HIPK4 is essential for murine spermiogenesis
- Department of Chemical and Systems Biology, Stanford University School of Medicine, United States
- Stanford University Mass Spectrometry, Stanford University, United States
- Chan Zuckerberg Biohub, Stanford University, United States
- Cell Science Imaging Facility, Stanford University School of Medicine, United States
- Department of Obstetrics and Gynecology, Reproductive Endocrinology and Infertility, Stanford University School of Medicine, United States
- Transgenic, Knockout, and Tumor Model Center, Stanford University School of Medicine, United States
- Department of Developmental Biology, Stanford University School of Medicine, United States
- Department of Chemistry, Stanford University, United States
Figures
Figure 1
Key steps of spermiogenesis.
(A) Schematic representation of murine male germ cells transitioning from round spermatids to elongated spermatozoa. These steps occur within the testis seminiferous epithelium and are conserved in …
Figure 2 with 2 supplements
HIPK4 is expressed in spermatids and required for male fertility in mice.
(A) Hipk4 expression in various murine tissues as determined by qPCR analysis of the Origene TissueScan Mouse Normal cDNA array. Data are normalized to Gapdh. (B–C) Hipk4 expression in adult mouse (B…
Figure 2—figure supplement 1
Hipk4 mRNA expression during the first wave of murine spermatogenesis.
Hipk4 transcript labeling by in situ hybridization in formalin-fixed, paraffin-embedded testis sections from wild-type C57BL/6NJ mice of the designated ages. Arrowheads indicate the loss of Hipk4 …
Figure 2—figure supplement 2
HIPK4 protein expression in adult murine seminiferous tubules.
(A-B) Immunofluorescence images of cryosectioned testes from adult wild-type (A) and HIPK4 knockout (B) mice. Stages for each testis section are shown. Scale bars: 20 µm.
Figure 3 with 2 supplements
HIPK4 knockout mice exhibit oligoasthenoteratozoospermia.
(A) Concentrations of epididymal sperm ± s.d. P values for the indicated statistical comparison are shown. (B) Percentage of epididymal sperm that were motile ± s.d. P values for the indicated …
Figure 3—figure supplement 1
Morphologies of wild-type and Hipk4 mutant sperm.
Phase contrast images of sperm obtained from mice with the designated genotypes. Sperm were treated with Diff-Quik stain prior to imaging, and multiple examples of homozygous mutant sperm are shown …
Figure 3—figure supplement 2
HIPK4 is not essential for sperm capacitation or acrosomal exocytosis.
(A) Western blot detection of soluble phosphotyrosine-containing proteins in sperm before or after capacitation with TYH medium + BSA (Cap buffer). (B) Percentage of wild-type or Hipk4 knockout …
Figure 4 with 3 supplements
HIPK4 regulates the acrosome–acroplaxome complex.
(A,A’) PAS-stained sections of seminiferous tubules from WT or Hipk4-/- mice at stages IX-XII of spermatogenesis. Green arrowheads point to the heads of representative elongating spermatids (Step …
Figure 4—figure supplement 1
Comparison of the seminiferous epithelium and epididymis of WT and Hipk4 knockout mice.
(A-B) Fixed, paraffin-embedded testis sections from WT (A) and Hipk4–/– (B) mice stained with PAS reagents. Stages for the tubules shown in each micrograph are indicated. (C-D) Fixed, …
Figure 4—figure supplement 2
HIPK4 null spermatids exhibit acrosome–acroplaxome defects.
(A) TEM images of step 4–8 spermatids from adult WT and Hipk4–/– mice. (B) TEM images of step 10 Hipk4–/– spermatids. (C) Higher magnification TEM images of the groove belt region in step 10 WT and H…
Figure 4—figure supplement 3
HIPK4 does not regulate the localization of anterior LINC complexes or manchette dynamics.
(A) Immunofluorescence imaging of the inner nuclear membrane protein DPY19L2 (red) in dissociated spermatids. The acrosome is labeled with FITC-PNA (green), and nuclei are stained with Hoechst 33342 …
Figure 5
Wild-type and HIPK4 knockout testes have similar transcriptomes.
(A) Summary of the types of RNA that were increased (up-regulated) or decreased (down-regulated) in Hipk4 knockout testes compared to wild-type, as measured by quadruplicate microarray analysis of …
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Figure 5—source data 1
Testis microarray data comparing WT and HIPK4 KO transcripts .
- https://cdn.elifesciences.org/articles/50209/elife-50209-fig5-data1-v1.xlsx
Figure 6
HIPK4 overexpression remodels the actin cytoskeleton in cultured cells.
(A–B) Brightfield images of NIH-3T3 cells retrovirally transduced with FLAG-tagged Hipk4 or kinase-dead Hipk4 K40S. (C) HIPK4-expressing NIH-3T3 cell with multiple nuclei. (D–E) Phalloidin and …
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Figure 6—source data 1
Phosphoproteome of HIPK4-expressing NIH-3T3 cells.
- https://cdn.elifesciences.org/articles/50209/elife-50209-fig6-data1-v1.xlsx
Figure 7 with 1 supplement
Loss of HIPK4 function alters actin dynamics in the acroplaxome.
(A–C) Fluorescence imaging of enzymatically dissociated spermatids co-labeled with FITC-PNA and (A) anti-bβ actin, (B) anti-CAPZA3, or (C) anti-CAPZB3. Nuclei were stained with Hoeschst 33342. …
Figure 7—figure supplement 1
Phalloidin staining of F-actin dynamics in the acroplaxome.
(A) Cryosectioned seminiferous tubules (stage X/XI) stained with Alexa Fluor-647-phalloidin to stain F-actin and an Alexa Fluor-488-anti-EB3 antibody to stain the manchette. Basal and apical …
Author response image 1
Two-dimensional structured illumination microscopy (SIM) of wild-type and Hipk4-/- spermatids.
Spermatids were isolated from the testes of wild-type and Hipk4-/- mice, fixed, and then stained with a rabbit polyclonal anti-β-actin antibody and Alexa Fluor 488-conjugated anti-rabbit IgG …
Additional files
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Supplementary file 1
Key Resources Table.
- https://cdn.elifesciences.org/articles/50209/elife-50209-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50209/elife-50209-transrepform-v1.docx
