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BOR - Papers in Press, published online ahead of print February 6, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026161
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BIOLOGY OF REPRODUCTION 70, 1685–1692 (2004)
DOI: 10.1095/biolreprod.103.026161
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Stimulation of Xenopus laevis Oocyte Maturation by Methyl-ß-Cyclodextrin1

Susan E. Sadler2, and Nicole D. Jacobs

Department of Biological Sciences, University of Denver, Denver, Colorado 80208


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cholesterol-depleting drug methyl-ß-cyclodextrin (Me-ß- CD) was tested for its effects on amphibian oocyte maturation, cholesterol depletion, and low-density membrane recovery. Progesterone-induced oocyte maturation was accelerated by pretreatment of cells with 5–50 mM Me-ß-CD in a dose-dependent manner. Treatment of oocytes with 50 mM Me-ß-CD alone was sufficient to induce germinal vesicle breakdown, stimulate formation of meiotic spindles, and stimulate phosphorylation of mitogen-activated protein kinase over time courses longer than those observed after progesterone treatment. After short-term (30 min) labeling of oocytes with [3H]cholesterol, 30–90 min of treatment with 5–50 mM Me-ß-CD removed 50%–70% of cell- associated label, and cholesterol depletion was not observed with {alpha}-cyclodextrin. After long-term (20–23 h) labeling of oocytes with [3H]cholesterol, Me-ß-CD treatment resulted in dose- dependent cholesterol depletion in the 5–50 mM range, and 50 mM Me-ß-CD removed approximately 50% of cell-associated label after 9 h. Treatment of oocytes with 5–50 mM Me-ß-CD also decreased recovery of low-density membrane by detergent- free sucrose gradient centrifugation. These results implicate cholesterol and low-density membrane domains in the signaling mechanisms leading to germinal vesicle breakdown in amphibian oocytes.

gametogenesis, meiosis, progesterone, signal transduction, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulating evidence supports a role for low-density membrane (LDM) microdomains (rafts and caveolae) in cell signaling and in transmembrane and transcellular processing (for reviews, see [1–4]). Cholesterol-enriched LDM containing immunodetectable caveolin-like protein has been recovered from Xenopus laevis oocytes [5]. Comparison of past observations in the oocyte system with more recent evidence in other cells suggests the intriguing possibility that LDM might be involved in triggering oocyte maturation. Isolated amphibian oocytes can be induced to mature (reinitiate the meiotic cell cycle) in vitro in response to treatment with the presumed natural steroid progesterone [6] or with insulin or insulin-like growth factor 1 [7, 8]. One early oocyte response to treatment with inducing hormone is a decrease in intracellular cAMP [9] that can be accounted for at least in part by inhibition of adenylyl cyclase [10, 11] and stimulation of phosphodiesterase type III [12–14]. Inhibition of adenylyl cyclase by progesterone does not involve pertussis toxin-sensitive G{alpha}i subunits of the heterotrimeric G protein complex [15], is correlated with slowing of guanine nucleotide exchange, and shares certain features with P site agonist action [16]. In other cell systems, P site adenosine action may be associated with caveolae [17].

Gallo et al. [18] reported that microinjection of an affinity-purified antibody that inhibits G{alpha}s activity is sufficient to stimulate oocyte maturation with a time course similar to that for oocytes treated with progesterone. Immunogold electron microscopic examination revealed clumped arrays of G{alpha}s on internal yolk platelet membranes and the plasma membrane, suggesting that oocyte G proteins are present in patches that are reminiscent of membrane rafts. Mouse oocytes have also been released from meiotic arrest after injection of anti-G{alpha}s antibody [19]. Other experimental evidence suggests that Gß{gamma} subunits are the principal mediators of progesterone action in amphibian oocytes [20]. Heterotrimeric G proteins interact with caveolin, the signature protein of caveolae in mammalian cell systems, and association of G{alpha} with caveolin within caveolar structures may impose negative regulation thus stabilizing the membrane- bound pool of G{alpha} in its inactive form [21]. Even though the direct interaction of G protein subunits with caveolin has been debated [22, 23], it is widely accepted that organization of adenylyl cyclase and G proteins into plasma membrane subdomains may coordinate and optimize signal transduction.

The liquid-ordered biophysical characteristics of rafts and caveolae are stabilized by the interactions of cholesterol, sphingolipids, and gangliosides, and various cholesterol-depleting drugs disrupt caveolae structure and function [24–27]. To test what effect cholesterol depletion might have on steroid-induced oocyte maturation, we used methyl-ß-cyclodextrin (Me-ß-CD), a cyclic oligosaccharide consisting of seven ß(1-4)glucopyranose units that form a torus- or ring-shaped structure with an external hydrophilic face and internal hydrophobic core. The ß-cyclodextrins act as external cholesterol sponges that remove membrane cholesterol by adhesional contact with the cell surface without intercalating into the plasma membrane [28]. We originally hypothesized that if the organizing influence of cholesterol- rich membrane microdomains were required for progesterone-induced inhibition of adenylyl cyclase, then cholesterol depletion would inhibit the steroid-induced response. However, the opposite effect was observed. Millimolar concentrations of Me-ß-CD accelerated progesterone-induced oocyte maturation, and treatment of oocytes with 50 mM Me- ß-CD was sufficient to induce germinal vesicle breakdown (GVBD) and stimulate phosphorylation of mitogen-activated protein kinase (MAPK), leading to chromosome condensation and spindle formation in the absence of added steroid. The cell division response was correlated with the ability of Me-ß-CD to deplete cholesterol after overnight loading of oocytes with radiolabeled cholesterol and to disrupt LDM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Animals and Oocyte Isolation

Mature Xenopus laevis females (Xenopus I, Dexter, MI) were housed in aquatic tanks in a temperature controlled room at 16°C, fed ground beef twice weekly, and maintained on daily cycles of 14L:10D. Frogs were primed by injection of 35 IU of eCG (PMSG; Calbiochem, La Jolla, CA) into the dorsal lymph sac 3–7 days before surgical removal of ovary. Prior to surgery, frogs were anesthetized by partial immersion in 200 ml of solution containing 0.12% tricaine (3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO) in 25 mM Hepes (Calbiochem), pH 7.0. Pieces of ovary were stored at room temperature in oocyte Ringers (83 mM NaCl, 1 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 10 mM Hepes, pH 7.9). Oocytes (stages V or VI according to Dumont [29]) were manually dissected using watchmaker's forceps under a stereomicroscope and stored in oocyte Ringers at room temperature until used for drug or hormone treatment.

Hormone/Drug Treatments and Monitoring of GVBD

Groups of oocytes were incubated in the indicated volumes of Ringers- HCO3 (73 mM NaCl, 10 mM NaHCO3, 1 mM MgCl2, 0.5 mM CaCl2, 1 mM KCl, 25 mM Hepes, pH 7.9) in the absence or presence of 1.5 µM progesterone (Calbiochem; diluted from a 0.5 mg/ml stock solution in ethanol stored at room temperature) or indicated concentrations of Me-ß- CD (Sigma) or {alpha}-cyclodextrin ({alpha}-CD; Sigma). GVBD was routinely monitored by tracking white spot formation followed by fixation of oocytes in 2% trichloroacetic acid, dissection using watchmaker's forceps under the stereomicroscope, and examination for the presence or absence of an intact nucleus (germinal vesicle). The results are expressed as the percentage of the sample population responding (%GVBD) as a function of time of treatment or drug treatment.

Histochemical Detection of Meiotic Spindles

Oocytes were fixed in 1% acetic acid in 95% ethanol for 24–48 h, dehydrated through an alcohol series (50%, 70%, 95%, and 100%), cleared in Neo-Clear (a xylene substitute; Harleco, EM Science, Gibbstown, NJ), embedded in Paraplast (Fisher Scientific, Pittsburgh, PA), and sectioned at 9 µm. Sections were float mounted on Superfrost/Plus slides (FisherBrand, Fisher Scientific) and warmed on a slide warmer for 1 h. After clearing in Neo-Clear and rehydrating down an alcohol series (100%, 95%, 70%, 50%), sections were stained for 2 h in freshly made aqueous 0.1% hematoxylin (Sigma). After staining, mounted sections were gently rinsed with deionized water for 2 min, immersed in 0.1% sodium bicarbonate for 1 min, and counterstained in 0.5% methyl green (0.5% dilution into 70% ethanol of 2% stock solution in water). Slides were then rinsed in 70% alcohol, immersed in 95% alcohol for 1 min, and transferred through two changes of 100% alcohol before being cleared through three changes of Neo-Clear and mounted using Permount (Sigma).

Assessment of MAPK Phosphorylation and ERK1/2 Levels

Phosphorylation of MAPK was observed by immunoblot detection as modified from the method of Haccard et al. [30]. After oocytes were incubated in Ringers-HCO3 in the presence of 1.5 µM progesterone or 50 mM Me-ß-CD for increasing times, groups of three (or six) oocytes were removed from the incubation medium, washed through two dishes of Ringers-HCO3, and dropped into 1.5-ml conical tubes. The Ringers-HCO3 buffer was removed and replaced with 30 µl (or 60 µl) of ice-cold homogenization buffer (80 mM ß-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 1 mM dithiothreitol, 100 µM sodium-orthovanadate, 10 mM NaF, 10 µg/ml leupeptin, 1 mM PMSF, 10 µg/ml aprotinin), and samples were homogenized using a plastic pestle. After centrifugation (10 000 x g for 5 min at 4°C), clear supernatant was removed from each tube and mixed with electrophoresis sample buffer. Proteins contained in 0.6 oocyte equivalents were resolved by SDS-PAGE (12.5% acrylamide) using the method of Laemmli [31] and were electroblot transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was blocked with I-Block (Tropix, Bedford, MA), and phosphorylated MAPK was immunoblot detected using a 1:2500 dilution of polyclonal anti-active MAPK (V8031; Promega, Madison, WI) as the primary antibody and 1:10 000 goat anti-rabbit alkaline phosphatase conjugate (Tropix) as the secondary antibody. Chemiluminescent detection was performed using CDP-Star (Tropix) with exposure to RX film (Fuji, Tokyo, Japan). Total ERK1/2 (extracellular regulated protein kinase, the gene product for MAPK irrespective of phosphorylation state) was assessed after stripping the immunoblot membrane in 10 ml of 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol at 70°C for 30 min, washing three times (5 min each) in Tris-buffered saline (150 mM NaCl, 10 mM Tris- HCl, pH 7.5), and repeating immunodetection starting with blocking and using 1:2500 polyclonal anti-ERK1/2 (V114A; Promega) as primary antibody.

[3H]Cholesterol Loading and Depletion

Loading and extraction of labeled cholesterol was performed by modification of the method of Rodal et al. [32]. Oocytes were incubated for 30 min (short term) or 20–21 h (long term) in 2-ml volumes of Ringers- HCO3 containing 1 mg/ml insulin-free BSA (Intergen, Purchase, NY) and 5 µCi [1,2-3H(N)]cholesterol (40 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO). Groups of cells were washed and transferred to 1- ml volumes of Ringers-HCO3 plus indicated concentrations of Me-ß-CD or {alpha}-CD. At indicated times, groups of 10 or 20 cells were washed through two large Petri dishes containing ~60 ml of Ringers-HCO3 at room temperature. Cell-associated radioactivity was measured by detergent extraction or cell solubilization. For detergent extraction, washed oocytes were dropped into 1.5-ml conical tubes, residual buffer was removed, and cells were homogenized in 250 µl of solution containing 2% Nonidet P40 (Roche, Mannheim, Germany) and 0.2% SDS (Boehringer Mannheim, Indianapolis, IN). Samples were microfuged at 15 000 x g for 5 min, and tritiated label in the resulting supernatant was measured. Alternatively, after loading and depletion, oocytes were washed and dropped into glass scintillation vials, excess buffer was removed, and cells were solubilized in 500 µl of TS-2 Tissue Solubilizer (Research Products International Corp., Mount Prospect, IL) by warming to 50°C. Supernatants or dissolved cells were mixed in 5 ml of Biosafe II scintillation cocktail (Research Products International), and radioactivity was quantified by liquid scintillation spectrometry using an LS 6500 counter (Beckman Instruments, Palo Alto, CA). When cells were solubilized with TS-2 Tissue Solubilizer, 150 µl of 10% acetic acid was added to the scintillation cocktail mixture before liquid scintillation spectrometry.

Measurement of Whole Cell and Cortical-Associated Label after Loading of Oocytes with [3H]Cholesterol

After incubation for either 30 min or 20 h in 2-ml volumes of Ringers- HCO3 containing 1 mg/ml insulin-free BSA and 5 µCi [3H]cholesterol, groups of 40 or 60 oocytes were washed sequentially through two dishes of Ringers-HCO3. Randomly selected groups of 10 oocytes were used to determine total counts per minute associated with whole cells. Cortical complexes (plasma membranes associated with vitelline envelope and surrounding follicle cells) were manually dissected as described previously [11]. Groups of cells or cortices were transferred to glass scintillation vials with minimal fluid transfer and solubilized in 500 µl TS-2 Tissue Solubilizer. Membrane-associated radioactivity was quantified by liquid scintillation spectrometry after addition of 150 µl of 10% acetic acid and 5 ml of Bio-Safe II scintillation cocktail.

Sucrose Gradient Centrifugation to Resolve LDM

After 6 h of treatment with indicated concentrations of Me-ß-CD, groups of oocytes (250 or 300 per group in different experiments) were used as starting material for separation of LDM from high-density membrane using a detergent-free method modified from that of Song et al. [33], as previously described [5].

Data Presentation

Numeric data were plotted and curves were fit to the data using SigmaPlot 8.0 (SPSS, Inc., Chicago, IL). Figures are representative of reproducible experiments using oocytes from different donor females. Figure captions and table footnotes describe how error bars were generated from data combined from multiple experiments. The significance of differences between treatment groups was evaluated using the Student t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To test the effect of cholesterol depletion on the time course of progesterone-induced GVBD, groups of 25–30 oocytes were incubated in increasing concentrations of Me- ß-CD in Ringers-HCO3 at room temperature for 4 h, washed, and incubated with 1.5 µM progesterone. Treatment of oocytes with concentrations of Me-ß-CD <5 mM had no apparent effect on the time course of GVBD (data not shown). Treatment of oocytes with 5, 25, or 50 mM Me-ß-CD accelerated the GVBD response in a dose-dependent fashion. The GVBD50 (time required for 50% of oocytes to display white spot formation) was approximately 5.5 h in a group of oocytes treated with progesterone alone, and the GVBD50 was accelerated to approximately 2 h after pretreatment of oocytes with 50 mM Me-ß-CD (Fig. 1A). To combine GVBD data from different experiments, GVBD50 values in the absence of Me-ß-CD were each normalized to 1, and effects of increasing concentrations of Me-ß-CD were expressed as the GVBD50 in the presence of drug divided by the GVBD50 in the absence of drug. The GVBD response was accelerated by increasing concentrations of cyclodextrin; 50 mM Me-ß-CD reduced the observed GVBD50 ratio to 0.42 ± 0.10 (Fig. 1B). Thus, treatment of oocytes with 50 mM Me-ß-CD for 4 h before transfer to progesterone resulted in a GVBD response that was more than twice as fast as that observed for cells that were not pretreated with Me-ß-CD. This effect was apparently due to drug action on the oocyte. Incubation of oocytes in 50 µg/ml of freshly prepared pronase E (type XXV; Sigma) in oocyte Ringers for 5 min at room temperature with gentle swirling and then washing with 10 mg/ml insulin-free BSA in oocyte Ringers to damage/remove follicle cells did not block Me-ß-CD action but rather accelerated the GVBD response in cells incubated in the absence or presence of Me-ß-CD before progesterone treatment (data not shown). Treatment of oocytes for 5 h with 50 µM lovastatin, an inhibitor of cholesterol synthesis, did not increase the sensitivity of oocytes to Me-ß-CD (data not shown).



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  		FIG. 1. Dose-dependent acceleration of progesterone-induced GVBD by Me-ß-cyclodextrin. Groups of 25–30 oocytes were incubated in the absence or presence of indicated concentrations of Me-ß-CD in Ringers- HCO3 for 4 h. After washing, groups of cells were transferred to 2-ml volumes of Ringers-HCO3 containing 1.5 µM progesterone at time zero and observed for the appearance of white spots as indication of GVBD. A) Results of a representative experiment using pronase-treated cells are expressed as the percentage of the population responding as a function of time after hormone treatment. B) GVBD50 values from five different experiments (two with protease-treated cells and three without protease treatment) using oocytes from different donor females were combined after normalizing GVBD50 values in the absence of Me-ß-CD to 1.0. The accelerating action of each concentration of drug is expressed as a fractional ratio calculated as the GVBD50 observed in the presence of Me-ß- CD (GVBD50 + Me-ß-CD) divided by the GVBD50 observed for companion cells in the absence of Me-ß-CD (GVBD50 – Me-ß-CD). Indicated values (mean ± SEM, n = 5) are significantly less than those for oocytes incubated in absence of Me-ß-CD (§P <= 0.05, §§P <= 0.025)

Me-ß-CD also was tested for its ability to stimulate oocyte maturation in the absence of added steroid. Figure 2A compares the time course of GVBD induced by exposure of oocytes to 1.5 µM progesterone with the time course of GVBD after treatment of a companion group of oocytes with 50 mM Me-ß-CD. Cyclodextrin induced GVBD with a time course (GVBD50 ~ 8.5 h) that was slower than that needed for companion cells to respond to progesterone (GVBD50 ~ 6 h). Progesterone-induced responses were easily scored by observing formation of distinctive pigment-ringed white spots on the oocyte animal pole (Fig. 2B, upper left), but treatment of oocytes with 50 mM Me- ß-CD resulted in stippling of the melanin pigment in the animal pole and blurring of white spots (Fig. 2B, upper right), rendering visualization of white spot formation an unreliable indicator of oocyte maturation. Therefore, acid fixation and dissection of oocytes were necessary to verify GVBD. In numerous experiments, when oocytes were incubated with Me-ß-CD alone, 5 mM drug never induced GVBD, 25 mM Me-ß-CD induced a marginal response (5%–40% GVBD in some groups of cells, data not shown), and treatment of oocytes with 50 mM Me-ß-CD generally resulted in 80%–100% response after 6–12 h (Fig. 2, A and C).



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  		FIG. 2. Oocyte responses to progesterone or Me-ß-cyclodextrin. A) Time courses of GVBD. At time zero, one group of 25 oocytes was placed into a 2-ml volume of Ringers-HCO3 containing 1.5 µM progesterone. A companion group of 400 oocytes was placed into 7.5 ml of Ringers-HCO3 containing 50 mM Me-ß-CD and gently swirled at room temperature. At indicated times, progesterone-treated cells were scored for GVBD by appearance of white spots, and groups of Me-ß-CD-treated cells (25 oocytes in each group) were acid-fixed and subsequently dissected to assess GVBD. Results are representative of four different experiments using oocytes from different donor frogs. B) White spots and meiotic spindle formation. Top: Stereomicroscopic appearance of white spots in oocytes treated with 1.5 µM progesterone (Prog) or 50 mM Me-ß-CD. Bar = 1 mm. Arrows point to faint white spots in oocytes treated with Me-ß-CD. Bottom: Evidence of meiotic spindles after GVBD induced by treatment of oocytes with 1.5 µM progesterone (Prog) or 50 mM Me-ß-CD. Oocytes were fixed, sectioned, and stained as described in Materials and Methods. Bar = 0.1 mm. C) Test for time of exposure required for oocytes to respond to Me-ß-cyclodextrin. At time zero, 400 oocytes were placed into a 7.5-ml volume of Ringers-HCO3 containing 50 mM Me-ß-CD and gently swirled at room temperature. At the time points indicated, groups of 25 oocytes were washed and placed into 2-ml volumes of Ringers- HCO3. At the 16-h time point, all groups were acid-fixed and dissected under a stereomicroscope to visualize the presence or absence of the nucleus. The percentage of cells in each group without a visible nucleus (%GVBD) is plotted as a function of time of exposure to Me-ß-CD

The 50 mM Me-ß-CD concentration was sometimes toxic to the oocytes. Different sets of oocytes harvested from different donor females displayed different abilities to withstand the effects of high-dose Me-ß-CD. During this study, 2 of 35 experiments had to be abandoned because most oocytes in the high-dose treatment groups did not survive. In other experiments, 5%–10% or fewer of oocytes incubated with Me-ß-CD became white (apparently dead) and swelled. Dead cells were always discarded and were not included in the final analysis. The 50 mM Me-ß-CD was hyperosmotic, as indicated by wrinkling of the oocyte surface, but the cells quickly recovered when washed and placed in Ringers-HCO3. However, the ability of Me-ß-CD to accelerate the progesterone response and induce oocyte maturation was not due to nonspecific hyperosmotic effects, because treatment of oocytes with 50 mM sucrose in Ringers-HCO3 did not affect the time course of the progesterone-induced response or induce oocyte maturation (data not shown).

GVBD induced by Me-ß-CD was accompanied by formation of meiotic spindles. These spindles were observed in oocytes that had undergone GVBD in response to either progesterone (Fig. 2B, lower left) or Me-ß-CD (Fig. 2B, lower right). In Figure 2B, the spindle in the progesterone- treated oocyte is positioned just below the white spot (area of pigment displaced by nuclear migration), but a white spot is not evident in the Me-ß-CD-treated cell. Condensed chromosomes in the meiotic spindle were more clearly evident after fluorescent staining (unpublished data).

To determine what period of exposure to Me-ß-CD is required for the GVBD response, oocytes were exposed to 50 mM Me-ß-CD, and subsets of cells were removed after increasing times, washed, and incubated in Ringers-HCO3 in the absence of Me-ß-CD. At the 16-h time point, all groups of oocytes were acid fixed and dissected to determine GVBD. Approximately 7 h of exposure to high-dose Me-ß-CD was required for 50% of the oocytes to display GVBD at the 16-h time point, and a full 16 h of exposure to Me-ß-CD was required for the maximal (90%) GVBD response (Fig. 2C). This finding contrasts with the very short exposure to progesterone required to stimulate the oocyte maturation response. A 15-min exposure of oocytes to 1.5 µM progesterone was sufficient to stimulate 100% GVBD after 5–6 h (data not shown).

GVBD induced by Me-ß-CD was accompanied by phosphorylation of MAPK. Figure 3 shows the time course of MAPK phosphorylation after treatment of oocytes from three different donor animals with progesterone or Me-ß- CD. Progesterone-induced phosphorylation of MAPK was evident after 2 or 3 h of hormone treatment. Phosphorylation of MAPK in Me-ß-CD-treated oocytes was detected after 5 or 6 h of drug treatment. To evaluate sample loading efficiency, the immunoblot for phospho-MAPK (frog C) was stripped and reblotted with anti-ERK1/2 (Fig. 3C, right). Comparison of the anti-ERK1/2 blot (Fig. 3C, right) and the anti-phospho-MAPK blot (Fig. 3C, left) verifies that the observed increases in immunoblot-detected phospho-MAPK were not due to unequal sample loading between lanes. Thus, when compared with progesterone effects, the slower time course of Me-ß-CD-induced GVBD (Fig. 2A) and meiotic spindle formation (Fig. 2B) is correlated with slower phosphorylation of MAPK (Fig. 3), consistent with a normal but slower progression of triggering events leading to meiotic cell division after treatment of oocytes with the cholesterol-depleting drug.



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  		FIG. 3. MAPK phosphorylation after treatment of oocytes with progesterone or Me-ß-CD. Phosphorylated MAPK (apparent molecular mass of 44 kDa) was detected at increasing times after treatment of oocytes from three different donor frogs (A, B, and C) with 1.5 µM progesterone or 50 mM Me-ß-CD. To verify that the immunoblot pattern was not an artifact of lane loading, the immunoblot membrane for frog C was stripped and immunoblotted a second time using anti-ERK1/2 antibody

If dose-dependent acceleration of progesterone-induced GVBD and the ability of high dose Me-ß-CD to induce GVBD are due to cholesterol depletion, then the observed physiological effects should be correlated with measurable changes in cholesterol levels. As detected with the enzyme- linked assay of Heider and Boyett [34], the total cholesterol content in amphibian oocytes was determined to be approximately 13 nmol of cholesterol per oocyte, and treatment of oocytes with up to 50 mM Me-ß-CD did not significantly lower cholesterol levels against this high background level (data not shown). As an alternative, the methods of Rodal et al. [32] were adapted to radiolabel oocyte cholesterol stores with [3H]cholesterol. Short-term (30 min) exposure at room temperature was used to label more rapidly exchanging surface cholesterol, and long-term (20–21 h) incubation with tritiated cholesterol was used to label more stable internal sites. As shown in Table 1, 78% ± 3% of cell-associated counts was recovered in cortical complexes after short-term (30 min) loading of oocytes with [3H]cholesterol. Long-term (20 h) incubation of oocytes with [3H]cholesterol resulted in increased total cell-associated counts, but only 40% ± 3% of label was recovered in cortical complexes, consistent with internalization of labeled sterol after longer incubation.


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  		TABLE 1. Measurement of whole-cell and cortex-associated label after short- and long-term loading of oocytes with [3H]cholesterol.a

After short- and long-term loading of oocytes with [3H]cholesterol, Me-ß-CD was used to deplete cell-associated radioactivity as an indicator of drug action. When oocytes were incubated with [3H]cholesterol for 30 min and then washed and exposed to 50 mM Me-ß-CD for increasing times (Fig. 4A, left), approximately 50% of the label was removed from oocytes after 15 min and 70% was removed after 90 min of cyclodextrin treatment. This cholesterol depletion was specific for Me-ß-CD and was not observed for {alpha}-CD, a smaller inactive congener that does not trap cholesterol in the same way as does Me-ß-CD [35]. Although 50 mM Me-ß-CD removed 60%–70% of surface- labeled cholesterol after 60 min, measurable depletion of surface cholesterol was not dose dependent in the 5–50 mM concentration range (Fig. 4A, right). When oocytes were incubated with [3H]cholesterol for 20–21 h, labeled pools of cholesterol became more resistant to cyclodextrin action. As shown in Figure 4B (left), 1 h of cyclodextrin treatment after overnight labeling of cholesterol pools removed only 25% of cell-associated [3H]cholesterol compared with 60% depletion after short-term labeling (Fig. 4A). Extended (9 h) incubation in the presence of Me-ß-CD resulted in dose-dependent removal of labeled cell-associated [3H]cholesterol in the 5–50 mM range, with 50 mM sufficient to deplete approximately 60% of cell-associated counts (Fig. 4B, right). Resolution of oocyte-associated label by thin-layer chromatography after long-term loading verified that >95% of cell-associated radioactivity co-migrated with the cholesterol standard (data not shown).



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  		FIG. 4. Cholesterol depletion after short-term or long-term labeling of oocytes with [3H]cholesterol. Groups of 200–280 oocytes were incubated in a 2-ml volume of Ringers-HCO3 plus 1 mg/ml BSA in the presence of 5 µCi [3H]cholesterol. A) Short-term (30 min) incubation. Left: Groups of 80 oocytes were washed and placed into 1-ml volumes of Ringers-HCO3 in the absence or presence of 50 mM {alpha}-CD or 50 mM Me-ß-CD. At indicated times, duplicate sets of 10 oocytes from each treatment group were detergent extracted and analyzed for tritium by liquid scintillation counting. Values are mean ± half-range (n = 2 samples in single representative experiment). Right: Groups of 25 oocytes were labeled with [3H]cholesterol for 30 min before treatment with increasing concentrations of Me-ß-CD in 1 ml Ringers-HCO3 for 60 min. In each experiment, duplicate sets of 10 oocytes were either homogenized and detergent extracted or dissolved in tissue solubilizer, and tritiated label was measured. §Significantly less than values for oocytes incubated in absence of Me-ß- CD (P <= 0.005). B) Long-term (20–21 h) incubation. Left: One group of 150 cells was placed into 2 ml of Ringers-HCO3. A companion group was placed into 2 ml of Ringers-HCO3 containing 50 mM Me-ß-CD. At indicated times, sets of 20 oocytes from each treatment group were washed in Ringers-HCO3, and duplicate sets of 20 oocytes were dropped into glass scintillation vials and solubilized before measurement of associated radioactivity. Values are mean ± half-range (n = 2 samples in single representative experiment). Right: After overnight labeling, groups of 25 oocytes were placed into 2-ml volumes of Ringers-HCO3 containing indicated concentrations of Me-ß-CD. After 9 h, the groups were washed, duplicate sets of 20 oocytes were dropped into glass scintillation vials and solubilized, and associated radiolabel was quantified. Indicated values are significantly less than those for oocytes incubated in the absence of Me-ß-CD (§P <= 0.025, §§P <= 0.01). In all panels, radioactivity associated with Me-ß-CD treatment groups is expressed as the relative percentage of control by comparison with groups of oocytes that were incubated equivalent times in the absence of Me-ß-CD. In right panels, each bar presents the mean ± SEM from three separate experiments using oocytes from different frogs

Because in other cell systems cholesterol depletion disrupts caveolae and signaling mechanisms associated with LDM microdomains [26, 27, 32], we tested the effect on LDM of extended treatment of oocytes with high-dose Me- ß-CD. When different groups of oocytes were incubated in the absence or presence of 5, 25, or 50 mM Me-ß-CD for 6 h before resolution of LDM by discontinuous sucrose gradient centrifugation, the amount of LDM recovered from oocytes was decreased (Fig. 5, top) in a dose-dependent fashion (Fig. 5, bottom). Because the GVBD response had been initiated after 6 h of incubation in the presence of 50 mM Me-ß-CD (see Fig. 2), it was important to determine whether the loss of recovery of LDM was a consequence of the progression of membrane changes leading to GVBD. To address this question, LDM recovery was determined in different groups of cells at times when approximately 50% of each group displayed GVBD in response to different inducing agents. The reduced recovery of LDM that was observed in cells treated with Me-ß-CD was not seen in cells treated with progesterone or insulin-like growth factor 1 (data not shown). Therefore, the observed disruption of LDM (reduced recovery of LDM as measured by reduced absorbance at 280 nm during fractionation) appears to result from Me-ß-CD treatment and apparently is not a biophysical consequence of the progression of oocyte to egg.



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  		FIG. 5. Effect of Me-ß-cyclodextrin on recovery of oocyte LDM by discontinuous sucrose gradient centrifugation. Groups of oocytes (either 250 or 300 cells per treatment group in different experiments) were manually dissected and incubated in the absence or presence of increasing concentrations of Me-ß-CD in 2-ml volumes of Ringers-HCO3 for 6 h at room temperature with gentle swirling. LDM in each set of cells was resolved by discontinuous sucrose gradient centrifugation. Profiles in the four upper panels show absorbance at 280 nm in a representative experiment as a function of relative positions on each gradient, with the top of each gradient at the left of each panel. On each panel, the position of LDM is indicated by the horizontal bar at a fixed height. Areas under the absorbance curves for LDM peaks were quantified by cutting out the peaks from print copies and weighing them on an analytical balance. The relative weights of LDM peaks from oocytes incubated in the presence of increasing concentrations of Me-ß-CD are plotted as a bar graph in the lower panel, with the weight of the LDM peak from oocytes incubated in the absence of Me-ß-CD normalized to 100 arbitrary units. *Mean ± half-range of means from two experiments using oocytes from different frogs; **Mean ± SEM from five different experiments using oocytes from different frogs. §Significantly less than area under LDM peaks in the absence of Me-ß-CD (P <= 0.01)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Me-ß-CD has been used extensively as a pharmacological tool to deplete cellular cholesterol, disrupt cholesterol- rich membrane domains, and investigate coincident changes in signaling phenomena and cellular responses. In cultured mammalian cells, 5–100 mM cyclodextrin has been used, and longer exposure to high concentrations is sometimes toxic to cells [36]. Therefore, the toxic response observed in some groups of frog oocytes in the present study was neither unexpected nor unusual. Lower doses of cyclodextrin exert maximal effects in mammalian cell lines with various time lines for effective cholesterol depletion and consequent effects. After long-term (24 h) labeling of mouse L-cell fibroblasts with [3H]cholesterol, 90% of cellular label was released after 8 h of incubation in 10 mM Me-ß-CD [25]. After short-term (15 min) labeling of HEp- 2 cells, 90% of label was removed after 15 min of treatment with 15 mM Me-ß-CD, and after long-term (20 h) labeling of HEp-2 cells with [3H]cholesterol, 50% of label was removed by 15 mM Me-ß-CD after 15 min and 70% was removed after 60 min [32]. In contrast, when the frog oocyte surface pools of cholesterol were labeled by 30 min of exposure to [3H]cholesterol, only 50% of cell-associated counts was removed after 15 min of incubation with 50 mM Me-ß-CD, and approximately 70% of label was removed after 90 min of incubation (Fig. 4A, left). Following long-term loading, only 30% of label was removed by 50 mM Me-ß-CD after 1 h and only 50% was removed after 9 h (Fig. 4B, left). At least three factors could contribute to the increased resistance of frog oocytes to cholesterol depletion as compared with cultured mammalian cells: 1) the frog oocytes were loaded and depleted at room temperature (21–22°C) instead of 37°C, 2) oocytes are surrounded by the glycoprotein vitelline envelope and follicle cells that may reduce contact of the drug with the oocyte surface or impede cholesterol efflux, and 3) the lipid constitution of amphibian cells may be sufficiently different from that of mammalian cells such that the cholesterol in LDM domains is more stable and therefore less easily labeled and removed than the cholesterol in mammalian cell rafts and caveolae, as suggested by the analysis of cholesterol-rich domains in amphibian eggs by Luria et al. [37]. Because damaging/removing follicle cells with pronase treatment accentuated the ability of cyclodextrin to accelerate progesterone-induced GVBD (Fig. 1), the presence of residual follicle cells apparently reduces drug contact with the oocyte surface. However, additional experimentation is required to more thoroughly assess how cholesterol depletion affects follicle cells and to determine whether more complete removal of follicle cells enhances the effectiveness of cyclodextrin treatment.

After short-term loading and depletion (Fig. 4A, right), the level of depletion was not correlated with the dose- dependent acceleration of steroid-induced GVBD (Fig. 1) and the ability of Me-ß-CD to induce GVBD in the absence of steroid. If removal of easily depleted oocyte surface cholesterol were responsible for the maturation response, then treatment of oocytes with 5 mM Me-ß-CD should have been as effective as treatment with 50 mM Me-ß-CD for inducing GVBD, but treatment of oocytes with 5 mM Me- ß-CD is not sufficient to induce the oocyte maturation response. The presence of the vitelline envelope confounds the interpretation of the cholesterol loading and depletion data. Even though almost 80% of cell-associated counts was recovered in the cortical complex after short-term loading (Table 1), it has not been determined whether the label is trapped in the perivitelline space (between plasma membrane and vitelline envelope) or incorporated into oocyte membrane lipid proper. Only after long-term (overnight) loading and long-term (9 h) treatment with Me-ß-CD was dose-dependent depletion of labeled cholesterol stores (Fig. 4B, right) correlated with the stepped acceleration of the maturation response (Fig. 1). Because long-term loading results in reduced recovery of counts in the cortical complex (Table 1), consistent with incorporation of sterol into internal membrane stores, long-term loading and depletion may reflect drug effect on internal membrane stores. Further experimentation is required to more fully investigate this possibility.

In the analysis of classical receptor-mediated drug action, dose-response curves for binding or effect generally approach a maximum over a 100-fold concentration range (2 log units) of inducing agent. Although the stepped acceleration of the progesterone-induced time course for GVBD with increasing concentrations of Me-ß-CD (Fig. 1) suggests dose dependence, the sensitivity of oocytes to Me- ß-CD-induced GVBD (from zero to maximal response over less than a 10-fold concentration range between 5 and 50 mM) is more indicative of a threshold effect. Once a critical biochemical endpoint has been achieved, the cells respond. The threshold for response may be depletion of membrane cholesterol by 50%. In cultured HEp-2 cells, Rodal et al. [32] observed that invaginated caveolae only formed when cholesterol levels were >50% of control levels. Treatment of oocytes with 50 mM Me-ß-CD removes >50% of the cholesterol from long-term labeled stores (Fig. 4B, right) and reduces recovery of LDM to approximately 50% that of control (Fig. 5, bottom). In other cell systems, raft- or caveolae-associated cholesterol is less easily extracted with cyclodextrin than is non-raft- or non-caveolae-associated cholesterol [28, 38]. Because dose-dependent depletion of cholesterol after long-term loading is correlated with significant loss of LDM, disruption of liquid-ordered cholesterol-rich domains may be involved in triggering the Me- ß-CD-induced response. Because the oocyte LDM fraction contains mostly internal membrane [5], the A280 profile of LDM during gradient fractionation reflects primarily internal membrane, and reduction of LDM recovery after treatment of oocytes with Me-ß-CD (Fig. 5) may reflect effects on both internal and surface membranes. Even though caveolar structures on the amphibian oocyte plasma membrane have not been microscopically identified, LDM isolated from oocytes has been shown to be associated with immunodetectable caveolin-like protein [5], suggesting the existence of caveolae-like membrane microdomains. These data support a model in which there is a requisite threshold level of cholesterol-rich LDM structure required to maintain the G2/M meiotic arrest in amphibian oocytes, sequestering and orienting signal transduction proteins in either active or inactive form. Cholesterol depletion disrupts the cholesterol-rich microdomains, releasing and/or activating signal transduction mechanisms that lead to MAPK phosphorylation [39] and subsequent GVBD. Further experimentation is required to determine which signaling proteins, including G proteins, are affected by cholesterol depletion and whether the effects of inducing hormones are mediated by LDM rafts.

To our knowledge this is the first report of induction by Me-ß-CD of meiotic maturation (phosphorylation of MAPK, GVBD, and formation of meiotic spindles) in vertebrate oocytes. Although membrane cholesterol and the effects of cholesterol depletion have not yet been tested in mammalian oocytes, caveolin and LDM may play a general role in gamete development and function. Caveolin-1 has been reported to be involved in meiotic cell cycle progression in the nematode Caenorhabditis elegans as indicated by RNA interference [40]. Ablation of caveolin-1 by RNA interference resulted in increased germ cell progression through meiotic development and increased egg laying by the injected hermaphrodite. Cholesterol depletion with ß- cyclodextrin caused a similar phenotype, suggesting that cholesterol-dependent maintenance of caveolar-like membrane structures is involved in sustaining meiotic arrest in the nematode. Additional experiments are required to more fully decipher the role(s) for low-density cholesterol-rich membrane in maintaining meiotic arrest, organizing oocyte signal transduction proteins, and contributing to the biochemical changes leading to meiotic maturation in amphibian and mammalian oocytes.


   ACKNOWLEDGMENTS
 
We thank James Maller for supplying the microtome used to section oocytes and the method for staining meiotic spindles, Linda Barlow for the use of her microscope-mounted camera system to photograph oocytes, and David Christophel for the use of his microscope-camera system to photograph meiotic spindles.


   FOOTNOTES
 
1 This work was supported by NIH grant 1 R15 GM60922 to S.E.S. Preliminary data from these studies were presented as a poster at the 2001 annual meeting of the American Society for Cell Biology. Back

2 Correspondence: Susan E. Sadler, University of Denver, Department of Biological Sciences, 2101 East Wesley, University Park, Denver, CO 80208. FAX: 303 871 3471; ssadler{at}du.edu Back

Received: 2 December 2003.

First decision: 29 December 2003.

Accepted: 2 February 2004.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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