Single-cell RNA-seq unravels alterations of the human spermatogonial stem cell compartment in patients with impaired spermatogenesis

Abstract

Summary Despite the high incidence of male infertility, only 30% of infertile men receive a causative diagnosis. To explore the regulatory mechanisms governing human germ cell function in normal and impaired spermatogenesis (crypto), we performed single-cell RNA sequencing (>30,000 cells). We find major alterations in the crypto spermatogonial compartment with increased numbers of the most undifferentiated spermatogonia (PIWIL4 + ). We also observe a transcriptional switch within the spermatogonial compartment driven by increased and prolonged expression of the transcription factor EGR4. Intriguingly, the EGR4-regulated chromatin-associated transcriptional repressor UTF1 is downregulated at transcriptional and protein levels. This is associated with changes in spermatogonial chromatin structure and fewer A dark spermatogonia, characterized by tightly compacted chromatin and serving as reserve stem cells. These findings suggest that crypto patients are disadvantaged, as fewer cells safeguard their germline’s genetic integrity. These identified spermatogonial regulators will be highly interesting targets to uncover genetic causes of male infertility.

Stage-specific gene and transcript dynamics in human male germ cells

“…To identify novel germ cell-specific marker genes, we focused on the top 100 DEGs per group comparison with elevated expression in SPG, SPC, SPD, and normal samples. After evaluating the expression of all genes for their germ cell-specificity in our published scRNA-seq dataset of 3 patients with normal spermatogenesis (Di Persio et al ., 2021) (Fig. 3A), we show 3 genes per group comparison as examples.…”

“…To evaluate gene expression of selected genes of interest at single-cell level, we generated uniform manifold approximation and projection (UMAP) plots (McInnes et al ., 2020) based on our previously published dataset (Di Persio et al ., 2021) using the tool Seurat (Stuart et al ., 2019; Hao et al ., 2021).…”

“…For example, comparing testicular tissues with SCO and spermatogonial arrest phenotypes, which only differ in the presence of spermatogonia, von Kopylow et al, (2010) were able to identify transcripts specifically expressed by spermatogonia. The authors identified the spermatogonial markers FGFR3 and UTF1, which are currently considered specific markers for different spermatogonial subpopulations (Guo et al, 2018;Sohni et al, 2019;Di Persio et al, 2021). Chalmel et al (2012) expanded on this approach by including samples from different developmental stages and arrest phenotypes, thereby extracting the transcriptional profiles of additional germ cell types.…”

The conserved genetic program of male germ cells uncovers ancient regulators of human spermatogenesis

“…An average of 44 million reads were generated per sample. The expression pattern of RNF113B was determined in a control testis by retrieving the corresponding data from our recently published single-cell RNA- Seq dataset of normal human spermatogenesis 31 .…”

“…Early (round) spermatids were practically absent in both species, with cellular debris accumulating in the post-meiotic region of the fruit fly gonad. By analyzing our previously published single-cell RNA-Seq dataset of normal human spermatogenesis 37 , we observed that RNF113B was predominantly expressed at meiotic entry, peaking at the diplotene stage of prophase I ( Fig. 3d ).…”