Estrogen-related receptor gamma and hearing function: evidence of a role in humans and mice

Abstract

Since estrogen is thought to protect pre-menopausal women from age-related hearing loss, we investigated whether variation in estrogen-signalling genes is linked to hearing status in the 1958 British Birth Cohort. This analysis implicated the estrogen-related receptor gamma (ESRRG) gene in determining adult hearing function and was investigated further in a total of 6134 individuals in 3 independent cohorts: (i) the 1958 British Birth Cohort; (ii) a London ARHL case-control cohort; and (iii) a cohort from isolated populations of Italy and Silk Road countries. Evidence of an association between the minor allele of single nucleotide polymorphism (SNP) rs2818964 and hearing status was found in females, but not in males in 2 of these cohorts: p = 0.0058 (London ARHL) and p = 0.0065 (Carlantino, Italy). Furthermore, assessment of hearing in Esrrg knock-out mice revealed a mild 25-dB hearing loss at 5 weeks of age. At 12 weeks, average hearing thresholds in female mice((-/-)) were 15 dB worse than in males((-/-)). Together these data indicate ESRRG plays a role in maintenance of hearing in both humans and mice.

Fig. 1

ABR hearing thresholds in Esrrg KO mice. (A) Genotyping by Southern blotting of offspring from Esrrg heterozygote hybrid crosses produces predicted fragments of 20 kb for the WT allele and 11 kb for the null allele. (B) ABR thresholds on click stimuli were recorded at 5 (n = 6 Esrrg KO vs. 11 controls), 12 (n = 9 Esrrg KO vs. 8 controls), and 18 (n = 3 Esrrg KO vs. 8 controls) weeks of age. Esrrg KO mice exhibit significant hearing loss compared to controls (MV ± SE, **p = 0.001; *p < 0.05, Mann–Whitney rank sum test). (C) Sex-specific difference in hearing thresholds for 5 (n = 2 female vs. 4 male) and 12 (n = 3 female vs. 6 male) weeks of age. Esrrg KO female mice exhibit greater hearing loss compared to males at 12 weeks (MV ± SE, *p < 0.05, Mann–Whitney rank sum test). (D) Sample recordings for Esrrg WT, Esrrg Het and Esrrg KO mice; hearing thresholds in the Esrrg KO (voltage scale doubled) are approximately 40 dB worse compared to the WT and Het controls.

Fig. 2

Expression of ESRRG in mouse inner ear. (A–D) Results of immunofluoresence with anti-ESRRG in mouse inner ear at P36: anti-ESRRG (green), DAPI (blue), and Phalloidin staining to f-actin (red). (A) Low magnification of mid-apical coil. Strong ESRRG immunoreactivity is localized to Reissner's membrane (rm), Hensen's cells (hc) can be seen in the inner (ihc) and outer (ohc) hair cell region (solid arrows). Weaker immunoreactivity is shown in the spiral ganglion neurons (sgn). (B) Mid magnification of the opposite mid-cochlear coil showing ESRRG immunoreactivity in the organ of Corti localizes to the inner (ipc) and outer (opc) pillar cells and supporting cell bodies and their processes intervening the outer hair cells. (C) High magnification of the organ of Corti. (D) Mid magnification of the opposite apical coil showing strong immunoreactivity in Hensen's cells. Scale bar is 20 μm. (E) Detection of Esrrg mRNAs in P2-P30 rat kidney, spleen, and cochlea tissue using reverse transcription–polymerase chain reaction. (RT-PCR). Samples were tested with (+) or without (−) reverse transcriptase (RT) enzyme. No bands were detected in RT (−), in the absence of RNA template or negative control for PCR (water only). Gapdh served as a positive control for mRNA quality.