Comparative Characteristics of Morphological Feature and Localization of Meiotic Spindle in Human Oocytes before and after Cryopreservation

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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Meiotic spindle recovery is faster in vitrification of human oocytes compared to slow freezing

Fertility and Sterility ◽

10.1016/j.fertnstert.2008.03.013 ◽

2009 ◽

Vol 91 (6) ◽

pp. 2399-2407 ◽

Cited By ~ 62

Author(s):

Patrizia Maria Ciotti ◽

Eleonora Porcu ◽

Leonardo Notarangelo ◽

Otello Magrini ◽

Antonia Bazzocchi ◽

...

Keyword(s):

Slow Freezing ◽

Meiotic Spindle ◽

Human Oocytes

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Visualization and Examination of the Meiotic Spindle in Human Oocytes with Polscope

Journal of Assisted Reproduction and Genetics ◽

10.1023/b:jarg.0000046202.00570.1d ◽

2004 ◽

Vol 21 (10) ◽

pp. 349-353 ◽

Cited By ~ 23

Author(s):

Janos Konc ◽

Katalin Kanyó ◽

Sandor Cseh

Keyword(s):

Meiotic Spindle ◽

Human Oocytes

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Prolonged ovarian storage of mature Drosophila oocytes dramatically increases meiotic spindle instability

eLife ◽

10.7554/elife.49455 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Ethan J Greenblatt ◽

Rebecca Obniski ◽

Claire Mical ◽

Allan C Spradling

Keyword(s):

Meiotic Chromosome ◽

Chromosome Instability ◽

Oocyte Quality ◽

Ribosome Profiling ◽

Meiotic Spindle ◽

Human Embryos ◽

Prolonged Storage ◽

Translational Efficiency ◽

Human Oocytes ◽

In The Wild

Human oocytes frequently generate aneuploid embryos that subsequently miscarry. In contrast, Drosophila oocytes from outbred laboratory stocks develop fully regardless of maternal age. Since mature Drosophila oocytes are not extensively stored in the ovary under laboratory conditions like they are in the wild, we developed a system to investigate how storage affects oocyte quality. The developmental capacity of stored mature Drosophila oocytes decays in a precise manner over 14 days at 25°C. These oocytes are transcriptionally inactive and persist using ongoing translation of stored mRNAs. Ribosome profiling revealed a progressive 2.3-fold decline in average translational efficiency during storage that correlates with oocyte functional decay. Although normal bipolar meiotic spindles predominate during the first week, oocytes stored for longer periods increasingly show tripolar, monopolar and other spindle defects, and give rise to embryos that fail to develop due to aneuploidy. Thus, meiotic chromosome segregation in mature Drosophila oocytes is uniquely sensitive to prolonged storage. Our work suggests the chromosome instability of human embryos could be mitigated by reducing the period of time mature human oocytes are stored in the ovary prior to ovulation.

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Prolonged ovarian storage of mature Drosophila oocytes dramatically increases meiotic spindle instability

10.1101/675389 ◽

2019 ◽

Author(s):

Ethan J. Greenblatt ◽

Rebecca Obniski ◽

Claire Mical ◽

Allan C. Spradling

Keyword(s):

Meiotic Chromosome ◽

Chromosome Instability ◽

Functional Decline ◽

Small Heat Shock Protein ◽

Oocyte Quality ◽

Ribosome Profiling ◽

Meiotic Spindle ◽

Mrna Levels ◽

Dependent Manner ◽

Human Oocytes

SummaryMore than 95% of fertilized Drosophila oocytes from outbred stocks develop fully regardless of maternal age, in contrast to human oocytes, which frequently generate non-viable aneuploid embryos. Since Drosophila oocytes are normally stored only briefly prior to ovulation, unlike their human counterparts, we investigated the effects of storage on oocyte quality. Using a novel system to acquire oocytes held for known periods, we analyzed by ribosome profiling how translation and cellular function change over time. Oocyte developmental capacity decays in a precise temperature-dependent manner over 1-4 weeks, due to a progressive inability to complete meiosis. Meiotic metaphase genes, the Fmr1 translational regulator, and the small heat shock protein chaperones Hsp26 and Hsp27 are preferentially translated during storage, and oocytes lacking Hsp26 and Hsp27 age prematurely. However translation falls generally 2.3-fold with age despite constant mRNA levels, and this inability to maintain translational equilibrium correlates with oocyte functional decline. These findings show that meiotic chromosome segregation in Drosophila oocytes is uniquely sensitive to prolonged quiescence, and suggest that the extended storage of mature human oocytes contributes to their chromosome instability. If so, then these problems may be more amenable to intervention than previously supposed.

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