Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation

Abstract

Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol homeostasis within the COC. Modulation of membrane cholesterol by MβCD improved survival of bovine oocytes and preserved integrity of GM1-related rafts after vitrification.

Fig 1. Effect of the exposure to cryoprotectans, vitrification, and cholesterol modulation during vitrification, on oocyte viability.

(A) Partially denuded oocytes were incubated in VAD-FMK-FITC to detect in situ activated caspases and subsequently washed in the presence of propidium iodide to assess membrane integrity. Oocytes showing brilliant green fluorescence were considered caspase positive and cumulus cells showing green/orange fluorescence were considered positive for either caspases or both markers. (B) Bright field, scale bar: 25 μm. (C) Percentage of oocytes caspase positive in the fresh control group (f ctr), in the toxicity control group (exposed to vitrification and warming solutions; t ctr), in the vitrified control group (v ctr) and in treatment groups in which cholesterol was added to oocytes prior to vitrification and removed after warming (+/-chol) for periods of 45 minutes and 2 hours for both processes (enrichment-depletion). Data represent the mean ± SEM of 8 independent experiments with ~20 COCs for each condition. Caspase variable was compared using Mix Lineal Generalized Models (MLGM) with Binomial family, logit link and LSD Fisher contrast. Different letters (a-c) denote significant differences (P<0.05).

Fig 2. Incorporation of cholesterol into cumulus-oocyte complexes estimated by BODIPY-cholesterol labeling in living cells.

(A) Cumulus-oocyte complexes (COCs), cumulus-free oocytes (ZP+) and ZP-free oocytes (ZP-) were incubated with 1 μM BODIPY-cholesterol after MβCD/cholesterol treatment of COCs for 45 minutes (lower panels). Upper panels show control conditions. Right panels show bright field; scale bar: 25μm. (B) Fluorescence intensity in ZP-free oocytes quantified with Image J software. Bars represent the mean ± SEM of 3 replicates from a total of 25 control oocytes and 28 cholesterol-loaded oocytes. Comparison of mean values was performed using Student t test. Asterisks denote significant differences (P<0.01). BPY-chol: BODIPY-cholesterol; chol: cholesterol; MβCD: methyl-β-cyclodextrin.

Fig 3. Effect of MβCD treatment on ZP-free and ZP-intact bovine oocytes.

(A) BODIPY-cholesterol fluorescence in ZP-free oocytes directly exposed or not to 15mM MβCD for 45 minutes (upper panels). Bright field showing the distribution of lipid droplets (LD), scale bar: 25 μm (middle panels). Nile Red staining of oocyte lipid droplets (lower panels). Images are representative of 28 control oocytes and 29 MβCD-treated oocytes for BODIPY-cholesterol, and 28 control oocytes and 32 MβCD-treated oocytes for Nile Red. (B) Fluorescence intensity of Nile Red in ZP-free oocytes quantified with Image J software. Comparison of mean values was performed using Student t test. Asterisk denote significant differences (P<0.05). (C) Bright field showing the distribution of LD in ZP-intact oocytes exposed or not to 15mM MβCD for 45 minutes. Control oocytes showed the same pattern (40/40) while 16/45 oocytes showed the cortex devoid of LD in the MβCD-treated group. Scale bar: 25 μm. Inserts show Nile Red staining. BPY-chol: BODIPY-cholesterol; chol: cholesterol; MβCD: methyl-β-cyclodextrin.

Fig 4. Free cholesterol, cholesterol esters and total cholesterol determined by Amplex® Red in oocytes and cumulus cells from autumn and spring compared to those from winter and summer.

Free cholesterol and total cholesterol (free cholesterol plus cholesterol esters) levels were measured through a fluorometric method in oocytes and cumulus cells from autumn-spring (A,C) and winter-summer (B,D). Total cholesterol was determined adding cholesterol esterase after free cholesterol measurement at time zero. Values are mean ± SEM of 8 replicates for autumn-spring and 8 replicates for winter-summer with n = 45–50 COCs/condition (ctr, +chol, +/-chol), total experiments N = 16. Comparison of means was performed using Bonferroni test for autumn-spring samples and LSD Fisher for winter-summer samples. Different letters (a-b) denote significant differences (p<0.05) among treatments within each seasonal period/cellular type. ctr: control; +chol: cells loaded with cholesterol for 45 minutes; +/-chol: cells loaded with cholesterol for 45 minutes and subsequently depleted of cholesterol for another 45 minutes.

Fig 5. Effect of vitrification on the raft marker lipid GM1.

(A) Control and cholesterol-loaded COCs were vitrified with the surface device Cryotech®. After warming, cholesterol-loaded oocytes were exposed to empty MβCD during the recovery period. GM1 was detected in living cumulus-free oocytes by using the fluorescent-labeled cholera toxin B subunit (upper panels). Lower panels show bright field; scale bar: 25μm (B) Fluorescence intensity of cholera toxin B subunit-GM1 binding (CTB-GM1) in cumulus-free oocytes quantified with ImageJ software. Bars represent the mean ± SEM of 3 replicates from a total of 32 fresh control oocytes (f ctr), 36 vitrified control oocytes (v ctr) and 39 vitrified cholesterol-loaded/cholesterol-removed oocytes (+/-chol). Comparison of means was performed using Bonferroni test. Different letters (a-b) denote significant differences (p<0.05). (C) Fluorescence intensity of BODIPY-cholesterol (BPY-chol) in ZP-free oocytes quantified with ImageJ software. Bars represent the mean ± SEM of 3 replicates from a total of 28 fresh control oocytes (f ctr), 26 vitrified control oocytes (v ctr) and 22 vitrified cholesterol-loaded/cholesterol-removed oocytes (+/-chol). Comparison of means was performed by ANOVA.