Establishment and Characterization of Testis Organoids with Proliferation and Differentiation of Spermatogonial Stem Cells into Spermatocytes and Spermatids

• Published in: Cells (Basel, Switzerland) Vol. 13; no. 19; p. 1642
• Main Authors: Zhang, Dong, Jin, Wencong, Cui, Yinghong, He, Zuping
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 02.10.2024; MDPI
• Subjects: Acrosin; Adult Germline Stem Cells - cytology; Adult Germline Stem Cells - metabolism; Animals; Antibodies; c-Kit protein; Cell Differentiation; Cell interactions; Cell Proliferation; Drug screening; Enzymes; Epigenetic inheritance; Epigenetics; Ethanol; Ethylenediaminetetraacetic acid; Gametes; Immunohistochemistry; Infertility; Male; Medical research; Mice; Mice, Nude; mitosis and meiosis; MLH1 protein; Molecular modelling; Neonates; Organogenesis; Organoids; Organoids - cytology; Organoids - metabolism; Ovaries; Protein gene product 9.5; Reproductive system; self-assembly; Sertoli cells; Sox9 protein; Spermatids; Spermatids - cytology; Spermatids - metabolism; Spermatocytes; Spermatocytes - cytology; Spermatocytes - metabolism; Spermatogenesis; Spermatogonia - cytology; Spermatogonia - metabolism; Stem cells; Stem Cells - cytology; Stem Cells - metabolism; Testes; testicular single cells; Testis - cytology; Testis - metabolism; testis organoids; Transplantation; Tubules; China; Grand Island; United States > US; self-assembly; mitosis and meiosis; testis organoids; testicular single cells; transplantation
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells13191642

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility.