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Regular Article |
a Department of Anatomy and Cellular Biology, Sackler School of Biomedical Sciences,
b and the Department of Obstetrics and Gynecology, Tufts University School of Medicine and New England Medical Center, Boston, Massachusetts 02111
c Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, gamete biology, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mouse oocyte's ability to undergo CG secretion arises very late in oocyte maturation, before ovulation and after completion of oocyte growth [4–6]. During this period, the oocyte undergoes meiotic maturation from the fully grown, prophase I-arrested, germinal vesicle (GV)-stage to the MII-arrested stage, at which point the mammalian egg is normally fertilized. Additionally, the oocyte undergoes cytoplasmic maturation, which results in development of the abilities to release and respond to [Ca2+]i and which culminates in an MII-stage egg that is maximally competent to be activated by a fertilizing sperm. A subset of human eggs that have matured meiotically to the MII-stage fail to activate following sperm and Ca2+ injection [7, 8], suggesting that cytoplasmic immaturity, or the inability to support egg activation, can be separated from nuclear maturity and may account for some cases of failed fertilization.
Previously published reports indicate that preovulatory GV-stage oocytes have a higher incidence of zona penetration or polyspermy (reviewed in [9]), indicating that the low level of fertilization-induced CG release is not sufficient to establish the zona block to polyspermy. During oocyte maturation, the ability to release [Ca2+]i is acquired and correlates with maturation-associated changes in the amount of critical type-1 inositol 1,4,5-trisphosphate (IP3) receptors [10, 11], sensitivity to IP3 [12, 13], and reorganization of the endoplasmic reticulum (ER) [14–16], which stores and releases [Ca2+]i. These changes are thought to account for decreased [Ca2+]i release by preovulatory oocytes relative to that by MII-stage eggs in response to fertilization and numerous [Ca2+]i release agonists [13, 16, 17]. However, if reduced [Ca2+]i release is responsible for the failure of CG exocytosis in preovulatory oocytes is not known.
Recently, we found that, during meiotic maturation, the oocyte also undergoes a substantial increase in the ability to respond to [Ca2+]i, which is sufficient to account for the aforementioned failure of CG exocytosis. When [Ca2+]i oscillations similar to those of fertilized MII-stage eggs were experimentally induced in preovulatory GV-stage oocytes, little, if any, CG secretion was detected compared to that in MII-stage eggs [18]. Thus, the development of competence to undergo activation events, such as CG secretion, requires a maturation-associated change (or changes) downstream of [Ca2+]i release, likely including Ca2+-dependent proteins and/or effectors.
One such effector of Ca2+ signaling that is activated on [Ca2+]i elevation in mouse eggs [19–21] is Ca2+/calmodulin-dependent protein kinase II (CaMKII), which has been implicated in meiotic resumption [20, 22] and in regulation of CG exocytosis [21]. In addition, CaMKII regulates translocation of synaptic vesicles in neurons [23, 24]. Therefore, CaMKII is an important candidate Ca2+ effector for CG exocytosis.
Our overall strategy was to identify the blocked step in the secretory pathway, the identity of which would narrow down the likely candidate biochemical effectors responsible, which could then be investigated. To identify the specific step that is deficient or inhibited in the secretory pathway, our first objective was to test three hypotheses by asking whether deficiencies are present in one of the following steps of the secretory mechanism: translocation, docking, and/or fusion. Morphologically "docked" CGs were defined as those in direct contact with the plasma membrane. The experimental strategy was to induce [Ca2+]i oscillations, to perform electron microscopy, and then to measure the distance of the CGs to the plasma membrane. The [Ca2+]i oscillations were induced by in vitro fertilization (IVF) or sperm cytosolic fraction (SF) injection. These treatments result in extensive CG exocytosis only in mature MII-stage eggs, whereas repetitive [Ca2+]i oscillations result at both stages [13, 25, 26]. The SF injection induces MII-like [Ca2+]i oscillations in GV-stage oocytes [25], whereas fertilization induces fewer [Ca2+]i oscillations in GV-stage oocytes relative to that in MII-stage eggs [13]. The final location and distance of CGs after stimulation would indicate if a block existed at the step of translocation, of docking, or of fusion. Because maturation-associated changes in the ER [14–16, 27–30] may be involved in the development of secretory competence, a second objective was to examine the proximity between CGs and cortical ER structures. A final objective of this study was to determine the amount and activity of a candidate biochemical regulator of translocation, CaMKII, during oocyte maturation.
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MATERIALS AND METHODS |
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The GV-stage oocytes and ovulated MII-arrested eggs were obtained from 6- to 10-wk-old female CF-1 mice (Charles River, Wilmington, MA; Harlan, Indianapolis, IN) following standard gonadotropin injection. For GV-stage oocytes, mice were primed with 5 IU of eCG, and cumulus-enclosed, fully grown oocytes were collected 44–48 h later by teasing apart ovaries with needles in Earle balanced salt solution containing 0.3%–0.6% (w/v) BSA or 0.1%–0.4% (w/v) polyvinylpyrolidine with 25 mM Hepes buffer at pH 7.3. Cumulus cells were removed by repeated pipetting. For MII-stage eggs, mice were primed with 5 IU of eCG and 44–48 h later with 5 IU of hCG, and eggs were collected 13–15.5 h post-HCG injection in the above medium. Cumulus cells were removed by treatment with 0.01% hyaluronidase for 3–5 min at 37°C.
IVF of Zona-Free Oocytes and Eggs
Sperm from the caudal region of the epididymides were collected in IVF medium [31] with 1% BSA and allowed to capacitate for 90 min at 37°C and 5% CO2. Zonae were removed from oocytes and eggs with brief treatment in acid Tyrode solution and allowed to recover in IVF medium with 1% BSA for 60 min before insemination. The IVF of zona-free eggs and oocytes allowed for a high percentage of fertilization, which was approximately synchronous. Eggs and oocytes were inseminated with 200 000 sperm/ml. To verify fertilization, eggs and oocytes were stained with 0.001% Hoescht 33258 for 5–10 min and then examined by epifluorescence microscopy for a decondensing sperm head.
Sperm Cytosolic Fraction Microinjection
Microinjections were performed in TL-Hepes with 2.5% sucrose (w/v) as previously described [32, 33]. Micromanipulations were performed using Narishige manipulators (Medical Systems, Great Neck, NY) mounted on a Nikon Diaphot microscope (Nikon, Inc., Garden City, NJ). Cytosolic sperm extracts were prepared from boar semen as described by Wu et al. [25, 26]. Briefly, semen samples were washed twice with TL-Hepes medium, and the sperm pellet was resuspended in a solution containing 75 mM KCl, 20 mM Hepes, 500 µM EGTA, 10 mM glycerophosphate, 1 mM dithiothreitol (DTT), 200 µM PMSF, 10 µg/ml of pepstatin, and 10 µg/ml of leupeptin, pH 7.0. The sperm suspension was sonicated for 15–25 min at 4°C (XL2020; Heat Systems, Inc., Farmingdale, NY). The lysate was then centrifuged twice at 10 000 x g for 45 min at 4°C and followed by ultracentrifugation. The clear supernatant was used as the cytosolic fraction. Ultrafiltration membranes (Centricon; Amicon, Beverly, MA) were used to wash the supernatant (75 mM KCl and 20 mM Hepes, pH 7.0) and to concentrate these extracts to 25 mg/ml of protein, followed by ammonium sulfate precipitation. Aliquots of sperm extracts were frozen at -80°C. A final protein concentration of 1 mg/ml in the pipette was used. The total amount of protein injected into an individual egg was 12.5–25 pg.
Fixation and Electron Microscopy
Eggs and oocytes were fixed 120–150 min posttreatment in 3% gluteraldehyde and 0.5% paraformaldehyde in 0.05 M cacodylate buffer for 20 min at 25°C for electron microscopy. Samples were then washed in buffer and postfixed with 1% osmium tetroxide and 0.8% potassium ferricyanide (to enhance membrane staining) for 60 min. Next, samples were washed in buffer and water and stained with 0.5% uranyl acetate for 15 min at 4°C. Finally, samples were dehydrated and embedded in EMbed 812-araldite (Electron Microscopy Sciences, Port Washington, PA). Silver sections (thickness, 
60–90 nm) were stained with 7.5% uranyl acetate in 50% ethanol for 45–60 sec and with Sato triple lead solution for 5–10 min. A Philips CM-10 electron microscope (Philips/FEI Co., Hillsboro, OR) was utilized. For each group of GV-stage oocytes, 9–10 oocytes were analyzed, and for each oocyte, 10 micrographs from nonadjacent sections were obtained. The distance of each CG to the plasma membrane was determined by taking the shortest perpendicular measurement from the plasma membrane to the CG surface closest to the plasma membrane.
Western Blot Analysis
Mouse anti-
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and anti-ß CaMKII were obtained from Stressgen (Victoria, BC, Canada) and Zymed Laboratories (San Francisco, CA), respectively. Mouse IgG was obtained from Sigma (St. Louis, MO). Antibodies were biotinylated with immunopure NHS-LC-biotin (Pierce, Rockford, IL) following the manufacturer's instructions. The SDS-PAGE was performed, and the proteins were transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA). Membranes were blocked overnight in PBS + 0.1% Tween-20 and 6% nonfat dried milk (Carnation, Nestle, Glendale, CA). Blots were incubated with primary antibody (1:1000 w:v) overnight, washed, and incubated with Vectastain (Vector Laboratories, Burlingame, CA) for 1 h, washed for more than 1 h, and followed by use of the SuperSignal ECL detection system (Pierce). Blots were exposed to film, and multiple exposures (10 sec to 15 min) were taken in the linear range of detection. Band quantification was performed by controlled densitometry with the Millipore Bio-Image System (Millipore) and 2D Analysis software (Millipore).
CaMKII Activity Assays
The SignaTECT CaMKII assay system (Promega, Madison, WI) was used according to the manufacturer's instructions. Purified CaMKII (Upstate Biotechnology, Lake Placid, NY) was used as a positive control. Briefly, eggs or oocytes were collected and frozen in extraction buffer (20 mM Tris-HCl, 2 mM EDTA, 2 mM EGTA, 20 µg/ml of soybean trypsin inhibitor, 10 µg/ml of aprotinin, 5 µg/ml of leupeptin, 2 mM DTT, 25 mM benzamidinem, and 1 mM PMSF) on dry ice and stored at -80oC until use. Samples were added to a reaction mix of 50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM DTT with 1 mM CaCl2, 1µM calmodulin and 0.02 mg/ml BSA, 0.1 mM ATP, [-32P]ATP (3000 Ci/mmol, 10 mCi/ml; NEN Life Science, Boston, MA), and 0.1 mM biotinylated peptide substrate and then incubated at 32°C for 2 min. The reaction was stopped with the addition of 2.5 mM guanidine hydrochloride. Next, 10–25 µl were spotted onto a SAM2 Biotin Capture Membrane, washed, dried, and exposed to a storage phosphor screen (Kodak, Rochester, NY). Results were visualized and quantified using the STORM 860 imaging system and ImageQuant version 1.2 software (Molecular Dynamics, Sunnyvale, CA). Identical circles were used to quantify the average count for each spot, and samples of equivalent numbers of eggs or oocytes were compared within the same experiment to determine the relative difference between GV and MII stages. Relative differences were pooled from different experiments. Increasing amounts of purified CaMKII (1.56–25 ng) were used to verify that the average count detected in the assay was linear with respect to the amount of CaMKII. Autocamtide-2, a specific peptide substrate for CaMKII [34] obtained from Biomol (Plymouth Meeting, PA), was used as a control for the kinase assay experiments as a competitor peptide to the biotinylated peptide substrate.
Statistical Comparisons
Statistical comparison of mean CG distances to the plasma membrane was performed using one-way ANOVA. Observed differences were examined using the unpaired t-test with Microsoft Excel software. Statistical comparison of CaMKII activity of GV-stage oocytes and MII-stage eggs was performed using the unpaired t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine at which step secretion is blocked, the extent of translocation, docking, and fusion in preovulatory GV-stage oocytes was analyzed following fertilization and SF injection. Three important aspects of the experimental design should be emphasized (see Introduction and Discussion for references). First, it was necessary to provide these oocytes with sufficient time after stimulation to complete any of the aforementioned steps. Because mature MII-stage eggs (positive controls) require 60–120 min for nearly complete CG exocytosis (90%), a time of 120–150 min was used. Second, many studies have previously demonstrated that GV-stage oocytes from preovulatory follicles are not competent to release the contents of CGs into the perivitelline space. Unlike MII-stage eggs, such GV-stage oocytes release only 0%–10% of CGs. Thus, >90% of CGs will not be lost during the 120-min poststimulation period but, instead, will remain for analysis for the extent of translocation, docking, and fusion. (Note that incomplete fusion could technically occur and would be detected by electron microscopy in the present study, but not necessarily in previous fluorescence studies of CGs.) Third, in both the preovulatory GV-stage oocytes and MII-stage eggs in the mouse, nearly all CGs are localized to the cell cortex within 2 µm of the plasma membrane.
After stimulation and incubation, oocytes and eggs were fixed, and the positions of CGs relative to the plasma membrane were determined using electron microscopy. In untreated MII-stage eggs, CGs were observed in a nondocked state (Fig. 1A). As expected, and as previously reported, no CGs were observed in the cortex of SF-injected MII-stage eggs (Fig. 1B), because CGs are secreted in response to SF-induced [Ca2+]i oscillations. Due to CG secretion in MII-stage eggs, translocation, docking, and fusion had already taken place; thus, no distance data for MII-stage eggs are reported. In contrast, in the secretion-incompetent GV-stage oocytes, CGs were observed in a morphologically nontranslocated and nondocked state in all three groups: control (Fig. 1C), fertilized, and SF-injected (Fig. 1D).
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Measurements were taken of individual CG distances to the plasma membrane in GV-stage oocytes. If a sufficient number of CGs in treated GV-stage oocytes underwent full or partial translocation, then a reduction in the mean CG distance from the plasma membrane would be expected. However, the mean CG distance (±SEM) in fertilized GV-stage oocytes, 0.30 ± 0.013 µm, was not significantly different from that for control oocytes, 0.30 ± 0.016 µm (Fig. 2). The mean distance (±SEM) for SF-injected GV-stage oocytes was also not reduced relative to that for controls but was greater, being 0.42 + 0.019 µm (Fig. 2). Comparable distributions of individual CG distances were observed among the three groups (Fig. 3). A bimodal distribution, indicating translocation of a small subset of CGs, was not observed. Therefore, these data indicate that preovulatory GV-stage oocytes are incompetent to undergo CG translocation on [Ca2+]i oscillations.
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CGs in Preovulatory GV-Stage Oocytes and Mature MII-Stage Eggs Are in Close Proximity to the ER Network
In the process of analyzing CG distances to the plasma membrane, we observed numerous membranous organelles in the cortex, consistent with the structure and distribution of the ER. Interestingly, many sectioned membrane profiles were in very close apposition to CGs and/or the plasma membrane in both GV-stage oocytes and MII-stage eggs (Fig. 4).
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Changes in CaMKII Amount and Activity Correlate with Acquisition of Secretory Competence During Oocyte Maturation
Because oocyte maturation is accompanied by changes in the amounts of various proteins [35, 36], the amount of a candidate regulator of CG translocation, CaMKII, at the GV and MII stages was examined. Two monoclonal antibodies that collectively recognize all four isoforms of CaMKII were used to detect total CaMKII in mouse oocytes and eggs. In SDS-PAGE, the ß, , and isoforms of CaMKII all migrate at 59–61 kDa, unlike the isoform, which migrates at 50 kDa. In Western blots of oocytes and egg lysates, a single specific band at approximately 60 kDa was detected that corresponded with one of two primary bands (50 and 60 kDa) detected in mouse brain lysate (Fig. 5A). Although CaMKII was detected in brain lysate, no corresponding band was observed in eggs or oocytes. No bands migrating at 50–60 kDa were observed on blots probed with nonspecific mouse IgG (Fig. 5A) or with the secondary reagent alone (data not shown). Quantification of the Western blots indicated a mean 150% increase in CaMKII in MII-stage eggs relative to that in GV-stage oocytes (Fig. 5B).
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Further analysis with an antibody that specifically recognizes the ß isoform revealed a 60-kDa band in MII-stage egg lysates, whereas no bands between 50–60 kDa were detected with an antibody that recognizes the , , and isoforms. Thus, ß CaMKII appears to be the predominant isoform in MII-stage eggs. This analysis was repeated three times; results were similar to those in Figure 5A, lane 2.
The maturation-associated increases determined by Western blot analysis should be accompanied by an increase in total CaMKII activity in MII-stage eggs relative to GV-stage oocytes. To test this prediction, saturating levels of Ca2+ and calmodulin were used to examine total CaMKII activity under maximally activating conditions (henceforth referred to as maximal activity). A representative experiment is shown in Figure 6A. In negative control samples, omission of Ca2+ and calmodulin resulted in low levels of kinase activity (Fig. 6A). For an additional negative control, the specific CaMKII substrate competitor, autocamtide-2, was added to samples of GV-stage oocytes or MII-stage eggs and inhibited CaMKII phosphorylation of the biotinylated substrate detected by this assay (data not shown). In assays comparing equal numbers of eggs and oocytes, the CaMKII activity of MII-stage eggs was 110% greater than that of GV-stage oocytes (Fig. 6B). Thus, the relative maturation-associated increases in CaMKII activity (110%) and amount (150%) are similar.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have demonstrated that, during maturation, a reorganization and increase occur in cortical ER in the eggs of worms [42], sea urchins [29, 30], frogs [27, 28], hamsters [16], and mice [14, 15]. These changes have been suggested to be responsible, in part, for the maturation-associated development of the [Ca2+]i-release mechanism. Additionally, in Xenopus eggs, a cortical network of ER develops that nearly completely surrounds individual CGs during maturation [27, 28]. This tight apposition between the ER and CGs has not been observed by electron microscopy in MII-stage mouse eggs [14, 43]. However, in this study, CGs were observed in close proximity to ER structures in both GV-stage oocytes and MII-stage eggs. The elongate and tubular ER profiles observed by electron microscopy likely correspond to similar profiles of cortical, filamentous DiI staining at both stages during mouse oocyte maturation [15, 44]. Our results suggest that secretory incompetence is not due to a lack of proximity of the CGs to the ER.
One hypothesis to account for the observed translocation deficiency is that the cytoskeleton participates in or promotes CG secretion in MII-stage eggs, but not in GV-stage oocytes. Alternatively, cytoskeletal elements could block translocation in oocytes. For example, microtubules, which are present in the cortex of GV-stage oocytes [45, 46] but are primarily absent in the cortex of MII-stage eggs, may inhibit CG translocation in GV-stage oocytes. However, to our knowledge, no evidence exists for CG association with microtubules or for a physical barrier of microtubules. A role for microfilaments rather than for microtubules in CG secretion and translocation has also been proposed. During oogenesis, CGs are translocated to the cortex by an actin microfilament-dependent mechanism [47, 48]. In MII-stage eggs, microfilament reorganization has been implicated in CG secretion [49–51], but the role of these microfilaments is unknown. Also, disassembly of cortical actin alone is insufficient to induce CG exocytosis [48], unlike in adrenal chromaffin cells, in which exocytosis is preceded by a disassembly of the cortical actin cytoskeleton that, presumably, acts as a barrier to prevent secretion [52].
An alternate hypothesis is that oocytes are deficient in a secretory machinery protein (or proteins) or a Ca2+-dependent signaling component necessary for CG translocation and subsequent secretion. We chose to examine a suitable candidate effector, CaMKII, that is also present in somatic secretory cells, including neurons [23, 24] and pancreatic cells [53]. The secretory mechanism in these cells has been much more thoroughly studied and shares many of the same basic secretory machinery proteins with eggs [54]. In neurons [23, 24], CaMKII is proposed to promote translocation by regulating the availability of synaptic vesicles for exocytosis through the phosphorylation of synapsin-1, which appears to untether vesicles from the actin cytoskeleton. Interestingly, the development of secretory responsiveness in pancreatic cells is also accompanied by an increase in the activity of CaMKII to phosphorylate exogenous synapsin-1 [55].
In vertebrate eggs, CaMKII is an important candidate Ca2+-effector molecule in egg-activation events. It regulates cell-cycle resumption in MII-stage Xenopus eggs by inactivating maturation-promoting factor and cytostatic factor [22]. In mouse eggs, artificial induction (via Ca2+ ionophore or ethanol) of CG secretion and exit from the MII stage are inhibited by CaMKII antagonists [20, 21]. In addition, CaMKII is activated on experimental elevation of [Ca2+]i in mouse eggs [19–21]. It is important to note that, although evidence to date indicates a role for CaMKII in meiotic resumption and CG release, full confirmation regarding a physiological role for CaMKII during mammalian fertilization requires further investigation and should be an active area of future research.
To our knowledge, the present study provides the first demonstration by Western blot analysis of CaMKII in mammalian eggs. Having demonstrated that CaMKII is present in MII-stage mouse eggs, the present study also found that the amount and maximal activity of CaMKII is greater in MII-stage eggs than in GV-stage oocytes. Because these maturation-associated changes in CaMKII amount and activity were determined in pooled samples of oocytes, it cannot be excluded that a subset of GV-stage oocytes contain MII-like levels of CaMKII yet are still incompetent to undergo secretion. These findings provide a testable hypothesis that these maturation-associated changes in CaMKII are responsible, in part, for the acquisition of activation competence in mammalian eggs, because CaMKII is likely involved in several important events at fertilization. Studies are in progress to determine if the activity of CaMKII in physiologically activated GV-stage oocytes is less than that in MII-stage eggs and if exogenous CaMKII can rescue GV-stage oocytes from secretory incompetence.
An alternate Ca2+-effector molecule is protein kinase C (PKC). The Ca2+-dependent and -independent isoforms of PKC are present in mouse [56] and rat [57] eggs. Although PKC is activated at fertilization [58] and phorbol esters induce CG secretion during both the GV and MII stages [5], PKC activity does not appear to be required for fertilization-induced CG exocytosis [59]. Also, phorbol ester-induced CG secretion in pig oocytes occurs by a Ca2+-independent mechanism [60]. Therefore, the extent to which PKC is a normal physiological effector for CG secretion is unresolved.
Further work is needed to determine if a causal relationship exists between the observed CaMKII increases and the development of translocation or secretory competence. At this time, it cannot be ruled out that the docking and/or fusion steps in CG secretion are also blocked in GV-stage oocytes. That PKC agonists can stimulate significant CG exocytosis in GV-stage oocytes suggests that the entire secretory machinery is not inhibited [5]. We suggest that this secretory incompetence at the step of translocation in preovulatory oocytes is a protective mechanism guarding against global CG release and premature modification of the zona pellucida, which could inhibit fertilization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Lalor Foundation (to A.L.A.), NICHD (HD 24191 to T.D.), and the U.S. Department of Agriculture (99-2371 and 97-2919 to R.A.F.). 
2 Correspondence and current address: Tom Ducibella, Dept. of Obstetrics and Gynecology, New England Medical Center, Box 36, Boston, MA 02111. tom.ducibella{at}tufts.edu
Accepted: June 29, 2001.
Received: April 18, 2001.
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