The sensitivity of the DNA damage checkpoint prevents oocyte maturation in endometriosis

Abstract

Mouse oocytes respond to DNA damage by arresting in meiosis I through activity of the Spindle Assembly Checkpoint (SAC) and DNA Damage Response (DDR) pathways. It is currently not known if DNA damage is the primary trigger for arrest, or if the pathway is sensitive to levels of DNA damage experienced physiologically. Here, using follicular fluid from patients with the disease endometriosis, which affects 10% of women and is associated with reduced fertility, we find raised levels of Reactive Oxygen Species (ROS), which generate DNA damage and turn on the DDR-SAC pathway. Only follicular fluid from patients with endometriosis, and not controls, produced ROS and damaged DNA in the oocyte. This activated ATM kinase, leading to SAC mediated metaphase I arrest. Completion of meiosis I could be restored by ROS scavengers, showing this is the primary trigger for arrest and offering a novel clinical therapeutic treatment. This study establishes a clinical relevance to the DDR induced SAC in oocytes. It helps explain how oocytes respond to a highly prevalent human disease and the reduced fertility associated with endometriosis.

Figure 1. Incubation in follicular fluid from patients with endometriosis delays or prevents oocyte maturation in mouse.

(a) Mouse oocytes were incubated in media containing human follicular fluid and later observed for Nuclear Envelope Breakdown (NEB) and Polar Body Extrusion (PBE). (b) Rate of PBE following 16 h incubation in media containing follicular fluid. Groups without common letters indicate statistical difference (Fisher’s exact test with Bonferroni correction, P < 0.05). Number of oocytes are shown in parenthesis, bars represent standard error. Experiments were repeated 7–10 times using follicular fluid from 3–7 different patients. (c,d) Following timelapse imaging of mouse oocytes in follicular fluid the time of NEB (c) and PBE (d) were determined and plotted cumulatively. Dashed vertical lines indicate the mean timing of PBE.

Figure 2. Endometriosis follicular fluid generates reactive oxygen species and causes DNA damage in oocytes.

(a) Representative brightfield and fluorescence images of oocytes loaded with the ROS indicator 2′7′-DHDCFDA and exposed to H₂O₂, Control-FF, Endo-FF or No-FF. Scale bar represents 50 μm. (b) Fluorescence readings from oocytes shown in (a) Normalised to No-FF group. Number of oocytes shown in parenthesis, data from 3 independent experiments. (c) Representative images showing merged (top) chromatin (middle) and γH2AX (bottom) staining in oocytes. The nuclear region (top row, white box) is enlarged in the Hoechst and γH2AX images. Oocytes treated with UV light (positive control) or incubated in, Control-FF, Endo-FF or No-FF as indicated. (d) Number of γH2AX foci per oocyte from (c). (b,d) Groups without common letters indicate statistical difference (b), P < 0.001; (d), P < 0.05; ANOVA with Tukey’s post-hoc test).

Figure 3. Endometriosis follicular fluid causes metaphase arrest, but does not disrupt spindles or metaphase chromosome alignment.

(a) Oocytes fixed after 16 h incubation in follicular fluid were stained for tubulin and chromatin and then categorised morphologically for progression through meiosis (GV, germinal vesicle; PM, prometaphase; M, metaphase; A, anaphase; T, telophase; MII, metaphase II). Fisher’s exact test with Bonferroni multiple comparison correction was used to test significance. (b,c) Spindle dimensions of metaphase arrested oocytes in Endo-FF and time matched controls showing spindle length (b) and width (c). Number of oocytes are indicated in parenthesis. Groups without common letters indicate statistical difference (P < 0.05; ANOVA, Tukey’s post-hoc test). Error is standard deviation. Representative micrographs shows measurement (white arrow) with tubulin and chromatin (red and green respectively). (d,e) Scatter plots showing metaphase bivalent stretch, displacement and θ for each bivalent in the Control-FF (d) and Endo-FF (e) groups. Colour coding of points corresponds to the maximum number of standard deviations from the mean position in any axis, where the mean and s.d. are defined by No-FF metaphase oocytes (green, <1 s.d.; yellow, <2 s.d.; orange, <3 s.d.; red, ≥3 s.d.; see Figure S3 and methods for detail). Micrographs show representative images (Anti-centromeric Antibody, red; Hoechst, green). Bar charts show the percentage of bivalents according to the number of s.d. from the mean.

Figure 4. ROS produced by endometrial follicular fluid blocks oocyte maturation.

(a) PBE rate following incubation in Control-FF or Endometriosis-FF with or without ATMi (40 μM). (b) PBE rate following incubation of oocytes in Control-FF or Endometriosis-FF with or without reversine (100 nM). (c) PBE rate after 24 hour culture following microinjection of morpholino against Mad2, a control morpholino, or no morpholino in the presence or absence of Endo-FF. (d,e) PBE rate of oocytes cultured in Control-FF or Endo-FF with or without addition of resveratrol (2 μM, d) or melatonin (100 μM, e).(f) Relative 2′7′-DHDCFDA fluorescence in oocytes incubated in Endo-FF with or without addition of melatonin (100 μM). (a–f) Number of oocytes shown in parenthesis, data from 2 or 3 independent experiments. Groups without common letters indicate statistical difference (P < 0.05). Error bars represent standard errors (a–e) or standard deviation (f). Statistical test used were Fisher’s exact test with Bonferroni correction (a–e), or unpaired t-test (f). (g) Proposed model showing the mechanism by which oocyte maturation is prevented or delayed in Endo-FF.