scholarly journals Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI

2003 ◽  
Vol 18 (6) ◽  
pp. 1289-1293 ◽  
Author(s):  
L. Rienzi
Keyword(s):  
Body Position ◽  
Polar Body ◽  
Meiotic Spindle ◽  
Developmental Potential

P–805 Artificial oocytes: from somatic cells to fertile pups

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
O Kocur ◽  
A Trout ◽  
P Xie ◽  
A Petrini ◽  
Z Rosenwaks ◽  
...  
Keyword(s):  
Genetic Variance ◽  
Imaging System ◽  
Polar Body ◽  
Somatic Cells ◽  
Human Reproduction ◽  
Meiotic Spindle ◽  
Mouse Strains ◽  
Age Related ◽  
Pregnant Mice ◽  
Post Implantation

Abstract Study question We analyzed the efficacy of generating artificial oocytes using somatic cells (SCs) from two mouse strains (B6D2F1 and FVB) and followed their full pre-/post-implantation development. Summary answer While artificial oocytes generated from the new strain (FVB) had higher fertilization rates, those from the standard strain (B6D2F1) provided expanded blastocysts and fertile pups. What is known already B6D2F1 is a popular hybrid mouse strain for cloning and transgenic creation due to its geno-/pheno-typic uniformity and high oocyte yield and quality. Indeed, B6D2F1 oocytes have a distinct metaphase II (MII) spindle complex, making them an ideal candidate to generate ooplasts used in SC nuclear transfer (SCNT). However, because they lack genetic variance, they are less suitable for reciprocal SCNT studies. In contrast, FVB mice have single nucleotide polymorphisms and indels on each chromosome that can aid in tracing the pedigree of progeny. Study design, size, duration A total of 10 experiments were performed over the course of 3 months, using 30 stimulated mice. SCs were retrieved from cumulus oophorus harvested from FVB and B6D2F1 mice. SCs from both strains were injected into enucleated MII B6D2F1 oocytes. Unmanipulated B6D2F1 oocytes were piezo-ICSI inseminated, serving as controls. The occurrence of haploidization, fertilization, and full preimplantation development was compared. Some blastocysts were transferred into pseudo-pregnant CD–1 mice to obtain offspring. Participants/materials, setting, methods Oocyte enucleation was performed under Oosight™ visualization and cytochalasin B exposure. An FVB or B6D2F1 SC was transferred into the perivitelline space of the ooplast with Sendai virus to promote fusion. Haploidization was monitored by pseudo-meiotic spindle formation followed by extrusion of a pseudo-polar body after insemination. Conceptuses were cultured in a time-lapse imaging system, with piezo-ICSI controls. Expanded blastocysts were transferred into uterine horns of pseudo-pregnant mice. Offspring were mated to test their fertility. Main results and the role of chance FVB (n = 278) and B6D2F1 (n = 905) SCs at G0 phase, with a diameter <10 mm, were chosen for SCNT and transferred into enucleated B6D2F1 ooplasts. Enucleation of 1,212 oocytes yielded a survival rate of 97.6%. Both FVB and B6D2F1 SCNT resulted in similar survival rates of 100% and 98.5%, respectively. Successful haploidization, determined by the presence of a pseudo-meiotic spindle 2 hours after SCNT, was also comparable, with 59.9% of FVB and 63.7% of B6D2F1. Survival after piezo-ICSI was also comparable between FVB- and B6D2F1-reconstituted oocytes, with rates of 64.3% and 60.3%, respectively, albeit lower than the control (75.2%, P < 0.00001). FVB embryos fertilized at a rate of 88.7%, comparable to the control zygotes at 85.8%, while B6D2F1 conceptuses demonstrated a lower fertilization rate (70.8%, P < 0.00001). Blastulation of FVB- and B6D2F1-derived embryos was 15.1% and 24.0%, respectively, while the control was 80.7% (P < 0.00001). Whole-genome karyotyping of 9 B6D2F1-derived blastocysts confirmed 5 of the samples to be euploid. FVB blastocysts (N = 8) and B6D2F1 blastocysts (N = 81) were transferred into pseudo-pregnant mice, resulting in 3 fertile offspring only from the B6D2F1 conceptuses. Limitations, reasons for caution This is still a limited number of observations, and pups were delivered only from the B6D2F1 strain. The utilization of a strain with higher genetic variance may help facilitate offspring fingerprinting. Wider implications of the findings: This study demonstrates the ability to generate artificial genotyped conceptuses, yielding live offspring. The identification of a feasible donor cell, together with optimization of cell cycle stage and standardization of post-implantation development, will help promote this technique for human reproduction in couples with age-related infertility or poor ovarian reserve. Trial registration number N/A

scholarly journals Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

1999 ◽  
Vol 146 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Stéphane Brunet ◽  
Angélica Santa Maria ◽  
Philippe Guillaud ◽  
Denis Dujardin ◽  
Jacek Z. Kubiak ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Spindle ◽  
Homologous Chromosomes ◽  
Mouse Oocytes ◽  
Sister Chromatids ◽  
M Phase ◽  
Continuous Presence ◽  
Set Up ◽  
Polar Body Extrusion ◽  
Reductional Division

During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.

Evaluation of oocyte quality: morphological, cellular and molecular predictors

2007 ◽  
Vol 19 (1) ◽  
pp. 1 ◽  
Author(s):  
Qiang Wang ◽  
Qing-Yuan Sun
Keyword(s):  
Transforming Growth Factor ◽  
Polar Body ◽  
Morphological Characteristics ◽  
Transforming Growth Factor Β ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Oocyte Competence ◽  
Cumulus Oocyte Complex ◽  
Glucose 6 Phosphate Dehydrogenase

Mounting evidence that oocyte quality profoundly affects fertilisation and subsequent embryo development drives the continued search for reliable predictors of oocyte developmental competence. In the present review, we provide an overall summary and analysis of potential criteria that can be used to evaluate oocyte quality. These criteria are specifically classified as morphological and cellular/molecular predictors. Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus–oocyte complex, polar body and/or meiotic spindle. Although the use of morphological characteristics as predictors of oocyte quality is controversial, such a grading system can provide valuable information for the preselection of oocytes with higher developmental competence and, therefore, may maximise embryo developmental outcome. Several intrinsic markers (such as mitochondrial status and glucose-6-phosphate dehydrogenase l activity) and extrinsic markers (such as apoptosis of follicular cells and levels of the transforming growth factor-β superfamily in follicular fluid or serum) have also been reported as useful indicators of oocyte competence and embryo quality. Compared with the morphological parameters, these cellular and molecular predictors of oocyte quality may prove to be more precise and objective, although further studies and refinement of techniques are needed.

scholarly journals Oocyte Positional Recognition for Automatic Manipulation in ICSI

Micromachines ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 429 ◽  
Author(s):  
Mozafar Saadat ◽  
Amir Hajiyavand ◽  
Ajai-pal Singh Bedi
Keyword(s):  
Body Position ◽  
Polar Body ◽  
Recognition System ◽  
Size Measurement ◽  
Transform Method ◽  
Position Detection ◽  
Polar Bodies ◽  
Fitting Method ◽  
Injection Mechanism ◽  
Intracellular Structure

Polar body position detection is a necessary process in the automation of micromanipulation systems specifically used in intracytoplasmic sperm injection (ICSI) applications. The polar body is an intracellular structure, which accommodates the chromosomes, and the injection must not only avoid this structure but be at the furthest point away from it. This paper aims to develop a vision recognition system for the recognition of the oocyte and its polar body in order to be used to inform the automated injection mechanism to avoid the polar body. The novelty of the paper is its capability to determine the position and orientation of the oocyte and its polar body. The gradient-weighted Hough transform method was employed for the detection of the location of the oocyte and its polar body. Moreover, a new elliptical fitting method was employed for size measurement of the polar bodies and oocytes for the allowance of morphological variance of the oocytes and their polar bodies. The proposed algorithm has been designed to be adaptable with typical commercial inverted microscopes with different criteria. The successful experimental results for this algorithm produce maximum errors of 5% for detection and 10% for reporting respectively.

scholarly journals Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes

2013 ◽  
Vol 200 (5) ◽  
pp. 567-576 ◽  
Author(s):  
Kexi Yi ◽  
Boris Rubinstein ◽  
Jay R. Unruh ◽  
Fengli Guo ◽  
Brian D. Slaughter ◽  
...  
Keyword(s):  
Symmetry Breaking ◽  
Cytoplasmic Streaming ◽  
Feedback Loop ◽  
Polar Body ◽  
Meiotic Spindle ◽  
Directed Motion ◽  
Family Protein ◽  
Polar Body Extrusion ◽  
Spindle Position ◽  
Meiosis I

Polar body extrusion during oocyte maturation is critically dependent on asymmetric positioning of the meiotic spindle, which is established through migration of the meiosis I (MI) spindle/chromosomes from the oocyte interior to a subcortical location. In this study, we show that MI chromosome migration is biphasic and driven by consecutive actin-based pushing forces regulated by two actin nucleators, Fmn2, a formin family protein, and the Arp2/3 complex. Fmn2 was recruited to endoplasmic reticulum structures surrounding the MI spindle, where it nucleated actin filaments to initiate an initially slow and poorly directed motion of the spindle away from the cell center. A fast and highly directed second migration phase was driven by actin-mediated cytoplasmic streaming and occurred as the chromosomes reach a sufficient proximity to the cortex to activate the Arp2/3 complex. We propose that decisive symmetry breaking in mouse oocytes results from Fmn2-mediated perturbation of spindle position and the positive feedback loop between chromosome signal-induced Arp2/3 activation and Arp2/3-orchestrated cytoplasmic streaming that transports the chromosomes.

Participation of the ubiquitin-proteasome pathway in rat oocyte activation

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Xin Tan ◽  
An Peng ◽  
Yong-Chao Wang ◽  
Yue Wang ◽  
Qing-Yuan Sun
Keyword(s):  
Meiotic Division ◽  
Polar Body ◽  
Meiotic Spindle ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Second Polar Body

The role of the ubiquitin-proteasome pathway (UPP) in mitosis is well known. However, its role in meiotic division is still poorly documented, especially in the activation of mammalian oocytes. In this study, the role of proteasome in the spontaneous and parthenogenetic activation of rat oocytes was investigated. We found that ALLN, an inhibitor of proteasome, when applied to metaphase II oocytes, inhibited spontaneous activation, blocked extrusion of the second polar body (PB) and caused the withdrawal of the partially extruded second PB. ALLN also inhibited the parthenogenetic activation induced by cycloheximide, but had no effect on the formation of pronuclei in activated eggs. In metaphase and anaphase, ubiquitin and proteasome localized to the meiotic spindle, concentrating on both sides of the oocyte–second PB boundary during PB extrusion. This pattern of cellular distribution suggests that UPP may have a role in regulating nuclear division and cytokinesis. Ubiquitin was seen to form a ring around the pronucleus, whereas proteasome was evenly distributed in the pronuclear region. Taken together, our results indicate that (1) UPP is required for the transitions of oocytes from metaphase II to anaphase II and from anaphase II to the end of meiosis; and (2) the UPP plays a role in cytokinesis of the second meiotic division.

scholarly journals Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 859-867 ◽  
Author(s):  
Xiao-Qian Meng ◽  
Ke-Gang Zheng ◽  
Yong Yang ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Actin Filaments ◽  
Laser Scanning ◽  
Polar Body ◽  
Laser Scanning Microscopy ◽  
Meiotic Spindle ◽  
Confocal Laser ◽  
Cell Cortex ◽  
First Polar Body

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

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