Abstract
Purpose :
A wide array of biological and technical variables can affect electroretinogram (ERG) outcomes, including gender. In turn, the prevalence of many retinal diseases exhibit strong gender dependencies. When we examined mouse scotopic ERG data by sex, trends were obvious under resting conditions. Collectively, these findings led us to determine if these functional differences could be observed at a molecular level. Hence, we used a mass spectrometry (MS) based approach to characterize potential sex-based differences in the steady state retina of the normal adult mouse.
Methods :
30-week-old outbred Swiss-Webster ND4 mice were used. Scotopic ERGs were obtained in 8 male and 8 female ketamine-xylazine anesthetized mice. Retinae from 5 male and 5 female mice of the same strain and age were processed for MS3 analysis on a Fusion Orbitrap Mass Spectrometer (Thermo Fisher) using Tandem-Mass-Tags for quantification. Three technical repeats were performed for each biological sample. Proteins of interest were validated using western blot analysis. We used Proteome Discoverer 2.2 SEQUEST-HT scoring for initial analysis. For characterization, only high scoring peptides were considered with a FDR<1%. Statistical analysis was performed using StataSE15, and bioinformatics by Ingenuity Pathway Analysis (Qiagen).
Results :
ERG results indicate that male mice have significantly higher b-wave amplitudes for 3/7 of the highest light intensities (figure), and higher a-wave amplitudes for the highest two intensities (not shown). MS identified 4,264 proteins, 4,093 of which were quantified. 68 proteins were differentially expressed, between genders, by at least 1.5-fold and 32 of those at least 2-fold. Two pheromone proteins, previously unreported in the retina, were identified and differed more than 4-fold by gender. Additionally, there were 17 crystallin proteins quantified, of which 12 were differentially expressed; 10 of the 12 were more abundant in male retinal proteomes.
Conclusions :
Distinct molecular phenotypes likely contribute to the significantly greater resting scotopic ERG amplitudes observed in male relative to female mice, given that the expression levels of a large number of proteins involved in structure, development, signaling, and metabolism exhibited sex-dependent differences. Our findings may provide phenotypic insights into retinal function, as well as why many retinal diseases exhibit unique gender disparities.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.