Mouse HFM1/Mer3 is required for crossover formation and complete synapsis of homologous chromosomes during meiosis

Abstract

Faithful chromosome segregation during meiosis requires that homologous chromosomes associate and recombine. Chiasmata, the cytological manifestation of recombination, provide the physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Formation of most crossover (CO) events requires the assistance of a group of proteins collectively known as ZMM. HFM1/Mer3 is in this group of proteins and is required for normal progression of homologous recombination and proper synapsis between homologous chromosomes in a number of model organisms. Our work is the first study in mammals showing the in vivo function of mouse HFM1. Cytological observations suggest that initial steps of recombination are largely normal in a majority of Hfm1(-/-) spermatocytes. Intermediate and late stages of recombination appear aberrant, as chromosomal localization of MSH4 is altered and formation of MLH1foci is drastically reduced. In agreement, chiasma formation is reduced, and cells arrest with subsequent apoptosis at diakinesis. Our results indicate that deletion of Hfm1 leads to the elimination of a major fraction but not all COs. Formation of chromosome axial elements and homologous pairing is apparently normal, and Hfm1(-/-) spermatocytes progress to the end of prophase I without apparent developmental delay or apoptosis. However, synapsis is altered with components of the central region of the synaptonemal complex frequently failing to extend the full length of the chromosome axes. We propose that initial steps of recombination are sufficient to support homology recognition, pairing, and initial chromosome synapsis and that HFM1 is required to form normal numbers of COs and to complete synapsis.

Figure 1. Hfm1 gene target design and expression of Hfm1 in mutant mice.

(a) The mouse Hfm1 gene-targeting construct. A trapping cassette was inserted into intron 2 of the Hfm1gene. F1/R1 and F1/R2 represent primers used for genotyping wild-type and knockout mice, respectively. Exons are shown as numbered black bars. (b) Genotyping strategy for identification of Hfm1^−/− mice. (c) Predicted alternative splicing variants of Hfm1 (Hfm1 001, 005, 201 and 202). Exons are shown as red boxes and blue boxes represent portions of the HFM1 protein with recognized domains. Black arrows indicate the positions of primers used for RT-PCR analysis. (d) Transcript levels in testes of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice measured by real time RT-PCR. (e) Percentage of litter obtained from Hfm1 heterozygous couples. (f) Hfm1^−/− males display significantly smaller testes than wild-type littermates. (g) Quantification of testis weight for wild-type, heterozygous and homozygous mice.

Figure 2. Hfm1^−/− mice show profound defects in gametogenesis.

(a) Histological sections of wild-type and Hfm1^−/− testes. TUNEL assay detecting apoptotic cells is also shown (middle and lower rows). Arrows mark examples of cells after incorporation of Fluorescein-dUTP. The bottom row shows higher magnification images of diakinesis spermatocytes. (b) Histological sections of wild-type and Hfm1^−/− ovaries. CL and F identify corpus lutea and follicles respectively. (c) Single spermatocytes showing a typical metaphase I plate arrangement of chromosomes in wild-type but lagging chromosomes scattered throughout the nucleus in Hfm1^−/−.

Figure 3. Abnormal accumulation of γH2AX, RAD51, and MSH4 foci in meiotic chromosomes of Hfm1^−/− spermatocytes.

(a) Chromosome spreads of wild-type and Hfm1^−/− spermatocytes immunostained for SYCP3 and γH2AX. Magnification bar represents 5 µm. (b) Quantification of wild-type and Hfm1^−/− spermatocytes showing additional γH2AX signal. (c) Representative spermatocytes of wild-type and Hfm1^−/− mice at different stages of prophase I immunostained for SYCP3 and RAD51. Magnification bar represents 5 µm. (d) Quantification of the number of RAD51 foci per cell at the indicated stages. Lep, leptotene; E-Zyg, early zygotene; L-Zyg, late zygotene; Pach, pachytene and Dip, diplotene. Horizontal lines denote means. See Table 1 for summary of means, standard deviations, and results of statistical tests. (e) Spread nuclei of wild-type and Hfm1^−/− spermatocytes immunostained for SYCP3 and MSH4. (f) Quantification of MSH4 foci per cell at the indicated stages. Horizontal lines denote means. See Table 1 for summary of means, standard deviations, and results of statistical tests.

Figure 4. Reduced number of COs in HFM1-deficient spermatocytes.

(a) Spread nuclei of wild-type and Hfm1^−/− pachytene spermatocytes immunostained for SYCP3 and MLH1. Magnification bar represents 5 µm. (b) Quantification of autosomal MLH1 foci per pachytene cells. See Table 1 for summary of means, standard deviations, and results of statistical tests. (c) Metaphase spreads of wild-type and Hfm1^−/− spermatocytes. Note the reduced number of bivalents (arrowheads) and increased number of univalents (tailed arrows) in Hfm1^−/− cells. X and Y indicate the sex chromosomes. (d) Quantification (mean ± standard deviation) of metaphase bivalents per cell. (e) Representative diplotene wild-type and Hfm1^−/− spermatocytes stained with anti-SYCP3 and CREST (a centromere marker) antibody. Note the increase in univalent chromosomes of knockout cells (arrowheads). Tailed arrows indicate examples of forming chiasmata in wild-type spermatocytes. Magnification bar represents 5 µm. (f) Quantification of cells in (e).

Figure 5. Effect of Hfm1 deletion on sex chromosome association.

(a) Representative pachytene wild-type and Hfm1^−/− spermatocytes immunostained for SYCP3 showing tightly or loosely associated X and Y chromosomes. Magnification bar represents 5 µm. (b) Quantification of cells in (a). Bars represent standard deviation obtained from 4 wild-type and 3 Hfm1^−/− mice.

Figure 6. Meiotic prophase I progress in wild-type and Hfm1^−/− mice.

(a) Representative pachytene wild-type and Hfm1^−/− spermatocytes stained with SYCP3 and CREST. Magnification bar represents 5 µm. (b) Co-immunostaining of SYCP3 and SYCP1 in wild-type and Hfm1^−/− spermatocytes with no apparent synaptic defects at different stages of prophase I. Note that diakinesis is the last stage of spermatogenesis detected for Hfm1^−/− cells. (c) Composition of spermatocyte population in wild-type and Hfm1^−/− spermatocytes.

Figure 7. Homologous chromosomes pair normally and achieve initial synapsis in Hfm1^−/− spermatocytes.

(a) Immunostaining of SYCP3 in wild-type and Hfm1^−/− spermatocytes. Examples of pachytene-like spermatocytes are shown. Arrows mark sites of synaptic defects. XY and X-Y indicate positions of tightly and loosely associated sex chromosomes, respectively. (b) Magnified chromosomes show details of synaptic defects. (c) Quantification of synaptic anomalies in meiotic chromosomes of Hfm1^−/− spermatocytes. a - Irrespective of whether interstitial asynapsis was also present. b - % calculated with respect to total chromosomes displaying anomalies. (d) Electron microscopy of silver stained pachytene-like spermatocytes. Arrows mark sites of synaptic defects. (e) Example of tangled chromosomes in diplotene Hfm1^−/− spermatocytes. Arrows indicate sites of entangled chromosome axes. Magnification bar represents 5 µm.

Figure 8. Synaptic defects in Hfm1^−/− spermatocytes.

(a) Defects in SYCP1, TEX12 and SYCE1 deposition on synaptonemal complex of Hfm1^−/− pachytene-like spermatocytes. Wild-type is shown for comparison. XY and X-Y represents the sex chromosomes. Arrows indicate examples of chromosomes with patches of central element and transverse filament components of the synaptonemal complex. (b) Magnified chromosomes display details of patchy staining for SYCP1 and SYCE1 in Hfm1^−/− spermatocytes. Arrows indicate SYCP1 and SYCE1.

Figure 9. Hfm1^−/− spermatocytes contain a small number of bivalents with wild-type levels of SYCP1 staining and uninterrupted synapsis.

(a) Hfm1^−/− nuclei with 1 (left) and 3 (right) bivalents displaying wild-type levels of SYCP1 staining. Arrows indicate chromosomes with wild-type like distribution and signal intensity for SYCP1. (b) Quantification of SYCP1 immunosignal for wild-type and Hfm1^−/− spermatocytes. 50 chromosomes were randomly picked for wild-type and 100 chromosomes for Hfm1^−/− spermatocytes. Hfm1 I and Hfm1 II represent chromosome populations from Hfm1^−/− spermatocytes with and without reduced immunofluorescence with respect to wild-type. Plotted values were obtained using the Metamorph program to determine average intensity per chromosomal area, which was corrected for background fluorescence and normalized by the length of the chromosome.

Figure 10. The meiotic role of mouse HFM1.

The proposed model summarizes observations of this and previous studies. (a) HFM1 is required for normal progression of meiotic recombination. Deletion of HFM1 in spermatocytes leads to the absence of most but not all COs. (b) Initial stages of recombination are sufficient to support homologous chromosomes pairing and initial synapsis. However, in the absence of CO-specific intermediates synapsis cannot be completed along the full lengths of bivalents.