Fig 2: Multiple defects are observed in spermatozoa from male homozygous Cep128 KI mice.a Male homozygous KI mice showed hyperplasia of sperm flagella (n = 3 biologically independent WT mice or KI mice; scale bars, 20 µm). b Dislocated arrangements of MTDs 1?3 and ODFs 1?3 were exhibited in the majority of the midpieces. ODF outer dense fibers, MTD peripheral microtubule doublets. (n = 3 biologically independent WT mice or KI mice; scale bars, 150 nm). c Impaired development of the connecting piece during spermiogenesis in male homozygous Cep128 KO and KI mice. In spermatid steps 9–14, PCs with incomplete structure were present in both male homozygous KO and KI mice. Signs of defective Sc formation were occasionally visible in spermatids from male homozygous KI mice, but not in KO mice. In contrast, the Bp was almost normal in both male homozygous KO and KI mice. PC proximal centriole, Sc segmented column, Bp basal plate. (n = 3 biologically independent WT mice, KO mice or KI mice; scale bars, 500 nm).

Fig 3: Anomalies in the morphology and ultrastructure of spermatozoa from male Cep128 KO mice.a Spermatozoa from male KO mice showed prominent deformities in the head, neck, and tail regions under light microscopy (n = 3 biologically independent WT mice or KO mice; scale bars, 20 µm). b The primary defect in the midpiece and principal pieces was the absence of the CP, while missing and misplaced MTDs were observed in the end piece. CP central-pair microtubules, MTD peripheral microtubule doublets. (n = 3 biologically independent WT mice or KO mice; scale bars, 150 nm).

Fig 4: Functional analysis of the CEP128R222Q variant in hRPE1 cells.a Western blot analysis showed an increased abundance of CEP128 protein with the CEP128R222Q variant. NC (negative control), cells transfected with negative control pENTER-Flag plasmid. Three independent experiments were performed. (Two-sided Student’s t test; error bars, mean ± SEM). The p values are indicated in the graphs. b Overexpression of CEP128R222Q compromised ciliary growth and disrupted cellular nuclear morphology. Three independent experiments were performed. Dotted box denoted the basal body and cilia. n the number of cells analyzed. NC (negative control), cells transfected with negative control pENTER-Flag plasmid. (Two-sided Student’s t test; error bars, mean ± SEM; green, CEP128; red, Ac-Tubulin; blue, DAPI; scale bars, 10 µm). The p values are indicated in the graphs. c Decreased levels of NIN, centriolin, and CEP170 were detected in the cells transfected with WT-CEP128 or CEP128R222Q when compared to the NC by western blot analysis. NC (negative control), cells transfected with negative control pENTER-Flag plasmid. Three independent experiments were performed. (Two-sided Student’s t test; error bars, mean ± SEM). The p values are indicated in the graphs. WT-CEP128 and CEP128R222Q downregulated the mRNA levels for NIN, centriolin, and CEP170 (d) by suppressing the expression of their transcription factors (e). NC (negative control), cells transfected with negative control pENTER-Flag plasmid. Three independent experiments were performed. (Two-sided Student’s t test; error bars, mean ± SEM). The p values are indicated in the graphs. Source data are provided as a Source Data file.

Fig 5: Proposed model of CEP128 modulation of male reproduction.The imbalance observed in CEP128 levels included increased CEP128 abundance or loss of CEP128 protein, which suppressed the normal functioning of genes/proteins essential for sperm production and morphology as well as fertilizing capability. Normal sperm eventually failed to form, impairing typical reproductive function.