In addition to the proband, two other male individuals in this family were affected, his brother (IV-8) and great uncle (II-5), who had never conceived during their marriages or while engaging in unprotected sexual intercourse. A semen analysis of this patient revealed an extremely reduced sperm count and motility as well as an abnormal sperm morphology (table S1). The somatic cell karyotype (46, XY), bilateral testicular size, secondary sex characteristics, and hormone levels were normal.

RESULTS Identification of a CEP78 splicing mutation in a large family with male infertilityĪ 28-year-old man from a consanguineous family who was diagnosed with 7 years of primary infertility was recruited for our study. Together, we elucidated CEP78 as a causative gene of male infertility in both humans and mice, providing unexplored insights into the roles of centrosomal proteins in reproductive biology.

In addition to the infertile phenotype, we observed deficient function of the retina and outer hair cells of the cochlea (OHCs) in Cep78 KO mice, as CEP78 mutations have been suggested to cause CRDHL syndrome in humans. Functionally, we showed that CEP78 promotes the expression of the deubiquitinating enzyme ubiquitin specific peptidase 16 (USP16), which stabilizes Tektin expression via the ubiquitination pathway. We further generated Cep78 knockout (KO) mice, and the phenotype of Cep78 KO male mice resembled the infertile phenotype of the patients, showing marked defects in sperm count, morphology, and motility. The patients presented a characteristic MMAF phenotype, including coiled, bent, irregular, short, or/and absent flagella and defects in sperm flagellar ultrastructure. In the present study, we identified the previously unreported homozygous splicing mutation c.1069+1G>A in CEP78 in an infertile family through whole-exome sequencing (WES). To date, only the centrosomal proteins of testis specific 10 (TSGA10) and CEP128 have been shown to be associated with male sterility in both humans and mice ( 14– 16). Loss-of-function variants in centrosomal protein of 135 kDa ( CEP135) and CEP112 account for multiple morphological abnormalities of the sperm flagella (MMAF) and acephalic spermatozoa phenotypes, respectively, in humans ( 6, 7), and Cep55, spermatogenesis associated 6 ( Spata6), Cep131, Cep164, centrin 1 ( Cetn1), and spermatogenesis and centriole associated 1 like ( Spatc1l) are suggested to be involved in mouse infertility ( 8– 13). Therefore, defects in the functions of centrosomal proteins are presumed to be linked to male infertility, but the relevant findings are limited. The molecular composition of the centrosome is complex, consisting of hundreds of proteins known as centrosomal proteins. Second, in fertilization, the centrosome forms the major microtubule-organizing center that contributes to the movement and fusion of the male and female pronuclei after completing replication and splitting, the sperm centrosome organizes the bipolar array that forms the mitotic spindle required for cleavage ( 5). First, during spermatogenesis, the centrosome is required to form the integral spermatozoon structure, including the sperm flagellum and head-tail coupling apparatus. The centrosome is essential for reproductive biology. The centrosome is a subcellular organelle composed of two centrioles embedded in pericentriolar material that is present in most eukaryotic cells and plays a critical role in various cellular processes, including cell division, motility, and cilia formation ( 4). Collectively, our findings identified nonfunctional CEP78 as an indispensable factor contributing to male infertility and revealed a role for this gene in regulating retinal and outer hair cell function in mice. Cep78 knockout mice also exhibited impairments in retina and outer hair cells of the cochlea. Mechanistically, CEP78 might regulate USP16 expression, which further stabilizes Tektin levels via the ubiquitination pathway. The infertility of the patients and knockout mice could not be rescued by an intracytoplasmic sperm injection treatment. We further created Cep78 knockout mice, which showed an extremely low sperm count, completely aberrant sperm morphology, and approximately null sperm motility.

Here, we identified a pathogenic splicing mutation in CEP78 in male infertile patients with severely reduced sperm number and motility, and the typical multiple morphological abnormalities of the sperm flagella phenotype. However, the role of centrosomal proteins in male infertility remains poorly defined.

Centrosomal protein dysfunction might cause ciliopathies.

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