Metabolic Control of Sertoli Cell Function: Implications for Germline Maintenance and Infertility

Martin A. Estermann¹, Karina F. Rodriguez¹ and Humphrey Yao¹

1.Reproductive Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709, USA


Abstract Text:
Infertility affects approximately 15% of couples worldwide, with male infertility accounting for nearly half of these cases. Although dietary and metabolic factors are known to influence reproductive health, the molecular mechanisms linking metabolism to fertility remain poorly understood. Our research investigates how cellular metabolism governs embryonic testis differentiation, and its long-term impact on adult testis function and fertility using mice as a model. We focused specifically on Sertoli cells, the somatic cell lineage in the testis that nurtures the germline. Previous studies have revealed that embryonic Sertoli cells rely on glucose to maintain their identity, and the expression of SOX9, their master regulatory transcription factor. In this study, we sought to address how does glucose metabolism regulate Sertoli cell differentiation and function? By performing a targeted ex vivo drug screening in embryonic mouse testes, we discovered that glucose metabolism in Sertoli cells predominantly feeds into the hexosamine biosynthetic pathway (HBP), rather than glycolysis. The HBP facilitates protein O-GlcNAcylation, a process involving the addition of a O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues on proteins. Protein O-GlcNAcylation, mediated by the enzyme O-GlcNAc transferase (OGT), acts as a nutrient sensor, regulating key cellular processes including transcription, translation, signal transduction and metabolism. Moreover, O-GlcNAcylation can affect the translocation, DNA binding, transactivation, and stability of transcription factors, regulating gene expression. To explore the role of O-GlcNAcylation in testis development, we performed transcriptomic analysis on embryonic testis treated ex vivo with the OGT inhibitor OSMI-1. This revealed significant defects in Sertoli cell differentiation and germ cell survival. To further explore the role of O-GlcNAcylation in vivo, we generated a Sertoli cell-specific Ogt conditional knockout mice (cKO), where Ogt was deleted during fetal development. By 8 weeks of age, Ogt cKO mice were infertile, exhibited reduced testis size and had seminiferous tubules devoid of germ cells. These findings confirmed that protein O-GlcNAcylation in Sertoli cells is essential for germline maintenance and fertility. Proteomics analysis of O-GlcNAcylated proteins in Sertoli cells revealed enrichment of targets involved in gene expression, protein translation, and cytoskeletal regulation. Among these, we identified and validated the O-GlcNAcylation of the Sertoli cell master regulator SOX9, providing a mechanistic link between glucose metabolism and Sertoli cell differentiation. In summary, our results propose a model in which Sertoli cells prioritize glucose metabolism through the HBP to sustain protein O-GlcNAcylation, thereby maintaining their differentiation and functional support of the germline. Disruption of this process compromises Sertoli cell function, germline survival, and fertility. These findings highlight the essential role of the hexosamine biosynthetic pathway and protein O-GlcNAcylation in supporting testicular function, offering valuable new insights into the metabolic regulation of reproductive health.