Research ArticleDISEASES AND DISORDERS

Disruption in ACTL7A causes acrosomal ultrastructural defects in human and mouse sperm as a novel male factor inducing early embryonic arrest

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Science Advances  28 Aug 2020:
Vol. 6, no. 35, eaaz4796
DOI: 10.1126/sciadv.aaz4796
  • Fig. 1 Identification of a homozygous ACTL7A mutation in two brothers with infertility.

    (A) Pedigree of a family with an inherited ACTL7A mutation. The open square and circles denoted the unaffected male and female family members, respectively. The filled squares denote the affected male members, and the double line indicates a consanguineous marriage. Individuals marked with an asterisk were investigated by WES. M, mutation; W, wild type. (B) The ACTL7A mutation was verified by Sanger sequencing. The homozygous ACTL7A mutation of both brothers (subjects II:1 and II:2) was inherited from heterozygous parental carriers (subjects I:1 and I:2). The dotted rectangle indicates the position of the mutation. (C) Location and conservation of the ACTL7A mutation. The conservation of the mutated amino acid is indicated by the alignment of this sequence with that of six mammalian species.

  • Fig. 2 Homozygous mutation in ACTL7A caused the ultrastructural impairment of sperm acrosomes in the two infertile brothers.

    (A) The signal of ACTL7A disappeared in the case of the spermatozoa from the infertile brothers carrying the homozygous ACTL7A mutation. The localization of ACTL7A was identified using polyclonal antibodies against ACTL7A, followed by the secondary antibodies (donkey anti-rabbit–Cy3; red). The acrosome and sperm nuclei were stained with PNA (green) and DAPI (blue), respectively. Scale bars, 2 μm. (B) The acrosome signals were distributed nonuniformly in sperm of brother 1. Acrosin (red) was a marker of acrosomal matrix. PNA (green) labels the outer acrosomal membrane. (C) The ultrastructure of sperms from the brothers with the ACTL7A mutation revealed acrosome detachment. The red arrowhead indicates the acrosome. The arrow indicates the plasma membrane. ac, acrosome; n, nucleus; pm, plasma membrane. Scale bars, 200 nm. Photo credit: Aijie Xin, Fudan University.

  • Fig. 3 Homozygous knock-in mutation in Actl7a leads to male infertility in the mouse model.

    (A) Genotyping of the constructed Actl7a knock-in (KI) mutation in mice. WT, wild type; +/KI, heterozygous mutation; KI/KI, homozygous mutation. (B) The ACTL7A protein was completely absent in the testes and sperms of Actl7aKI/KI mice. α-Tubulin was used as a loading control. (C) The Actl7aKI/KI male mice were completely infertile. The fertility assessment experiments were performed for the male WT (WT; n = 5) mice and the male mice with heterozygous (+/KI; n = 5) and homozygous (KI/KI; n = 5) mutations. One-way ANOVA, **P < 0.01; NS, not significant. Data are means ± SEM.

  • Fig. 4 Homozygous knock-in mutation in mouse Actl7a causes acrosomal defects due to fusion failure of proacrosomal vesicle.

    (A) The location of acrosome changed in sperm from the Actl7aKI/KI mice. Acrosin (red) and PNA represented acrosomal matrix and the outer acrosomal membrane, respectively. Sperm nuclei were stained with DAPI (blue). Scale bars, 2 μm. (B) TEM analysis revealed the detachment of the acrosome (ac) from the sperm nuclei (n) in the sperms from the Actl7aKI/KI mice. The red arrowhead indicates the acrosome. The arrow indicates the plasma membrane (pm). (C) Acrosome biogenesis of WT and Actl7aKI/KI mouse spermatid. (a to d) Four phases of acrosome biogenesis in WT. Golgi phase (a), cap phase (b), acrosomal phase (c), and maturation phase (d). (a′ to d′) Higher magnification of the boxed region from (a) to (b). Numerous proacrosomal vesicles derived from the trans-face of the Golgi (a and a′) attached to the acroplaxome forming a cap over the nucleus (b and b′). Acrosome further developed and flattened over half of the nucleus in acrosomal phase (c and c′) and continued to maturation (d and d′). (e to e′) Four corresponding phases in Actl7aKI/KI mouse. (e′ to h′) Higher magnification of the boxed region from (e) to (h). Big and atypical proacrosomic vesicles generated during Golgi phase (e and e′). The proacrosomal vesicle fusion failed (f and f′) and accumulated gradually (g and g′), eventually causing the abnormal acrosome detached from the nuclear envelope (h and h′). pv, proacrosomal vesicles; g, Golgi; n, nucleus; ag, acrosomal granule; apx, acroplaxome. Photo credit: Aijie Xin, Fudan University.

  • Fig. 5 Altered expression and distribution of PLCζ in the sperm from ACTL7A/Actl7a-mutated males and overcoming male infertility by AOA.

    (A) The PLCζ protein was significantly reduced in both human and mouse sperm affected by ACTL7A/Actl7a mutations. α-Tubulin was used as a loading control. (B) Confocal immunofluorescence images revealed the mislocalization of PLCζ on ACTL7A/Actl7a-mutated sperm. Single-sperm immunofluorescence analysis for PLCζ (red) and PNA (green) was performed in both human and mouse spermatozoa with ACTL7A/Actl7a mutations. Nuclei were stained with DAPI (blue). (C) The oocytes were fertilized by the sperms from the Actl7a-mutated male mice after AOA. ICSI failed to enable the sperms from the Actl7aKI/KI mice to fertilize the normal oocytes (lane 2). However, AOA using SrCl2 rendered the sperms from Actl7aKI/KI mice capable of successfully fertilizing the oocytes, leading to the development of the resulting embryos into blastulas (lane 3). (D) Representative images of the offspring (dotted circle) of the Actl7aKI/KI male mice born after the combination of ICSI and AOA. (E) Representative images of the embryonic development from days 0 to 3 following ICSI with AOA in case of brother 2 and his wife. Numbers 1 to 4 represent the four matured MII oocytes acquired after subjecting the oocytes from brother 2’s wife to COH. The development of the four embryos from days 0 to 3 is shown horizontally. Embryos 1 and 2 developed normally and were subsequently transplanted. Photo credit: Ronggui Qu, Fudan University.

  • Table 1 Homozygous ACTL7A mutation identified in two brothers affected by male infertility.

    Brothers 1 and 2
    GeneACTL7A
    Variant coordinates*Chr9:111625335, G>A
    TranscriptNM_006687
    cDNA mutationc.733G>A
    Protein alterationp.Ala245Thr
    Mutation typeMissense, homozygous
    Allele frequencies in human populations
      Allele frequency in gnomAD4.068 × 10−6
      Number of homozygotes0
    Conservation†
      Phastcons1
      Phylop4.084
    Functional prediction
      PROVEANDeleterious
      PolyPhen-2Probably damaging
      MutationTasterDisease causing

    *Variant coordinate is based on the human genome assembly GRCh37/h19.

    †The Phastcons value is close to 1 when a nucleotide is conserved, and the predicted conserved sites are assigned positive scores by Phylop.

    Supplementary Materials

    • Supplementary Materials

      Disruption in ACTL7A causes acrosomal ultrastructural defects in human and mouse sperm as a novel male factor inducing early embryonic arrest

      Aijie Xin, Ronggui Qu, Guowu Chen, Ling Zhang, Junling Chen, Chengqiu Tao, Jing Fu, Jianan Tang, Yanfei Ru, Ying Chen, Xiandong Peng, Huijuan Shi, Feng Zhang, Xiaoxi Sun

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