BRCA2 deficiency is a potential driver for human primary ovarian insufficiency

Abstract

Reproductive problem has been one of the top issues for women health worldwide in recent decades. As a typical female disease, primary ovarian insufficiency (POI) results in a loss of ovarian follicles and oocytes that thus destroys women fertility. However, due to the complex of POI etiology and rare resource of human POI oocytes, few biomarkers have been identified in clinics and no effective strategy could be applied to treat POI patients. In the search of possible association between DNA damage and POI by Smart-Seq2 and RT2 profiler PCR array, we find that BRCA2, a core DNA repair gene for homologous recombination shows significantly lower expression in two POI patient oocytes. In line with this, we generated oocyte-specific knockout mouse model driven by Gdf9-Cre. The Brca2-deficient mice are infertile because of the arrested follicle development and defective oocyte quality caused by the accumulation of DNA damage. Notably, ectopic expression of Brca2 in Brca2-deficient oocytes could partially restore the oocyte maturation and chromosome stability. Collectively, our data assign a definite deficiency to BRCA2 as a POI driver during follicle development and oocyte maturation, and provide a potential fertility treatment strategy for POI patients induced by BRCA2 deficiency.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. Expression level of DNA damage response genes in human POI oocytes.

a The gene panel on the RT2 Profiler PCR Array. These genes are crucial factors in BER, NER, MMR, DSB repair, or other relative pathways in DNA damage response. b Quality control test of the whole panel of the array. Five control genes (ACTB, B2M, GAPDH, HPRT1, and RPLPO) were contained on the array as quality control. Average CT value of the control genes were summarized from real-time qPCR of the array. All the included oocytes showed qualified CT value except oocyte POI-10 and oocyte POI-13. These two oocytes were thus excluded from the subsequent experiments. c Expression level of the 84 DNA damage response genes among the individual human oocytes. POI-04 and POI-20 oocytes showed significantly lower expression of BRCA2, while POI-17 oocyte expressed lower XRCC2

Fig. 2. Deletion of Brca2 in oocytes leads to female infertility.

a Schematic illustration of deletion of Brca2 exons and creation of Brca2 knockout allele by Gdf9-Cre-mediated recombination in oocytes. b Immunoblotting analysis of Brca2 protein expression in control and Brca2-deficient oocytes. The amount of Gapdh was used as an internal control. Molecular mass is given in kilodaltons. Two hundered germinal vesicle oocytes isolated from ovaries of 2-month-old mice were used for each lane of the blots. For each experiment, at least five mice of each genotype were used. c Comparison of breeding between control and Brca2-deficient females. The female mice were cocaged with fertile male mice. The breeding assay was recorded for at least 6 months

Fig. 3. Deletion of Brca2 in oocytes impairs ovarian development.

a Representative images of ovarian size from control and Brca2-deficient mice. Scale bar, 200 μm. b Average weights of ovaries obtained from control and Brca2-deficient females. Data are shown as mean ± SEM. c–f Histology of ovarian sections from young adult control and Brca2-deficient females. Ovaries from 2-month-old mice were embedded in paraffin, and sections of 8 μm thickness were prepared and stained with H&E. Black arrows in (d) show the growing follicles at different developmental stages; yellow arrows in (f) indicate the developmentally arrested follicles with degenerating oocytes. CL, corpus luteum. Scale bars in (c) and (e), 250 μm; scale bars in (d) and (f), 50 μm. g Quantification analysis of primordial follicles between control and Brca2-deficient ovaries. h Quantification analysis of activated follicles between control and Brca2-deficient ovaries. Brca2-deficient ovary contained a significantly increased number of activated follicles with developmental arrest and degraded oocytes compared with the control ovary

Fig. 4. Deletion of Brca2 compromises oocyte meiotic maturation.

a Representative images of in vivo matured eggs from control and Brca2-deficient mice. Scale bar, 20μm. b The number of eggs obtained from control and Brca2-deficient mice. c The rate of fragmented eggs retrieved from control and Brca2-deficient mice. d Immunofluorescence of phosphorylated H2AX (γH2AX) in control and Brca2-deficient oocytes. e Fluorescence intensity of γH2AX signal in control and Brca2-deficient oocytes were summarized. f Representative images of GV oocytes obtained from control and Brca2-deficient ovaries. Scale bar, 20 μm. g The rate of developmentally arrested oocytes in control and Brca2-deficient mice. h Representative images of MII oocytes matured in vitro from control and Brca2-deficient GV oocytes. Scale bar, 20 μm. i The rate of first polar body extrusion of control and Brca2-deficient oocytes matured in vitro. j Representative images of spindle morphology and chromosome alignment in control and Brca2-deficient oocytes after in vitro maturation. Oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize spindles and counterstained with Hoechst to visualize chromosomes. Scale bar, 20 μm. k The percentage of oocytes with aberrant spindle in control and Brca2-deficient groups. l The percentage of oocytes with misaligned chromosome in control and Brca2-deficient groups. Statistical data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance

Fig. 5. Ectopic expression of Brca2 recovers the quality of Brca2-deficient oocytes.

a Immunoblotting analysis of Brca2-HA protein expression in Brca2-deficient oocytes. The blot was probed with anti-Brca2 antibody. The amount of Gapdh was used as an internal control. Two hundered germinal vesicle oocytes isolated from ovaries of 2-month-old mice were used for each lane of the blots. For each experiment, at least five mice of each genotype were used. b Representative images of MII oocytes matured in vitro from control (Brca2-proficient), Brca2-deficient, and Brca2-rescued GV oocytes. Scale bar, 20 μm. c The rate of first polar body extrusion of control, Brca2-deficient, and Brca2-rescued oocytes matured in vitro. d Representative images of spindle morphology and chromosome alignment in control, Brca2-deficient, and Brca2-rescued oocytes after in vitro maturation. Oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize spindles and counterstained with Hoechst to visualize chromosomes. Scale bar, 20 μm. (e, f) The rates of disorganized spindles and misaligned chromosomes in control, Brca2-deficient and Brca2-rescued oocytes. g Immunofluorescence of γH2AX in control, Brca2-deficient and Brca2-rescued oocytes. h Fluorescence intensity of γH2AX signal in control, Brca2-deficient and Brca2-rescued oocytes were summarized. Statistical data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance