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.
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 histomorphometric analyses of testicular tissues. We found major alterations in the crypto spermatogonial compartment with increased numbers of the most undifferentiated spermatogonia (PIWIL4+ State 0 cells). We also observed a transcriptional switch within the spermatogonial compartment driven by the increased and prolonged expression of the transcription factor EGR4. Intriguingly, EGR4-regulated genes included the chromatin-associated transcriptional repressor UTF1, which was downregulated. Histomorphometrical analyses showed that these transcriptional changes were mirrored at the protein level and accompanied by a change in the chromatin structure of spermatogonia. This resulted in a reduction of Adark spermatogonia - characterized by tightly compacted chromatin and serving as reserve stem cells. These findings suggest that crypto patients are at a disadvantage especially in cases of gonadotoxic damage as they have less cells safeguarding the genetic integrity of the germline. We hypothesize that the more relaxed chromatin status of spermatogonia is dependent on decreased UTF1 expression caused by EGR4 activation. These identified regulators of the spermatogonial compartment will be highly interesting targets to uncover genetic causes of male infertility.
The process of spermatogenesis—when germ cells differentiate into sperm—is tightly regulated, and misregulation in gene expression is likely to be involved in the physiopathology of male infertility. The testis is one of the most transcriptionally rich tissues; nevertheless, the specific gene expression changes occurring during spermatogenesis are not fully understood. To better understand gene expression during spermatogenesis, we generated germ cell–specific whole transcriptome profiles by systematically comparing testicular transcriptomes from tissues in which spermatogenesis is arrested at successive steps of germ cell differentiation. In these comparisons, we found thousands of differentially expressed genes between successive germ cell types of infertility patients. We demonstrate our analyses’ potential to identify novel highly germ cell–specific markers (TSPY4 and LUZP4 for spermatogonia; HMGB4 for round spermatids) and identified putatively misregulated genes in male infertility (RWDD2A,CCDC183,CNNM1,SERF1B). Apart from these, we found thousands of genes showing germ cell–specific isoforms (includingSOX15,SPATA4,SYCP3,MKI67). Our approach and dataset can help elucidate genetic and transcriptional causes for male infertility.
Cell differentiation processes are highly dependent on cell stage-specific gene expression, including timely production of alternatively spliced transcripts. One of the most transcriptionally rich tissues is the testis, where the process of spermatogenesis, or generation of male gametes, takes place. To date, germ cell-specific transcriptome dynamics remain understudied due to limited transcript information emerging from short-read sequencing technologies. To fully characterize the transcriptional profiles of human male germ cells and to understand how the human spermatogenic transcriptome is regulated, we compared whole transcriptomes of men with different types of germ cells missing from their testis. Specifically, we compared the transcriptomes of testis lacking germ cells (Sertoli cell-only phenotype; SCO; n=3), with an arrest at the stage of spermatogonia (SPG; n=4), spermatocytes (SPC; n=3), and round spermatids (SPD; n=3), with the transcriptomes of testis with normal and complete spermatogenesis (Normal; n=3). We found between 839 and 4,138 differentially expressed genes (DEGs, log2 fold change ≥ 1) per group comparison, with the most prevalent changes observed between SPG and SPC arrest samples, corresponding to the entry into meiosis. We detected highly germ cell-type specific marker genes among the topmost DEGs of each group comparison. Moreover, applying state-of-the-art bioinformatic analysis we were able to evaluate differential transcript usage (DTU) during human spermatogenesis and observed between 1,062 and 2,153 genes with alternatively spliced transcripts per group comparison. Intriguingly, DEGs and DTU genes showed minimal overlap (< 8%), suggesting that stage-specific splicing is an additional layer of gene regulation in the germline. By generating the most complete human testicular germ cell transcriptome to date, we unravel extensive dynamics in gene expression and alternative splicing during human spermatogenesis.
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