The Neonatal and Adult Human Testis Defined at the Single-Cell Level

Sohni, Abhishek; Tan, Kun; Song, Hye Won; Burow, Dana; Rooij, Dirk G. de; Laurent, Louise C.; Hsieh, Tung-Chin; Rabah, Raja; Hammoud, Saher Sue; Vicini, Elena; Wilkinson, Miles

doi:10.1016/j.celrep.2019.01.045

Cited by 267 publications

(417 citation statements)

References 62 publications

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“…To validate newly-identified noncanonical primate markers expressed by SPG1 and SPG2 undifferentiated spermatogonia (TCF3, MAGEB2, CDK17, MORC1, DNAJB6, PIWIL4 and FMR1), we co-stained with UCHL1, a broad undifferentiated spermatogonia marker ( Figure 3A-D, S3A-E, upper panels ), or CKIT, a differentiating spermatogonia marker ( Figure 3A-D, S3A-E, lower panels) in human testes tissue. TCF3 and PIWIL4 have been described in previous papers 22,23,28 , while MAGEB2, CDK17, MORC1, DNAJB6, and FMR1 are new markers described in this study. Consistent with our scRNAseq data predictions, our protein markers exhibit overlap with UCHL1+ spermatogonia cells lining the basement membrane of the human testis ( Figure 3A-D, upper panels ), but less overlap with cKIT+ differentiating spermatogonia ( Fig 3A-D, lower panels ); confirming that SPG1 and SPG2 are undifferentiated spermatogonial cell populations.…”

Section: Resultssupporting

confidence: 61%

“…In order to functionally characterize the colonization potential of the SPG1 population, we enriched for these cells by FACS using the SPG1 cell surface marker TSPAN33 ( Figure 2E, Figure 3J ) that was also described in the human undifferentiated spermatogonia clusters of two previous studies 22,23 . Unsorted, TSPAN33-negative, and TSPAN33-positive cells were xenotransplanted into recipient nude mice ( Figure 3I-J ).…”

Section: Resultsmentioning

confidence: 99%

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Single-cell RNA sequencing of human, macaque, and mouse testes uncovers conserved and divergent features of mammalian spermatogenesis

Shami

Zheng

Munyoki

et al. 2020

Preprint

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Spermatogenesis is a highly regulated process that produces sperm to transmit genetic information to the next generation. Although extensively studied in mice, our current understanding of primate spermatogenesis is limited to populations defined by state-specific markers defined from rodent data. As between-species differences have been reported in the process duration and cellular differentiation hierarchy, it remains unclear how molecular markers and cell states are conserved or have diverged from mice to man. To address this challenge, we employ single-cell RNA-sequencing to identify transcriptional signatures of major germ and somatic cell-types of the testes in human, macaque and mice. This approach reveals differences in expression throughout spermatogenesis, including the stem/progenitor pool of spermatogonia, classical markers of differentiation, potential regulators of meiosis, the kinetics of RNA turnover during spermatid differentiation, and germ cell-soma communication. These datasets provide a rich foundation for future targeted mechanistic studies of primate germ cell development and in vitro gametogenesis. potential communications with the germline. Overall, this study provides the first single-cell comparative analysis of the spermatogenesis program between primates and rodents. Such a new resource is expected to improve our knowledge base for future studies of germ cell development in primates, and ultimately improve our understanding of the intrinsic and extrinsic evolutionary changes of the gametogenesis program. Knowledge gained from these data will inform fertility restoration efforts, including SSC culture and in vitro gametogenesis. ResultsSingle-cell sequencing identifies major germ and somatic cell types of adult human and macaque testes. Using the Drop-seq platform, we generated single cell transcriptome data from ~14K and ~22K adult human and rhesus macaque testes, respectively. Using these datasets, our overall strategy was to first identify major cell types and states in the adult human and rhesus macaque testes separately, then combine them, along with our previous reported mouse data 12 , to define evolutionarily conserved or diverged programs within germ cells and the somatic cells of the testis (Figure 1A). The doublet rates for the human and macaque datasets is estimated to be <2%, therefore, allowing reliable analysis of individual cells (Figure S1A). After QC filtering (see Methods), 13,837 and 21,574 cells were retained from human and macaque, respectively, with an average of 3,210 unique molecular identifiers (UMIs) per cell, and 1,304 genes detected per cell. This sequencing depth and the number of detected genes was sufficient to define major cell types 13,14 . Systematic comparisons of technical batches (such as Human 1.1-1.5, Figure S1B-C) and biological replicates (4 humans and 5 macaques, Figure S1D-E) confirmed high batch-to-batch or individual-to-individual concordance, despite the variable proportions of cell types in different samples, likely due to differences i...

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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Single-cell RNA sequencing of human, macaque, and mouse testes uncovers conserved and divergent features of mammalian spermatogenesis

Shami

Zheng

Munyoki

et al. 2020

Preprint

Self Cite

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“…Because our study focuses on representative stages, it will be important to further dissect these mechanisms in order to fully understand the complex and well-coordinated nature of spermatogenesis. Recent studies using single cell analyses have revealed new details on the shifting, transitory transcriptomic and epigenomic environments of progressive cell types in human and mouse spermatogenesis 54–59 . Such dynamism is achievable through the functional interplay of complex combinations of TFs and enhancers, as well as other regulatory elements.…”

Section: Discussionmentioning

confidence: 99%

Super-enhancer switching drives a burst in germline gene expression at the mitosis-to-meiosis transition

Maezawa

Yukawa

Rothenberg

et al. 2020

Preprint

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26The testis has the most diverse and complex transcriptome of all organs due to bursts in 27 expression of thousands of germline-specific genes. Much of this unique gene expression takes 28 place when mitotic germ cells differentiate and enter into meiotic prophase. Here, we 29 demonstrate that the genome-wide reorganization of super-enhancers (SEs) drives bursts of 30 germline genes after the mitosis-to-meiosis transition. At the mitosis-to-meiosis transition, 31 mitotic SEs dissolve while meiotic SEs are established. Meiotic SEs are associated with the 32 activation of key germline genes, defining the cellular identity of germ cells. This SE switching 33 is regulated by the establishment of meiotic SEs via A-MYB (MYBL1), a key transcription 34 factor for germline genes, and by the resolution of mitotic SEs via SCML2, a germline-specific 35 Polycomb protein required for spermatogenesis-specific gene expression. Prior to the entry 36 into meiosis, meiotic SEs are preprogrammed in mitotic spermatogonia, serving to direct the 37 unidirectional differentiation of spermatogenesis. We identify key regulatory factors for both 38 mitotic and meiotic enhancers, revealing a molecular logic for the concurrent activation of 39 mitotic enhancers and suppression of meiotic enhancers in the somatic and/or mitotically 40 proliferating phase.41replicates ( Fig. 1b, Supplementary Fig. 1). (While generated for and analyzed in this study, our 93 H3K27ac ChIP-seq data for wild-type PS and RS was initially introduced in another study that 94 analyzed active enhancers on the sex chromosomes 23 .) Consistent with the massive, dynamic 95 transcriptional change occurring at the mitosis-to-meiosis transition, we observed H3K27ac peaks 96 in mitotically proliferating spermatogonia (blue shadow), H3K27ac peaks unique to meiotic 97 spermatocytes (red shadow), and constitutive peaks (gray shadow: Fig. 1b). 99For the quantitative comparison of active enhancers during spermatogenesis, we analyzed 100 H3K27ac ChIP-seq peaks ±1 kb outside transcription start sites (TSSs); hereafter, we refer to such 101 peaks as 'distal peaks' and peaks within ±1 kb as 'proximal peaks.' Through the use of MACS2 24 , 102 a program that identifies the significant enrichment of ChIP-seq signals, we detected 11,433 distal 103 H3K27ac ChIP-seq peaks that were present in at least one stage of spermatogenesis (for these 104 analyses, we permitted only distal peaks with a normalized enrichment value of ≥4; see Methods;105 Supplementary Table 1). The distal peaks were categorized into 9 clusters through k-means 106 clustering; we further organized these into three classes as follows (Fig. 1c). The first class (705 107 peaks, comprising clusters 1-3) represents constitutive active enhancers, i.e., those observed 108 throughout spermatogenesis. The second class (2,524 peaks, comprising clusters 4 and 5) represents 109 enhancers that are active in the mitotic proliferation phase of spermatogenesis (i.e., the 'mitotic 110 phase') but are inactive in meiotic and postmeiotic p...

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“…MAGE-B2 was also shown to be enriched in spermatogonia in human testis by single cell RNAsequencing (Guo et al, 2018;Sohni et al, 2019;Xia et al, 2020).…”

Section: The Mouse Ortholog Of Human Mage-b2 (Mage-b4) Regulates Stemmentioning

confidence: 97%

Enhanced stress tolerance through reduction of G3BP and suppression of stress granules

Lee

Klein

Tacer

et al. 2020

Preprint

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Stress granules (SG) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SG act as a protective mechanism to cope with acute stress, but persistent SG have cytotoxic effects that are associated with several age-related diseases.Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP.MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Importantly, knockout of the MAGE-B2 mouse ortholog confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermo-sensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress through fine-tuning phase separation and could aid in the development of male fertility therapies.

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The Neonatal and Adult Human Testis Defined at the Single-Cell Level

Cited by 267 publications

References 62 publications

Single-cell RNA sequencing of human, macaque, and mouse testes uncovers conserved and divergent features of mammalian spermatogenesis

Single-cell RNA sequencing of human, macaque, and mouse testes uncovers conserved and divergent features of mammalian spermatogenesis

Super-enhancer switching drives a burst in germline gene expression at the mitosis-to-meiosis transition

Enhanced stress tolerance through reduction of G3BP and suppression of stress granules

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