Cholesterol depletion disorganizes oocyte membrane rafts altering mouse fertilization

Abstract

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.

Figure 1. Effect of cholesterol depletion mediated by MβCD on oocyte survival.

Zona-free mouse oocytes were incubated with different concentrations of MβCD for 30 min at 37°C. (A) Differential interference contrast micrographs of depleted oocytes after MβCD treatment. Are also illustrated by inserted pictures healthy and dead oocytes demonstrating or not their viability by the trypan blue exclusion test. (B) Percentages of living oocytes after cholesterol depletion. Data represent the mean ± SEM of at least 3 independent experiments from a total of 101 control oocytes, 49 oocytes depleted at 5 mM, 92 oocytes depleted at 15 mM and 29 oocytes depleted at 30 mM of MβCD. Comparison of mean values was performed using Bonferroni test. Different letters (a-c) denote significant differences (P<0.05).

Figure 2. Effect of cholesterol disrupting agents on mouse fertilization.

Zona-free mouse oocytes were incubated with either different concentrations of MβCD for 30 min at 37°C to remove cellular cholesterol or 200 µg/ml of Nystatin to sequestrate cholesterol into complexes. Cholesterol repletion was carried out incubating MβCD-treated oocytes with MβCD/Chol complexes. After depletion/repletion and sequestration treatments, oocytes were washed and inseminated. (A) Effect of cholesterol depletion and repletion on the fertilization rateand (B) fertilization index. (C) Effect of Nystatin induced cholesterol sequestration on the fertilization index. Data in A and B represent the mean ± SEM of at least 3 independent experiments from a total of 101 control oocytes, 49 oocytes depleted at 5 mM, 92 oocytes depleted at 15 mM and 52 oocytes depleted/repleted at 15 mM of MβCD. Data in C represent the mean ± SEM of 3 independent experiments from a total of 33 control oocytes and 72 Nystatin-treated oocytes. Comparison of mean values was performed using LSD or Student’s t tests. Different letters (a-c) denote significant differences (P<0.05).

Figure 3. Effect of cholesterol depletion and repletion on polar body extrusion.

Zona-free mouse oocytes were incubated with 15 mM of MβCD for 30 min at 37°C to remove cellular cholesterol. Cholesterol repletion was carried out incubating MβCD-treated oocytes with MβCD/Chol complexes. After depletion/repletion treatments, oocytes were washed and inseminated. (A) Percentage of expulsed polar bodies (PB) after fertilization of cholesterol depleted oocytes. (B) Effect of cholesterol depletion and repletion on the extrusion of the second polar body visualized by DAPI staining. Inserts show a zoom of the regions indicated by asterisks. White arrowheads indicate PB and red arrowheads indicate meiosis arrest. Data in A represent the mean ± SEM of 3 independent experiments from a total of 55 control oocytes and 57 oocytes depleted at 15 mM MβCD. Asterisks (**) indicate significant differences with respect to control (P<0.01).

Figure 4. Subcellular localization of BODIPY-Cholesterol in the mouse oocyte.

Zona-intact ovulated oocytes were incubated with the fluorescent cholesterol probe for 15 min at 37°C. (A,B) Oocytes continuously incubated with BPY-Chol were imaged at 15 and 50 min. (D,E) Pulse-chase experiment. After incubation, BPY-Chol was washed and followed in time. (C,F) Fluorescence intensity quantified with ImageJ software. The bars represent the mean ± SEM of a total of 15 oocytes for continuous exposition experiment and 15 oocytes for pulse-chase experiment. Comparison of mean values for each subcellular compartment over time was performed using Student t test. Asterisks denote significant differences (P<0.01). Fluorescence of oocytes measured after 90 min was normalized to 100%.

Figure 5. Effect of cholesterol depletion and repletion on oocyte cholesterol content.

Zona-intact ovulated oocytes were pretreated with 15 mM MβCD for 30 min at 37°C to remove cholesterol. Cholesterol repletion was carried out incubating MβCD-treated oocytes with MβCD/Chol complexes. After depletion/repletion treatment, oocytes were washed and incubated with BPY-Chol for 15 min at 37°C. (A) Control, (B) depleted and, (C) depleted/repleted oocytes labeled with BPY-Chol. (D) Fluorescence intensity quantified with ImageJ software. Bars represent the mean ± SEM of 3 independent experiments from a total of 14 control oocytes, 23 depleted oocytes, and 17 depleted/repleted oocytes. Comparison of mean values was performed using Bonferroni test. Different letters (a-b) denote significant differences (P<0.05).

Figure 6. Presence of the raft markers GM1, Flotillin-2 and Caveolin-1 in the mouse oocyte.

(A) Plasma membrane localization of the raft marker lipid GM1 assessed by incubation of living oocytes with CTB-AF⁴⁸⁸. (B) Indirect immunofluorescence detection in fixed oocytes and immunoblot detection in whole oocyte lysates of flotillin-2 and (C) caveolin-1. Fluorescence staining was performed in a total of 35 oocytes for GM1, 13 oocytes for flotillin-2 (Flot-2), and 10 oocytes for caveolin-1 (Cav-1). For the Western blots, numbers to the left of each panel indicate the molecular weight of the protein. A total of 120 (Flot-2) and 470 oocytes (Cav-1) were pooled and lysed. 3T3 cell lysates were used as positive controls.

Figure 7. Electron micrographs of caveolae-like microdomains in the mouse oocyte.

Ultrastructural plasma membrane with a caveolae-like invagination. OC: Oocyte Cytoplasm; PM: Plasma Membrane; PVS: PeriVitelline Space; C: Caveola; ZP: Zona Pellucida.

Figure 8. Effect of cholesterol depletion on c-Src and CD9 subcellular localization. Src-family kinase role on second polar body extrusion.

(A) Cortex localization of the raft-associated tyrosine kinase c-Srcassessed by indirect immunofluorescence. (B) Cytoplasmic relocation of the c-Src kinase after MβCD treatment. No primary antibody controls were negative. Staining of a total of 14 control oocytes and 6 MβCD-treated oocytes. Within each group, oocytes showed the same staining pattern. (C) Plasma membrane localization of the CD9 tetraspanin, a non-raft protein. (D) CD9 remained at the plasma membrane after MβCD treatment. Staining of a total of 18 control oocytes and 18 MβCD-treated oocytes. In both groups, oocytes showed the same staining pattern. (E) Effect of Src-family kinase inhibition assessed by incubation of oocytes with PP2 on the extrusion of the second polar body (PB). Data represent the mean ± SEM of 3 independent experiments from a total of 77 control oocytes and 85 or 69 oocytes treated with PP2 at 10 or 100 µM, respectively. (F) DAPI-stained images illustrating 1- a blocked telophase, 2- the beginning of the formation of the PB, 3- its almost complete formation, and 4- an extruded PB. Comparison of mean values was performed using Bonferroni test. Different letters (a-b) denote significant differences (P<0.05). *: oocyte chromatin; S: sperm decondensed chromatin; PB: Polar Body.