Biology subjects

Neuhaus, N.

Publications and source records attributed to Neuhaus, N..

10 recordsLinked to original sources

bioRxiv2026

A single-nucleus multiomic and spatial atlas of gene regulation in human spermatogenesis

Gametogenesis, the production of oocytes and sperm, ensures the faithful transmission of genetic material to the next generation. In males, spermatogenesis occurs within the testis, where germ cells progress through a highly ordered developmental programme in close association with supporting somatic cells. Defects in this process cause infertility, yet the gene regulatory mechanisms coordinating normal and dysfunctional human spermatogenesis remain incompletely defined. Here we generate a single-nucleus multiomic and spatial atlas of human spermatogenesis by profiling chromatin accessibility and gene expression in the same nuclei and integrating these data with spatial transcriptomics of intact testicular tissue. We infer high-confidence gene regulatory networks that resolve stage-specific activity of known and novel candidate regulators across germline and somatic compartments, and map spatially restricted signalling interactions within the seminiferous tubule niche. Integration with infertility-associated genetic variation links non-coding risk loci to candidate enhancers and target genes in cell-type-specific regulatory contexts. Finally, profiling clinical cryptozoospermia samples as in vivo perturbations supports the ability of this network to capture downstream transcriptional consequences of disease-associated regulatory disruption. Together, these data provide a spatially resolved regulatory framework for human spermatogenesis and a foundation for interpreting the molecular basis of male infertility.

genomics↗

bioRxiv2026

Spermatogonial stem cells and their differentiation roadmap throughout marmoset development

Sperm transfer genetic information from one generation to the next. These cells originate from gonocytes, which are specified during early embryonal development. To date however, there is limited information on the transcriptional gatekeepers governing the key events leading to sperm production: transition of gonocytes out of pluripotency around the time of birth, formation of the spermatogonial compartment prior to puberty, and initiation of germ cell differentiation at the time of puberty. We address this knowledge gap by employing the marmoset monkey (Callithrix jacchus) as a model for human postnatal testicular development. We analysed the transcriptional profiles of [~]48,000 neonatal, pre-pubertal, pubertal, and adult testicular cells and correlated these with histomorphometric measurements. We uncovered a transcriptional state linking gonocytes and spermatogonia characterized by CITED2 expression. Moreover, we propose NANOS2 and DPPA4 as regulators of spermatogonial plasticity from pre-puberty onwards and identify molecular gatekeepers of male germ cell differentiation.

developmental biology↗

bioRxiv2026

Chromatoid body integrates piRNA, SMG6 and m⁶A pathways to control mRNAs in the male germline

Spermatogenesis requires tightly controlled transcriptome regulation, supported by the PIWI-interacting RNA (piRNA) and nonsense-mediated decay (NMD) pathways, both concentrated in the chromatoid body (CB) of haploid spermatids. We previously showed that the NMD endonuclease SMG6 interacts with the piRNA-binding protein PIWIL1, and that loss of either factor results in overlapping mRNA dysregulation, suggesting functional cooperation. Here, we demonstrate that SMG6 and PIWIL1 assemble with shared RNA-regulatory proteins and bind common mRNA targets in the testis. Functional experiments in GC-2spd cells revealed cooperative regulation of selected transcripts, including Ccny and Taf1d, and established that SMG6 is required for piRNA-guided degradation of these targets, implicating its endonuclease activity in the piRNA pathway. Target mRNAs were mA-methylated, and this modification shaped their expression levels, SMG6 binding, and CB localization. Moreover, intact target mRNAs accumulated in CBs isolated from Smg6-cKO testes, indicating defective CB-associated decay. Together, these findings uncover a key role for the CB in mRNA regulation through coordinated action of the NMD and piRNA pathways.

cell biology↗

bioRxiv2024

Insights into gene conversion and crossing-over processes from long-read sequencing of human, chimpanzee and gorilla testes and sperm

Homologous recombination rearranges genetic information during meiosis to generate new combinations of variants. Recombination also causes new mutations, affects the GC content of the genome and reduces selective interference. Here, we use HiFi long-read sequencing to directly detect crossover and gene conversion events from switches between the two haplotypes along single HiFi-reads from testis tissue of humans, chimpanzees and gorillas as well as human sperm samples. Furthermore, based on DNA methylation calls, we classify the cellular origin of reads to either somatic or germline cells in the testis tissue. We identify 1692 crossovers and 1032 gene conversions in nine samples and investigate their chromosomal distribution. Crossovers are more telomeric and correlate better with recombination maps than gene conversions. We show a strong concordance between a human double-strand break map and the human samples, but not for the other species, supporting different PRDM9-programmed double-strand break loci. We estimate the average gene conversion tract lengths to be similar and very short in all three species (means 40-100 bp, fitted well by a geometric distribution) and that 95-98% of non-crossover events do not involve tracts intersecting with polymorphism and are therefore not detectable. Finally, we detect a GC bias in the gene conversion of both single and multiple SNVs and show that the GC-biased gene conversion affects SNVs flanking crossover events. This implies that gene conversion events associated with crossover events are much longer (estimated above 500 bp) than those associated with non-crossover events. Highly accurate long-read sequencing combined with the classification of reads to specific cell types provides a new, powerful way to make individual, detailed maps of gene conversion and crossovers for any species.

evolutionary biology↗

bioRxiv2023

Genome-wide DNA methylation changes in human spermatogenesis

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global-decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatid-specific methylome. Hypomethylated regions in spermatids were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.

genetics↗

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

TRIM71 deficiency causes germ cell loss during mouse embryogenesis and promotes human male infertility

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling the complex molecular network orchestrating germ cell development. TRIM71 is a stem cell-specific factor essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype, which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of developing germ cells in the testes seminiferous tubules. Infertility originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, in vitro growth competition assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells, a human GCT-derived cell line which we used as a surrogate model for proliferating PGCs, showed that TRIM71 promotes NCCIT cell proliferation and survival. Our data collectively suggest that germ cell loss in cKO mice results from combined defects during the specification and maintenance of PGCs prior to their sex determination in the genital ridges. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with SCO phenotype. Our work reveals for the first time an association of TRIM71 variants with human male infertility, and uncovers further developmental roles for TRIM71 in the generation and maintenance of germ cells during mouse embryogenesis.

developmental 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

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↗