CENP-E haploinsufficiency causes chromosome misalignment and spindle assembly checkpoint activation in the spermatogonia

Jie Chen; Jie-Jie He; Shan Wu; Zhao-Yang Deng; Yu-Peng Liu; Jian-Fan Chen; Yue Xu; Han-Kai Fang; Ya-Lan Wei; Zhen-Yu She

doi:10.1111/andr.13834

CENP-E haploinsufficiency causes chromosome misalignment and spindle assembly checkpoint activation in the spermatogonia

Andrology. 2024 Dec 30. doi: 10.1111/andr.13834. Online ahead of print.

Authors

Jie Chen^{1

2}, Jie-Jie He^{1

2}, Shan Wu^{1

2}, Zhao-Yang Deng^{1

2}, Yu-Peng Liu^{1

2}, Jian-Fan Chen^{1

2}, Yue Xu^{1

2}, Han-Kai Fang^{1

2}, Ya-Lan Wei^{3

4}, Zhen-Yu She^{1

2}

Affiliations

¹ Department of Cell Biology and Genetics, The School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
² Key Laboratory of Stem Cell Engineering and Regenerative Medicine, Fujian Province University, Fuzhou, Fujian, China.
³ Medical Research Center, Fujian Maternity and Child Health Hospital, Fuzhou, Fujian, China.
⁴ College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University, Fuzhou, Fujian, China.

PMID: 39737806
DOI: 10.1111/andr.13834

Abstract

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.

Keywords: CENP‐E; chromosome alignment; kinesin; spermatogonia; spindle assembly checkpoint.

Abstract

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