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Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry
Published on: December 28, 2021
EP300-interacting inhibitor of differentiation 3 is required for spermatogenesis in mice
Ping Zhang1,2, Longsheng Zhang3, Li Yu3
1International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Background:
Mammalian spermatogenesis is a highly complex process of cell proliferation, meiosis, and differentiation. A series of genes are expressed in an orderly and precise manner to ensure spermatogenesis, with chromatin undergoing intricate changes throughout. EP300-interacting inhibitor of differentiation 3 (Eid3) is a testis-enriched gene, but its role in male reproduction remains unclear.
Objective:
To investigate the role of EID3 in male spermatogenesis and explore the potential underlying mechanism.
Materials And Methods:
We generated Eid3 knockout mouse model using the CRISPR-Cas9 system. We measured the expression of EID3 in mouse tissues and testicular cell populations by qRT-PCR and western blot. Histological analysis, including hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining, together with computer-assisted sperm analysis (CASA), were performed to evaluate the effect of EID3 on spermatogenesis in mice. Light and ultrastructural microscopy were used to evaluate the morphology and structure of the Eid3-/- spermatozoa. We used western blot and immunofluorescence to further analyze the function of EID3 in spermiogenesis.
Results:
Eid3-/- mouse showed a significant decrease in sperm count, motility, and morphology. Loss of EID3 impaired the normal meiotic process and induced apoptosis of abnormally developing spermatocytes, ultimately resulting in the decrease of sperm cell number. Additionally, EID3 deficiency led to a decrease in histone acetylation levels in spermatids, impaired histone-to-protamine transition and chromatin condensation process, and ultimately resulted in abnormal sperm morphology.
Discussion And Conclusions:
This study confirms for the first time that EID3 is crucial for meiosis and chromatin condensation during spermatogenesis, and EID3 deficiency leads to a significant decrease in sperm parameters. Given the high expression paradigm of Eid3 in human testis, EID3 likely plays a role in human reproduction. Future research could provide a new target for the clinical diagnosis and treatment of male infertility.
