The study, published in BMC Genomics by a research group led by Xinxin Cao, Lihui Zhang, Kaidong Liu, Jinshan Zhao and Hegang Li, focused on dairy goat embryos at 55 to 65 days of gestation, a critical window during which the gonads commit to their developmental trajectories. The team analyzed three groups of embryos: genetic females, genetic males, and intersex fetuses affected by polled intersex syndrome. Using single-cell RNA sequencing, the researchers profiled the gene expression of thousands of individual cells, then combined this with tissue morphology and immunofluorescence staining to confirm where key proteins were actually located within the developing organs.

Single-cell RNA sequencing works by dissociating a tissue into its component cells and capturing the messenger RNA from each one, allowing researchers to see which genes are active in which cells rather than averaging signals across a whole organ. In this study, that approach revealed striking cellular heterogeneity within the embryonic gonads. The team identified distinct populations of germ cells, the precursors of eggs and sperm, alongside a diverse cast of somatic cells, including stromal cells, granulosa cells, endothelial cells, immune cells, and, in males, Sertoli cells, interstitial cells and epithelial cells. The composition and proportions of these populations differed dramatically between female, male and intersex gonads.

One of the most consequential findings concerns where the different gonadal lineages come from. Through computational reconstruction of differentiation trajectories, a technique known as pseudotime analysis that orders cells along a developmental path based on their gene expression, the researchers found evidence that stromal cells may initiate the development of female and intersex gonads, while epithelial cells appear to serve as the origin of male gonadal cells. This suggests that the cellular ancestry of a gonad may be set remarkably early, and that in intersex embryos the developmental program may be steered toward a male-like trajectory from its starting point.

The molecular culprits behind sex reversal are familiar names in developmental biology, but the study adds new nuance. The researchers documented aberrant expression of SOX9, AMH and DMRT1, a trio of genes that together drive testis formation. SOX9 is the master switch of testis development; AMH, produced by Sertoli cells, triggers the regression of female reproductive structures; and DMRT1 reinforces the male program. In the intersex gonads, these genes were misexpressed in ways consistent with a partial female-to-male conversion. The finding confirms that the same core genetic circuitry implicated in disorders of sex development in humans is also at work in goats with polled intersex syndrome.

Perhaps the most exciting aspect of the study is the identification of new candidate regulators. The researchers flagged PHLDB2, ZNRF3 and TEAD1 as novel genes potentially involved in orchestrating sex reversal. ZNRF3 is known from other contexts to participate in Wnt signaling regulation, a pathway with well-established roles in ovary determination, while TEAD1 sits at the heart of the Hippo signaling pathway, which governs organ size and cell fate. Their emergence from an unbiased single-cell screen suggests that the network controlling gonadal sex determination is even more intricate than the canonical SOX9-centered model implies, and opens new avenues for experimental validation.

The study also delivered a surprise from the immune compartment. The team detected TREM2-positive macrophages within the goat embryonic gonads, a cell population that had not previously been characterized in this setting. Macrophages are best known as immune scavengers, but growing evidence implicates them in shaping tissue environments during organogenesis. The researchers propose that multiple genes expressed in these and other gonadal cells may modulate sex reversal by altering the gonadal microenvironment, the local signaling milieu in which germ cells and somatic cells make their fate decisions. In other words, sex determination may not be a purely cell-autonomous process but one influenced by the surrounding cellular ecosystem.

In a finding that borders on the unexpected, the analysis also implicated circadian rhythm-related genes in early gonadal development and the formation of intersex traits. Circadian genes are classically associated with daily biological rhythms, yet they have increasingly been shown to influence developmental timing in various tissues. Their differential expression across female, male and intersex gonads hints that the internal clock machinery may intersect with the sex determination network, a connection that, if confirmed experimentally, would add an entirely new dimension to how scientists think about disorders of sexual development.

The practical implications for livestock breeding are immediate. Polled intersex syndrome arises in goats bred for the polled, or hornless, trait, because the genetic mutation responsible for hornlessness is tightly linked to the intersex condition. Understanding precisely which cells and genes go awry during early gonad formation could enable breeders to select against the syndrome more effectively, protecting productivity in dairy herds. The single-cell atlas generated by this study provides a molecular reference that could support marker-assisted selection or even gene-editing strategies aimed at decoupling the polled trait from intersex development.

But the study’s ambitions extend well beyond the barnyard. The authors emphasize that polled intersex syndrome in goats serves as a valuable natural model for human reproductive defects, including disorders of sex development that affect roughly one in thousands of newborns worldwide. Because the goat gonad develops through mechanisms broadly comparable to those in humans, the cellular and molecular pathways mapped here, from stromal and epithelial origins to SOX9, AMH and DMRT1 dysregulation, from immune modulation to circadian influences, offer a framework for investigating why and how human sex development sometimes diverges. As single-cell technologies continue to mature, studies like this one demonstrate that even a familiar farm animal can illuminate some of the most fundamental and intimate questions in human biology: how a tiny embryonic organ decides what it will become, and what happens when that decision goes awry.

Subject of Research: Single-cell transcriptomic analysis of intersex gonad initiation and early sex differentiation in dairy goat embryos

Article Title: Single-cell RNA sequencing analysis reveals new insights for intersex gonad initiation and early sex differentiation of dairy goats

Article References: Cao, X., Zhang, L., Liu, K., Zhang, Y., Liu, X., Xu, D., Lin, X., Campo, F. P., Sun, M., Qin, Z., Gao, X., He, J., Zhao, J., & Li, H. (2026). Single-cell RNA sequencing analysis reveals new insights for intersex gonad initiation and early sex differentiation of dairy goats. BMC Genomics. https://doi.org/10.1186/s12864-026-13404-4

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13404-4

Keywords: single-cell RNA sequencing, polled intersex syndrome, dairy goats, sex differentiation, gonad development, SOX9, AMH, DMRT1, pseudotime analysis, germ cells, macrophages, circadian rhythm genes