Study On Prestin Expression In Outer Hair Cells Of Spag6 Gene Knockout Mice

BackgroundDeafness, the most common sensory or functional defective disease, is tremendously attributed to genetic maladies. Nevertheless, multitudinous genes relating to hearing disorder still haven’t been identified hitherto. Therefore, it is the hot spot of researches in hearing dysfunction that to discover new pathogenic gene and to elucidate its mechanism responsible for deafness. Sperm-associated antigen 6 (Spag6) gene encodes SPAG6, a microtubule-related protein, which is firstly found in infertile males. Previous study found that Spag6 gene knockout mice were terribly infertile, whose sperm had marked motility defects with frequent loss of the sperm central pair of microtubules within flagellar structures. And SPAG6 ubiquitously expresses in tissue and organs containing ciliated cells, such as central nervous system, respiratory system, hemopoietic system and even in tumors. Any deficiencies of Spag6 gene could render severe diseases, such as hydrocephalus, pneumonia and development retardation. These aforementioned findings highlight that SPAG6 is significant for the maintaining of multiple physiological functions in vivo.As is well-known, hair cells in organ of Corti are fundamental for hearing generation. The cylindrical outer hair cells (OHCs) are able to rapidly alter their length and stiffness in response to acoustic mechanical stimulation, which derives from a putative molecular motor designated prestin. Prestin is one highly specialized anion transporting carrier protein, which is belonged to a solute carrier proteint family (SLC26), exclusively expressing in the lateral wall of outer hair cells (OHCs). As the molecular basis for electromotility of the OHCs and for the cochlear amplifier, this protein is also designated the motor protein. The expression of prestin is impressionable to different conditions, like, the application of ototoxic drugs, noise exposure and direct interactions with several specific proteins. Thus the normal hearing status depending on prestin is quite sensitive to variety of interferences.Noticeably, patients afflicted with primary ciliary dyskinesia often have hearing impairment simultaneously, which hints that some genes encoding microtubule-related protein play significant roles for the normal auditory function. On the basis that SPAG6 widely expresses in ciliated cells and potentially involves in inner ear development, it is reasonable to hypothesize that this microtubule-related protein exists in cochlear hair cells and may be crucial for the normal function of hair cells. In our preliminary researches, we found that Spag6 gene knockout mice (Spag6-/-) had severe hearing loss in comparison to the wild type (Spag6+/+) and heterozygous (Spag6+/-) mice. Therefore, if SPAG6 expresses in OHCs, it may be associated with the process of electromotility and correlated to prestin. Additionally, so far as we know, there are sparing findings about microtubule-related proteins interacting with prestin. In this regard, the present works were designed to determine whether SPAG6 existed in cochlear hair cells and, if so, to study the presumable correlations between prestin and SPAG6.ObjectiveTo investigate whether SPAG6 exists in cochlear hair cells, and, if so, to study the presumable correlations between prestin and SPAG6.MethodsNeonatal wild type mice (Spag6+/+), heterozygous mice (Spag6+/-) as well as gene knockout mice (Spag6-/-) were born by the intercross of the parental heterozygous mice (Spag6+/-) male and female mice. Researches were conducted by different mice groups according to their genotypes. The morphological features of hair cells in basilar membrane and the distribution of SPAG6 in organ of Corti were investigated by immunofluorescent staining through a confocal microscope. The amount of prestin in different Spag6 mutant mice (Spag6+/+, Spag6+/-, Spag6-/-) were measured by Western blotting and Real-time Q-PCR. Co-immunoprecipitation tests were performed to confirm the presumed interaction between prestin and SPAG6.Results1. SPAG6 expressed in the cuticular plate in outer hair cells (OHCs) and co-localized with prestin in the lateral wall of OHCs.2. In comparison to Spag6+/+and Spag6+/-mice, Spag6-/- mice showed apparent morphological abnormity of OHCs and lower intensity of prestin fluorescence.3. The expression of prestin in Spag6 gene knockout mice (Spag6-/-) reduced significantly at both protein and mRNA levels.4. Co-immunoprecipitation tests demonstrated the interaction between prestin and SPAG6.Conclusions:The genetic traits and gross appearances of these Spag6 mutant mice were consistent with the previous findings, which laid a feasible foundation for the following experiments. SPAG6 is found to be indispensible for the stability of OHCs by maintaining the normal expression of prestin. Spag6 gene knockout mice afflicted severe hearing loss, which implied that Spag6 gene associated with deafness.