Sheep Gene Mutation Linked to Year-Round Breeding Shows Gain-of-Function in Cells

A single-letter change in the sheep melanocortin 4 receptor gene, MC4R, is strongly associated with year-round breeding in domestic sheep, and new laboratory experiments suggest the mutation makes the receptor more active at the cellular level. The finding, published in Genetics Selection Evolution, combines whole-genome resequencing of hundreds of sheep with functional tests in cultured cells, offering the first functional characterization of this variant in the species.

Breed classifications were drawn primarily from the reference work Animal Genetic Resources in China: Sheep and Goats, with support from published studies of reproductive seasonality.

Using fixation index (FST) scans and a genome-wide association study that treated estrus pattern as a binary trait, the team pinpointed a region on sheep chromosome 23 containing the MC4R gene. Within that region, a missense mutation designated I173M (g.59480440G > C, p.Ile173Met) emerged as the strongest selected site. The mutant allele was significantly more frequent in year-round estrus breeds than in seasonal breeds, with a difference reported at P < 0.0001. The signal was corroborated by nucleotide diversity and Tajima’s D statistics, and the gene was also flagged in the same screen alongside BMPR1B, a gene associated with lambing.

The I173M substitution replaces isoleucine with methionine at position 173 of the receptor protein, a site the authors found to be highly conserved across 13 vertebrate species, with sheep MC4R sharing 92.77 percent similarity with the human protein. Structural modeling predicted that the change alters the receptor’s three-dimensional shape, and the mutation was mapped to the fourth transmembrane domain, a region previously shown to be important for ligand binding in other studies. Notably, the variant was absent from all 33 Mouflon samples examined, suggesting it likely arose after sheep domestication, although the authors caution that the available evidence cannot definitively distinguish a single ancestral origin from multiple independent mutations.

In laboratory tests, the mutation did not change how much receptor protein was produced or where it ended up. When HEK293T cells were transfected with either wild-type or mutant MC4R constructs, western blotting and flow cytometry showed no significant difference in protein expression or cell-surface localization, and immunostaining confirmed that both receptor versions trafficked normally to the cell membrane. The functional difference lay elsewhere: in how strongly the receptor signaled.

That difference showed up in receptor activity assays. In the absence of any agonist, the mutant receptor showed significantly higher basal, or constitutive, cAMP activity than the wild-type receptor, which itself was more active than an empty vector control. When cells were stimulated with the natural ligands alpha-MSH and beta-MSH across six concentrations, the mutant receptor responded more potently. With alpha-MSH, cAMP activity of the mutant was fourfold higher than wild type, and with beta-MSH the EC50 dropped from 0.46 ± 0.07 micromolar for the wild-type receptor to 0.05 ± 0.02 micromolar for the mutant, indicating substantially greater sensitivity. The MAPK/ERK pathway showed a parallel pattern: alpha-MSH activated the mutant receptor with an EC50 of 0.26 ± 0.03 micromolar versus 0.98 ± 0.03 micromolar for wild type, a fourfold improvement in activation efficiency, while beta-MSH produced a significant increase in maximal response. The authors characterize I173M as a gain-of-function mutation.

Transcriptome sequencing of cells expressing the mutant receptor identified 53 differentially expressed genes, 26 upregulated and 27 downregulated, with enrichment of pathways including GnRH signaling, ovarian steroidogenesis, the cAMP signaling pathway and the MAPK signaling pathway. Genes such as NR4A2, IL5RA and CAMK2A were upregulated in mutant-transfected cells. The authors interpret this as evidence that the mutation amplifies signaling through pathways known to participate in reproductive regulation.

To probe effects on ovarian function, the team transfected sheep primary ovarian granulosa cells, derived from 5-month-old Hu sheep, with wild-type or mutant MC4R plasmids. Both constructs enhanced cell proliferation and viability relative to empty-vector controls, as measured by EdU incorporation and CCK-8 assays, although the difference between the two genotypes was not statistically significant. Apoptotic regulators Caspase-3 and P53 were downregulated in transfected cells relative to controls, again without a genotype-dependent difference, while flow cytometry with Annexin V/PI staining showed significantly reduced apoptosis in mutant-transfected cells compared with controls. Steroidogenic pathway genes, including 3βHSD, CYP19A1, StAR, FSHR and LHR, were upregulated in both transfected groups, but FSHR, LHR and StAR mRNA levels were significantly higher in mutant-transfected cells than in wild-type-transfected cells, suggesting the variant potentiates gonadotropin-responsive steroidogenic networks.

The authors place these results in a broader context of MC4R’s known roles in mammalian reproduction. MC4R is widely expressed in the brain, where it regulates food intake and energy expenditure, and previous work in rodents has linked it to LH release, ovulation and estrous cycle regularity. In mice, MC4R knockout disrupts follicular dynamics and reduces corpus luteum counts, and reinsertion of the gene in Kiss1 neurons restores estrous cyclicity. In sheep, prior studies had connected MC4R mainly to growth traits, meat quality and feed intake, making the present association with estrus seasonality a new direction for the gene in this species.

The researchers are careful to note the limits of what the study demonstrates. Functional validation was conducted entirely in vitro, in HEK293T cells and cultured granulosa cells, and the study lacks direct in vivo evidence that MC4R regulates sheep estrus. The breeds analyzed may not fully represent population diversity, and unbalanced sample sizes across groups may introduce population structure effects on the GWAS and FST results. Estrus regulation also involves multiple genes, hormone levels and environmental factors beyond MC4R. The authors suggest that future work measuring hormone levels after receptor activation in vivo, overexpressing MC4R in ovarian granulosa cell lines, and conducting transcriptomic and metabolomic analyses of sheep with different I173M genotypes would firm up the variant’s role. Even so, they argue that the strong population association, the gain-of-function signaling profile and the enhanced steroidogenic gene expression in granulosa cells together position MC4R I173M as a promising candidate marker for marker-assisted breeding aimed at improving reproductive efficiency in seasonally estrous sheep breeds.

Subject of Research: A genetic variant in the sheep MC4R gene associated with year-round estrus and its cellular mechanisms

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

Article References: Yang, Y., Kang, Y., Liu, P., Cao, C., Li, Y., Li, R., Lan, X., & Pan, C. (2026). Cellular mechanism of gain-of-function mutation I173M in sheep MC4R gene identified in year-round and seasonal estrus breeds through whole-genome resequencing. Genetics Selection Evolution, 58(1), Article 72. https://doi.org/10.1186/s12711-026-01091-4

Image Credits: AI Generated

DOI: 10.1186/s12711-026-01091-4

Keywords: sheep, MC4R, I173M mutation, year-round estrus, whole-genome resequencing, gain-of-function, granulosa cells, cAMP signaling, MAPK/ERK, reproductive genetics, GWAS, steroidogenesis