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The Journal of Cell Biology Oct 2024The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this...
The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.
Topics: Animals; Dyneins; Anaphase; Drosophila melanogaster; Drosophila Proteins; Microtubules; Molecular Dynamics Simulation; Mutation; Spindle Apparatus; Humans; Mutation, Missense
PubMed: 38949648
DOI: 10.1083/jcb.202310022 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... May 2024Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility...
OBJECTIVE
Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development. Premature ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles, constituting a significant cause of female infertility. In recent years, with the help of the rapid development in genetic sequencing technology, it has been demonstrated that genetic factors play a crucial role in the onset of POI. Among the population suffering from POI, genetic studies have revealed that genes involved in processes such as meiosis, DNA damage repair, and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified. FA complementation group M () is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including -. Abnormalities in the genes are associated with female infertility and gene knockout mice also exhibit phenotypes similar to those of POI. During the genetic screening of POI patients, this study identified a suspicious variant in . This study aims to explore the pathogenic mechanisms of the genes of the FA pathway and their variants in the development of POI. We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals.
METHODS
One POI patient was included in the study. The inclusion criteria for POI patients were as follows: women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L (with a minimum interval of 4 weeks inbetween tests), alongside clinical symptoms of menstrual disorders, normal chromosomal karyotype analysis results, and exclusion of other known diseases that can lead to ovarian dysfunction. We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG). Subsequently, the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis. Plasmids containing wild-type and mutant genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP - group, the EGFP - group, and the EGFP group, respectively. To validate the production of truncated proteins, cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody. In order to investigate the impact on DNA damage repair, immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP WT group and the EGFP -MUT group to examine whether the variant affected FANCM's ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in both the EGFP - group and the EGFP - group, which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody.
RESULTS
In a POI patient from a consanguineous family, we identified a homozygous variant in the gene, c.1152-1155del:p.Leu386Valfs*10. The patient presented with primary infertility, experiencing irregular menstruation since menarche at the age of 16. Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone (AMH) level of 0.07 ng/mL. Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia. The patient's parents were a consanguineous couple, with the mother having regular menstrual cycles. The patient had two sisters, one of whom passed away due to osteosarcoma, while the other exhibited irregular menstruation, had been diagnosed with ovarian insufficiency, and remained childless. Bioinformatics analysis revealed a deletion of four nucleotides (c.1152-1155del) in the exon 6 of the patient's gene. This variant resulted in a frameshift at codon 386, introducing a premature stop codon at codon 396, which ultimately led to the production of a truncated protein consisting of 395 amino acids. experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization, with the protein being present only in the cytoplasm. Following treatment with mitomycin C, there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid (<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant.
CONCLUSIONS
The homozygous variant in the gene, c.1152-1155del:p.Leu386Valfs*10, results in the production of a truncated FANCM protein. This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex, preventing its entry into the nucleus and the subsequent recognition of DNA damage. Consequently, the localization of the FA core complex on chromatin is disrupted, impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs. By disrupting the rapid proliferation and meiotic division processes of primordial germ cells, the reserve of oocytes is depleted, thereby triggering premature ovarian insufficiency in females.
Topics: Female; Primary Ovarian Insufficiency; Humans; Mutation; Fanconi Anemia; Adult; Infertility, Female; DNA Helicases
PubMed: 38948269
DOI: 10.12182/20240560207 -
EMBO Reports Jun 2024Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a...
Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.
PubMed: 38943004
DOI: 10.1038/s44319-024-00187-6 -
Aquatic Toxicology (Amsterdam,... Jun 2024Environmental estrogens (EEs) are found extensively in natural waters and negatively affect fish reproduction. Research on the reproductive toxicity of EEs mixtures in...
Environmental estrogens (EEs) are found extensively in natural waters and negatively affect fish reproduction. Research on the reproductive toxicity of EEs mixtures in fish at environmentally relevant concentrations is scarce. In this study, adult male zebrafish were exposed for 60 days to EES (a mixture of EEs), EE2-low (5.55 ng/L, with an estrogenic potency equal to EES), and EE2-high (11.1 ng/L). After exposure, the expression levels of vtg1, vtg3, and esr1 in the livers in EES-treated fish remained unaltered, whereas they were significantly increased in EE2-treated fish. Both EE2-high and EES exposures notably reduced the gonad somatic index and sperm count. A disrupted spermatogenesis was also observed in the testes of EE2-high- and EES-exposed fish, along with an alteration in the expression of genes associated with spermatogonial proliferation (pcna, nanog), cell cycle transition (cyclinb1, cyclind1), and meiosis (aldh1a2, cyp26a1, sycp3). Both EE2 and EES significantly lowered plasma 11-ketotestosterone levels in males, likely by inhibiting the expression level of genes for its synthesis (scc, cyp17a1 and cyp11b2), and increased 17β-estradiol (E2) levels, possibly through upregulating the expression of cyp19a1a. A significant increase in tnfrsf1a expression and the tnfrsf1a/tnfrsf1b ratio in EE2-high and EES-treated males also suggests increased apoptosis via the extrinsic pathway. Further investigation showed that both EE2-high and EES diminished the sexual behavior of male fish, accompanied with reduced E2 levels in the brain and the expression of genes in the kisspeptin/gonadotropin-releasing hormone system. Interestingly, the sexual behavior of unexposed females paired with treated males was also reduced, indicating a synergistic effect. This study suggests that EES have a more severe impact on reproduction than EE2-low, and EEs could interfere not only with spermatogenesis in fish, but also with the sexual behaviors of both exposed males and their female partners, thereby leading to a more significant disruption in fish reproduction.
PubMed: 38941808
DOI: 10.1016/j.aquatox.2024.107008 -
Sheng Li Xue Bao : [Acta Physiologica... Jun 2024Oocyte maturation and early embryonic development are key steps in the reproductive physiology of female mammals, and any error in this process can adversely affect... (Review)
Review
Oocyte maturation and early embryonic development are key steps in the reproductive physiology of female mammals, and any error in this process can adversely affect reproductive development. Recent studies have shown that epigenetic modifications of histones play important roles in the regulation of oocyte meiosis and quality assurance of early embryonic development. Histone deacetylase 11 (HDAC11) is the smallest known member of the histone deacetylases (HDACs) family, and inhibition of HDAC11 activity significantly suppresses the rate of oocyte maturation, as well as the development of 8-cell and blastocyst embryos at the embryonic stage. This paper focuses on recent progress on the important role of HDAC11 in the regulation of mammalian oocyte maturation and early embryonic development, hoping to gain insights into the key roles played by epitope-modifying proteins represented by HDAC11 in the regulation of mammalian reproduction and their molecular mechanisms.
Topics: Animals; Oocytes; Embryonic Development; Histone Deacetylases; Female; Humans; Oogenesis; Mammals; Meiosis
PubMed: 38939938
DOI: No ID Found -
Heliyon Jun 2024Sirtuin 5 (Sirt5), a member of the Sirtuin family, is involved in various intracellular biological processes. However, the function of Sirt5 in oocyte maturation has not...
Sirtuin 5 (Sirt5), a member of the Sirtuin family, is involved in various intracellular biological processes. However, the function of Sirt5 in oocyte maturation has not been clearly elucidated. In this study, we observed that Sirt5 was persistently expressed during the meiotic division of mouse oocytes, with a notable decline in expression in aging oocytes. Sirt5 inhibition led to the failure of the first polar body extrusion and induced cell cycle arrest, indicative of unsuccessful oocyte maturation. Furthermore, Sirt5 inhibition was associated with the extrusion of abnormally large polar bodies, suggesting disrupted asymmetric oocyte division. Mechanistically, the inhibition of Sirt5 resulted in aberrant spindle assembly and disordered chromosome alignment in oocytes. Moreover, Sirt5 inhibition caused the spindle to be centrally located in the oocyte without migrating to the cortical region, consequently preventing the formation of the actin cap. Further investigation revealed that Sirt5 inhibition notably diminished the expression of phosphorylated cofilin and profilin1, while increasing cytoplasmic F-actin levels. These findings suggest that Sirt5 inhibition during oocyte maturation adversely affects spindle assembly and chromosome alignment and disrupts actin dynamics impairing spindle migration and contributing to the failure of symmetric oocyte division and maturation.
PubMed: 38933958
DOI: 10.1016/j.heliyon.2024.e32466 -
Plants (Basel, Switzerland) Jun 2024Ogura cytoplasmic male sterility (CMS) is considered the rapeseed ( L.) with the most potential to be utilized as a heterosis system worldwide, but it lacks sufficient...
Ogura cytoplasmic male sterility (CMS) is considered the rapeseed ( L.) with the most potential to be utilized as a heterosis system worldwide, but it lacks sufficient restorers. In this study, root tip cell (RTC) mitotic and pollen mother cell (PMC) meiosis observations were compared to ensure the number of chromosomes and the formation of a chromosomal bridge using restorer lines R2000, CLR650, and Zhehuhong (a new restorer) as the experimental material. Further, molecular markers of exogenous chromosomal fragments were detected and the sequence and expression differences of restorer genes in the three lines were determined to identify the distinctive characteristics of Zhehuhong. The results showed that the number of chromosomes in Zhehuhong was stable (2n = 38), indicating that the exogenous radish chromosome segment had been integrated into the chromosome of Zhehuhong. Molecular marker detection revealed that Zhehuhong was detected at most loci, with only the RMA05 locus being missed. The exogenous radish chromosome segment of Zhehuhong differed from R2000 and CLR650. The pollen mother cells of Zhehuhong showed chromosome lagging in the meiotic metaphase I, meiotic anaphase I, and meiotic anaphase II, which was consistent with R2000 and CLR650. The restorer gene in Zhehuhong had 85 SNPs compared with R2000 and 119 SNPs compared with CLR650, indicating the distinctive characteristic of in Zhehuhong. In terms of the spatial expression of , the highest level was detected in the anthers in the three restorer lines. In addition, in terms of temporal expression, the gene expression of Zhehuhong was highest at a bud length of 4 mm. Our results clearly indicated that Zhehuhong is a new restorer line for the Ogura CMS system, which can be used further in rapeseed heterosis utilization.
PubMed: 38931135
DOI: 10.3390/plants13121703 -
Plants (Basel, Switzerland) Jun 2024(Laxm.) is a leguminous plant with significant ecological benefits, but its embryonic development mechanism remains unclear. We investigated the flower bud...
(Laxm.) is a leguminous plant with significant ecological benefits, but its embryonic development mechanism remains unclear. We investigated the flower bud differentiation, megaspore and microspore formation, gametophyte development, and embryo and endosperm development in . Our aim was to elucidate the relationship between the external morphology and internal development processes of male and female floral organs during growth, as well as the reproductive factors influencing fruiting. The results indicated that although the pistil develops later than the stamen during flower bud differentiation, both organs mature synchronously before flowering. pollen exhibits three germination grooves, a reticulate outer wall, and papillary structures on the anther surface. In vivo pollination experiments revealed abnormal spiral growth of pollen tubes within the style and the occurrence of callus plugs, which may reduce the seed setting rate. The anther wall development follows the dicotyledonous type, with tetrads formed through microspore meiosis exhibiting both left-right symmetry and tetrahedral arrangements. has a single ovule, and the embryo sac develops in the monosporic polygonum type. After dormancy, the zygote undergoes multiple divisions, progressing through spherical, heart-shaped, and torpedo-shaped embryo stages, culminating in a mature embryo. A mature seed comprises cotyledons, hypocotyl, embryo, radicle, and seed coat. Phylogenetic tree analysis reveals a close genetic relationship between and other leguminous plants from the genera and . This study provides valuable insights into the regulation of flowering and hybrid breeding in leguminous plants and offers a new perspective on the development of floral organs and seed setting rates.
PubMed: 38931095
DOI: 10.3390/plants13121661 -
Genes Jun 2024Chromosomal translocations can result in phenotypic effects of varying severity, depending on the position of the breakpoints and the rearrangement of genes within the...
Chromosomal translocations can result in phenotypic effects of varying severity, depending on the position of the breakpoints and the rearrangement of genes within the interphase nucleus of the translocated chromosome regions. Balanced translocations are often asymptomatic phenotypically and are typically detected due to a decrease in fertility resulting from issues during meiosis. Robertsonian translocations are among the most common chromosomal abnormalities, often asymptomatic, and can persist in the population as a normal polymorphism. We serendipitously discovered a Robertsonian translocation between chromosome 21 and chromosome 22, which is inherited across three generations without any phenotypic effect, notably only in females. In situ hybridization with alpha-satellite DNAs revealed the presence of both centromeric sequences in the translocated chromosome. The reciprocal translocation resulted in a partial deletion of the short arm of both chromosomes 21, and 22, with the genes remaining present in the middle part of the new metacentric chromosome. The rearrangement did not cause alterations to the long arm. The spread of an asymptomatic heterozygous chromosomal polymorphism in a population can lead to mating between heterozygous individuals, potentially resulting in offspring with a homozygous chromosomal configuration for the anomaly they carry. This new karyotype may not produce phenotypic effects in the individual who presents it. The frequency of karyotypes with chromosomal rearrangements in asymptomatic heterozygous form in human populations is likely underestimated, and molecular karyotype by array Comparative Genomic Hybridization (array-CGH) analysis does not allow for the identification of this type of chromosomal anomaly, making classical cytogenetic analysis the preferred method for obtaining clear results on a karyotype carrying a balanced rearrangement.
Topics: Humans; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 22; In Situ Hybridization, Fluorescence; Karyotype; Karyotyping; Phenotype; Translocation, Genetic
PubMed: 38927657
DOI: 10.3390/genes15060722 -
Biomolecules May 2024Potato is the most important non-cereal crop worldwide, and, yet, genetic gains in potato have been traditionally delayed by the crop's biology, mostly the genetic... (Review)
Review
Potato is the most important non-cereal crop worldwide, and, yet, genetic gains in potato have been traditionally delayed by the crop's biology, mostly the genetic heterozygosity of autotetraploid cultivars and the intricacies of the reproductive system. Novel site-directed genetic modification techniques provide opportunities for designing climate-smart cultivars, but they also pose new possibilities (and challenges) for breeding potato. As potato species show a remarkable reproductive diversity, and their ovules have a propensity to develop apomixis-like phenotypes, tinkering with reproductive genes in potato is opening new frontiers in potato breeding. Developing diploid varieties instead of tetraploid ones has been proposed as an alternative way to fill the gap in genetic gain, that is being achieved by using gene-edited self-compatible genotypes and inbred lines to exploit hybrid seed technology. In a similar way, modulating the formation of unreduced gametes and synthesizing apomixis in diploid or tetraploid potatoes may help to reinforce the transition to a diploid hybrid crop or enhance introgression schemes and fix highly heterozygous genotypes in tetraploid varieties. In any case, the induction of apomixis-like phenotypes will shorten the time and costs of developing new varieties by allowing the multi-generational propagation through true seeds. In this review, we summarize the current knowledge on potato reproductive phenotypes and underlying genes, discuss the advantages and disadvantages of using potato's natural variability to modulate reproductive steps during seed formation, and consider strategies to synthesize apomixis. However, before we can fully modulate the reproductive phenotypes, we need to understand the genetic basis of such diversity. Finally, we visualize an active, central role for genebanks in this endeavor by phenotyping properly genotyped genebank accessions and new introductions to provide scientists and breeders with reliable data and resources for developing innovations to exploit market opportunities.
Topics: Solanum tuberosum; Plant Breeding; Apomixis; Reproduction; Genes, Plant; Phenotype; Tetraploidy; Genotype
PubMed: 38927018
DOI: 10.3390/biom14060614