Research ArticleCELL BIOLOGY

SPO16 binds SHOC1 to promote homologous recombination and crossing-over in meiotic prophase I

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Science Advances  23 Jan 2019:
Vol. 5, no. 1, eaau9780
DOI: 10.1126/sciadv.aau9780
  • Fig. 1 Identification of mammalian SPO16.

    (A and B) Immunoprecipitation (IP) experiments showing the interaction of SPO16 with the XPF-like domain of SHOC1 (amino acids 975 to 1156). HA, hemagglutinin. (C to E) Levels of Spo16 mRNA in multiple mouse tissues (C), in developing testes (D), and in male germ cells at different stages (E). Gapdh served as the loading control. sgA, spermatogonia type A; sgB, spermatogonia type B; pre-L, preleptonema; L-Z, leptonema to zygonema; P, pachynema; RS, round spermatids; ES, elongated spermatids; ov, ovary. (F) Immunofluorescent staining of exogenously expressed, GFP-tagged SPO16 protein (green) and SYCP3 (red) on the nuclear surface spreads of WT or Dmc1−/− testes electroporated with plasmids encoding SPO16-GFP. SYCP3 marks the meiotic chromosome axes. Arrows indicate the sex bodies. Enlarged images show the partially or fully synapsed homologous pairs and the regions of which are bordered with a dashed line. L, leptotene; EZ, early zygonema; MZ, midzygonema; LZ, late zygonema; EP, early pachynema; Z-like, zygonema-like. Scale bar, 10 μm. (G) Quantification of GFP foci detected in WT and Dmc1−/− spermatocytes at indicated stages. Z*, zygonema-like. Numbers of spermatocytes analyzed (n) are indicated. Median focus numbers are marked. Error bar indicates SEM. ***P < 0.001 by two-tailed Student’s t tests. (H) Costaining of FLAG-SPO16 (green) with RAD51 (red) on the nuclear surface spreads of WT testes electroporated with plasmids encoding FLAG-SPO16. RAD51 marks early recombination nodules. Scale bar, 10 μm. (I) Costaining of SPO16-GFP (green) with RPA2 (red) on nuclear spreads prepared from WT testes electroporated with plasmids encoding SPO16-GFP. Scale bar, 10 μm. (J) Costaining of FLAG-SPO16 (green) with SYCP1 (red) on the nuclear surface spreads of WT testes electroporated with plasmids encoding FLAG-SPO16. Scale bar, 10 μm.

  • Fig. 2 Deletion of Spo16 led to massive germline loss and infertility in both males and females.

    (A) Schematic diagram of the CRISPR-Cas9 strategy to generate null allele for Spo16. Exons, sgRNA, and primers are indicated. The detailed sequence is shown in fig. S2D. Chr, chromosomes. (B) Genotyping results to distinguish the WT (+) allele and the null allele (−), which contains 19-bp insertion. Mk, DNA marker. (C) Representative image of testes derived from Spo16+/− and Spo16−/− males at the age of PD42. (D) Weights of testes derived from WT and Spo16−/− males at indicated ages. Numbers of testes analyzed (n) are indicated. Error bars indicate SEM. n.s., not significant. Dashed line shows the weight of the knockout testes at PD90. ***P < 0.001 by two-tailed Student’s t tests. (E) H&E staining results of paraffin-embedded testes from WT and Spo16−/− males. Stages of seminiferous tubules in control testes are indicated. Scale bar, 50 μm. (F) Morphology of Spo16+/− and Spo16−/− ovaries at PD42. (G) Immunohistochemistry (IHC) staining of MVH showing the oocytes in WT and Spo16−/− ovaries at E17.5 and PD1. Scale bar, 50 μm.

  • Fig. 3 Meiocytes null for SPO16 failed to achieve complete synapsis.

    (A) Staining of the synapsed chromosome marker SYCP1 (green) with SYCP3 (red) on the nuclear surface spreads of spermatocytes derived from WT and Spo16−/− males at PD42. P-like, pachynema-like. Scale bar, 10 μm. (B) Meiotic prophase progression in WT and Spo16−/− testes. Error bars indicate SEM. P*, pachynema-like. The numbers of spermatocytes analyzed (n) are indicated. (C) Staining of the unsynapsed chromosome marker HORMAD1 (green) with SYCP3 (red) on the nuclear surface spreads of spermatocytes derived from WT and Spo16−/− males at PD42. Scale bar, 10 μm. (D) Meiotic prophase I progression of female PGCs in WT and Spo16−/− ovaries. The numbers of spermatocytes analyzed (n) are indicated. (E and F) SYCP1 (E) and HORMAD1 (F) were stained together with SYCP3 on the nuclear surface spreads of female PGCs derived from WT and Spo16−/− ovaries at E17.5. Scale bars, 10 μm.

  • Fig. 4 SPO16 deletion caused defects in homolog pairing and meiotic recombination.

    (A) γH2AX (green) staining marked the sex body of WT spermatocytes at pachytene stage and PSB of pachytene-like Spo16−/− spermatocytes. Green dashed lines in the magnified images indicate the area of γH2AX staining. Scale bar, 10 μm. (B) Quantification of chromosomes within the sex body or PSB. Numbers of spermatocytes analyzed (n) are indicated. ***P < 0.001 by two-tailed Student’s t tests. (C) Staining of TRF1 (marker of telomeres; green) and CREST (marker of centromeres; red) showing nonhomologous pairing and asynapsis in Spo16−/− spermatocytes. Scale bar, 10 μm. (D and E) Quantification of TRF1 (D) and CREST (E) dots in WT and Spo16−/− spermatocytes at pachytene and pachytene-like stages, respectively. Numbers of spermatocytes analyzed (n) are indicated. Median focus numbers are marked. Error bars indicate SEM. **P < 0.01 and ***P < 0.001 by two-tailed Student’s t tests. (F to H) Early recombination markers, RAD51 and DMC1, were detected on the nuclear surface spreads of WT and Spo16−/− spermatocytes (F), and the quantifications of RAD51 and DMC1 foci are shown in (G) and (H), respectively. Scale bar, 10 μm. Numbers of spermatocytes analyzed (n) are indicated. ***P < 0.001 by two-tailed Student’s t tests. (I and J) MLH1 (late recombination marker; green) was detected on the nuclear surface spreads of WT and Spo16−/− spermatocytes and PGCs (I) at indicated stages, and the quantification of MLH1 foci is shown in (J). Scale bar, 10 μm. ***P < 0.001 by two-tailed Student’s t tests.

  • Fig. 5 Assembly of ZMM foci during meiotic recombination.

    (A) Western blot showing the levels of SHOC1 and TEX11 in WT, Spo16−/−, and Shoc1−/− testes at the age of PD21. (B and C) SHOC1 was detected on the nuclear surface spreads of WT, Spo16−/−, Dmc1−/−, and Spo16−/−;Dmc1−/− spermatocytes at indicated stages (C), and the quantification of SHOC1 foci is shown in (B). Scale bar, 10 μm. ***P < 0.001 by two-tailed Student’s t tests. (D and E) Immunostaining of TEX11 on the nuclear surface spreads of WT, Spo16−/−, Dmc1−/−, and Spo16−/−;Dmc1−/− spermatocytes at indicated stages. The quantification of TEX11 foci is shown in (D). Scale bar, 10 μm. ***P < 0.001 by two-tailed Student’s t tests. (F and G) Immunostaining results and quantification of MSH4 foci were detected on the nuclear surface spreads of WT, Spo16−/−, Dmc1−/−, and Spo16−/−;Dmc1−/− spermatocytes at indicated stages. Scale bar, 10 μm. ***P < 0.001 by two-tailed Student’s t tests.

  • Fig. 6 SPO16 was required for the stabilization for RPA-SPATA22-MEIOB complex.

    (A and B) SPATA22 was detected on the nuclear surface spreads of WT, Spo16−/−, Dmc1−/−, and Spo16−/−;Dmc1−/− spermatocytes (A), and the quantification is shown in (B). Scale bar, 10 μm. **P < 0. 01 and ***P < 0.001 by two-tailed Student’s t tests. (C and D) RPA1 was detected on the nuclear surface spreads of WT and Spo16−/− spermatocytes (C), and the quantification of RPA1 foci is shown in (D). Scale bar, 10 μm. ***P < 0.001 by two-tailed Student’s t tests. (E) Schematic diagram showing a proposed model of the roles of mammalian SHOC1-SPO16-TEX11 complex in meiotic recombination in meiotic prophase I. After strand invasion, SHOC1 is recruited to the joint molecules or D loops. This is a step crucial for the stabilization of these structures and promoting DNA synthesis and elongation of the DNA ends. After that, SHOC1 further recruits SPO16 and TEX11 to facilitate the repair of DSBs through a DSBR pathway with the formation of dHJs. In the absence of SPO16 or TEX11, the DSBs are repaired via a SDSA pathway. KO, knockout.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau9780/DC1

    Fig. S1. MmSPO16 has a conserved XPF-like domain.

    Fig. S2. Localization of SPO16 and generation of knockout mice.

    Fig. S3. SPO16 deletion leads to massive germline loss.

    Fig. S4. Insufficient meiotic prophase progression in Spo16−/− testes.

    Fig. S5. Insufficient meiotic recombination in SPO16-deleted spermatocytes and oocytes.

    Fig. S6. Detection of RPA complex in WT and SPO16-deleted spermatocytes.

    Table S1. Genes specifically expressed in meiotic prophase I.

    Table S2. Homology of MmSPO16 to known proteins.

    Table S3. Primer sequences.

    Table S4. Antibody information.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. MmSPO16 has a conserved XPF-like domain.
    • Fig. S2. Localization of SPO16 and generation of knockout mice.
    • Fig. S3. SPO16 deletion leads to massive germline loss.
    • Fig. S4. Insufficient meiotic prophase progression in Spo16−/− testes.
    • Fig. S5. Insufficient meiotic recombination in SPO16-deleted spermatocytes and oocytes.
    • Fig. S6. Detection of RPA complex in WT and SPO16-deleted spermatocytes.
    • Table S1. Genes specifically expressed in meiotic prophase I.
    • Table S2. Homology of MmSPO16 to known proteins.
    • Table S3. Primer sequences.
    • Table S4. Antibody information.

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