Molecular and epigenetic regulation of seasonal reproduction in Terai tree frog (Polypedates teraiensis)

Abstract

Seasonal breeders predominantly use photoperiod as the predictable environmental cue to time their reproduction. Terai tree frogs are long-day seasonal breeders, but the molecular mechanism is unknown. We tested the role of different photoperiodic conditions on expression levels of candidate genes involved in seasonal reproduction and epigenetic regulation. Four experiments were performed. In experiment 1, frogs were exposed to long (LD: 16L:8D) or short photoperiod (SD: 8L:16D). In experiment 2, animals were procured at four different phases of breeding, i.e., during April (emergence just after hibernation), June (breeding phase), August (post-breeding), and October (just before hibernation). In experiments 3 and 4, frogs were exposed to equinox photoperiod but different (10, 100, or 500 lx) light intensities (exp. 3) or wavelength (red: 640 nm, green: 540 nm, blue: 450 nm or white; exp. 4). After 2 weeks, animals were euthanized, and their brain was harvested. mRNA levels of transcripts involved in photoperiodic transduction (Eya3 and Opn5), reproduction (Tshß, GnRH, Dio2, and Dio3), and epigenetics regulation (Dnmt1, Dnmt3a, Hdac1, Hdac3, and Tet2) were measured. Results show that LD promotes the upregulation of Eya3, Opn5, Tshß, GnRH, and Dio2. Differential expression of Opn5 during LD and SD suggests its involvement in light perception. Dio3 levels were upregulated in SD (exp.1) and during the post-breeding phase (exp. 2). These results employ the limited role of light intensity and spectrum in reproduction. This is the first study showing molecular machinery involved in the amphibian system's seasonal reproduction and epigenetic regulation.