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Micrographs demonstrating the presence of germ cells in testicular tissues of selected Klinefelter patients. a-d show PAS-stained sections and e-h depict sections following immunohistochemical stainings for VASA/DDX4. Germ cell types within the seminiferous tubules are indicated using black arrows for spermatogonia, white arrows for spermatocytes, and arrow heads for spermatids. Seminiferous tubules devoid of germ cells are marked by asterisks. Normal qualitatively normal spermatogenesis, KS Klinefelter syndrome, + with germ cells, − without germ cells, SCO Sertoli cell-only. Scale bars represent 50 μm
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Background:
The most common sex chromosomal aneuploidy in males is Klinefelter syndrome, which is characterized by at least one supernumerary X chromosome. While these men have long been considered infertile, focal spermatogenesis can be observed in some patients, and sperm can be surgically retrieved and used for artificial reproductive technique...
Contexts in source publication
Context 1
... testicular tissue samples with and without germ cells were identified by histological analysis following PAS staining ( Fig. 1a-d) and immunohistochemical detection of the germ cell marker VASA/DDX4 (Fig. 1e-h). Individual tubules showing focal spermatogenesis could be detected in selected Klinefelter tissues (Fig. 1b, f). This was in contrast to control samples with qualitatively normal spermatogenesis (Fig. 1a, e), which showed spermatogenesis in the majority ...
Context 2
... testicular tissue samples with and without germ cells were identified by histological analysis following PAS staining ( Fig. 1a-d) and immunohistochemical detection of the germ cell marker VASA/DDX4 (Fig. 1e-h). Individual tubules showing focal spermatogenesis could be detected in selected Klinefelter tissues (Fig. 1b, f). This was in contrast to control samples with qualitatively normal spermatogenesis (Fig. 1a, e), which showed spermatogenesis in the majority of seminiferous tubules and samples with the complete absence of germ cells ...
Context 3
... testicular tissue samples with and without germ cells were identified by histological analysis following PAS staining ( Fig. 1a-d) and immunohistochemical detection of the germ cell marker VASA/DDX4 (Fig. 1e-h). Individual tubules showing focal spermatogenesis could be detected in selected Klinefelter tissues (Fig. 1b, f). This was in contrast to control samples with qualitatively normal spermatogenesis (Fig. 1a, e), which showed spermatogenesis in the majority of seminiferous tubules and samples with the complete absence of germ cells displaying a Sertoli cell-only phenotype (Fig. 1d, ...
Context 4
... identified by histological analysis following PAS staining ( Fig. 1a-d) and immunohistochemical detection of the germ cell marker VASA/DDX4 (Fig. 1e-h). Individual tubules showing focal spermatogenesis could be detected in selected Klinefelter tissues (Fig. 1b, f). This was in contrast to control samples with qualitatively normal spermatogenesis (Fig. 1a, e), which showed spermatogenesis in the majority of seminiferous tubules and samples with the complete absence of germ cells displaying a Sertoli cell-only phenotype (Fig. 1d, ...
Context 5
... focal spermatogenesis could be detected in selected Klinefelter tissues (Fig. 1b, f). This was in contrast to control samples with qualitatively normal spermatogenesis (Fig. 1a, e), which showed spermatogenesis in the majority of seminiferous tubules and samples with the complete absence of germ cells displaying a Sertoli cell-only phenotype (Fig. 1d, ...
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... profiling was performed for selected marker genes using qPCR. Successful separation of germ cells from testicular somatic cells could be demonstrated by significantly higher expression of six germ cell marker genes (Additional file 1: Figure S1; FGFR3, UTF1, RHOXF2/2B, MAGEA4, VASA/DDX4, RHOXF1). ...
Context 7
... from patients with an SCO phenotype yielded only an AT fraction (Additional file 1: Figure S2C). In KS samples with germ cells (KS+; Additional file 1: Figure S1D), both SN and AT fraction (KS+; Additional file 1: Figure S1E) were obtained and the presence of germ cell cluster was microscopically confirmed. In the KS− group, only AT fractions could be recovered (Additional file 1: Figure S2F). ...
Context 8
... from patients with an SCO phenotype yielded only an AT fraction (Additional file 1: Figure S2C). In KS samples with germ cells (KS+; Additional file 1: Figure S1D), both SN and AT fraction (KS+; Additional file 1: Figure S1E) were obtained and the presence of germ cell cluster was microscopically confirmed. In the KS− group, only AT fractions could be recovered (Additional file 1: Figure S2F). ...
Similar publications
Cryptorchidism is an undeniable risk factor for testicular germ cell tumors (TGCTs) and is also commonly associated with Klinefelter syndrome (KS) patients. Embryonal cell carcinoma usually shows strong expression of CD30 and OCT3/4, with patchy staining of PLAP1. Most patients with nonseminomatous GCTs (NSGCTs) can achieve total remission with pro...
Background:
Long-term infertility can be attributed to many factors, with the genetic factor being the most overlooked due to its many nonspecific morphological or endocrine signs. We present a rare case of a patient with progressive testicular failure associated with 48,XXYY syndrome.
Case presentation:
A 39-year-old Arab man presented to our f...
Citations
... Patients with KS undergo a substantial loss of germ cells before puberty 8 and the underlying mechanisms have remained unclear. Owing to the considerable depletion of germ cells in pubertal patients with KS, the challenges in timely diagnosing prepubertal patients with KS and the technological limitations, only limited molecular abnormalities in gonadal somatic cells have been documented [8][9][10][11][12][13][14][15][16] . The fundamental causes and initiating events of infertility in KS, particularly the early developmental defects in germ cells and associated molecular dysregulation, remain unclear. ...
The dosage of X-linked genes is accurately regulated with the development of fetal germ cells (FGCs)1,2. How aberrant dosage of X-linked genes impairs FGC development in humans remains poorly understood. FGCs of patients with Klinefelter syndrome (KS), who have an extra X chromosome, provide natural models for addressing this issue³. Here we demonstrate that most human FGCs in KS are arrested at an early stage, characterized by the upregulation of genes related to pluripotency, the WNT pathway and the TGF-β pathway, along with the downregulation of genes involved in FGC differentiation. The limited KS FGCs that are capable of reaching the late stage remain relatively naive. X chromosomes are not inactivated and the dosage of X-linked genes is excessive in KS FGCs. X-linked genes dominate the differentially expressed genes and are enriched in critical biological processes associated with the developmental delay of KS FGCs. Moreover, aberrant interactions between Sertoli cells and FGCs disrupt the migration of late FGCs to the basement membrane in KS. Notably, inhibition of the TGF-β pathway improves the differentiation of KS FGCs. Our findings elucidate how the extra X chromosome impairs the development of male FGCs and reveal the initial molecular events preceding germ cell loss in KS.
... Data sources and sample metadata are detailed in Supplementary Tables S1 and S2, respectively. To mitigate batch effects, we excluded from consideration samples which did not use an Illumina sequencer (e.g., [39]); sequenced too few cells for use with a common informatic workflow, detailed below [11,40,41] (<100 cells; these samples are typically from studies employing the whole-transcript but low-throughput Fluidigm C1/SmartSeq or SmartSeq2 methods [42]); used FACS-sorted, enriched, or otherwise pre-selected cell populations rather than enzymatically digested testes tissue [24,43] (both because the selection process may activate the cells and because the meaningful integration of datasets would not be possible, these samples having biased cell populations); and those where the donor had sex chromosome aneuploidy (e.g., [44]), testosterone-suppression, non-obstructive azoospermia, or otherwise impaired fertility. ...
... This resolution was empirically chosen on the basis of both a Clustree v0.5.0 [55] dendrogram and manual review (using the interactive browser cellxgene v0.18.0 (https://github.com/chanzuckerberg/cellxgene, accessed on 13 May 2023)), with clusters made at resolutions in the range 0 to 0.5, at intervals of 0.05, interpreted in the context of known testes cell markers (given in Supplementary Table S4 and sourced from [10,16,25,40,41,43,44]) and, to facilitate an unbiased annotation of cluster identities, an allagainst-all differential expression analysis. ...
... Other versions of the SPG atlas for different clustering resolutions are shown in Supplementary Figure S8 and presented in Supplementary Tables S6 and S8. Using an unsupervised clustering approach, we identified 10 distinct cell clusters, which we annotated using both established biomarkers for the major somatic and germ cell types (Figure 1; biomarkers sourced from previous publications [10,16,25,40,41,43,44] and detailed in Supplementary Table S4) and an all-against-all differential gene expression analysis (Supplementary Table S5). Following batch correction (see Section 2), the atlas showed no residual batch effects on the basis of sample accession, study of origin, donor age, or cell cycle phase (Supplementary Figure S1), nor an overtly disproportionate contribution made by any individual sample on the basis of number of genes, mitochondrial genes, or UMIs per cell (Supplementary Figure S2 Table S5). ...
Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling, it has been proposed that undifferentiated human spermatogonia can be subclassified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc) RNA-sequencing libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat, and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate ‘state 0B’ with a suppressive transcriptomic programme that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells—which are mitotically arrested—can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mice (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive lifespan while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes.
... 132 Samples were reprocessed using a common Kallisto/Bustools 39 and Seurat 40 workflow with 133 conservative parameters (see Materials and Methods) to generate an integrated atlas of 60,427 134 testicular cells (Figure 1), each expressing on average 2877 genes/cell (Supplementary Table 3). 135 Using an unsupervised clustering approach, we identified 10 distinct cell clusters, which we 136 annotated using both established biomarkers for the major somatic and germ cell types (Figure 1; 137 biomarkers sourced from previous publications 10,16,25,[41][42][43][44] and detailed in Supplementary Table 4) 138 and an all-against-all differential gene expression analysis (Supplementary Table 5). Following 139 batch correction (see Materials and Methods), the atlas showed no residual batch effects on the 140 basis of sample accession, study of origin, donor age or cell cycle phase (Supplementary Figure 141 1), nor an overtly disproportionate contribution made by any individual sample on the basis of 142 number of genes, mitochondrial genes, or UMIs per cell (Supplementary Figure 2). ...
... typically from studies employing the whole-transcript but low-throughput Fluidigm C1/SmartSeq or 594 SmartSeq2 methods 106 ); used FACS-sorted, enriched, or otherwise pre-selected cell populations 595 rather than enzymatically-digested testes tissue 24,43 (both because the selection process may 596 activate the cells and because the meaningful integration of datasets would not be possible, these 597 samples having biased cell populations); and those where the donor had sex chromosome 598 aneuploidy (e.g. 41 ), testosterone-suppression, non-obstructive azoospermia, or otherwise impaired 599 fertility. 600 ...
... Supplementary Table 4 and sourced from 10,16,25,[41][42][43][44] ) and, to facilitate an unbiased annotation of 633 cluster identities, an all-against-all differential expression analysis. 634 ...
Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling it has been proposed that undifferentiated human spermatogonia can be sub-classified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc)RNA-seq libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate 'state 0B' with a suppressive transcriptomic program that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells - which are mitotically arrested - can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mouse (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive life while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes.
... Since XIST is expressed at high levels in men with KS (reviewed in Winge et al., 2020), the additional X-chromosome is expected to undergo inactivation as well. With the emergence of single-cell RNA sequencing (scRNAseq), three studies including samples from a total of six men with KS (Laurentino et al., 2019;Mahyari et al., 2021;Zhao et al., 2020), all indicated a central role of the Sertoli cells in the testicular pathology of men with KS (Winge et al., 2020), which supports earlier morphological studies from 1969 (Skakkebaek, 1969). In the latter, two distinct types of Sertoli cell-only (SCO) tubules, termed type A and B, were identified, with type A Sertoli cells resembling adult mature Sertoli cells and type B Sertoli cells resembling immature Sertoli cells (Skakkebaek, 1969). ...
Klinefelter syndrome (47,XXY) causes infertility with a testicular histology comprising two types of Sertoli cell-only tubules, representing mature and immature-like Sertoli cells, and occasionally focal spermatogenesis. Here, we show that the immature Sertoli cells highly expressed XIST and have two X-chromosomes, while the mature Sertoli cells lack XIST expression and have only one X-chromosome. Sertoli cells supporting focal spermatogenesis also lack XIST expression and the additional X-chromosome, while the spermatogonia expressed XIST despite having only one X chromosome. XIST was expressed in Sertoli cells until puberty, where a gradual loss was observed. Our results suggest that a micro-mosaic loss of the additional X-chromosome is needed for Sertoli cells to mature and to allow focal spermatogenesis.
... With the rapid advancement of high-resolution genomic techniques, such as single-cell RNA sequencing (scRNAseq), the understanding of the molecular mechanisms associated with the testicular phenotype of NOA has improved (Wang et al., 2018;Laurentino et al., 2019;Zhao et al., 2020;Alfano et al., 2021;Mahyari et al., 2021;Di Persio et al., 2021;Chen et al., 2022). Collectively, they demonstrate an altered cellular composition in the testes, with immature LCs and SCs and enrichment of proinflammatory macrophages in both males with iNOA and KS (Di Persio and Neuhaus, 2023). ...
... Samples were included from six publicly available scRNAseq studies of testicular tissue from adult males (Guo et al., 2018;Laurentino et al., 2019;Zhao et al., 2020;Mahyari et al., 2021;Di Persio et al., 2021;Nie et al., 2022). Combined, these comprised data from 29 males, including six with KS, four with iNOA, one patient with an AZF microdeletion (AZFdel), one patient with OA, 14 controls (CTRL), and three with CO. ...
... To investigate the testicular microvasculature of males with KS at the cellular level, we included samples from six publicly available scRNAseq studies of testicular tissue from adult males (Guo et al., 2018;Laurentino et al., 2019;Zhao et al., 2020;Mahyari et al., 2021;Di Persio et al., 2021;Nie et al., 2022). We divided the samples into four groups based on the reproductive conditions of the individuals: males with KS, males with NOA (iNOA, AZFdel), controls (CTRL, OA), and males with CO ( Fig. 1, upper panel). ...
STUDY QUESTION
Does Klinefelter syndrome (KS) lead to a distinct gene expression pattern at single-cell level in the testes that could provide insight into the reported microvascular dysfunction in the testes?
SUMMARY ANSWER
A distinct gene expression pattern within microvascular-associated cells of males with KS suggests excessive endothelial cell (EC) activation, disorganized vessel formation, and the presence of immature vessels with compromised integrity.
WHAT IS KNOWN ALREADY
Recent studies show that males with KS exhibit microvascular dysfunction in their testes, which affects blood flow and is associated with lower circulating levels of testosterone.
STUDY DESIGN, SIZE, DURATION
A comparative cross-sectional study of males with KS (n = 6), non-obstructive azoospermia (NOA) (n = 5), cryptozoospermia (n = 3), and controls (n = 15) was carried out.
PARTICIPANTS/MATERIALS, SETTING, METHODS
We analyzed publicly available single-cell RNA sequencing data of testicular cells from males with KS, males with NOA, males with cryptozoospermia, and controls. The integration of these datasets allowed us to analyze gene expression profiles and communication patterns among the cell types within the testis and to identify capillary ECs to investigate changes at the microvascular level.
MAIN RESULTS AND THE ROLE OF CHANCE
Rooted in changes at the single-cell level, our study demonstrates a shift in gene expression forming the foundation for altered cellular communication, microvascular remodeling, and pro-inflammatory responses within the testes of males with KS. We identified genes that were dysregulated in capillary ECs from males with KS (Padj < 0.05). Specifically, the unique microvascular gene expression in males with KS indicated enhanced capillary EC activation and increased inflammatory cross-talk, leading to impaired vessel maturation and increased EC barrier permeability.
LIMITATIONS, REASONS FOR CAUTION
Our study is constrained by an unbalanced design, with varying sample sizes and number of cells within each group. We acknowledge the restricted access to clinical information. In addition, our findings were deduced from changes in gene expression, which limits us to infer potential biological consequences arising from these alterations. Furthermore, the absence of a pre-pubertal age group limits the generalizability of our findings and warrants further investigation.
WIDER IMPLICATIONS OF THE FINDINGS
This study offers novel insights into the testicular pathophysiology in KS and underscores the potential contribution of microvascular dysfunction to the hypogonadism and infertility observed in males with KS. While this study aims to better understand the microvascular dysfunction in KS, the precise connections to testosterone deficiency and testicular atrophy remain to be fully elucidated.
STUDY FUNDING/COMPETING INTEREST(S)
A.S. was supported by the Independent Research Fund Denmark (0134-00130B). C.H.G. was supported by Novo Nordisk Foundation (NNF15OC0016474, NNF20OC0060610), ‘Fonden til lægevidenskabens fremme’, the Familien Hede Nielsen foundation and the Independent Research Fund Denmark (0134-00406A). E.B.J. was supported by Aarhus University and E.B.J. and C.H.G by the Independent Research Fund Denmark (2096-00165A). J.M.K. was supported by Lundbeckfonden (R307-2018-3667), Carlsberg Fonden (CF19-0687), Novo Nordisk Fonden (0073440) and Steno Diabetes Center Aarhus (SDCA). The authors declare no conflicts of interest.
TRIAL REGISTRATION NUMBER
N/A.
... Until now, various research regarding other types of infertile groups, especially oligozoospermia, have illustrated that abnormal methylation of imprinted genes might disrupt spermatozoa production and/or lead to male factor infertility [54][55][56][57]. MEST, as an imprinted gene, severally has been reported as hypermethylated in male infertility [26,52,[58][59][60]. ...
Male infertility accounts for nearly 40%–50% of all infertile cases. One of the most prevalent disorders detected in infertile men is errors in the MEST differentially methylated region (DMR), which has been correlated with poor sperm indexes. The aim of our study was to characterize the methylation pattern of the MEST gene, along with assessing seminal factors and chromatin condensation in sperm samples from both infertile patients and fertile cases, all of whom were candidates for intracytoplasmic sperm injection. We collected forty-five semen specimens from men undergoing routine spermiogram analysis at the Infertility Treatment Center. The specimens consisted of 15 samples of normospermia as the control group, 15 individuals of asthenospermia, and 15 individuals of oligoasthenoteratospermia as the cases group. Standard semen analysis and the chromatin quality and sperm maturity tests using aniline blue staining were performed. The DNA from spermatozoa was extracted and treated with a sodium bisulfite–based procedure. The methylation measure was done quantitatively at the DMRs of the MEST gene by quantitative methylation-specific polymerase chain reaction (qMSP). The mean percentages of total motility, progression, and morphology in normospermia were significantly higher than oligoasthenoteratospermia and asthenospermia, and they were substantially higher in asthenospermia compared to oligoasthenoteratospermia (P ≤ 0.05). The mean percentages of histone transition abnormality and MEST methylation in oligoasthenoteratospermia were significantly higher than asthenospermia and normospermia (P ≤ 0.05). A negative correlation existed between the histone transition abnormality and MEST methylation with sperm parameters. In conclusion, chromatin integrity, sperm maturity, and MEST methylation may be considered important predictors for addressing male factor infertility. Therefore, we suggest that male infertility may be linked to methylation of the imprinted genes.
... Differences were found in the degree of DNA methylation in the imprinted region, demonstrating that KS is associated with the degree of methylation of imprinted genes [92]. It was also found that Sertoli cells were the most affected cells in KS patients [93]. Mahyari et al. used 10X Genomics and sparse matrix decomposition to compare multiple cell populations in KS patient samples [94,95]. ...
Spermatogenesis, a key part of the spermiation process, is regulated by a combination of key cells, such as primordial germ cells, spermatogonial stem cells, and somatic cells, such as Sertoli cells. Abnormal spermatogenesis can lead to azoospermia, testicular tumors, and other diseases related to male infertility. The application of single-cell RNA sequencing (scRNA-seq) technology in male reproduction is gradually increasing with its unique insight into deep mining and analysis. The data cover different periods of neonatal, prepubertal, pubertal, and adult stages. Different types of male infertility diseases including obstructive and non-obstructive azoospermia (NOA), Klinefelter Syndrome (KS), Sertoli Cell Only Syndrome (SCOS), and testicular tumors are also covered. We briefly review the principles and application of scRNA-seq and summarize the research results and application directions in spermatogenesis in different periods and pathological states. Moreover, we discuss the challenges of applying this technology in male reproduction and the prospects of combining it with other technologies.
... These include a study on blood from a single 47,XXY male, finding distinct cell subpopulations with gene expression association with various phenotypical traits (57). However, more scRNAseq studies have been performed on testicular tissue (n = 3), identifying dysregulated testicular pathways in KS patients (58)(59)(60). These studies have established that Sertoli cells are the most affected testicular cell type in KS (59,61), with a subset lacking XIST expression, resulting in loss of X chromosome inactivation (58). ...
Sex chromosome abnormalities (SCAs) are chromosomal disorders with either a complete or partial loss or gain of sex chromosomes. The most frequent SCAs include Turner syndrome (TS; 45,X), Klinefelter syndrome (KS; 47,XXY), Trisomy X syndrome (47,XXX) and Double Y syndrome (47,XYY). The phenotype seen in SCAs is highly variable and may not merely be due to the direct genomic imbalance from altered sex chromosome gene dosage, but also to additive alterations in gene networks and regulatory pathways across the genome as well as individual genetic modifiers. This review summarizes the current insight into the genomics of SCAs. In addition, future directions of research that can contribute to decipher the genomics of SCA are discussed such as single-cell omics, spatial transcriptomics, system biology thinking, human induced pluripotent stem cells and animal models, and how these data may be combined to bridge the gap between genomics and the clinical phenotype.
... We evaluated the expression of the genes of interest in previously published, well-characterized, and publicly available single-cell RNA (scRNA) datasets, namely one dataset by Di Persio et al., 26 (GEO: GSE153947 for adult human testes with CS, n = 3), two datasets by Hermann et al., 27 (GEO: GSE109037 for adult murine testes, n = 3; GEO: GSE109033 for adult human testes with CS, n = 3) and a fourth dataset by Laurentino et al., 28 (GEO: GSE130151 for an adult human testis with Klinefelter syndrome and residual spermatogenesis, n = 1). Details of the individual data analysis can be found in the original publications. ...
... The expression of the genes of interest was visualized from the already processed data. For the Di Persio 26 and Laurentino 28 datasets, expression data were plotted onto the Uniform manifold approximation and projections (UMAP) and visualized using the FeaturePlot function on the R (v3.6.0) package Seurat (v3.0.2). ...
... To further specify WWC expression in residual somatic cells, a published scRNA dataset of testicular tissue of a patient with Klinefelter syndrome was analyzed. 28 While the testicular WWC1 mRNA level was very low in this sample, expression of WWC2 and WWC3 could be detected in clusters identified mainly in peritubular myoid cells and Sertoli cells ( Figure 3E-H). ...
The family of WWC proteins is known to regulate cell proliferation and organ growth control via the Hippo signaling pathway. As WWC proteins share a similar domain structure and a common set of interacting proteins, they are supposed to fulfill compensatory functions in cells and tissues. While all three WWC family members WWC1, WWC2, and WWC3 are found co‐expressed in most human organs including lung, brain, kidney, and liver, in the testis only WWC2 displays a relatively high expression. In this study, we investigated the testicular WWC2 expression in spermatogenesis and male fertility. We show that the Wwc2 mRNA expression level in mouse testes is increased during development in parallel with germ cell proliferation and differentiation. The cellular expression of each individual WWC family member was evaluated in published single‐cell mRNA datasets of murine and human testes demonstrating a high WWC2 expression predominantly in early spermatocytes. In line with this, immunohistochemistry revealed cytosolic WWC2 protein expression in primary spermatocytes from human testes displaying full spermatogenesis. In accordance with these findings, markedly lower WWC2 expression levels were detected in testicular tissues from mice and men lacking germ cells. Finally, analysis of whole‐exome sequencing data of male patients affected by infertility and unexplained severe spermatogenic failure revealed several heterozygous, rare WWC2 gene variants with a proposed damaging function and putative impact on WWC2 protein structure. Taken together, our findings provide novel insights into the testicular expression of WWC2 and show its cell‐specific expression in spermatocytes. As rare WWC2 variants were identified in the background of disturbed spermatogenesis, WWC2 may be a novel candidate gene for male infertility.
... Klinefelter syndrome (KS) makes up the most common part of male chromosome disorders [120], which is also widely studied by scRNA-seq studies [55,78,121]. Importantly, usually the studies on KS patients were considered as a part of works on NOA patients because of the popular azoospermic phenotype [122]. ...
... Nevertheless, due to the special karyotype of KS (one or more extra X [123]), the changes and relative mechanisms in KS testes were distinguishable from the above-mentioned iNOA patients [124]. Interestingly, for scRNA-seq data of KS testicular samples with the presence of germ cells, there seemed to be no huge changes at the transcriptional level of KS germ cells compared with normal germ cells [121]. But such a conclusion was drawn indirectly based on the co-clustering of KS germ cells with normal germ cells, rather than differential expression analysis (because there was only one KS sample sequenced with only 39 germ cells identified in Laurentino's study [121]). ...
... Interestingly, for scRNA-seq data of KS testicular samples with the presence of germ cells, there seemed to be no huge changes at the transcriptional level of KS germ cells compared with normal germ cells [121]. But such a conclusion was drawn indirectly based on the co-clustering of KS germ cells with normal germ cells, rather than differential expression analysis (because there was only one KS sample sequenced with only 39 germ cells identified in Laurentino's study [121]). Unlike the other type of male infertility (in which the scientist could deeply analyze germ cell changes), Laurentino's study was almost the "best" work of KS germ cells because most of the scRNA-seq samples on KS from other teams lacked spermatogenic cells and showed a SCOS or even tubular atrophy pattern [55,78]. ...
So far there has been no comprehensive review using systematic literature search strategies to show the application of single-cell RNA sequencing (scRNA-seq) in the human testis of the whole life cycle (from embryos to aging males). Here, we summarized the application of scRNA-seq analyses on various human testicular biological samples. A systematic search was conducted in PubMed and Gene Expression Omnibus (GEO), focusing on English researches published after 2009. Articles related to GEO data-series were also retrieved in PubMed or BioRxiv. 81 full-length studies were finally included in the review. ScRNA-seq has been widely used on different human testicular samples with various library strategies, and new cell subtypes such as State 0 spermatogonial stem cells (SSC) and stage_a/b/c Sertoli cells (SC) were identified. For the development of normal testes, scRNA-seq-based evidence showed dynamic transcriptional changes of both germ cells and somatic cells from embryos to adults. And dysregulated metabolic signaling or hedgehog signaling were revealed by scRNA-seq in aged SC or Leydig cells (LC), respectively. For infertile males, scRNA-seq studies revealed profound changes of testes, such as the increased proportion of immature SC/LC of Klinefelter syndrome, the somatic immaturity and altered germline autophagy of patients with non-obstructive azoospermia, and the repressed differentiation of SSC in trans-females receiving testosterone inhibition therapy. Besides, the re-analyzing of public scRNA-seq data made further discoveries such as the potential vulnerability of testicular SARS-CoV-2 infection, and both evolutionary conservatism and divergence among species. ScRNA-seq analyses would unveil mechanisms of testes' development and changes so as to help developing novel treatments for male infertility.
