The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse

Abstract

In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIβ-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.

Keywords: Double-strand breaks; Meiosis; RAD9A; Spermatogenesis.

Fig. 1.

Levels of RAD9A protein in mouse testis. (A) RAD9A protein abundance detected by immunoblotting of total testis extracts (50 µg/lane) isolated from juvenile mice of ascending age, adult (Ad) wild-type testis and wild-type mouse ES cells. Beta-actin was a loading control. Mouse age (postnatal day, pnd) is indicated under each lane. (B) Localization of RAD9A protein in adult mouse testis determined by immunohistochemistry in sections from adult Rad9a^f/+ testis at 40× (left panel or at 100×, right panel). RAD9A signal (brown) was detected in the nucleus of pachytene spermatocytes (red arrows) and Sertoli cells (green arrowheads). Roman numerals indicate the stage of the mouse seminiferous epithelial cycle. Hematoxylin (blue) marks the nuclei. Spermatids were only weakly labeled. (C) Immunostaining for RAD9A, SYCP3 and DMC1 in squash preparations of adult seminiferous tubules. Left panels show RAD9A (green) and DAPI (blue; revealing nuclear localization); middle panels, SYCP3 (far red), RAD9A (green); right panels, DMC1 (red), RAD9A (green). Upper panels: leptotene/zygotene stage; lower panels: pachytene stage. In leptotene/zygotene spermatocytes, there are bright DMC1 foci at DSBs and the RAD9A signal is diffuse with some speckled aggregates. In pachytene, RAD9A localizes primarily to the XY body (arrowhead), with a few additional regions staining positive, in distinct foci not associated with chromatin. (D) Colocalization of RAD9A (green) with SUMO1 (red). SUMO1 was not detected in zygotene spermatocytes (upper panel). In pachytene spermatocytes (lower panel) RAD9A localized primarily to the XY body (arrowhead) with a few additional foci as in C. Colocalization of RAD9A and SUMO1 in a pachytene spermatocyte confirms localization of RAD9A to the XY body (arrowhead).

Fig. 2.

Testis-specific deletion of Rad9a leads to reduced testis size and loss of germ cells. (A) Quantitative RT-PCR for Rad9a mRNA in whole testis from Rad9a^f/+, Rad9a^f/del and Rad9a^f/delCre⁺ mice. Arbp is used as an internal control. The twofold reduction in Rad9a mRNA level in the heterozygous-like Rad9a^f/del mouse testis is similar to that in Rad9a^+/− ES cells (Hopkins et al., 2004). In the Rad9a^f/delCre⁺ mutant, Rad9a message was reduced to less than a third of that in the Rad9a^f/+ control. Expression of Rad9a in Sertoli cells probably accounts for the levels detected. (B) Testes from Rad9a^f/+, Rad9a^f/del and Rad9a^f/delCre⁺ mice. Rad9a^f/+ and Rad9a^f/del mouse testes were equivalent in size. However, the Rad9a^f/delCre⁺ mouse testis was one-third the size of the other two. (C) Histological examination of testicular sections from adult Rad9a^f/+ (left), Rad9a^f/del (middle) and Rad9a^f/delCre⁺ (right) littermates stained with periodic acid-Schiff (PAS). The testicular section from a sterile Rad9a^f/delCre⁺ male shows depletion of germ cell populations in most tubules, vacuolization, agametic tubules (asterisks) and lack of round or elongating spermatids. Tubules with one layer of cells were found at stages where there should normally be three to four layers.

Fig. 3.

Spermatocytes in Rad9a^f/delCre⁺ testis can escape floxed allele excision. (A,B) Immunofluorescence was performed on testicular sections from adult Rad9a^f/+ (A) and Rad9a^f/delCre⁺ (B) mice. Left panels: detection of RAD9A (green), SYCP3 (red) and nuclei (by DAPI staining; blue). Right panels: for clarity, only RAD9A and SYCP3 are shown. Pachytene spermatocytes (red SYCP3 on chromosomes) in Rad9a^f/+ testis formed an uninterrupted concentric circle with bright RAD9A signal (green) on the XY body (A, right panel, white arrows). RAD9A-positive spermatocytes were readily detected in Rad9a^f/delCre⁺ tubules indicating lack of ‘flox’ allele excision (B, right panel, white arrows). A few spermatocytes with interrupted chromosome cores and lower intensity of SYCP3 appear RAD9A-negative (white arrowheads). Rad9a^f/+ tubules were filled with structured layers including round and elongated haploid spermatids, and fewer ‘DAPI only’ haploid nuclei were detected in the lumen of Rad9a^f/delCre⁺ tubules, confirming that these cells have escaped excision of the conditional Rad9a allele.

Fig. 4.

Elevated levels of apoptosis in adult testes of infertile Rad9a^f/delCre⁺ mice. (A–C) Testes from adult Rad9a^f/+ (A) and Rad9a^f/delCre⁺ (B,C) littermates were stained using the TUNEL technique, and sections were counterstained with Hematoxylin. Higher numbers of apoptotic cells appear in Rad9a^f/delCre⁺ testes with intermediate (B) as opposed to nearly complete germ cell loss (C). Tubules were staged according to Russell et al. (Russell et al., 1990) and stages indicated with Roman numerals. Asterisks after Roman numerals indicate that mutant tubules were staged with the closest possible approximation to a normal seminiferous tubule cycle. In the Rad9a^f/delCre⁺ testes prophase I spermatocytes were observed in tubules of all stages (red arrowheads). (D) Quantification of TUNEL-stained apoptotic cells was performed in round cross sections of tubules. Data were collected from at least 100 tubules per section from three different sections and average total apoptotic cells/100 tubules (gray columns) or apoptotic index (black columns) are shown. Numbers for the Rad9a^f/+ genotype were from two randomly picked males in this group (see Table 1; results from Rad9a^f/delCre⁺ males are listed individually).

Fig. 5.

Rad9a^f/delCre⁺ spermatocytes contain residual, unrepaired DNA DSBs. (A) Co-immunofluorescence on paraffin-embedded testis tissue from pnd13 Rad9a^f/+ (left panel) and Rad9a^f/delCre⁺ (right panel) littermates. Pachytene spermatocytes have fully synapsed chromosomes marked by SYCP3 (green) and correctly formed XY bivalents stained with γH2AX (red). Rad9a^f/delCre⁺ spermatocytes often have excessive γH2AX staining resembling an apoptotic response due to DNA damage (arrowheads). DAPI was used to stain nuclei. (B) Examples of typical Rad9a^f/+ and Rad9a^f/delCre⁺ leptotene (top panels) and zygotene (middle panels) spermatocytes, with partially synapsed chromosomes as indicated by interrupted SYCP3 signal and ‘clouds’ of γH2AX localized to sites of obligatory DNA DSBs. Lower panels: normal localization of γH2AX at the XY body of a Rad9a^f/+ (left panel) and Rad9a^f/delCre⁺ (right panel) pachytene spermatocytes. Autosomes in RAD9A-deficient pachytene spermatocytes retained γH2AX indicating persistent DNA damage (right panel). (C) Double immunolabeling of DMC1 (red) and SYCP3 (green) was performed on spermatocyte spreads from Rad9a^f/+ (top panel) and two different Rad9a^f/delCre⁺ (middle and bottom panel) testes. DAPI was used to stain nuclei. Mid-pachytene stage was determined by the extent of axial element formation and chromosome compaction and lack of thickening at the ends. In Rad9a^f/+ pachytene spermatocytes, DMC1 is cleared from autosomes, indicating complete repair of DNA DSBs. Multiple DMC1 foci were present on at least three autosomes in Rad9a^f/delCre⁺ spermatocytes at mid-pachytene (arrowheads). Dashed ovals indicate the location of the sex body.

Fig. 6.

Localization of TOPBP1 in Rad9a^f/+ and Rad9a^f/delCre⁺ spermatocytes. Rad9a^f/+ (top row) and confirmed RAD9-deficient Rad9a^f/delCre⁺ (bottom row) nuclei labeled with anti-TOPBP1 (green), anti-SYCP3 (red), anti-RAD9A (far red, not shown) and DAPI (blue) are shown. Both Rad9a^f/+ (A) and Rad9a^f/delCre⁺ (E) leptotene spermatocytes contain multiple TOPBP1 foci throughout the nucleus. In Rad9a^f/+ (Bi,Bii) and Rad9a^f/delCre⁺ (Fi,Fii) zygotene spermatocytes, TOPBP1 is found along the unsynapsed regions of chromosome axes (blue arrowheads in Bi, Bii and Fii) and presumably on the X and Y (dashed oval in Bii). TOPBP1 signal in Rad9a^f/+ (Ci,Cii) and RAD9A-deficient (Gi,Gii) pachytene nuclei was observed at the unsynapsed regions of the X and Y (dashed ovals, X and Y label); however, it was quite weak in Rad9a^f/delCre⁺ pachytene-like spermatocytes (Gi,Gii,H) as opposed to a strong signal encompassing the entire sex body in Rad9a^f/+ spermatocytes (Ci,Cii). RAD9A-deficient nuclei retain diffuse TOPBP1 staining along the SC of autosomes; white arrowheads indicate interruptions in the SC in RAD9A-deficient pachytene spermatocytes (also insets in Gi,Gii); yellow arrowheads in H indicate paired homologs with partial asynapsis (top inset); green arrowhead in Gi shows TOPBP1 retained on autosomes in pachytene stage; green arrowhead in H indicates a mixed XY+autosomal domain (also H, bottom inset). Diplotene spermatocytes appeared normal in spreads from Rad9a^f/+ testis (D) and were not found in RAD9A-deficient spermatocytes from Rad9a^f/delCre⁺ testes.