Spatial Transcriptomics Reveals Inhba/Smad2/E2f4 Axis in Lrp2 high Thecal Cell Proliferation in Androgen-Induced PCOS Mice

Man Luo^1,2Xiaona Tian¹Li Li¹Guomei Zhang¹Wenzhi Liu¹Linlin Mei¹Haoran Li¹Hongjuan Li¹Dongmei Zhang¹Mengsi Zhou^1,2Cheng Xiao^3,4Jianping Ye^5,6Xiaofeng Yang^1*

• ¹Department of Obstetrics and Gynecology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, China
• ²Zhengzhou Key Laboratory of Endocrine Metabolism and Immunity in Polycystic Ovary Syndrome, Zhengzhou, China
• ³Institute of Muscle Biology and Growth, Forschungsinstitut fur Nutztierbiologie (FBN), Dummerstorf, Germany
• ⁴Institute of Agricultural and Environmental Sciences, Universitat Rostock, Rostock, Germany
• ⁵Institute of Trauma and Metabolism, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, China
• ⁶Academy of Medical Sciences, Zhengzhou, China

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by elevated androgen levels and impaired follicular development. A hallmark of PCOS is the excessive proliferation of thecal cells (TCs), which are critical for androgen production. However, the molecular mechanisms underlying this aberrant cellular expansion remain incompletely understood.A DHEA-induced mouse model was used to recapitulate the hormonal and ovarian features of human PCOS. Spatial transcriptomics was employed to profile gene expression in ovarian tissue at cellular resolution. Differential expression analysis, pathway enrichment, and spatial co-localization were performed to identify regulatory networks. Functional assays were conducted in cultured TCs using siRNA-mediated knockdown of target genes, and cell proliferation and cell cycle progression were evaluated using EdU incorporation and flow cytometry.Spatial transcriptomic profiling revealed widespread transcriptional changes in the ovaries of PCOS mice, including a marked expansion of a TCs subpopulation with high Lrp2 expression. This subset exhibited enhanced activity in genes involved in androgen synthesis and cell cycle regulation. A signaling axis comprising Inhba, Smad2, and E2f4 was identified as a key regulator of this proliferative response, with all three genes co-expressed in the affected regions. Knockdown of any component of this axis significantly suppressed TCs proliferation in vitro, with the greatest effect observed upon Inhba silencing.The Inhba/Smad2/E2f4 axis contributes to thecal cell hyperplasia and androgen excess in PCOS, and may serve as a mechanistic entry point for further investigation into the regulation of TCs proliferation in this disorder.