Buffalo milk commands a devoted following for its rich, mellow flavor and dense nutritional profile, anchoring iconic cheeses and butter across Asia, the Mediterranean, and beyond. Yet behind the creamy appeal lies a stubborn scientific problem: the water buffalo, the world’s second most important milk-producing species after cattle, has lagged far behind dairy cows in genetic and molecular research. Breeds across Asia and Africa, where most of the global buffalo population lives, produce less milk over shorter lactations with longer calving intervals, and their genetic improvement has been slow. A team of researchers in China and Egypt has now zeroed in on one molecular player that may help close that gap: the estrogen receptor alpha gene, known as ESR1.
In a study published in the journal Archives Animal Breeding, Zhixiang Wang of Guangxi University and colleagues systematically tested how ESR1 controls the workhorse cells of milk production, the buffalo mammary epithelial cells. Using two complementary genetic manipulation techniques in cultured cells, small interfering RNA to silence the gene and a lentiviral vector to overexpress it, the team showed that ESR1 sits at a regulatory crossroads governing milk fat synthesis, milk protein production, cell proliferation, and cell survival. When ESR1 activity was dialed down, the cells grew more slowly and produced less fat and protein machinery; when it was dialed up, the opposite occurred, with the effects on gene expression mirroring each other almost perfectly in the two experimental directions.
The experimental material came from lactating Murrah buffaloes at the Buffalo Research Institute of the Chinese Academy of Agricultural Sciences in Nanning, Guangxi. The researchers collected mammary gland tissue from mid-lactation animals aged five to six years, then isolated mammary epithelial cells using a tissue explant culture method. Fibroblast contamination was removed through repeated rounds of differential adhesion purification, and the resulting cell populations were verified as genuine mammary epithelial cells by confirming that they expressed characteristic marker genes such as CSN2, CSN3, and LALBA at levels far higher than in buffalo fibroblast controls. The purified cells displayed the expected oval epithelial morphology and a typical growth curve, moving from a lag phase into rapid logarithmic growth before reaching confluence.
With a validated cell system in hand, the team designed siRNA molecules targeting the buffalo ESR1 messenger RNA and delivered them into the cells, confirming successful uptake through green fluorescence signals and verifying silencing efficiency by quantitative real-time PCR. Proliferation was then measured in two independent ways: the CCK-8 assay, which tracks metabolic activity as a proxy for cell number over 24, 48, and 72 hours, and the EdU assay, which directly labels cells in the process of copying their DNA. Both approaches converged on the same conclusion. Suppressing ESR1 significantly reduced proliferative activity, while the absorbance values in the interference group dropped markedly at 24 and 48 hours compared with controls.
The gene expression data revealed how deeply ESR1’s influence runs through the milk synthesis machinery. Silencing ESR1 significantly downregulated a suite of genes responsible for lipid uptake, transport, and storage, including PPARG, LPL, PLIN2, VLDLR, ADFP, and CD36, although the fatty acid synthesis gene ACACA remained unchanged. Genes tied to milk protein synthesis also fell, among them the casein genes CSN1S1 and CSN1S2 and key components of the mTOR signaling pathway such as RPS6, RHEB, EIF4E, and AKT1. Proliferation-associated genes, including STAT5A, MTOR, ELF5, CCND1, EGF, and IGF1, were likewise suppressed. Meanwhile, the pro-apoptotic genes BAX and CASP9 rose while the anti-apoptotic gene BCL2 fell, indicating that losing ESR1 pushes the cells toward self-destruction.
Overexpression told the mirror-image story. When the researchers infected the cells with a lentiviral vector carrying the buffalo ESR1 gene, ESR1 transcript levels rose dramatically, and the EdU and CCK-8 assays showed significantly enhanced proliferation at 48 and 72 hours. The lipid transport genes PPARG, LPL, PLIN2, VLDLR, ADFP, and CD36 all climbed, as did the casein genes and the mTOR pathway components. The proliferation genes STAT5A, MTOR, ELF5, CCND1, EGF, and IGF1 were upregulated, BCL2 increased, and the pro-apoptotic genes BAX and CASP9 declined. The complete symmetry between the knockdown and overexpression results strengthens the case that ESR1 is a genuine driver of these cellular programs rather than a passive bystander.
The fat-related findings fit neatly with earlier work in other species. Milk fat synthesis depends on three linked stages: uptake of fatty acids from circulating lipoproteins, their synthesis and modification, and storage in lipid droplets before secretion. LPL breaks down triglycerides in chylomicrons and very-low-density lipoproteins into free fatty acids for cellular uptake, CD36 ferries long-chain fatty acids across the membrane, and VLDLR delivers fatty acids and cholesterol through endocytosis. The authors note that ESR1 can recruit the co-activator PPARGC1B, which boosts the transcriptional activity of PPARG, a master regulator of lipid metabolism that in turn upregulates CD36 and LPL. The synchronized expression patterns observed in the buffalo cells suggest that ESR1 amplifies the lipid metabolic network largely through this PPARG-centered cascade, while its positive effects on the lipid droplet proteins PLIN2 and ADFP may help stabilize the droplets that milk fat secretion requires.
The protein synthesis results point to a two-pronged mechanism. In dairy cow cells, previous research showed that ESR1 indirectly upregulates CSN1S1 and CSN1S2 through an ERα–NFκB1–mTOR cascade, and the buffalo data align closely with that model. The PI3K-AKT-mTOR pathway is a central hub for milk protein synthesis, and ESR1’s positive regulation of AKT1, RHEB, RPS6, and EIF4E suggests it feeds energy and building blocks into the protein translation machinery through this route. Intriguingly, ESR1 also negatively regulated PDK1, an enzyme that shunts pyruvate away from the mitochondria into glycolysis. The authors propose that when high ESR1 expression lowers PDK1 levels, the pyruvate dehydrogenase complex regains activity, allowing pyruvate to enter the tricarboxylic acid cycle. That metabolic shift would enhance oxidative phosphorylation, supplying the ATP and amino acid precursors that the energy-hungry mTOR-driven protein synthesis program demands.
The study’s authors are careful to frame the work as a foundation rather than a finished tool. Profiling ESR1 expression patterns and identifying functional genetic variants could yield candidate molecular markers for marker-assisted selection and genomic selection programs, allowing breeders to screen for buffalo individuals with inherently high milk yield and improved composition. But they also flag real risks: artificially excessive activation of mammary cell proliferation and milk synthesis could impose metabolic stress and negative energy balance on lactating animals, and because ESR1 is a pleiotropic receptor with essential roles in systemic reproductive regulation, overactivation might disturb endocrine homeostasis, impair fertility, or raise susceptibility to mammary gland disorders. In vivo studies to define the optimal range of ESR1 activity are the clear next step. For now, the study delivers something buffalo genetics has long lacked: a mechanistic, experimentally validated picture of how a single estrogen-responsive gene orchestrates the cellular engines of one of the world’s most cherished milks.
Subject of Research: Regulation of lactation traits by the estrogen receptor alpha gene ESR1 in buffalo mammary epithelial cells
Article Title: The role of ESR1 in regulating lactation traits in buffalo mammary epithelial cells
Article References: Wang, Z., Chen, X., Qin, C., Jiang, J., Li, L., Liu, Q., Teng, C., Xiong, J., Wang, L., Amin, A., & Li, Z. (2026). The role of ESR1 in regulating lactation traits in buffalo mammary epithelial cells. Archives Animal Breeding, 69(2), 347-361. https://doi.org/10.5194/aab-69-347-2026
Image Credits: AI Generated
Keywords: ESR1, estrogen receptor alpha, buffalo, mammary epithelial cells, lactation, milk fat synthesis, milk protein synthesis, mTOR signaling, siRNA, gene overexpression, cell proliferation, animal breeding
News Source: Juliet Wilcox. (October 9, 2026). Estrogen Receptor Gene ESR1 Emerges as a Master Switch for Buffalo Milk Production. Scienmag.
