Sutherland JM , Sobinoff AP , Gunter KM , Fraser BA , Pye V , Bernstein IR , Boon E , Siddall NA , De Andres LI , Hime GR , Holt JE , Graf T , McLaughlin EA .

	Figure 1. Quantitative PCR (qPCR) Analysis of Musashi during ovary maturation. (A) QPCR of Msi1 in ovary time-course from post-natal day (PND) 2 through to sexual maturity at PND35 and in isolated granulosa cells (GC). MRNA expression relative to Cyclophilin A (2eΔCt); values are mean + SEM; (ovary: n = 3, GC: n = 2). (B) QPCR of Msi2 in ovary time-course from PND 2 through to PND35 and in isolated granulosa cells (GC). Fold change relative to Cyclophilin A (2eΔCt); values are mean + SEM; (ovary: n = 3, GC: n = 2).
	Figure 2. Immunoblot analysis of Musashi during ovary maturation. (A) Protein densitometry of full-length MSI1 and MSI2 in ovary time-course from post-natal day (PND) 2 through to sexual maturity at PND35. Protein expression relative to TUBA1A. For original immunoblots see Supplemental Figure S2. n = 1. (B) Immunoblot of MSI1 and MSI2 in isolated granulosa cells (GC), germinal vesicle oocytes (GV), and in vitro matured metaphase II oocytes (MII). Positive expression of MSI1 was detected at the appropriate size of 39 kDa for isoform 1 and 34 kDa for isoform 2 (only in oocytes). Positive expression of MSI2 was detected at ~37 kDa. Protein expression was measured relative to TUBA1A loading control at 55 kDa. For immunoblots with molecular weight marker, see Supplemental Figure S2.
	Figure 3. Immunolocalization of Musashi during ovary maturation. Mouse ovary sections were probed for antibodies against MSI1 (red) and MSI2 (green), and counter-stained with DNA marker DAPI (blue). Based on ovary age and follicle composition, images were classified developmentally into primordial, primary, pre-antral, and antral. Sections were visualized via epifluorescent microscopy. Insets in primordial and primary sets show zoomed image of follicle-type. Scale bar in merged images = 40 μm.
	Figure 4. Immunocytochemistry of Musashi in germinal vesicle (GV) and metaphase II (MII) oocytes. Mouse oocytes were probed for antibodies against MSI1 (red) and MSI2 (green), and counter-stained with DNA marker Hoechst (blue). Representative images show Z-stack of total oocyte. Bright-field (BF) images were taken alongside confocal immunofluorescent microscopy. In BF images GV = germinal vesicle, ZP = zona pellucida, PB = polar body, SP = MII spindle. Scale bar = 20 μm. Consistent for n = 10.
	Figure 6. Assessment of Msi2Gt/Gt ovarian and oocyte health. (A) Immunofluorescence of TUNEL and CASPASE-3 in Msi2Gt/Gt and Wt adult ovary. Representative images demonstrate an increase in TUNEL (green) and CASPASE-3 (red) in Msi2Gt/Gt pre-antral (^) and antral (+) follicles, verified via quantitative analysis n = 4. Scale bar = 50 μm; (B) Msi2Gt/Gt and Wt MII oocyte quality. MII oocytes collected from the oviductal ampullae of super-ovulated adult mice were fixed and subjected to immunocytochemistry for α-tubulin (green) to identify spindles and Hoechst (blue) to identify polar body DNA and oocyte chromosomes. Representative images show typical MII staining pattern in Wt depicting polar body (PB) formation and normal chromosome and spindle alignment (SP). Msi2Gt/Gt images depict irregular spindle assembly and chromosome alignment and oocyte apoptosis. n = 3 biological replicates, 3 mice/replicate, *: denotes p < 0.05. Scale bar = 20 μm.
	Figure 7. Assessment of Msi2Gt/Gt fertility. (A) Sperm-zona pellucida binding in Msi2Gt/Gt (n = 8) and Wt (n = 9) MII oocytes. MII oocytes were collected from the oviductal ampullae of super-ovulated adult mice and subjected to sperm zona-binding with capacitated mature epididymal spermatozoa from control adult males. Following incubation and washing, the number of sperm that remained bound to the zona pellucida was counted. ***: denotes p < 0.001. (B) Average litter size in Msi2Gt/Gt and Wt females. Breeding pairs were assembled, following genotyping, at ~5 weeks and pairs were kept together for up to 6 months. The average number of live births per litter was recorded. n = 3 individual Wt × Wt breeding pairs, n = 10 individual Msi2Gt/Gt (female) and Wt (male) pairs. * denotes p < 0.05.

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