Extra-retinal regulation of avian reproduction: A role for VA-opsin and neuropsin

1. Introduction:
Photoperiod is well established as a major cue regulating the timing of seasonal reproduction across vertebrate taxa. While detection of photic cues in mammalian species has been tied to melanopsin in retinal ganglion cells, detection of photic cues in avian species has been localized to the hypothalamus. Multiple candidate photoreceptive opsins have been localized to the deep brain (VA-opsin, neuropsin and melanopsin), however, despite decades of research the identity of the photoreceptors necessary for the expression of seasonal breeding in birds remains unresolved. Using advancements in viral vector and RNA silencing techniques this study sought to establish causal involvement of the two leading photoreceptor candidates: VA-opsin and Neuropsin
2. Methods:
A novel AAV2-viral construct expressing GFP and silencing RNA constructs targeting VA-opsin and neuropsin was injected into the third ventricle of photosensitive adult male Japanese quail (n=28) via stereotaxic surgery. Following surgery birds were held on short days (7L:17D) for three weeks for recovery and to allow expression of viral constructs. Birds were then transferred to long day (18L:6D) for 4 weeks to allow for full development of reproductive competence in control animals. Animals were then euthanized and tissues collected for subsequent analysis. Morphometric measurements were collected prior to photostimulation, immediately after, and weekly thereafter. All work was conducted in compliance with local animal care and use guidelines (Roslin Institute Study P140, PPL 7007909).
3. Approach for statistical analysis:
Morphometric data were analysed by linear mixed effects modelling of output by the main effects of time, treatment and their interaction, with repeated measures for individual animals to control for the effects of repeated sampling. Tissue and molecular analyses were statistically analysed by ANOVA.
4. Results and conclusions:
RNA silencing of opsin expression resulted in increased rates of cloacal gland growth compared to sham controls. Treatment with RNAi targeting VA-opsin resulted in a decreased rate of mass gain compared to all other groups. However, there was no difference in final testes mass between treatments, with all animals showing enlarged testes characteristic of breeding preparation. Taken together these finds suggest that VA and neuropsin may provide tonic inhibition of some but not all aspects of seasonal breeding in birds.