Melatonin Regulates Sheep Granulosa Cell Proliferation and Apoptosis Through a Novel Micropeptide, IGF1Raa

Melatonin is a hormone primarily secreted by the pineal gland, best known for regulating circadian rhythms. It also plays a critical role in ovarian physiology by modulating granulosa cell (GC) function during follicular development, although the underlying molecular mechanisms remain incompletely understood. Recent advances in ribosome sequencing (Ribo-seq) have revealed that many small open reading frames (sORFs), previously overlooked in conventional annotations, actually encode functional micropeptides. However, whether melatonin exerts its ovarian effects through such micropeptides has remained unexplored. In the present study, we identified a previously unannotated sORF within the 5'untranslated region (UTR) of sheep IGF1R mRNA and demonstrated that this sORF encodes a 32-amino-acid micropeptide, which we named IGF1Raa. Knockdown and overexpression of IGF1Raa in sheep GCs, assessed by CCK-8, Edu, flow cytometry, and ELISA, revealed that IGF1Raa negatively regulates GC proliferation and E2 secretion and promotes apoptosis. Co-ip assays and RNA-seq show that IGF1Raa physically associates with PPP2R2B, a regulatory subunit of protein phosphatase 2A (PP2A), and modulates PP2A protein expression. Consequently, IGF1Raa regulates the phosphorylation of AMPK (Thr172) and its downstream effector Raptor (Ser792), thereby controlling the AMPK/mTOR signaling cascade. Importantly, melatonin suppresses IGF1Raa expression, and overexpression of IGF1Raa partially abrogates melatonin's stimulatory effects on GC proliferation and steroidogenesis. Collectively, our findings identify IGF1Raa as a melatonin-responsive micropeptide that governs GC function through the PP2A/AMPK/mTOR axis, providing new insights into the regulatory network of ovarian physiology and offering potential therapeutic targets for reproductive disorders.