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doi: 10.1242/10.1242/dev.00410

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Impaired meiotic DNA-damage repair and lack of crossing-over during spermatogenesis in BRCA1 full-length isoform deficient mice

Xiaoling Xu1,2,*, Olga Aprelikova3, Peter Moens4, Chu-Xia Deng2 and Priscilla A. Furth1,5

1 Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA
2 Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
3 Laboratory of Biosystems and Cancer, 37/5016, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
4 Department of Biology, York University, Toronto, Ontario, M3J 1P3, Canada
5 Department of Oncology, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA



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  		Fig. 1. Size and histology of testes from p53+/- control (C) and Brca1{Delta}/{Delta}p53+/- mutant (M) mice. Ages of mice are indicated. (A,D,G,J,M) Control mice; (B,E,H,K,N) Brca1 mutant mice. No significant difference in size was detected between control and mutant testes at postnatal day 10 (P10); however, mutant testes are smaller at P16 and older. Arrows indicate spermatozoa (A), diplotene spermatocytes (G), round spermatocytes (J) and differentiated spermatids (M). Mutant testes lack these cells. Arrowheads in H,K,N indicate condensed nuclei, which are TUNEL positive (see Fig. 3).

 


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  		Fig. 2. Loss of the full-length isoform of Brca1 does not interfere with the mitotic phase of spermatogenesis. Immunolocalization of GCNA in p53+/- control (A,C,E) and Brca1{Delta}/{Delta}p53+/- mutant (B,D,F) testes at P10 (A,B), P16 (C,D) and P21 (E,F). In control testes, early pachytene spermatocytes located inside the lumen (arrows) and premeiotic cells located along the base of tubules (arrowheads) are GCNA positive. By contrast, spermatocytes inside the lumen of Brca1 mutant testes are all positive for GCNA (arrows and arrowheads, B,D,F). Anti-BrdU (G,H), and anti-phosphorylated H3 (I,J) staining in testes from 6-week-old control (G,I) and mutant (H,J) mice. Arrows in G-J indicate BrdU-positive cells (G,H) and phosphorylated H3-positive cells (I,J).

 


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  		Fig. 3. Absence of the Brca1 full-length isoform results in p53-dependent and p53-independent apoptosis. Percentages of seminiferous tubules containing TUNEL-positive cells (A) and percentages of TUNEL-positive cells (B) at P10, P16 and P21 in p53+/- and Brca1{Delta}11/{Delta}11p53+/- testes. TUNEL-positive cells in Brca1{Delta}11/{Delta}11p53+/+ (P19) (C), Brca1{Delta}11/{Delta}11p53+/- (P21) (E) and Brca1{Delta}11/{Delta}11p53-/- (P21) (G) testes. Corresponding H&E-stained histological images are shown in D,F,H.

 


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  		Fig. 4. Scp3 immunofluorescence staining of primary spermatocyte chromosomes from p53+/- control (A,C,E) and Brca1{Delta}11/{Delta}11p53+/- mutant (B,D,F) mice. (A,B) Zygotene, (C,D) pachytene and (E,F) diplotene spermatocytes. Arrows in C,D indicate paired XY chromosomes. Arrows in E indicate chiasmata in diplotene spermatocytes of control mice. No normal diplotene chromosome spreads were found in mutant spermatocytes, although some cells show fragmented cores/SCs (F). (G) Comparison of pachytene and diplotene stage spermatocytes at P16 and P21. Two-thousand meiosis I spermatocytes were counted. Data are plotted as average percentage (mean±s.d.) determined from four pairs of mutant and control mice. There were no statistically significant differences in the numbers of pachytene stage spermatocytes between mutant and control mice (synapses; P>=0.08). A significant difference in the numbers of diplotene stage spermatocytes was observed (cross-over; P>=0.002).

 


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  		Fig. 5. Localization of Mlh1 in Brca1 mutant mice. (A,B) Scp3 and Mlh1 double immunofluorescence staining of chromosomes prepared from p53+/- control (A) and Brca1{Delta}11/{Delta}11p53+/- mutant (B) spermatocytes at the pachytene stage. Three pairs of mutant and control mice at P16, P18 and P21 were examined. There were no significant differences in Mlh1 foci formation between Brca1 mutant and control mice in (P<=0.0001). (C) Scp3 and Mlh1 double immunofluorescence localization of chromosomes prepared from Brca1{Delta}11/{Delta}11p53+/- oocytes. More than 10 mutant and control embryos at E16.5-17.5 were examined and no obvious differences were found. (D) Western blot analysis of Mlh1 and Pms2 in p53+/- (C) and Brca1{Delta}11/{Delta}11p53+/- (M) testes. Genotypes, ages and antibodies are indicated.

 


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  		Fig. 6. Altered localization of Rad51, but not Dmc1, in Brca1 mutant spermatocytes. Rad51 (A,B) and Dmc1 (C,D) double immunofluorescence localization of p53+/- and Brca1{Delta}11/{Delta}11 p53+/- primary spermatocytes chromosomes at the pachytene (A,B) and zygotene (C,D) stages. Three pairs of mutant and control mice at P16, P18 and P21 were examined. There were significant differences in Rad51 foci formation between Brca1 mutant and control mice (compare A with B; P<=0.0001). (E) Western blot analysis of Rad51 and Dmc1 expression in p53+/- (C) and Brca1{Delta}11/{Delta}11 p53+/- (M) testes. Genotypes, ages and antibodies are indicated.

 


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  		Fig. 7. Absence of the Brca1 full-length isoform disrupted {gamma}H2AX localization. Scp3 and {gamma}H2AX double immunofluorescence localization of control and mutant spermatocytes. (A-D,F-I) Localization of {gamma}H2AX in unirradiated testes. Localization is similar at the leptotene (Lep) and early zygotene (E. Zyg) stages in control and mutant spermatocytes. Starting from the late zygotene (L. Zyg) stage, {gamma}H2AX localizes to the XY body in wild-type spermatocytes (arrows, C,D). However, in 90% of spermatocytes from Brca1 mutant mice, {gamma}H2AX fails to localize to the XY body and remains as multiple foci at the late zygotene stage (H) and early pachytene (E. Pac) stage (I). (E,J) Localization of {gamma}H2AX in early pachytene stage spermatocytes 10 minutes after 80 Gy irradiation. The XY body cannot be detected.

 


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  		Fig. 8. Representative electrophoretic gels of RT-PCR products obtained using RNA isolated from mouse testes (A,B) and embryos (C). Genotypes, ages of the mice and primers used are indicated.

 


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  		Fig. 9. Molecular events leading to defective spermatogenesis in Brca1 full-length isoform-deficient mice. Two major functions of Brca1 during spermatogenesis are empahasized: (1) recruiting DNA damage-repair proteins to damage sites; and (2) regulating RNA expression of genes involved in DNA-damage repair. Absence of the full-length isoform of Brca1 results in impaired DNA-damage repair and leads to the termination of spermatocyte development at the pachytene stage by p53-dependent and p53-independent apoptosis.

 

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