J. Cell Sci. 39, i-i 2
Printed in Great Britain © Company of Biologists Limited
OOCYTE MATURATION: ABERRANT POSTFUSION RESPONSES OF THE RABBIT PRIMARY
OOCYTE TO PENETRATING SPERMATOZOA
M. BERRIOS* AND J. M. BEDFORDf
Departments of Obstetrics and Gynecology and Anatomy, Cornell University
Medical College, New York, N.Y. 10021, U.S.A.
SUMMARY
Primary oocytes cannot be fertilized normally; they begin to develop this capacity as meiosis
resumes. To elucidate the changes involved in acquisition of their fertilizability, rabbit primary
oocytes displaying a germinal vesicle (GV oocytes) were placed in Fallopian tubes inseminated
previously with spermatozoa, recovered 2-5 h later and examined by light and electron microscopy. At least 4 aspects of GV oocyte/sperm interaction were abnormal. Although the vestments and oolemma seem normally receptive to spermatozoa, fusion with the oolemma of the
primary oocyte did not elicit exocytosis of cortical granules, and consequently multiple entry
of spermatozoa into the ooplasm was common. Secondly, the GV oocyte cortex failed to achieve
a normal engulfment of the anterior part of the sperm head. It sank into the ooplasm capped by
only a small rostral vesicle or left the stable inner acrosomal membrane as a patch in the
oolemma. Only rarely then was there significant dispersion of the sperm chromatin, and this
remained surrounded by nuclear envelope. The persistence of this envelope constitutes a
further aberrant feature, for it disappears immediately in secondary oocytes and was absent in
primary oocytes in which germinal vesicle breakdown had occurred. The results are discussed
with particular reference to current ideas about male pronucleus formation.
INTRODUCTION
Primary oocytes possessing a germinal vesicle (GV oocytes) cannot be fertilized
normally, and their fertilizability is acquired during the resumption of meiosis in the
period preceding ovulation (see Donahue, 1972). Little is known of the basis of this
immaturity. Spermatozoa can penetrate the cumulus oophorus and zona pellucida of
the GV oocyte taken from a non-stimulated follicle (Chang, 1955; Thibault, 1973;
Iwamatsu & Chang, 1972; Yanagimachi, 1974; Niwa & Chang, 1975; Mahi &
Yanagimachi, 1976; Moore & Bedford, 1978); and in the rabbit these barriers are
traversed as easily as those of the ovulated egg (Overstreet & Bedford, 1974). However,
it has been concluded from phase-contrast microscope observations that spermatozoa
reaching the perivitelline space do not fuse with and are not incorporated into the vitellus
of rabbit primary oocytes (Chang, 1955; Overstreet & Bedford, 1974), implying that
resumption of meiosis may bring about functional changes in the oolemmal surface
(see also, Yanagimachi & Nicolson, 1976). A subsequent ultrastructural demonstration that the hamster primary oocyte incorporates spermatozoa readily and in
• Present address: The Rockefeller University, New York, N.Y. 10021, U.S.A.
f To whom requests for reprints should be addressed.
z
M. Berrios and J. M. Bedford
apparently normal fashion in vivo (Moore & Bedford, 1978) raises the possibility
that spermatozoa can enter that of the rabbit, and that this may have been missed in
light-microscope examination because of the persistence of the normal shape and size
of the penetrating sperm head. For the nuclei of spermatozoa incorporated during
resumption of meiosis tend not to decondense because of an inability of the immature
ooplasm to induce this (Thibault & Gerard, 1970; Usui & Yanagimachi, 1976;
Thibault, 1977).
The present ultrastructural study demonstrates that the oolemma of the rabbit GV
oocyte is receptive to spermatozoa. However, cortical granule exocytosis, the mode of
incorporation of the sperm head, and the behaviour of its nuclear envelope and
chromatin within such oocytes were abnormal.
MATERIALS AND METHODS
Rabbit semen collected from New Zealand White males with an artificial vagina was gently
centrifuged for about 5 min. The sperm pellet was resuspended in M-199 (GIBCO, N.Y.)
containing 10 % heated rabbit serum and aliquots of 20 /il containing about 8 x io6 spermatozoa
were inseminated into the Fallopian tubes of non-stimulated females. Nine to eleven hours
later, oocytes were released from large to medium size antral follicles in the ovaries of other
non-stimulated females. Those invested by cumulus oophorus, were transferred through 2
changes of fresh M-199 P' u s i ° % serum, and then instilled with a pipette via aflankincision
into the tubal ampulla of the inseminated female. This manipulation allows normal fertilization
of mature rabbit oocytes, but where the block to polyspermy is in any way defective, the high
sperm number tends to reveal this. Two, three, four or five hours later the oocytes were
recovered by flushing the oviducts with M-199. They were examined first briefly under a
stereo-dissecting microscope, and those that had perivitelline spermatozoa and ooplasm of
normal appearance were selected. The oocytes were then fixed for I h according to Barros &
Berrios (1977) and after dehydration were embedded in Epon 812, or in Spurr (Spurr, 1969).
Fig. 1. Diagrammatic representation (oblique sections) of the major differences in the
mode of sperm incorporation by mature ova and the immature GV rabbit oocyte,
respectively. Figs. A-C represent events in the mature ova within the first hour
after sperm/egg fusion. The initial phase is characterized by disappearance of the
nuclear envelope of the spermatozoon, the beginnings of dispersion of chromatin in
the posterior region of the sperm head, by loss of cortical granules and by protrusion
of the tail (not shown) and the rostral half of the sperm head from the surface of the
oolemma (A). This stage is followed (B) by envelopment of the cranial half of the sperm
head by folds of oolemma, and by more extensive dispersion of the chromatin. Finally
(c) the membranes surrounding the anterior surface of the nucleus become completely
enclosed and, as aflattenedvesicle of dual origin, are reflected away from the chromatin
whose dispersion continues to completion.
Although the phase of fusion itself seems similar in the GV oocyte (D), fusion does
not induce exocytosis of cortical granules lying at the surface, the nuclear envelope of
the spermatozoon does not disappear, and its chromatin remains condensed. Subsequently, the sperm head simply sinks into the ooplasm (E) leaving the inner acrosomal
membrane at the surface or sequestered eventually as a vesicle composed very largely
of this membrane alone (F). The nuclear envelope expands somewhat but persists as
an encompassing membrane, and the extent of chromatin dispersion within it is
generally minimal. Some obvious decondensation of the nuclear chromatin does
occur eventually within the GV oocyte and the investing membrane becomes more
complex, apparently through the addition of further membrane, the source of which is
not clear. The structure e in E, F, probably represents the detached equatorial segment of the acrosome.
Response of immature oocytes to spermatozoa
OVULATED OVUM
FOLLICULAR OOCYTE
(germinal vesicle)
Fig. i. For legend see opposite.
M. Berrios and J. M. Bedford
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Response of immature oocytes to spermatozoa
5
In 10 separate trials, a total of 80 rabbit oocytes displaying perivitelline spermatozoa were so
prepared, and a i-/(m section was taken of 48 of these. Spermatozoa were actually observed
fusing with or inside the oolemma in 20 oocytes, germinal vesicle breakdown having occurred
in only 3 of these. Thin sections were cut, and ultrathin grey sections, stained with uranyl
acetate and lead citrate, were examined in a JEOL, JEM-100 B transmission electron microscope.
OBSERVATIONS
The oolemma of the immature GV oocyte of the rabbit is receptive to spermatozoa,
at least 17 of those sectioned displaying spermatozoa at various stages of fusion. It
evidently cannot mount a block to polyspermy, since most oocytes had more than one
spermatozoon fusing with or inside the oolemma. This is corroborated by the visible
failure of exocytosis of cortical granules situated immediately beneath the oocyte
surface (Figs. 2, 3, 6, 7). Establishment of the exocytosis response is not an immediate
correlate of germinal vesicle breakdown, for the 3 oocytes displaying this were
polyspermic and there were always intact cortical granules not far from spermatozoa
fusing with the oolemma.
The GV oocyte displayed a second defective response to spermatozoa, characterized
by an absence of the normal oolemmal engulfment that in the mature ovum incorporates
the rostral portion of the sperm head into the ooplasm (Fig. 1 B, C). After an apparently
normal fusion phase (Figs. 2, 3) the head often simply sank into the ooplasm of the
GV oocyte (Figs. 1 D-F, 4, 5), without any engulfment of its anterior region. This left
the inner acrosomal membrane, identifiable by electron-dense subacrosomal material,
at the oocyte surface (Fig. 5). In some cases there was a limited oolemmal' phagocytic'
response to the inner acrosomal membrane, but most of the small rostral vesicle so
formed (Fig. 6) appeared to be derived mainly from inner acrosomal membrane, with
only little contribution from the oolemma. A lamellar structure that appeared to be
the equatorial segment of the sperm acrosome was often visible in the ooplasm
(Figs. 4, 7). If this is the equatorial segment, as seems likely, its separate situation in
several cases suggests that it may become pinched off as a free entity in the immature
oocyte, instead of remaining associated with the inner acrosomal membrane/oolemmal
complex as it does within mature ova.
A somewhat different picture of incorporation was presented by the 3 oocytes fixed
Figs. 2, 3. Longitudinal sections through rabbit sperm heads at an early stage of their
incorporation into GV oocytes in vivo. Note the persisting nuclear envelope of the
spermatozoon around the exposed posterior region of the sperm head. Intact cortical
granules remain immediately beneath the oolemma (arrows). Fig. 2, x 17400; Fig. 3,
x 23400.
Figs. 4, 5. Sections showing a subsequent stage in which the rabbit sperm head has
sunk into the ooplasm of the GV oocyte without engulfment of its rostral region
(cf. Fig. IB, E), SO leaving the inner acrosomal membrane (distinguished by an
electron density brought by associated subacrosomal material) as a part of the oocyte
surface. Note the persistence of the sperm nuclear envelope and, in Fig. 4, what may
be equatorial segment (arrow). Fig. 4, x 24000; Fig. 5, x 25000.
M. Berrios andj. M. Bedford
Response of immature oocytes to spermatozoa
after germinal vesicle breakdown. For the configuration of membranes around some
sperm heads within these oocytes suggested that they had been incorporated normally
(see Fig. 1 c). That oocytes begin to respond differently to spermatozoa as germinal
vesicle breakdown occurs is indicated also by the behaviour of the sperm nuclear
envelope. This envelope always persisted in spermatozoa that entered GV oocytes
(Figs. 1-7) but was not seen here in follicular oocytes in which germinal vesicle
breakdown had occurred. Within many of the GV oocytes recovered after 4-5 h in the
oviduct, the sperm nuclear envelope had expanded grossly and extra membrane within
an essentially intact outer envelope (Fig. 8) could possibly represent internal projections of it. However, the source of this extra membrane is uncertain.
As could be anticipated from previous light-microscope studies (see Thibault, 1977),
the sperm chromatin did not disperse at the rate seen after penetration of mature ova.
In many GV oocytes where the envelope of the sperm nucleus appeared intact, there
was only slight erosion of sperm chromatin (Figs. 2-7), but a significant decondensation often occurred in others where the nuclear envelope had expanded (Fig. 8), even
though germinal vesicle breakdown had not taken place. It is stressed that the presence
or absence of the sperm nuclear envelope is not the only important factor mediating
chromatin dispersion. For where this envelope was absent in penetrated oocytes,
recovered after 4-5 h, in which germinal vesicle breakdown had occurred, there was
often minimal dispersion of the exposed sperm chromatin. This was true whether
sperm/oocyte interaction was at an early stage or was complete, with the sperm head
lying totally within the ooplasm. In contrast to the failure of both the nuclear envelope
and chromatin of the spermatozoon to disperse within the ooplasm of the GV oocyte,
the stable - S - S - crosslinked material that occupies the post-acrosomal region of the
sperm head disappeared consistently even at the earlier stage of their interaction
(Figs. 2, 3). This did not occur immediately in the material less exposed beneath the
equatorial segment and inner acrosomal membrane, but the equatorial segment had
divested itself of its associated perinuclear material where it had been reflected away
from the sperm nucleus (Figs. 4, 7).
DISCUSSION
Although there is much interest currently in the factors responsible for the resumption of meiosis and synthetic activities of the oocyte (see, for example, Bloom &
Mukherjee, 1972; Wassarman & Letourneau, 1976; Motlik, Kopecny & Pivko, 1978),
Fig. 6. Section of a rabbit sperm head incorporated by a GV oocyte. In this case, the
rostral part of the nucleus is capped by a 'mini' vesicle which, its electron-dense
character suggests, is composed almost entirely of inner acrosomal membrane. Note
the persistent cortical granules (arrows) and the sperm nuclear envelope that is
essentially intact, x 24300.
Fig. 7. Transverse section of a sperm head within a GV oocyte. It exhibits almost no
dispersion of chromatin and is surrounded by an intact nuclear envelope. A lamellar
structure (arrow) that resembles and may well be the equatorial segment of the
acrosome, lies adjacent to it. x 28000.
7
M. Berrios andj. M. Bedford
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Response of immature oocytes to spermatozoa
the changes that finally enable it to support normal fertilization have not been
elucidated. Past studies performed in several domestic and laboratory mammals
demonstrate that the follicular cell investment and zona pellucida of the immature
oocyte are readily penetrated, and Overstreet & Bedford (1974) have shown that these
barriers in rabbit GV oocytes are already as penetrable as those of ovulated ova. But
their conclusion and that of Chang (1955) that spermatozoa cannot enter the vitellus
of the immature GV oocyte, are not correct. As shown here for the rabbit and
previously in the hamster (Moore & Bedford, 1978), the oolemma of the primary
oocyte is receptive to multiple sperm entry before and after breakdown of the germinal
vesicle. This accords with indications in this laboratory obtained with markers for
terminal sugars (lectins) and for surface anion distribution (cationized ferritin and
ferric oxide colloid), that there are no significant changes in the molecular character
of the rabbit oocyte surface as it passes from diplotene to metaphase II (N. L. Cross,
unpublished observations).
Although the penetrability ot the primary oocyte vestments and the fusogenic
potential of its oolemma seem functionally normal, several specific post-fusion events
are not. First, fusion with a fertilizing spermatozoon does not seem to elicit cortical
granule exocytosis. There is a comparable failure of exocytosis in intact GV oocytes of
the hamster in vivo (Moore & Bedford, 1978) in immature amphibian eggs (e.g.
Katagiri, 1974), and in Arbacia eggs (Longo, 1978). This failure could be due to an
immaturity of the granule itself (since a protease component has been implicated in its
release (Longo & Schuel, 1973)), to an uncoupled state of the oolemma and cortical
granules, and /or to a failure of elevation of ooplasmic Ca2+ upon stimulation of the
oolemma. This ion seems to be a trigger in several stimulus-secretion coupling systems
(see Douglas, 1978), and micro-injection of Ca2+ brings about cortical exocytosis in
Rarrn pipiens oocytes (Hollinger & Schuetz, 1976).
The existence of a functional immaturity of the cortical cytoskeleton as a second
aberrant feature of the GV oocyte is suggested by its coincident failure to develop the
normal engulfment response seen in mature ova (cf. Fig. IB with A). Unlike exocytosis
and male pronucleus formation, this aspect of maturation is probably not shared with
oocytes of non-eutherian vertebrates, and thus may be a relatively recent evolutionary
development. For the sparse evidence in chicken (Okamura & Nishiyama, 1978),
lamprey (Nicander & Sjoden, 1971) and newt (Picheral, 1977) suggests that their
spermatozoa are incorporated by mature ova simply through fusion of the inner
acrosomal membrane with the oolemma, essentially according to the pattern described
in some invertebrates (see Colwin & Colwin, 1967).
A third aspect of oocyte immaturity, inability to disperse sperm chromatin, has been
Fig. 8. Visible remains of the chromatin of a sperm nucleus lying within a GV oocyte
recovered after 4 h in the Fallopian tube. Notwithstanding the persistent immature
state of the oocyte, significant dispersion of the chromatin has occurred within a
complex of membranes believed to represent modified sperm nuclear membrane. The
outer envelope of the complex appears intact but the internal membranes, which
could be projections of this, seem discontinuous, x 25900.
9
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M. Berrios and J.M. Bedford
attributed by Thibault (1973, 1977) to lack of a male pronucleus growth factor. The
present study indicates that chromatin dispersal requires a complex of activities rather
than only one 'factor'. A striking feature, difficult to discern in the light microscope, is
persistence of the envelope of the rabbit sperm nucleus incorporated by the GV oocyte,
for there is no sign of this at even the early stage of sperm interaction with the mature
rabbit ovum (Bedford, 1972), nor was it apparent here where germinal vesicle breakdown had occurred. This envelope is a barrier that must hinder exchange between
ooplasm and sperm chromatin, and its dissolution seems necessary for development
of the male pronucleus. But its disappearance is not the only key to pronucleus
formation. For absence of the envelope of the sperm nucleus did not assure a normal
rate of dispersion of the sperm chromatin in the rabbit oocyte, nor does it in the
hamster (Moore & Bedford, 1978). Since significant decondensation occurred
occasionally within 4 h in some membrane-bounded sperm nuclei in GV oocytes,
particularly where the envelope had expanded, it seems that different activities are
required for disassembly of the envelope on the one hand, and sperm chromatin on
the other.
The fact that there is probably more than one activity to consider in male pronucleus formation (i.e. nuclear envelope disassembly, - S - S - cleavage, protamine
substitution, and possibly proteases required for decondensation) suggests that it is a
biochemically complex phenomenon. It should not be assumed either that the different
activities that bring about these facets of pronucleus formation appear synchronously
at some moment following gonadotrophin stimulation. For although Thibault (1977)
reports that the rabbit oocyte will support complete pronucleus formation only if
recovered 6-5 h or more after treatment with human chorionic gonadotrophin, the GV
oocyte clearly can bring about some chromatin dispersion within 4 h (Fig. 8). It has
seemed likely that such dispersion must involve a reduction of disulphide bonds
(Calvin & Bedford, 1971), and since the post-acrosomal - S - S - crosslinked material
and that beneath the acrosome disappeared regardless of the stage of oocyte maturity,
some ability to cleave - S - S - crosslinks may exist in even GV oocytes. It has seemed
reasonable that a protease also may be involved in sperm chromatin decondensation,
but the idea has been advanced that such a protease exists in the sperm nucleus
(Zirkin & Chang, 1977). However, thiol-induced proteolytic activity that disperses
sperm chromatin in vitro is removed merely by centrifugation of spermatozoa through
sucrose, and is acrosomal in origin (Young, 1979). That an acrosomal protease could
possibly operate in this way (Marushige & Marushige, 1975, 1978) is very unlikely,
not least because decondensation may begin before the acrosome of the fertilizing
spermatozoon has any association with the oolemma (Bedford, 1972). Thus, at present,
there is no evidence that the factors that transform the sperm nucleus are not intrinsic
to the ooplasm.
In conclusion, the rabbit GV oocyte cannot be fertilized normally because it is unable
to achieve exocytosis of its cortical granules and so mount a block to polyspermy; it
cannot achieve the engulfment response to the rostral region of the fertilizing sperm
head that typifies gamete interaction in Eutheria; and it cannot disperse the envelope
or the chromatin of the sperm nucleus in the coordinated fashion needed for male
Response of immature oocytes to spermatozoa
11
pronucleus formation. A normal mode of sperm incorporation and dispersion of the
sperm nuclear envelope may be accomplished by oocytes in which germinal vesicle
breakdown has occurred, within 4 h of collection, but these fail still to achieve
exocytosis or a normal rate of sperm nucleus decondensation. The relationship
between breakdown of the germinal vesicle and these separate aspects is not necessarily
constant among different mammals. Preliminary results (unpublished) show that the
guinea-pig GV oocyte has similar deficiencies in this respect to that of the rabbit. On
the other hand the hamster GV oocyte can engulf the sperm head normally in vivo and
disassemble the sperm nuclear envelope, though there is no cortical exocytosis or
significant nuclear dispersion (Moore & Bedford, 1978), and that of the dog can
induce decondensation of the sperm nucleus before germinal vesicle breakdown
(Mahi & Yanagimachi, 1976). Since some of these maturation events appear similar
to those in the sea urchin (Longo, 1978) and anuran amphibians (Smith & Ecker,
1969; Katagiri, 1974; Elinson, 1977), it may be valid to probe some aspects of the
biochemistry of mammalian oocyte maturation by extrapolation from analyses made
in other groups.
We are grateful to Miu Ying Fong and Angela Cantone for excellent technical help. This
study was supported by The Ford Foundation and by The National Institutes of Health
(HD-07527). M.B. has been a Ford Foundation Fellow.
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(Received 28 November 1978 - Revised 13 February 1979)