DNA repair in primordial follicle oocytes following cisplatin treatment

Abstract

Purpose: Genotoxic chemotherapy and radiotherapy can cause DNA double stranded breaks (DSBs) in primordial follicle (PMF) oocytes, which then undergo apoptosis. The development of effective new fertility preservation agents has been hampered, in part, by a limited understanding of DNA repair in PMF oocytes. This study investigated the induction of classical DSB repair pathways in the follicles of wild type (WT) and apoptosis-deficient Puma^-/- mice in response to DSBs caused by the chemotherapy agent cisplatin.

Methods: Adult C57BL/6 WT and Puma^-/- mice were injected i.p. with saline or cisplatin (5 mg/kg); ovaries were harvested at 8 or 24 h. Follicles were counted, and H2A histone family member (γH2AX) immunofluorescence used to demonstrate DSBs. DNA repair protein RAD51 homolog 1 (RAD51) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) immunofluorescence were used to identify DNA repair pathways utilised.

Results: Puma^-/- mice retained 100% of follicles 24 h after cisplatin treatment. Eight hours post-treatment, γH2AX immunofluorescence showed DSBs across follicular stages in Puma^-/- mice; staining returned to control levels in PMFs within 5 days, suggesting repair of PMF oocytes in this window. RAD51 immunofluorescence eight hours post-cisplatin was positive in damaged cell types in both WT and Puma^-/- mice, demonstrating induction of the homologous recombination pathway. In contrast, DNA-PKcs staining were rarely observed in PMFs, indicating non-homologous end joining plays an insignificant role.

Conclusion: PMF oocytes are able to conduct high-fidelity repair of DNA damage accumulated during chemotherapy. Therefore, apoptosis inhibition presents a viable strategy for fertility preservation in women undergoing treatment.

Keywords: Apoptosis; Chemotherapy; DNA repair; Fertility; Follicle; Oocyte.

Fig. 1

Follicular quantification in Puma^-/- mice by follicular stage and treatment group. Primordial (a), transitional (b), primary (c), secondary (d), antral (e), and atretic (f) follicles were counted in ovaries harvested 24 h after Puma^-/- mice were treated with either saline or 5 mg/kg cisplatin (cisplat) (N = 3/treatment group). Data are expressed as mean follicles per animal ± SEM; statistical comparisons were made using Student’s unpaired t test

Fig. 2

γH2AX-positive follicles by follicular stage and treatment group in Puma^-/- mice. Following treatment with saline or cisplatin (cisplat), γH2AX-positive follicles were counted, and expressed as a percentage of the total follicles seen in each respective follicular stage. This was performed at three time-points post-treatment: 8 h (a), 24 h (b), and 5 days (c). Data are expressed as mean percentage ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s unpaired t test)

Fig. 3

γH2AX staining in follicles by follicular stage and treatment group in Puma^-/- mice. γH2AX-stained follicles of all stages were visualised by confocal microscopy at 8 hours, 24 hours, and 5 days following treatment with saline or cisplatin (cisplat). Red = γH2AX; green = c-Kit; blue = DAPI. Representative images of positively staining primordial, transitional, primary, secondary, and antral follicles at 8 hours following cisplatin treatment (a). Scale bars = 10 μm (primordial, transitional, primary follicles); 50 μm (secondary); 100 μm (antral). Follicles staining positively for γH2AX were further analysed to assess the cell types affected at each follicular stage, and expressed as a total percentage of γH2AX-positive follicles for that stage. Analysis was conducted at 8 hours following treatment (b) with saline (i) or cisplatin (ii); 24 h following treatment (c) with saline (i) or cisplatin (ii); 5 days following treatment (D) with saline (i) or cisplatin (ii)

Fig. 4

RAD51-positive follicles by follicular stage and treatment group in WT and Puma^-/- mice. Eight hours following treatment with saline or cisplatin (cisplat), RAD51-positive follicles were counted, and expressed as a percentage of the total follicles seen in each respective follicular stage. This was performed in WT mice (a) and Puma^-/- mice (b). Data are expressed as mean percentage ± SEM; P < 0.05, **P < 0.01 (Student’s unpaired t test)

Fig. 5

RAD51 staining in follicles by follicular stage and treatment group in WT and Puma^-/- mice. RAD51-stained follicles of all stages were visualised by confocal microscopy at 8 hours following treatment with saline or cisplatin (cisplat). Red = RAD51; green = c-Kit; blue = DAPI. Representative images of positively staining primordial, transitional, primary, secondary, and antral follicles of Puma^-/- mice, 8 hours following treatment with cisplatin (A). Scale bars = 10 μm (primordial, transitional, primary follicles); 50 μm (secondary); 100 μm (antral). Follicles staining positively for RAD51 were further analysed to assess the cell types affected at each follicular stage, and expressed as a total percentage of RAD51-positive follicles for that stage. Analysis was conducted in WT mice (B) 8 hours following treatment with saline (i) or cisplatin (ii); and in Puma^-/- mice (C) 8 hours following treatment with saline (i) or cisplatin (ii)

Fig. 6

DNA-PKcs-positive follicles by follicular stage and treatment group in WT and Puma^-/- mice. Eight hours following treatment with saline or cisplatin (cisplat), DNA-PKcs-positive follicles were counted, and expressed as a percentage of the total follicles seen in each respective follicular stage. This was performed in WT mice (a) and Puma^-/- mice (b). Data are expressed as mean percentage ± SEM; P < 0.05, **P < 0.01 (Student’s unpaired t test)