Supplementary Information for
Yesilaltay A, Dokshin GA, Bussoc D, Wang L, Galiani D, Chavarria T, Vasile E, Quilaqueo L, Orellana JA, Walzer D, Shalgi R, Dekel N, Albertini DF, Rigotti A, Page DC, and Krieger M
Excess cholesterol induces mouse egg activation and may cause female infertility
PNAS 111, E1972 (2014)
- Supporting Information Includes:
- Supplementary Materials and Methods
- Figure S1. Visualization of spontaneous pronucleus formation in SR-BI KO eggs. Eggs were harvested from SR-BI KO females~20 h after hCG administration(induction of superovulation) and denuded by hyaluronidase treatment. The eggs were stained to visualize chromosomes (DAPI; blue), microtubules (tubulin;green), centromeres (anticentromere antibody) (Fig. 3), and the nuclear membrane (lamin B1; red). Deconvolved andz-projected images were collected.A representative image of an egg with a lamin B1-stained pronucleus (asterisk) is shown. The arrow indicates the second PB. (Scale bar: 10µm.
- Figure S2. Visualization of spontaneously formed second PBs in SR-BI KO eggs. Eggs were harvested from SR-BI KO females~20 h after hCG administration anddenuded by hyaluronidase treatment. The eggs were stained to visualize chromosomes (DAPI; blue), microtubules (tubulin) (Fig. 3), centromeres [anti-centromere antibody (ACA); red], and the nuclear membrane (lamin B1) (Fig. S1). Deconvolved andz-projected images were collected. Representative imagesof PBs from eggs at various stages of meiosis (schematically indicated inUpper) are shown. The images shown here are from the same eggs shown in Fig. 3. Thepresence of only 20 chromatids (measured by counting ACA staining puncta) confirms that the observed PBs were second PBs (first PBs would contain40 chromatids). (Scale bar: 10µm.
- Figure S3. MPF and MAPK activities of ovulated eggs from control and SR-BI KO mice. Ovulated eggs were harvested fromSR-BI+/+,SR-BI+/-, and SR-BI KO (SR-BI-/-) females~18 h after hCG administration (induction of superovulation), denuded by hyaluronidase treatment, and incubated for 6 h in M16 medium at37 °C. The eggs were washed and pooled into sets of four eggs each:SR-BI+/+, five pools;SR-BI+/-, seven pools; andSR-BI-/-(KO), nine pools. None of the eggsfrom theSR-BI+/+andSR-BI+/-mice exhibited second PBs (0%), whereas second PBs were observed in some of the SR-BI KO eggs (100% of eggs in six pools) butnot in other SR-BI KO eggs (all eggs in three pools; 0%). Relative (A) MPF and (B) MAPK activities were determined as incorporation of32P from [?-32P]ATP intothe MPF substrate histone H1 (~30–32 kDa) and the MAPK substrate MBP (~21.5 kDa) as described inSI Materials and Methods. The values for one-way ANOVAfor MPF and MAPK activities were P=0.002. *Pvalue=0.0001 for comparisons withSR-BI+/-;†Pvalue=0.0001 (unpaired Studentttest) for comparisons withSR-BI+/+.Cshows an autoradiograph from the assay used to calculate the values inAandB(SI Materials and Methods). The no egg ctrl lane represents a controlsample that did not contain eggs.
- Figure S4. Relative levels of cholesterol in eggs determined by filipin fluorescence. Oocytes were harvested fromSR-BI+/+and SR-BI KO females~16 h after hCGadministration (induction of superovulation) and denuded by hyaluronidase treatment.SR-BI+/+eggs were incubated for 10 min at 37 °C in either M16 mediumalone (n=6 eggs imaged) or M16 containing 0.5 mM cholesterol/MßCD (Chol/MßCD;n=6). Eggs from SR-BI KO mice (n=7) were not treated with Chol/MßCD.All eggs were then fixed, washed, stained by incubation with 0.5 mg/mL Filipin III complex in PBS for 1 h at room temperature, washed, and imaged byfluorescence microscopy as described inSI Materials and Methods(above). Bars represent the means±SEMs in arbitrary units (AUs) relative to the fluorescenceintensity of the untreatedSR-BI+/+control that was defined as 1.0 in each experiment. The value for one-way ANOVA analysis of all samples wasP=0.0026,with SR-BI KO eggs exhibiting a significantly higher filipin fluorescence level compared with the untreatedSR-BI+/+control (68% increase).SR-BI+/+eggstreated with Chol/MßCD showed a 38% increase in fluorescence compared with untreatedSR-BI+/+cells. *P=0.004 for one-sample Studentttest;†P=0.019 forStudentttest;#P=0.001 for posthoc Tukey test.
- Figure S5. SrCl2-induced oscillations in [Ca2+]iin a WT C57BL/6 egg. Eggs isolated from the oviducts of hormone-primed WT C57BL/6 females~14–16 h after hCGadministration were denuded by hyaluronidase treatment. The eggs were loaded with the calcium-sensitive fluorescent dye Fura-2AM. For SrCl2activation,eggs were placed at time=0inCa2+-free M2 medium supplemented with 5 mM SrCl2at 37 °C for the duration of the experiment. Untreated control eggs werealso examined. Relative [Ca2+]iwas measured with an inverted microscope as a function of time as the ratio of fluorescence intensities excited by 340- and380-nm light (details inSI Materials and Methods). Data for individual SrCl2-treated (black line) and untreated control (light gray line) eggs are shown.
- Figure S6. Effects of cholesterol loading for 25 min on the relative [Ca2+]iof eggs from WT C57BL/6 females. Eggs from WT C57BL/6 females were harvested~14–16 h after hCG administration, stripped of cumulus cells by hyaluronidase treatment, and loaded with the calcium-sensitive fluorescent dye Fura-2AM. At time=0, the eggs were placed in M2 medium supplemented with (treated) or without (control) 0.125 mM cholesterol/MßCD (Chol/MßCD) at 37 °C. A diagram of theexperimental protocols is shown in the box in the upper right. The eggs were then placed in a fluorescence microscope for an initial period of imaging at37 °C(imaging #1) that began shortly after the addition of Chol/MßCD. Relative [Ca2+]imeasured using an inverted microscope as a function of time was determinedas the ratio of fluorescence intensities excited by 340-nm and 380-nm light [sequentially excited every (A)5or(B) 60 s]. After 25 min of Chol/MßCD exposure,the eggs were washed and placed in Chol/MßCD-free M2 medium and repositioned in the microscope, and the imaging continued for an additional (A)15minor (B)~2.5 h at 37 °C (imaging #2). Fluorescence data could not be collected during the 5–10 min required to wash away the Chol/MßCD, position, and refocuson the eggs for additional imaging in Chol/MßCD-free medium (indicated by dotted lines in the diagram in the upper right). InA, we present some of the datafrom a subset of eggs treated with Chol/MßCD (n=4), in which the peak of [Ca2+]ioccurred before the wash period from one untreated control egg (blue).Slight experiment to experiment variations in the times at which imaging began or ended are indicated by wavy lines in the diagram in the upper right. Afterimaging, eggs were cultured in Chol/MßCD-free M2 medium at 37 °C for an additional~5–24 h to assess second PB extrusion and cleavage to the two-cell stage.Bshows results for four representative individual eggs treated with Chol/MßCD and one (blue) of four untreated control eggs from the same experiment.Second PB extrusion was assessed at the end of the imaging period.
- Figure S7. Effects of the cholesterol loading on viability and activation of controlSR-BI+/-eggs. Eggs fromSR-BI+/-mice harvested~16 h after hCG adminis-tration (induction of superovulation) in three independent experiments were denuded by hyaluronidase treatment. All eggs were then preincubated for15 min at 37 °C in M16 medium without (-) or with (+) 0.5 mM cholesterol-loaded MßCD (cholesterol) and then washed one time in M16 medium. The oocyteswere then incubated for 6 h in M16 medium at 37 °C in the absence (-) or presence (+) of 5 mM SrCl2/2 mM EGTA. The total numbers of oocytes examined were105 (untreated), 88 (SrCl2/EGTA), and 90 (cholesterol). After 6 h of incubation, we determined (A) the percentage of oocytes that remained viable and (B)thepercentage of those oocytes exhibiting second PB extrusion. The values for one-way ANOVA were (A)P=0.86 and (B)P=0.0001. SrCl2/EGTA and cholesteroltreatments both yielded statistically significant differences in PB extrusion compared with the untreatedSR-BI+/-control as indicated.#P<0.001 (posthocTukey test). (C) The oocytes were visualized using phase contrast microscopy. Solid white arrowheads indicate PBs, and open arrowhead indicates cumulus cells.(Magnification: 100×. Scale bar: 100µm.)
- Table S1. Completion of MI