Single-Cell Sequencing Reveals Androgen Deficiency Arrests Meibocyte Differentiation and Impairs EMC-Mediated Lipid Metabolism

Abstract

Purpose To investigate the cellular dynamics of androgen-deficient meibomian gland dysfunction (MGD) and to elucidate the molecular mechanisms limiting the structural regenerative efficacy of topical dihydrotestosterone (DHT) treatment. Methods Single-cell RNA sequencing (scRNA-seq) was performed on meibomian glands from sham-operated controls, castrated mice, and castrated mice receiving topical 0.08% DHT. We performed comparative analyses to quantify changes in cell composition, reconstruct differentiation trajectories, and systematically profile alterations in metabolic pathways and organelle homeostasis networks. Results Castration arrested meibocyte differentiation and redirected cell fate commitment. Pseudotime trajectory analysis revealed a pronounced shift from the meibocyte lineage toward ductal and progenitor fates, accompanied by comprehensive downregulation of lipogenic genes (e.g., Fasn, Awat2). This fate reprogramming was coupled with disrupted endoplasmic reticulum (ER) homeostasis, including suppression of the ER membrane protein complex (EMC) and impaired lipid assembly machinery. Topical DHT treatment partially restored meibocyte differentiation but failed to fully recover gland morphology. Furthermore, rapid metabolic reactivation following DHT treatment outpaced ER capacity recovery, inducing proteostatic stress. Conclusions Androgen deficiency drives MGD through dual mechanisms: reprogramming of meibocyte cell fate toward non-lipogenic lineages and suppression of EMC-mediated stabilization of ER-resident lipogenic enzymes. The resulting temporal mismatch between lipogenic reactivation and ER capacity recovery upon DHT treatment induces proteostatic stress, limiting the efficacy of androgen monotherapy and identifying the EMC–ER axis as a candidate therapeutic target for combinatorial intervention.