Single-Nucleus Transcriptomics Reveals Granulosa Cell Heterogeneity and Microenvironmental Remodeling Across Bovine...

Area of Science:

• Reproductive Biology
• Genomics
• Veterinary Science

Background:

• Ovarian function is vital for dairy cattle fertility, but its cellular and molecular underpinnings across various physiological and dysfunctional states are not fully understood.
• Hormonal profiles differ significantly between ovarian states, with specific estrogen to progesterone ratios characterizing follicular, luteal, and cystic conditions.

Purpose of the Study:

• To comprehensively characterize the cellular and molecular landscape of the bovine ovary across distinct physiological and pathological states using single-nucleus RNA sequencing.
• To identify cell-type-specific transcriptional and metabolic variations associated with different ovarian functions and dysfunctions in dairy cows.

Main Methods:

• Single-nucleus RNA sequencing (snRNA-seq) was performed on ovarian tissues from 18 Holstein cows representing follicular, luteal, mid-gestation pregnancy, inactive, and luteal cystic states.
• Analysis included cell-type annotation, granulosa cell (GC) subclustering, trajectory inference, co-expression analysis, ligand-receptor/ligand-target prediction, and transcriptome-based metabolic flux estimation.
• Hormone measurements in serum and follicular fluid were conducted to correlate endocrine profiles with ovarian states.

Main Results:

• Twenty-seven ovarian cell clusters and five granulosa cell (GC) subtypes were identified, showing state-associated variations in composition and gene expression.
• Luteal cystic ovaries exhibited increased immune cell representation and inflammation-related gene signatures, while inactive ovaries showed reduced intercellular communication.
• GCs displayed state-dependent differences in transcriptional programs for proliferation, steroidogenesis, extracellular matrix organization, inflammation, and metabolism, with metabolic inference revealing pathway variations.

Conclusions:

• This study provides a valuable single-nucleus transcriptomic resource for bovine ovarian research, detailing cellular and molecular differences across multiple ovarian states.
• Candidate cell populations and molecular pathways involved in ovarian function and dysfunction, particularly in cystic and inactive states, have been identified for future investigation.
• Understanding these cellular and molecular features is critical for improving fertility and reproductive health in dairy cattle.