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

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. [ABSTRACT FROM AUTHOR]