1. Multidrug-resistant transport activity protects oocytes from chemotherapeutic agents and changes during oocyte maturation 
Lynae M. Brayboy, M.D., Nathalie Oulhen, Ph.D., Jeannine Witmyer, Ph.D., Jared Robins, M.D., Sandra Carson, M.D., Gary M. Wessel, Ph.D. 
Fertility and Sterility 
Volume 100, Issue 5, Pages e7 (November 2013)
DOI: /j.fertnstert
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Dynamic expression of multidrug-resistant (MDR) transporter mRNA and protein during oocyte maturation in mouse. (A) MDR-1 RNA levels increase during oogenesis. Quantitative polymerase chain reaction was used to measure the RNA levels of MDR-1 in mouse oocytes at the indicated developmental stages: germinal vesicle (GV), meiosis I (MI), and meiosis II (MII). All values were normalized against the β-actin RNA and represented as a fold change relative to the amount of RNA present in the GV oocytes. Significance was assessed between each developmental stage with the use of Student t test (P<.05). ∗Significant differences were obtained between GV and MII and between MI and MII. (B) MDR-1 protein expression. Western blot with the use of an antibody against MDR-1 on GV, MI, and MII mouse oocytes. Fifty oocytes were loaded in each group. (C) MDR-1 is expressed throughout the oocytes. Immunofluorescence with the use of an antibody against MDR-1 on mouse oocytes at (a) GV, (b) MI, and (c) MII. The corresponding differential interference contrast images are respectively shown in panels d–f. Pictures were taken at ×200 magnification. Scale bar = 50 μm.
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3. Figure 2
Multidrug-resistant (MDR) transporter activity changes with oocyte maturation stage. (A) Mouse oocytes were incubated with calcein AM without (control: g, i, k) or with PSC 833 (PSC: h, j, l). The corresponding differential interference contrast images are respectively shown in a to f, at 200x magnification. Scale bar, 50 μm. (B) The fluorescence resulting from the calcein in the whole oocytes was quantified using metamorph. 12 GV, 7 MI, and 8 MII were used for the quantification in the control; 9 GV, 4 MI, and 7 MII were analyzed after the PSC833 treatment. Significance was assessed between control (without PSC) and PSC833 treated oocytes (with PSC) for each developmental stage using Student t test, P<.005. Significant differences (∗) were obtained for each stage.
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4. Figure 3
Multidrug-resistant (MDR) transporter activity is essential for the survival of mouse oocytes exposed to a chemotherapeutic agent. (A) Cell death was measured with the use of the Live/Dead assay. GV, MI, and MII mouse oocytes were incubated for 12 hours with 25 mmol/L cyclophosphamide only (panels b, k, t; e, n, w; and h, q, z) or in the presence of 25 mmol/L cyclophosphamide and 25 μmol/L PSC 833 (panels c, l, u; f, o, x; and i, r, α). Untreated oocytes were used as control (panels a, j, s; d, m, v; and g, p, y). The Live/Dead assay was performed by simultaneously monitoring the fluorescence after addition of calcein AM (panels j–r) and ethidium homodimer 1 (panels s–α). Calcein AM (green) is retained in live cells, whereas ethidium homodimer 1 (red) enters dead cells. The corresponding differential interference contrast images are shown in panels a–i. Scale bar = 100 μm. (B) The fluorescence was quantified with the use of Metamorph. For each oocyte, only a small area was considered for the fluorescence measurement to avoid the surrounding granulosa cells. Cell viability is represented on the graph with the use of a relative unit: ratio of green/red fluorescence measured in each oocyte. The number of oocytes used for each condition is indicated in parentheses. Significance was assessed between oocytes treated with cyclophosphamide without or with PSC 833 for each developmental stage with the use of Student t test (P<.005). ∗Significant differences were obtained for each stage.
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5. Figure 4
Multidrug-resistant (MDR) transporter activity is essential for the survival of human oocytes exposed to a chemotherapeutic agent. (A) Cell death was measured with the use of the Live/Dead assay. Human oocytes were incubated for 12 hours with 80 mmol/L cyclophosphamide only (panels b, e, and h) or in the presence of 80 mmol/L cyclophosphamide and 10 μmol/L PSC 833 (panels c, f, and i). Untreated oocytes were used as control (panels a, d, and g). The Live/Dead assay was performed by simultaneously monitoring the fluorescence after addition of calcein AM (panels d–f) and ethidium homodimer 1 (panels g–i). Calcein AM (green) is retained in live cells, whereas ethidium homodimer 1 (red) enters dead cells. The differential interference contrast images are respectively shown in panels a–c. Scale bar = 100 μm. (B) The fluorescence was quantified with the use of Metamorph. For each oocyte, only a small area was considered for the fluorescence measurement to avoid the surrounding granulosa cells. Cell viability is represented on the graph with the use of a relative unit: ratio of green/red fluorescence measured in each oocyte. The number of oocytes used for each condition is indicated in parentheses. Significance was assessed between oocytes treated with cyclophosphamide without or with PSC 833 with the use of Student t test (P<.005). ∗Significant difference was obtained in the presence of PSC 833.
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6. Supplemental Figure 1
Oocyte multidrug-resistant (MDR) transporter transcriptome in wild-type (WT) mice and Taf4B (knockout [KO] subfertile mice). rpkm = reads per kilobase length of transcript per million reads.
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7. Supplemental Figure 2
Multidrug-resistant (MDR) transporter transcriptome in human oocytes.
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8. Supplemental Figure 3
Titration of the MDR-1 antibody on mouse oocytes. Geminal vesicle oocytes were used to define the optimal concentration of MDR-1 antibody. (A) The control represents oocytes that were treated only with the secondary antibody, in the absence of MDR-1 primary antibody. Three dilutions of MDR-1 antibody were tested: (B) 1:100, (C) 1:250, and (D) 1:500. The differential interference contrast images are respectively shown in (E–H). ×200 magnification. Scale bar = 50 μm.
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9. Supplemental Figure 4
Immunofluorescence control on mouse oocytes. Immunofluorescence without MDR-1 primary antibody on (A) GV, (B) MI, and (C) MII oocytes. The corresponding differential interference contrast images are shown in (D–F). Pictures were taken with the same microscope settings as in Figure 3 (laser intensity, pin-hole opening). ×200 magnification. Scale bar = 50 μm.
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10. Supplemental Figure 5
Multidrug-resistant (MDR) transporter activity changes with oocyte maturation stage in FVBN mouse. (A) Oocytes were incubated with calcein AM without (control) or with PSC 833 (PSC). Pictures were taken at ×200 magnification. Scale bar = 50 μm. (B) The fluorescence was quantified with the use of Metamorph. Ten GV and 6 MI oocytes were used for the quantification in the control, and 8 GV and 10 MI oocytes were analyzed after the PSC treatment. Significance was assessed between control (without PSC) and PSC-treated oocytes for each developmental stage with the use of Student t test (P<.05). ∗Significant differences were obtained in GV and MI oocytes.
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11. Supplemental Figure 6
Human oocytes die in the presence of cyclophosphamide and PSC 833. Human oocytes were treated for 12 hours with (A) 80 mmol/L cyclophosphamide only or (B) 80 mmol/L cyclophosphamide and 10 μmol/L PSC 833. Cell death was analyzed after incubation of the oocytes in 0.04% trypan blue. Scale bar = 100 μm.
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12. Supplemental Figure 7
Multidrug-resistant (MDR) transporter activity in granulosa cells. (A) Geminal vesicle oocytes surrounded by granulosa cells were incubated with calcein AM. (B) The corresponding differential interference contrast image. ×200 magnification. Scale bar = 50 μm.
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