Research ArticleDEVELOPMENTAL BIOLOGY

The histone modification reader ZCWPW1 is required for meiosis prophase I in male but not in female mice

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Science Advances  14 Aug 2019:
Vol. 5, no. 8, eaax1101
DOI: 10.1126/sciadv.aax1101
  • Fig. 1 ZCWPW1 expression and dynamic localization in meiotic germ cells.

    (A) Western blotting of ZCWPW1 in isolated male germ cells, demonstrating that the Zcwpw1 expression level increased from the leptotene stage to the round spermatid stage and then disappeared in the elongated spermatid. β-Actin was used as the control. (B) Western blotting of ZCWPW1 in the cytoplasmic and nuclear fractions of PD35 wild-type testes shows that ZCWPW1 was only expressed in the nuclei. Lamin B1 was used as the marker of nuclear fractions. (C to K) Chromosome spreads of spermatocytes from the testes of PD35 Zcwpw1+/+ and Zcwpw1−/− males immunostained for ZCWPW1 (green) and SYCP3 (red). ZCWPW1 was diffuse (arrows) from the leptotene to zygotene stages (C to F, arrows). In pachytene cells, ZCWPW1 was localized in the XY body (G and H, white dashed circles). (I to K) In Zcwpw1−/− spermatocytes, no ZCWPW1 signal was detected. (L to T) Chromosome spreads of oocytes from E17.5 Zcwpw1+/+ and Zcwpw1−/− ovaries immunostained for ZCWPW1 (green) and SYCP3 (red). ZCWPW1 was diffuse (arrows) from the leptotene to pachytene stages (L to Q, arrows). (R to T) In Zcwpw1−/− oocytes, no ZCWPW1 signal was detected.

  • Fig. 2 ZCWPW1 is needed for maintaining fertility in a sex-dependent manner.

    (A) Schematic representation of the CRISPR-Cas9 genome editing system to the Zcwpw1-deficient mice. (B) Western blotting showed that ZCWPW1 was deleted in PD16 Zcwpw1−/− testes compared to wild type. β-Actin was used as the loading control. (C and D) Co-immunoprecipitation analysis of ZCWPW1 and H3K4me3 from PD18 testis protein extracts. ZCWPW1 immunoprecipitated with H3K4me3. (E). Testes from PD35 Zcwpw1+/+ and Zcwpw1−/− male mice. Zcwpw1−/− male mice had reduced testis size at PD35 (n = 3, Welch’s t test analysis; P < 0.0001). (F) Hematoxylin and eosin (H&E) staining of adult Zcwpw1+/+ testis. (G) H&E staining of adult Zcwpw1−/− testis sections showed complete arrest of spermatogenesis. Arrows, apoptotic spermatocytes; arrowhead, empty seminiferous tubules; asterisk, seminiferous tubules lack of postmeiotic spermatocytes. (H) Cumulative numbers of pups per female during the defined time period. n = 3 mice for each genotype. (I to N) Histological analysis of ovaries from Zcwpw1+/+ and Zcwpw1−/− females. (I and J). Morphological studies of ovaries showed that at 3-month Zcwpw1−/− ovaries (J) exhibited similar morphologies as Zcwpw1+/+ ovaries (I). (K and L). At 6 months of age, Zcwpw1−/− ovaries (L) contained fewer but healthy follicles and corpora lutea (CL). (M and N). At around 8 months of age, the mutant ovaries (N) had no oocytes or follicles. (Photo credit: Miao Li and Tao Huang, Shandong Provincial Hospital Affiliated with Shandong University).

  • Fig. 3 Disrupted chromosomal synapsis in Zcwpw1−/− spermatocytes.

    (A to F) Chromosome spreads of spermatocytes from the testes of PD35 wild-type (A to C) and Zcwpw1−/− (D to F) males were immunostained for SYCP1 (green) and SYCP3 (red). Arrows indicate synapsed chromosomes, and arrowheads indicate the single chromosome. (G). The numbers of synapsed chromosome pairs in wild-type spermatocytes and Zcwpw1−/− spermatocytes. In Zcwpw1−/− spermatocytes, the average number of synapsed chromosome pairs was 8. (H) Frequencies of meiotic stages in Zcwpw1+/+ and Zcwpw1−/− spermatocytes. The numbers marked in the bars represent the percentage of cells at indicated meiosis stage. For each genotype, three mice were analyzed. P values were calculated by Student’s t test. (I to L) SIM images of spermatocyte chromosome spreads immunostained for SYCP3 (red) and N-SYCP1 (green) from PD25 testes. Arrows indicate the synapsed region, and arrowheads indicate the AEs. (I’ to L’) Magnified views of the synapsed region show that N-SYCP1 was localized in the central region of SCs in a continuous pattern (arrows). (M to R) Chromosome spreads of spermatocytes from Zcwpw1+/+ (M to O) and Zcwpw1−/− (P and Q) males were immunostained for SYCP3 (red) and γH2AX (green). Representative images of spermatocytes at the leptotene, zygotene, and pachytene stages are shown. (S and T) Zcwpw1+/+ and Zcwpw1−/− cells immunostained for SYCP3 (red) and breast cancer 1 (BRCA1; green). Representative images of spermatocytes at pachytene (S, arrow indicating XY body) and pachytene-like (T, arrows indicating BRCA1 signal) stages are shown. (U and V) Zcwpw1+/+ and Zcwpw1−/− spermatocytes immunostained for SYCP3 (red) and MLH1 (green, arrows). Zcwpw1−/− spermatocytes lack MutL homolog 1 (MLH1) signal (V).

  • Fig. 4 Zcwpw1 is essential for male meiotic recombination.

    (A to U) Immunostaining for SYCP3 (red) and recombination protein (green) was performed on Zcwpw1+/+ and Zcwpw1−/− spermatocytes from PD25 mice. Representative images of spermatocytes at the leptotene, zygotene, and pachytene (pachytene-like) stages are shown. Each dot represents the number of DNA repair protein foci per cell, with black dots indicating Zcwpw1+/+ spermatocytes and red dots indicating Zcwpw1−/− spermatocytes. Solid lines show the mean and SD of foci number in each group of spermatocytes. (A to G) RPA2 foci. (H to N) RAD51 foci. (O to U) DMC1 foci. P values were calculated by Student’s t test. (V to X) Immunostaining for SYCP3 (red) and TRF1 (green) was performed on Zcwpw1+/+ and Zcwpw1−/− spermatocytes. Representative images of spermatocytes at the pachytene (pachytene-like) stages are shown. Zcwpw1−/− spermatocytes had comparable numbers of TRF1 foci on equator images as compared to Zcwpw1+/+ spermatocytes. n.s., no statistical significance.

  • Fig. 5 Successful but delayed meiosis prophase I in Zcwpw1−/− oocytes.

    (A) Frequencies of meiotic stages in E17.5 Zcwpw1+/+ and Zcwpw1−/− oocyte chromosome spreads. The numbers marked in the bars represent the percentage of cells at the indicated meiosis stage. For each genotype, three mice were analyzed. P values were calculated by Student’s t test. (B) Frequencies of meiotic stages in PD1 Zcwpw1+/+ and Zcwpw1−/− oocyte chromosome spreads. The numbers marked in the bars represent the percentage of cells at the indicated meiosis stage. For each genotype, three mice were analyzed. P values were calculated by Student’s t test. (C to H) Chromosome spreads of E17.5 ovaries from wild-type (C to E) and Zcwpw1−/− (F to H) females were immunostained for SYCP3 (red) and SYCP1 (green). Representative images of oocytes at the leptotene, zygotene, and pachytene stages are shown. Arrows indicate the synapsed chromosomes. (I to L) Chromosome spreads of E17.5 ovaries immunostained for SYCP3 (red) and N-SYCP1 (green) using SIM at the indicated stages. Arrows indicate the synapsed region. (I’ to L’) Magnified views of the synapsed region show that N-SYCP1 localized in the central regions of SCs in a continuous pattern (arrows). (M to Y) Immunostaining for SYCP3 (red) and RAD51/DMC1/MLH1 (green) was performed on Zcwpw1+/+ and Zcwpw1−/− oocytes from E17.5 females. Representative images of oocytes in zygotene and pachytene stages are shown. Each dot represents the number of RAD51/DMC1/MLH1 foci per cell, with black dots indicating the Zcwpw1+/+ oocytes and red dots indicating Zcwpw1−/− oocytes. Solid lines show the mean and SD of foci number in each group. (M to Q) RAD51 foci. (R to V) DMC1 foci. (W to Y) MLH1 foci.

  • Fig. 6 The knockout of Zcwpw1 leads to POI.

    (A and B) Representative Zcwpw1+/+ (A) and Zcwpw1−/− (B) ovary sections from E13.5 females immunostained for mouse vasa homolog (MVH) with hematoxylin counterstaining. (C) Oocyte counts (relative numbers) showed that there were similar numbers of oocytes in E13.5 Zcwpw1+/+ and Zcwpw1−/− females. MVH-positive cells were counted. (D and E) Representative Zcwpw1+/+ (D) and Zcwpw1−/− (E) ovary sections from PD1 females immunostained for MVH with hematoxylin counterstaining. (F) Relative oocyte counts showed that Zcwpw1−/− ovaries contained significantly fewer oocytes than Zcwpw1+/+ ovaries. (G and H) Representative Zcwpw1+/+ (G) and Zcwpw1−/− (H) ovary sections from PD8 females immunostained for MVH with hematoxylin counterstaining. (I) Relative oocyte counts showed that Zcwpw1−/− ovaries contained significantly fewer follicles than Zcwpw1+/+ ovaries. MVH-positive oocyte nuclei with characteristic surrounding granulosa cell layers were counted. In all cases, counts were made for every section (8 μm per section) and summed to calculate the total number of oocytes per ovary. For each genotype, six ovaries from three mice were analyzed. P values were calculated by Student’s t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax1101/DC1

    Fig. S1. Zcwpw1 is generally expressed in different tissues.

    Fig. S2. The SC structure appeared normal in Zcwpw1−/− spermatocytes.

    Fig. S3. Representative images of leptotene and zygotene stages in Zcwpw1−/− spermatocytes.

    Fig. S4. The SC structure appeared normal in Zcwpw1−/− oocytes.

    Fig. S5. Protein profiling analysis of PD14 wild-type and Zcwpw1−/− testes.

    Table S1. List of 94 differentially expressed proteins in PD14 Zcwpw1+/+ and Zcwpw1−/− testes by HPLC-MS.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Zcwpw1 is generally expressed in different tissues.
    • Fig. S2. The SC structure appeared normal in Zcwpw1−/− spermatocytes.
    • Fig. S3. Representative images of leptotene and zygotene stages in Zcwpw1−/− spermatocytes.
    • Fig. S4. The SC structure appeared normal in Zcwpw1−/− oocytes.
    • Fig. S5. Protein profiling analysis of PD14 wild-type and Zcwpw1−/− testes.
    • Table S1. List of 94 differentially expressed proteins in PD14 Zcwpw1+/+ and Zcwpw1−/− testes by HPLC-MS.

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