Chemical Tags on an Estrogen Receptor Gene May Help Time Puberty in Sheep

Puberty is one of the most consequential transitions in the life of a farm animal. The moment a ewe lamb reaches reproductive competence shapes her age at first mating, the length of her productive life, and ultimately the economics of an entire flock. For decades, researchers have focused on the hormonal cascade that drives this transition, tracing the maturation of the hypothalamic-pituitary-gonadal axis and the rising pulses of gonadotropin-releasing hormone that awaken the ovary. Now, a team at Tarim University in China has turned attention to a quieter layer of control: chemical modifications to DNA itself that may help decide when the reproductive machinery switches on.

The new study, published in Frontiers in Animal Science, examined Duolang sheep, an indigenous breed from the Xinjiang region prized for its early sexual maturity, year-round estrous cycles, and relatively high reproductive output. First estrus in this breed typically arrives at around five months of age, making the animals an attractive model for dissecting the molecular events that accompany the onset of fertility. The researchers focused on ESR2, the gene encoding estrogen receptor beta, a protein abundant in ovarian granulosa cells that mediates estrogen’s local effects on follicular development, granulosa cell differentiation, and ovulation. When ESR2 function is lost in experimental animals, granulosa cells respond poorly to gonadotropins, follicular maturation falters, and ovulation is impaired.

The logic behind the study rests on a well-established principle of epigenetics. DNA methylation, the addition of methyl groups to cytosine bases within CpG dinucleotides, tends to cluster in promoter regions called CpG islands. When these islands are heavily methylated, transcription factors may struggle to bind and chromatin may become less accessible, generally suppressing gene transcription. Because epigenetic regulation has already been implicated in the developmental activation of the female reproductive axis, the team asked a straightforward question: does methylation of the ESR2 promoter change as Duolang ewes pass through puberty, and do those changes track the gene’s activity?

To find out, the researchers collected ovarian tissue from fifteen healthy ewes divided into three groups of five: prepubertal animals at ninety days of age, pubertal animals sampled within twenty-four hours of their first observed estrus, and early postpubertal animals sampled within seven days after estrous behavior subsided. Puberty was defined behaviorally, by voluntary acceptance of mounting, restlessness, increased vocalization, vulvar swelling, and mucous discharge during daily ram-exposure checks. The team first cloned and sequenced a 2,871-base-pair stretch of the ESR2 promoter, identifying four candidate transcription start sites, a single TATA box, sixty-seven predicted transcription factor binding sites, and two CpG islands. They then used bisulfite sequencing, a technique that converts unmethylated cytosines to uracil while leaving methylated cytosines intact, to measure methylation at nine sites in the first CpG island and twenty-two sites in the second.

The results revealed a striking asymmetry between the two regulatory regions. Methylation of CpG island 1 declined steadily across the three stages, falling from 82.22 percent in prepubertal ovaries to 78.71 percent at puberty and 71.11 percent in the early postpubertal period, an absolute drop of more than eleven percentage points. CpG island 2 behaved differently, dipping from 79.09 percent to 74.45 percent at puberty before rebounding to 79.82 percent afterward, a level significantly higher than the pubertal value. Meanwhile, ESR2 messenger RNA expression climbed progressively: significantly higher at puberty than before it, and significantly higher still after puberty than at either earlier stage.

The correlation analysis sharpened the picture. In the prepubertal group, methylation of CpG island 1 showed only a weak, statistically insignificant relationship with ESR2 expression. But in the pubertal and postpubertal groups, the correlation turned strongly negative, with coefficients of -0.956 and -0.969 respectively, meaning that lower methylation was consistently paired with higher gene expression. At the level of individual CpG sites, the story grew more nuanced: some positions within each island showed nominal associations with expression in one direction, others in the opposite direction, and the average methylation of an island did not always capture what was happening at specific sites. The authors are careful to note that these site-level findings rest on unadjusted P values and should be treated as candidate associations rather than confirmed regulatory elements.

To probe whether demethylation could actually drive ESR2 expression, the team turned to cultured ovarian granulosa cells, verified as more than 95 percent pure by immunofluorescent staining for the follicle-stimulating hormone receptor. Treating the cells with 0.5 micromolar 5-aza-2′-deoxycytidine, a drug that inhibits DNA methyltransferases and strips methyl groups genome-wide, significantly increased ESR2 mRNA expression after thirty-six hours and boosted cell viability at later time points. On its face, the experiment seems to close the loop: remove methylation, and the gene wakes up.

But the researchers are refreshingly candid about the limits of that inference. Because 5-Aza-dC acts globally rather than on the ESR2 promoter specifically, and because promoter methylation was not remeasured after treatment, the rise in expression cannot be attributed directly to demethylation of either CpG island. Nor was the ERβ protein itself quantified, leaving open whether the transcriptional change translated into more receptor. The authors also acknowledge a series of constraints on the tissue study: only five animals per stage, behavioral rather than endocrine confirmation of pubertal status, just five bisulfite-sequenced clones per animal per island, and whole-ovary samples in which shifts in the proportions of granulosa, theca, stromal, and luteal cells could masquerade as regulatory changes. The expression stage-pattern, they note, may equally reflect changes in follicular composition and the ovarian endocrine environment rather than a causal role for ESR2 in triggering puberty.

Context from other species both supports and complicates the interpretation. In Holstein cows, prenatal heat exposure has been linked to site-specific methylation of the ESR2 untranslated region and altered expression of ovarian steroid receptors in adult dominant follicles, hinting that this gene’s epigenetic state is sensitive to developmental conditions. An integrated methylome and transcriptome analysis of Hu sheep ovaries found a negative relationship between methylation near transcription start sites and gene expression, and large-scale demethylation has been observed in proliferating mouse granulosa cells during follicular development. Genome-wide studies of gilt ovaries and goat hypothalami, however, show that methylation changes during reproductive maturation are tissue-specific, region-specific, and stage-dependent, meaning the declining pattern seen in Duolang CpG island 1 should not be assumed to be a universal signature of puberty.

What the study ultimately delivers is a well-mapped starting point rather than a mechanism. It establishes that the ESR2 promoter carries dynamic, stage-specific methylation patterns in a breed defined by early fertility, that one of those patterns inversely tracks gene expression precisely when the ovary is transitioning to reproductive activity, and that pharmacological demethylation is associated with increased ESR2 transcription in granulosa cells. The candidate CpG sites identified here, particularly those within island 1 that correlated negatively with expression, now form a shortlist for promoter-reporter assays, targeted methylation editing, and transcription-factor binding experiments. If those follow-up studies hold up, epigenetic markers of reproductive maturation could eventually inform selection programs aimed at breeding sheep that reach puberty sooner and reproduce more prolifically, turning a chemical footnote on DNA into a practical tool for livestock genetics.

Subject of Research: ESR2 promoter DNA methylation and mRNA expression in sheep ovaries during puberty onset

Article Title: ESR2 promoter methylation and mRNA expression patterns in Duolang sheep ovaries across pubertal stages

Article References: Gu, R., Liu, L., Wu, L., Wang, M., & Xing, F. (2026). ESR2 promoter methylation and mRNA expression patterns in Duolang sheep ovaries across pubertal stages. Frontiers in Animal Science, 7, Article 1932991. https://doi.org/10.3389/fanim.2026.1932991

Image Credits: AI Generated

DOI: 10.3389/fanim.2026.1932991

Keywords: DNA methylation, ESR2, estrogen receptor beta, puberty, Duolang sheep, ovary, granulosa cells, epigenetics, CpG islands, bisulfite sequencing, reproductive maturation, 5-Aza-dC