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Journal of Ovarian Research Jun 2024Agar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of...
BACKGROUND
Agar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP.
METHODS
Human metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software.
RESULTS
We identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP.
CONCLUSIONS
Our findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest.
Topics: Humans; Oocytes; Female; Zona Pellucida; Transcriptome; Single-Cell Analysis; Sequence Analysis, RNA; Gene Expression Profiling; Adult
PubMed: 38926883
DOI: 10.1186/s13048-024-01463-8 -
Current Protocols Jun 2024Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in...
Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in principle, it is based on denaturation and hybridization of a DNA probe and its complementary sequence; however, it is subject to continuous optimization. Here we share how in-house FISH can be optimized using different control tissues to visualize and ultimately validate common and novel genetic abnormalities unearthed by next-generation sequencing (NGS). Seven specific FISH probes were designed and labeled, and conditions for eight tissue types and one patient-derived tumor organoid were optimized. Formalin-fixed paraffin-embedded (FFPE) tissue slides were used for each experiment. Slides were first deparaffinized, then placed in a pretreatment solution followed by a digestion step. In-house FISH probes were then added to the tissue to be denatured and hybridized, and then washed twice. To obtain optimal results, probe concentration, pepsin incubation time, denaturation, and the two post-hybridization washes were optimized for each sample. By modifying the above conditions, all FISH experiments were optimized in separate tissue types to investigate specific genomic alterations in tumors arising in those tissues. Signals were clear and distinct, allowing for visualization of the selected probes. Following this protocol, our lab has quickly optimized 11 directly labeled in-house FISH probes to support genetic aberrations nominated by NGS, including most recent discoveries through whole-genome sequencing analyses. We describe a robust approach of how to advance in-house labeled FISH probes. By following these guidelines, reliable and reproducible FISH results can be obtained to interrogate FFPE slides from benign, tumor tissues, and patient-derived tumor organoid specimens. This is of most relevance in the era of NGS and precision oncology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Metaphase FISH optimization Support Protocol 1: In-house probe labeling and preparation Support Protocol 2: Metaphase spread preparation Basic Protocol 2: Optimization of FISH on formalin-fixed paraffin-embedded tissue.
Topics: In Situ Hybridization, Fluorescence; Humans; Precision Medicine; Paraffin Embedding; Neoplasms; High-Throughput Nucleotide Sequencing; DNA Probes
PubMed: 38923415
DOI: 10.1002/cpz1.1093 -
PLoS Genetics Jun 2024Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events...
Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events such as homolog pairing and chromosome segregation are not fully understood in many species. Here, we employ Oligopaint DNA FISH to investigate mechanisms of meiotic homolog pairing and chromosome segregation in the holocentric pantry moth, Plodia interpunctella, and compare our findings to new and previous studies in the silkworm moth, Bombyx mori, which diverged from P. interpunctella over 100 million years ago. We find that pairing in both Bombyx and Plodia spermatogenesis is initiated at gene-rich chromosome ends. Additionally, both species form rod shaped cruciform-like bivalents at metaphase I. However, unlike the telomere-oriented chromosome segregation mechanism observed in Bombyx, Plodia can orient bivalents in multiple different ways at metaphase I. Surprisingly, in both species we find that kinetochores consistently assemble at non-telomeric loci toward the centers of chromosomes regardless of where chromosome centers are located in the bivalent. Additionally, sister kinetochores do not seem to be paired in these species. Instead, four distinct kinetochores are easily observed at metaphase I. Despite this, we find clear end-on microtubule attachments and not lateral microtubule attachments connecting these separated kinetochores to the meiotic spindle. These findings challenge the classical view of segregation where paired, poleward-facing kinetochores are required for accurate homolog separation in meiosis I. Our studies here highlight the importance of exploring fundamental processes in non-model systems, as employing novel organisms can lead to the discovery of novel biology.
PubMed: 38913752
DOI: 10.1371/journal.pgen.1011329 -
Methods in Molecular Biology (Clifton,... 2024While interphase and metaphase-directed molecular cytogenetics is a standard technique in routine tumor (cyto)genetics, fluorescence in situ hybridization-based banding...
While interphase and metaphase-directed molecular cytogenetics is a standard technique in routine tumor (cyto)genetics, fluorescence in situ hybridization-based banding (FISH-banding) approaches are less commonly applied. In research FISH-banding showed its excellence in the characterization of simple and complex chromosomal aberrations; however, in routine settings, it is still only little applied. The main argument against FISH-banding is, that it shall be associated with comparatively high costs. However, if applied advisedly FISH-banding can even save costs, as in one or two chromosome-specific FISH experiments; otherwise, cryptic, not resolvable chromosomal rearrangements may be resolved quickly. Here the protocol for the only yet commercially available FISH-banding approach-the multicolor banding (MCB/ mBAND)-is outlined.
Topics: In Situ Hybridization, Fluorescence; Humans; Neoplasms; Chromosome Banding; Chromosome Aberrations
PubMed: 38913311
DOI: 10.1007/978-1-0716-3946-7_11 -
Methods in Molecular Biology (Clifton,... 2024Chromosome banding can be defined as the lengthwise variation in staining properties along a chromosome stained with a dye. Chromosome banding became more practical in...
Chromosome banding can be defined as the lengthwise variation in staining properties along a chromosome stained with a dye. Chromosome banding became more practical in the early 1970s and is an essential technique used in karyotyping to identify human chromosomes for both clinical and research purposes. Most importantly, karyotyping is now considered a mandatory investigation of all newly diagnosed leukemias. Some banding methods, such as Giemsa (G)-, reverse (R)-, and centromere (C)-banding, still contribute greatly by being used as a routine procedure in clinical cytogenetic laboratory nowadays. Each chromosome has a unique sequence of bar code-like stripes, allowing the identification of individual homologues and the recognition of structural abnormalities through analyzing the disruption of the normal banding pattern at specific landmarks, regions, and bands as described in the ideogram. Since the quality of metaphases obtained from malignant cells is generally inferior to normal constitutional cells for karyotyping, a practical and accurate chromosome identification training guide is indispensable for a trainee or newly employed cytogenetic technologist in a cancer cytogenetic laboratory. The most common and currently used banding methods and chromosome recognition guide for distinguishable bands of each chromosome are described in detail in this chapter with an aim to facilitate quick and accurate karyotyping in cancer cells.
Topics: Humans; Chromosome Banding; Karyotyping; Chromosomes, Human; Metaphase
PubMed: 38913307
DOI: 10.1007/978-1-0716-3946-7_7 -
Animal Reproduction 2024In reproductive technologies, uncovering the molecular aspects of oocyte and embryo competence under different conditions is crucial for refining protocols and enhancing...
In reproductive technologies, uncovering the molecular aspects of oocyte and embryo competence under different conditions is crucial for refining protocols and enhancing efficiency. RNA-seq generates high-throughput data and provides transcriptomes that can undergo additional computational analyses. This study presented the transcriptomic profiles of matured oocytes and blastocysts produced from buffalo crossbred (), coupled with gene co-expression and module preservation analysis. Cumulus Oophorus Complexes, obtained from slaughterhouse-derived ovaries, were subjected to maturation to yield metaphase II oocytes (616) or followed fertilization and culture to yield blastocysts for sequencing (526). Oocyte maturation (72%, ±3.34 sd) and embryo development (21.3%, ±4.18 sd) rates were obtained from three embryo production routines following standard protocols. Sequencing of 410 metaphase II oocytes and 70 hatched blastocysts (grade 1 and 2) identified a total of 13,976 genes, with 62% being ubiquitously expressed (8,649). Among them, the differentially expressed genes (4,153) and the strongly variable genes with the higher expression (fold-change above 11) were highlighted in oocytes (, , , , and ) and blastocysts (, , , , , and ) as representative indicators of molecular quality. Additionally, genes exclusively found in oocytes (224) and blastocysts (2,200) with specific biological functions were identified. Gene co-expression network and module preservation analysis revealed strong preservation of functional modules related to exosome components, steroid metabolism, cell proliferation, and morphogenesis. However, cell cycle and amino acid transport modules exhibited weak preservation, which may reflect differences in embryo development kinetics and the activation of cell signaling pathways between buffalo and bovine. This comprehensive transcriptomic profile serves as a valuable resource for assessing the molecular quality of buffalo oocytes and embryos in future embryo production assays.
PubMed: 38912163
DOI: 10.1590/1984-3143-AR2023-0131 -
International Journal of Biological... 2024Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61...
Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.
Topics: Protein Serine-Threonine Kinases; Humans; Polo-Like Kinase 1; Mitosis; Cell Cycle Proteins; Proto-Oncogene Proteins; Cysteine-Rich Protein 61; Microtubules; F-Box-WD Repeat-Containing Protein 7; HeLa Cells; Phosphorylation; Ubiquitin-Protein Ligases; Microtubule-Associated Proteins
PubMed: 38904029
DOI: 10.7150/ijbs.93335 -
BioRxiv : the Preprint Server For... Feb 2024During meiosis, homologous chromosomes segregate so that alleles are transmitted equally to haploid gametes, following Mendel's Law of Segregation. However, some selfish...
During meiosis, homologous chromosomes segregate so that alleles are transmitted equally to haploid gametes, following Mendel's Law of Segregation. However, some selfish genetic elements drive in meiosis to distort the transmission ratio and increase their representation in gametes. The established paradigms for drive are fundamentally different for female vs male meiosis. In male meiosis, selfish elements typically kill gametes that do not contain them. In female meiosis, killing is predetermined, and selfish elements bias their segregation to the single surviving gamete (i.e., the egg in animal meiosis). Here we show that a selfish element on mouse chromosome 2, , drives using a hybrid mechanism in female meiosis, incorporating elements of both male and female drivers. If is destined for the polar body, it manipulates segregation to sabotage the egg by causing aneuploidy that is subsequently lethal in the embryo, so that surviving progeny preferentially contain . In heterozygous females, orients randomly on the metaphase spindle but lags during anaphase and preferentially remains in the egg, regardless of its initial orientation. Thus, the egg genotype is either euploid with or aneuploid with both homologs of chromosome 2, with only the former generating viable embryos. Consistent with this model, heterozygous females produce eggs with increased aneuploidy for chromosome 2, increased embryonic lethality, and increased transmission of . In contrast to a male meiotic driver, which kills its sister gametes produced as daughter cells in the same meiosis, eliminates "cousins" produced from meioses in which it should have been excluded from the egg.
PubMed: 38903120
DOI: 10.1101/2024.02.22.581453 -
BioRxiv : the Preprint Server For... May 2024Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this...
Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.
PubMed: 38903107
DOI: 10.1101/2024.05.23.595547 -
Communications Biology Jun 2024AMPK is a well-known energy sensor regulating cellular metabolism. Metabolic disorders such as obesity and diabetes are considered detrimental factors that reduce...
AMPK is a well-known energy sensor regulating cellular metabolism. Metabolic disorders such as obesity and diabetes are considered detrimental factors that reduce fecundity. Here, we show that pharmacologically induced in vitro activation (by metformin) or inhibition (by dorsomorphin) of the AMPK pathway inhibits or promotes activation of ovarian primordial follicles in cultured murine ovaries and human ovarian cortical chips. In mice, activation of primordial follicles in dorsomorphin in vitro-treated ovaries reduces AMPK activation and upregulates Wnt and FOXO genes, which, interestingly, is associated with decreased phosphorylation of β-catenin. The dorsomorphin-treated ovaries remain of high quality, with no detectable difference in reactive oxygen species production, apoptosis or mitochondrial cytochrome c oxidase activity, suggesting safe activation. Subsequent maturation of in vitro-treated follicles, using a 3D alginate cell culture system, results in mature metaphase eggs with protruding polar bodies. These findings demonstrate that the AMPK pathway can safely regulate primordial follicles by modulating Wnt and FOXO genes, and reduce β-catenin phosphorylation.
Topics: Animals; Female; Mice; Ovarian Follicle; AMP-Activated Protein Kinases; Pyrimidines; Pyrazoles; Humans; Up-Regulation; Forkhead Transcription Factors; Wnt Proteins; beta Catenin; Phosphorylation; Mice, Inbred C57BL; Metformin; Wnt Signaling Pathway
PubMed: 38902324
DOI: 10.1038/s42003-024-06418-9