Cellular mechanism of gain-of-function mutation I173M in sheep MC4R gene identified in year-round and seasonal estrus breeds through whole-genome resequencing

Abstract

Abstract Background Investigating key genes regulating year-round estrus can improve sheep reproductive performance and industry efficiency. Although several candidate genes have been proposed, the key regulators underlying estrus seasonality remain unclear. Therefore, this study aimed to identify critical genetic variants associated with year-round estrus using whole-genome resequencing data from multiple sheep breeds. Results In this study, we analyzed whole-genome resequencing data from 392 sheep across six year-round and ten seasonal estrus breeds, and identified the Melanocortin 4 receptor ( MC4R ) gene as a key regulator of estrus seasonality. A missense mutation in the MC4R gene, I173M (g.59480440G > C; p.Ile173Met), was significantly enriched in year-round estrus breeds. Transcriptome analysis following MC4R I173M transfection revealed differential expression of genes enriched in reproductive pathways. Subsequent functional experiments demonstrated that the I173M mutation increased MC4R constitutive activity, activated cAMP and MAPK/ERK signaling, and promoted both granulosa cell proliferation and the expression of steroidogenic regulatory genes. Cells harboring the I173M mutation exhibited substantially amplified transcription levels of gonadotropin signaling components. Specifically, follicle-stimulating hormone receptor ( FSHR ) and luteinizing hormone receptor ( LHR ) were expressed at much higher levels relative to wild-type (WT) controls. Conclusions Our study provides evidence that MC4R functions as a molecular modulator of estrous cyclicity, with the I173M missense mutation identified as a critical functional variant driving constitutive estrous activity in ovine species.