Biology subjects

Almstrup, K.

Publications and source records attributed to Almstrup, K..

8 recordsLinked to original sources

bioRxiv2026

Mapping the human sperm proteome across compartments, donors and individual cells

The human spermatozoon is among the most specialized cells in the body, yet where its proteins reside, where they come from and how they vary between and within men remain largely unresolved. Here we present the deepest proteomic atlas of human spermatozoa to date, identifying 10,828 proteins across six donors. Abundances correlate well between individuals and are anchored by a conserved core of mitochondrial energy-metabolism proteins, while a smaller fraction varies between donations from the same man. By mechanically separating heads from tails, we assigned thousands of previously unlocalized proteins to a compartment, and by comparing seminal fluid from vasectomized and non-vasectomized men we distinguished sperm- and epididymis-derived proteins from accessory-gland secretions. Using a single-cell analysis workflow we further show that the proteomes of individual spermatozoa can be measured, recovering up to ~1,000 protein groups per cell. In a cohort with total fertilization failure, we find recurrent mitochondrial rather than sperm-egg fusion dysregulation. Together or data provides novel insights into the dynamics of the sperm proteome and male infertility.

cell biology↗

bioRxiv2024

Accurate calling of low-frequency somatic mutations by sample-specific modeling of error rates

Calling rare somatic variants from NGS data is more challenging than calling inherited variants, especially if the somatic variant is only present in a small fraction of the cells in the sequenced biopsy. In this case, having a good estimate of the error rate of a specific base in a particular read becomes essential. In paired-end sequencing, where some DNA fragments are shorter than twice the read length, the overlapping regions of the read pairs are an ideal resource for training models to discern context-dependent base error rates, as any discordant bases in the overlaps must be caused by a sequencing error or an alignment error. We have created a new tool named BBQ (an acronym for Better Base Quality) that uses overlapping reads to estimate the error rate conditional on the mutation type, sequence context, and base quality. We also estimate how much the error rate of concordant bases in overlapping reads is decreased compared to bases in non-overlapping reads. Results show that overlapping reads can remove sequencing errors induced by DNA damage and that the increased quality of overlapping reads differs between samples and mutation types, reflecting different damage patterns between samples. We use the error models to call rare somatic variants. Sequencing data from a testis biopsy and a cell-free DNA sample serve as a proof-of-concept for rare germ cell mutation calling and for detecting rare cancer mutations. We find that using the sample-specific error models of BBQ allows us to call rare somatic variants with fewer false positives than existing tools such as Mutect2 and Strelka2.

bioinformatics↗

bioRxiv2024

Within-ejaculate haploid selection reduces disease biomarkers in human sperm

The germline is widely regarded as a checkpoint against the inheritance of damaged genomes, yet the mechanisms that could enact such filtering remain poorly resolved. Of the millions of sperm in a human ejaculate, only one fertilises the egg, creating a strong opportunity for selection among gametes. Combining within-ejaculate selection on sperm quality with whole-genome sequencing and proteomics in healthy donors, we find that this selection is biased against molecular signatures of age-related disease. The most reproducibly diverging genes are tumour suppressors, and genes diverging under sperm longevity-based selection are enriched for senescence-associated genes involved in genome maintenance and oxidative stress response. High-quality sperm are further depleted of inflammation- and cancer-associated proteins. This genomic signature is conserved in zebrafish, in which longer-lived sperm sire longer-lived offspring. We propose that within-ejaculate selection acts as a pre-fertilisation filter against age-related disease alleles, with implications for offspring lifespan and healthspan.

molecular biology↗

bioRxiv2024

Estimating gene conversion tract length and rate from PacBio HiFi data

Gene conversions are broadly defined as the transfer of genetic material from a donor to an acceptor sequence and can happen both in meiosis and mitosis. They are a subset of non-crossover events and, like crossover events, gene conversion can generate new combinations of alleles and counteract mutation load by reverting germline mutations through GC-biased gene conversion. Estimating gene conversion rate and the distribution of gene conversion tract lengths remains challenging. We present a new method for estimating tract length, rate and detection probability of non-crossover events directly in HiFi PacBio long read data. The method can be used to make inference from sequencing of gametes from a single individual. The method is unbiased even under low single nucleotide variant (SNV) densities and does not necessitate any demographic or evolutionary assumptions. We test the accuracy and robustness of our method using simulated datasets where we vary length of tracts, number of tracts, the genomic SNV density and levels of correlation between SNV density and NCO event position. Our simulations show that under low SNV densities, like those found in humans, only a minute fraction ([~]2%) of NCO events are expected to become visible as gene conversions by moving at least one SNV. We finally illustrate our method by applying it to PacBio sequencing data from human sperm.

genomics↗

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↗

bioRxiv2024

X-chromosome loss rescues Sertoli cell maturation and spermatogenesis in Klinefelter syndrome

Klinefelter syndrome (47,XXY) causes infertility with a testicular histology comprising two types of Sertoli cell-only tubules, representing mature and immature-like Sertoli cells, and occasionally focal spermatogenesis. Here, we show that the immature Sertoli cells highly expressed XIST and have two X-chromosomes, while the mature Sertoli cells lack XIST expression and have only one X-chromosome. Sertoli cells supporting focal spermatogenesis also lack XIST expression and the additional X-chromosome, while the spermatogonia expressed XIST despite having only one X-chromosome. XIST was expressed in Sertoli cells until puberty, where a gradual loss was observed. Our results suggest that a micro-mosaic loss of the additional X-chromosome is needed for Sertoli cells to mature and to allow focal spermatogenesis.

genetics↗

bioRxiv2023

A Practical Approach to Estimating the Effect of Synchrotron Radiation on Sperm Motility and Viability

Studying the dynamics of sperm tail beating in 3D is important for understanding decreasing fertility trends related to motility. Syn-chrotron X-ray tomography (SXRT) shows promising results for imaging live biological samples in 3D and time, making it a candidate for imaging sperm tail beating patterns. However, the dose of ionizing radiation (IR) that the cells would receive during image acquisition is of concern as this is likely to affect essential cellular functions. The effect of IR on motility at the dose rates encountered in a synchrotron has never been assessed. Here, results are presented of analyzing the movement and viability of sperm cells exposed at varying durations at the TOMCAT beamline (Swiss Light Source (SLS)) which has the field of view and spatial resolution needed to reconstruct sperm cells coupled with sub-second exposure times. The results indicate that motility is affected long before viability and that sperm cell dynamics are affected very quickly by the beam.

biophysics↗

bioRxiv2021

The molecular evolution of spermatogenesis across mammals

The testis is a key male reproductive organ that produces gametes through the process of spermatogenesis. Testis morphologies and spermatogenesis evolve rapidly in mammals, presumably due to the evolutionary pressure on males to be reproductively successful1,2. The rapid evolution of the testis was shown to be reflected at the molecular level based on bulk-tissue work3-8, but the molecular evolution of individual spermatogenic cell types across mammalian lineages remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from eleven species that cover the three major mammalian lineages (eutherians, marsupials, egg-laying monotremes) and birds (the evolutionary outgroup), and include seven key primates. Our analyses reveal that the rapid evolution of the testis is driven by accelerated fixation rates of gene expression changes, amino acid altering substitutions, and newly emerged genes in late spermatogenic stages - likely facilitated by reduced pleiotropic constraints, haploid selection, and a transcriptionally permissive chromatin environment. We identify temporal expression changes of individual genes across species, which may have contributed to the emergence of species-specific phenotypes, but also conserved expression programs underlying ancestral spermatogenic processes. Sex chromosome analyses show that genes predominantly expressed in spermatogonia (i.e., germ cells fueling spermatogenesis) and Sertoli cells (i.e., somatic supporting cells) independently accumulated on X chromosomes across mammals during evolution, presumably due to male-beneficial selective forces. Further work uncovered that the process of meiotic sex chromosome inactivation (MSCI) also occurs in monotremes and hence is common to the different mammalian sex chromosome systems, contrary to previous inferences9. Thus, the general mechanism of meiotic silencing of unsynapsed chromatin (MSUC), which underlies MSCI, represents an ancestral mammalian feature. Together, our study illuminates the cellular and molecular evolution of mammalian spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

evolutionary biology↗