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BioRxiv : the Preprint Server For... May 2024The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While...
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.
PubMed: 38826327
DOI: 10.1101/2024.05.22.595167 -
The Biological Bulletin Aug 2023AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, (), a () gene, identified in a subtractive screen for maternal ascidian cDNAs in , an...
AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, (), a () gene, identified in a subtractive screen for maternal ascidian cDNAs in , an ascidian species with a tadpole larva. The gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed but is not detected in eggs or embryos of the closely related tailless species . There is a large insertion in in the genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the gene found in other ascidians. Thus, the ascidian genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that is lacking the tyrosine kinase domain in the tailless , although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic tyrosine kinase is another described pseudogene from and appears not to be necessary for adult development.
Topics: Animals; Urochordata; Protein-Tyrosine Kinases; Amino Acid Sequence; Zygote; Pseudogenes; Phylogeny
PubMed: 38820291
DOI: 10.1086/730536 -
Frontiers in Oncology 2024To review the outcome of PGT-M in hormone-related hereditary tumor syndrome and evaluate the effect of ovarian induction on tumor growth in those patients.
PURPOSE
To review the outcome of PGT-M in hormone-related hereditary tumor syndrome and evaluate the effect of ovarian induction on tumor growth in those patients.
METHODS
Medical records of PGT-M were retrospectively analyzed in patients with hormone-related heritage tumors in our reproductive center. A total of eleven women with hereditary breast and ovarian cancer (HBOC) (including BRCA1/2 mutation carriers), and Lynch syndrome (including MMR gene mutation carriers) were included. Thirteen IVF/PGT-M cycles were performed. Eleven for PGT-M and two for fertility preservation. The ovulation protocol, numbers of oocytes retrieved and two pronuclei (2PN) zygotes, PGT-M results, and clinical outcomes were analyzed. Tumor progression was also estimated by comparing transvaginal ultrasound (TVS), MR, CT, or colonoscopy according to the follow-up requirements of different tumors.
RESULTS
Eleven IVF/PGT-M cycles were performed with an antagonist protocol; Two cycles were performed with a mild stimulation protocol. The total dose of gonadotropin (Gn) was 1827 IU per patient (range from 1200 to 2625 IU). The median number of oocytes retrieved was 13 (range from 4 to 30), and the median number of 2PN zygotes was 8 (range from 2 to 16). A total of 32 embryos underwent PGT-M, and 9 (28.1%) embryos were suitable for transfer. Six transfer cycles were performed, and 5 cycles got clinical pregnancy (83%) with five newborns (83%). The follow-up examinations conducted 10-18 months after PGT-M/delivery revealed no new lesions or tumor progression.
CONCLUSION
PGT-M results can provide important information for improving the consultation of hormone-related heritage tumor patients regarding their fertility preservation and reproductive options. Ovarian induction for women with hormone-related hereditary tumor syndrome is not associated with tumor progression.
PubMed: 38800375
DOI: 10.3389/fonc.2024.1378019 -
BioRxiv : the Preprint Server For... May 2024Regulation of transcription during embryogenesis is key to development and differentiation. To study transcript expression throughout embryogenesis at single-molecule...
Regulation of transcription during embryogenesis is key to development and differentiation. To study transcript expression throughout embryogenesis at single-molecule resolution, we developed a high-throughput single-molecule fluorescence in situ hybridization (smFISH) method that relies on computational methods to developmentally stage embryos and quantify individual mRNA molecules in single embryos. We applied our system to , a zygotically transcribed gene essential for hermaphrodite development and dosage compensation. We found that is rapidly activated during early embryogenesis by increasing both the number of mRNAs produced per transcription site and the frequency of sites engaged in transcription. Knockdown of and , a subunit of the dosage compensation complex (DCC), increased the number of active transcription sites for the X chromosomal gene but not the autosomal gene , suggesting that the DCC reduces the frequency of transcription. The temporal resolution from staging of embryos showed that the deletion of a single DCC recruitment element near the gene causes higher mRNA expression after the start of dosage compensation, which could not be resolved using mRNAseq from mixed-stage embryos. In summary, we have established a computational approach to quantify temporal regulation of transcription throughout embryogenesis and demonstrated its potential to provide new insights into developmental gene regulation.
PubMed: 38798598
DOI: 10.1101/2024.05.15.594414 -
Vaccines Apr 2024The WHO reported an estimated 249 million malaria cases and 608,000 malaria deaths in 85 countries in 2022. A total of 94% of malaria deaths occurred in Africa, 80% of... (Review)
Review
The WHO reported an estimated 249 million malaria cases and 608,000 malaria deaths in 85 countries in 2022. A total of 94% of malaria deaths occurred in Africa, 80% of which were children under 5. In other words, one child dies every minute from malaria. The RTS,S/AS01 malaria vaccine, which uses the circumsporozoite protein (CSP) to target sporozoite infection of the liver, achieved modest efficacy. The Malaria Vaccine Implementation Program (MVIP), coordinated by the WHO and completed at the end of 2023, found that immunization reduced mortality by only 13%. To further reduce malaria death, the development of a more effective malaria vaccine is a high priority. Three malaria vaccine targets being considered are the sporozoite liver infection (pre-erythrocytic stage), the merozoite red blood cell infection (asexual erythrocytic stage), and the gamete/zygote mosquito infection (sexual/transmission stage). These targets involve specific ligand-receptor interactions. However, most current malaria vaccine candidates that target two major parasite population bottlenecks, liver infection, and mosquito midgut infection, do not focus on such parasite ligands. Here, we evaluate the potential of newly identified parasite ligands with a phage peptide-display technique as novel malaria vaccine antigens.
PubMed: 38793735
DOI: 10.3390/vaccines12050484 -
Antioxidants (Basel, Switzerland) Apr 2024Various antioxidants are tested to improve the viability and development of cryopreserved oocytes, due to their known positive health effects. The aim of this study was...
Various antioxidants are tested to improve the viability and development of cryopreserved oocytes, due to their known positive health effects. The aim of this study was to find whether astaxanthin (AX), a xanthophyll carotenoid, could mitigate deteriorations that occurred during the vitrification/warming process in bovine oocytes. Astaxanthin (2.5 µM) was added to the maturation medium during the post-warm recovery period of vitrified oocytes for 3 h. Afterward, the oocytes were fertilized in vitro using frozen bull semen and presumptive zygotes were cultured in the B2 Menezo medium in a co-culture with BRL-1 cells at 38.5 °C and 5% CO until the blastocyst stage. AX addition significantly reduced ROS formation, lipid peroxidation, and lysosomal activity, while increasing mitochondrial activity in vitrified oocytes. Although the effect of AX on embryo development was not observed, it stimulated cell proliferation in the blastocysts derived from vitrified oocytes and improved their quality by upregulation or downregulation of some genes related to apoptosis (, ), oxidative stress (, ), and development () compared to the vitrified group without AX. Therefore, the antioxidant properties of astaxanthin even during short exposure to bovine vitrified/warmed oocytes resulted in improved blastocyst quality comparable to those from fresh oocytes.
PubMed: 38790660
DOI: 10.3390/antiox13050556 -
Cells May 2024The possibility of detecting the developmental competence of individually cultured embryos through analysis of spent media is a major current trend in an ART setting....
The possibility of detecting the developmental competence of individually cultured embryos through analysis of spent media is a major current trend in an ART setting. However, individual embryo culture is detrimental compared with high-density group culture due to the reduced concentration of putative embryotropins. The main aim of this study was to identify an individual culture system that is not detrimental over high-density group culture in the bovine model. Blastocyst rates and competence were investigated in a conventional (GC) group, semi-confined group (MG), and individual culture (MS) in a commercial microwell device. Main findings showed that: (1) individual embryos can be continuously cultured for 7 days in ~70 nL microwells (MS) without detrimental effects compared with the GC and MG; (2) MS and MG blastocysts had a reduced number of TUNEL-positive cells compared to GC blastocysts; (3) though blastocyst mean cell numbers, mitochondrial activity, and lipid content were not different among the three culture conditions, MS blastocysts had a higher frequency of small-sized lipid droplets and a reduced mean droplet diameter compared with GC and MG blastocysts. Overall, findings open the way to optimize the development and competence of single embryos in an ART setting.
Topics: Animals; Cattle; Blastocyst; Zygote; Embryo Culture Techniques; Embryonic Development; Female; Mitochondria
PubMed: 38786090
DOI: 10.3390/cells13100868 -
Current Issues in Molecular Biology Apr 2024Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological...
Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized-egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the gene without any off-target effect detected. The production of knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats.
PubMed: 38785502
DOI: 10.3390/cimb46050234 -
IScience Jun 2024Maternal-to-zygotic transition (MZT) is central to early embryogenesis. However, its underlying molecular mechanisms are still not well described. Here, we revealed the...
Maternal-to-zygotic transition (MZT) is central to early embryogenesis. However, its underlying molecular mechanisms are still not well described. Here, we revealed the expression dynamics of 5,000 proteins across four stages of zebrafish embryos during MZT, representing one of the most systematic surveys of proteome landscape of the zebrafish embryos during MZT. Nearly 700 proteins were differentially expressed and were divided into six clusters according to their expression patterns. The proteome expression profiles accurately reflect the main events that happen during the MZT, i.e., zygotic genome activation (ZGA), clearance of maternal mRNAs, and initiation of cellular differentiation and organogenesis. MZT is modulated by many proteins at multiple levels in a collaborative fashion, i.e., transcription factors, histones, histone-modifying enzymes, RNA helicases, and P-body proteins. Significant discrepancies were discovered between zebrafish proteome and transcriptome profiles during the MZT. The proteome dynamics database will be a valuable resource for bettering our understanding of MZT.
PubMed: 38784018
DOI: 10.1016/j.isci.2024.109944 -
HGG Advances May 2024While most dizygotic twins have a dichorionic placenta, rare cases of dizygotic twins with a monochorionic placenta have been reported. The monochorionic placenta in...
While most dizygotic twins have a dichorionic placenta, rare cases of dizygotic twins with a monochorionic placenta have been reported. The monochorionic placenta in dizygotic twins allows in utero exchange of embryonic cells, resulting in chimerism in the twins. In practice, this chimerism is incidentally identified in mixed ABO blood types or in the presence of cells with a discordant sex chromosome. Here, we applied whole-genome sequencing to one triplet and one twin family to precisely understand their zygotic compositions, using millions of genomic variants as barcodes of zygotic origins. Peripheral blood showed asymmetrical contributions from two sister zygotes, where one of the zygotes was the major clone in both twins. Single-cell RNA sequencing of peripheral blood tissues further showed differential contributions from the two sister zygotes across blood cell types. In contrast, buccal tissues were pure in genetic composition, suggesting that in utero cellular exchanges were confined to the blood tissues. Our study illustrates the cellular history of twinning during human development, which is critical for managing the health of chimeric individuals in the era of genomic medicine.
PubMed: 38773773
DOI: 10.1016/j.xhgg.2024.100301