Research ArticleFertility

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo

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Science Signaling  26 Mar 2019:
Vol. 12, Issue 574, eaao7232
DOI: 10.1126/scisignal.aao7232
  • Fig. 1 Male mice deficient in Hspa4l are infertile, and their sperm heads show a rounded morphology.

    (A) Male mice of the indicated genotype (Hspa4l+/+ or Hspa4l−/−) were mated with the indicated female mice, and the resulting litter sizes was determined. Data are means ± SD. ***P < 0.001 by two-tailed Student’s t test. (B) Representative images of the heads of sperm in the cauda epididymis of wild-type and Hspa4l-deficient mice. Scale bar, 10 μm. (C and D) Fertilization ratios with Hspa4l−/− sperm in vitro. We analyzed in vitro fertility using two male mice of each genotype with ZP-intact oocytes (C) or ZP-free oocytes (D) in five or three independent experiments, respectively. Data are means ± SD. *P < 0.05, ***P < 0.001 by two-tailed Student’s t test.

  • Fig. 2 Ppp1cc2 forms a chaperone-substrate complex with Hsc70, which includes Hspa4l, and Ppp1cc2 dephosphorylates the pSer56of Prm2.

    (A) Chromatin-binding fractions from sperm of mice of the indicated genotypes were analyzed by Western blotting (IB) with antibodies (Abs) against the indicated proteins. Blots are representative of three experiments. (B) Ppp1cc2 to Prm2 ratios in the chromatin-binding fractions. Results indicate the relative Ppp1cc2:Prm2 ratio in Hspa4l-deficient sperm compared to that in wild-type sperm. Data are means ± SD of three independent experiments. ***P = 0.0003 by Welch’s t test. (C) Interaction between Ppp1cc2 and Hsc70. Sperm cell lysates from the indicated mice were subjected to immunoprecipitation (IP) with control or anti-Ppp1cc antibodies and then analyzed by Western blotting with antibodies against the indicated proteins. Blots are representative of three experiments. IgG, immunoglobulin G. (D) Interaction between Hspa4l and Hsc70. Sperm cell lysates from the wild-type mice were subjected to immunoprecipitation with control or anti-Hspa4l antibodies and then analyzed by Western blotting with antibodies against the indicated proteins. Blots are representative of three experiments. (E) Interaction between Ppp1cc2 and Prm2. Sperm cell lysates from wild-type mice were subjected to GST pull-down experiments with the indicated fusion proteins. The samples were then analyzed by Western blotting with anti-GST antibody. Blots are representative of three experiments. (F) Ppp1cc2 dephosphorylates Ser56 of Prm2. Phosphatase assays were performed using the indicated fusion proteins as substrates. After the reactions were complete, phosphorylated or total substrates were detected by biotinylated Phos-tag or Coomassie blue staining (CBB), respectively. Data are representative of three experiments. (G and H) Results of three independent phosphatase assays using pGST-Prm2 (G) or pGST-Prm2 S56A (H). These substrates were dephosphorylated by samples immunoprecipitated by an anti-Ppp1cc antibody from the chromatin-binding fraction of epididymal sperm as described in Materials and Methods. Results indicate the relative ratios of phosphorylated proteins after the reaction using an anti-Ppp1cc antibody to those using a control antibody. Data are means ± SD. For (G), P = 0.0323; for (H), P = 0.2785 by Welch’s t test.

  • Fig. 3 Conversion of the codon encoding Ser56 to a codon encoding alanine in the Prm2 locus rescues the infertile phenotype of Hspa4l−/−mice.

    (A) Hspa4l−/−; Prm2S56A/S56A male mice are fertile. Male mice of the indicated genotypes were mated with wild-type (wt) female mice, and the resulting litter sizes were determined. Data are means ± SD. ***P < 0.001 by two-tailed Student’s t test. “#” indicates Welch’s t test. (B) Analysis of in vitro fertility. Two male mice of each the indicated genotypes were used. Data are means ± SD of three independent experiments. **P < 0.01, ***P < 0.001 by two-tailed Student’s t test. (C and D) Substitution of Ser56 with alanine in Prm2 rescues the abnormal morphology of Hspa4l−/− sperm. (C) Results are from two male mice of each of the indicated genotypes. Data are means ± SD of three independent experiments. *P < 0.05, ***P < 0.001 by two-tailed Student’s t test. (D) Representative images showing the morphology of the sperm from mice of the indicated genotypes. Scale bar, 10 μm.

  • Table 1 Development of oocytes after intracytoplasmic injection of sperm.

    Results are from three independent experiments using two male mice of each genotype.

    GenotypeNo. of
    injected
    oocytes
    No. of
    two-cell
    No. of
    embryo
    transfer
    No. of
    progeny
    Hspa4l +/+4940 (91.6%)*4012 (30.0%)**
    Hspa4l −/−20024 (12.0%)*240 (0.0%)**

    *P = 1.10420691732578 × 10−10.

    **P = 0.0140905514994987 by Fisher’s exact probability test.

    • Table 2 Development of oocytes injected with round spermatids.

      Results are from three independent experiments using two male mice of each genotype.

      GenotypeNo. of
      injected
      oocytes
      No. of
      two-cell
      No. of
      embryo
      transfer
      No. of
      progeny
      Hspa4l +/+16671 (42.8%)*6316 (25.4%)**
      Hspa4l −/−21076 (36.2%)*6020 (33.3%)**

      *P = 0.434569594957852.

      **P = 0.570464814844937 by Fisher’s exact probability test.

      Supplementary Materials

      • www.sciencesignaling.org/cgi/content/full/12/574/eaao7232/DC1

        Fig. S1. Generation of Hspa4l−/− mice.

        Fig. S2. Hspa4l−/− male mice of a mixed strain are not sterile.

        Fig. S3. Histological analysis of testes.

        Fig. S4. The numbers of spermatogonia and sperm are normal in Hspa4l−/− mice.

        Fig. S5. Subcellular localization of Ppp1cc2.

        Fig. S6. Ppp1cc2 forms a chaperone-substrate complex with Hsp70.

        Fig. S7. Subcellular analysis of the interaction of Ppp1cc2 with Hsc70.

        Fig. S8. Generation of Prm2S56A/wt and Prm2S56D/wt mice and demonstration that Prm2S56A/S56A and Prm2S56D/S56D male mice are fertile.

        Fig. S9. No mutations in potential off-target sites.

        Fig. S10. No mutations in neighboring sites of mutated regions.

        Fig. S11. Morphological analysis of sperm.

        Table S1. Sperm binding assays.

        Table S2. Development of oocytes in culture after fertilization with sperm from Hspa4l−/− mice.

        Table S3. Development of oocytes in culture after intracytoplasmic injection of sperm.

        Table S4. Development of oocytes in culture after injection with round spermatids.

        Table S5. Development of oocytes in culture after fertilization with sperm from Prm2S56A/S56A mice.

        Table S6. Development of oocytes in culture after fertilization with sperm from Prm2S56D/S56D mice.

        Table S7. Development of oocytes in culture after fertilization with sperm from Hspa4l−/−; Prm2S56A/S56A mice.

        Table S8. Development of oocytes in culture after fertilization with sperm from Hspa4l−/−; Prm2S56D/S56D mice.

        Table S9. Primers used for sequencing potential off-target sites.

      • This PDF file includes:

        • Fig. S1. Generation of Hspa4l−/− mice.
        • Fig. S2. Hspa4l−/− male mice of a mixed strain are not sterile.
        • Fig. S3. Histological analysis of testes.
        • Fig. S4. The numbers of spermatogonia and sperm are normal in Hspa4l−/− mice.
        • Fig. S5. Subcellular localization of Ppp1cc2.
        • Fig. S6. Ppp1cc2 forms a chaperone-substrate complex with Hsp70.
        • Fig. S7. Subcellular analysis of the interaction of Ppp1cc2 with Hsc70.
        • Fig. S8. Generation of Prm2S56A/wt and Prm2S56D/wt mice and demonstration that Prm2S56A/S56A and Prm2S56D/S56D male mice are fertile.
        • Fig. S9. No mutations in potential off-target sites.
        • Fig. S10. No mutations in neighboring sites of mutated regions.
        • Fig. S11. Morphological analysis of sperm.
        • Table S1. Sperm binding assays.
        • Table S2. Development of oocytes in culture after fertilization with sperm from Hspa4l−/− mice.
        • Table S3. Development of oocytes in culture after intracytoplasmic injection of sperm.
        • Table S4. Development of oocytes in culture after injection with round spermatids.
        • Table S5. Development of oocytes in culture after fertilization with sperm from Prm2S56A/S56A mice.
        • Table S6. Development of oocytes in culture after fertilization with sperm from Prm2S56D/S56D mice.
        • Table S7. Development of oocytes in culture after fertilization with sperm from Hspa4l−/−; Prm2S56A/S56A mice.
        • Table S8. Development of oocytes in culture after fertilization with sperm from Hspa4l−/−; Prm2S56D/S56D mice.
        • Table S9. Primers used for sequencing potential off-target sites.

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