The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice
- Center for Reproductive Medicine, Shandong University, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, China
- Shandong Technology Innovation Center for Reproductive Health, China
- CUHK-SDU Joint Laboratory on Reproductive Genetics, School of Biomedical Sciences, Chinese University of Hong Kong, China
- Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital, Affiliated to Shandong First Medical University, China
- School of Basic Medical Sciences, Shandong University, China
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, China
- Genome Tagging Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, China
- Research Unit of Gametogenesis and Health of ART-Offspring, Chinese Academy of Medical Sciences, China
Figures
Figure 1 with 2 supplements
Ssh2 knockout (KO) causes severe reproductive defects and male infertility in mice.
(A) Schematic representation of the generation of Ssh2 KO mice using CRISPR/Cas9. (B) Validation of Ssh2 KO by western blotting in testicular lysates from wild-type (WT) and Ssh2 KO 8-week-old mice …
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Figure 1—source data 1
Observational datasets and original blots.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig1-data1-v2.zip
Figure 1—figure supplement 1
PCR genotyping for wild-type (WT) and Ssh2 knockout (KO) mice.
PCR genotyping demonstrating the absence of the WT band in Ssh2 KO mice.
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Figure 1—figure supplement 1—source data 1
Original gels.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Fluorescence-activated cell sorting (FACS) assessment in spermatogenic cells from wild-type (WT) and Ssh2 knockout (KO) murine testes.
Distribution of spermatogenic cells by FACS. The number of spermatids (round/elongated spermatids in WT testes and round spermatids in Ssh2 KO testes) showed no significant variation in Ssh2 KO …
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Figure 1—figure supplement 2—source data 1
Original images.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig1-figsupp2-data1-v2.zip
Figure 2 with 2 supplements
Spermatogenic arrest and enhanced germ cell apoptosis result from Ssh2 knockout (KO).
(A) Periodic acid Schiff (PAS)-hematoxylin-stained sections of seminiferous epithelia from wild-type (WT) mice and Ssh2 KO mice (8 weeks of age, n=3), indicating spermatogenic arrest at steps 2–3 of …
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Figure 2—source data 1
Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL)-related observational datasets and original images/blots.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig2-data1-v2.zip
Figure 2—figure supplement 1
SSH2 is not required for meiotic progression in mouse spermatogenesis.
Testes from nearly 3-week-old wild-type (WT) mice (left panels) and Ssh2 knockout (KO) mice (right panels) were sampled for preparing chromosome spreads immunostained for SYCP1 and SYCP3. …
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Figure 2—figure supplement 1—source data 1
Original images.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
Comparison of spermatogenesis in wild-type (WT) and Ssh2 knockout (KO) mice.
Comparison of spermatogenesis in WT and Ssh2 KO mice assessed in testicular sections stained for hematoxylin at postnatal day (PD)7, PD14, PD21, PD28, PD35, and PD60 accompanied by epididymal …
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Figure 2—figure supplement 2—source data 1
Original images.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig2-figsupp2-data1-v2.zip
Figure 3
Acrosome biogenesis is disrupted during spermiogenesis in Ssh2 knockout (KO) mice.
(A) Analysis of spermiogenesis and acrosome biogenesis in wild-type (WT) and Ssh2 KO mice (8 weeks of age, n=3) by fluorescence imaging of spermatids labeled with peanut agglutinin (PNA) lectin …
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Figure 3—source data 1
Original images.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig3-data1-v2.zip
Figure 4
SSH2 accumulates at the acrosomal region of spermatids.
(A) Immunoblotting against SSH2 in wild-type murine testes sampled at the indicated postnatal days (PD). The expression of SSH2 at PD14, PD21, PD28, PD35, and PD56 was measured. GAPDH was used as …
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Figure 4—source data 1
Original images/blots.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig4-data1-v2.zip
Figure 5 with 1 supplement
Ssh2 knockout (KO) results in disturbed filamentous actin (F-actin) remodeling in spermatids.
(A) Immunofluorescence detection of F-actin (red) and peanut agglutinin (PNA) lectin (green) during spermatogenesis in testicular sections from wild-type (WT) (top lane) and Ssh2 KO (bottom lane) …
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Figure 5—source data 1
Original images.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig5-data1-v2.zip
Figure 5—figure supplement 1
Disordered filamentous actin (F-actin) in Ssh2 knockout (KO) testes.
Fluorescence imaging of phalloidin-labeled F-actin (red)-stained seminiferous tubules from wild-type (WT) (left panels) and Ssh2 KO (right panels) murine testes at postnatal day (PD)21, PD35, and …
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Figure 5—figure supplement 1—source data 1
Original images.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig5-figsupp1-data1-v2.zip
Figure 6
Ssh2 knockout (KO) spermatids exhibit defects in proacrosomal vesicle transport.
(A) Immunofluorescence staining of the vesicular trafficking-related Golgi-specific protein GOPC (red) in wild-type (WT) and Ssh2 KO round spermatids in testicular sections co-stained with peanut …
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Figure 6—source data 1
Observational datasets and original images.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig6-data1-v2.zip
Figure 7 with 1 supplement
Ssh2 knockout (KO) spermatids display impaired COFILIN phospho-regulation that disrupts filamentous actin (F-actin) remodeling.
(A) Immunoblotting against SSH2, p-COFILIN, COFILIN, LIMK1, and LIMK2 in wild-type (WT) and Ssh2 KO testes sampled on postnatal day (PD)82. GAPDH was used as the loading control. (B) Testicular …
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Figure 7—source data 1
Original images/blots.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig7-data1-v2.zip
Figure 7—figure supplement 1
COFILIN-associated protein expression level in wild-type (WT) and Ssh2 knockout (KO) testes.
Protein extracts from testes isolated from WT and Ssh2 KO mice at postnatal day (PD)15, PD30, and PD82 were used for western blot analysis for LIMK1, LIMK2, p-COFILIN, and COFILIN. GAPDH was used as …
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Figure 7—figure supplement 1—source data 1
Original blots.
- https://cdn.elifesciences.org/articles/83129/elife-83129-fig7-figsupp1-data1-v2.zip
Author response image 1
Representative images of ultrastructural analysis of actin organization in WT and Ssh2 KO spermatids.
No intact F-actin structure was observed in WT round spermatids with few aggregated actin bundles (as red arrowheads indicated) detected in Ssh2 KO spermatids under TEM. The regions outlined by …
