Figure - available via license: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Content may be subject to copyright.
Source publication
Despite the high incidence of male infertility, about 70% of infertile men do not receive a causative diagnosis. To gain insights into the regulatory mechanisms governing human germ cell function in normal and impaired spermatogenesis (cryptozoospermic patients, crypto), we combined single cell RNA sequencing (>30.000 cells), proteome, and histomor...
Similar publications
Spermatogenesis is a complex and dynamic processwhich is precisely controlledby genetic and epigenetic factors. With the development of new technologies (e.g., single-cell RNA sequencing), increasingly more regulatory genes related to spermatogenesis have been identified. In this review, we address the roles and mechanisms of novel genes in regulat...
Citations
... Single-cell sequencing studies have identified various 'states' of germ cells, with progression of development of differentiation from State 0 to State 4, where each state is characterized by a unique set of markers, although at times partially overlapping (6,81). UTF1 and PIWIL4 are examples of markers expressed in the earliest state, with PIWIL4 being the more specific marker (6,82,83). Another newly identified candidate marker for undifferentiated spermatogonia, carrying SSC characteristics, involves the LPPR3 protein (6). ...
Fertility preservation via biobanking of testicular tissue retrieved from testicular biopsies is now generally recommended for boys who need to undergo gonadotoxic treatment prior to the onset of puberty, as a source of spermatogonial stem cells (SSCs). SSCs have the potential of forming spermatids and may be used for therapeutic fertility approaches later in life. Although in the past 30 years many milestones have been reached to work towards SSC-based fertility restoration therapies, including transplantation of SSCs, grafting of testicular tissue and various in vitro and ex vivo spermatogenesis approaches, unfortunately, all these fertility therapies are still in a preclinical phase and not yet available for patients who have become infertile because of their treatment during childhood. Therefore, it is now time to take the preclinical research towards SSC-based therapy to the next level to resolve major issues that impede clinical implementation. This review gives an outline of the state of the art of the effectiveness and safety of fertility preservation and SSC-based therapies and addresses the hurdles that need to be taken for optimal progression towards actual clinical implementation of safe and effective SSC-based fertility treatments in the near future.
... Further to this, EGR4 expression has also been isolated in human testes, where it is localised within stem cells that survive across the lifespan to produce spermatozoa [42]. Di Persio et al. [43] also demonstrated that EGR4 is upregulated in the most undifferentiated spermatogonial cells, and that it is an upstream regulator of several transcription factors involved in cell proliferation and differentiation. Our research shows that EGR4-S was upregulated with the oncogenic transformation of mammary cells in vitro and was detected in cancer tissue but not in patient-matched normal tissue, suggesting that the expression of this splice variant is a characteristic of the cancer cell. ...
The zinc finger transcription factor EGR4 has previously been identified as having a critical role in the proliferation of small cell lung cancer. Here, we have identified a novel, shortened splice variant of this transcription factor (EGR4-S) that is regulated by Heat Shock Factor-1 (HSF1). Our findings demonstrate that the shortened variant (EGR4-S) is upregulated with high EGFR, HER2, and H-Rasv12-expressing breast cell lines, and its expression is inhibited in response to HER pathway inhibitors. Protein and mRNA analyses of HER2+ human breast tumours indicated the novel EGR4-S splice variant to be preferentially expressed in tumour tissue and not detectable in patient-matched normal tissue. Knockdown of EGR4-S in the HER2-amplified breast cancer cell line SKBR3 reduced cell growth, suggesting that EGR4-S supports the growth of HER2+ tumour cells. In addition to chemical inhibitors of the HER2 pathway, EGR4-S expression was also found to be suppressed by chemical stressors and the overexpression of HSF1. Under these conditions, reduced EGR4-S levels were associated with the observed lower cell growth rate, but the augmentation of properties associated with higher metastatic potential. Taken together, these findings identify EGR4-S as a potential biomarker for HER2 pathway activation in human tumours that is regulated by HSF1.
... Publications of high-quality single-cell RNA sequencing (scRNA-seq) transcriptomes obtained from human testicular tissues with intact spermatogenesis [22][23][24][25] have greatly advanced our knowledge with regard to the transcriptional changes associated with human germ cell differentiation. Moreover, comparative analyses of scRNA-seq results from men with intact and severely impaired spermatogenesis have provided insight into the molecular mechanisms associated with failure of germ cell differentiation [25,26]. In order to assess the presence of aberrant epigenetic patterns in TGCs and the potential association with aberrant transcriptional profiles, we combined the two powerful approaches of WGBS data with scRNA-seq in samples with intact and impaired spermatogenesis. ...
... To ascertain the relevance of the DMR-associated genes, we made use of the available scRNA-seq data obtained by our group from three patients with normal spermatogenesis (CTR) and three patients with cryptozoospermia (CZ) [26]. We subset the germ cells and obtained 14,098 and 5,939 cells from the CTR and CZ groups, respectively (Fig. 3a). ...
... To assess whether DNA methylation changes are associated with changes in gene expression levels, we used our recently published differential gene expression analysis of CTR and CZ testicular samples [26]. For this, germ cells were divided into three knot groups (Additional file 2: Fig. S6A) and tradeSeq was used to find trajectory-based differential expression [34]. ...
Background
Several studies have reported an association between male infertility and aberrant sperm DNA methylation patterns, in particular in imprinted genes. In a recent investigation based on whole methylome and deep bisulfite sequencing, we have not found any evidence for such an association, but have demonstrated that somatic DNA contamination and genetic variation confound methylation studies in sperm of severely oligozoospermic men. To find out whether testicular germ cells (TGCs) of such patients might carry aberrant DNA methylation, we compared the TGC methylomes of four men with cryptozoospermia (CZ) and four men with obstructive azoospermia, who had normal spermatogenesis and served as controls (CTR).
Results
There was no difference in DNA methylation at the whole genome level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 differentially methylated regions (DMRs), 238 of which were hypermethylated in CZ (binominal test, p < 2.2 × 10 –16 ). The DMRs were enriched for distal regulatory elements ( p = 1.0 × 10 –6 ) and associated with 132 genes, 61 of which are differentially expressed at various stages of spermatogenesis. Almost all of the 67 DMRs associated with the 61 genes (94%) are hypermethylated in CZ (63/67, p = 1.107 × 10 –14 ). As judged by single-cell RNA sequencing, 13 DMR-associated genes, which are mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter is hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, eight of which downregulated in CZ, germ cell-specific enhancers may be affected.
Conclusions
We found that impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome. We hypothesize that the described DNA methylation changes may reflect or contribute to premature abortion of spermatogenesis and therefore not appear in the mature, motile sperm.
