Fig 1.
Oocyte maturation after in vitro (A,B) and in vivo (C,D) preovulatory aging (PreOA). A) Decrease in MII oocytes after PreOA in follicle culture (n = 572) compared to control (n = 721). B) Increased percentage of oocytes arrested at GV (Control: n = 33, PreOA: n = 66) and GVBD stage (Control: n = 111, PreOA: n = 204) or degenerated (Control: n = 7, PreOA: n = 76) in preovulatory-aged in vitro-cultured oocytes compared to controls. C) Decrease in mean number of ovulated MII oocytes retrieved per mouse in control mice receiving cetrorelix (+Cetro Control; n = 38) compared to controls not receiving cetrorelix (-Cetro Control; n = 38). Preovulatory aging (n = 32) further reduced the oocyte number compared to the +Cetro Control group. D) Percentage of degenerated oocytes showed no significant differences between the–Cetro Control, +Cetro Control and PreOA group. Significant difference to controls: * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 2.
Relative abundance of the maternal effect proteins SMARCA4 (A, B), NLRP5 (C, D) and YBX2 (E, F) in control and in vitro preovulatory-aged (PreOA) GV oocytes. A, A’, C, C’, E and E’) Representative images of oocytes stained with antibodies against SMARCA4, NLRP5 and YBX2. YBX2 staining (E, E’) is shown in images of oocytes examined by a glow mode by confocal laser scanning microscopy. The coloring of the scale in E corresponds to relative expression levels. B, D, F) Box plot analysis of relative abundance of SMARCA4 (B), NLRP5 (D) and YBX2 (F) proteins in PreOA oocytes compared to controls. Scale bar in E’ = 20 μm and also applies to A, A’, C, C’ and E. Significant difference to control: ** p < 0.01, *** p < 0.001.
Fig 3.
Ybx2 mRNA levels (A) and YBX2 protein localization and abundance in control and preovulatory-aged (PreOA) MII oocytes grown in vitro (B-D) or in vivo (E-G). SCC: Spindle chromosome complex; SCMC: Subcortical maternal complex. Arrowheads in B’, C’, and E’: spindle domain right and left of chromosomes that is enriched for YBX2. Scale bar in F = 20 μm and also applies to B, C and E. Scale bar in F’ = 10 μm and applies to B’, C’ and E’. Significant difference to control: * p < 0.05, *** p < 0.001.
Fig 4.
Distribution and abundance of histone 3 lysine 9 trimethylation (H3K9me3) in control and preovulatory-aged (PreOA) MII oocytes grown in vitro (A-C) or in vivo (D-F). Arrowheads in A’ indicate pericentromeric heterochromatin enriched for H3K9me3. A, B, D, E: DAPI stained MII chromosomes. A’, B’, D’, E’: anti-H3K9me3 staining. Arrow in B, B’: Unaligned chromosome. Scale bar in in E = 5 μm and also applies to A-E’. Significant difference to control: *** p < 0.001.
Fig 5.
Spindle abnormalities and chromosome alignment in in vitro control and preovulatory-aged (PreOA) MII oocytes.
Spindles (green) and chromosomes (red) in control (A) and PreOA MII oocytes (B) and the percentage of oocytes with spindle and chromosome abnormalities (C). Scale bar in B = 10 μm and also applies to A. Significant differences between groups: ** p < 0.01; *** p < 0.001.