Comparison of embryo developmental potential of in vitro matured metaphase i (IVM-MI) oocytes according to meiotic spindle position

SummaryBovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8h of initial culture) or metaphase I (12h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.

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Overheating is detrimental to meiotic spindles within in vitro matured human oocytes

Zygote ◽

10.1017/s0967199404002631 ◽

2004 ◽

Vol 12 (1) ◽

pp. 65-70 ◽

Cited By ~ 30

Author(s):

Xiao-Fang Sun ◽

Wei-Hua Wang ◽

David L. Keefe

Keyword(s):

High Temperature ◽

Light Microscope ◽

Polarized Light ◽

Germinal Vesicle ◽

Meiotic Spindle ◽

Human Oocytes ◽

Living State ◽

Meiotic Spindles ◽

Metaphase I

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24–36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 °C for up to 20 min. After heating, oocytes were returned to 37 °C and then imaged for another 20 min at 37 °C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 °C during 40 min of examination and their maximum retardance (1.72–1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 °C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 °C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 °C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 °C and 40 °C, respectively. Heating to 40 °C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 °C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 °C is crucial for normal spindle morphology.

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Development of mouse embryos derived from oocytes reconstructed by metaphase I spindle transfer

Zygote ◽

10.1017/s0967199403001072 ◽

2003 ◽

Vol 11 (1) ◽

pp. 53-59 ◽

Cited By ~ 6

Author(s):

Yong Cheng ◽

Lei Lei ◽

Duan-Cheng Wen ◽

Zi-Yu Zhu ◽

Qing-Yuan Sun ◽

...

Keyword(s):

Polar Body ◽

Mouse Embryos ◽

Meiotic Spindle ◽

Division Rate ◽

Daughter Cells ◽

Maturation Medium ◽

First Polar Body ◽

Morula Stage ◽

Metaphase I

Abnormal oocyte spindle is frequently associated with the infertility of aged women. Directly manipulating the metaphase I (MI) spindle may be a feasible method to overcome this kind of problem. Here, we report that the MI meiotic spindle can be removed from MI mouse oocytes and will autonomously divide into two daughter cells with the same size, morphology and an equal number of chromosomes after culture for 5 h in maturation medium. The division rate of the MI spindle reached 56% after 10-15 h of culture. After transferring the MI meiotic spindle into synchronous ooplasm by electrofusion, about 61% of the reconstructed oocytes continued to complete the first meiosis and extruded a normal first polar body. The matured reconstructed oocytes can also be fertilised. Approximately 50% of the 2-cell embryos developed to the morula stage after in vitro culture.

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O-171 Altered meiotic spindle morphology and composition in in vitro matured oocytes

Human Reproduction ◽

10.1093/humrep/deab127.052 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

P Karamtzioti ◽

G Tiscornia ◽

D Garcia ◽

A Rodriguez ◽

I Vernos ◽

...

Keyword(s):

Metaphase Plate ◽

Spindle Pole ◽

Meiotic Spindle ◽

Developmental Potential ◽

Software Analysis ◽

Spindle Microtubules ◽

Non Parametric

Abstract Study question How does the meiotic spindle tubulin PTMs of MII oocytes matured in vitro compare to that of MII oocytes matured in vivo? Summary answer MII cultured in vitro present detyrosinated tubulin in the spindle microtubules, while MII oocytes matured in vivo do not. What is known already A functional spindle is required for chromosomal segregation during meiosis, but the role of tubulin post-translational modifications (PTMs) in spindle meiotic dynamics remains poorly characterized. In contrast with GVs matured in vitro within the cumulus oophorous, in vitro maturation of denuded GVs to the MII stage (GV-MII) is associated with spindle abnormalities, chromosome misalignment and compromised developmental potential. Although aneuploidy rates in GV-MII are not higher than in vivo matured MII, disorganized chromosomes may contribute to compromised developmental potential. However, to date, spindle PTMs morphology of GV-MII has not been compared to that of in vivo cultured MII oocytes. Study design, size, duration GV (n = 125), and MII oocytes (n = 24) were retrieved from hormonally stimulated women, aged 20 to 35 years old. GVs were matured to the MII stage in vitro in G-2 PLUS medium for 30h; the maturation rate was 68,2%; the 46 GV-MII oocytes obtained were vitrified, stored, and warmed before fixing and subjecting to immunofluorescent analysis. In vivo matured MII oocytes donated to research were used as controls. Participants/materials, setting, methods Women were stimulated using a GnRH antagonist protocol, with GnRH agonist trigger. Trigger criterion was ≥2 follicles ≥18mm; oocytes were harvested 36h later. Spindle microtubules were incubated with antibodies against alpha tubulin and tubulin PTMs (acetylation, tyrosination, polyglutamylation, Δ2-tubulin, and detyrosination); chromosomes were stained with Hoechst 33342 and samples subjected to confocal immunofluorescence microscopy (ZEISS LSM780), with ImageJ software analysis. Differences in spindle morphometric parameters were assessed by non-parametric Kruskal–Wallis and Fisher’s exact tests. Main results and the role of chance Qualitatively, Δ2-tubulin, tyrosination and polyglutamylation were similar for both groups. Acetylation was also present in both groups, albeit in different patterns: while in vivo matured MII oocytes showed acetylation at the poles, GV-MII showed a symmetrical distribution of signal intensity, but discontinuous signal on individual microtubule tracts, suggesting apparent islands of acetylation. In contrast, detyrosination was detected in in vivo matured MII oocytes but was absent from GV-MII. Regarding spindle pole morphology, of the four possible phenotypes described in the literature (double flattened and double focused; flattened-focused, focused-flattened, with the first word characterizing the cortex side of the spindle), we observed double flat shaped spindle poles in 86% of GV-MII oocytes (25/29) as opposed to 40.5% (15/37) for the in vivo matured MII oocytes (p = 0.0004, Fisher’s exact test). Further morphometric analysis of the spindle size (maximum projection, major and minor axis length) and the metaphase plate position (proximal to distal ratio, angle) revealed decreased spindle size in GV-MII oocytes (p = 0.019, non parametric Kruskal- Wallis test). Limitations, reasons for caution Oocytes retrieved from hyperstimulation cycles could be intrinsically impaired since they failed to mature in vivo. Our conclusions should not be extrapolated to IVM in non-stimulated cycles, as in this model, the cumulus oophorus is a major factor in oocyte maturation and correlation with spindle dynamics has been inferred. Wider implications of the findings The metaphase II spindle stability compared to the mitotic or metaphase I meiotic one justifies the presence of PTMs such as acetylation and glutamylation, which are found in stable, long-lived microtubules. The significance of the absence of detyrosinated microtubules in the MII-GV group remains to be determined Trial registration number not applicable

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Dynamics and clinical relevance of maternal mRNA clearance during the oocyte-to-embryo transition in humans

Nature Communications ◽

10.1038/s41467-020-18680-6 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Qian-Qian Sha ◽

Wei Zheng ◽

Yun-Wen Wu ◽

Sen Li ◽

Lei Guo ◽

...

Keyword(s):

Mrna Decay ◽

Meiotic Spindle ◽

Preimplantation Embryos ◽

Human Embryos ◽

Developmental Potential ◽

Maternal Mrna ◽

Before And After ◽

Maternal Transcripts ◽

Human Preimplantation Embryos

Abstract Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3ʹ-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.

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Meiotic spindle position and second polar body orientation enable the prediction of embryonic developmental potential and embryo euploidy

Fertility and Sterility ◽

10.1016/j.fertnstert.2018.07.648 ◽

2018 ◽

Vol 110 (4) ◽

pp. e225

Author(s):

S. Kim ◽

J. Eum ◽

W. Choi ◽

S. Kwon ◽

S. Bang ◽

...

Keyword(s):

Polar Body ◽

Body Orientation ◽

Meiotic Spindle ◽

Developmental Potential ◽

Second Polar Body ◽

Spindle Position

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Evaluation of the developmental potential of metaphase I oocytes from stimulated intracytoplasmic sperm injection cycles

Reproduction Fertility and Development ◽

10.1071/rd10228 ◽

2011 ◽

Vol 23 (3) ◽

pp. 433 ◽

Cited By ~ 3

Author(s):

Mei Li ◽

Yuan Li ◽

Shui-Ying Ma ◽

Huai-Liang Feng ◽

Hui-Jun Yang ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Control Group ◽

High Quality ◽

Developmental Potential ◽

Short Period ◽

Group A ◽

Group B ◽

Group D ◽

Metaphase I

The objective of the present study was to evaluate the developmental potential and clinical application value of metaphase I (MI) oocytes obtained from stimulated intracytoplasmic sperm injection (ICSI) cycles. ICSI was performed on MI oocytes immediately after denudation (Group A), or on in vitro-matured (IVM) oocytes following culture; oocytes in culture were further divided into two groups, being cultured for either 3–5 h (Group B) or 24–28 h (Group C). Metaphase II oocytes from the same cycle(s) isolated for ICSI served as the control group (Group D). The rates of normal fertilisation, cleavage and high-quality embryos were compared among the four groups. High-quality embryos were transferred whenever possible, and pregnancy rates were evaluated. Results showed that normal fertilisation rates for Groups B, C and D were significantly higher than that of Group A (68.6%, 57.8%, 74.5% and 30.1%, respectively; P < 0.01). The rate of high-quality embryos in Group B was comparable with Group D; the rate for Group C was significantly lower than that of the other groups (P < 0.05). Two clinical pregnancies were achieved after transfer of embryos from IVM oocytes. In vitro maturation of MI oocytes for a short period of time may increase the number of available embryos; however, overnight in vitro culture of MI oocytes did not improve results.

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Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076408 ◽

1983 ◽

Vol 41 ◽

pp. 552-555

Author(s):

Conly L. Rieder ◽

S. Bowser ◽

R. Nowogrodzki ◽

K. Ross ◽

G. Sluder

Keyword(s):

Small Sample Size ◽

Small Sample ◽

Meiotic Spindle ◽

Serial Sections ◽

Mitotic Apparatus ◽

Thin Sections ◽

Cell Cycles ◽

Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

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