| Part I Hair Cell Polarity Defects and Hearing Loss in Sperm-Associated Antigen 6 (Spag6)-Deficient MiceObjectiveSpag6 encodes an axoneme central apparatus protein that is required for normal flagellar and cilia motility. Recent findings suggest that Spag6 also plays a role in ciliogenesis, orientation of cilia basal feet, and planar polarity. Sensory cells of the inner ear display unique structural features that underlie their mechanosensitivity and resolution, and represent perhaps the most distinctive form of a type of cellular polarity, known as planar cell polarity (PCP). However, the role for Spag6 in the inner ear has not yet been explored. Given recent information suggesting roles for Spag6 in PCP, it is conceivable that SPAG6 affects the structure or function of the hair cells in the inner ear. To explore this possibility, we characterized hair cell structure and function in Spag6 knock-out mice.Methods1. Auditory brainstem response (ABR) and cochlear microphonics (CM) were performed respectively. For ABR, mice were stimulated with clicks and 4kHz, 8kHz,16kHz,32kHz pure tones. For CM, mice were evoked by clicks in the closed field.2. Inner ear whole mounts were immunostained to examine the microtubule system beneath the cuticular plates of the hair cells.3. Inner ear wholemounts were stained to observe FZD6 protein distribution.4. Inner ear whole mounts were immunostained to observe the morphology of the hair bundles and polarity. The deviation degree was analyzed.5. Inner ear whole mounts immunostaining and scanning electron microscope(SEM) were used to observe the pair of centrioles and the relationship between the kinocilium and hair bundle. The kinocilia deviation degrees were analyzed.Results1. ABR results showed that Spag6-/- mice (n=7) had severe hearing loss, the average ABR threshold (click) of the Spag6-/- mice was 70.71±4.14 dB, while Spag6+/+ littermates (n=9) was 30.00±2.21 dB. Analysis of hearing function in response to pure tones (4, 8, 16, 32 kHz) confirmed that hearing in Spag6-/- mice was diminished across the entire analyzed frequency spectrum. The SPLs required to elicit a response in the Spag6-deficient mice were significantly higher than that in the wild-type controls (*P<0.05). Consistently, the CM response to 80dB could not be elicited in all the Spag6-/- mice (n=3), while the average CM threshold of Spag6+/+ littermates (n=4) was 40.00±4.08dB. The difference between the wild-type and mutant mice was significant (*P<0.05).2. In the wild-type mice, the apical microtubules radiated along the periphery of the cell cortex from the base of kinocilium where basal bodied were located. In the Spag6-/- mice, the apical microtubules were disturbed and distributed randomly in the cuticular plate.3. The mutants showed an abnormal distribution of FZD6 protein compared to the wild-type mice. FZD6 protein lost its asymmetric and polarized distribution, and was no longer concentrated in the apical medial region of the hair cells and pillar cells, but seemed to be distributed in the entire membrane in Spag6-deficient mice4. An accumulation of hair cells were observed in the apical region of the cochlea and a widened sensory epithelium near the apex in the Spag6-/- mice, while abnormal hair cell accumulation was not observed in wild-type mice. In wild-type mice, three rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs) were regularly aligned. The stereocilia form bilaterally symmetrical "Λ""-shaped hair bundles. In Spage mice, alignment of hair cells was abnormal and the orientations of the hair cell bundles were no longer consistent with the mediolateral axis, but orientated randomly, and hair cells were disordered. Compared to the wild-type mice, the hair bundle deviation degrees were abnormally larger in Spag6-/- mice.5. In wild-type mice, the position of the kinocilium predicts the orientation of the bundle. However, the kinocilium in mutants was not properly positioned and frequently deviated from the normal position. In wild-type hair cells, pairs of centrioles were aligned along the mediolateral axis, and the maternal centriole was associated with the base of the kinocilium. However, in Spag6-/- mice, the two centrioles of the basal body and kinocilium were dislocated; the centrioles were no longer aligned mediolaterally along the axis of bundle polarity. Compared to the wild-type mice, the kinocilia deviation degrees were abnormally larger in Spag6-/-mice.Conclusions Our findings demonstrate a role for Spag6 in auditory function in mice, througha role in the regulation of PCP in the auditory epithelium.Part II Otitis Media in Sperm-Associated Antigen 6 (Spag6-Deficient MiceObjectiveSpag6 encodes an axoneme central apparatus protein, which is required not only for normal flagellar and cilia motility, but plays a role in ciliogenesis, orientation of cilia basal feet, and planar polarity. In the middle ear and Eustachian tube, polarized ciliary system play an important role in providing protection against infection of the middle ear, which can transport and clean serous fluid and mucus of the middle ear. Cilia defects can cause otitis media (OM). However, the role for Spag6 in the middle ear and Eustachian tube has not yet been explored. In the present study, the function of Spag6 in the middle ear and Eustachian tube was examined using Spag6-deficient mice.Methods1. RT-PCR and immunofluorescence were used to detect the expression of Spag6 gene in the wild-type and mutant mice.2. Otoscope and HE section were used to obtain a clear idea of the middle ear.3. Scanning electron microscope was used to observe the ciliary epithelium of the middle ear.4. Transmission electron microscope was used to observe the orientation of the basal body feet and the two central microtubules of all the cilia.5. Immunofluorescence was used to observe the FZD6 protein distribution in the middle ear ciliary epithelium.6. PCR, bacterial culture and colony counting were used to identify and quantify pathogens in the middle ear. Mucicarmine staining and realtime-PCR were used to observe the goblet cells and mucus factor Mucin5ac and Mucin5b. The angle between the midline of the skull base and the bony part of the Eustachian tube was measured.Results1. In the wild-type mice, Spag6 mRNA was detected in the middle ear and SPAG6 protein was located in the cilia of the ciliated columnar epithelium; No Spag6 mRN A or protein was detected in the mutant mice.2. Otoscope, HE section results showed that obvious otitis media manifestations were observed in the middle ear of the mutant mice.3. SEM result showed that cilia of the wild-type mice (at P25) were well-organized in the middle ear epithelial cells. However, there was significant reduction in cilia density of the Spag6-deficient mice (at P25), and the cilia were largely arrayed in a disordered fashion. Cilia were present in the promontorium tympani of the wild-type mice, but, they prematurely disappeared in the Spag6-deficient mice.4. In the 25 day old wild-type mice, orientation of basal feet of middle ear epithelial cells was similar, with the basal feet all pointing in the same orientation. However, in the mutant mice (at P25), the orientation of basal feet appeared to be random. Consistent with the basal feet results, in wild-type mice, the orientation of the two central microtubules of all the cilia was uniform, as shown by the same orientation of lines connecting the two microtubules in all the axonemes. However, in Spag6-deficient mice, the orientation of the two central microtubules was evidently random, with the lines connecting central microtubules pointing in different directions in some axonemes.5. Immunofluorescence showed that the FZD6 protein distributed asymmetrically in the membrane of the control mice; while in the mutants, FZD6 protein lost its polarized distribution.6. PCR and bacterial culture techniques showed that Moraxella catarrhalis were detected in both of the mutant mice and control mice, and there was no significant difference in bacterial colony number between the mutant mice and control mice. There was no obvious difference between the mutant and control mice in the goblet cell density, mucus factor expression levels and Eustachian tube angles.ConclusionsIn summary, our findings demonstrate an association between Spag6 mutation and the pathogenesis of OM in mice. OM appears to develop as a result of ciliary dysfunction leading to effusions and progressive middle ear inflammation. The structural abnormalities in cilia orientation may be a consequence of Spag6 contribution to the regulation of PCP in the middle ear and Eustachian tubes. |
