Sperm Differentiation Research Articles

Transient receptor potential a1b regulates primordial germ cell numbers and sex differentiation in developing zebrafish.

Temperature is a leading environmental factor determining the sex ratio of some animal populations, such as fish, amphibians, and reptiles. However, the underlying mechanism by which temperature affects gender is still poorly understood. Transient receptor potential a1b (Trpa1b) belongs to the ion channel family of transient receptor potentials and exhibits dual thermosensitivity to heat and cold. In this study, we have unveiled a novel function of the trpa1b gene. Zebrafish generated through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with Trpa1b-null manifest a male-biased sex ratio. The quantity of primordial germ cells (PGCs) in zebrafish is closely linked to gender determination and gonadal development. Yet the role of the trpa1b gene in zebrafish reproductive development remains unexplored in the literature. Our investigation revealed a significant reduction in PGCs in Trpa1b mutant zebrafish compared to their wild-type counterparts 24-h postfertilization (hpf). Transcriptome sequencing of tissues near the reproductive crest of embryos at 1.25 days postfertilization (dpf) revealed differential changes in PGC-related marker genes and genes related to sperm cell development and differentiation. The relative expression of ddx4 and sycp3 genes was significantly downregulated, whereas amh was significantly upregulated at 20 dpf in trpa1b-/- zebrafish. The results of this study provide valuable insights and references for studying the molecular mechanism of sex determination in zebrafish. Undoubtedly, these results will further enhance our understanding of gender differentiation and gonadal development in fish and other vertebrates.

Integrated cytological and transcriptomic analyses provide new insights into restoration of pollen viability in synthetic allotetraploid Brassica carinata.

Upregulation of genes involved in DNA damage repair and sperm cell differentiation leads to restoration of pollen viability in synthetic allotetraploid B. carinata after chromosome doubling. Apart from the well-known contribution of polyploidy to crop improvement, polyploids can also be induced for other purposes, such as to restore the viability of sterile hybrids. The mechanism related to viability transition between the sterile allodiploid and the fertile allotetraploid after chromosome doubling are not well understood. Here, we synthesised allodiploid B. carinata (2n = 2x = 17) and allotetraploid B. carinata (2n = 4x = 34) as models to investigate the cytological and transcriptomic differences during pollen development. The results showed that after chromosome doubling, the recovery of pollen viability in allotetraploid was mainly reflected in the stabilisation of microtubule spindle morphology, normal meiotic chromosome behaviour, and normal microspore development. Interestingly, the deposition and degradation of synthetic anther tapetum were not affected by polyploidy. Transcription analysis showed that the expression of genes related to DNA repair (DMC1, RAD51, RAD17, SPO11-2), cell cycle differentiation (CYCA1;2, CYCA2;3) and ubiquitination proteasome pathway (UBC4, PIRH2, CDC53) were positively up-regulated during pollen development of synthetic allotetraploid B. carinata. In summary, these results provide some refreshing updates about the ploidy-related restoration of pollen viability in newly synthesised allotetraploid B. carinata.

Comparison of the Level of Vitamins A, D and E of Plasma Seminal of Azoospermia and Normozoospermia.

<b>Background and Objective:</b> Biological exploration of male infertility is important for its treatment. Seminal plasma, by its composition, presents numerous molecules that can be exploited in the investigation of new sperm biomarkers. The evaluation of new biomarkers of azoosperm seminal plasma aims to identify vitamins A, D and E which can serve as discriminating biochemical markers in the exploration of azoospermia. <b>Materials and Methods:</b> Thirty normozoospermic and 30 azoospermic sperm samples were collected by masturbation after three days of sexual abstinence from consulting patients at the Pasteur Institute of Côte d'Ivoire. After centrifugation of the sperm, the seminal plasma was collected and were analyzed for vitamin A, D and E. After extracting the vitamins from the seminal plasma, they were quantified using high-performance liquid chromatography. <b>Results:</b> The concentration of vitamin A in seminal plasma from normal samples was 1.66±1.81 and 0.28±0.52 mg/L in pathological samples. The average vitamin D concentration in seminal plasma of normospermia was 0.27±0.40 and 0.08±0.12 mg/L in seminal plasma of azoospermia. For vitamin E, the results obtained show an average concentration of 2.56±3.58 mg/L in normal ejaculate and 0.33±0.51 mg/L in pathological ejaculate. Only vitamins A and E showed a significant difference in the two categories of sperms. <b>Conclusion:</b> The determination of the concentration of vitamins A, D and E in seminal plasma showed that only vitamins A and E can serve as a biomarker for the differentiation of normozoospermic and azoospermic sperm.

O-305 Mapping the entire functionally active seminal microbiota and its association with semen quality parameters

Abstract Study question Does semen harbour functionally active microorganisms, and whether the microbial composition associate with semen quality parameters? Summary answer Seminal fluid harbours functionally alive microorganisms including bacteria, viruses, archaea, viroids and fungi whose composition and metabolic functions associate with semen quality parameters. What is known already Seminal fluid has been shown to possess different microbes, where different bacteria have been associated with spermatogenesis, seminal parameters and infertility. Nevertheless, previous results are inconclusive and the core microorganismal composition in semen is not determined and whether the identified bacterial DNA sequences refer to alive/functionally active microbes is not clear. Further, there is limited knowledge of the potential host-microbe interactions. Study design, size, duration In total 78 men with age between 18 and 45 years donated semen for the study, 59 men with good quality semen parameters and 19 men with low semen quality and diagnosed asthenospermia, oligoasthenozoospermia or oligoasthenoteratozoospermia comprised control and case groups, respectively. Participants/materials, setting, methods Semen quality parameters were analysed following WHO, 2021 guidelines. Seminal fluid underwent NextSeq total RNA sequencing, separating human and non-human sequences with Hisat2 aligner. Taxonomic classification of microorganisms was obtained using Kraken2. Microbial species and human RNA-Seq analyses, comparing control and infertility groups, utilized metagenomeSeq and edgeR R packages. Human and microbial pathways were annotated using MetaCyc, followed by GSEA analysis. The integration of metabolic pathways between host and microbes was investigated. Main results and the role of chance With our total RNAseq analysis, we mapped the entire alive microbiota composing of 6453 microorganisms within the seminal fluid samples. Microbes such as bacteria, fungi, viruses, viroids and archea were identified. When analysing microbiota associations with seminal parameters, 404 microbial species (Lactobacillus, Gardnerella, Prevotella, Atopobium, Pseudomonas among others) were significantly differently present among infertile men vs. good quality semen group. The host transcriptomic analysis identified 2569 differentially expressed genes in infertile vs. good quality semen groups, involved in sperm differentiation, spermatid development, reproduction, cilium movement and meiosis. Metabolic pathway analysis detected possible metabolic activity in the host-microbiota crosstalk in FAO-PWY: fatty acid & beta-oxidation, LIPASYN-PWY: phospholipases, PWY66-429: fatty acid biosynthesis, PWY-3781: aerobic respiration I pathways. Limitations, reasons for caution Bigger sample size of infertile men would be needed to validate the study findings. Wider implications of the findings We confirm the presence of active microbes in semen with implications in seminal quality parameters such as motility, sperm count and morphology. Our results contribute to better understanding of seminal microbiota dysregulation in infertility with broader implications for reproductive health, suggesting functional links between seminal microbiota and sperm parameters. Trial registration number NA

Whole transcriptome analysis to identify non-coding RNA regulators and hub genes in sperm of non-obstructive azoospermia by microarray, single-cell RNA sequencing, weighted gene co-expression network analysis, and mRNA-miRNA-lncRNA interaction analysis

BackgroundThe issue of male fertility is becoming increasingly common due to genetic differences inherited over generations. Gene expression and evaluation of non-coding RNA (ncRNA), crucial for sperm development, are significant factors. This gene expression can affect sperm motility and, consequently, fertility. Understanding the intricate protein interactions that play essential roles in sperm differentiation and development is vital. This knowledge could lead to more effective treatments and interventions for male infertility.Materials and methodsOur research aim to identify new and key genes and ncRNA involved in non-obstructive azoospermia (NOA), improving genetic diagnosis and offering more accurate estimates for successful sperm extraction based on an individual’s genotype.ResultsWe analyzed the transcript of three NOA patients who tested negative for genetic sperm issues, employing comprehensive genome-wide analysis of approximately 50,000 transcript sequences using microarray technology. This compared gene expression profiles between NOA sperm and normal sperm. We found significant gene expression differences: 150 genes were up-regulated, and 78 genes were down-regulated, along with 24 ncRNAs up-regulated and 13 ncRNAs down-regulated compared to normal conditions. By cross-referencing our results with a single-cell genomics database, we identified overexpressed biological process terms in differentially expressed genes, such as “protein localization to endosomes” and “xenobiotic transport.” Overrepresented molecular function terms in up-regulated genes included “voltage-gated calcium channel activity,” “growth hormone-releasing hormone receptor activity,” and “sialic acid transmembrane transporter activity.” Analysis revealed nine hub genes associated with NOA sperm: RPL34, CYB5B, GOL6A6, LSM1, ARL4A, DHX57, STARD9, HSP90B1, and VPS36.ConclusionsThese genes and their interacting proteins may play a role in the pathophysiology of germ cell abnormalities and infertility.

Experimental Evaluation of a Direct Fitness Effect of the De Novo Evolved Mouse Gene Pldi.

De novo evolved genes emerge from random parts of noncoding sequences and have, therefore, no homologs from which a function could be inferred. While expression analysis and knockout experiments can provide insights into the function, they do not directly test whether the gene is beneficial for its carrier. Here, we have used a seminatural environment experiment to test the fitness of the previously identified de novo evolved mouse gene Pldi, which has been implicated to have a role in sperm differentiation. We used a knockout mouse strain for this gene and competed it against its parental wildtype strain for several generations of free reproduction. We found that the knockout (ko) allele frequency decreased consistently across three replicates of the experiment. Using an approximate Bayesian computation framework that simulated the data under a demographic scenario mimicking the experiment's demography, we could estimate a selection coefficient ranging between 0.21 and 0.61 for the wildtype allele compared to the ko allele in males, under various models. This implies a relatively strong selective advantage, which would fix the new gene in less than hundred generations after its emergence.

CCDC157 is essential for sperm differentiation and shows oligoasthenoteratozoospermia-related mutations in men.

Oligoasthenoteratospermia (OAT), characterized by abnormally low sperm count, poor sperm motility, and abnormally high number of deformed spermatozoa, is an important cause of male infertility. Its genetic basis in many affected individuals remains unknown. Here, we found that CCDC157 variants are associated with OAT. In two cohorts, a 21-bp (g.30768132_30768152del21) and/or 24-bp (g.30772543_30772566del24) deletion of CCDC157 were identified in five sporadic OAT patients, and 2 cases within one pedigree. In a mouse model, loss of Ccdc157 led to male sterility with OAT-like phenotypes. Electron microscopy revealed misstructured acrosome and abnormal head-tail coupling apparatus in the sperm of Ccdc157-null mice. Comparative transcriptome analysis showed that the Ccdc157 mutation alters the expressions of genes involved in cell migration/motility and Golgi components. Abnormal Golgi apparatus and decreased expressions of genes involved in acrosome formation and lipid metabolism were detected in Ccdc157-deprived mouse germ cells. Interestingly, we attempted to treat infertile patients and Ccdc157 mutant mice with a Chinese medicine, Huangjin Zanyu, which improved the fertility in one patient and most mice that carried the heterozygous mutation in CCDC157. Healthy offspring were produced. Our study reveals CCDC157 is essential for sperm maturation and may serve as a marker for diagnosis of OAT.

MORN2 regulates the morphology and energy metabolism of mitochondria and is required for male fertility in mice

BackgroundMitochondria produce adenosine triphosphate through respiratory activities to power sperm differentiation and motility, and decreased mitochondrial respiratory activity can result in poor sperm motility and asthenospermia. The mitochondrial sheath is a component of the mid-piece of the sperm flagellum, and dysfunction of the sheath can reduce sperm motility and cause male infertility. The membrane occupation and recognition nexus-motif protein 2 (MORN2) is testis enriched in mice, and the MORN motif was reported to play a role in the regulation of bioelectrical signal homeostasis in cardiomyocytes.MethodsWe generated Morn2–/– mice using CRISPR/Cas9 and evaluated the potential functions of MORN2 in spermiogenesis through histological analysis, fertility examination, RT-PCR, CASA, immunofluorescence, TUNEL, electron microscopy analysis, mitochondrial energy metabolism analysis, etc.ResultsThe Morn2–/– mice were infertile, and their sperm showed severe motility defects. Morn2–/– sperm also had abnormal morphology characterized by bent heads, aberrant mitochondrial sheath formation, lower mitochondrial membrane potential, higher levels of reactive oxygen species, and decreased mitochondrial respiratory activity.ConclusionsOur study demonstrates that MORN2 is essential for male fertility and indicates that MORN2 functions in mitochondrial sheath formation and regulates mitochondrial respiratory activity.

ScRNA-seq Reveals Novel Genetic Pathways and Sex Chromosome Regulation in Tribolium Spermatogenesis.

Spermatogenesis is critical to sexual reproduction yet evolves rapidly in many organisms. High-throughput single-cell transcriptomics promises unparalleled insight into this important process but understanding can be impeded in nonmodel systems by a lack of known genes that can reliably demarcate biologically meaningful cell populations. Tribolium castaneum, the red flour beetle, lacks known markers for spermatogenesis found in insect species like Drosophila melanogaster. Using single-cell sequencing data collected from adult beetle testes, we implement a strategy for elucidating biologically meaningful cell populations by using transient expression stage identification markers, weighted principal component clustering, and SNP-based haploid/diploid phasing. We identify populations that correspond to observable points in sperm differentiation and find species specific markers for each stage. Our results indicate that molecular pathways underlying spermatogenesis in Coleoptera are substantially diverged from those in Diptera. We also show that most genes on the X chromosome experience meiotic sex chromosome inactivation. Temporal expression of Drosophila MSL complex homologs coupled with spatial analysis of potential chromatin entry sites further suggests that the dosage compensation machinery may mediate escape from meiotic sex chromosome inactivation and postmeiotic reactivation of the X chromosome.

RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis

Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double-strand break (DSB) formation, and disruption of meiotic gene expression and DSB repair in germ cells lacking NELF.

Cytological and transcriptomic analyses provide insights into the pollen fertility of synthetic allodiploid Brassica juncea hybrids.

Imbalanced chromosomes and cell cycle arrest, along with down-regulated genes in DNA damage repair and sperm cell differentiation, caused pollen abortion in synthetic allodiploid Brassica juncea hybrids. Interspecific hybridization is considered to be a major pathway for species formation and evolution in angiosperms, but the occurrence of pollen abortion in the hybrids is common, prompting us to recheck male gamete development in allodiploid hybrids after the initial combination of different genomes. Here, we investigated the several key meiotic and mitotic events during pollen development using the newly synthesised allodiploid B. juncea hybrids (AB, 2n=2× =18) as a model system. Our results demonstrated the partial synapsis and pairing of non-homologous chromosomes concurrent with chaotic spindle assembly, affected chromosome assortment and distribution during meiosis, which finally caused difference in genetic constitution amongst the final tetrads. The mitotic cell cycle arrest during microspore development resulted in the production of anucleate pollen cells. Transcription analysis showed that sets of key genes regulating cyclin (CYCA1;2 and CYCA2;3), DNA damage repair (DMC1, NBS1 and MMD1), and ubiquitin-proteasome pathway (SINAT4 and UBC) were largely downregulated at the early pollen meiosis stages, and those genes involved in sperm cell differentiation (DUO1, PIRL1, PIRL9 and LBD27) and pollen wall synthesis (PME48, VGDH11 and COBL10) were mostly repressed at the late pollen mitosis stages in the synthetic allodiploid B. juncea hybrids (AB). In conclusion, this study elucidated the related mechanisms affecting pollen fertility during male gametophyte development at the cytological and transcriptomic levels in the synthetic allodiploid B. juncea hybrids.

Role of MOB4 in Cell Proliferation and Neurogenesis

Signaling pathways that integrate a large set of inputs (both extra- and intracellular) to control cell proliferation are essential during both development and adult stages to guarantee organism homeostasis. Mobs are small adaptor proteins that participate in several of these signaling pathways. Here, we review recent advances unravelling Mob4 cellular functions, a highly conserved non-catalytic protein, that plays a diversity of roles in cell proliferation, sperm cell differentiation and is simultaneously involved in synapse formation and neural development. In addition, the gene is often overexpressed in a large diversity of tumors and is linked to poor clinical outcomes. Nevertheless, Mob4 molecular functions remain poorly defined, although it integrates the core structure of STRIPAK, a kinase/phosphatase protein complex, that can act upstream of the Hippo pathway. In this review we focus on the recent findings of Mob4 functions, that have begun to clarify its critical role on cell proliferation and the development of tissues and individuals.

Cascading effects of hypobaric hypoxia on the testis: insights from a single-cell RNA sequencing analysis.

Most mammals tolerate exposure to hypobaric hypoxia poorly as it may affect multiple regulatory mechanisms and inhibit cell proliferation, promote apoptosis, limit tissue vascularization, and disrupt the acid-base equilibrium. Here, we quantified the functional state of germ cell development and demonstrated the interaction between the germ and somatic cells via single-cell RNA sequencing (scRNA-seq). The present study elucidated the regulatory effects of hypobaric hypoxia exposure on germ cell formation and sperm differentiation by applying enrichment analysis to genomic regions. Hypobaric hypoxia downregulates the genes controlling granule secretion and organic matter biosynthesis, upregulates tektin 1 (TEKT1) and kinesin family member 2C (KIF2C), and downregulates 60S ribosomal protein 11 (RPL11) and cilia- and flagella-associated protein 206 (CFAP206). Our research indicated that prosaposin-G protein-coupled receptor 37 (PSAP-GPR37) ligands mediate the damage to supporting cells caused by hypobaric hypoxic exposure. The present work revealed that hypoxia injures peritubular myoid (PTM) cells and spermatocytes in the S phase. It also showed that elongating spermatids promote maturation toward the G2 phase and increase their functional reserve for sperm-egg binding. The results of this study provide a theoretical basis for future investigations on prophylactic and therapeutic approaches toward protecting the reproductive system against the harmful effects of hypobaric hypoxic exposure.

Histone Variant H3.3 Controls Arabidopsis Fertility by Regulating Male Gamete Development.

Reprograming of chromatin structures and changes in gene expression are critical for plant male gamete development, and epigenetic marks play an important role in these processes. Histone variant H3.3 is abundant in euchromatin and is largely associated with transcriptional activation. The precise function of H3.3 in gamete development remains unclear in plants. Here, we report that H3.3 is abundantly expressed in Arabidopsis anthers and its knockout mutant h3.3-1 is sterile due to male sterility. Transcriptome analysis of young inflorescence has identified 2348 genes downregulated in h3.3-1 mutant, among which 1087 target genes are directly bound by H3.3, especially at their 3' ends. As a group, this set of H3.3 targets is enriched in the reproduction-associated processes including male gamete generation, pollen sperm cell differentiation and pollen tube growth. The function of H3.3 in male gamete development is dependent on the Anti-Silencing Factor 1A/1B (ASF1A/1B)-Histone regulator A (HIRA)-mediated pathway. Our results suggest that ASF1A/1B-HIRA-mediated H3.3 deposition at its direct targets for transcription activation forms the regulatory networks responsible for male gamete development.

The RNA-binding protein Adad1 is necessary for germ cell maintenance and meiosis in zebrafish.

The double stranded RNA binding protein Adad1 (adenosine deaminase domain containing 1) is a member of the adenosine deaminase acting on RNAs (Adar) protein family with germ cell-specific expression. In mice, Adad1 is necessary for sperm differentiation, however its function outside of mammals has not been investigated. Here, through an N-ethyl-N-nitrosourea (ENU) based forward genetic screen, we identified an adad1 mutant zebrafish line that develops as sterile males. Further histological examination revealed complete lack of germ cells in adult mutant fish, however germ cells populated the gonad, proliferated, and entered meiosis in larval and juvenile fish. Although meiosis was initiated in adad1 mutant testes, the spermatocytes failed to progress beyond the zygotene stage. Thus, Adad1 is essential for meiosis and germline maintenance in zebrafish. We tested if spermatogonial stem cells were affected using nanos2 RNA FISH and a label retaining cell (LRC) assay, and found that the mutant testes had fewer LRCs and nanos2-expressing cells compared to wild-type siblings, suggesting that failure to maintain the spermatogonial stem cells resulted in germ cell loss by adulthood. To identify potential molecular processes regulated by Adad1, we sequenced bulk mRNA from mutants and wild-type testes and found mis-regulation of genes involved in RNA stability and modification, pointing to a potential broader role in post-transcriptional regulation. Our findings suggest that the RNA regulatory protein Adad1 is required for fertility through regulation of spermatogonial stem cell maintenance in zebrafish.

P-021 A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals

Abstract Study question The present study aimed to investigate the role of HUWE1 in non-obstructive azoospermia (NOA) etiology and germ cell differentiation. Summary answer An SNP mutation in HUWE1 promoter region downregulates HUWE1 expression in NOA patients, the genetic polymorphisms of HUWE1 are related to the pathogenesis of NOA. What is known already HUWE1, a ubiquitin ligase, is crucial to the development of male reproductive system and embryonic development. However, the specific role of HUWE1 in regulating germ cell differentiation is unclear, and clinical evidence for the relationship between HUWE1 and the pathogenesis of male infertility is lacking. Study design, size, duration 190 NOA Chinese patients were recruited and analyzed single nucleotide polymorphisms (SNPs) in HUWE1 gene. Participants/materials, setting, methods Regulation of HUWE1 gene expression by RARα factor was evaluated by performing ChIP assay, EMSA, and siRNA-mediated RARα knockdown. We used C18-4 cells to determine whether HUWE1 participated in RA-mediated RARα signaling and performed luciferase assay, CCK-8 assay, immunofluorescence staining, RT-qPCR, and western blot analysis. The expression level of HUWE1 and RARa was evaluated in clinical samples obtained from NOA or obstructive azoospermia (OA) patients using RT-qPCR. Main results and the role of chance We explored the mechanism by which polymorphism of the HUWE1 promoter leads to spermatogenesis disorders, and focused on the identification of mutations in the SNP site of the HUWE1 regulatory sequence among patients with NOA, which may influence HUWE1 expression. The results of in vitro experiments showed that RARα binds to the HUWE1 promoter region and regulates its expression. However, the regulatory effect of RARα weakened following introduction of the mutation into the HUWE1 regulatory sequence. The expression of spermatogonial differentiation-related gene STRA8 was impaired, and γH2AX foci accumulated following inhibition of HUWE1 gene expression using siRARα. Finally, to determine whether HUWE1 expression was important in patients with NOA, its expression was verified using testicular puncture samples collected from patients with NOA and the results revealed that HUWE1 expression was abnormal (P < 0.01). In addition, RARα expression was significantly downregulated (P < 0.001). Overall, our result support the hypothesis that HUWE1 expression is induced by the RA/RARα signaling pathway and it inhibit γH2AX foci accumulation, thereby considerably promoting sperm differentiation and meiotic prophase. Limitations, reasons for caution The effect of SNPs in HUWE1 on male infertility is further studied in a wider population. Wider implications of the findings Our study of HUWE1 and RA/RARα signaling pathway provides new information on the regulatory mechanism of spermiogenesis, suggesting that SNPs in HUWE1 promoter are etiological factors in patients with NOA and providing a rationale for further drug development to target HUWE1 in these patients. Trial registration number the National Key Research and Development Program of China (2018YFC1003603)

Selective sweeps identification in distinct groups of cultivated rye (Secale cereale L.) germplasm provides potential candidate genes for crop improvement

BackgroundDuring domestication and subsequent improvement plants were subjected to intensive positive selection for desirable traits. Identification of selection targets is important with respect to the future targeted broadening of diversity in breeding programmes. Rye (Secale cereale L.) is a cereal that is closely related to wheat, and it is an important crop in Central, Eastern and Northern Europe. The aim of the study was (i) to identify diverse groups of rye accessions based on high-density, genome-wide analysis of genetic diversity within a set of 478 rye accessions, covering a full spectrum of diversity within the genus, from wild accessions to inbred lines used in hybrid breeding, and (ii) to identify selective sweeps in the established groups of cultivated rye germplasm and putative candidate genes targeted by selection.ResultsPopulation structure and genetic diversity analyses based on high-quality SNP (DArTseq) markers revealed the presence of three complexes in the Secale genus: S. sylvestre, S. strictum and S. cereale/vavilovii, a relatively narrow diversity of S. sylvestre, very high diversity of S. strictum, and signatures of strong positive selection in S. vavilovii. Within cultivated ryes we detected the presence of genetic clusters and the influence of improvement status on the clustering. Rye landraces represent a reservoir of variation for breeding, and especially a distinct group of landraces from Turkey should be of special interest as a source of untapped variation. Selective sweep detection in cultivated accessions identified 133 outlier positions within 13 sweep regions and 170 putative candidate genes related, among others, to response to various environmental stimuli (such as pathogens, drought, cold), plant fertility and reproduction (pollen sperm cell differentiation, pollen maturation, pollen tube growth), and plant growth and biomass production.ConclusionsOur study provides valuable information for efficient management of rye germplasm collections, which can help to ensure proper safeguarding of their genetic potential and provides numerous novel candidate genes targeted by selection in cultivated rye for further functional characterisation and allelic diversity studies.

Genome-wide association study reveals candidate markers related to field fertility and semen quality traits in Holstein-Friesian bulls

In vitro assessment of bull semen quality is routinely used in bull semen processing centres in order to ensure that semen destined to be used in the field has passed minimum standards. Despite these stringent quality control checks, individual bulls that pass the quality control checks can still vary in field fertility by up to 25%. A genome-wide association study was undertaken to determine genetic markers associated with prefreeze and post-thaw bull sperm quality traits as well as field fertility. Genome-wide association analysis was performed using a single nucleotide polymorphism (SNP) regression mixed linear model in WOMBAT. Genes within a 250 Kb span of a suggestive (P ≤ 1 × 10−5) SNP were considered as candidate genes. One SNP was associated with adjusted pregnancy rate, and 21 SNPs were associated across the seven semen quality traits (P ≤ 1 × 10−5). Functional candidate genes include SIPA1L2 which was associated with adjusted pregnancy rate. This encodes a Rap GTPase-activating protein involved in Rap1 signalling pathway and was previously found to play a role in the process of sperm differentiation. Gene ontology (GO) analysis also identified significantly enriched biological processes involved protein tyrosine kinase activity including genes such as DYRK1A, TEC and TXK that were associated with sperm motility prior to freezing. Another candidate gene associated with post-thaw sperm motility was FHDC1 which coordinates actin filament and microtubule dynamics. The induced 11 GO terms in the ejaculates rejected after freezing trait were related to ATPase, phosphatase and hydrolase activity. These results reveal novel specific genomic regions and candidate genes associated with economically important phenotypes such as field fertility and semen quality traits.

DOT1L regulates chromatin reorganization and gene expression during sperm differentiation

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone‐to‐protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79‐methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked‐out (KO) in postnatal male germ cells, we found that Dot1l‐KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l‐KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l‐KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.

DNALI1 interacts with the MEIG1/PACRG complex within the manchette and is required for proper sperm flagellum assembly in mice.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.