The role of sperm associated antigen 6 gene in morphological changes of inner ear development and signal regulation of auditory organs in mice

Abstract

Aim: In order to analyze the effects of the deletion of the Sperm Associated Antigen 6 (Spag6) gene on the inner ear and the auditory system of mice. Method: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology was utilized, and the Spag6 gene knockout mouse model was constructed. Self-breeding was carried out to obtain the F1 generation Spag6 homozygous mouse (Spag−/−), the Spag6 heterozygous mouse (Spag+/−) and the Spag6 wild mouse (Spag+/+) were obtained. Polymerase Chain Reaction (PCR) technology was used to verify the three genotypes of mice. The mean hearing threshold of Spag−/− mice, Spag+/− mice and Spag+/+ mice after different intensity of sound stimulation was detected. The inner ear cochlea tissues of Spag−/− mice and Spag+/+ mice were collected and paraffin sections were made. Morphological differences of inner ear tissues in mice were analyzed by hematoxylin – eosin (HE) staining. Immunofluorescence staining was used to analyze the number of hair cells in the inner ear of mice. Apoptosis of mouse inner ear hair cells was analyzed by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining Result: It was found that the hearing of Spag−/− mice decreased significantly compared with that of Spag+/+ mice (P < 0.01), and the hearing of Spag+/− mice decreased significantly compared with that of Spag+/− mice (P < 0.05). After HE staining and immunofluorescence staining, hair cells in the inner ear cochlea of Spag−/− mice were found to be defective. The apoptosis detection results of TUNEL staining indicated that the number of apoptosis of hair cells in inner ear cochlea of Spag−/− mice was significantly higher than that of Spag+/+ mice, and the average optical density of Corti apparatus of Spag−/− mice was significantly higher than that of Spag+/+ mice (P < 0.01). Conclusion: The results showed that the loss of the Spag6 gene accelerated the apoptosis of hair cells in the cochlear tissue of the inner ear of mice, thereby affecting the auditory system of mice. The significance of this study is to lay the foundation for future studies on the effects of Spag6 on deafness. Keywords: Spag6 genes knock-out, Auditory brainstem response, Hair cells, Cell apoptosis, Corti instrument