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Journal of Biomedical Optics Jun 2024Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond...
SIGNIFICANCE
Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye.
AIM
This work is aimed to (1) achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle.
APPROACH
We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy.
RESULTS
We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization.
CONCLUSIONS
This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.
Topics: Animals; Mice; Oocytes; Humans; Female; Lasers; Spindle Apparatus; Microscopy, Confocal; Metaphase; Microscopy, Polarization
PubMed: 38812963
DOI: 10.1117/1.JBO.29.6.065002 -
Open Biology May 2024The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The...
The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.
Topics: Aurora Kinase B; Phosphorylation; Humans; Histones; Mitosis; Protein Phosphatase 1; Cell Cycle Proteins; HeLa Cells; Spindle Apparatus; Centromere; Nuclear Proteins
PubMed: 38806145
DOI: 10.1098/rsob.230460 -
Journal of Ovarian Research May 2024The key to enhancing the efficacy of antagonistic regimens in pregnancy is to better synchronize follicular growth during cycles of controlled ovarian stimulation (COS),...
BACKGROUND
The key to enhancing the efficacy of antagonistic regimens in pregnancy is to better synchronize follicular growth during cycles of controlled ovarian stimulation (COS), especially in patients with diminished ovarian reserve (DOR). During in vitro fertilization-embryo transfer (IVF-ET) treatment, luteal phase estrogen pretreatment may enhance follicular development synchronization and yield of mature oocytes. However, the effect of estrogen pretreatment in DOR patients with elevated basal follicle-stimulating hormone (FSH) levels has not been well studied.
METHODS
We retrospectively analyzed the clinical data of patients with elevated basal FSH levels and DOR (401 cycles) who underwent IVF/intracytoplasmic monosperm injection (ICSI)-assisted conception. Both groups were treated with a flexible gonadotropin-releasing hormone (GnRH) antagonist regimen and were further divided into two groups according to whether they received luteal estrogen pretreatment. There were 79 patients in the estrogen pretreatment group and 322 patients in the control group. On the second day of the menstrual cycle, gonadotropin (Gn) stimulation of the ovaries was initiated. The general characteristics, clinical, biological parameters and outcomes of the two groups were compared.
RESULTS
The basic profiles of the two groups were similar (P > 0.05). More patients in the pretreatment group showed FSH rebound after gonadotropin (Gn) initiation, resulting in a significantly higher number of Gn days and total Gn than those in the control group (P < 0.05). There was no statistically significant difference in the number of days of antagonist use, follicle output rate (FORT), number of metaphase II(MII)eggs obtained, number of Two pronuclei (2PN) fertilized, number of D quality embryos, blastocyst formation rate, fresh embryo clinical pregnancy rate, cumulative pregnancy rate, and non-transferable embryo rate between the two groups (P > 0.05).
CONCLUSIONS
The use of luteal phase estrogen pretreatment in patients with elevated basal FSH combined with DOR resulted in high FSH levels after the release of negative feedback, which was detrimental to early follicular growth, did not increase the follicular output rate, may have increased the use and duration of controlled ovarian stimulation drugs, and did not increase the number of eggs gained or improve clinical outcomes.
Topics: Humans; Female; Retrospective Studies; Adult; Follicle Stimulating Hormone; Ovulation Induction; Ovarian Reserve; Estrogens; Fertilization in Vitro; Pregnancy; Gonadotropin-Releasing Hormone; Pregnancy Rate; Embryo Transfer
PubMed: 38802887
DOI: 10.1186/s13048-024-01415-2 -
JBRA Assisted Reproduction May 2024Following the advancement of medically assisted reproduction (MAR) technology, and the rationale to extend the culture to the blastocyst stage, performing elective... (Review)
Review
Following the advancement of medically assisted reproduction (MAR) technology, and the rationale to extend the culture to the blastocyst stage, performing elective single embryo transfer (eSET), gamete quality and assessment have acquired large relevance in ART. Embryo quality is strictly correlated with gametes quality and culture conditions. Oocyte maturity assessment is therefore imperative for fertilization and embryo evolution. Mature oocytes at the metaphase II stage result in a higher fertilization rate compared to immature oocytes. Indeed, oocyte morphology evaluation represents an important and challenging task that may serve as a valuable prognostic tool for future embryo development and implantation potential. Different grading systems have been reported to assess human embryos, however, in many cases, it is still a major challenge to select the single embryo to transfer with the highest implantation potential. Further, eSET has conferred a challenge to embryologists, who must try to enhance embryo culture and selection to provide an adequate success rate, whilst reducing the overall number of embryos transferred. Above the standard morphological assessment, there are several invasive or non-invasive approaches for embryo selection such as preimplantation genetic testing, time-lapse technology, proteomics and metabolomics, as well as oxygen utilization and analysis of oxidative stress in culture medium. This short review is not designed to be a comprehensive review of all possible features that may influence oocyte quality. It does give, however, a brief overview and describes the prognostic value of the morphological characteristics of human oocytes on their developmental capacity following ART treatments.
PubMed: 38801314
DOI: 10.5935/1518-0557.20240034 -
JBRA Assisted Reproduction May 2024One of the techniques that has gained much attention is the in vitro maturation of oocytes for patients who use assisted reproduction techniques. However, its results...
OBJECTIVE
One of the techniques that has gained much attention is the in vitro maturation of oocytes for patients who use assisted reproduction techniques. However, its results are still inferior to controlled ovarian stimulation methodologies. Understanding the maturation mechanisms based on analyses can help improve this methodology's results. The work aims to identify the central genes differentially expressed in oocytes after in vitro maturation in the germinal vesicle and metaphase II stages.
METHODS
This work is a computational analysis. The entire search will be conducted using the Gene Expression Omnibus (GEO) database. To carry out and obtain the data present in the work, an advanced research search was carried out in the GEO database within the period from January 1, 2013, to January 1, 2023. A total of 27 genomic data were available in the GEO database, of which only two were used.
RESULTS
Two datasets were identified on the Gene Expression Omnibus database platform: registration data GSE158802 and GSE95477. From the analysis, we identified five downregulated and thirty-six upregulated genes; the central genes that correlated with the main gene proteins found were CLTA and PANK1.
CONCLUSIONS
There was a differential regulation of gene expression. The most central ones are related to energy capture.
PubMed: 38801311
DOI: 10.5935/1518-0557.20240030 -
BioRxiv : the Preprint Server For... May 2024Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our...
UNLABELLED
Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our ability to efficiently generate a GFP-marked acentric X chromosome fragment in neuroblasts to identify forces acting on chromosome arms that drive congression and alignment. We find acentrics efficiently align on the metaphase plate, often more rapidly than kinetochore-bearing chromosomes. Unlike intact chromosomes, the paired sister acentrics oscillate as they move to and reside on the metaphase plate in a plane distinct and significantly further from the main mass of intact chromosomes. Consequently, at anaphase onset acentrics are oriented either parallel or perpendicular to the spindle. Parallel-oriented sisters separate by sliding while those oriented perpendicularly separate via unzipping. This oscillation, together with the fact that in monopolar spindles acentrics are rapidly shunted away from the poles, indicates that distributed plus-end directed forces are primarily responsible for acentric migration. This conclusion is supported by the observation that reduction of EB1 preferentially disrupts acentric alignment. In addition, reduction of Klp3a activity, a gene required for the establishment of pole-to-pole microtubules, preferentially disrupts acentric alignment. Taken together these studies suggest that plus-end forces mediated by the outer pole-to-pole microtubules are primarily responsible for acentric metaphase alignment. Surprisingly, we find that a small fraction of sister acentrics are anti-parallel aligned indicating that the kinetochore is required to ensure parallel alignment of sister chromatids. Finally, we find induction of acentric chromosome fragments results in a global reorganization of the congressed chromosomes into a torus configuration.
ARTICLE SUMMARY
The kinetochore serves as a site for attaching microtubules and allows for successful alignment, separation, and segregation of replicated sister chromosomes during cell division. However, previous studies have revealed that sister chromosomes without kinetochores (acentrics) often align to the metaphase plate, undergo separation and segregation, and are properly transmitted to daughter cells. In this study, we discuss the forces acting on chromosomes, independent of the kinetochore, underlying their successful alignment in early mitosis.
PubMed: 38798431
DOI: 10.1101/2023.11.14.567057 -
Mathematical Biosciences May 2024This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase...
This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase is introduced and developed in the context of the cell cytoplasm and liquid crystalline structures. Prophase, prometaphase and metaphase are then briefly described in order to focus on anaphase, which is the main study of this paper. The entities involved are modelled in terms of liquid crystal defects and microtubules are represented as defect flux lines. The mathematical techniques employed make extensive use of energy considerations based on the work that was developed by Dafermos (1970) from the classical Frank-Oseen nematic liquid crystal energy (Frank, 1958; Oseen, 1933). With regard to liquid crystal theory we introduce the concept of regions of influence for defects which it is believed have important implications beyond the subject of this paper. The results of this paper align with observed biochemical phenomena and are explored in application to HeLa cells and Caenorhabditis elegans. This unified approach offers the possibility of gaining insight into various consequences of mitotic abnormalities which may result in Down syndrome, Hodgkin lymphoma, breast, prostate and various other types of cancer.
PubMed: 38795952
DOI: 10.1016/j.mbs.2024.109219 -
Genes Apr 2024Cytogenetic studies are essential in the diagnosis and follow up of patients with bone marrow failure syndromes (BMFSs), but obtaining good quality results is often...
Cytogenetic studies are essential in the diagnosis and follow up of patients with bone marrow failure syndromes (BMFSs), but obtaining good quality results is often challenging due to hypocellularity. Optical Genome Mapping (OGM), a novel technology capable of detecting most types chromosomal structural variants (SVs) at high resolution, is being increasingly used in many settings, including hematologic malignancies. Herein, we compared conventional cytogenetic techniques to OGM in 20 patients with diverse BMFSs. Twenty metaphases for the karyotype were only obtained in three subjects (15%), and no SVs were found in any of the samples. One patient with culture failure showed a gain in chromosome 1q by fluorescence in situ hybridization, which was confirmed by OGM. In contrast, OGM provided good quality results in all subjects, and SVs were detected in 14 of them (70%), mostly corresponding to cryptic submicroscopic alterations not observed by standard techniques. Therefore, OGM emerges as a powerful tool that provides complete and evaluable results in hypocellular BMFSs, reducing multiple tests into a single assay and overcoming some of the main limitations of conventional techniques. Furthermore, in addition to confirming the abnormalities detected by conventional techniques, OGM found new alterations beyond their detection limits.
Topics: Humans; Male; Female; Middle Aged; Adult; Aged; In Situ Hybridization, Fluorescence; Chromosome Mapping; Bone Marrow Failure Disorders; Chromosome Aberrations; Adolescent; Cytogenetic Analysis; Bone Marrow Diseases; Karyotyping; Young Adult
PubMed: 38790188
DOI: 10.3390/genes15050559 -
Health Science Reports May 2024Coronavirus disease 2019 (COVID-19) is a major public health problem that requires preventative vaccines. However, there is vaccine hesitancy among women of reproductive...
BACKGROUND AND AIMS
Coronavirus disease 2019 (COVID-19) is a major public health problem that requires preventative vaccines. However, there is vaccine hesitancy among women of reproductive age in Iraq. This study aimed to investigate SARS-CoV-2 vaccination effects on intracytoplasmic sperm injection (ICSI) and related fertility parameters.
METHODS
The study population comprised 54 infertile patients undergoing the ICSI procedure at a fertility clinic: vaccinated ( = 17) and non-vaccinated ( = 37). SARS-CoV-2-IgG/mL was assayed in follicular fluid from patients. Fertility parameters were assessed using oocyte and embryo quality and pregnancy outcomes between study groups, with respect to the time interval from vaccination to ova pick up.
RESULTS
There were no significant differences between non-vaccinated and vaccinated groups in respect of oocytes quality with regard to the mean number of picked up oocytes ( = 0.564), abnormal oocyte ( = 0.827), oocytes metaphase I and II ( = 0.306; = 0.165), germinal vesicles ( = 0.076), grade I, II, and III fertilized oocytes ( > 0.05), and for maturation rate ( = 0.13). There were also no significant differences ( > 0.05) in embryo quality parameters with the mean number of grade I, II, and III fertilized oocytes and the fertilization rate, the number of transferred embryo (0.086). There were no significant differences between vaccinated and unvaccinated groups with respect to follicular fluid SARS-CoV-2-IgG ( = 0.854), and pregnancy outcomes ( = 0.550).
CONCLUSIONS
The COVID-19 mRNA vaccine has no effect on ICSI, fertility parameters, and pregnancy outcome.
PubMed: 38784253
DOI: 10.1002/hsr2.2142 -
International Journal of Molecular... Apr 2024Numerous post-translational modifications are involved in oocyte maturation and embryo development. Recently, lactylation has emerged as a novel epigenetic modification...
Numerous post-translational modifications are involved in oocyte maturation and embryo development. Recently, lactylation has emerged as a novel epigenetic modification implicated in the regulation of diverse cellular processes. However, it remains unclear whether lactylation occurs during oocyte maturation and embryo development processes. Herein, the lysine lactylation (Kla) modifications were determined during mouse oocyte maturation and early embryo development by immunofluorescence staining. Exogenous lactate was supplemented to explore the consequences of modulating histone lactylation levels on oocyte maturation and embryo development processes by transcriptomics. Results demonstrated that lactylated proteins are widely present in mice with tissue- and cell-specific distribution. During mouse oocyte maturation, immunofluorescence for H3K9la, H3K14la, H4K8la, and H4K12la was most intense at the germinal vesicle (GV) stage and subsequently weakened or disappeared. Further, supplementing the culture medium with 10 mM sodium lactate elevated both the oocyte maturation rate and the histone Kla levels in GV oocytes, and there were substantial increases in Kla levels in metaphase II (MII) oocytes. It altered the transcription of molecules involved in oxidative phosphorylation. Moreover, histone lactylation levels changed dynamically during mouse early embryogenesis. Sodium lactate at 10 mM enhanced early embryo development and significantly increased lactylation, while impacting glycolytic gene transcription. This study reveals the roles of lactylation during oocyte maturation and embryo development, providing new insights to improving oocyte maturation and embryo quality.
Topics: Animals; Histones; Oocytes; Mice; Embryonic Development; Female; Protein Processing, Post-Translational; Oogenesis; Lysine; In Vitro Oocyte Maturation Techniques; Gene Expression Regulation, Developmental
PubMed: 38732042
DOI: 10.3390/ijms25094821