(238) Mislocalization of Centrobin Triggers Promiscuous Centriole Amplification During Spermiogenesis

Yaron Dayani ^1;2; Marnie W. Skinner ^1;2;3; Paula Nhan ¹; Philip W. Jordan ^1;3

1. Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD

2. The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD

3. Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD

Abstract Text:

In mitotically dividing cells, Centrobin (CNTROB) has been implicated in centriole duplication and centrosome function. It is enriched at the daughter/immature centriole and is essential for proper centriole duplication, as its depletion leads to centrosomes with incomplete or absent centrioles. Additionally, Centrobin was identified to interact with DNA repair protein BRCA2 and has been linked to processes such as DNA damage response, cytokinesis, and cell cycle progression. To further investigate its role, we generated a Centrobin mutant mouse model lacking exon 10 using CRISPR-Cas9 genome editing. This mutation introduces a frameshift in the translated region, resulting in a truncated protein lacking all amino acids encoded by exon 10 and beyond. Phenotypic analysis revealed that homozygous mutant females were fertile with normal litter sizes, and the mutant allele was segregated according to Mendelian inheritance. In contrast, homozygous mutant males exhibited infertility despite normal gross morphology, health, and growth. Testes from mutant males showed a reduced testes-to-body weight ratio compared to heterozygous controls and defects in post-meiotic stages of spermatogenesis, with significantly diminished spermatozoa in the epididymis. Immunofluorescence microscopy revealed that wild-type Centrobin localizes to the centrosome, proximal to centrioles, whereas the truncated protein was mislocalized. Key meiotic processes, including the first centriole duplication, homologous recombination, synaptonemal complex formation and disassembly, bipolar spindle formation during meiosis I, and the second-round centriole duplication during meiosis II, were unaffected in mutant spermatocytes. However, during spermiogenesis, excess centriole components were observed in haploid spermatids, suggesting aberrant rounds of centriole duplication in post-meiotic stages. These findings implicate Centrobin as a critical regulator of centriole homeostasis during spermatogenesis. Ongoing studies aim to elucidate the functional relationship between Centrobin and polo-like kinase 4 (PLK4), the master regulator of centriole duplication. This work provides new insights into the molecular mechanisms underlying male infertility and centriole regulation during spermatogenesis.