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Neonatal and adult human testis defined at the single-cell level

ABSTRACT: Spermatogenesis has been well studied in rodents and invertebrates, but remains poorly understood in humans. As a step towards illuminating human spermatogenesis, we used single-cell RNA-sequencing (scRNAseq) analysis to analyze neonatal and adult human testes. Clustering analysis of neonatal testes revealed 3 germ subsets, including cells with characteristics of primordial germ cells (PGCs), and more differentiated cells with gene expression profiles similar with adult spermatogonial stem cells (SSCs). We identified markers for these neonatal subsets, including protein markers for the PGC-like (PGCL) subset. Clustering analysis of the adult testis revealed 9 germ and 3 somatic cell subsets. Among the germ cell clusters are 4 undifferentiated spermatogonia (SPG) states, each marked by specific genes. One of the SPG states has characteristics suggesting it is enriched for SSCs. We identified protein markers specific for this state, including cell-surface proteins that we used to enrich for these cells. We mapped the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also defined somatic cell subsets in the human testis and traced their developmental trajectories. Together, our data provides a blueprint for understanding the development of the male germline and supporting somatic cells in humans. The germ cell subset markers we identified are candidates to be used for clinical applications, including SSC therapy for treating infertility.

ORGANISM(S): Homo sapiens

PROVIDER: GSE124263 | GEO | 2018/12/22

REPOSITORIES: GEO

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Project description:In the adult mammalian testis, spermatogenic differentiation starts from a minute population of spermatogonial stem cells (SSCs). SSCs are generated after birth from the fetal gonocytes, themselves derived from the primordial germ cells (PGCs), which are specified during the first days after implantation. Transcriptome profiling of purified preparations evidenced the preferential accumulation in SSCs of transcripts of PU.1 (Sfpi1), a regulatory gene previously identified in hematopoietic progenitors. In situ immunolabeling and RNA determination showed a complex pattern of expression in the adult testis, first in SSCs and early spermatogonia followed by de novo expression in pachytene spermatocytes. Spermatogenesis in a null mutant (PU.1(G/G)) was arrested at the prenatal stage, with reduced numbers of gonocytes owing to a defect in proliferation already noticeable at E12.5. Transcripts of several germinal markers, including vasa (Mvh, Ddx4), Oct4 (Pou5f1), Dazl and Taf4b, were detected, whereas stella (PGC7, Dppa3) was not. Germ cells of PU.1(G/G) newborn testes grafted in nude mice did not initiate the postnatal replicative stage, whereas grafts of their wild-type littermates underwent complete spermatogenesis. During embryonic development, PU.1 transcription was initiated as early as the blastocyst stage, with a generalized expression at E6.5 in the embryonic ectoderm. PU.1 therefore appears to play a determinant role in at least two distinct lineages and, given its wide range of expression, possibly in other stem cells. Experiment Overall Design: Our starting point was the establishment of a transcriptome profile of purified preparations of SSCs, on the assumption that crucial genes would appear among those expressed at elevated levels in the stem cells. Work on adult SSCs is made difficult by their small number. We previously reported an efficient procedure, in which a truncated version of the Stra8 promoter is used to express in SSCs a neutral heterologous surface protein including two domains of the human CD4 antigen (huCD4) (Giuili et al., 2002). Once fractionated by immunomagnetic sorting, the huCD4-positive fraction is homogeneous for the expression of known SSC markers and highly efficient in the colonization of a sterile recipient.

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Neonatal and adult human testis defined at the single-cell level

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