Germinal Vesicle Breakdown Research Articles

Increased in vitro production of bovine embryos resulting from oocyte maturation in the presence of triciribine, a specific inhibitor of AKT

The aim of this study was to evaluate the effect of different concentrations of triciribine, a selective Akt inhibitor, on various aspects of oocyte maturation and on the IVF of bovine embryos. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with: 0 (control), 1, 5, 10, and 20 μM of triciribine. The nuclear maturation was assessed by staining with acetic orcein, while the cytoplasmic maturation was evaluated by mitochondrial (MitoTracker® Red CMXRos) and lipid droplets distribution (LipidTOX). COCs were fertilized in vitro and cultured for nine days. Cleavage rates, blastocyst production, and hatching rates were determined on days three, seven, and nine of in vitro culture, respectively. Oocytes from COCs treated with 1 μM of triciribine were stained at 3, 6, and 9 h of IVM to determine the inhibitor's involvement in germinal vesicle breakdown. Analysis of variance (ANOVA) of the data was performed and the means were compared using the SNK test at a 5 % significance level. Exposure of COCs to 1, 5, and 10 μM of triciribine did not alter the number of matured oocytes (P < 0.05), a concentration of 20 μM reduced the number of oocytes in MII with a consequent increase in oocytes in MI (P < 0.05). This concentration markedly reduced the number of oocytes with peripheral cortical granules and the rates of cleavage and blastocysts (P < 0.05). On the other hand, when COCs were matured in the presence of 1 μM, there was an increase in the blastocyst rate (P < 0.05), but without altering the timing of meiosis resumption (P < 0.05). It is concluded that the Akt pathway participates in the nuclear and cytoplasmic events of in vitro maturation of bovine oocytes, but through mechanisms that do not interfere with germinal vesicle breakdown. Modulation of Akt activity in bovine COCs during IVM with 1 μM of triciribine increases the in vitro production of bovine embryos.

How the Oocyte Nucleolus Is Turned into a Karyosphere: The Role of Heterochromatin and Structural Proteins.

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.

Methylmercury chloride inhibits meiotic maturation of mouse oocytes in vitro by disrupting the cytoskeleton

Methylmercury chloride (MMC) is a persistent heavy metal contaminant that can bioaccumulate in humans via the food chain, exerting detrimental effects on health. Nevertheless, the specific influence of MMC on oocyte meiotic maturation has yet to be elucidated. This research demonstrated that MMC exposure during the in vitro cultivation of mouse oocytes did not influence germinal vesicle breakdown but markedly decreased oocyte maturation rates. Subsequent analysis indicated that MMC exposure resulted in aberrant spindle morphology and disorganized chromosome alignment, alongside continuous activation of the spindle assembly checkpoint (SAC). However, MMC exposure didn't alter the localization pattern of microtubule-organizing center-associated proteins. MMC exposure considerably diminished the acetylation level of α-tubulin, signifying reduced microtubule stability. Additionally, MMC exposure disrupted the dynamic alterations of F-actin. MMC exposure didn't affect mitochondrial localization, mitochondrial membrane potential, adenosine triphosphate content or the concentrations of reactive oxygen species. Nonetheless, MMC exposure triggered DNA damage and modified histone modification levels. Consequently, the defects in oocyte maturation induced by MMC exposure can be attributed to impaired cytoskeleton dynamics and DNA damage. This study offers the first comprehensive elucidation of the negative impacts of MMC on oocyte maturation, highlighting the potential reproductive health risks associated with MMC exposure.

CtIP regulates G2/M transition and bipolar spindle assembly during mouse oocyte meiosis

CtBP-interacting protein (CtIP) is known for its multifaceted roles in DNA repair and genomic stability, directing the homologous recombination-mediated DNA double-stranded break (DSBs) repair pathway via DNA end resection, an essential error-free repair process vital for genome stability. Mammalian oocytes are highly prone to DNA damage accumulation due to prolonged G2/prophase arrest. Here, we explore the functions of CtIP in meiotic cell cycle regulation via a mouse oocyte model. Depletion of CtIP by siRNA injection results in delayed germinal vesicle breakdown and failed polar body extrusion. Mechanistically, CtIP deficiency increases DNA damage and decreases the expression and nuclear entry of CCNB1, resulting in marked impairment of meiotic resumption, which can be rescued by exogenous CCNB1 overexpression. Furthermore, depletion of CtIP disrupts MTOCs coalescence at spindle poles as indicated by failed accumulation of γ-tubulin, p-Aurora kinase A, Kif2A, and TPX2, leading to abnormal spindle assembly and prometaphase arrest. These results provide valuable insights into the important roles of CtIP in the G2/M checkpoint and spindle assembly in mouse oocyte meiotic cell cycle regulation.

Canine oocyte nuclear maturation with Nano-ozone (NZS) supplementation: The alterations of antioxidant, and oxidant status and CDK1, cyclin B1 expressions

This study aims to evaluate the effects of nano-ozone solution (NZS) on canine oocyte nuclear maturation, associated with the alterations of antioxidant and oxidant status and cyclin-dependent kinase 1 (CDK1), cyclin B1 gene expressions. Oocytes were cultured in four distinct concentrations of NZS (0.5, 1, 2, and 5 µg/mL) and parthenogenetically activated. The rates of oocytes arrested at the Germinal Vesicle (GV), Germinal Vesicle Breakdown (GVBD), Metaphase I (MI), and Metaphase II (MII) stages were statistically different among groups (P < 0.05). The oocytes cultured in 1 µg/mL NZS yielded the best oocyte maturation rate at the MI and MII stages; however, the lowest maturation and high degeneration rates were observed in Group E. The measurements of Malondialdehyde (MDA), reduced Glutathione (GSH), Superoxide Dismutase (SOD), and Ferric Reducing/Antioxidant Power assay (FRAP) were performed from IVM culture media. No statistical difference was observed in SOD and MDA results (P > 0.05). GSH levels were statistically significant between Group A-Group E (p = 0.003), Group B-Group E (p = 0.045), and Group E-Group D (p = 0.021). The culture media in Group D and Group E had high FRAP concentrations and significantly differed between groups (P < 0.05). CDK1, and cyclin B1 genes, which are subunits of maturation-promoting factor (MPF), are upregulated in Group B and Group C, while are downregulated in oocytes of Group E.This study showed that low, controlled doses of NZS (1 µg/mL) supplementation could improve the meiotic competence of canine oocytes and lead to positive response in expressions of CDK1 and cyclin B1 on the gene level.

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclinB1 and Securin stabilization in meiotic oocyte.

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosisI. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphaseI (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclinB1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphaseII (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

Effects of orange peel extract (Citrus sinensis) treatment on zebrafish oocytes (Danio rerio) exposed to heat stress.

Heat stress (HS) can negatively impact oocytes by disrupting mitochondrial activity, increasing the production of reactive oxygen species, and decreasing antioxidant levels. This study investigated the impact of orange peel (OP) exposure on zebrafish oocytes (ZOs) diameter, survival rate, and germinal vesicle breakdown (GVBD) during HS. We investigated the antioxidant effect of flavonoids (concentration = 328.58 ppm) derived from OP (Citrus sinensis) added to in vitro maturation (IVM) media of ZOs (Danio rerio) under non-heat stress (NHS) and HS conditions to mimic in vivo HS conditions due to the global warming phenomenon on females. ZO in stage 3 (n = 1080) was treated with 4 μL of OP extract (not treated/control) under HS: 32°C (Heat stress 32°C solution/Heat stress 32°C orange peel [HS32S/HS32O]) and 34°C (Heat stress 34°C solution/Heat stress 34°C orange peel [HS34S/HS34O]); and NHS: 28°C (Non-heat stress solution/Non-heat stress orange peel [NHSS/NHSO]), during maturation. After 24 h of maturation, we observed the oocyte diameter, survival rate, and GVBD rate. The data were analyzed with IBM Statistics 23 software using two-way analysis of variance and Kruskal-Wallis (p < 0.05). The highest oocyte diameter data were in NHS treated with OP extract (NHSO) group (0.759 ± 0.01; mean ± standard error) compared with HS group using and without OP extract (HS32S [0.583 ± 0.02]; HS32O [0.689 ± 0.02]; HS34S [0.554 ± 0.02]; and HS34O [0.604 ± 0.02]). The survival rate of OP treated group, namely, NHSO (93% ± 3%), HS32O (85% ± 2%), and HS34O (80% ± 2%) was higher than that of the group without treatment (NHSS [83% ± 3%], HS32S [71% ± 6%], and HS34S [63% ± 3%]). ZO treated with OP extract (NHSO [93% ± 3%], HS32O [85% ± 2%], and HS34O [80% ± 2%]) showed a higher GVBD rate than the group without treatment (NHSS [83% ± 3%], HS32S [71% ± 6%], and HS34S [63% ± 3%]). It revealed that OP can enhance the oocyte diameter, survival rate, and GVBD rate of ZO under NHS and HS. Further investigation should be conducted to determine the effect of OP extract (C. sinensis) on in vivo conditions in females as an alternative treatment to face global warming.

Wnt-5a/Ca2+ pathway modulates endogenous current and oocyte structure of Xenopus laevis

Wnt signaling plays an essential role in cellular processes like development, maturation, and function maintenance. Xenopus laevis oocytes are a suitable model to study not only the development but also the function of different receptors expressed in their membranes, like those receptors expressed in the central nervous system (CNS) including Frizzled 7. Here, using frog oocytes and recordings of endogenous membrane currents in a two-electrode path configuration along with morphological observations, we evaluated the role of the non-canonical Wnt-5a ligand in oocytes. We found that acute application of Wnt-5a generated changes in endogenous calcium-dependent currents, entry oscillatory current, the membrane's outward current, and induced membrane depolarization. The incubation of oocytes with Wnt-5a caused a reduction of the membrane potential, potassium outward current, and protected the ATP current in the epithelium/theca removed (ETR) model. The oocytes exposed to Wnt-5a showed increased viability and an increase in the percentage of the germinal vesicle breakdown (GVBD), at a higher level than the control with progesterone. Altogether, our results suggest that Wnt-5a modulates different aspects of oocyte structure and generates calcium-dependent endogenous current alteration and GVDB process with a change in membrane potential at different concentrations and times of the exposition. These results help to understand the cellular effect of Wnt-5a and present the use of Xenopus oocytes to explore the mechanism that could impact the activation of Wnt signaling.

Propylparaben exposure impairs G2/M and metaphase-anaphase transition during mouse oocyte maturation

Propylparaben (PrPB) is a known endocrine disrupting chemicals that is widely applied as preservative in pharmaceuticals, food and cosmetics. PrPB has been detected in human urine samples and human serum and has been proven to cause functional decline in reproduction. However, the direct effects of PrPB on mammalian oocyte are still unknown. Here, we demonstrationed that exposure to PrPB disturbed mouse oocyte maturation in vitro, causing meiotic resumption arrest and first polar body extrusion failure. Our results indicated that 600 μM PrPB reduced the rate of oocyte germinal vesicle breakdown (GVBD). Further research revealed that PrPB caused mitochondrial dysfunction and oxidative stress, which led to oocyte DNA damage. This damage further disturbed the activity of the maturation promoting factor (MPF) complex Cyclin B1/ Cyclin-dependent kinase 1 (CDK1) and induced G2/M arrest. Subsequent experiments revealed that PrPB exposure can lead to spindle morphology disorder and chromosome misalignment due to unstable microtubules. In addition, PrPB adversely affected the attachment between microtubules and kinetochore, resulting in persistent activation of BUB3 amd BubR1, which are two spindle-assembly checkpoint (SAC) protein. Taken together, our studies indicated that PrPB damaged mouse oocyte maturation via disrupting MPF related G2/M transition and SAC depended metaphase-anaphase transition.

Bisphenol M inhibits mouse oocyte maturation in vitro by disrupting cytoskeleton architecture and cell cycle processes

Bisphenol M (BPM), an alternative to bisphenol A (BPA), is commonly utilized in various industrial applications. However, BPM does not represent a safe substitute for BPA due to its detrimental effects on living beings. This research aimed to assess the influence of BPM exposure on the in vitro maturation of mouse oocytes. The findings revealed that BPM exposure had a notable impact on the germinal vesicle breakdown (GVBD) rate and polar body extrusion (PBE) rate throughout the meiotic progression of mouse oocytes, ultimately resulting in meiotic arrest. Investigations demonstrated that oocytes exposure to BPM led to continued activation of spindle assembly checkpoint. Further studies revealed that securin and cyclin B1 could not be degraded in BPM-exposed oocytes, and meiosis could not realize the transition from the MI to the AI stage. Mechanistically, BPM exposure resulted in abnormal spindle assembly and disrupted chromosome alignment of oocytes. Additionally, abnormal positioning of microtubule organizing center-associated proteins implied that MTOC may be dysfunctional. Furthermore, an elevation in the acetylation level of α-tubulin in oocytes was observed after BPM treatment, leading to decreased microtubule stability. In addition to its impact on microtubules, BPM exposure led to a reduction in the expression of the actin, signifying the disruption of actin assembly. Further research indicated a heightened incidence of DNA damage in oocytes following BPM exposure. Besides, BPM exposure induced alterations in histone modifications. The outcomes of this experiment demonstrate that BPM exposure impairs oocyte quality and inhibits meiotic maturation of mouse oocytes.

Melatonin mitigates PNMC-induced disruption of spindle assembly and mitochondrial function in mouse Oocytes

3-methyl-4-nitrophenol (PNMC), a degradation product of organophosphorus insecticides and a byproduct of fuel combustion, exerting endocrine-disrupting effects. However, its impact on the meiotic process of oocytes remains unclear. In the present study, we investigated the effects of PNMC on meiotic maturation of mouse oocytes in vitro and related mechanisms. Morphologically, PNMC-exposure affected germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) in mouse oocytes. Proteomic analysis suggested that PNMC-exposure altered oocyte protein expression that are associated with cytoskeleton, mitochondrial function and oxidative stress. Further studies demonstrated that PNMC-exposure disrupted spindle assembly and chromosome alignment, caused sustained activation of spindle assembly checkpoint (SAC), and arrested meiosis in oocytes. Specifically, PNMC-exposure interfered with the function of microtubule organizing centers (MTOCs) by significantly reducing phosphorylated mitogen activated protein kinase (p-MAPK) expression and disrupting the localization of Pericentrin and p-Aurora A, leading to spindle assembly failure. Besides, PNMC-exposure also increased α-tubulin acetylation, decreased microtubule stability. Moreover, PNMC-exposure impaired mitochondrial function, evidenced by abnormal mitochondrial distribution, decreased mitochondrial membrane potential and ATP levels, release of Cytochrome C into the cytoplasm, and elevated ROS levels. As a result, exposure to PNMC caused DNA damage and early apoptosis in oocytes. Fortunately, melatonin was able to promote oocyte maturation by removing the excessive ROS and enhancing mitochondrial function. These results highlight the adverse effects of PNMC on meiotic maturation, and underscore the protective role of melatonin against PNMC-induced damage.

Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase of Caenorhabditis elegans Meiosis.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

O-070 Cas9-based chromosome labelling reveals large-scale age-dependent centromere reorganisation in human oocytes

Abstract Study question Can centromeres be labelled in live human oocytes using Cas9 system and is the labelling efficiency affected by advancing female age? Summary answer Centromeres in human oocytes can be labelled using Cas9 system. Signal intensity decreases in oocytes from older patients, suggesting centromeric DNA unravelling as oocytes age. What is known already Aneuploidy in human oocytes is associated with infertility, pregnancy loss or genetic diseases in offspring. It arises due to abnormal chromosome segregation during meiosis, with the risk increasing with advancing female age. This is attributed to age-dependent loss of pericentromere-localised cohesion which holds sister chromatids together. Our work revealed that shugoshin-2 (Sgo2), a protein involved in protection of sister chromatid cohesion is also lost with age. This can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. This hints at more extensive alterations to centromere organisation with advanced maternal age. Study design, size, duration This study uses human oocytes deselected from fertility treatment donated for research purposes with informed consent (HFEA RO155). The oocytes were collected fresh at germinal vesicle (GV), metaphase I (MI) or metaphase II (MII) stage, injected with Cas9 labelling system and live imaged or fixed. Participants/materials, setting, methods Centromeres were labelled in human oocytes using a dead (nuclease-deficient) (d)Cas9-EGFP protein and alpha satellite-specific small guide RNAs (sgRNA). Human oocytes were injected with dCas9-EGFP:sgAlphaSat and live-imaged using dual colour confocal microscopy with SPY555-DNA dye to also visualise chromosomes. High resolution imaging results were analysed with in-house automated kinetochore tracking software. Additional oocytes were fixed and processed for immunofluorescence to determine the high resolution localisation of dCas9-EGFP:sgAlphaSat and Sgo2 molecules. Main results and the role of chance We found that centromeres in human oocytes are efficiently labelled when using Cas9 system. During both meiosis I and II the dCas9-EGFP:sgAlphaSat probe co-localised with pericentromeric bridge and subcentromeric pools of Sgo2. We observed that the dCas9-EGFP:sgAlphaSat signal fades with advancing female age. Quantification reveals that in meiosis II the loss is abrupt at around 35 years of age, resembling Sgo2 behaviour. Initial live imaging indicates that already compromised signal in older oocytes is completely lost about 4hrs after germinal vesicle breakdown, suggesting that advancing age renders centromeres more vulnerable as meiosis I progresses. We have successfully tracked the centromeres in high resolution imaging using the automated software, which allows for detailed investigation of human meiosis chromosome dynamics. This method has been previously validated and successfully used for other cell systems. Limitations, reasons for caution Availability of human oocytes for research purposes is low, limiting our sample size. Moreover, most oocytes used for this study were deselected from treatment of patients diagnosed with infertility, potentially affecting their quality. Wider implications of the findings Our results reveal a large-scale change in centromeric DNA structure in human oocytes with increasing age. Live centromere labelling is a first successful attempt to bivalent tracking in human meiosis with potential to provide insight into age-related loss of fertility in women. Trial registration number n/a

O-007 Leveraging warning thresholds of oocyte immaturity rate to identify patients at risk of pathological endophenotypes. Analysis of 16,155 retrievals with implications for rescue-IVM

Abstract Study question How to develop a robust strategy to pinpoint women exhibiting potential oocyte maturity defects, making them candidates for in-depth genetic investigations and rescue-IVM interventions? Summary answer Oocyte immaturity rate warning-limit was 51%. Within three retrievals, 7.8%, 1.5%, and 0.3% of the patients might exceed it once, twice and three times, respectively. What is known already Oocyte maturity involves cytoplasmic and nuclear aspects. While cytoplasmic maturity is challenging to define and monitor in IVF, nuclear maturation is evaluated through germinal vesicle breakdown and first polar body extrusion. Consensus suggests 10-20% immaturity rates post-OS is acceptable, but some patients exceed. From a diagnostic standpoint, women surpassing these rates may be eligible for additional genetic assessments; from a clinical standpoint, instead, exploring rescue-IVM is promising. However, guidelines for both practices are currently lacking. Study design, size, duration Retrospective study including retrievals with ≥1 cumulus-oocyte-complex (COC; years:2008-2022). The weighted-mean immaturity-rate (20%) and the outliers for COCs retrieved (&amp;gt;22) were defined on the whole dataset (N = 16155). Confounders upon immaturity-rates were outlined among first retrievals with ≥5 COCs (N = 7963). Warning immaturity-rate limit was calculated as “weighted-average+2SD” among first retrievals with 5-22 COCs (N = 7523). Conservative and estimated prevalence of facing immaturity-rates higher than the warning limit across multiple retrievals were also calculated. Participants/materials, setting, methods All retrievals were conducted 35.5±0.5 hours after trigger and cumulus-cells were removed to assess oocyte maturity 3.7±1.2 hours later. Rescue-IVM was not conducted at our center, and immature oocytes were discarded. OS, time between pick-up and denudation, and maternal characteristics were tested as putative confounders through linear regressions. Main results and the role of chance Among all retrievals conducted between 2008 and 2022, the weighted-mean oocyte immaturity-rate was 20%, suggesting that normally ≥1 immature oocyte(s) might be obtained with ≥5 COCs. Use of agonist trigger versus hCG (unstandardized coefficient-B: -2.1%,95%CI from -2.8% to -1.4%,adjusted-p&amp;lt;0.001), length of OS (unstandardized coefficient-B:-0.6%,95%CI from -0.8% to -0.4%,adjusted-p&amp;lt;0.001), and ratio COCs:follicles &amp;gt;16 mm at ovulation induction (unstandardized coefficient-B:+4.6%, 95%CI from +3.8% to + 5.3%,adjusted-p&amp;lt;0.001) were associated with the immaturity-rates in a multivariate linear regression. Among first retrievals with ≥5 and ≤22 COCs, the immaturity-rates warning limit was defined as 51%. In 3.6% (N = 286) of the 7962 first retrievals with ≥5 COCs the immaturity-rates were ≥51%. The same prevalence for second and third retrievals were 3.8% (N = 86/2232) and 2.1% (N = 14/667). The conservative prevalence of patients with immaturity-rates ≥51% once, twice and three times among three consecutive retrievals were 4.5% (N = 361/7962), 0.3% (N = 23/7962), and 0.03% (N = 2/7962). The same rates, assuming all patients would conduct three consecutive retrievals, were 7.8% (N = +257), 1.5% ( = +93), and 0.3% (N = +21). Out of the 667 patients that indeed conducted 3 retrievals between 2008-2022, these rates were 7.6% (N = 51), 0.9% (N = 6) and 0.4% (N = 3), supporting the reliability of the predicted estimates. Limitations, reasons for caution Both germinal-vesicles and metaphase-I immature oocytes were not deemed clinically useful here, although this is controversial. Well-designed studies to systematically monitor rescued oocytes’ developmental, chromosomal, and reproductive competence are still needed, especially in patients subject to high immaturity-rates. Wider implications of the findings Oocyte immaturity-rates ≥51% with ≥5 COCs especially when consistently experienced across multiple retrievals, is a statistically-sound criterion to candidate patients to whole-exome-screening. This aims at outlining gene associations with defective oocyte maturation. The clinical effectiveness of rescue-IVM should be primarily studied in these women (prevalence ≤1%). Trial registration number None

Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase of C. elegans meiosis.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in C. elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

Toxicological effects of nodularin on the reproductive endocrine system of female zebrafish (Danio rerio)

Nodularin is a potent cyanotoxin that has been detected in aquatic environments as well as in the body of aquatic organisms throughout the world, but its effects on the reproductive system are yet to be explored. The present study investigated the toxic effects of environmentally relevant concentrations of nodularin on the reproductive endocrine system of female zebrafish (Danio rerio). After exposure to nodularin for 14 days, decreased gonadosomatic Index (GSI), germinal vesicle breakdown (GVBD), and decreased level of follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2) level and increased testosterone (T) content in female zebrafish suggested that nodularin may disrupt both oocyte growth and maturation. In support of this data, alteration in different marker gene expression on the hypothalamic-pituitary-gonadal-liver (HPGL) axis was observed. Transcriptional levels of genes related to steroidogenesis including cytochrome P450 aromatase (cyp19a1a) in the ovary and primary vitellogenin genes (vtg1, vtg2, and vtg3) in the liver were down-regulated and marker genes for oxidative stress (sod, cat, and gpx) were up-regulated on HPGL axis. These findings revealed for the first time that nodularin is a potent endocrine-disrupting compound posing oxidative stress and causes reproductive endocrine toxicity in female zebrafish, emphasizing the importance of assessing its environmental risks.

A rare case of intra-ovarian oocyte maturation.

The intra-ovarian presence of ootids, i.e. female gametes that have completed meiosis, is considered exceptional in the animal kingdom. The present study explores the first such case to be reported in a sea cucumber (Echinodermata: Holothuroidea). In the overwhelming majority of animals, including holothuroids, oocytes (i.e. immature female gametes) that are developing in the ovary undergo a primary arrest at the prophase stage of meiosis, which may last from days to decades. In free-spawning taxa, this arrest is normally lifted only during or shortly before transit in the gonoduct, when gamete release (spawning) is imminent. However, oocytes of the holothuroid Chiridota laevis were discovered to have resumed the second meiotic division including the completion of germinal vesicle breakdown and polar-body expulsion inside the ovary, effectively reaching the ootid stage concomitantly with ovulation (i.e. escape from follicle cells) prior to spawning. The potential drivers and significance of this exceptionally rare case of full intra-ovarian oogenic maturation are discussed.

Zinc deficiency deteriorates ovarian follicle development and function by inhibiting mitochondrial function

Zinc (Zn) is a crucial trace element essential for human growth and development, particularly for reproductive health. Previous research has shown a decrease in serum zinc concentration with age and individuals with conditions such as polycystic ovary syndrome (PCOS) and diabetes mellitus. However, the specific effects of zinc deficiency on the female reproductive system, especially ovarian function, are not fully understood. In our study, we observed a significant reduction in the total number of follicles and mature follicles in the zinc deficiency group. This reduction correlated with decreased level of anti-Mullerian hormone (AMH) and abnormal gene expression affecting hormone secretion regulation. Furthermore, we found that zinc deficiency disrupted mitochondrial dynamics, leading to oxidative stress in the ovaries, which further inhibited autophagy and increased ovarian apoptosis. These changes ultimately resulted in the failure of germinal vesicle breakdown (GVBD) and reduced oocyte quality. Meanwhile, administration of zinc glycine effectively alleviated the oocyte meiotic arrest caused by dietary zinc deficiency. In conclusion, our findings demonstrated that dietary zinc deficiency can affect hormone secretion and follicle maturation by impairing mitochondrial function and autophagy.

Real-Time Observation of Germinal Vesicle Migration During Oocyte Meiotic Cell Division Using Ovarian Fluorescent Transgenic Zebrafish.

The transgenic (TG) zebrafish allows researchers to bio-image specific biological phenomena in cells and tissues in vivo. We established TG lines to monitor changes in the ovaries of live fish. The original TG line with ovarian fluorescence was occasionally established. Although the cDNA integrated into the line was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka β-actin promoter, the expression of EGFP is restricted to the oocytes and gills in adult fish. Furthermore, we found that germinal vesicles (GVs) in oocytes of the established line can be observed by relatively strong fluorescence around the GV. In this study, we tried to capture the dynamic processes of germinal vesicle breakdown (GVBD) during meiotic cell division using the GV fluorescent oocytes. As a result, GV migration and GVBD could be monitored in real time. We also succeeded in observing actin filaments involved in the migration of GV to the animal pole. This strain can be used for education in the process of oocyte meiotic cell division.

Administration of Arginine Vasotocin and modified Isotocin improve artificial propagation and post-spawning survival of female yellow catfish

Low rate of spawning and high percentage of death after artificial propagation are frequently observed in catfish, which do harms to the long-term population stability and genetic breeding programs in aquaculture. In this study, yellow catfish (Pelteobagrus fulvidraco), an important freshwater species in China was used as a research model, since the farmed female yellow catfish had poor reproduction performance and a high death rate after treatment with the traditional exogenous hormones, luteinizing hormone releasing hormone analogues2 (LHRH-A2) and human chorionic gonadotropin (HCG). We tried to investigate the roles of the Arginine Vasotocin (AVT)/ Isotocin (IT) neuropeptide family in reproduction and design an optimal strategy for inducing artificial propagation in yellow catfish. Two copies of pro-avt and pro-it were detected in hypothalamus and ovaries of yellow catfish, and their mRNA levels gradually increased during spring-summer season. Furthermore, the transcript levels of pro-avt and pro-it in hypothalamus significantly increased after injection of LHRH-A2 and HCG. Although AVT could not induce germinal vesicle breakdown (GVBD) in female yellow catfish, administration of AVT significantly stimulated premature ovulation in a dose-dependent manner and induced the expression of ovulation-related genes, ptgs2a and acvr1. Furthermore, there is a significant up-regulation of serum prostaglandin E2 (PGE2) and luteinizing hormone (LH) in female fish after AVT treatment, whereas no difference was observed in follicle stimulating hormone (FSH). Since IT of yellow catfish was insoluble in saline, a rigid peptide segment was added at the end of IT (the modified IT was named IT-M), which could greatly improve its solubility and induce ovulation in female yellow catfish. Subsequently, we demonstrated that adding carp pituitary extraction (CPE) to the second injection of traditional methods could efficiently improve the spawned fish, eggs production, fertilization rate, hatching rate and post-spawning survival of female yellow catfish, despite at a relatively high cost in hormones. We further discovered that IT-M + AVT could substitute for HCG + CPE in the second injection and had high efficiency in artificial propagation, reducing the hormone costs to less than one-third. Altogether, we provide an effective and affordable combination of exogenous hormones to improve reproduction performances and post-spawning survival rate in yellow catfish.