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First published online 3 December 2003
doi: 10.1242/dev.00888

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BRCA2 deficiency in mice leads to meiotic impairment and infertility

¹ Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, 1050 Boyles Street, Frederick, MD 21702, USA
² Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA
³ Department of Epidemiology and Preventive Medicine, School of Medicine, University of Maryland, Baltimore, MD 21201, USA
⁴ Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA

View larger version (15K): [in a new window]   Fig. 1. (A) The 165 kb BAC RP11-777I19 insert. The insert contains 80 kb of the BRCA2 gene as well as 30 kb of the upstream region and 55 kb of the flanking downstream region. In addition to BRCA2, based on the sequence information (Accession Numbers Z73359 and 74739) the BAC insert is predicted to contain two novel genes of unknown function. (B) Southern analysis of EcoRV digested DNA isolated from littermates obtained from a cross between Brca2 Ko/+; Tg/+ and Brca2 Ko/+ mice. WT, wild-type allele; MT, mutant allele. Presence of a 8 kb band (lower panel) on BamHI-digested DNA shows the presence of the BAC transgene (Tg); (C) Table showing the offspring of various genotypes obtained from a cross between Brca2 Ko/+; Tg/+ and Brca2 Ko/+ mice, in expected Mendelian ratios.

View larger version (146K): [in a new window]   Fig. 2. In situ hybridization analyses comparing the expression of the human BRCA2 transgene and endogenous mouse Brca2. (A-D) Sagittal sections of a E9.5 transgenic embryo. (E-H) Sections of the testis of a 3-week-old transgenic male. (I-L) Sections of the ovary of 3-week-old transgenic female. (M-P) Sections of the ovary of a 10-week-old transgenic female. (A,E,I M) Bright-field pictures of Hematoxylin and Eosin stained sections adjacent to those used in the in situ hybridization studies; (D,H,L,P) respective sections hybridized with human BRCA2 sense probe as negative control. (B) Mouse Brca2 expression is detected in the E9.5 embryo in the brain in the neuroepithelium of the ventricular region and the neural tube (first branchial arches). (C) The BRCA2 transgene is expressed at reduced levels compared with the endogenous gene but the spatial expression pattern is identical to that of the endogenous gene. (F) Brca2 expression is detected using antisense probe in the spermatocytes present in the seminiferous tubules. (G) The human BRCA2 transgene expression is found at a level just above background. (J) In the ovary of a 3-week-old female, the mouse Brca2 gene is found to be highly expressed in follicles at various stages of maturation. (K) No specific signal is observed in the follicles by human BRCA2 antisense probe. (N) Brca2 expression is detected in the follicles in granulosa cells and oocytes (arrow) of 10-week-old ovary. (O) Expression of the human transgene is not detected in the ovary. A, antral follicle; ba, branchial arch; CL, corpus lutea, GC, granulosa cells; ne, neuralepithelium; nt, neural tube; O, oocyte; Pa, preantral follicle; Pr, primary follicle; Sp, spermatocytes; vl, ventricular layer. Scale bars: 100 µm.

View larger version (136K): [in a new window]   Fig. 3. Histology of testes at various ages. Testes from control (Brca2 Ko/+; Tg/+, A and C) and rescued males (Brca2 Ko/Ko; Tg/+, B,D,E) at 3 weeks (A,B), 2 months (C,D) and 7 months (E) of age were analyzed. (A,B) In young control males at 3 weeks of age, the seminiferous tubules are filled with spermatocytes. The rescued testes show presence of several seminiferous tubules with spermatocytes but there are few that have reduced number of spermatocytes. (C,D) At 2 months, adult male control testes contain spermatocytes at various stages of maturation as well as spermatozoa in the seminiferous tubules. In rescued males, the spermatocytes appear to be arrested in meiotic prophase I and no postmeiotic cells are visible. Several seminiferous tubules containing only Sertoli cells are observed. There appears to be a relative increase in the number of Sertoli cells per seminiferous tubule in the mutants (see insets, arrows indicate the Sertoli cells). (E) In rescued testes, by 7 months of age, most of the seminiferous tubules are completely devoid of germ cells and none contain normal looking spermatocytes, while control tubules exhibit normal spermatogenesis. (F,G) In rescued testes, there is an increase in apoptosis, as indicated by the TUNEL-positive staining cells (brown precipitate). Very few spermatocytes show brown staining in 3-week-old control males where as majority of the spermatocytes show positive staining in the rescued testes. In the control testis, some tubules show TUNEL-positive cells next to the basal lamina of the tubules, indicating that they are the spermatogonial cells. S, spermatozoa; Sp, spermatocytes. Scale bars: 50 µm.

View larger version (85K): [in a new window]   Fig. 4. Analysis by immunofluorescence of the localization of BRCA2, H1t, SPO11 and -H2AX proteins during male meiosis. Localization of these proteins was compared between control (Brca2 Ko/+; Tg/+, A,C,E,G,I) and rescued (Brca2 Ko/Ko; Tg/+, B,D,F,H,J) in preparations of surface-spread chromatin from spermatocytes. Nuclei were stained with antisera against SYCP3 (in red, except in C and D where SYCP3 is in green). (A) Mouse BRCA2 is present in the nuclei during zygonema, but (B) no signal is detected in the rescued spermatocytes as they lack a functional mouse Brca2 gene. (C,D) The human protein is not detected in control or rescued spermatocytes. (E,F) Nuclei were examined for presence of male germ cell-specific, mid-pachytene marker, histone H1t. Control cells show positive staining but rescued cells lack the staining. (G,H) Both control and rescued spermatocytes exhibit expression of SPO11 protein during leptotene/zygotene stage, with diminished staining by pachynema in control spermatocytes (arrow). (I,J) Localization of phosphorylated histone H2AX (-H2AX), a marker for double-strand break formation, is seen in both genotypes. During pachytene stage, the staining of -H2AX disappears except in the sex chromosomes (arrow). Rescued spermatocytes do not show this typical pachytene staining pattern, further evidence that they do not reach this stage.

View larger version (64K): [in a new window]   Fig. 5. Immunofluorescent analysis of localization of RAD51, DMC1 and RPA proteins (green) during male meiosis. Expression of these proteins was compared between control (Brca2 Ko/+; Tg/+, A,D, F) and rescued (Brca2 Ko/Ko; Tg/+, B,E,G) spermatocytes. Nuclei are also stained with SYCP3 antisera (in red). (A,B) RAD51 foci are visible on chromatin of control spermatocytes during the zygotene stage, but most of the rescued spermatocytes showed little or no RAD51 foci. (C) Semi-quantitative analysis of RAD51 foci per spermatocyte at the leptotene and zygotene stages reveals the reduction in number of foci in rescued spermatocytes. The majority of control spermatocytes had 100-250 foci per nuclei (>100 group). In the rescued spermatocytes, 58% had no foci while majority of nuclei in the 1-100 group had fewer than 10 foci, although a small number had 20-30 foci per nuclei. (D,E) Similar to RAD51, there is marked reduction in the number of DMC1 foci in rescued spermatocytes compared with control cells. (F,G) RPA protein is present in abundant foci in rescued spermatocytes compared with control spermatocytes.

View larger version (121K): [in a new window]   Fig. 6. Histology of ovaries obtained from control and rescued females at various ages. (A,C,E) Ovaries from control females (Brca2 Ko/+; Tg/+); (B,D,F) ovaries from rescued females (Brca2 Ko/Ko; Tg/+). (A,B) Ovaries from postnatal day 2 females stained with Weigert's Hematoxylin-picric acid Methyl Blue; (C,D) Three-week-old females; (E,F) Six-week-old females. The 3- and 6-week-old ovary sections are stained with Hematoxylin and Eosin. A marked reduction in the follicles was seen in the 6-week-old rescued ovaries. CL, corpus lutea; P, primordial follicles; Pr, primary follicles; Pa, preantral follicles; A, antral follicles; O, oocyte. Scale bars: 50 µm. (G) Table shows the number of follicles found in 3-week-old control and rescued female ovaries. The number of primordial and primary follicles were significantly reduced in rescued females.

View larger version (21K): [in a new window]   Fig. 7. Results of in vitro maturation of oocytes. GV-intact oocytes were collected from control and rescued female mice and allowed to undergo meiotic maturation in vitro. (A,B) The percentages of oocytes that had emitted the first polar body (PB1), undergone germinal vesicle breakdown (GVBD) but not emitted the first polar body, emitted an oversized polar body (oversized PB1), or remained GV-intact after 16 hours (A) or 19 hours (B). Values above the bars show the actual numbers of oocytes with the given appearance per total number of oocytes in a group.

View larger version (55K): [in a new window]   Fig. 8. Phenotypes of oocytes after in vitro maturation. Examples of the various phenotypes observed in matured control and rescued oocytes (red labeling indicates F-actin detected by phalloidin, and blue labeling indicates DNA detected by DAPI). (A) Normal mature `PB1' egg, with the first polar body emitted (indicated by PB1) and the egg chromosomes organized on the metaphase II spindle. An actin-rich cap overlies the meiotic spindle (asterisk). (B,C) Two examples of `oversized polar body' phenotype. Twenty-eight percent (7/25) of the rescued eggs had this phenotype, and in six of these seven, all of the DNA was present in the polar body-like structures (arrows), with no DNA present in the egg. These polar body-like structures ranged in size from very large, containing approximately one-third of the oocyte cytoplasm (B) to somewhat more normal in size (C; compare with A). (D) An oocyte that has undergone GVBD but not emitted PB1, with chromatin near the center of the egg that appears to have failed to condense properly. In this cell, there is no actin-rich cap and instead the actin is symmetrically localized around the cell cortex, as it is in a GV-intact oocyte. (E) An oocyte that has undergone GVBD but not emitted PB1, with condensed chromatin that remains near the center of the egg (rather than having migrated to the cell periphery). This DNA appears to be poorly organized, not organized on a metaphase I spindle. As in the cell in D, the actin in this cell is symmetrically localized. (F) An oocyte that has undergone GVBD but not emitted PB1, with the metaphase I spindle having migrated to the cell periphery. The asterisk indicates the actin-rich cap over the metaphase I spindle. This oocyte appears poised to undergo PB1 emission; it is not clear if it was arrested at this stage or was simply slower than the rest of its cohort. (G) An egg that has emitted PB1 (out of the plane of focus of this image), but the egg DNA looks abnormal, as a tightly condensed wad of DNA rather than chromosomes organized on the metaphase II spindle. (Only the DAPI staining in shown in this image to show the DNA wad more clearly.) (H) A nearly normal metaphase II egg, except the chromosomes are not completely aligned on the metaphase II plate; the arrowhead indicates chromatin cluster that is not aligned. (Only the DAPI staining in shown in this image, in order to show the straggler chromosome more clearly.) (I) Percentages of the control and rescued oocytes that displayed each of the eight observed phenotypes after 19 hours in culture medium lacking dbcAMP. Values over the bars indicate the actual numbers of oocytes per total control or rescued oocytes that displayed that phenotype.