that entry and retention of some
proteins within the PVS is selective. The granular filamentous matrix in the PVS space of unfertilized oocytes is clearly retained as is the oviductal OGP. OGP
has been suggested to play numerous roles in fertilization. In the PVS, it may aid in fertilization by increasing fertilization rate and decreasing polyspermy
(Kouba et al., 2000), although this latter point has not
been confirmed in all studies (McCauley et al., 2001).
Fig. 2. Transmission electron micrographs showing the granulefilament matrix in the perivitelline space of unfertilized human (A),
opossum (B), and rabbit (C) oocytes. The matrix is particularly well
developed in the rabbit PVS. ZP ⫽ zona pellucida
zona is bathed in oviductal fluid. All mammalian oviducts that have been examined, except possibly rats
(Arias et al., 1994) and mares (Buhi et al., 1996), syn-
WHAT IS THE SIGNIFICANCE OF THE
MATRIX IN THE PVS OF UNFERTILIZED
OOCYTES?
It is not known yet if the granular-filamentous matrix in the PVS of unfertilized oocytes plays any role in
oogenesis or fertilization. The matrix is rich in hyaluronan, which would tend to draw water into the space.
Indeed the size of the PVS appears to be related to the
amount of granular-filamentous matrix present. For
example, in opossums, which have an abundant matrix
in the PVS, the space before fertilization is relatively
large (Talbot and DiCarlantonio, 1984b), while in rodents that have a very modest matrix the space is small
(Dandekar and Talbot, 1992). The size of the space
before fertilization could affect how the fertilizing
sperm approaches the oolemma. In mammals, gamete
membrane fusion occurs between the sperm’s plasma
membrane overlying the equatorial segment of the acrosome and the tips of the oocyte microvilli (Bedford
and Cooper, 1978; Talbot and Chacon, 1982). For example, in rodents a sperm passing through the zona
and entering the PVS would tend to have its head
forced sideways so that the fusogenic part of the
sperm’s plasma membrane contacted the tips of the
oocyte microvilli, thereby facilitating fusion (Barros
and Franklin, 1968; Yanagimachi and Noda, 1970).
Little work has been done to determine what effect
hyaluronan in the PVS would have on gamete membrane fusion. In ultrastructural studies, hyaluronan
appears to be anchored to the oolemma as well as
present in the PVS (Dandekar et al., 1992, 1992; Kan,
1990). Somatic cell fusion can be inhibited by hyaluro-
352
P. TALBOT AND P. DANDEKAR
nan (Kujawa et al., 1986), and it is possible that the
presence of hyaluronan in the PVS and on the oolemma
could impede gamete membrane fusion. Even after acrosome loss, sperm carry substantial amounts of hyaluronidase that become redistributed to the inner acrosomal membrane (Cowan et al., 1991, 1986) where it
could readily encounter hyaluronan in the PVS. Thus,
it is plausible that sperm hyaluronidase functions in
degrading hyaluronan in the PVS and on the oolemma,
thereby facilitating gamete membrane fusion. This
could be particularly important in species having a
well-developed hyaluronan-containing matrix in the
PVS. It is interesting that during conventional in vitro
fertilization of porcine oocytes, exogenous hyaluronan
increased monospermy (Suzuki et al., 2000), which
could be due to impeding gamete membrane fusion. If
the cortical reaction releases an inhibitor(s) of hyaluronidase, then the presence of undegraded hyaluronan in the PVS and on the oolemma could block fusion
of additional sperm. This idea, however, has not yet
been investigated experimentally, and it is complicated
by the observation that mammalian sperm appear to
have more than one form of hyaluronidase (Baba et al.,
2002).
THE PVS AFTER FERTILIZATION
After fertilization, the PVS of rodents increases significantly in size (Fig. 1B). Fertilization initiates the
cortical reaction resulting in release of the cortical
granule contents into the PVS (Cherr and Ducibella,
1990; Hoodbhoy and Talbot, 1994). Some contents are
thought to diffuse through the zona pellucida and set
up the zona reaction as a block to polyspermy (Cherr
and Ducibella, 1990; Cran and Esper, 1990). Several
proteinases have been identified that are thought to
fulfill this role by modifying the zona (Gwatkin et al.,
1973) or more specifically the zona protein ZP2 (Moller
and Wassarman, 1989) and preventing further binding
of sperm to the zona. In addition, N-acetylglucosaminidase is present in mouse cortical granules and has been
shown experimentally to cause the loss in sperm-binding activity leading to the zona block to polyspermy
(Miller et al., 1993). Finally, a 32-kD protein was recently shown to be a cortical granule protein. The function of this protein is not yet established; however, in in
vitro experiments, a monoclonal antibody to this protein did not affect fertilization or polyspermy (Gross et
al., 2000).
It has also become apparent from electron microscopic and confocal scanning laser microscopic examination of fertilized rodent and human oocytes that not
all cortical granule components diffuse into the zona
pellucida. Some of the cortical granule contents dehisce
and remain in the PVS to form a new extracellular
matrix, referred to as the cortical granule envelope
(Fig. 3A–C) (Dandekar and Talbot, 1992). At the TEM
level, this envelope is made of numerous electron dense
granules, distinct in size and texture from those found
around unfertilized oocytes (Dandekar et al., 1992,
1995; Dandekar and Talbot, 1992). Initially upon release, the granule contents remain aggregated (Fig.
3A) even though they are no longer surrounded by a
membrane. The granules eventually disperse to fill the
PVS (Fig. 3B), and the thickness of the cortical granule
envelope can be quite substantial (Fig. 3B,C). In the
Fig. 3. Transmission electron micrographs showing the cortical
granule envelope in the PVS of fertilized human (A), mouse (B), and
opossum oocytes (C). In the human, cortical granules have been
exocytosed and are in the process of dispersing (arrows). In the mouse,
the granules have already dispersed and formed the envelope (arrows). In the opossum, the envelope (*) is fully formed beyond the tips
of the microvilli. ZP ⫽ zona pellucida
POLYSPERMY AND THE PVS
353
marsupial for example, it is as thick as the zona itself
(Fig. 3C). The cortical granule envelope remains in the
PVS throughout preimplantation development and is
lost only after blastocysts hatch from the zona pellucida (Talbot and DiCarlantonio, 1984a).
COMPOSITION OF THE CORTICAL
GRANULE ENVELOPE
The cortical granule envelope has not yet been isolated from mammalian oocytes and analyzed biochemically. However, it has been shown that the envelope is
stabilized by ruthenium red, a polycation (Dandekar et
al., 1992, 1995; Dandekar and Talbot, 1992), suggesting that the envelope is negatively charged probably
due to glycosylation. Its charge might preclude diffusion of the envelope molecules through the zona,
thereby retaining it in the PVS throughout preimplantation development. The idea that the envelope is rich
in carbohydrate is supported by the observation that
lectins such as ConA and LCA bind to the envelope of
fertilized hamster oocytes (Hoodbhoy and Talbot,
2001). Since the same lectins bind the cortical granules
of unfertilized oocytes but do not bind the PVS of unfertilized oocytes recovered from the oviduct, it is reasonable to conclude that the envelope originates from
glycosylated molecules released from the cortical granules during the cortical reaction. An antibody, ABL2,
that recognizes the cortical granules of unfertilized
mouse and hamster oocytes also binds to material in
the PVS and on the oolemma of fertilized hamster
oocytes (Fig. 4A,B) (Hoodbhoy et al., 2001). Binding of
ABL2 to PVS material and to the surface of dividing
blastomeres continues until the time of blastocyst
hatching. ABL2 recognizes two bands of 56 and 62 kDa
on Western blots of unfertilized oocytes. These bands
are decreased in amount in zona free fertilized oocytes
but equal in amount to unfertilized oocytes when fertilized oocytes are examined zona intact. ABL2 also
labels cortical granules of rat, pig and bovine oocytes
and it reacts with bands at 56 and 62 kDa on Western
blots of these species. The relationship between these
two proteins is not yet known, but the immunocytochemical and biochemical observations with the ABL2
antibody are consistent with p62/56 being components
of the cortical granule envelope. To date, these are the
only proteins that have been identified as putative
cortical granule envelope components. Because some
ABL2 antibody binds to the oolemma after fertilization,
it is possible that one protein is present in the envelope
while the other is located on or in the oolemma. In
mice, new synthesis of ABL2 binding antigens has been
reported after fertilization beginning at about the twocell stage with greatest synthesis at the eight-cell stage
(Johnson and Calarco, 1980; Polak-Charcon et al.,
1985). Likewise in hamsters, greater labeling was seen
in the PVS space and on the blastomere surface of
eight-cell embryos than at the one-cell stage (Hoodbhoy
et al., 2001). These observations suggest that the cortical granule envelop material recognized by the ABL2
antibody is released and assembled at fertilization but
that it is replenished and its amount is augmented
during preimplantation development. p62/p56 have not
yet been identified biochemically; however, interestingly, antibodies to sea urchin hyalin also react with
the cortical granules of unfertilized hamster oocytes
Fig. 4. Confocal scanning laser micrographs of an unfertilized
hamster oocyte (A) and 2-cell preimplantation embryo (B) labeled
with the ABL2 antibody. The cortical granules are labeled in the
unfertilized oocyte while the cortical granule envelop in the PVS
appears to be labeled in the 2-cell stage embryo. Micrographs provided courtesy of Dr. Tanya Hoodbhoy.
and with the cortical granule envelope of fertilized
oocytes and preimplantation embryos (Hoodbhoy et al.,
2000). On Western blots, the anti-hyalin antibody also
recognizes p62/p56. Hyalin is a complex protein that is
released into the PVS of sea urchin oocytes at fertilization, and plays a role in normal development and
morphogenesis of sea urchins (Adelson and Humphreys, 1988; Fink and McClay, 1985; McClay and
Fink, 1982). Mechanical removal of hyalin from sea
urchin oocytes retards their growth. However, urchins
can synthesis new hyalin that, when released, enables
development to continue (Citkowitz, 1971). The ABL2
antigens p62/p58 are much smaller than sea urchin
hyalin, but the immunological data suggest that hyalin
and the ABL2 antigens share at least one epitope.
There is not currently evidence that the ABL2 antigens
block polyspermy. When injected in vivo into the oviduct at various times before fertilization, the ABL2
antibody did not increase the incidence of polyspermy
(Hoodbhoy et al., 2001). However, this could be a null,
not negative, result as we do not know that the ABL2
antibodies are function blocking with respect to
polyspermy, and it is possible that other cortical granule components blocked polyspermy even though the
354
P. TALBOT AND P. DANDEKAR
ABL2 antigens were inactivated by antibodies. Further
work will be required to determine if p62/56 do play a
role in blocking polyspermy.
ASSEMBLY OF THE CORTICAL
GRANULE ENVELOPE
Little is known about the assembly of the cortical
granule envelope. When cortical granules undergo exocytosis, their contents are released into the PVS by
exocytosis (Dandekar et al., 1992; Dandekar and Talbot, 1992; Szollosi, 1967). The contents are easily identifiable as they remain transiently aggregated and
form an electron dense clump about the size of the
cortical granules inside oocytes. However, the granules
in the PVS do appear to be slightly expanded, apparently due to partial dispersion of their contents. Although assembly has not been studied in detail beyond
ultrastructural observation, the clumped material appears to eventually disperse to form an envelope that
completely surrounds the fertilized oocyte (Dandekar
et al., 1992, 1995; Dandekar and Talbot, 1992). The
granules of the undispersed contents and the dispersed
contents look similar. In an interesting study, Cran
and Cheng (1986) observed that the dispersion of cortical granules in the PVS of pig oocytes following ionophore-induced exocytosis was related to the amount of
calcium in the culture medium. At 1.8 mM calcium,
exocytosis failed to occur. At 4.72 mM calcium, cortical
granules were released but did not disperse, and at
7.64 mM calcium, released granules dispersed into the
PVS. Thus, calcium appears to be important in dispersion of the cortical granule material, which would be
the initial step in enabling the cortical granule envelop
to assemble.
DOES THE CORTICAL GRANULE ENVELOPE
BLOCK POLYSPERMY?
There are several mechanisms by which the cortical
granule envelope could block polyspermy. After fertilization, the PVS enlarges significantly with the formation of the cortical granule envelope (Talbot and DiCarlantonio, 1984a). In unfertilized oocytes, the PVS is
small, and sperm are likely to come in contact with the
oolemma upon entering the PVS. The enlarged PVS of
the fertilized oocyte could pose an orientation problem
for sperm after penetrating the zona. When the PVS is
large, it may be more difficult for the side of the sperm
head, where the fusogenic portion of the plasma membrane resides, to engage and bind to the microvilli of
the oolemma. Thus bringing the gametes into proper
alignment for fusion may be less likely to occur in
fertilized oocytes with a large PVS than in unfertilized
oocytes with a smaller PVS where the sperm head
would be more likely to encounter the oolemma.
It is well established that some components in mammalian cortical granules can enter the zona pellucida
and modify it to prevent polyspermy (Barros and
Yanagimachi, 1971, 1972; Gwatkin et al., 1973; Wolf
and Hamada, 1977). While the zona reaction clearly
appears to be a major block to polyspermy in many
mammals (Cherr and Ducibella, 1990; Yanagimachi,
1994), the possibility that the cortical granule envelope
provides an additional block should be considered.
When more than one sperm enters the PVS simultaneously, the only remaining sites for a polyspermy
block are in the PVS itself or at the oolemma. The
cortical granule envelope itself could provide a direct
physical block to supernumerary sperm in the PVS and
prevent them from binding to the oolemma. The envelope might also contain molecules that directly affect
sperm motility or fusability after entering the PVS.
Because the cortical granule envelope is not usually
investigated directly in studies of fertilization, we
know very little about the role it plays in blocking
polyspermy. Nevertheless, some experiments have
been done that suggest the envelope functions in blocking polyspermy. In pig oocytes undergoing in vivo fertilization, cortical granule contents disperse in the PVS
following fertilization (Cran and Cheng, 1986). However, following in vitro fertilization, the granule contents do not all disperse properly and remain as aggregates near the plasma membrane. As discussed previously, proper dispersion of granules contents depends
on having sufficient levels of calcium in the culture
medium (Cran and Cheng, 1986). In vitro fertilization
in pigs is also accompanied by high levels of
polyspermy that could be due to failure of cortical granules to disperse. Their contents would, therefore, not be
able to interact with the zona or to form the cortical
granule envelope thereby setting up blocks to
polyspermy. The idea that the cortical granule envelope functions in blocking polyspermy is further supported by the observation that human oocytes that
undergo in vitro fertilization (IVF) are often polyspermic and that such oocytes often show a large number of
undispersed cortical granules in their PVS (Sathananthan and Trounson, 1982). These are granules that
have undergone exocytosis but whose contents have
not dispersed to form a cortical granule envelope. We
have often observed a similar failure of the cortical
granule contents to disperse in in vitro fertilized human oocytes (Dandekar et al., 1992). Although these
oocytes were not checked for polyspermy, it has been
reported by a number of labs that polyspermy is a
complication of human IVF (Diamond et al., 1985;
Soupart and Strong, 1975). In the pig studies, the failure of the cortical granule contents to disperse was
related to too little calcium in the culture medium
(Cran and Cheng, 1986). If a similar condition exists in
human IVF culture media, polyspermy might be favored over monospermy because of incomplete formation of the cortical granule envelope and/or failure of
the zona reaction to occur.
Some interesting data relating to polyspermy and
the PVS have been obtained from human oocytes undergoing SUZI (subzonal insemination) (Tesarik and
Mendoza, 1994). When acrosome reacted sperm were
injected into the PVS of aged human oocytes, very few
sperm were able to fuse with the oolemma. This could
be due to a polyspermy preventing mechanism at the
oolemma. However, sperm readily penetrate human
oocytes if the zona is removed (Soupart and Strong,
1975; Tesarik, 1989). Unfortunately, the cortical granule status of the oocytes in the SUZI study was not
known, but a partial or complete cortical reaction could
have occurred as the oocytes were aged. These data
with human oocytes subjected to SUZI would be consistent with the idea that the cortical granule envelope
was at least partially formed in aged oocytes and prevented sperm fusion with the oolemma. Removal of the
POLYSPERMY AND THE PVS
zona results in dispersion of the cortical granule envelope (Talbot and DiCarlantonio, 1984a), which could
explain why zona free oocytes readily permit sperm
fusion (Soupart and Strong, 1975; Tesarik, 1989). It is
also important to note that the oolemma of hamsters
does not lose its ability to fuse with sperm until the
8-cell stage (Usui and Yanagimachi, 1976; Zuccotti et
al., 1991), suggesting that if a block to polyspermy
exists in hamsters after passage through the zona, the
block would be in the PVS.
Unlike many other mammals, rabbits do not appear
to have a strong block to polyspermy at the zona level
(Austin, 1961; Braden et al., 1954). Numerous sperm
can enter the PVS during fertilization yet oocytes remain monospermic. This suggests that rabbits have a
powerful block to polyspermy at the level of the oolemma and indeed the charge density of the rabbit
oolemma, as assessed by labeling with charged ferric
colloid particles, increases after fertilization (Cooper
and Bedford, 1971). However, a block to polyspermy in
rabbits could likewise indicate a strong block at the
level of the cortical granule envelope. In an interesting
study on rabbits, McCulloh et al. (1987) found that
zona enclosed oocytes were better able to prevent
polyspermy than zona free oocytes, even though there
is no block to polyspermy at the level of the zona in
rabbits. This could be due to the presence of the cortical
granule envelope in the PVS of zona intact oocytes.
This envelope would be lost after zona removal thereby
making the oocytes more vulnerable to polyspermy. It
would be interesting to compare the envelope of rabbits
and other mammals to determine if a block to
polyspermy exists at this level.
Further experimental evidence for the existence of a
PVS block to polyspermy has come from a re-insemination study using mice (Maluchnik and Borsuk,
1994). Fertilized oocytes freed of the zona before extrusion of the second polar body were capable of fusing
with sperm. However, these same oocytes had not been
able to bind motile sperm in the PVS prior to zona
removal. These data support the idea that a block to
polyspermy exists at the level of the PVS. Supplementary motile sperm do not bind to the oolemma; however, sperm can fuse with the oolemma if the zona and
the cortical granule envelope are removed. While these
data identify a block to polyspermy at the level of the
PVS, they do not identify what in the PVS does the
blocking. The major component of the PVS after fertilization is the cortical granule envelope, which would
have been removed in the re-insemination experiments. These data support the idea that the cortical
granule envelope does play a role in blocking
polyspermy by preventing supplementary sperm from
binding to the oolemma.
While the cortical granule envelope appears to play a
role in blocking polyspermy when more than one sperm
gains access to the PVS, it is not yet know if p62/p56
actually perform this function or if other envelope components block polyspermy. In vivo experiments in
which the ABL2 antibody or an antibody to sea urchin
hyalin were injected into the ampulla of hamster oviducts prior to fertilization did not result in an increased incidence of polyspermy (Hoodbhoy et al., 2000,
2001). However, this could be a null result as it is
possible that p62/p56 were neutralized by the antibody
355
but backed up by another protein(s) in the cortical
granule envelope, or it is possible that these antibodies
were not capable of blocking this putative function of
p62/p56, or that other proteins in the cortical granule
envelope block polyspermy. Additional studies will
need to be done to establish if p62/p56 do have a role in
blocking polyspermy.
DOES SMOKING AFFECT BLOCKS
TO POLYSPERMY
It is well established that smokers have an increased
incidence of infertility and that there may be multiple
mechanisms that account for this (Phipps et al., 1987;
Stillman et al., 1986). Moreover, numerous studies
done in human in vitro fertilization labs generally confirm that smoking is detrimental to reproductive processes (Klonoff-Cohen et al., 2001). Cigarette smoke
contains over 4,000 chemicals (EPA, 1992), many of
which are recognized toxicants and some of which are
only now being identified as potentially dangerous (Ji
et al., 2002). It is probable that many of the toxicants in
cigarette smoke gain access to oocytes. Cotinine, a major metabolite of nicotine, has been reported to be detectable at high levels in the follicular fluid of both
active and passive smokers (Zenzes et al., 1996). Nicotine has been reported to increase the incidence of
polyspermy in sea urchin oocytes (Longo and Anderson, 1970; Rothschild, 1954). The mechanism for this
action is not yet established; however, sea urchin oocytes do have nicotinic acetylcholine receptors in their
plasma membranes (Ivonnet and Chambers, 1997),
suggesting nicotine could act directly on the plasma
membrane to affect polyspermy. In a structural study
of sea urchin eggs undergoing fertilization while exposed to nicotine, it was observed that the cortical
reaction and formation of the activation calyx occurs
more quickly in nicotine treated oocytes than in controls (Longo and Anderson, 1970). It is not clear if this
effect was due to multiple sperm fusing with the oocytes thereby initiating the reaction at several places
or if the acceleration of the cortical reaction and calyx
formation is a direct consequence of nicotine. The effects of nicotine on polyspermy in mammalian oocytes
are not well characterized. However, since cotinine, a
major metabolite of nicotine, has been reported to accumulate in follicular fluid (Zenzes et al., 1996) and
could thereby gain access to the PVS, the possibility
that chemicals in cigarette smoke affect polyspermy
should be investigated further. It would be interesting,
for example, to determine if smokers undergoing in
vitro fertilization have a higher incidence of
polyspermy than non-smokers.
SUMMARY AND CONCLUSIONS
The PVS of mammalian oocytes is a dynamic zone
that undergoes changes in size and composition during
development. An extracellular matrix first appears in
the PVS of follicular oocytes after the LH surge, and
later the PVS is modified by the addition of oviduct
secretory glycoprotein. The effects of the matrix prior
to fertilization have not been investigated but could
affect fertilization, as hyaluronan, a molecule that can
inhibit fusion, is present in the PVS of unfertilized
oocytes. Following fertilization, a new matrix, the cortical granule envelope, appears in the PVS. In oocytes
356
P. TALBOT AND P. DANDEKAR
where the cortical granule contents do not completely
disperse to form this matrix, polyspermy levels are
increased. This could be because granule contents involved in the zona reaction are not dispersed and hence
are not able to enter the zona and/or because the cortical granule envelope does not form properly, and supplementary sperm entering the PVS are not blocked
from binding to the oolemma. Most work on
polyspermy blocks have focused on the zona pellucida
and on the plasma membrane of the oocyte. Often in
studies of the plasma membrane block, zona free oocytes are used so that the cortical granule envelope
would not be present. New work is needed to examine
the functions of the cortical granule envelope directly
and to determine if it plays a role in blocking
polyspermy. Clearly the PVS is an interesting and complex compartment surrounding mammalian oocytes.
Much work remains to be done to define the matrices
present in this space, how some components are selectively retained in the space while other pass freely
away from the space through the zona, and what roles
the matrix in the PVS plays in fertilization and preimplantation development.
ACKNOWLEDGMENTS
We gratefully acknowledge Karen Riveles for her
suggestions on the manuscript. We thank Dr. Tanya
Hoodbhoy for providing Figures 4A,B and Min Liu for
helping to obtain the images in Figure 1A–C.
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