Category: Oocyte

DISCUSSION

Me-p-CD has been used extensively as a pharmacological tool to deplete cellular cholesterol, disrupt cholesterolrich 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. Therefore, the toxic response observed in some groups of frog oocytes in the present study was neither unexpected nor unusual. Lower doses of cyclo-dextrin 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-p-CD. After short-term (15 min) labeling of HEp-2 cells, 90% of label was removed after 15 min of treatment with 15 mM Me-p-CD, and after long-term (20 h) labeling of HEp-2 cells with [3H]cholesterol, 50% of label was removed by 15 mM Me-p-CD after 15 min and 70% was removed after 60 min. 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-p-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-p-CD after 1 h and only 50% was removed after 9 h (Fig. 4B, left).

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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-p-CD in Ringers-HCO3 at room temperature for 4 h, washed, and incubated with 1.5 |xM progesterone. Treatment of oocytes with concentrations of Me-p-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-p-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-p-CD (Fig. 1A). To combine GVBD data from different experiments, GVBD50 values in the absence of Me-p-CD were each normalized to 1, and effects of increasing concentrations of Me-p-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-p-CD reduced the observed GVBD50 ratio to 0.42 ± 0.10 (Fig. 1B). Thus, treatment of oocytes with 50 mM Me-p-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-p-CD. This effect was apparently due to drug action on the oocyte. Incubation of oocytes in 50 |xg/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-p-CD action but rather accelerated the GVBD response in cells incubated in the absence or presence of Me-p-CD before progesterone treatment (data not shown). Treatment of oocytes for 5 h with 50 |xM lovastatin, an inhibitor of cholesterol synthesis, did not increase the sensitivity of oocytes to Me-p-CD (data not shown).

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METHODS

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 ) 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.

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INTRODUCTION

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 ). Cholesterol-enriched LDM containing immunodetectable caveolin-like protein has been recovered from Xenopus laevis oocytes. 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 or with insulin or insulin-like growth factor 1. One early oocyte response to treatment with inducing hormone is a decrease in intracellular cAMP that can be accounted for at least in part by inhibition of adenylyl cyclase and stimulation of phosphodiesterase type III. Inhibition of adenylyl cyclase by progesterone does not involve pertussis toxin-sensitive Gai subunits of the heterotrimeric G protein complex, is correlated with slowing of guanine nucleotide exchange, and shares certain features with P site agonist action. In other cell systems, P site adenosine action may be associated with caveolae.

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