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

Abstract

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...

Tcf21+mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration

“…To get a sense of cellular interactions of Tcf21 + cells within the testis, we calculated Interaction Scores for highly variable and previously defined ligand-receptor (L-R) pairs in germ cells and somatic cells or among somatic cells of the testis (Figure 6C, Table S3). We previously showed that the Tcf21 + population has more potential interactions with spermatogonial populations than other germ cells (Shami et al, 2020) (Figure 6C, right panel), Similarly, we find that spermatogonia can convey information back to the Tcf21 + population (Figure 6C, left panel) via Pdgfa, various ADAMs, calmodulins, FGFs, and guanine nucleotide binding proteins (Table S3).…”

“…We recently employed single-cell RNAseq (scRNAseq) to generate a molecular atlas of the mouse testis (Green et al, 2018, Sohni et al, 2019 (Shami et al, 2020). Our analysis of 35,000 cells identified not only all somatic cell types known to be present in the adult mouse testis but also an unexpected Tcf21 + population (Figure 1A) (Green et al, 2018).…”

“…We used a previously curated list of ligand-receptor pairs in human as reference, identifying corresponding orthologues in mouse as previously described (Shami et al, 2020). We selected pairs with either highly variable ligand gene among the 7 somatic cell centroids, or highly variable receptor gene among the 26 germ cell centroids (6 SPG and 20 non-SPG clusters).…”

“…For each ligand-receptor pair we calculated its apparent signaling strength as an "Interaction Score", defined as the product of the mean expression level of the ligand in one cell type and that of the receptor in another cell type. In all, we calculated such an Interaction Score matrix of cell type pairs for germ(ligand)-soma(receptor) interaction, soma (ligand)-soma (receptor) interaction and soma (ligand)-germ(receptor) interaction (reproduced with permission from (Shami et al, 2020), respectively. To extract the general signaling pattern for each interaction matrix, we defined "strong interactions" for each matrix by keeping the highest 5% Interaction Scores for each matrix.…”

Genome medicine in male infertility: From karyotyping to single‐cell analysis

“… 29 Tan et al listed most of the single‐cell transcriptome analyses that have been conducted on mouse, human, and macaque testes 30 . By modifying their report, single‐cell transcriptome analysis performed using human testicular samples is summarized in Table 1, 31–38 which included eight publications and our current project. The majority of them have focused on germ cells, and the major method to isolate and harvest a single cell type, which is the most important and difficult part of single‐cell analysis, was enzymatic digestion combined with flow cytometric sorting or physical filtering.…”