Clinical efficacy of treatments against non-obstructive azoospermia (NOA), which affects 1% of men, are currently limited by the incomplete understanding of NOA pathogenesis and normal spermatogenic microenvironment. Here, we profile >80,000 human testicular single-cell transcriptomes from 10 healthy donors spanning the range from infant to adult and 7 NOA patients. We show that Sertoli cells, which form the scaffold in the testicular microenvironment, are severely damaged in NOA patients and identify the roadmap of Sertoli cell maturation. Notably, Sertoli cells of patients with congenital causes (Klinefelter syndrome and Y chromosome microdeletions) are mature, but exhibit abnormal immune responses, while the cells in idiopathic NOA (iNOA) are physiologically immature. Furthermore, we find that inhibition of Wnt signaling promotes the maturation of Sertoli cells from iNOA patients, allowing these cells to regain their ability to support germ cell survival. We provide a novel perspective on the development of diagnostic methods and therapeutic targets for NOA.
SummaryGeneration of functional spermatids from azoospermia patients is of unusual significance in the treatment of male infertility. Here, we report an efficient approach to obtain human functional spermatids from cryptorchid patients. Spermatogonia remained whereas meiotic germ cells were rare in cryptorchid patients. Expression of numerous markers for meiotic and postmeiotic male germ cells was enhanced in human spermatogonial stem cells (SSCs) of cryptorchidism patients by retinoic acid (RA) and stem cell factor (SCF) treatment. Meiotic spreads and DNA content assays revealed that RA and SCF induced a remarkable increase of SCP3-, MLH1-, and CREST-positive cells and haploid cells. Single-cell RNA sequencing analysis reflected distinct global gene profiles in embryos derived from round spermatids and nuclei of somatic cells. Significantly, haploid spermatids generated from human SSCs of cryptorchid patients possessed fertilization and development capacity. This study thus provides an invaluable source of autologous male gametes for treating male infertility in azoospermia patients.
Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.
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