Biology subjects

Dugas, M.

Publications and source records attributed to Dugas, M..

5 recordsLinked to original sources

bioRxiv2022

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

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.

molecular biology↗

bioRxiv2021

EGR4-dependent decrease of UTF1 is associated with failure to reserve spermatogonial stem cells in infertile men

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. One Sentence SummaryReserve spermatogonial stem cell depletion in infertile men is regulated by an EGR4-dependent UTF1 decrease, which changes chromatin morphology.

molecular biology↗

bioRxiv2020

Benchmarking atlas-level data integration in single-cell genomics

Cell atlases often include samples that span locations, labs, and conditions, leading to complex, nested batch effects in data. Thus, joint analysis of atlas datasets requires reliable data integration. Choosing a data integration method is a challenge due to the difficulty of defining integration success. Here, we benchmark 38 method and preprocessing combinations on 77 batches of gene expression, chromatin accessibility, and simulation data from 23 publications, altogether representing >1.2 million cells distributed in nine atlas-level integration tasks. Our integration tasks span several common sources of variation such as individuals, species, and experimental labs. We evaluate methods according to scalability, usability, and their ability to remove batch effects while retaining biological variation. Using 14 evaluation metrics, we find that highly variable gene selection improves the performance of data integration methods, whereas scaling pushes methods to prioritize batch removal over conservation of biological variation. Overall, BBKNN, Scanorama, and scVI perform well, particularly on complex integration tasks; Seurat v3 performs well on simpler tasks with distinct biological signals; and methods that prioritize batch removal perform best for ATAC-seq data integration. Our freely available reproducible python module can be used to identify optimal data integration methods for new data, benchmark new methods, and improve method development.

bioinformatics↗

bioRxiv2020

The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis

BackgroundIn the past 15 years, numerous studies have described aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in sperm of infertile patients, but the prevalence and genomic extent of abnormal methylation patterns have remained unknown. ResultsUsing deep bisulfite sequencing (DBS), we screened swim-up sperm samples from 40 normozoospermic and 93 oligoasthenoteratozoospermic (OAT) patients for H19 and MEST methylation. Based on this screening, we defined three patient groups: normal controls (NC), abnormally methylated infertile (AMI; n=7) and normally methylated infertile (NMI; n=86). Whole genome bisulfite sequencing (WGBS) of five NC and five AMI samples revealed abnormal methylation levels of all 50 imprinting control regions in each AMI sample. To investigate whether this finding reflected epigenetic germ line mosaicism or the presence of residual somatic DNA, we made a genome-wide inventory of soma-germ cell specific DNA methylation. We found that >2,000 germ cell-specific genes are promoter-methylated in blood and that AMI samples had abnormal methylation levels at these genes, consistent with the presence of somatic cell DNA. The comparison between the five NC and six NMI samples revealed 19 differentially methylated regions (DMRs), none of which could be validated in an independent cohort of 40 men. Previous studies reported a higher incidence of epimutations at single CpG sites in the CTCF-binding region 6 of H19 in infertile patients. DBS analysis of this locus, however, revealed an association between DNA methylation levels and genotype (rs2071094), but not fertility phenotype. ConclusionsOur results suggest that somatic DNA contamination and genetic variation confound methylation studies in sperm of infertile men. While we cannot exclude the existence of rare patients with slightly abnormal sperm methylation at non-recurrent CpG sites, the prevalence of aberrant methylation in swim-up purified sperm of infertile men has likely been overestimated, which is reassuring for patients undergoing assisted reproduction.

genetics↗

bioRxiv2019

Biallelic mutations in M1AP are a frequent cause of meiotic arrest leading to male infertility

Male infertility affects [~]7% of men in Western societies, but its causes remain poorly understood. The most clinically severe form of male infertility is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis, but so far only few genes have been reported to cause germ cell arrest in males. To address this gap, whole exome sequencing was performed in 60 German men with complete meiotic arrest, and we identified in three unrelated men the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 arresting protein. Then, with collaborators from the International Male Infertility Genomics Consortium (IMIGC), we screened a Dutch cohort comprising 99 infertile men and detected the same homozygous variant c.676dup in a man with hypospermatogenesis predominantly displaying meiotic arrest. We also identified two Portuguese men with NOA carrying likely biallelic loss-of-function (LoF) and missense variants in M1AP among men screened by the Genetics of Male Infertility Initiative (GEMINI). Moreover, we discovered a homozygous missense variant p.(Pro389Leu) in M1AP in a consanguineous Turkish family comprising five infertile men. M1AP is predominantly expressed in human and mouse spermatogonia up to secondary spermatocytes and previous studies have shown that knockout male mice are infertile due to meiotic arrest. Collectively, these findings demonstrate that both LoF and missense M1AP variants that impair its protein cause autosomal-recessive meiotic arrest, non-obstructive azoospermia and male infertility. In view of the evidence from several independent groups and populations, M1AP should be included in the growing list of validated NOA genes.

genetics↗