FancJ (Brip1) loss-of-function allele results in spermatogonial cell depletion during embryogenesis and altered processing of crossover sites during meiotic prophase I in mice

Abstract

Fancj, the gene associated with Fanconi anemia (FA) Complementation Group J, encodes a DNA helicase involved in homologous recombination repair and the cellular response to replication stress. FANCJ functions in part through its interaction with key DNA repair proteins, including MutL homolog-1 (MLH1), Breast Cancer Associated gene-1 (BRCA1), and Bloom syndrome helicase (BLM). All three of these proteins are involved in a variety of events that ensure genome stability, including the events of DNA double strand break (DSB) repair during prophase I of meiosis. Meiotic DSBs are repaired through homologous recombination resulting in non-crossovers (NCO) or crossovers (CO). The frequency and placement of COs are stringently regulated to ensure that each chromosome receives at least one CO event, and that longer chromosomes receive at least one additional CO, thus facilitating the accurate segregation of homologous chromosomes at the first meiotic division. In the present study, we investigated the role of Fancj during prophase I using a gene trap mutant allele. Fancj (GT/GT) mutants are fertile, but their testes are very much smaller than wild-type littermates, predominantly as a result of impeded spermatogonial proliferation and mildly increased apoptosis during testis development in the fetus. This defect in spermatogonial proliferation is consistent with mutations in other FA genes. During prophase I, early events of synapsis and DSB induction/repair appear mostly normal in Fancj (GT/GT) males, and the FANCJ-interacting protein BRCA1 assembles normally on meiotic chromosome cores. However, MLH1 focus frequency is increased in Fancj (GT/GT) males, indicative of increased DSB repair via CO, and is concomitant with increased chiasmata at diakinesis. This increase in COs in the absence of FANCJ is associated with increased localization of BLM helicase protein, indicating that BLM may facilitate the increased rate of crossing over in Fancj (GT/GT) males. Taken together, these results demonstrate a critical role for FANCJ in spermatogenesis at two stages: firstly in the proliferative activity that gives rise to the full complement of testicular spermatogonia and secondly in the establishment of appropriate CO numbers during prophase I.

Keywords: BACH1; BRIP1; Fanconi anemia; Gametogenesis; MLH1; Meiotic prophase I.

Fig. 1

Testis weights and sperm numbers are significantly reduced in Fancj^GT/GT male mice compared to wildtype and heterozygous littermates. Testis weights (a) and epididymal spermatozoa counts (b) for Fancj^+/+ (filled circles, n=3), Fancj^GT/+ (filled triangles, n=6), Fancj^GT/GT (filled squares, n=6) male mice. Testis weights are for pairs of testes and expressed in mg ± s.d. Sperm counts are presented as entire epididymal spermatozoa counts per mouse. Unpaired t tests were used to compare testis weights and sperm numbers between Fancj^+/+ and Fancj^GT/GT: ***p<0.0001, **p<0.05. No statistically significant differences were found between Fancj^+/+ and Fancj^GT/+ males for either testis weights or sperm counts. c–d Testicular histology for Fancj^+/+ (c) and Fancj^GT/GT (d) males, as shown by H&E staining of paraffin-embedded testis sections. Sg, spermatogonia; Sc, spermatocytes; St, spermatids; S, spermatozoa. e, f Testicular TUNEL staining to reveal apoptotic cells in Fancj^+/+ (e) and Fancj^GT/GT (f) males

Fig. 2

Impaired establishment of the primordial germ cell population in Fancj^GT/GT males during fetal development. Testis sections from Fancj^GT/+ (black bars) and Fancj^GT/GT (gray bars) males from e14.5 through until day 5 pp stained for the germ cell marker, TRA98 (a), and processed for apoptotic cell counts using the TUNEL assay (b). Values given are numbers of cells per ×20 view±standard deviation. Statistically significant differences are highlighted with asterisks: *p<0.05; **p<0.001; ***p<0.0001 (unpaired t test)

Fig. 3

Normal chromosome synapsis during prophase I in Fancj^GT/GT males. Synapsis is observed by the localization of the synaptonemal complex proteins SYCP1 (red) and SYCP3 (green) on meiotic chromosome spread preparations from Fancj^+/+ (a–d) and Fancj^GT/GT (e–f) males. Synapsed chromosome cores are indicated where red and green signals overlap to produce a yellow signal

Fig. 4

Prophase I progression in Fancj^+/+ and Fancj^GT/GT males. Double strand break processing was monitored by staining for γH2AX to highlight the location and frequency of DSBs (a–d, m–p) and RAD51 (e–h, q–t) to highlight initial events of DSB processing. Additionally, BRCA1 is known to participate in various events during prophase I and localizes to chromosome cores (i–l, u–x). In addition, BRCA1 binds to FANCJ. However, loss of FANCJ in the Fancj^GT/GT males does not affect the normal localization of BRCA1 during prophase I

Fig. 5

Elevated and persistent BLM focus frequency at pachynema in Fancj^GT/GT males. BLM is localized in red and SYCP3 in green on meiotic chromosome spread preparations from Fancj^+/+ (a–d) and Fancj^GT/GT (e–f) males through prophase I. Upper panels show the three-color images, and lower panels show just the BLM staining

Fig. 6

Elevated class I crossovers in Fancj^GT/GT males. a–b MLH1 localization (green) on meiotic chromosome cores stained with SYCP3 (red) in spermatocyte spreads from Fancj^+/+ (a) and Fancj^−/− (b) males. c–d Giemsa-stained diakinesis preps showing chiasmata configurations from Fancj^+/+ (c) and Fancj^−/− (d) males. e Quantitation and statistical comparison of MLH1 focus frequency and chiasmata counts from at least 3 mice for each genotype. Fancj^+/+ shown with filled/open circles, Fancj^−/− shown with filled/open squares. ***p<0.0001 by Mann–Whitney U test

Fig. 7

Western blot analysis of whole testis lysates from Fancj^+/+ and Fancj^GT/GT males reveals specific alterations in DNA MMR proteins and BLM. a–g Quantitation of protein levels for BRCA1, BLM, MLH1, MLH3, MSH4, MSH5, and MUS81 normalized to β-tubulin, showing significant increases in protein levels in Fancj^GT/GT (gray bars) compared to Fancj^+/+ (black bars) testis extracts for all except BRCA1 and MUS81 (p<0.0001, unpaired t test). Representative Western blots are provided in panel h (at least 3 separate westerns were performed from two biological replicates) along with their respective controls (actin or tubulin, depending on the size of the protein of interest). Quantitation of each target protein is based on normalization to actin or tubulin