Cross-Species Single-Cell Atlas Reveals Conserved and Divergent Programs in Mammalian Fetal Ovarian Development
核心洞察
Researchers generated a bovine single-cell RNA-seq dataset spanning six gestational stages (E38–E112) and integrated it with human and mouse data to create a 107,930-cell cross-species atlas of early ovarian development.
Germ cell developmental trajectories showed strong conservation across species, with shared regulators including POU5F1 (搜索), NANOG (搜索), and SYCP family members, while granulosa cells displayed pronounced species-specific divergence.
A bovine-specific cell population expressing steroidogenic markers (CYP17A1 (搜索), HSD3B1 (搜索)) was identified, suggesting a unique early steroidogenic lineage not observed in human or mouse fetal ovaries.
A comprehensive cross-species single-cell transcriptomic analysis has mapped the cellular and molecular landscape of early female gonadal development across cattle, humans, and mice, revealing both deeply conserved regulatory programs and striking species-specific divergence in ovarian cell differentiation.
The study, which integrated newly generated bovine single-cell RNA sequencing (scRNA-seq) data with publicly available human and mouse datasets, provides a systematic framework for understanding how early ovarian development is regulated at the molecular level across mammals. The integrated atlas comprised 107,930 high-quality single cells after quality control, spanning six bovine gestational stages (E38–E112), six human post-conception weeks (PCW6–PCW16), and five mouse embryonic stages (E11.5–E18.5).
Eleven shared gonadal cell types and a bovine-specific population
Unsupervised clustering based on 16,073 one-to-one orthologous genes identified 11 major cell types across all three species, including germ cells, granulosa cells, mesenchymal cells, mesothelial cells, epithelial cells, erythroid cells, endothelial cells, immune cells, perivascular cells, glomerular mesonephros cells, and neural cells. Germ cells were identified by high expression of DAZL and DDX4, while granulosa cells were marked by KITLG and AMHR2.
Notably, the analysis also uncovered an unclassified bovine-specific cell population detected at all sampled stages, with higher relative abundance at early gestational time points (E38, E46, and E73). Further characterization using a support vector machine (SVM) classifier revealed that this population partially expressed the mesenchymal lineage-associated marker PDGFRA and showed clear expression of CYP17A1 (搜索), along with partial expression of steroidogenesis-related genes including INSL3, HSD3B1 (搜索), and STAR.
"This population may represent either a bovine-specific early steroidogenic lineage in the female gonad or a theca precursor population with species-specific temporal state," the researchers noted, highlighting that neither human nor mouse fetal ovaries exhibit comparable steroidogenic gene expression during the equivalent developmental window.
Epigenetic dynamics in germ cells and granulosa cells
Analysis of epigenetic regulators revealed dynamic changes in both germ cells and granulosa cells across species. In female germ cells, the maintenance DNA methyltransferase DNMT1 and the DNA demethylation factor TET1 showed high expression at early developmental stages followed by decreased expression at later stages, consistent with the well-characterized wave of global DNA demethylation during germ cell development.
Histone modification regulators also exhibited species-specific patterns. EZH2, a core component of Polycomb repressive complex 2 (PRC2), showed sustained but gradually decreasing expression in mouse female germ cells, while SUZ12 was upregulated in bovine germ cells (E46–E83) but showed decreasing expression in mouse. In granulosa cells, DNMT1 showed a progressive decrease following sex determination in bovine and human, while TET1 displayed a gradual increase after E73 in bovine, suggesting a shift from DNA methylation maintenance toward increased demethylation activity during granulosa cell development.
Conserved germ cell trajectory with species-specific pathway enrichment
Pseudotime trajectory analysis of germ cells revealed a consistent developmental progression across all three species, transitioning from pluripotency-associated states toward meiotic initiation and differentiation. Early development was marked by high expression of POU5F1 (搜索), NANOG (搜索), TFAP2C, and SOX17, which were gradually downregulated as maturation regulators such as KIT and ELAVL2 were activated. Meiotic entry was characterized by upregulation of SYCP1 (搜索), SYCP2, SYCP3, TEX30, ZCWPW1, and SMC1B.
A total of 34 conserved, highly dynamic genes were identified across species, including NANOG (搜索), POU5F1 (搜索), SYCP1 (搜索), and JUN. However, substantial species-specific expression dynamics were also observed, with 162 bovine-specific, 36 human-specific, and 566 mouse-specific genes. Gene Ontology analysis revealed that bovine germ cells were enriched for pathways related to cell morphogenesis and reproductive processes, human germ cells for cell division and DNA replication, and mouse germ cells for meiosis-related pathways including homologous chromosome pairing and segregation.
Transcription factor regulon analysis identified conserved active regulons including NANOG (搜索), WT1, GATA4, GATA6, PBX1 (搜索), MAF, and HMGA2. PBX1 showed the strongest signal in cattle, while species-specific regulons included ELF1, ELK3, and RFX2 in bovine, MAFF and TEAD4 in mouse, and NFE2L3, STAT1, and E2F1 in human.
Granulosa cells display pronounced species divergence
In contrast to germ cells, granulosa cells exhibited more pronounced cross-species divergence. SVM-based classification trained on human data showed that mouse and bovine granulosa cells had low prediction probabilities (below 0.2 and 0.4, respectively), indicating substantial transcriptomic divergence.
Top-weighted genes driving granulosa cell discrimination included LAMA2 and MMP14 in bovine, associated with extracellular matrix organization; RHOB and ARHGAP28 in human, implicating Rho GTPase-mediated cytoskeletal signaling; and SOX4 and FST in mouse, related to TGF-β/Activin signaling.
Despite this divergence, the AP-1 transcription factor family members FOS (搜索) and JUNB (搜索) emerged as the most enriched common active regulons across all three species. FOXL2 (搜索), a critical granulosa cell determinant, was detected at late pseudotime in human and mouse but not in bovine, suggesting a delayed granulosa cell maturation program in cattle.
Cell-cell communication reveals germ cells as signaling sources
Cell-cell communication analysis using CellChat revealed that interactions involving germ cells were relatively weak compared with those among gonadal somatic cell populations, a pattern consistently observed across all three species. When germ cells were designated as signaling sources, conserved ligand-receptor pairs were identified, including MDK-SDC4 interactions between germ cells and epithelial cells in all three species, and NECTIN3-NECTIN2 interactions between germ cells and multiple somatic cell types in cattle and mouse.
In contrast, when germ cells were treated as signal receivers, overall interaction strength was reduced and only species-specific pathways were identified. WNT5A-FZD3 and KITL-KIT interactions were observed between bovine granulosa cells and germ cells, while THBS1-CD47 signaling was detected specifically between mouse granulosa cells and germ cells. No significant enrichment of ligand-receptor pairs was found between human somatic cells and germ cells in this direction.
"This pattern raises the possibility that early germ cells may contribute to shaping their local microenvironment rather than functioning solely as passive recipients of niche signals," the authors noted.
A framework for cross-species reproductive biology
The study establishes a generalizable framework for cross-species single-cell transcriptomic integration and quantitative assessment of transcriptional conservation. The SVM-based classifier demonstrated that germ cells, immune cells, endothelial cells, and erythroid cells exhibit greater cross-species similarity, while granulosa cells, mesenchymal cells, and epithelial cells display clearer species-specific divergence.
The researchers acknowledged several limitations, including the reliance on approximate developmental stage alignment across species and the restriction to one-to-one orthologous genes, which limits the ability to capture species-specific genes or gene family expansions that may play important roles in lineage diversification.