- Email: [email protected]
Premature cortical granule loss does not prevent sperm penetration of mouse eggs
DEVELOPMENTAL
Premature
BIOLOGY
71,
22-32 (1979)
Cortical Granule Loss Does Not Prevent Sperm Penetration Mouse Eggs
of
DON P. WOLF, SANTO V. NICOSIA, AND MITSUMA HAMADA’ Division of Reproductive Biology, Department of Obstetrics and Gynecology, and Departments of Biochemistry and Biophysics and Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received December 18, 1978; accepted in revised form February 5, 1979 The role of cortical granules in the mouse egg’s plasmalemma block to polyspermy was investigated by examining the effect of premature granule loss on egg fertility. Granule loss, quantitated by transmission electron microscopic examination, was induced in zona-free eggs by exposure to the divalent cation ionophore, A23187, or by mechanical removal of zonae. Egg exposure to ionophore led to the loss of approximately 50% of the egg’s complement of granules in the absence of nuclear activation (parthenogenesis), while complete cortical granule loss accompanied the parthenogenetic activation seen in a limited population of mechanically stimulated eggs. Aged eggs underwent nuclear activation without a dramatic reduction in granule complements. The fertility of treated zona-free eggs was identical to that of controls, as measured by the percentage of eggs penetrated and by the mean number of sperm recovered per egg. Moreover, both ionophore-treated and aged eggs subsequently underwent a normal sperm-induced block response. Exposure of zona-intact eggs to ionophore was also without effect on egg fertility. These results indicate that cortical granules are not involved in the plasmalemma block to polyspermy in the mouse.
additional sperm and in the physicochemical isolation of the embryo. Extensive studies conducted in amphibians (Grey et al., 1974, 1976; Wolf et al., 1976) and marine invertebrates (Epel, 1977; Schuel, 1979, for review) suggest that a block results when specific sperm binding sites on the egg surface are altered directly by cortical granule components or indirectly when the fertilization envelope is formed. In mammals, the importance of cortical granules in the egg’s block to polyspermy is uncertain. While these structures are released at fertilization in all mammalian eggs examined, species differences are apparent in block responses. The hamster undergoes a highly efficient penetration block at the zona pellucida and a late, possibly unimportant block at the egg plasmalemma, whereas the rabbit egg utilizes only the latter reaction in maintaining monospermy. The mouse and rat undergo moderately effective blocks at both levels (Austin,
INTRODUCTION
At ovulation the eggs of most mammals are arrested at the metaphase stage of the second meiotic division. Nuclear activation, the resumption of the division process, is normally triggered by sperm but can be induced artificially by aging (Kaufman, 1975), cold or osmotic shock (Austin and Braden, 1954; Solter et al., 1971), electrical stimulation (Tarkowski et al., 1970; Gwatkin et al., 1973; Gulyas, 1976; Zamboni et al., 1976), calcium ionophore (Steinhardt, 1974), and pricking (Uehara and Yanagimachi, 1977). In response to sperm and to some parthenogenetic treatments, the egg also undergoes a cortical reaction which includes the exocytotic release of cortical granules. Granule contents are subsequently involved in blocking penetration of ’ Present address: Sandoz Research Laboratory, Reproduction Section, Minimitsuru, Yamanashi, Japan. 22 0012.1606/79,‘070022-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
WOLF, NICOSIA, AND HAMADA
1961). In the mouse and human, it appears that some cortical granules are lost even before eggs are exposed to sperm, leading to suggestions that granules may be involved in other phenomena, such as the induction of an acrosome reaction (Nicosia et al., 1977; Rousseau et al., 1977). Direct evidence for granule involvement in an egg block to polyspermy is available; “cortical granule preparations,” which may contain not only released cortical granule contents but substances originating elsewhere in the egg as well as hydrolysis products, have been obtained by electrical stimulation or by sperm-induced activation of zona-free eggs (Barros and Yanagimachi, 1972; Gwatkin et al., 1973; Wolf and Hamada, 1977), and these preparations contain an activity that can reduce the fertility of intact eggs, presumably by acting on the zona. Moreover, the premature loss of cortical granules, triggered in intact hamster eggs in Ca”‘-Mg’+-free medium by the calcium ionophore, A23187, leads to a zona reaction (Steinhardt et al., 1974). At the plasmalemma level, cortical granule loss has been correlated with reduced penetration of the et al., zona-free hamster egg (Gwatkin 1976), and crude cortical granule preparations can reduce the fertility of zona-free mouse eggs (Wolf and Hamada, 1977). A logical sequel to these experiments involves the subcellular localization of the fertilityreducing activity of cortical granules, an impractical undertaking in mammals. Therefore, we sought an alternative approach to clarify the role of cortical granules in the egg’s plasmalemma block response. The existence and timing of the block to polyspermy in zona-free mouse eggs have been described previously (Wolf, 1978). In the present study, cortical granule loss was triggered prematurely in zona-free mouse eggs, and egg fertility was subsequently quantitated. Our results indicate that cortical granules are not involved in the plasmalemma block response of mouse eggs.
Cortical
Granules
in Mouse Eggs
MATERIALS
23
AND METHODS
Eggs were recovered from superovulated females 13-15 hr after human chorionic gonadotropin administration (HCG; 10 IU; Sigma, St. Louis, MO.) from 6- to 12-weekold Swiss mice primed with pregnant mare serum gonadotropin (10 IU; Organon, Oss, Holland) (Wolf and Inoue, 1976). Cumulus cells were dispersed by transferring eggs into drops of culture medium containing 0.1% hyaluronidase (Sigma, Type I) under silicone oil (Dow-Corning 200 Fluid, Midland, Mich.). A Krebs-Ringer bicarbonate medium, supplemented with glucose, pyruvate, lactate, and bovine serum albumin (Sigma, Fraction V; 20 mg/ml), was employed; and eggs were maintained at 37”C, as detailed previously (Wolf and Inoue, 1976). Zonae were removed mechanically from cumulus-free eggs (Wolf et al., 1976). Zona-free eggs were then washed and examined periodically for the presence of polar bodies. First polar bodies were invariably lost during zona removal. A small population of eggs, which constituted the spontaneously activated eggs employed in this study, extruded a second polar body within 30 min. Eggs were recovered 20 hr postHCG for the parthenogenetic treatment (Kaufman, 1975) which involved exposing aged eggs to culture medium with or without hyaluronidase (0.1% for lo-15 min) and incubating for an additional 2 hr. Epididymal sperm were capacitated by preincubation in culture medium for at least 1 hr; eggs were inseminated under oil in microdishes approximately 40 min after activation and incubated in the same medium in closed containers for 2-4 hr (Wolf and Inoue, 1976). Zona-intact or -free eggs were exposed at 37°C to ionophore A23187 (21.8 a) in complete culture medium. In this case, eggs were inseminated usually in the presence of ionophore. In scoring sperm penetration, eggs were washed free of loosely bound sperm, mounted on slides, fixed, stained with acetolacmoid, and examined at 400 x with phase-contrast optics.
24
DEVELOPMENTAL BIOLOGY
Eggs were considered penetrated if they contained a sperm tail and an enlarged sperm head or a male pronucleus within the cytoplasm. Evidence for parthenogenetic activation included the presence of a second polar body and of a single pronucleus. Stock solutions (1-5 mg/ml) of the ionophore A23187 (courtesy of Dr. Robert Hammill, Eli Lilly, Indianapolis, Ind.) were prepared in dimethylformamide and stored in the dark at -20°C. Occasionally, the activity of stock solutions was monitored by pigment changes induced in unfertilized Xenopus eggs (Steinhardt et al., 1974). For electron microscopy, eggs were processed by sequential fixation in 3% glutaraldehyde (280 mOsm) and 1% osmium tetroxide in 0.1 M cacodylate buffer. After alcohol dehydration, eggs were embedded for 48 hr at 60°C in Epon 812 (Luft, 1961). Thin sections, 60-70 nm thick, were mounted on Formvar-coated, 100~pm-mesh grids, stained with lead citrate (Reynolds, 1963) and uranyl acetate, and examined with a Hitachi HU-12A electron microscope operated at 75 kV. Cortical granule preparations were quantitated in serial sections or in multiple sections through representative regions of the entire egg by the procedure of Nicosia et al. (1977), which included counts of both electron light and dark granules, as described by these authors. The statistical significance of the results was evaluated by Student’s t test. RESULTS
Cortical Granules in Activated Zona-Free Eggs An unfertilized tubal egg control, fixed within 30 min of recovery from the oviduct, contained about 35 granules/100 pm of plasmalemma (Table l), compared with an average value of 32.7 reported previously for three zona-intact eggs (Nicosia et al., 1977).
Eggs exposed to ionophore (3.6 $MJ for 30-45 min contained fewer granules (P < 0.01) than the control, the value (approx
VOLUME 71,1979 TABLE
1
QUANTITATION OF CORTICAL GRANULE COMPLEMENTS IN ZONA-FREE EGGS BEFORE AND AFTER PENETRATION” Egg We
Unpenetrated
Penetrated
Treatment
Cortical granules/100 am plasmalemma
Control Ionophore treated Spontaneously activated Control Ionophore treated Spontaneously activated
34.72 + 4.56 18.82 + 6.79’ 4.16 f 3.45 6.69 f 4.41 3.34 f 2.44 5.30 f 1.68
n These results (means + SD) were derived from analysis of a single egg in each category, except in unpenetrated ionophore-treated (N = 2) and spontaneously activated (N = 4) eggs. ‘Displaced granules (400-500 nm away from the egg plasma membrane) in ionophore-treated eggs were included in this quantitation.
19) in Table 1 representing an average obtained from two eggs. Many granules in treated eggs were translocated away from their extreme cortical positions (see below). These observations have also been corroborated by qualitative observations in additional eggs. Parthenogenetic activation did not accompany the ionophore-induced response. Zona-intact or -free mouse eggs exposed for 30 min to ionophore concentrations as high as 8 fl retained intact metaphase spindles for 3 hr, as determined by light microscopy. This finding agrees with the results of Masui et al. (1977). The loss of cortical granules in spontaneously activated eggs (N = 4) was nearly complete, the value in Table 1 being dramatically less than that of the unfertilized control (approx 4) (P < 0.001) and comparable with values determined for penetrated eggs. The four eggs examined in this category were derived from three separate experiments. In vivo aged eggs recovered 20 hr after HCG administration and exposed to hyaluronidase retained complete complements of cortical granules, although extensive
WOLF, NICOSIA, AND HAMADA
quantitation was not done and granules were often displaced slightly inward from the plasmalemma. Nuclear activation of aged eggs occurred in controls (without hyaluronidase) as readily as in those exposed to the enzyme. Each treatment examined in this study elicited a different egg response. Eggs underwent nuclear activation in response to zona removal or aging, concomitant with cortical granule release in the former case, while exposure to ionophore selectively induced cortical granule release.
Morphology of Activated Eggs During the quantitation of granules by transmission electron microscopic evaluation of thin sections, the morphological characteristics of eggs were also studied. Uninseminated zona-free eggs, which served as controls, were similar ultrastructurally to the zona-intact eggs described previously (Nicosia et al., 1977), in that cortical granules of varying electron density were present in their usual ectoplasmic location, immediately below a “rough” plasmalemma characterized by prominent microvilli (Figs. 1A and B). Granules were distributed throughout the egg cortex, except in the area overlying the meiotic region, which was covered by a “smooth” plasmalemma. This polarity in surface topography was less pronounced in these eggs than that observed previously with zonaintact eggs (Nicosia et al., 1977) due to a reduction in the number and in the size of microvilli. Zona removal by mechanical means may alter surface morphology; however, no functional significance has been attached to it (Nicosia et al., 1978). Golgi complexes were inactive, as indicated by the absence of visible intracisternal material. Ionophore- treated eggs differed from controls both in the number (Table 1) and in the location of cortical granules; many of the remaining granules were located in deep cortical regions several hundred nanome-
Cortical Granules in Mouse Eggs
25
ters (400-500 nm) ‘away from the plasmalemma (Fig. 1C). Golgi complexes were inactive but showed moderate dilatation and a peculiar concentric arrangement of peripheral cisternal spaces (Fig. 1D). Polarity in surface topography appeared to be more pronounced than in control eggs due to the presence of more prominent microvilli. Spontaneously activated eggs abstricted a second polar body which was covered by a smooth membrane overlying a lOO- to 200-nm-thick filamentous band. This structure contained free chromatin material and was free of cortical granules. While second polar bodies are often characterized by the presence of membrane-enclosed chromatin (Zamboni, 1971), eggs were fixed here within minutes of abstriction before nuclear membrane formation had been completed. The egg exhibited a predominantly smooth, albeit uneven, surface with microvilli present only near the midbody (Figs. 1E and F). The few cortical granules present were in a normal cortical location. Golgi complex cisternae were empty, and the cytoplasm contained occasional lipid droplets, secondary lysosomes, and myelin bodies. No gross differences in the number of cortical granules located deep within the cytoplasm were observed among these egg groups.
Fertility
of Activated Eggs
The influence of premature granule loss on egg fertility was examined by inseminating control unactivated and experimentally activated eggs with capacitated epididymal sperm. Eggs were not inseminated immediately after exposure to the activation treatment, but rather after an interval of about 40 min, since approximately 40 min are required for the block response in zonafree mouse eggs (Wolf, 1978). The results in Table 2 clearly indicate that premature granule loss did not influence egg fertility, as experimental levels of penetration and polyspermy and the mean number of sperm
26
DEVELOPMENTAL BIOLOGY VOLUME71,1979
FIG. 1.
WOLF, NICOSIA, AND HAMADA TABLE
27
Cortical Granules in Mouse Eggs 2
PENETRATION CHARACTERISTICS OF ACTIVATED ZONA-FREE MOUSE EGGS Activation
Mechanical Pretreatment
Penetrated (%I
stimulus
removal of zonae with ionophore
(LUnactivated zona-free * Eggs extruding second ’ Eggs exposed to ~1.8 within 50 min. ’ The number of eggs is
Control eggs” Experimental* Control eggs” Experimental”
81 87 100 100
Polyspermic (8)
(73/90)” (34/39) (64/64) (116/116)
38 47 88 91
(28/73) (16/34) (56/64) (106/116)
Mean number sperm/ egg 1.38 1.74 3.41 3.37
eggs inseminated in culture medium, as described in Materials and Methods. polar bodies within 30 min of zona removal and therefore classified as activated. pM A23187 for at least 10 min at 37°C and then inseminated with lo” sperm/ml included in parentheses.
per egg were similar to those of the corresponding controls. Differences in the mean number of sperm per egg between ionophore-treated and spontaneously activated eggs reflect, in part, endogenous variability in sperm preparations (Wolf, 1978), although an ionophore-dependent enhancement in penetration has also been observed (Wolf, manuscript in preparation). The parthenogenetic activation of zonaintact eggs associated with aging may involve limited cortical granule loss but is not usually associated with an efficient block response (Merchant and Chang, 1971; Flechon et al., 1975). This observation was confirmed in the present study, where zonaintact eggs recovered 20 hr after HCG administration were aged in vitro for 2 hr, at which time 44% had activated. Those eggs, when inseminated after zona removal, were highly fertile (103/llO; 94%). Exposure of intact hamster eggs to ionophore leads to granule release and to a zona reaction, a block to sperm penetration at the zona pellucida (Steinhardt et al., 1974).
In order to examine the ability of ionophore to induce a zona reaction in the mouse, we exposed zona-intact eggs to ionophore (20min preincubation in 3.6 fl A23187) and subsequently monitored egg fertility. Ionophore exposure was not associated with reduced penetration, since the mean number of sperm per egg for treated and control categories was 1.05 and 1.25, respectively. In this case, granules released in the presence of a zona had no apparent influence on a plasmalemma block, minimizing the importance of the dilution of cortical granule material that undoubtedly occurs in zona-free eggs. Morphology
of Penetrated
Eggs
Sperm penetration of eggs was confirmed in inseminated controls by the presence of sperm remnants (tail, inner acrosomal membrane cap) within the egg cytoplasm. These eggs displayed a nearly complete loss of cortical granules and the disappearance of polarity in the distribution of surface microvilli (Table 1, Figs. 2A and B). Only
FIG. 1. Representative micrographs depicting cortical configurations in zona-free eggs before insemination. (A-B) Control eggs. Cortical granules (cg) are present in normal number and location below a ruffled plasmalemma. Heterogeneity of granules is indicated by their different electron density (B). A. x 4500; B, x 14,800. (C-D) Ionophore-treated eggs. Granules are restricted to focal cortical regions and many of them (arrows) are displaced away from their characteristic subplasmalemmal location. Note prominent microvilli (mv) and concentrically arranged Golgi cisternae (go). C, x 8100; D, x 16,900. (E-F) Spontaneously activated eggs. The cortex of this egg type is almost devoid of granules and is covered by a predominantly smooth plasmalemma, except in the region of second polar body abstriction (spb). E, X 5600; F, x 2000.
28
DEVELOPMENTAL
BIOLOGY
FIG. 2.
VOLUME 71,1979
WOLF,
NICOSIA,
AND
HAMADA
the egg surface near the site of second polar body abstriction contained numerous microvilli, while the remaining surface was generally smooth. Most sperm attached to the penetrated egg had undergone an acrosome reaction, as had all sperm that had penetrated the egg. Cortical granules retained by ionophoretreated eggs disappeared following sperm penetration. These eggs showed about the same number of granules (Table 1, Figs. 2C and D) as penetrated controls. There was no morphological evidence for a further inward retraction of dislocated granules. Penetration in spontaneously activated eggs devoid of cortical granules (Fig. 2E) was also confirmed by high-resolution analysis (Fig. 2F). Pronuclei formed in these eggs with a timing and frequency similar to those in control eggs (Fig. 2G). Kinetics
of Sperm Penetration
With zona-free eggs which undergo premature loss of cortical granules and incorporate sperm like untreated controls, it could be argued that the egg does not prematurely block sperm penetration because cortical granule material is lost or diluted. Therefore, the kinetics of penetration were examined on aged and ionophore-treated eggs to see if premature granule loss prevented or precluded a sperm-triggered block to penetration (Fig. 3). Limited availability of material prevented the use of spontaneously activated eggs. Sperm penetration of aged or ionophore-treated eggs followed the same time course as that of unstimulated eggs reported previously
Cortical Granules in Mouse Eggs
29
FIG. 3. Kinetics of sperm penetration into zonafree mouse eggs. Eggs were aged in uirv (20 hr postHCG administration; crosses) or pretreated with ionophore (1.8 a, 20 min; open circles) before insemination with approximately 2 x lo” capacitated epididymal sperm/ml. Each point represents the mean of at least 25 eggs.
(Wolf, 1978). Thus, a delay of approximately 30 min was observed before penetration began, and the process was completed by 2 hr, when penetration levels reached constant levels. The addition of a second aliquot of capacitated sperm at 120 min to ionophore-pretreated eggs did not influence subsequent penetration levels. These results indicate that aged, activated eggs or eggs which have prematurely lost half of their cortical granules show a normal time course of penetration and a normal block to penetration. DISCUSSION
Results from the present study indicate that: (a) A premature, nearly complete loss of cortical granules occurs in a limited population of mouse eggs denuded of their zonae pellucidae by mechanical means; (b) exposure of eggs to the calcium ionophore, A23187, in the presence of extracellular cal-
FIG. 2. Representative micrographs of zona-free eggs after insemination. Eggs were incubated with capacitated epididymal spermatozoa for several hours before fixation. (A-B) Control egg. Note the uniformly smooth plasmalemma and the absence of cortical granules in A (X 6700) and the sperm tail (arrow) in proximity of a Golgi complex (go) in B (X 14,100). (C-D) This egg was preincubated with ionophore for 10 min before exposure. 20-30 min later, to sperm. The cortical morphology is similar to that of penetrated control eggs, except possible for an increased number of lipid droplets (1). C, x 7700. A sperm tail (arrow) is present within a focally elevated cortical area which still contains a few cortical granules (cg). D. x 10,500. (E-G) Spontaneously activated egg inseminated approximately 40 min after activation. Cortical granules are essentially absent, and the egg plasmalemma is predominantly smooth. Note the fertilizing sperm tail (sp) in the cortex of one egg (F) and both male (mp) and female Cfp) pronuclei in the egg depicted in G. Go, Golgi complex. E. x 6250: F, x :{5.400: G. x 2500.
30
DEVELOPMENTAL
BIOLOGY
cium and magnesium, leads to partial granule loss with the remaining granules, some dislocated inward, capable of discharging after sperm penetration; (c) granule loss, as induced by ionophore, occurs without concomitant nuclear activation; and (d) premature loss of granules by itself neither triggers the egg block to sperm penetration nor prevents a sperm-induced block from being established. Two categories of eggs “lost” cortical granules following experimental manipulation. In the most dramatic case, spontaneous activation after zona removal, all four eggs examined showed a nearly total loss of these structures. Cortical granule release has been triggered in hamster eggs by pricking (Uehara and Yanagimachi, 1977), and it is possible that in activated mouse eggs, mechanical removal of zonae simulates pricking. Critical to the interpretation of the present findings is the fate of the missing granules. Their disappearance could result from extensive inward migration, disintegration in situ, or release either intact or after fusion with the egg plasma membrane by an exocytotic mechanism. Dislocation of granules inward from their extreme cortical positions did occur to a limited extent in ionophore-treated eggs; however, this mechanism cannot account entirely for granule loss and does not apply at all to spontaneously activated eggs. Similarly, little, if any, evidence exists for cytoplasmic disintegration of granules or for their release as intact structures. Cortical granule release occurs within minutes of sperm penetration in hamster (Barros and Yanagimachi, 1972) and mouse (Nicosia et al., 1977) eggs. At about the same time, depending on the species, alterations occur in the zona pellucida and/or in the egg plasmalemma that reduce the receptivity of these structures to sperm. The existence, for at least several hours, of a block to polyspermy at the egg plasmalemma of mouse eggs inseminated in vitro with capacitated epididymal sperm has
VOLUME 71,1979
been documented (Wolf, 1978). Based on these observations, on the direct experimental evidence cited in the Introduction, and by analogy to marine invertebrates, cortical granules have been implicated in the mammalian egg’s block responses. The possibility of a more generalized role for granules has been considered but seems unlikely, as early development of parthenogenotes occurs even in the presence of granules (Steinhardt et al., 1974), and some marine invertebrates retain a complete complement of granules during early embryonic growth (Rebhun, 1962; Paul, 1975). The present results argue against a cortical granule involvement in the plasmalemma block in the mouse, since premature loss of most granules did not influence egg fertility. Moreover, ionophore-treated eggs, either zona-intact or -free, which should have undergone a 50% reduction in granules showed a high fertility; and zona-free eggs preexposed to ionophore were capable of a plasmalemma block in response to sperm penetration. Since presumed granule loss in treated, zona-intact eggs did not result in a premature plasmalemma block, the dilution of cortical granule components that occurs with zona-free eggs is not a critical factor. Moreover, either the granules remaining in ionophore-treated zona-free eggs provide sufficient material for the block response or else these structures are not involved in the sperm-triggered reaction. We previously reported (Wolf and Hamada, 1977) that preexposure of zona-free eggs to a crude cortical granule preparation decreased the mean number of sperm penetrating these eggs. This observation is consistent with cortical granule involvement in the egg’s plasmalemma block response; the decrease, although statistically significant, was limited, and the cortical granule preparation was undoubtedly impure. The latter fact may be especially significant in view of the nonspecific sensitivity of zona-free mouse eggs to attack by proteases (Wolf et al., 1976).
The role of cortical granules in the plasmalemma block in other mammals is unclear. Cortical granule loss, as monitored by light microscopy, can be induced readily in hamster eggs by electrical stimulation (Gwatkin et al., 1973; Gulyas, 1976), pricking (Uehara and Yanagimachi, 1977), or exposure agents to membrane-active (Gwatkin et al., 1976) or ionophore (Steinhardt et al., 1974). However, the consequences of granule loss to egg fertility in this species are unclear. Decreased sperm binding to and penetration of the zona-free egg have been correlated with cortical granule loss induced by neuraminidase treatment (Gwatkin et al., 1976). Under comparable conditions in a second laboratory, neuraminidase treatment of zona-free eggs did not influence egg fertility (Hirao and Yanagimachi, 1978). Interpretation of these results is further complicated by the results from reinsemination experiments which indicate that a plasmalemma block is not established in hamster eggs until 2.5-3 hr after penetration or long after cortical granules have discharged (Barros and Yanagimachi, 1972). Indeed, more recent experiments suggest that a plasmalemma block is never established in the hamster, as pronuclear and two-cell eggs stripped of their zonae are capable of fusing with capacitated acrosome-reacted sperm (Usui and Yanagimachi, 1976). The rabbit egg relies exclusively on a plasmalemma block to polyspermy, and cortical granule loss has been associated with egg penetration by the fertilizing sperm (Austin, 1961). However, we are unaware of any direct experimental evidence in this species for cortical granule involvement in a block response. In the absence of cortical granule involvement, the plasmalemma block in mammals may be electrically mediated. Rapid spermtriggered depolarization of the plasmalemma is a common response seen with eggs of several lower vertebrates and marine invertebrates, and recent “voltage clamp” experiments in the sea urchin dem-
onstrate that a block to sperm penetration is associated with this event (Jaffe, 1976). The electrophysiological properties 01’ mouse eggs have been characterized, including those changes that occur over long time intervals during development (Cross et al., 1973; Powers and Tupper, 1974: Okamoto et al., 1977). In general, the patterns of such changes observed during the course of oogenesis, fertilization, and early development are similar among widely divergent species. In view of the present findings and the above observations, it now seems appropriate and feasible to monitor changes in the electrical properties of zona-free mouse eggs at fertilization, in an effort to elucidate the mechanism by which these eggs maintain the monospermic condition. The authors express their appreciation to I1t-h. Charles Met;! and Herbert Schuel for tbnr c%tic,;tl review of the manuscript, to I)r. Richard Stark anti Ms. -Janice Sowinski for assistance in elr.~,trott tnwroscopic studies. and to Miss Pat Park for exwllettt secretarial and editorial assistance. This research ua.s supported by NIH Grants HII-076X anti HI)-i~til’2 (Project L’ and E. M. Core).
AUSTIN. C. K. (1961). ‘The Mammalian Kgg.” Hlac,hwell Scientific. Oxford. AUSTIN. C. H.. and HKAI)EN, A. W. H. ( 1964). Indwtion and inhibition of the second polar diviston tn the rat egg and subsequent fertilization Aus/. .1. BlOl. Ser. 7, 195-210. BAHKOS. C., and YANAGIMACHI. K. (19721. I'ol~ sperm?-preventing mechanisms tn the golden hanster egg. -I. Erp. Zool. 180, 251-266. Goss, M. H.. CROSS, I’. C.. and BRINSTFX, ti. I.. (1973). Changes in memhrane potential during mouse egg development. Derehp. BLOT.33, 412-4 1fj. EPEL, 11. (1977). The egg surface in relation to metabolic activation at fertilization. In “Imrnunohiolog~ of Gametes” IM. Kdidin and M. H. ,Johnson. etis.~. pp. 21.5-254. Cambridge liniverstty I’wss. (lam bridge. FLI~CHON, J.-E., HUNEAU. II., SOLARI, A., and ?‘HIBAULT, C. (1975). Reaction corticale et blocage de la polyspermie dans l’oeuf de lapine. Ann. Bid. AnLm. Biochim. Biophys. 15,9-l& GREY, H. I>., WOLF. II. I'., and HEDIIICK, .J. 1,. (197.1) Formation and structure of the fertilization envy Lope in Xeno~x~s l~ews. Det~lo~. Hwl. 36, 44-f; 1. GREY, R. I).. WORKIP;(:. I’. K., and HEI)KI(.K .I. I,. (197fi). Evidence that the fertilization tw\~t~lopt~
32
DEVELOPMENTAL BIOLOGY
blocks sperm entry in eggs of Xenopus laeuis: Interaction of sperm with isolated envelopes. Deuelop. Biol. 54, 52-60. GULYAS, B. J. (1976). Ultrastructural observations on rabbit, hamster and mouse eggs following electrical stimulation in uitro. Amer. J. Anat. 147, 203-218. GWATKIN, R. B. L., RASMUSSON, G. H., and WILLIAMS, D. T. (1976). Induction of the cortical reaction in hamster eggs by membrane-active agents. J. Reprod. Fertil. 47, 299-303. GWATKIN, R. B. L., WILLIAMS, D. T., HARTMANN, J. F., and KNIAZUK, M. (1973). The zona reaction of hamster and mouse eggs: Production in vitro by a trypsin-like protease from cortical granules. J. Reprod. Fertil. 32, 259-265. HIRAO, Y., and YANAGIMACHI, R. (1978). Effects of various enzymes on the ability of hamster egg plasma membranes to fuse with spermatozoa. Gamete Res. 1, 3-12. JAFFE, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature (London) 261,68-71. KAUFMAN, M. H. (1975). The experimental induction of parthenogenesis in the mouse. In “The Early Development of Mammals” (M. Balls and A. E. Wilds, eds.), pp. 25-44. Cambridge University Press, Cambridge. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. MASUI, Y., MEYERHOF, P. G., MILLER, M. A., and WASSERMAN, W. J. (1977). Roles of divalent cations in maturation and activation of vertebrate oocytes. Differentiation 9, 49-57. MERCHANT, H., and CHANG, M. C. (1971). An electron microscopic study of mouse eggs matured in viuo and in uitro. Anat. Rec. 171, 21-38. NICOSIA, S. V., WOLF, D. P., and INOUE, M. (1977). Cortical granule distribution and cell surface characteristics in mouse ova. Develop. Biol. 57, 56-74. NICOSIA, S. V., WOLF, D. P., and MASTROIANNI, L., JR. (1978). Surface topography of mouse eggs before and after insemination. Gamete Res. 1, 145-155. OKAMOTO, H., TAKAHASHI, K., and YAMASHITA, N. (1977). Ionic currents through the membrane of the mammalian oocyte and their comparison with those in the tunicate and sea urchin. J. Physiol. 267,465495. PAUL, M. (1975). The polyspermy block in eggs of Urechis caupo. Exp. Cell Res. 90, 137-142. POWERS, R. D., and TUPPER, J. T. (1974). Some electrophysiological and permeability properties of the mouse egg. Deuelop. Biol. 38,320-331. REBHUN, L. I. (1962). Electron microscopic studies on the vitelline membrane of the surf clam, Spisula solidissima. J. Ultrastruct. Res. 6, 107-122.
VOLUME 71,1979
REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. ROUSSEAU, P., MEDA, P., LECART, C., HAUMONT, S., and FERIN, J. (1977). Cortical granule release in human follicular oocytes. Biol. Reprod. 16, 104-111. SCHUEL, H. (1978). The role of cortical granules in fertilization. Gamete Res. 1, 299-382. SOLTER, D. (1977). Organization and the antigenic properties of the egg membrane. In “Immunobiology of Gametes” (M. Edidin and M. H. Johnson, eds.), pp. 207-234. Cambridge University Press, Cambridge. SOLTER, D., BICZYSKO, W., GRAHAM, C., PIENKOWSKI, M., and KOPROWSKI, H. (1971). Ultrastructure of early development of mouse parthenogenones. J. Exp. Zool. 188, l-24. STEINHARDT, R. A., EPEL, D., CARROLL, E. J., and, YANAGIMACHI, R. (1974). Is calcium ionophore a universal activator for unfertilized eggs? Nature (London) 252,41-43. TARKOWSKI, A. K., WITKOWSKA, A., and NOWICKA, J. (1970). Experimental parthenogenesis in the mouse. Nature (London) 226, 162-165. UEHARA, T., and YANAGIMACHI, R. (1977). Activation of hamster eggs by pricking. J. Exp. Zool. 199,269274. USUI, N., and YANAGIMACHI, R. (1976). Behavior of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization and early development. J. Ultrastruct. Res. 57, 276-288. WOLF, D. P. (1978). The block to sperm penetration in zona-free mouse eggs inseminated in vitro. Develop. Biol. 64, l-10. WOLF, D. P., and HAMADA, M. (1977). Induction of zonal and oolemmal blocks to sperm penetration in mouse eggs with cortical granule exudate. Biol. Reprod. 17, 350-354. WOLF, D. P., and INOUE, M. (19761. Sperm concentration dependency in the fertilization and zonae sperm binding properties of mouse eggs inseminated in vitro. J. Exp. Zool. 196, 27-38. WOLF, D. P., INOUE, M., and STARK, R. A. (19761. Penetration of zona-free mouse eggs. Biol. Reprod. 15,213-221. WOLF, D. P., NISHIHARA, T., WEST, M., WYRICK, R. E., and HEDRICK, J. L. (1976). Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus laevis eggs. Biochemistry 15, 36713678. ZAMBONI, L. (1971). “Fine Morphology of Mammalian Fertilization,” first ed. Harper and Row, New York. ZAMBONI, L., PATTERSON, H., and JONES, M. (1976). Loss of cortical granules in mouse ova activated in uiuo by electric shock. Amer. J. Anat. 147, 95-101.
Premature
BIOLOGY
71,
22-32 (1979)
Cortical Granule Loss Does Not Prevent Sperm Penetration Mouse Eggs
of
DON P. WOLF, SANTO V. NICOSIA, AND MITSUMA HAMADA’ Division of Reproductive Biology, Department of Obstetrics and Gynecology, and Departments of Biochemistry and Biophysics and Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received December 18, 1978; accepted in revised form February 5, 1979 The role of cortical granules in the mouse egg’s plasmalemma block to polyspermy was investigated by examining the effect of premature granule loss on egg fertility. Granule loss, quantitated by transmission electron microscopic examination, was induced in zona-free eggs by exposure to the divalent cation ionophore, A23187, or by mechanical removal of zonae. Egg exposure to ionophore led to the loss of approximately 50% of the egg’s complement of granules in the absence of nuclear activation (parthenogenesis), while complete cortical granule loss accompanied the parthenogenetic activation seen in a limited population of mechanically stimulated eggs. Aged eggs underwent nuclear activation without a dramatic reduction in granule complements. The fertility of treated zona-free eggs was identical to that of controls, as measured by the percentage of eggs penetrated and by the mean number of sperm recovered per egg. Moreover, both ionophore-treated and aged eggs subsequently underwent a normal sperm-induced block response. Exposure of zona-intact eggs to ionophore was also without effect on egg fertility. These results indicate that cortical granules are not involved in the plasmalemma block to polyspermy in the mouse.
additional sperm and in the physicochemical isolation of the embryo. Extensive studies conducted in amphibians (Grey et al., 1974, 1976; Wolf et al., 1976) and marine invertebrates (Epel, 1977; Schuel, 1979, for review) suggest that a block results when specific sperm binding sites on the egg surface are altered directly by cortical granule components or indirectly when the fertilization envelope is formed. In mammals, the importance of cortical granules in the egg’s block to polyspermy is uncertain. While these structures are released at fertilization in all mammalian eggs examined, species differences are apparent in block responses. The hamster undergoes a highly efficient penetration block at the zona pellucida and a late, possibly unimportant block at the egg plasmalemma, whereas the rabbit egg utilizes only the latter reaction in maintaining monospermy. The mouse and rat undergo moderately effective blocks at both levels (Austin,
INTRODUCTION
At ovulation the eggs of most mammals are arrested at the metaphase stage of the second meiotic division. Nuclear activation, the resumption of the division process, is normally triggered by sperm but can be induced artificially by aging (Kaufman, 1975), cold or osmotic shock (Austin and Braden, 1954; Solter et al., 1971), electrical stimulation (Tarkowski et al., 1970; Gwatkin et al., 1973; Gulyas, 1976; Zamboni et al., 1976), calcium ionophore (Steinhardt, 1974), and pricking (Uehara and Yanagimachi, 1977). In response to sperm and to some parthenogenetic treatments, the egg also undergoes a cortical reaction which includes the exocytotic release of cortical granules. Granule contents are subsequently involved in blocking penetration of ’ Present address: Sandoz Research Laboratory, Reproduction Section, Minimitsuru, Yamanashi, Japan. 22 0012.1606/79,‘070022-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
WOLF, NICOSIA, AND HAMADA
1961). In the mouse and human, it appears that some cortical granules are lost even before eggs are exposed to sperm, leading to suggestions that granules may be involved in other phenomena, such as the induction of an acrosome reaction (Nicosia et al., 1977; Rousseau et al., 1977). Direct evidence for granule involvement in an egg block to polyspermy is available; “cortical granule preparations,” which may contain not only released cortical granule contents but substances originating elsewhere in the egg as well as hydrolysis products, have been obtained by electrical stimulation or by sperm-induced activation of zona-free eggs (Barros and Yanagimachi, 1972; Gwatkin et al., 1973; Wolf and Hamada, 1977), and these preparations contain an activity that can reduce the fertility of intact eggs, presumably by acting on the zona. Moreover, the premature loss of cortical granules, triggered in intact hamster eggs in Ca”‘-Mg’+-free medium by the calcium ionophore, A23187, leads to a zona reaction (Steinhardt et al., 1974). At the plasmalemma level, cortical granule loss has been correlated with reduced penetration of the et al., zona-free hamster egg (Gwatkin 1976), and crude cortical granule preparations can reduce the fertility of zona-free mouse eggs (Wolf and Hamada, 1977). A logical sequel to these experiments involves the subcellular localization of the fertilityreducing activity of cortical granules, an impractical undertaking in mammals. Therefore, we sought an alternative approach to clarify the role of cortical granules in the egg’s plasmalemma block response. The existence and timing of the block to polyspermy in zona-free mouse eggs have been described previously (Wolf, 1978). In the present study, cortical granule loss was triggered prematurely in zona-free mouse eggs, and egg fertility was subsequently quantitated. Our results indicate that cortical granules are not involved in the plasmalemma block response of mouse eggs.
Cortical
Granules
in Mouse Eggs
MATERIALS
23
AND METHODS
Eggs were recovered from superovulated females 13-15 hr after human chorionic gonadotropin administration (HCG; 10 IU; Sigma, St. Louis, MO.) from 6- to 12-weekold Swiss mice primed with pregnant mare serum gonadotropin (10 IU; Organon, Oss, Holland) (Wolf and Inoue, 1976). Cumulus cells were dispersed by transferring eggs into drops of culture medium containing 0.1% hyaluronidase (Sigma, Type I) under silicone oil (Dow-Corning 200 Fluid, Midland, Mich.). A Krebs-Ringer bicarbonate medium, supplemented with glucose, pyruvate, lactate, and bovine serum albumin (Sigma, Fraction V; 20 mg/ml), was employed; and eggs were maintained at 37”C, as detailed previously (Wolf and Inoue, 1976). Zonae were removed mechanically from cumulus-free eggs (Wolf et al., 1976). Zona-free eggs were then washed and examined periodically for the presence of polar bodies. First polar bodies were invariably lost during zona removal. A small population of eggs, which constituted the spontaneously activated eggs employed in this study, extruded a second polar body within 30 min. Eggs were recovered 20 hr postHCG for the parthenogenetic treatment (Kaufman, 1975) which involved exposing aged eggs to culture medium with or without hyaluronidase (0.1% for lo-15 min) and incubating for an additional 2 hr. Epididymal sperm were capacitated by preincubation in culture medium for at least 1 hr; eggs were inseminated under oil in microdishes approximately 40 min after activation and incubated in the same medium in closed containers for 2-4 hr (Wolf and Inoue, 1976). Zona-intact or -free eggs were exposed at 37°C to ionophore A23187 (21.8 a) in complete culture medium. In this case, eggs were inseminated usually in the presence of ionophore. In scoring sperm penetration, eggs were washed free of loosely bound sperm, mounted on slides, fixed, stained with acetolacmoid, and examined at 400 x with phase-contrast optics.
24
DEVELOPMENTAL BIOLOGY
Eggs were considered penetrated if they contained a sperm tail and an enlarged sperm head or a male pronucleus within the cytoplasm. Evidence for parthenogenetic activation included the presence of a second polar body and of a single pronucleus. Stock solutions (1-5 mg/ml) of the ionophore A23187 (courtesy of Dr. Robert Hammill, Eli Lilly, Indianapolis, Ind.) were prepared in dimethylformamide and stored in the dark at -20°C. Occasionally, the activity of stock solutions was monitored by pigment changes induced in unfertilized Xenopus eggs (Steinhardt et al., 1974). For electron microscopy, eggs were processed by sequential fixation in 3% glutaraldehyde (280 mOsm) and 1% osmium tetroxide in 0.1 M cacodylate buffer. After alcohol dehydration, eggs were embedded for 48 hr at 60°C in Epon 812 (Luft, 1961). Thin sections, 60-70 nm thick, were mounted on Formvar-coated, 100~pm-mesh grids, stained with lead citrate (Reynolds, 1963) and uranyl acetate, and examined with a Hitachi HU-12A electron microscope operated at 75 kV. Cortical granule preparations were quantitated in serial sections or in multiple sections through representative regions of the entire egg by the procedure of Nicosia et al. (1977), which included counts of both electron light and dark granules, as described by these authors. The statistical significance of the results was evaluated by Student’s t test. RESULTS
Cortical Granules in Activated Zona-Free Eggs An unfertilized tubal egg control, fixed within 30 min of recovery from the oviduct, contained about 35 granules/100 pm of plasmalemma (Table l), compared with an average value of 32.7 reported previously for three zona-intact eggs (Nicosia et al., 1977).
Eggs exposed to ionophore (3.6 $MJ for 30-45 min contained fewer granules (P < 0.01) than the control, the value (approx
VOLUME 71,1979 TABLE
1
QUANTITATION OF CORTICAL GRANULE COMPLEMENTS IN ZONA-FREE EGGS BEFORE AND AFTER PENETRATION” Egg We
Unpenetrated
Penetrated
Treatment
Cortical granules/100 am plasmalemma
Control Ionophore treated Spontaneously activated Control Ionophore treated Spontaneously activated
34.72 + 4.56 18.82 + 6.79’ 4.16 f 3.45 6.69 f 4.41 3.34 f 2.44 5.30 f 1.68
n These results (means + SD) were derived from analysis of a single egg in each category, except in unpenetrated ionophore-treated (N = 2) and spontaneously activated (N = 4) eggs. ‘Displaced granules (400-500 nm away from the egg plasma membrane) in ionophore-treated eggs were included in this quantitation.
19) in Table 1 representing an average obtained from two eggs. Many granules in treated eggs were translocated away from their extreme cortical positions (see below). These observations have also been corroborated by qualitative observations in additional eggs. Parthenogenetic activation did not accompany the ionophore-induced response. Zona-intact or -free mouse eggs exposed for 30 min to ionophore concentrations as high as 8 fl retained intact metaphase spindles for 3 hr, as determined by light microscopy. This finding agrees with the results of Masui et al. (1977). The loss of cortical granules in spontaneously activated eggs (N = 4) was nearly complete, the value in Table 1 being dramatically less than that of the unfertilized control (approx 4) (P < 0.001) and comparable with values determined for penetrated eggs. The four eggs examined in this category were derived from three separate experiments. In vivo aged eggs recovered 20 hr after HCG administration and exposed to hyaluronidase retained complete complements of cortical granules, although extensive
WOLF, NICOSIA, AND HAMADA
quantitation was not done and granules were often displaced slightly inward from the plasmalemma. Nuclear activation of aged eggs occurred in controls (without hyaluronidase) as readily as in those exposed to the enzyme. Each treatment examined in this study elicited a different egg response. Eggs underwent nuclear activation in response to zona removal or aging, concomitant with cortical granule release in the former case, while exposure to ionophore selectively induced cortical granule release.
Morphology of Activated Eggs During the quantitation of granules by transmission electron microscopic evaluation of thin sections, the morphological characteristics of eggs were also studied. Uninseminated zona-free eggs, which served as controls, were similar ultrastructurally to the zona-intact eggs described previously (Nicosia et al., 1977), in that cortical granules of varying electron density were present in their usual ectoplasmic location, immediately below a “rough” plasmalemma characterized by prominent microvilli (Figs. 1A and B). Granules were distributed throughout the egg cortex, except in the area overlying the meiotic region, which was covered by a “smooth” plasmalemma. This polarity in surface topography was less pronounced in these eggs than that observed previously with zonaintact eggs (Nicosia et al., 1977) due to a reduction in the number and in the size of microvilli. Zona removal by mechanical means may alter surface morphology; however, no functional significance has been attached to it (Nicosia et al., 1978). Golgi complexes were inactive, as indicated by the absence of visible intracisternal material. Ionophore- treated eggs differed from controls both in the number (Table 1) and in the location of cortical granules; many of the remaining granules were located in deep cortical regions several hundred nanome-
Cortical Granules in Mouse Eggs
25
ters (400-500 nm) ‘away from the plasmalemma (Fig. 1C). Golgi complexes were inactive but showed moderate dilatation and a peculiar concentric arrangement of peripheral cisternal spaces (Fig. 1D). Polarity in surface topography appeared to be more pronounced than in control eggs due to the presence of more prominent microvilli. Spontaneously activated eggs abstricted a second polar body which was covered by a smooth membrane overlying a lOO- to 200-nm-thick filamentous band. This structure contained free chromatin material and was free of cortical granules. While second polar bodies are often characterized by the presence of membrane-enclosed chromatin (Zamboni, 1971), eggs were fixed here within minutes of abstriction before nuclear membrane formation had been completed. The egg exhibited a predominantly smooth, albeit uneven, surface with microvilli present only near the midbody (Figs. 1E and F). The few cortical granules present were in a normal cortical location. Golgi complex cisternae were empty, and the cytoplasm contained occasional lipid droplets, secondary lysosomes, and myelin bodies. No gross differences in the number of cortical granules located deep within the cytoplasm were observed among these egg groups.
Fertility
of Activated Eggs
The influence of premature granule loss on egg fertility was examined by inseminating control unactivated and experimentally activated eggs with capacitated epididymal sperm. Eggs were not inseminated immediately after exposure to the activation treatment, but rather after an interval of about 40 min, since approximately 40 min are required for the block response in zonafree mouse eggs (Wolf, 1978). The results in Table 2 clearly indicate that premature granule loss did not influence egg fertility, as experimental levels of penetration and polyspermy and the mean number of sperm
26
DEVELOPMENTAL BIOLOGY VOLUME71,1979
FIG. 1.
WOLF, NICOSIA, AND HAMADA TABLE
27
Cortical Granules in Mouse Eggs 2
PENETRATION CHARACTERISTICS OF ACTIVATED ZONA-FREE MOUSE EGGS Activation
Mechanical Pretreatment
Penetrated (%I
stimulus
removal of zonae with ionophore
(LUnactivated zona-free * Eggs extruding second ’ Eggs exposed to ~1.8 within 50 min. ’ The number of eggs is
Control eggs” Experimental* Control eggs” Experimental”
81 87 100 100
Polyspermic (8)
(73/90)” (34/39) (64/64) (116/116)
38 47 88 91
(28/73) (16/34) (56/64) (106/116)
Mean number sperm/ egg 1.38 1.74 3.41 3.37
eggs inseminated in culture medium, as described in Materials and Methods. polar bodies within 30 min of zona removal and therefore classified as activated. pM A23187 for at least 10 min at 37°C and then inseminated with lo” sperm/ml included in parentheses.
per egg were similar to those of the corresponding controls. Differences in the mean number of sperm per egg between ionophore-treated and spontaneously activated eggs reflect, in part, endogenous variability in sperm preparations (Wolf, 1978), although an ionophore-dependent enhancement in penetration has also been observed (Wolf, manuscript in preparation). The parthenogenetic activation of zonaintact eggs associated with aging may involve limited cortical granule loss but is not usually associated with an efficient block response (Merchant and Chang, 1971; Flechon et al., 1975). This observation was confirmed in the present study, where zonaintact eggs recovered 20 hr after HCG administration were aged in vitro for 2 hr, at which time 44% had activated. Those eggs, when inseminated after zona removal, were highly fertile (103/llO; 94%). Exposure of intact hamster eggs to ionophore leads to granule release and to a zona reaction, a block to sperm penetration at the zona pellucida (Steinhardt et al., 1974).
In order to examine the ability of ionophore to induce a zona reaction in the mouse, we exposed zona-intact eggs to ionophore (20min preincubation in 3.6 fl A23187) and subsequently monitored egg fertility. Ionophore exposure was not associated with reduced penetration, since the mean number of sperm per egg for treated and control categories was 1.05 and 1.25, respectively. In this case, granules released in the presence of a zona had no apparent influence on a plasmalemma block, minimizing the importance of the dilution of cortical granule material that undoubtedly occurs in zona-free eggs. Morphology
of Penetrated
Eggs
Sperm penetration of eggs was confirmed in inseminated controls by the presence of sperm remnants (tail, inner acrosomal membrane cap) within the egg cytoplasm. These eggs displayed a nearly complete loss of cortical granules and the disappearance of polarity in the distribution of surface microvilli (Table 1, Figs. 2A and B). Only
FIG. 1. Representative micrographs depicting cortical configurations in zona-free eggs before insemination. (A-B) Control eggs. Cortical granules (cg) are present in normal number and location below a ruffled plasmalemma. Heterogeneity of granules is indicated by their different electron density (B). A. x 4500; B, x 14,800. (C-D) Ionophore-treated eggs. Granules are restricted to focal cortical regions and many of them (arrows) are displaced away from their characteristic subplasmalemmal location. Note prominent microvilli (mv) and concentrically arranged Golgi cisternae (go). C, x 8100; D, x 16,900. (E-F) Spontaneously activated eggs. The cortex of this egg type is almost devoid of granules and is covered by a predominantly smooth plasmalemma, except in the region of second polar body abstriction (spb). E, X 5600; F, x 2000.
28
DEVELOPMENTAL
BIOLOGY
FIG. 2.
VOLUME 71,1979
WOLF,
NICOSIA,
AND
HAMADA
the egg surface near the site of second polar body abstriction contained numerous microvilli, while the remaining surface was generally smooth. Most sperm attached to the penetrated egg had undergone an acrosome reaction, as had all sperm that had penetrated the egg. Cortical granules retained by ionophoretreated eggs disappeared following sperm penetration. These eggs showed about the same number of granules (Table 1, Figs. 2C and D) as penetrated controls. There was no morphological evidence for a further inward retraction of dislocated granules. Penetration in spontaneously activated eggs devoid of cortical granules (Fig. 2E) was also confirmed by high-resolution analysis (Fig. 2F). Pronuclei formed in these eggs with a timing and frequency similar to those in control eggs (Fig. 2G). Kinetics
of Sperm Penetration
With zona-free eggs which undergo premature loss of cortical granules and incorporate sperm like untreated controls, it could be argued that the egg does not prematurely block sperm penetration because cortical granule material is lost or diluted. Therefore, the kinetics of penetration were examined on aged and ionophore-treated eggs to see if premature granule loss prevented or precluded a sperm-triggered block to penetration (Fig. 3). Limited availability of material prevented the use of spontaneously activated eggs. Sperm penetration of aged or ionophore-treated eggs followed the same time course as that of unstimulated eggs reported previously
Cortical Granules in Mouse Eggs
29
FIG. 3. Kinetics of sperm penetration into zonafree mouse eggs. Eggs were aged in uirv (20 hr postHCG administration; crosses) or pretreated with ionophore (1.8 a, 20 min; open circles) before insemination with approximately 2 x lo” capacitated epididymal sperm/ml. Each point represents the mean of at least 25 eggs.
(Wolf, 1978). Thus, a delay of approximately 30 min was observed before penetration began, and the process was completed by 2 hr, when penetration levels reached constant levels. The addition of a second aliquot of capacitated sperm at 120 min to ionophore-pretreated eggs did not influence subsequent penetration levels. These results indicate that aged, activated eggs or eggs which have prematurely lost half of their cortical granules show a normal time course of penetration and a normal block to penetration. DISCUSSION
Results from the present study indicate that: (a) A premature, nearly complete loss of cortical granules occurs in a limited population of mouse eggs denuded of their zonae pellucidae by mechanical means; (b) exposure of eggs to the calcium ionophore, A23187, in the presence of extracellular cal-
FIG. 2. Representative micrographs of zona-free eggs after insemination. Eggs were incubated with capacitated epididymal spermatozoa for several hours before fixation. (A-B) Control egg. Note the uniformly smooth plasmalemma and the absence of cortical granules in A (X 6700) and the sperm tail (arrow) in proximity of a Golgi complex (go) in B (X 14,100). (C-D) This egg was preincubated with ionophore for 10 min before exposure. 20-30 min later, to sperm. The cortical morphology is similar to that of penetrated control eggs, except possible for an increased number of lipid droplets (1). C, x 7700. A sperm tail (arrow) is present within a focally elevated cortical area which still contains a few cortical granules (cg). D. x 10,500. (E-G) Spontaneously activated egg inseminated approximately 40 min after activation. Cortical granules are essentially absent, and the egg plasmalemma is predominantly smooth. Note the fertilizing sperm tail (sp) in the cortex of one egg (F) and both male (mp) and female Cfp) pronuclei in the egg depicted in G. Go, Golgi complex. E. x 6250: F, x :{5.400: G. x 2500.
30
DEVELOPMENTAL
BIOLOGY
cium and magnesium, leads to partial granule loss with the remaining granules, some dislocated inward, capable of discharging after sperm penetration; (c) granule loss, as induced by ionophore, occurs without concomitant nuclear activation; and (d) premature loss of granules by itself neither triggers the egg block to sperm penetration nor prevents a sperm-induced block from being established. Two categories of eggs “lost” cortical granules following experimental manipulation. In the most dramatic case, spontaneous activation after zona removal, all four eggs examined showed a nearly total loss of these structures. Cortical granule release has been triggered in hamster eggs by pricking (Uehara and Yanagimachi, 1977), and it is possible that in activated mouse eggs, mechanical removal of zonae simulates pricking. Critical to the interpretation of the present findings is the fate of the missing granules. Their disappearance could result from extensive inward migration, disintegration in situ, or release either intact or after fusion with the egg plasma membrane by an exocytotic mechanism. Dislocation of granules inward from their extreme cortical positions did occur to a limited extent in ionophore-treated eggs; however, this mechanism cannot account entirely for granule loss and does not apply at all to spontaneously activated eggs. Similarly, little, if any, evidence exists for cytoplasmic disintegration of granules or for their release as intact structures. Cortical granule release occurs within minutes of sperm penetration in hamster (Barros and Yanagimachi, 1972) and mouse (Nicosia et al., 1977) eggs. At about the same time, depending on the species, alterations occur in the zona pellucida and/or in the egg plasmalemma that reduce the receptivity of these structures to sperm. The existence, for at least several hours, of a block to polyspermy at the egg plasmalemma of mouse eggs inseminated in vitro with capacitated epididymal sperm has
VOLUME 71,1979
been documented (Wolf, 1978). Based on these observations, on the direct experimental evidence cited in the Introduction, and by analogy to marine invertebrates, cortical granules have been implicated in the mammalian egg’s block responses. The possibility of a more generalized role for granules has been considered but seems unlikely, as early development of parthenogenotes occurs even in the presence of granules (Steinhardt et al., 1974), and some marine invertebrates retain a complete complement of granules during early embryonic growth (Rebhun, 1962; Paul, 1975). The present results argue against a cortical granule involvement in the plasmalemma block in the mouse, since premature loss of most granules did not influence egg fertility. Moreover, ionophore-treated eggs, either zona-intact or -free, which should have undergone a 50% reduction in granules showed a high fertility; and zona-free eggs preexposed to ionophore were capable of a plasmalemma block in response to sperm penetration. Since presumed granule loss in treated, zona-intact eggs did not result in a premature plasmalemma block, the dilution of cortical granule components that occurs with zona-free eggs is not a critical factor. Moreover, either the granules remaining in ionophore-treated zona-free eggs provide sufficient material for the block response or else these structures are not involved in the sperm-triggered reaction. We previously reported (Wolf and Hamada, 1977) that preexposure of zona-free eggs to a crude cortical granule preparation decreased the mean number of sperm penetrating these eggs. This observation is consistent with cortical granule involvement in the egg’s plasmalemma block response; the decrease, although statistically significant, was limited, and the cortical granule preparation was undoubtedly impure. The latter fact may be especially significant in view of the nonspecific sensitivity of zona-free mouse eggs to attack by proteases (Wolf et al., 1976).
The role of cortical granules in the plasmalemma block in other mammals is unclear. Cortical granule loss, as monitored by light microscopy, can be induced readily in hamster eggs by electrical stimulation (Gwatkin et al., 1973; Gulyas, 1976), pricking (Uehara and Yanagimachi, 1977), or exposure agents to membrane-active (Gwatkin et al., 1976) or ionophore (Steinhardt et al., 1974). However, the consequences of granule loss to egg fertility in this species are unclear. Decreased sperm binding to and penetration of the zona-free egg have been correlated with cortical granule loss induced by neuraminidase treatment (Gwatkin et al., 1976). Under comparable conditions in a second laboratory, neuraminidase treatment of zona-free eggs did not influence egg fertility (Hirao and Yanagimachi, 1978). Interpretation of these results is further complicated by the results from reinsemination experiments which indicate that a plasmalemma block is not established in hamster eggs until 2.5-3 hr after penetration or long after cortical granules have discharged (Barros and Yanagimachi, 1972). Indeed, more recent experiments suggest that a plasmalemma block is never established in the hamster, as pronuclear and two-cell eggs stripped of their zonae are capable of fusing with capacitated acrosome-reacted sperm (Usui and Yanagimachi, 1976). The rabbit egg relies exclusively on a plasmalemma block to polyspermy, and cortical granule loss has been associated with egg penetration by the fertilizing sperm (Austin, 1961). However, we are unaware of any direct experimental evidence in this species for cortical granule involvement in a block response. In the absence of cortical granule involvement, the plasmalemma block in mammals may be electrically mediated. Rapid spermtriggered depolarization of the plasmalemma is a common response seen with eggs of several lower vertebrates and marine invertebrates, and recent “voltage clamp” experiments in the sea urchin dem-
onstrate that a block to sperm penetration is associated with this event (Jaffe, 1976). The electrophysiological properties 01’ mouse eggs have been characterized, including those changes that occur over long time intervals during development (Cross et al., 1973; Powers and Tupper, 1974: Okamoto et al., 1977). In general, the patterns of such changes observed during the course of oogenesis, fertilization, and early development are similar among widely divergent species. In view of the present findings and the above observations, it now seems appropriate and feasible to monitor changes in the electrical properties of zona-free mouse eggs at fertilization, in an effort to elucidate the mechanism by which these eggs maintain the monospermic condition. The authors express their appreciation to I1t-h. Charles Met;! and Herbert Schuel for tbnr c%tic,;tl review of the manuscript, to I)r. Richard Stark anti Ms. -Janice Sowinski for assistance in elr.~,trott tnwroscopic studies. and to Miss Pat Park for exwllettt secretarial and editorial assistance. This research ua.s supported by NIH Grants HII-076X anti HI)-i~til’2 (Project L’ and E. M. Core).
AUSTIN. C. K. (1961). ‘The Mammalian Kgg.” Hlac,hwell Scientific. Oxford. AUSTIN. C. H.. and HKAI)EN, A. W. H. ( 1964). Indwtion and inhibition of the second polar diviston tn the rat egg and subsequent fertilization Aus/. .1. BlOl. Ser. 7, 195-210. BAHKOS. C., and YANAGIMACHI. K. (19721. I'ol~ sperm?-preventing mechanisms tn the golden hanster egg. -I. Erp. Zool. 180, 251-266. Goss, M. H.. CROSS, I’. C.. and BRINSTFX, ti. I.. (1973). Changes in memhrane potential during mouse egg development. Derehp. BLOT.33, 412-4 1fj. EPEL, 11. (1977). The egg surface in relation to metabolic activation at fertilization. In “Imrnunohiolog~ of Gametes” IM. Kdidin and M. H. ,Johnson. etis.~. pp. 21.5-254. Cambridge liniverstty I’wss. (lam bridge. FLI~CHON, J.-E., HUNEAU. II., SOLARI, A., and ?‘HIBAULT, C. (1975). Reaction corticale et blocage de la polyspermie dans l’oeuf de lapine. Ann. Bid. AnLm. Biochim. Biophys. 15,9-l& GREY, H. I>., WOLF. II. I'., and HEDIIICK, .J. 1,. (197.1) Formation and structure of the fertilization envy Lope in Xeno~x~s l~ews. Det~lo~. Hwl. 36, 44-f; 1. GREY, R. I).. WORKIP;(:. I’. K., and HEI)KI(.K .I. I,. (197fi). Evidence that the fertilization tw\~t~lopt~
32
DEVELOPMENTAL BIOLOGY
blocks sperm entry in eggs of Xenopus laeuis: Interaction of sperm with isolated envelopes. Deuelop. Biol. 54, 52-60. GULYAS, B. J. (1976). Ultrastructural observations on rabbit, hamster and mouse eggs following electrical stimulation in uitro. Amer. J. Anat. 147, 203-218. GWATKIN, R. B. L., RASMUSSON, G. H., and WILLIAMS, D. T. (1976). Induction of the cortical reaction in hamster eggs by membrane-active agents. J. Reprod. Fertil. 47, 299-303. GWATKIN, R. B. L., WILLIAMS, D. T., HARTMANN, J. F., and KNIAZUK, M. (1973). The zona reaction of hamster and mouse eggs: Production in vitro by a trypsin-like protease from cortical granules. J. Reprod. Fertil. 32, 259-265. HIRAO, Y., and YANAGIMACHI, R. (1978). Effects of various enzymes on the ability of hamster egg plasma membranes to fuse with spermatozoa. Gamete Res. 1, 3-12. JAFFE, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature (London) 261,68-71. KAUFMAN, M. H. (1975). The experimental induction of parthenogenesis in the mouse. In “The Early Development of Mammals” (M. Balls and A. E. Wilds, eds.), pp. 25-44. Cambridge University Press, Cambridge. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. MASUI, Y., MEYERHOF, P. G., MILLER, M. A., and WASSERMAN, W. J. (1977). Roles of divalent cations in maturation and activation of vertebrate oocytes. Differentiation 9, 49-57. MERCHANT, H., and CHANG, M. C. (1971). An electron microscopic study of mouse eggs matured in viuo and in uitro. Anat. Rec. 171, 21-38. NICOSIA, S. V., WOLF, D. P., and INOUE, M. (1977). Cortical granule distribution and cell surface characteristics in mouse ova. Develop. Biol. 57, 56-74. NICOSIA, S. V., WOLF, D. P., and MASTROIANNI, L., JR. (1978). Surface topography of mouse eggs before and after insemination. Gamete Res. 1, 145-155. OKAMOTO, H., TAKAHASHI, K., and YAMASHITA, N. (1977). Ionic currents through the membrane of the mammalian oocyte and their comparison with those in the tunicate and sea urchin. J. Physiol. 267,465495. PAUL, M. (1975). The polyspermy block in eggs of Urechis caupo. Exp. Cell Res. 90, 137-142. POWERS, R. D., and TUPPER, J. T. (1974). Some electrophysiological and permeability properties of the mouse egg. Deuelop. Biol. 38,320-331. REBHUN, L. I. (1962). Electron microscopic studies on the vitelline membrane of the surf clam, Spisula solidissima. J. Ultrastruct. Res. 6, 107-122.
VOLUME 71,1979
REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. ROUSSEAU, P., MEDA, P., LECART, C., HAUMONT, S., and FERIN, J. (1977). Cortical granule release in human follicular oocytes. Biol. Reprod. 16, 104-111. SCHUEL, H. (1978). The role of cortical granules in fertilization. Gamete Res. 1, 299-382. SOLTER, D. (1977). Organization and the antigenic properties of the egg membrane. In “Immunobiology of Gametes” (M. Edidin and M. H. Johnson, eds.), pp. 207-234. Cambridge University Press, Cambridge. SOLTER, D., BICZYSKO, W., GRAHAM, C., PIENKOWSKI, M., and KOPROWSKI, H. (1971). Ultrastructure of early development of mouse parthenogenones. J. Exp. Zool. 188, l-24. STEINHARDT, R. A., EPEL, D., CARROLL, E. J., and, YANAGIMACHI, R. (1974). Is calcium ionophore a universal activator for unfertilized eggs? Nature (London) 252,41-43. TARKOWSKI, A. K., WITKOWSKA, A., and NOWICKA, J. (1970). Experimental parthenogenesis in the mouse. Nature (London) 226, 162-165. UEHARA, T., and YANAGIMACHI, R. (1977). Activation of hamster eggs by pricking. J. Exp. Zool. 199,269274. USUI, N., and YANAGIMACHI, R. (1976). Behavior of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization and early development. J. Ultrastruct. Res. 57, 276-288. WOLF, D. P. (1978). The block to sperm penetration in zona-free mouse eggs inseminated in vitro. Develop. Biol. 64, l-10. WOLF, D. P., and HAMADA, M. (1977). Induction of zonal and oolemmal blocks to sperm penetration in mouse eggs with cortical granule exudate. Biol. Reprod. 17, 350-354. WOLF, D. P., and INOUE, M. (19761. Sperm concentration dependency in the fertilization and zonae sperm binding properties of mouse eggs inseminated in vitro. J. Exp. Zool. 196, 27-38. WOLF, D. P., INOUE, M., and STARK, R. A. (19761. Penetration of zona-free mouse eggs. Biol. Reprod. 15,213-221. WOLF, D. P., NISHIHARA, T., WEST, M., WYRICK, R. E., and HEDRICK, J. L. (1976). Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus laevis eggs. Biochemistry 15, 36713678. ZAMBONI, L. (1971). “Fine Morphology of Mammalian Fertilization,” first ed. Harper and Row, New York. ZAMBONI, L., PATTERSON, H., and JONES, M. (1976). Loss of cortical granules in mouse ova activated in uiuo by electric shock. Amer. J. Anat. 147, 95-101.