Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs

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Developmental Biology 257 (2003) 166 –176

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Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs Manqi Deng,a Hidefumi Kishikawa,b,1 Ryuzo Yanagimachi,b Gregory S. Kopf,a,2 Richard M. Schultz,a,c and Carmen J. Williamsa,* a

Center for Research on Reproduction & Women’s Health and Department of Obstetrics & Gynecology, University of Pennsylvania, Philadelphia, PA 19104, USA b Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822, USA c Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA Received for publication 3 December 2002, revised 8 January 2003, accepted 14 January 2003

Abstract A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca2⫹ levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2f during formation of the new CGFD. Moreover, clamping intracellular Ca2⫹ did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Cortical granule; Oocyte maturation; Exocytosis; Fertilization

Introduction A cortical granule (CG)-free domain (CGFD) is observed in the region overlying the metaphase II (MII) spindle in rodent eggs (Gulyas, 1980; Nicosia et al., 1977; Okada et al., 1986). The plasma membrane in this region lacks microvilli and is enriched in cortical actin (Longo and Chen, 1985). Previous studies in the mouse have demonstrated that sperm are less likely to penetrate the egg in the * Corresponding author. Fax: ⫹1-215-573-7627. E-mail address: [email protected] (C.J. Williams). 1 Present address: Department of Urology, Osaka University Medical School, 2-2 Yamadaoka, Suita City, Osaka, Japan. 2 Present address: Women’s Health Research Institute, Wyeth Research, P.O. Box 8299, Philadelphia, PA 19101-8299, USA.

amicrovillar region (Nicosia et al., 1977; Wilson and Snell, 1998). Reduced sperm binding (and hence penetration) in this region would minimize the potential for damage to the spindle or perturbation in chromosome segregation by the fertilizing sperm. Redistribution and/or exocytosis of CGs during maturation are likely mechanisms for the formation of the CGFD. Evidence supporting CG redistribution comes from studies showing the formation of a region of increased CG density in the periphery of the CGFD (Ducibella et al., 1990; Okada et al., 1986). Evidence supporting CG exocytosis includes the observation that a decrease in the number of CGs occurs during maturation (from ⬃6000 to ⬃4000) (Ducibella et al., 1988; Okada et al., 1986), and this decrease is associated with the CG-mediated conversion of ZP2 to ZP2f (Ducibella

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et al., 1990). Moreover, CGs become increasingly competent to undergo exocytosis between metaphase I (MI) and MII (Ducibella and Buetow, 1994; Ducibella et al., 1990). The observation that the CGFD spatially overlaps with the MII chromatin and spindle suggests a close link between the two. Disruption of the spindle with microtubule inhibitors does not prevent formation of a CGFD over the chromatin (Connors et al., 1998). In fact, when these treatments result in a “scattering” of the chromatin, amicrovillar, actinrich CGFDs form over each of the smaller regions of the egg cortex containing chromatin (Connors et al., 1998; Longo and Chen, 1985; Van Blerkom and Bell, 1986). These findings suggest that a factor or factors specifically associated with the egg chromatin, and not the spindle itself, are responsible for altering the ultrastructure of the cortex and inducing a CGFD. What is not known is whether the chromatin induces localized CG exocytosis or redistribution to generate these CGFDs. We report here that inducing the formation of a CGFD in MII-arrested mouse eggs is a conserved property of chromatin from both animal and plant somatic cells and is heat-stable but lost following treatment with either DNase or a protease. Surprisingly, the chromatin-induced formation of a localized CGFD in a MII-arrested egg is due to CG redistribution and not CG exocytosis. This finding led us to reexamine the relative contributions of CG redistribution and exocytosis in the formation of the CGFD during mouse oocyte maturation. We find that the initial formation of the CGFD in the MI egg is due to CG redistribution and not exocytosis. Between MI and MII, CG redistribution continues to be the dominant factor in the further enlargement of the CGFD, although CG exocytosis that occurs during this time may also contribute.

Materials and methods Oocyte and egg collection and culture Female CF-1 mice (6 – 8 weeks old) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Fully grown, GV-intact oocytes and MII-arrested eggs were collected from gonadotropin-treated females as previously described (Mehlmann and Kline, 1994). The collection medium was modified Whitten’s medium (Whitten, 1971) containing 15 mM Hepes, pH 7.3, 7 mM NaHCO3, 10 ␮g/ml gentamicin, and 0.01% polyvinyl alcohol (PVA) (Whittens/Hepes/PVA) or CZB–Hepes (Kimura and Yanagimachi, 1995). For GV oocyte collection, the medium was supplemented with 0.25 mM dibutyryl cAMP to inhibit resumption of meiosis (Cho et al., 1974). Unless otherwise indicated, all cells were cultured in Whitten’s medium supplemented with 10 ␮g/ml gentamicin and 0.01% PVA (Whittens/PVA) at 37°C in a humidified atmosphere of 5% CO2 in air. For oocyte maturation experiments, oocytes were washed free of dibutyryl cAMP and then matured for 8 h in

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Whittens/PVA ⫾ 50 ␮M 1,2-bis(o-aminophenoxy)ethane N,N,N⬘,N⬘-tetraacetic acid acetoxymethyl ester (BAPTA/ AM; Molecular Probes, Eugene, OR). In one set of experiments, eggs were incubated in Whittens/PVA ⫾ 50 ␮M BAPTA/AM for 30 min, and then microinjected and cultured in medium containing 50 ␮M BAPTA/AM. Two to three hours after injection, the eggs were fixed and processed for CG and DNA staining (see below). Eggs were parthenogenetically activated by incubation for 5 min in Whittens/PVA containing 8% ethanol. Preparation of sperm heads and somatic nuclei for microinjection Sperm were collected from the caudae epididymides of 10- to 12-week-old B6D2F1/J males (Jackson Laboratories, Bar Harbor, ME) in 500 ␮l Whittens/PVA. The sperm were filtered through a layer of Kimwipes to remove large pieces of tissue and collected into an Eppendorf tube. The sperm were pelleted at 3000 rpm for 5 min at 4°C and then resuspended in ice-cold nuclear isolation medium (NIM) (Kimura et al., 1998) supplemented with 1% PVA (NIM/ PVA). To remove the sperm tails, the sperm suspension was sonicated four times for 15 s by using a Bransonic Ultrasonic Cleaner (Model 1210, Branson Utrasonic Corporation, Danbury, CT), then centrifuged as above, and the supernatant (that contained most of the sperm tails) was removed. The sperm heads were washed twice in 500 ␮l NIM/PVA and then resuspended in NIM/PVA containing 50% glycine and stored at ⫺20°C until use (up to 6 months). To destroy their ability to induce egg activation, sperm heads suspended in 400 ␮l NIM were heated in a water bath at 60 – 65°C for 30 – 40 min. The heat-treated sperm heads were washed twice in NIM/PVA, then resuspended in NIM/ PVA prior to use. In some experiments, the heat-treated sperm heads were permeabilized with 0.1% Triton X-100 and then treated with different reagents prior to microinjection. These treatments included 2 M NaCl in 25 mM Tris– HCl, pH 7.4, for 20 min, 500 units/ml DNase 1 (Amersham Pharmacia Biotech, Piscataway, NJ) in 40 mM Tris–HCl, 6 mM MgCl2, pH 7.5, at 37°C for 40 – 60 min, 100 ␮g/ml trypsin (Sigma, St. Louis, MO) in NIM at room temperature for 40 min, and a combination of 5% mixed alkyltrimethylammonium bromide (Sigma) and 20 mM dithiothreitol in NIM for 20 min to remove the perinuclear theca (Kimura et al., 1998). The treated sperm heads were washed once in 500 ␮l NIM/PVA, and then resuspended in NIM/PVA before injection into MII eggs. Somatic cell nuclei were isolated as follows from mouse cumulus cells, NIH 3T3 fibroblasts, round spermatids, and Arabidopsis thaliana protoplasts. Cumulus cells (obtained during egg collection) and NIH 3T3 cells were washed in PBS and resuspended in NIM/PVA. Round spermatids were collected from the testis as previously described (Kimura and Yanagimachi, 1998). Protoplasts were prepared freshly

Fig. 1. Effect of microinjection of exogenous chromatin on cortical granule localization. Either maturing or metaphase II-arrested eggs were microinjected with a single nucleus from cumulus cells, fibroblasts, heat-treated sperm or plant protoplasts. After 2– 4 h, the cells were stained for DNA and CGs, and representative images are shown. In this and all subsequent figures, DNA is shown in green, and CGs are shown in red. (A) Cumulus cell nucleus (n ⫽ 22). (B) Fibroblast nucleus (n ⫽ 15). (C) Sperm head (n ⫽ 18). (D) Protoplast nucleus (n ⫽ 7). MII, metaphase II egg chromatin; SP, inactive sperm chromatin; CC, cumulus cell chromatin; FB, fibroblast chromatin; PP, protoplast chromatin. In (A–C), the confocal section that included both sets of chromatin was near the egg surface, whereas the section in (D) was in the equatorial region. This accounts for the difference in the CG distribution. Fig. 2. Lack of Ca2⫹ oscillation-inducing activity in heat-treated sperm heads. Sperm heads were not treated (A, A⬘) or heat-inactivated (B, B⬘) prior to microinjection into eggs. The eggs were monitored for Ca2⫹ oscillations for 1–2 h (A, B), then fixed and stained for DNA and CGs (A⬘, B⬘). All untreated sperm heads (n ⫽ 9), but no heat-inactivated sperm heads (n ⫽ 11), caused Ca2⫹ oscillations. Representative Ca2⫹ tracings and confocal images are shown. MII indicates the position of the MII chromatin.

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Fig. 3. Effect of sperm head treatments on their ability to induce formation of a new CGFD. Sperm heads were either not treated (A, n ⫽ 33) or were treated with DNase (B, n ⫽ 21) or trypsin (C, n ⫽ 30) prior to microinjection into eggs. After 2 h, the eggs were fixed and stained for DNA and CGs, and representative confocal images are shown. MII, metaphase II egg chromatin; SP, sperm head DNA. Fig. 4. Effect of chelating egg intracellular Ca2⫹ on formation of a CGFD. MII-arrested eggs were loaded with BAPTA/AM (A, n ⫽ 45) or cultured in the same medium not containing BAPTA/AM (B, n ⫽ 55). The eggs then were microinjected with heat-inactivated sperm heads, and after 2 h were fixed and stained for DNA and CGs. The BAPTA-treated group was in the continuous presence of BAPTA/AM throughout the experiment, including the microinjection procedure. MII, metaphase II egg chromatin; SP, sperm head DNA.

from A. thaliana leaves as previously described (Chen and Halkier, 2000), then washed in PBS and resuspended in Whittens/Hepes/PVA. To isolate nuclei for microinjection, a 5-␮l drop of the suspension of intact somatic cells was placed on an inverted 100-mm petri dish lid adjacent to the drop of operation medium, covered with light mineral oil, and placed on the microscope stage. The cells were then aspirated in and out of a narrow bore pipette (5– 8 ␮m inner diameter) to break the plasma membrane and remove the cytoplasm as previously described (Kimura and Yanagimachi, 1995). Microinjection of eggs with sperm heads and somatic nuclei Microinjection pipettes were pulled by using a micropipette puller (model P-97; Sutter Instrument Co., Novato, CA) and then broken off at the appropriate tip location to form a flat tip of the appropriate inner diameter (5 ␮m for somatic cell nuclei and 8 ␮m for sperm heads) using a microforge (model MF-79; Narishige Co., Tokyo, Japan).

Microinjection of both sperm heads and nuclei was performed by using a piezo micromanipulator (model PMM; Prime Tech, Japan) as previously described (Kimura and Yanagimachi, 1995), except that all injections were done at room temperature, and for some experiments, the operation medium was Whittens/Hepes/PVA. In the case of eggs to be used for in vitro fertilization experiments, the operation medium contained heat inactivated 5% fetal calf serum (Life Technologies, Rockville, MD), and the eggs were washed free of serum immediately after microinjection. This was done to prevent ZP modifications due to mechanically induced CG release during microinjection. Care was taken to place the sperm heads or nuclei into the egg cortex as close to the plasma membrane as possible. After microinjection, the eggs were cultured for 2– 4 h and then fixed and processed for CG and DNA staining (see below). Detection of cortical granules and DNA For staining of intracellular CGs, the ZPs were removed by brief incubation in acid Tyrodes, pH 2.0, then the eggs were washed, fixed in 3.8% paraformaldehyde for 30 min at

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room temperature, and blocked in PBS containing 3 mg/ml BSA, 0.01% Tween 20 and 0.1 M glycine. CGs were detected with Lens culinaris agglutinin as previously described (Connors et al., 1998). For staining of extracellular CG exudate, ZP-intact eggs were fixed and then stained with FITC-conjugated UEA I as previously described (Lee et al., 1988). UEA I was used for these experiments to avoid nonspecific background staining of the ZP that occurs when Lens culinaris agglutinin is used. DNA was detected by mounting in Vectashield (Vector Laboratories, Burlingame, CA) containing 1.5 ␮g/ml DAPI. All cells were observed by using a Leica TCS NT confocal microscope at 400⫻ magnification. Images shown in the figures are confocal sections through the oocyte or egg region that included all chromatin present; therefore, some images are surface views and some are sections through the equatorial region of the egg. Quantification of the size of the CGFD was performed by using MetaMorph software (Universal Imaging Corp., West Chester, PA).

Calcium imaging Eggs were loaded with the Ca2⫹ indicator fura-2 (Molecular Probes, Eugene, OR) by incubation for 30 min in fura-2, acetoxymethyl ester (10 ␮M in Whittens/PVA containing 0.025% Pluronic F-127; Molecular Probes). The eggs were washed and then microinjected with either heatinactivated or untreated sperm heads. The injected eggs were placed on a temperature-controlled microscope stage in microdrops of Whittens/PVA under mineral oil and laminar flow of 5% CO2 in air. Cells were illuminated by using a 100-watt xenon arc lamp; light output was passed through a Lambda 10-2 filter wheel (Sutter Instrument Co., Novato, CA) to alternate excitation wavelengths between 340 and 380 nm. Emitted light passed through a fura-2 bandpass filter cube and was recorded by using a Princeton Instruments MicroMAX CCD camera (Roper Scientific, Trenton, NJ). The emitted fluorescence was averaged for each egg, and the 340/380 emission ratios were analyzed to determine alterations in intracellular Ca2⫹ using MetaFluor software (Universal Imaging Corp., West Chester, PA).

Zona pellucida conversion assay A biotin-based method to examine the conversion of ZP glycoprotein ZP2 to the proteolytically cleaved form, ZP2f, in individual eggs was performed as described previously (Moos et al., 1994). Briefly, ZPs were mechanically isolated, washed in 1% Triton X-100, rinsed, and biotinylated using 1 mM sulfouccinimidyl-6 (biotinamido) hexanoate (Pierce, Rockford, IL) in 0.1 M NaHCO3 (pH 8.3). The ZPs then were washed free of unbound biotin, transferred individually to 10 ␮l of SDS buffer and stored at ⫺20°C. The samples were electrophoretically separated, transferred, and processed for biotinylated protein detection by using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. The membrane then was dried and exposed to X-ray film. In vitro fertilization Eggs microinjected with inactivated or normal sperm heads, or control noninjected eggs, were cultured in Whittens/PVA for 2 h and then inseminated with capacitated sperm from B6SJLF1/J males (Jackson Laboratory, Bar Harbor, ME) as previously described (Moore et al., 1993). Three hours after insemination, the eggs were washed free of unbound sperm and then processed for DNA staining as above. The eggs were observed by using a Nikon TE 300 epifluorescence microscope. A sperm head injected egg was considered fertilized if two or more decondensing sperm heads were found in the cytoplasm of the egg, while noninjected eggs were considered fertilized if one or more decondensing sperm heads were seen. In all cases, the egg chromatin was distinctly visible.

Results Effect of microinjecting nuclei on cortical granule localization in mouse eggs To determine whether mouse egg chromatin-induced formation of CGFDs was a phenomenon specific to female germ cell chromatin, we microinjected male germ cell and somatic cell nuclei into eggs and then examined their effect on CG localization. Somatic cell nuclei from cumulus cells and fibroblasts were used, and mature sperm were used as a source of male germ cell chromatin. In the case of the mature sperm, to prevent CG exocytosis that occurs as a result of egg activation, the isolated sperm heads were heat-treated (Perry et al., 1999). Approximately 4 h after microinjection of nuclei isolated from cumulus cells or fibroblasts, the chromatin from these cells had undergone condensation and localization to the egg cortex. When these eggs were stained for CGs, the region of the cortex overlying and adjacent to the exogenous chromatin had developed a CGFD (Fig. 1A and B). Microinjected, heat-inactivated sperm heads decondensed and remained in the region of the egg cortex into which they had been injected. When these eggs were stained for CGs 2 h after microinjection of the sperm heads, a CGFD also was observed (Fig. 1C). Similar results were also observed when nuclei of round spermatids were injected (data not shown). The newly formed CGFDs easily could be distinguished from those associated with the egg chromatin because the egg chromosomes were well aligned on a metaphase plate. Of note, although the sperm heads decondensed, they did not transform into individual chromosomes prior to formation of the new CGFD. In addition, we frequently noted, as have others (Ducibella et

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al., 1990; Okada et al., 1986), an apparent increase in the CG density at the periphery of the CGFD of both the egg and exogenous chromatin (Fig. 1). To determine whether the CGFD-inducing factor was specific to mammalian cells, nuclei were isolated from A. thaliana protoplasts and microinjected into eggs. Similar to the mammalian cell nuclei, microinjection of the plant nuclei resulted in the formation of a CGFD (Fig. 1D). As a control, sperm tails and somatic cell cytoplasm were injected into the egg cortex by the same procedure as sperm head/nucleus injection, but these manipulations did not affect CG localization (data not shown). Effect of various treatments on the ability of sperm heads to induce changes in cortical granule localization To define further the factor(s) that induces formation of a new CGFD, we treated sperm heads in various ways, including extensive heating, and treatment with detergent, DNase, and protease. We used sperm heads for these experiments because they are easily isolated and manipulated, have relatively small amounts of cytoplasm, and after microinjection into eggs are simple to distinguish from the condensed metaphase II egg chromatin. To prevent sperm head-induced Ca2⫹ oscillations and CG exocytosis, the sperm heads were heat-treated prior to use (Perry et al., 1999). To ensure that the heat-treated sperm had lost completely their ability to induce Ca2⫹ oscillations in the eggs, Ca2⫹ imaging was performed after sperm head injection. The eggs injected with untreated control sperm heads exhibited Ca2⫹ oscillations (Fig. 2A), while eggs injected with heat-treated sperm heads had no Ca2⫹ increases (Fig. 2B). As expected, control sperm head injection resulted in extensive release of CGs (Fig. 2A⬘). Despite the lack of Ca2⫹ oscillations, heat-treated sperm heads induced formation of a CGFD in the region overlying the sperm head (Fig. 2B⬘). When these eggs were cultured for 5 h after injection, no morphological evidence of egg activation, including polar body extrusion and pronuclear formation, was observed in eggs injected with heat-treated sperm (n ⫽ 29), and this activity was retained even after heating to 100°C (data not shown). The activity was resistant to extraction procedures including treatment with 1% Triton X-100, 2 M NaCl, and a combination of 5% mixed alkyltrimethylammonium bromide and 20 mM dithiothreitol that has been shown previously to remove completely the sperm perinuclear theca (data not shown) (Kimura et al., 1998). In contrast, sperm heads treated with DNase to disrupt the integrity of the chromatin lost their ability to induce CGFDs (compare Fig. 3A with B). Similarly, trypsin treatment of sperm heads was sufficient to interfere with the ability of microinjected sperm heads to induce a CGFD (compare Fig. 3A with C). These findings were not due to placement of the sperm heads at an increased distance from the plasma membrane, as can be seen by the proximity of the sperm head to the plasma membrane in Fig. 3B and C.

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Cortical granule redistribution in response to heatinactivated sperm heads As mentioned in the Introduction, the generation of a new CGFD in response to sperm head microinjection could be explained by either localized CG exocytosis or CG redistribution. A local CG exocytosis would be Ca2⫹-dependent and result in the detection of the CG exudates on the plasma membrane with the corresponding conversion of ZP2 to ZP2f and a block to polyspermy. Although CG redistribution could be Ca2⫹ dependent, it would not result in these other changes. To discriminate between these two possibilities, we first utilized the Ca2⫹ chelator, BAPTA, to prevent alterations in the levels of cytoplasmic Ca2⫹. Heat-inactivated sperm induced formation of a CGFD in MII eggs despite BAPTA/AM treatment before, during, and after sperm head injection (Fig. 4). The Ca2⫹-buffering efficiency of BAPTA under the conditions used was confirmed by the observation that loading eggs with BAPTA/AM under identical conditions prior to insemination completely inhibited sperm induced Ca2⫹ oscillations, even though sperm– egg fusion was documented (data not shown). In addition to counting CGs, another indicator of CG exocytosis is the presence of CG exudate at the extracellular surface of the egg plasma membrane. This exudate can be visualized on the plasma membrane of nonpermeabilized eggs by using a lectin known as UEA I that stains the CG exudate without generating background staining of the ZP (Lee et al., 1988). To determine whether microinjected heat-inactivated sperm heads induced CG exocytosis, we stained nonpermeabilized eggs for CG exudate using UEA I. Eggs microinjected with untreated sperm heads and ethanol-activated eggs were used as positive controls for the procedure. Ethanol-activated eggs (n ⫽ 54) showed UEA I staining over the entire egg surface, except for the region that comprises the CGFD (Fig. 5A). Similarly, untreated sperm head-induced CG exocytosis resulted in UEA I staining that was particularly enhanced at the region of the plasma membrane overlying the sperm head, but not over the egg chromatin (n ⫽ 21) (Fig. 5B). In contrast, the untreated control eggs (n ⫽ 45) and eggs injected with inactivated sperm heads (n ⫽ 19) had no detectable UEA I staining (Fig. 5C and D). To verify these findings, biochemical assessment of the conversion of ZP2 to ZP2f was used as a second indicator of CG exocytosis in these same groups of eggs. Metaphase II eggs and eggs injected with either heat-inactivated sperm heads (n ⫽ 12) or buffer alone (n ⫽ 15) did not undergo ZP2 conversion (Fig. 6). In contrast, ethanol-activated eggs (n ⫽ 17), and eggs injected with untreated sperm heads (n ⫽ 16) underwent almost complete conversion of ZP2 to ZP2f (Fig. 6). As a physiological indicator of CG exocytosis, we performed in vitro fertilization on the sperm headinjected eggs to determine if the ZP block to polyspermy had occurred. Penetration of the ZP was documented by

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exocytosis could not account for the formation of this CGFD because there was no significant increase in the conversion of ZP2 to ZP2f in the treated oocytes. In contrast, between MI and MII, an increase in ZP2 to ZP2f conversion was observed and this increase was inhibited by BAPTA treatment (Fig. 7C). Nevertheless, the CGFD continued to enlarge between MI and MII in these BAPTAtreated eggs, but to a lesser extent than in control eggs (Fig. 8). Thus, CG exocytosis contributes to the enlargement of the CGFD between MI and MII. In toto, results of these experiments suggest that the initial formation of the CGFD in the MI egg results from CG redistribution and that although CG redistribution is the major mechanism for the further enlargement of the CGFD during the MI to MII transition, CG exocytosis is a contributing factor.

Discussion Fig. 5. Inability of heat-inactivated sperm heads to produce CG exudate on the egg plasma membrane. MII eggs were ethanol-activated (A), microinjected with active sperm heads (B), microinjected with heat-treated sperm heads (C), or untreated (D). After 2 h, the eggs were stained for DNA (green) and extracellular CG exudate (red). MII, MII egg chromatin; MPN, male pronucleus; FPN, female pronucleus, PB, second polar body; SP, sperm head DNA.

The results presented here indicate that the formation of a CGFD that occurs during maturation of mouse oocytes

determining whether any additional sperm heads, excluding the microinjected sperm heads, were decondensing in the egg cytoplasm. In agreement with results of the ZP2 conversion assay, which suggested the failure to establish the ZP block to polyspermy, when these groups of eggs were inseminated, penetration of the ZP by additional sperm occurred in most of the heat-treated sperm head-injected, and control noninjected metaphase II eggs (Table 1). As expected, only rarely were additional sperm able to penetrate the ZP of untreated sperm head-injected eggs (Table 1). Effect of BATPA on CGFD formation and ZP2 conversion during oocyte maturation The results described above indicated that the formation of the CGFD in the vicinity of the microinjected, heatinactivated sperm head was most likely due to CG redistribution and not CG exocytosis. This surprising finding led us to reexamine the role of CG redistribution in the formation of the CGFD during oocyte maturation. We first determined if the CGFD forms by MI in oocytes matured in the presence of BAPTA, which inhibits CG exocytosis following egg activation (Kline and Kline, 1992). Although the formation of the MI spindle was frequently perturbed, a CGFD formed despite BAPTA treatment (Fig. 7A and B). CG

Fig. 6. Effect of microinjection of heat-inactivated sperm heads on the conversion of ZP2 to ZP2f. Eggs were not microinjected (MII), microinjected with buffer (sham), ethanol activated (EtOH), or microinjected with inactivated sperm heads (IS) or untreated active sperm heads (AS), and then cultured for 2 h. The ZPs then were isolated and individually assessed for the conversion of ZP2 to ZP2f. (A) Representative gel image of individual biotinylated ZPs from the indicated treatment groups. (B) Quantification of the percentage conversion of ZP2 to ZP2f in each treatment group. Graph represents the mean ⫾ S.E. of a total of 11–17 ZPs in each group. In this and Fig. 7, the percentage of ZP2f ⫽ amount ZP2f/9 [amount ZP2 ⫹ amount ZP2f].

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Table 1 In vitro fertilization of eggs injected with heat-inactivated or untreated sperm Treatment group

# eggs counted*

# eggs with 1 sperm penetrated

# eggs with 2 sperm penetrated

# eggs with sperm penetrated after insemination (%)

Heat-treated sperm Untreated sperm Noninjected

40 53 51

21 4 42

8 0 8

29 (73%) 4 (8%) 50 (98%)

* Data are pooled from two independent experiments.

(and presumably other rodents) in response to chromatin is largely due to CG redistribution and not CG exocytosis. (Although it is formally possible that site-specific CG degradation induced by chromatin could lead to the appearance of a CGFD, we believe that this is most unlikely.) Nevertheless, limited CG exocytosis occurs between MI and MII (this study; and Okada et al., 1986, 1993), and we propose that this exocytosis, as well as the loss of CGs incorporated into the first polar body (Okada et al., 1986), contributes to the enlargement of the CGFD. This likely is a consequence of the maturation-associated acquisition of the ability of CGs to undergo exocytosis due in part to translocation of CGs to the cortex (Ducibella et al., 1990; Gulyas, 1980). Because CG exocytosis is a Ca2⫹dependent event (Abbott and Ducibella, 2001) and Ca2⫹/ calmodulin-dependent protein kinase II (CaMKII) is implicated in vesicle secretion (Easom, 1999; Schweitzer et al., 1995), the maturation associated increase in the amount of the inositol 1,4,5-trisphosphate type 1 receptor (Mehlmann et al., 1996) and CaMKII (Abbott et al., 2001) also could lead to an increase in constitutive levels of CG exocytosis between MI and MII. The ability to induce a CGFD by redistribution is a conserved property of chromatin from diverse sources, including male germ cells and somatic cells of plants and animals. Although we did not identify this activity, it remains associated with detergent-extracted sperm heads, is very heat-stable, and still is present following treatments that remove perinuclear theca material. Intact chromatin, however, is required, because either DNase or protease treatment results in a loss of the activity. These results suggest that the activity is tightly associated with intact chromatin. A possibility we considered is that Ca2⫹ tightly associated with condensed chromatin (Strick et al., 2001) induces localized CG redistribution. The experiments reported here utilizing the Ca2⫹ chelator, BAPTA, make this proposition unlikely. Treatment of eggs with amounts of BAPTA sufficient to prevent completely Ca2⫹ oscillations in response to a fertilizing sperm does not prevent the formation of a CGFD. Although very small local alterations in Ca2⫹ in the region of the chromatin may not be prevented by these treatments, it is not likely that such small Ca2⫹ shifts would induce such a major change in the subcellular distribution of CGs.

Chromatin could also acquire the ability to induce a CGFD after exposure to the egg cytoplasm. This could be accomplished by the direct physical association of a cytoplasmically derived activity, e.g., an egg protein, with the chromatin. Alternatively, the activity could be associated with an organelle(s) that becomes localized in the region of the chromatin. For example, in the mouse, mitochondria are enriched in the region of the MII chromatin (Calarco, 1995) and in the region surrounding individual microinjected bivalent chromatin (Van Blerkom and Bell, 1986). Although we do not observe an enrichment of mitochondria in the region of the injected sperm heads (data not shown), we cannot exclude the possibility that another organelle could be responsible. Nuclear vesicles that are likely of endoplasmic reticulum origin associate with chromatin during nuclear envelope assembly (Grant and Wilson, 1997). This association, however, appears to occur following metaphase, making it a less likely candidate organelle. Thus, although we defined several properties of the activity, its identity remains unknown. A maturation-associated decrease in the number of CGs is a common feature of oocytes from several species, e.g., human (Ducibella et al., 1995), but formation of a CGFD appears unique to rodents because a CGFD is not observed in cats (Byers et al., 1992), pigs (Kim et al., 1996; Wang et al., 1997), cows (Long et al., 1994), or humans (Santella et al., 1992). Coincident with development of the CGFD is the presence of a region highly enriched in cortical actin and devoid of microvilli that extends well beyond the limits of the meiotic spindle. These features are much less prominent in eggs of larger mammals (Sun et al., 2001). In addition to inducing CG redistribution, chromatin also induces the enrichment of cortical actin (Evans et al., 2000; Van Blerkom and Bell, 1986). As discussed in the Introduction, perhaps the development of this specialized region in rodent eggs in the vicinity of the chromatin is important to prevent sperm binding in the region of the MII spindle. Reduced sperm penetration in this region would minimize the potential for damage to the spindle or perturbation in chromosome segregation by the fertilizing sperm. This may be of particular importance in rodents in which the egg surface area is typically ⬃25% that of larger mammals (e.g., human, cow, pig) and therefore the risk is much higher in rodents of sperm entry in this region. The chromatin-induced formation of a region enriched in

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Fig. 7. Effect of BAPTA on CGFD formation and ZP2 conversion during oocyte maturation. GV oocytes were cultured to the MI stage in the presence (A, n ⫽ 27) or absence (B, n ⫽ 35) of BAPTA/AM, then were fixed and stained for DNA and CGs. Representative images are shown. (C) Quantification of the percentage conversion of ZP2 to ZP2f in each treatment group. Culture stages: GV, GV oocytes were incubated in IBMX-containing medium for 6 h; GV-MI, oocytes were incubated for 6 h; GV-MII, oocytes were incubated for 16 h. Stages in BAPTA indicate when during culture BAPTA-AM was present. Between 8 and 12 individual ZPs were analyzed in each group, and the data are expressed as the mean ⫾ S.E.M. Significant differences were found among the groups (P ⬍ 0.01, ANOVA). *, The difference is significant as compared with all other groups (P ⬍ 0.01, Fisher’s PLSD). **, The difference is significant as compared with all other groups (P ⬍ 0.01, Fisher’s PLSD).

cortical actin may be directly coupled to the formation of the CGFD by nucleating a regional reorganization of the egg cytoskeleton. CG redistribution could result from either active CG translocation using molecular motors or by CG exclusion during this reorganization. Treatment of eggs with nocodazole under conditions that completely disrupt the MII spindle has no effect on the formation of the CGFD following sperm head injection. Likewise, microfilament

disruption by cytochalasin D treatment has no effect (unpublished observations). It should be noted, however, that much of the egg cortical actin is refractory to cytochalasin D treatment (Brown et al., 2002; Connors et al., 1998), and thus, a role for microfilaments in CG redistribution remains unresolved. Experiments currently being performed using more potent modulators of actin polymerization may delineate the role of microfilaments in CG redistribution.

M. Deng et al. / Developmental Biology 257 (2003) 166 –176

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Fig. 8. Effect of BAPTA on the size of the CGFD. BAPTA-AM was absent (A) or present (B) in the culture medium from MI to MII, at which time the eggs were processed for CG localization and DNA staining. The experiment was conducted twice and shown are representative images. (C) Quantification of the relative size of the CGFD. The fraction of the circumference of the MII egg not containing CGs was determined. The experiment was conducted twice and a total of 20 eggs were analyzed. The data are expressed as the mean ⫾ S.E.M. The difference is significant (P ⬍ 0.01, t test).

Acknowledgments

References

We thank Marcela Michaut and Zhe Xu for assistance with the ZP2 conversion assay, Sixue Chen for preparation of plant protoplasts, and Shicheng Yang for providing NIH 3T3 cells. M.D. also thanks Marcela Michaut for discussions relating to the role of cortical granule redistribution in generating the cortical granule-free domain. This work was supported by a grant from the NIH (HD 22732) to G.S.K., R.M.S., and C.J.W., and grants from the Harold Castle Foundation and the Kosasa Family Foundation to R.Y. Part of this work was done in the Intermediate Voltage Electron Microscopy and Biomedical Image Analysis Facility at the University of Pennsylvania, supported by NIH Grant RR2483.

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