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Updated: May 7, 2025
Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry
Published on: December 28, 2021
CENP-E haploinsufficiency causes chromosome misalignment and spindle assembly checkpoint activation in the
Jie Chen1,2, Jie-Jie He1,2, Shan Wu1,2
1Department of Cell Biology and Genetics, The School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
Background:
The establishment of kinetochore-microtubule attachment is essential for error-free chromosome alignment and segregation during cell division. Defects in chromosome alignment result in chromosome instability, birth defects, and infertility. Kinesin-7 CENP-E mediates kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint in somatic cells, however, mechanisms of CENP-E in germ cells remain poorly understood.
Objectives:
This study aimed to explore the functions of CENP-E in the proliferation and self-renewal of spermatogonia.
Materials And Methods:
A CENP-E heterozygous knockout strain was established in C57BL/6J mice using the CRISPR/Cas9 and Cre/LoxP system. Hematoxylin-eosin staining was performed to study the histology. The inhibition of CENP-E in the GC-1 spg cells was performed using the specific inhibitor GSK923295. The expression and localization of spermatogonial marker proteins were determined by immunofluorescence using confocal microscopy in the control and CENP-E+/- heterozygous mouse testes. The protein expression level was analyzed using Western blot. The cell-cycle and apoptosis assay were measured using flow cytometry. In addition, karyotype analysis was performed using hypotonic preparation and chromosome spreading.
Results:
Here, we reveal that CENP-E haploinsufficiency results in chromosome misalignment, spindle disorganization, and metaphase arrest in spermatogonia, which leads to the loss of spermatogonia, chromosomal instability, and spermatogenic disorders. Notably, CENP-E ablation leads to the activation of spindle assembly checkpoint and aneuploidy, which impairs the proliferation and self-renewal of spermatogonia.
Discussion And Conclusion:
CENP-E depletion disrupts the recruitment of key checkpoint proteins, including BubR1, Bub1, KIF2C, and Aurora B, indicating a causal relationship between chromosome misalignment and spindle assembly checkpoint activation in spermatogonia. Our findings demonstrate that CENP-E regulates kinetochore-microtubule attachment, chromosome alignment, and spindle assembly checkpoint in spermatogonia.
Insights
CENP-E is crucial for spermatogonia proliferation and self-renewal. Its depletion causes chromosome misalignment and spindle assembly checkpoint activation, leading to spermatogenic disorders.
Area of Science:
- Cell Biology
- Genetics
- Reproductive Biology
Background:
- Accurate kinetochore-microtubule attachment is vital for chromosome segregation during cell division.
- Defects in chromosome alignment can cause infertility and birth defects.
- The role of Kinesin-7 CENP-E in germ cell division is not well understood.
Purpose of the Study:
- To investigate the function of CENP-E in spermatogonial proliferation and self-renewal.
- To elucidate the mechanisms by which CENP-E influences germ cell division.
Main Methods:
- CRISPR/Cas9 and Cre/LoxP systems were used to create CENP-E heterozygous knockout mice.
- Immunofluorescence, Western blot, and flow cytometry were employed to analyze spermatogonia.
- Histological and karyotype analyses were performed on mouse testes.
Main Results:
- CENP-E haploinsufficiency led to chromosome misalignment, spindle disorganization, and metaphase arrest in spermatogonia.
- CENP-E depletion activated the spindle assembly checkpoint and caused aneuploidy.
- These defects resulted in spermatogonial loss, chromosomal instability, and spermatogenic disorders.
Conclusions:
- CENP-E is essential for proper kinetochore-microtubule attachment and chromosome alignment in spermatogonia.
- CENP-E regulates the spindle assembly checkpoint, preventing aneuploidy.
- Disruption of CENP-E function impairs spermatogonial proliferation and self-renewal, leading to reproductive issues.
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