Immunofluorescence Staining of Spindles, Chromosomes, and Kinetochores in Human Oocytes

Riris, Solon; Cawood, Suzanne; Gui, Liming; Serhal, Paul; Homer, Hayden

doi:10.1007/978-1-62703-191-2_12

Cited by 4 publications

(3 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Whole oocytes were fixed and stained using a method previously described ( Riris et al, 2013 ). Briefly, oocytes were washed in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO 4 .7H 2 O; pH 6.9) with 0.25% Triton X-100, then fixed in 3.7% paraformaldehyde in PHEM for 30 min.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities

et al. 2016

View full text Add to dashboard Cite

The first meiotic division in human oocytes is highly error-prone and contributes to the uniquely high incidence of aneuploidy observed in human pregnancies. A successful meiosis I (MI) division entails separation of homologous chromosome pairs and co-segregation of sister chromatids. For this to happen, sister kinetochores must form attachments to spindle kinetochore-fibres emanating from the same pole. In mouse and budding yeast, sister kinetochores remain closely associated with each other during MI, enabling them to act as a single unified structure. However, whether this arrangement also applies in human meiosis I oocytes was unclear. In this study, we perform high-resolution imaging of over 1900 kinetochores in human oocytes, to examine the geometry and architecture of the human meiotic kinetochore. We reveal that sister kinetochores in MI are not physically fused, and instead individual kinetochores within a pair are capable of forming independent attachments to spindle k-fibres. Notably, with increasing female age, the separation between kinetochores increases, suggesting a degradation of centromeric cohesion and/or changes in kinetochore architecture. Our data suggest that the differential arrangement of sister kinetochores and dual k-fibre attachments may explain the high proportion of unstable attachments that form in MI and thus indicate why human oocytes are prone to aneuploidy, particularly with increasing maternal age.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence was performed using a method previously described ( Riris et al, 2013 ). Oocytes were incubated at 37°C for 1 h with primary antibodies diluted in blocking solution, followed by a 15 min wash in PBB with 0.05% Tween-20, then incubation with secondary antibodies for 1 h, followed by a final wash step.…”

Section: Methodsmentioning

confidence: 99%

Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Human oocytes were fixed and immunostained using protocols as described in detail and validated extensively previously [17][18][19][20][21]. Briefly, oocytes were washed in PIPES, HEPES, EGTA and Magnesium Sulphate (PHEM) buffer (pH 7.0) and pre-permeabilised in 0.25% Triton-X in PHEM.…”

Section: Immunostaining Of Gv− Oocytes and Quantification Of Dna Damagementioning

confidence: 99%

The Presence of Immature GV− Stage Oocytes during IVF/ICSI Is a Marker of Poor Oocyte Quality: A Pilot Study

et al. 2020

Self Cite

View full text Add to dashboard Cite

Here we investigate whether the presence of germinal vesicle-stage oocytes (GV− oocytes) reflects poor oocyte developmental competence (or quality). This was a prospective, non-randomised, cohort pilot-study involving 60 patients undergoing in vitro fertilization/ intracytoplasmic sperm injection for whom complete pregnancy outcome data were available. Patients in whom GV− oocytes were retrieved (GV+) at transvaginal oocyte retrieval (TVOR) were compared with those from whom no GVs were retrieved (GV−). We found that GV+ (n = 29) and GV− (n = 31) patients were similarly aged (35.4 vs. 36.4 years; p = 0.446). GV+ patients had a mean of 2.41 ± 2.03 GVs and comparable yields of MII oocytes to GV− patients (11 ± 6.88 vs. 8.26 ± 4.84; p = 0.077). Compared with GV− patients, GV+ patients had markedly lower implantation rates (11.8% vs. 30.2%; p = 0.022) as well as oocyte utilisation rates for clinical pregnancy (2.3% vs. 6.8%; p = 0.018) and live-birth (1.9% vs. 5.7%; p = 0.029). DNA damage levels measured using γH2AX immunostaining were not different in oocytes from women <36 years versus those ≥36 years (p = 0.606). Thus, patients who have GV− stage oocytes at TVOR exhibit poor oocyte quality reflected in reduced per-oocyte pregnancy success rates and uniformly high levels of oocyte DNA damage.

show abstract

The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction

et al. 2021

View full text Add to dashboard Cite

Immunofluorescence Staining of Spindles, Chromosomes, and Kinetochores in Human Oocytes

Cited by 4 publications

References 6 publications

Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities

Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities

The Presence of Immature GV− Stage Oocytes during IVF/ICSI Is a Marker of Poor Oocyte Quality: A Pilot Study

The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction

Contact Info

Product

Resources

About