Figure 1
From: Dysfunctional MDR-1 disrupts mitochondrial homeostasis in the oocyte and ovary
Characterization of mdr1a mutant. (A) Expression levels of MDR-1 in ovary lysates of wild type, mdr1a mutant, and mdr1a/mdr1b/bcrp−/− triple knockout (TKO) animals. Blots were cropped for display. Full-length blots are presented in (Fig. S6A). (B) Schematic of primers for the validation of the perturbation of mdr1a exon 23 in mdr1a mutants. Primers F1 and R1 were used to validate the perturbation of mdr1a exon 23 in the transcript (cDNA). Primers F2 and R2 were used to validate the insertion of MuLV LTR sequence in mdr1a locus (Genomic) in the genome of mdr1a mutant mice. (C) PCR amplification. With wild type ovarian cDNA template, F1/R1 amplified a 471 bp product (no perturbation). With mdr1a mutant cDNA template, F1/R1 amplified a larger product of ~500 bp (replacement of exon23 with a fragment of MuLV LTR sequence) and a smaller product of ~400 bp (deletion of exon 23). With wild type genomic DNA template, F2/R2 gave no amplification (absence of insertion). With mdr1a mutant genomic DNA template, F2/R2 amplified a ~550 bp product (MuLV LTR insertion). Actin was amplified as a positive control. Gels were cropped for display. Full-length gels are presented in (Fig. S6C,D). (D) Illustration of functional domains on MDR-1 protein. Blue represents transporter integral membrane type-1 fused domains. Orange represents ATP-binding cassette, ABC transporter-type domains. (E) Structure of mouse P-glycoprotein (PDB accession code: 3G5U). The region deleted by skipping of exon 23 is shown in black, including the second nucleotide binding domain (NBD2) and transmembrane helices TM11 and TM12. The two nucleotide binding domains, and N and C-terminals are labeled. The lipid bilayer is indicated by dotted lines. (F) Wild type and mdr1a mutant oocytes exhibit calcein-AM fluorescence from timepoint 0 to 4 (every 30 min), with positive control wild type oocytes incubated with 25 mM PSC 833. Scale bar: 200 µm. (G) Normalized fluorescence intensity (∆F/F) of the change in individual oocyte fluorescence intensity over time (n = 4). Mutant oocytes exhibit a slower decrease in fluorescent intensity compared to wild type oocytes. (H) Ovaries from animals treated with 75 mg/kg and 150 mg/kg cyclophosphamide show increases in cell death with LIVE/DEAD Cell Viability Assay at 24 hours. Cell death is most apparent in cells that morphologically appear to be somatic and not oocytes. mdr1a mutants display more cell death at 48 hours when exposed to cyclophosphamide compared to wild type controls.
