Search Results (203)

Background: Herbal medicine represents a rich yet complex source of bioactive compounds, offering both therapeutic potential and toxicological risks. Methods: In this study, we systematically evaluated the biological effects of three traditional herbal extracts—Mentha longifolia, Scrophularia orientalis, and Echium biebersteinii—using Caenorhabditis elegans as an in vivo model. Results: All three extracts significantly reduced worm survival, induced larval arrest, and triggered a high incidence of males (HIM) phenotypes, indicative of mitotic failure and meiotic chromosome missegregation. Detailed analysis of germline architecture revealed extract-specific abnormalities, including nuclear disorganization, ectopic crescent-shaped nuclei, altered meiotic progression, and reduced bivalent formation. These defects were accompanied by activation of the DNA damage response, as evidenced by upregulation of checkpoint genes (atm-1, atl-1), increased pCHK-1 foci, and elevated germline apoptosis. LC-MS profiling identified 21 major compounds across the extracts, with four compounds—thymol, carvyl acetate, luteolin-7-O-rutinoside, and menthyl acetate—shared by all three herbs. Among them, thymol and carvyl acetate significantly upregulated DNA damage checkpoint genes and promoted apoptosis, whereas thymol and luteolin-7-O-rutinoside contributed to antioxidant activity. Notably, S. orientalis and E. biebersteinii shared 11 of 14 major constituents (79%), correlating with their similar phenotypic outcomes, while M. longifolia exhibited a more distinct chemical profile, possessing seven unique compounds. Conclusions: These findings highlight the complex biological effects of traditional herbal extracts, demonstrating that both beneficial and harmful outcomes can arise from specific phytochemicals within a mixture. By deconstructing these extracts into their active components, such as thymol, carvyl acetate, and luteolin-7-O-rutinoside, we gain critical insight into the mechanisms driving reproductive toxicity and antioxidant activity. This approach underscores the importance of component-level analysis for accurately assessing the therapeutic value and safety profile of medicinal plants, particularly those used in foods and dietary supplements. Full article

The efficacy of in vitro fertilization (IVF) is significantly hindered by early embryonic developmental failure and oocyte maturation arrest. Recent findings in reproductive genetics have identified several oocyte-specific genes—TUBB8, KIF11, and CKAP5—as essential regulators of meiotic spindle formation and cytoskeletal dynamics. Mutations in these genes can lead to significant meiotic defects, fertilization failure, and embryo arrest. The links between genotype and phenotype, along with the underlying biological mechanisms, remain inadequately characterized despite the increasing number of identified variations. This systematic review was conducted in accordance with PRISMA 2020 guidelines. Relevant papers were retrieved from the PubMed and Embase databases using combinations of the keywords “TUBB8,” “KIF11,” “CKAP5,” “oocyte maturation arrest,” “embryonic arrest,” and “IVF failure.” Studies were included if they contained clinical, genomic, and functional data on TUBB8, KIF11, or CKAP5 mutations in women undergoing IVF. Molecular data, including gene variant classifications, inheritance models, in vitro tests (such as microtubule network analysis in HeLa cells), and assisted reproductive technology (ART) outcomes, were obtained. Eighteen trials including 35 women with primary infertility were included. Over fifty different variants were identified, the majority of which can be attributed to TUBB8 mutations. TUBB8 disrupted α/β-tubulin heterodimer assembly due to homozygous missense mutations, hence hindering meiotic spindle formation and leading to early embryo fragmentation or the creation of many pronuclei and cleavage failure. KIF11 mutations resulted in spindle disorganization and chromosomal misalignment via disrupting tubulin acetylation and microtubule transport. Mutations in CKAP5 impaired bipolar spindle assembly and microtubule stabilization. In vitro validation studies showed cytoskeletal disturbances, protein instability, and dominant negative effects in transfected animals. Donor egg IVF was the sole effective treatment; however, no viable pregnancies were documented in patients with pathogenic mutations of TUBB8 or KIF11. TUBB8, KIF11, and CKAP5 are essential for safeguarding oocyte meiotic competence and early embryonic development at the molecular level. Genetic differences in these genes disrupt microtubule dynamics and spindle assembly, resulting in various aspects of oocyte maturation and fertilization. Functional validation underscores the necessity of routine genetic screening for women experiencing unresolved IVF failure, as it substantiates their causal role in infertility. Future therapeutic avenues in ART may be enhanced by tailored counseling and innovative rescue methodologies like as gene therapy. Full article

The wolf fish Hoplias malabaricus is a Neotropical species characterized by remarkable karyotypic diversity, including seven karyomorphs (KarA-G) with distinct sex chromosome systems. This study investigated the homologous XY (KarF) and XY₁Y₂ (KarG) sex chromosome systems present in this species by integrating cytogenetics and genomics to examine sex chromosomes’ composition through characterization of repeatome (satellite DNA and transposable elements) and sex-linked markers. Our analysis indicated that both karyomorphs are little differentiated in their sex chromosomes content revealed by satDNA mapping and putative sex-linked markers. Both repeatomes were mostly composed of transposable elements, but neither intra- (male versus female) nor interspecific (KarF x KarG) variations were found. In both systems, we demonstrated the occurrence of sex-specific sequences probably located on the non-recombining region of the Y chromosome supported by the accumulation of sex-specific haplotypes of HmfSat10-28/HmgSat31-28. This investigation offered valuable insights by highlighting the composition of homologous XY and XY₁Y₂ multiple sex chromosomes. Although homologous, the large Y chromosome in KarF corresponds to two separate linkage groups (Y₁ and Y₂) in KarG implying a specific meiotic arrangement involving the X chromosome in a meiotic trivalent chain. This scenario likely influenced recombination rates and, as a result, the genomic composition of these chromosomes. Full article

Meiotic recombination is essential for chromosomal segregation and facilitates the exchange between homologs, which leads to the transmission of new combinations of linked alleles to the progeny. The eukaryotic meiotic machinery is generally highly conserved, but the frequency of crossover occurrence can vary dramatically across species and populations, between individuals, and across sexes. The chicken and the guinea fowl exhibit interspecific variation in the distribution of crossovers along their largest chromosomes. In many organisms, an association has been observed between the preferred crossover location and certain sequence parameters, such as high GC content, CpG islands, or gene promoters. Here, we compared the distribution of these genomic parameters with the recombination landscape, represented by MLH1 focus frequencies, in the two birds. We found an association between GC content density and recombination in the chicken, but the remaining parameters showed weak or no association with recombination, especially in the guinea fowl. We conclude that despite the different broad-scale crossover distribution, the investigated genomic parameters remained remarkably similar in these two species. We suggest that the density of these genomic features is more likely related to microscale variations in recombination rates, such as those determined by open chromatin configurations. Full article

In vitro maturation (IVM) of oocytes retrieved from ovum pick-up (OPU) or ovarian tissue (OT) is a standard approach for patients with specific conditions where prior hormonal stimulation is contraindicated. However, the developmental competence of oocytes matured in vitro is still inferior to that of oocytes matured in vivo. Capacitation IVM (CAPA-IVM) includes an extra step of pre-maturation culture (PMC) with c-type natriuretic peptide (CNP) as a meiotic arrestor to better synchronize cytoplasmic and nuclear maturity in oocytes by allowing the cytoplasm additional time to acquire essential components critical for optimal competency. This study aims to evaluate the effect of CAPA-IVM on equine oocyte quality and developmental competence. Immature cumulus–oocyte complexes (COCs) were retrieved from slaughterhouse ovaries and matured in vitro either in CAPA-IVM (short 6 h, long 24 h pre-maturation) or standard IVM. Mature oocytes from each group were analyzed for calcium-releasing potential (n = 52) and single-oocyte proteomics (n = 44), and embryo development (n = 229) was assessed after fertilization with piezo-drilled intracytoplasmic sperm injection (ICSI). Genetic analysis of developed blastocysts (n = 41) was performed to detect chromosomal aberrations. Our findings demonstrate that CAPA-IVM of equine COCs yields significantly higher maturation rates than controls. Moreover, short CAPA-IVM with six hours pre-maturation culture showed substantially higher embryo development potential than the control group (20/69 vs. 9/63, respectively). Genetic analysis revealed a high euploidy rate in equine blastocysts regardless of the maturation conditions. Live calcium imaging of the fertilized oocytes demonstrated that the majority of oocytes displayed non-continuous calcium oscillation patterns, irrespective of maturation conditions. Single-oocyte proteomics reveals a comparable proteomic landscape between mature oocytes subjected to short CAPA-IVM and standard IVM. However, we identified four enriched gene sets with positive enrichment scores after short CAPA-IVM, related to cytoskeleton regulation, ribosomal function, and cytosolic components. Our findings indicate that CAPA-IVM holds the potential to improve oocyte quality and competence in horses. However, further fine-tuning of culture conditions would benefit the effective use of these IVM systems. Moreover, given that the mare serves as an excellent model for human reproduction, the molecular trends identified in this study could provide valuable insights for advancing human artificial reproductive technologies. Full article

Well-spread pachytene chromosomes are critical for studying the location of meiotic proteins along individual chromosomes. However, producing good spreads in species with large genomes is challenging due to the tangling of pachytene chromosomes. Existing protocols often fail to achieve proper separation of large chromosomes in spreads. Here, we describe in detail an improved protocol that ensures the effective separation of large pachytene chromosomes and demonstrates its suitability for protein immunodetection. To develop the protocol, pollen mother cells at the middle–late pachytene stage from Allium fistulosum, a species with a large genome and chromosomes, were used. The protocol involved three main steps: fixing anthers in Clark’s solution (ethanol–acetic acid, 3:1), digestion in an enzyme mixture, and gentle squashing in 45% acetic acid. A clear ZYP1 signal on all separated chromosomes was observed. The high quality of well-spread pachytene chromosomes obtained with the modified protocol allowed for the easy extraction of individual chromosomes for more precise detection and analysis of the proteins of interest. Full article

Mercury, a prevalent heavy metal, negatively impacts oocyte maturation. However, the exact mechanism by which methylmercury chloride (MMC) affects this process remains elusive. The present study found that MMC administration triggered meiotic failure in oocytes by disrupting cumulus cell expansion, leading to compromised spindle apparatus and altered chromosomal architecture, which are crucial for oocyte development. This disruption is characterized by abnormal microtubule organization and defective chromosome alignment. Additionally, MMC exposure caused oxidative stress-induced apoptosis due to mitochondrial dysfunction, as indicated by decreased mitochondrial membrane potential, mitochondrial content, mitochondrial DNA copy number, and adenosine triphosphate levels. Proteomic analysis identified 97 differentially expressed proteins, including P62, an autophagy marker. Our results confirmed that MMC induced autophagy, particularly through the hyperactivation of the mitochondrial autophagy to remove damaged and normal mitochondria. The mitochondrial reactive oxygen species (ROS) scavenger Mito-TEMPO alleviated oxidative stress and mitochondrial autophagy levels, suggesting that mitochondrial ROS initiates this autophagic response. Notably, MMC activates mitochondrial autophagy via the monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signal pathway due to mitochondrial dysfunction. In vivo studies in mice revealed that MMC exposure decreased reproductive performance, attributed to excessive mitochondrial autophagy leading to reduced oocyte quality. Overall, these findings demonstrate that MMC exposure impairs oocyte maturation via the hyperactivation of mitochondrial autophagy induced by mitochondrial dysfunction. Full article

Background/Objectives: Preimplantation genetic testing methods to detect aneuploidy (PGT-A) based on genomewide single nucleotide polymorphism (SNP) data were scarce and did not meet our needs. Methods: Hence, we developed a novel method for this purpose. After the raw B-allele frequency (rBAF) values of Single Nucleotide Polymorfisms (SNPs) are obtained from a sample of interest with SNP array, the BAF values for specific categories of SNPs (cBAF) are visualized separately. Results: The analysis of the cBAF, rBAF and Log₂R profiles enables to distinguish all common types of chromosomal abnormalities without haplotyping. This was demonstrated by reanalyzing data from 359 embryos which had previously been analyzed with Karyomapping. We identified additional underrepresented maternal haplotypes in five samples that we could not detect with Karyomapping. In addition, we identified all chromosomes with meiotic-origin copy number gains (both parental homolog (BPH)) (n = 70) and all chromosomes with a non-mosaic copy number loss larger than 5 Mb (n = 93) that had been detected with Karyomapping. Conclusions: We conclude that the proposed method can be used to reliably detect meiotic-origin aneuploidy without haplotyping and, hence without the need for a phasing reference. Full article

Retinoic acid (RA) plays a critical role in initiating meiosis in primordial germ cells (PGC), yet the specific mechanisms of its interaction with PGC remain unclear. In this study, we used an in vitro feeder-free culture system with chicken PGC as a model to explore the mechanisms by which RA induces the entry of PGC into meiosis. Results demonstrated that exogenous RA treatment altered the cell cycle distribution of PGC, significantly increasing the proportion of cells in the G1 phase and decreasing those in the G2 phase, suggesting that RA may promote the transition of PGC from proliferation to differentiation. Giemsa staining further revealed that chromosomes in a subset of RA-treated PGC exhibited meiotic characteristics. Through combined RNA-seq and ATAC-seq analyses, we identified that CYP26A1, a gene involved in RA degradation, was significantly upregulated in the RA-treated group, with enhanced accessibility in its chromatin regions. This finding suggests a robust mechanism for self-regulation of RA levels within PGC, indicating that CYP26A1 may play a pivotal role in the degradation of exogenous RA in chicken PGC. This study elucidated the effects of RA on chicken PGC and provided new insights into the role of RA in germ cell differentiation. Full article

Zea mays (maize) is both an agronomically important crop and a powerful genetic model system with an extensive molecular toolkit and genomic resources. With these tools, maize is an optimal system for cytogenetic study, particularly in the investigation of chromosome segregation. Here, we review the advances made in maize chromosome segregation, specifically in the regulation and dynamic assembly of the mitotic and meiotic spindle, the inheritance and mechanisms of the abnormal chromosome variant Ab10, the regulation of chromosome–spindle interactions via the spindle assembly checkpoint, and the function of kinetochore proteins that bridge chromosomes and spindles. In this review, we discuss these processes in a species-specific context including features that are both conserved and unique to Z. mays. Additionally, we highlight new protein structure prediction tools and make use of these tools to identify several novel kinetochore and spindle assembly checkpoint proteins in Z. mays. Full article

The cell cycle, essential for growth, reproduction, and genetic stability, is regulated by a complex network of cyclins, Cyclin-Dependent Kinases (CDKs), phosphatases, and checkpoints that ensure accurate cell division. CDKs and phosphatases are crucial for controlling cell cycle progression, with CDKs promoting it and phosphatases counteracting their activity to maintain balance. The nucleolus, as a biomolecular condensate, plays a key regulatory role by serving as a hub for ribosome biogenesis and the sequestration and release of various cell cycle regulators. This phase separation characteristic of the nucleolus is vital for the specific and timely release of Cdc14, required for most essential functions of phosphatase in the cell cycle. While mitosis distributes chromosomes to daughter cells, meiosis is a specialized division process that produces gametes and introduces genetic diversity. Central to meiosis is meiotic recombination, which enhances genetic diversity by generating crossover and non-crossover products. This process begins with the introduction of double-strand breaks, which are then processed by numerous repair enzymes. Meiotic recombination and progression are regulated by proteins and feedback mechanisms. CDKs and polo-like kinase Cdc5 drive recombination through positive feedback, while phosphatases like Cdc14 are crucial for activating Yen1, a Holliday junction resolvase involved in repairing unresolved recombination intermediates in both mitosis and meiosis. Cdc14 is released from the nucleolus in a regulated manner, especially during the transition between meiosis I and II, where it helps inactivate CDK activity and promote proper chromosome segregation. This review integrates current knowledge, providing a synthesis of these interconnected processes and an overview of the mechanisms governing cell cycle regulation and meiotic recombination. Full article

Background/Objective: Balanced reciprocal translocations are structural chromosomal anomalies that involve a mutual exchange of segments between two non-homologous chromosomes with a consequent 50–80% risk of conceiving fetuses with unbalanced chromosomal anomalies. This study describes a 37-year-old woman, at 13 + 5 weeks of gestation, who is a balanced reciprocal translocation 46,XX,t(9;18)(q34;q11.2) carrier, with a high-risk non-invasive prenatal screening test, NIPT, for chromosome 18 aneuploidy. Methods: The highlighted aneuploidy was characterized with cytogenetic, FISH and SNP-array techniques. Results: Cytogenetic analysis, performed on flask-cultured amniocytes, indicated a 48,XX,+2mar karyotype on 50 metaphases. SNP array analysis showed a 15.3 Mb duplication of chromosome 18p (arr[hg19]18p11.32-p11.21(12,842-15,303,932)x4), consistent with a partial tetrasomy 18p, and a 926 kbp duplication of chromosome 9q (arr[GRCh37]9q34.3(140,118,286-141,044,489)x3), consistent with partial trisomy 9q. FISH analysis with a 9q34.3 probe was performed on flask-cultured amniocytes’ metaphases, highlighting the presence of a third signal on one of the two marker chromosomes (18p11.32-p11.21). Conclusions: The evidence of such partial aneuploidies suggests that different mutational events may be possible at meiotic segregation or probably post-meiotic segregation. The results obtained highlight the high sensitivity of the screening test, NIPT, with massive parallel sequencing, and the usefulness of cytogenetics, cytogenomics and molecular biology techniques, in synergy, to characterize and confirm positive NIPT results. Full article

Among eukaryotes, there are many examples of partial genome elimination during ontogenesis. A striking example of this phenomenon is the loss of entire avian chromosomes during meiosis, called a germline-restricted chromosome (GRC). The GRC is absent in somatic tissues but present in germ cells. It has been established that a prophase I male GRC is usually represented by a univalent surrounded by heterochromatin. In the present study, an immunocytochemical analysis of zebra finch spermatocytes was performed to focus on some details of this chromosome’s organization. For the first time, it was shown that a prophase I GRC contains the HORMAD1 protein, which participates in the formation of a full axial element. This GRC axial element has signs of a delay of core protein loading, probably owing to peculiarities of meiotic silencing of chromatin. The presence of repressive marks (H3K9me3 and H3K27me3) and the lack of RNA polymerase II, typically associated with active transcription, indicate transcriptional inactivation in the GRC body, despite the known activity of some genes of the GRC. Nevertheless, RPA and RAD51 proteins were found at some GRC sites, indicating the formation and repair of double-strand breaks on this chromosome. Our results provide new insights into the meiotic behavior and structure of a GRC. Full article

Oocyte meiotic maturation includes large-scale chromatin remodeling as well as cytoskeleton and nuclear envelope rearrangements. This review addresses the dynamics of key cytoskeletal proteins (tubulin, actin, vimentin, and cytokeratins) and nuclear envelope proteins (lamin A/C, lamin B, and the nucleoporin Nup160) in parallel with chromatin reorganization in maturing mouse oocytes. A major feature of this reorganization is the concentration of heterochromatin into a spherical perinucleolar rim called surrounded nucleolus or karyosphere. In early germinal vesicle (GV) oocytes with non-surrounded nucleolus (without karyosphere), lamins and Nup160 are at the nuclear envelope while cytoplasmic cytoskeletal proteins are outside the nucleus. At the beginning of karyosphere formation, lamins and Nup160 follow the heterochromatin relocation assembling a new spherical structure in the GV. In late GV oocytes with surrounded nucleolus (fully formed karyosphere), the nuclear envelope gradually loses its integrity and cytoplasmic cytoskeletal proteins enter the nucleus. At germinal vesicle breakdown, lamin B occupies the karyosphere interior while all the other proteins stay at the karyosphere border or connect to chromatin. In metaphase oocytes, lamin A/C surrounds the spindle, Nup160 localizes to its poles, actin and lamin B are attached to the spindle fibers, and cytoplasmic intermediate filaments associate with both the spindle fibers and the metaphase chromosomes. Full article

The speedy growth of copper oxide nanoparticle (CuO NP) manufacturing due to their wide application in industries has caused concerns due to their increased discharge into the environment from both purposeful and accidental sources. Their presence at an elevated concentration in the environment can cause potential hazards to the plant kingdom, specifically to staple food crops. However, limited research is available to determine the consequences of CuO NPs. The present study aimed to assess the morphological and cytological changes induced by CuO NPs on Pisum sativum L., a key staple food crop. Seeds of Pisum sativum were exposed to various concentrations of CuO NPs (0, 25, 50, 75, 100, and 125 ppm) for 2 h, and their effects on seed germination (SG), radicle length (RL), cell proliferation kinetics (CPK), mitotic index (MI), cell death (CD), micronucleus frequency (MNF), and chromosomal aberration frequency (CAF) were studied. The results indicate a significant reduction in SG, RL, CPK, and MI and a significant dose-dependent increase in CD, MNF, and CAF. CuO NP treatment has led to abnormal meiotic cell division, increased incidence of micronucleus frequency, and chromosomal aberration frequency. Additionally, the CuO NP-treated groups showed an increase in the percentage of aberrant meiotic cells such as laggard (LG), double bridge (DB), stickiness (STC), clumped nuclei (CNi), precocious separation (PS), single bridge (SB), and secondary association (SA). CuO NP treatment led to reductions in SG as follows: 55% at 24 h, 60.10% at 48 h, and 65% at 72 h; reductions in RL as follows: 0.55 ± 0.021 cm at 24 h, 0.67 ± 0.01 cm at 48 h, and 0.99 ± 0.02 cm at 72 h; reductions in CPK as follows: 34.98% at prophase, 7.90% at metaphase, 3.5% at anaphase, and 0.97% at telophase. It also led to a 57.45% increase in CD, a 39.87% reduction in MI, and a 60.77% increase in MNF at a higher concentration of 125 ppm. The findings of this study clearly show that CuO NPs have a genotoxic effect on the food crop plant Pisum sativum. Full article