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Human Reproduction (Oxford, England) Jun 2024Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes...
STUDY QUESTION
Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes?
SUMMARY ANSWER
Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes.
WHAT IS KNOWN ALREADY
Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis.
STUDY DESIGN, SIZE, DURATION
This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months.
PARTICIPANTS/MATERIALS, SETTING, METHODS
All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling.
MAIN RESULTS AND THE ROLE OF CHANCE
Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining.
LARGE SCALE DATA
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633).
LIMITATIONS, REASONS FOR CAUTION
It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation.
WIDER IMPLICATIONS OF THE FINDINGS
Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.
PubMed: 38876973
DOI: 10.1093/humrep/deae126 -
Reproductive Toxicology (Elmsford, N.Y.) Jun 2024Ketoconazole (KTZ) is widely used as a fungicide, but it is also known to target steroid hormone formation which may affect female reproductive health. Our study aims to...
Ketoconazole (KTZ) is widely used as a fungicide, but it is also known to target steroid hormone formation which may affect female reproductive health. Our study aims to investigate the effects of KTZ on in vitro matured bovine cumulus-oocyte complexes (COCs), as a model for female reproductive toxicity. Cumulus cells of in vitro maturing COCs produce progesterone and pregnenolone, but exposure to 10 M KTZ effectively blocked the synthesis of these hormones. Exposure to lower concentrations of KTZ (i.e. 10 M and 10 M) had no such effect on steroidogenesis compared to the 0.1 % v/v DMSO vehicle control. Classical parameters of in vitro COC maturation, such as oocyte nuclear maturation to the metaphase II stage and expansion of the cumulus investment, were not affected by any KTZ concentration tested. Apoptosis and necrosis levels were also not altered in cumulus cells or oocytes exposed to KTZ. Moreover, oocytes exposed to KTZ during maturation showed normal cleavage and early embryo development up to day 8 post fertilization; albeit a statistically significant decrease was observed in day 8 blastocysts produced from oocytes exposed to the lowest concentration of 10 M KTZ. When unexposed mature oocytes were fertilized, followed by embryo culture for 8 days under KTZ exposure, no adverse effects in embryo cleavage and blastocyst formation were observed. In conclusion, KTZ has no major impact on in vitro bovine oocyte maturation and blastocyst formation in our study, even at concentrations blocking steroidogenesis.
PubMed: 38876429
DOI: 10.1016/j.reprotox.2024.108637 -
Toxicology in Vitro : An International... Jun 2024Fangchinoline (FA) is an alkaloid derived from the traditional Chinese medicine Fangji. Numerous studies have shown that FA has a toxic effect on various cancer cells,...
Fangchinoline (FA) is an alkaloid derived from the traditional Chinese medicine Fangji. Numerous studies have shown that FA has a toxic effect on various cancer cells, but little is known about its toxic effects on germ cells, especially oocytes. In this study, we investigated the effects of FA on mouse oocyte maturation and its potential mechanisms. Our results showed that FA did not affect meiosis resumption but inhibited the first polar body extrusion. This inhibition is not due to abnormalities at the organelle level, such as chromosomes and mitochondrial, which was proved by detection of DNA damage and reactive oxygen species. Further studies revealed that FA arrested the oocyte at the metaphase I stage, and this arrest was not caused by abnormal kinetochore-microtubule attachment or spindle assembly checkpoint activation. Instead, FA inhibits the activity of anaphase-promoting complexes (APC/C), as evidenced by the inhibition of CCNB1 degeneration. The decreased activity of APC/C may be due to a reduction in CDC25B activity as indicated by the high phosphorylation level of CDC25B (Ser323). This may further enhance Maturation-Promoting Factor (MPF) activity, which plays a critical role in meiosis. In conclusion, our study suggests that the metaphase I arrest caused by FA may be due to abnormalities in MPF and APC/C activity.
PubMed: 38876226
DOI: 10.1016/j.tiv.2024.105876 -
Molecular Human Reproduction Jun 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 data-independent acquisition, 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.
Topics: Humans; Oocytes; Female; Meiosis; Proteostasis; Adult; Proteomics; Single-Cell Analysis; Maternal Age; Proteome; Proteasome Endopeptidase Complex; Middle Aged
PubMed: 38870523
DOI: 10.1093/molehr/gaae023 -
Toxicological Sciences : An Official... Jun 2024Inhalation exposures to dihydroxyacetone (DHA) occur through spray tanning and e-cigarette aerosols. Several studies in skin models have demonstrated that millimolar...
Inhalation exposures to dihydroxyacetone (DHA) occur through spray tanning and e-cigarette aerosols. Several studies in skin models have demonstrated that millimolar doses of DHA are cytotoxic, yet the genotoxicity was unclear. We examined the genotoxicity of DHA in cell models relevant to inhalation exposures. Human bronchial epithelial cells BEAS-2B, lung carcinoma cells A549, cardiomyocyte Ac16, and hepatocellular carcinoma HepG3 were exposed to DHA, and low millimolar doses of DHA were cytotoxic. IC90 DHA doses induced cell cycle arrest in all cells except the Ac16. We examined DHA's genotoxicity using strand break markers, DNA adduct detection by Repair Assisted Damage Detection (RADD), metaphase spreads, and a forward mutation assay for mutagenesis. Similar to results for skin, DHA did not induce significant levels of strand breaks. However, RADD revealed DNA adducts were induced 24 h after DHA exposure, with BEAS-2B and Ac16 showing oxidative lesions and A549 and HepG3 showing crosslink-type lesions. Yet, only low levels of reactive oxygen species or advanced glycation end products were detected after DHA exposure. Metaphase spreads revealed significant increases in chromosomal aberrations in the BEAS-2B and HepG3 with corresponding changes in ploidy. Finally, we confirmed the mutagenesis observed using the supF reporter plasmid. DHA increased the mutation frequency, consistent with methylmethane sulfonate, a mutagen and clastogen. These data demonstrate DHA is a clastogen, inducing cell-specific genotoxicity and chromosomal instability. The specific genotoxicity measured in the BEAS-2B in this study suggests that inhalation exposures pose health risks to vapers, requiring further investigation.
PubMed: 38867704
DOI: 10.1093/toxsci/kfae075 -
Zhongguo Zhen Jiu = Chinese Acupuncture... Jun 2024To assess the effect of acupuncture therapy (acupuncture for regulating menstruation and promoting pregnancy) on pregnancy outcomes in patients with diminished ovarian...
OBJECTIVE
To assess the effect of acupuncture therapy (acupuncture for regulating menstruation and promoting pregnancy) on pregnancy outcomes in patients with diminished ovarian reserve (DOR) undergoing in vitro fertilization-embryo transfer (IVF-ET).
METHODS
Eighty women with DOR were divided into an observation group (40 cases, 1 case dropped out) and a control group (40 cases, 1 case dropped out) according to whether acupuncture therapy was given or not. In the control group, IVF-ET was delivered. In the observation group, before IVF-ET, acupuncture therapy was given. Two groups of acupoints were used alternatively, including Baihui (GV 20), Shenting (GV 24), Benshen (GB 13), Zhongwan (CV 12), Guanyuan (CV 4), and bilateral Tianshu (ST 25), Shenshu (BL 23), Ciliao (BL 32), etc. Acupuncture was operated once every other day, three interventions a week, for 12 weeks. The primary outcome was clinical pregnancy rate (CPR). Secondary outcomes included the total days and amount of gonadotropin (Gn) used, the number of oocytes retrieved, the number of oocytes in metaphase of second meiosis (MⅡ), the number of transferable embryos, the number of high-quality embryos, the cycle cancellation rate, the positive rate of human choriogonadotropin (HCG), the embryo implantation rate, live birth rate (LBR), the basal serum levels of sex hormones (follicular stimulating hormone [FSH], estradiol (E2), FSH/luteinizing hormone [LH]) and antral follicle count (AFC).
RESULTS
CPR in the observation group was higher than that in the control group (53.8% [21/39] . 17.9% [7/39], <0.05). The results of the number of oocytes retrieved, the number of oocytes in MⅡ, the number of transferable embryos, the number of high-quality embryos, the positive rate of HCG, the embryo implantation rate, and LBR in the observation group were higher than those in the control group (<0.05). The serum level of FSH and FSH/LH in the observation group were lower thau those in the control group (<0.05). The differences were not significant statistically in the total days and amount of Gn used, the cycle cancellation rate, serum level of E2 and AFC between the two groups (>0.05). Logic regression analysis showed that CPR increased in the observation group when compared with that of the control group ( = 5.33, 95%: 1.90-14.97, = 0.001).
CONCLUSION
Acupuncture can improve the pregnancy outcomes of DOR women undergoing IVF-ET.
Topics: Humans; Female; Adult; Pregnancy; Fertilization in Vitro; Acupuncture Therapy; Ovarian Reserve; Embryo Transfer; Pregnancy Outcome; Infertility, Female; Cohort Studies; Acupuncture Points; Pregnancy Rate; Young Adult
PubMed: 38867627
DOI: 10.13703/j.0255-2930.20230716-k0002 -
Developmental Cell Jun 2024The forces that orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers...
The forces that orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers to measure the force on human mitotic spindles. Combining the spindle's measured resistance to rotation, the speed at which it rotates after laser ablating astral microtubules, and estimates of the number of ablated microtubules reveals that each microtubule contacting the cell cortex is subject to ∼5 pN of pulling force, suggesting that each is pulled on by an individual dynein motor. We find that the concentration of dynein at the cell cortex and extent of dynein clustering are key determinants of the spindle's resistance to rotation, with little contribution from cytoplasmic viscosity, which we explain using a biophysically based mathematical model. This work reveals how pulling forces on astral microtubules determine the mechanics of spindle orientation and demonstrates the central role of cortical dynein clustering.
PubMed: 38866013
DOI: 10.1016/j.devcel.2024.05.022 -
Ecotoxicology and Environmental Safety Jul 20242-Ethylhexyl diphenyl phosphate (EHDPP) is a representative organophosphorus flame retardant (OPFR) that has garnered attention due to its widespread use and potential...
2-Ethylhexyl diphenyl phosphate (EHDPP) is a representative organophosphorus flame retardant (OPFR) that has garnered attention due to its widespread use and potential adverse effects. EHDPP exhibits cytotoxicity, genotoxicity, developmental toxicity, and endocrine disruption. However, the toxicity of EHDPP in mammalian oocytes and the underlying mechanisms remain poorly understood. Melatonin is a natural free radical scavenger that has demonstrated cytoprotective properties. In this study, we investigated the effect of EHDPP on mouse oocytes in vitro culture system and evaluated the rescue effect of melatonin on oocytes exposed to EHDPP. Our results indicated that EHDPP disrupted oocyte maturation, resulting in the majority of oocytes arrested at the metaphase I (MI) stage, accompanied by cytoskeletal damage and elevated levels of reactive oxygen species (ROS). Nevertheless, melatonin supplementation partially rescued EHDPP-induced mouse oocyte maturation impairment. Results of single-cell RNA sequencing (scRNA-seq) analysis elucidated potential mechanisms underlying these protective effects. According to the results of scRNA-seq, we conducted further tests and found that EHDPP primarily disrupts mitochondrial distribution and function, kinetochore-microtubule (K-MT) attachment, DNA damage, apoptosis, and histone modification, which were rescued upon the supplementation of melatonin. This study reveals the mechanisms of EHDPP on female reproduction and indicates the efficacy of melatonin as a therapeutic intervention for EHDPP-induced defects in mouse oocytes.
Topics: Animals; Melatonin; Mice; Oocytes; Mitochondria; Female; Flame Retardants; Reactive Oxygen Species; Organophosphates; DNA Damage; Apoptosis; Organophosphorus Compounds
PubMed: 38865937
DOI: 10.1016/j.ecoenv.2024.116559 -
Proceedings of the National Academy of... Jun 2024Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation...
Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on but not on , and , while the unloading of RNF212B at the end of pachynema is dependent on and . Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for and exhibit an identical phenotype to that of single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.
Topics: Animals; Mice; Meiosis; Male; Female; Ubiquitin-Protein Ligases; Crossing Over, Genetic; Chromosome Pairing; Poly-ADP-Ribose Binding Proteins; Mice, Knockout; Humans; Ligases
PubMed: 38865271
DOI: 10.1073/pnas.2320995121 -
Molecular Biology of the Cell Jun 2024The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein...
The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and co-orientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that the SPC105R C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for lateral microtubule attachments and bi-orientation of homologs, which are critical for accurate chromosome segregation in meiosis I.
PubMed: 38865189
DOI: 10.1091/mbc.E24-02-0067