Global inflammatory response in in vitro organ cultured testes using single-cell RNA-sequencing

In vitro functional sperm production is important for understanding spermatogenesis and for the treatment of male infertility. Here, we describe similarities and differences between testis tissues in vivo and in vitro and clarify abnormalities in the early stage of in vitro spermatogenesis at single-cell resolution. While in vitro spermatogenesis progressed similarly to in vivo spermatogenesis until the early pachytene spermatocyte stage, a noticeable acute inflammatory response occurred in immune cells and non-immune testicular somatic cells immediately after cultivation. Inhibitor treatment revealed that NLRP3 inflammasome signaling is key to the inflammation. We observed damaged/dead germ cell accumulation in cultured testis, which may be due to dysfunctional phagocytosis by Sertoli cells. Our data revealed abnormal testicular milieu of in vitro cultured testes caused by tissue-wide sterile inflammation, in which the danger-associated molecular pattern-NLRP3 inflammasome axis may be a key element. ### Competing Interest Statement The authors have declared no competing interest. * dpp : days post-partum BTB : blood-testis barrier scRNAseq : single-cell RNA sequencing GO : gene ontology BHB : β-hydroxybutyrate NLRP3 : nucleotide-binding oligomerization domain leucine rich repeat, and pyrin domain-containing protein 3 DAMPs : damage-associated molecular patterns GSEA : gene set enrichment analysis PAMPs : pathogen-associated molecular patterns