True Nondisjunction of Whole Bivalents in Oocytes with Attachment and Congression Defects

2017 ◽  
Vol 151 (1) ◽  
pp. 10-17 ◽  
Author(s):  
Martin Sodek ◽  
Kristina Kovacovicova ◽  
Martin Anger
Keyword(s):  
Chromosome Segregation ◽  
Developmental Disorders ◽  
Polar Body ◽  
Direct Consequence ◽  
Early Embryos ◽  
First Polar Body ◽  
Polar Body Extrusion ◽  
Meiosis I

Chromosome segregation in mammalian oocytes is prone to errors causing aneuploidy with consequences such as precocious termination of development or severe developmental disorders. Aneuploidy also represents a serious problem in procedures utilizing mammalian gametes and early embryos in vitro. In our study, we focused on congression defects during meiosis I and observed whole nondisjoined bivalents in meiosis II as a direct consequence, together with a substantially delayed first polar body extrusion. We also show that the congression defects are accompanied by less stable attachments of the kinetochores. Our results describe a process by which congression defects directly contribute to aneuploidy.

scholarly journals Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability

Animals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 209 ◽  
Author(s):  
Ling Yang ◽  
Qingkai Wang ◽  
Maosheng Cui ◽  
Qianjun Li ◽  
Shuqin Mu ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Melatonin Receptors ◽  
Melatonin Treatment ◽  
In Vitro Development ◽  
First Polar Body ◽  
Mitochondrial Distribution ◽  
Polar Body Extrusion ◽  
Vitro Development

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10−5 M luzindole (melatonin receptor antagonist), 10−5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10−5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

scholarly journals 298RED DEER (CERVUS ELAPHUS) PARTHENOGENETIC BLASTOCYSTS PRODUCED USING IONOMYCIN/6DMAP ACTIVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 268 ◽  
Author(s):  
S.E. Beaumont ◽  
D.K. Berg ◽  
G.W. Asher
Keyword(s):  
Red Deer ◽  
Polar Body ◽  
Negative Control ◽  
Blastocyst Development ◽  
Cell Counts ◽  
First Polar Body ◽  
Significant Difference ◽  
Chi Square Analysis ◽  
Polar Body Extrusion

Successful activation of red deer oocytes is a necessary prerequisite for the cloning of red deer individuals with desirable genetic characteristics. To investigate this, an established biphasic protocol used for oocyte activation in sheep was investigated for suitability. The method chosen was 5μM Ionomycin for 5min followed by 2mM 6DMAP for 3h ( Loi P et al., 1998 Biol. Reprod. 58, 1177–1187). The medium used during activation and subsequent culture was Deer Synthetic Oviduct Fluid, which has been shown to support routine in vitro fertilization and blastocyst development (15%) of in vitro-matured red deer oocytes (DSOF, Berg D et al., 2003 Theriogenology 59, 189–205). Red deer abattoir-derived COCs were matured in vitro for 22h before random allocation across 3 treatment groups comprising a standard IVF group, the activation group and a negative control group exposed to medium only. Activation treatment oocytes were stripped of cumulus by vortexing in 0.1% hyaluronidase before selecting for first polar body extrusion. First-step activation was performed in medium comprising HEPES-buffered IVF-DSOF containing 4mM Ca2+. Second-step activation used 3mM Ca2+ early DSOF under 7% O2, 5% CO2, and 88% N2 at 38.5°C. Standard IVF was conducted at 23h post-IVM using 4mM Ca2+ IVF-DSOF and 0.5×106mL−1 final sperm concentration. Following activation and IVF, oocytes were washed 3 times in HEPES DSOF before culture for 7 days in sequential DSOF with late DSOF on Day 4 containing 1.5mM Ca2+. Cleavage was assessed 24h after activation, and all blastocysts were fixed for cell counts. Four replicates of each treatment were performed. Cleavage and blastocyst rates were examined by chi-square analysis and cell numbers by ANOVA. First polar body extrusion rate was 84%. Cleavage was similar between the activation treatment and IVF (P>0.05 ); but a significant difference was found in blastocyst development rates (P<0.05) with the Ionomycin and 6DMAP protocol being superior to the IVF treatment. Exposure to high Ca2+ media alone resulted in only 5% of the negative control oocytes cleaving to 2 cells. Results show that Ionomycin and 6DMAP are effective in activating red deer oocytes and DSOF is a suitable medium to produce parthenogenetic blastocysts.

scholarly journals Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes

Reproduction ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 559-570 ◽  
Author(s):  
Tamás Somfai ◽  
Manabu Ozawa ◽  
Junko Noguchi ◽  
Hiroyuki Kaneko ◽  
Katsuhiko Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Formation Rate ◽  
Metaphase Plate ◽  
Chromosome Analysis ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Formation ◽  
First Polar Body ◽  
Polar Body Extrusion

We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 μg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P < 0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P < 0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

Increased frequency of chromosome congression defects and aneuploidy in mouse oocytes cultured at lower temperature

2017 ◽  
Vol 29 (5) ◽  
pp. 968 ◽  
Author(s):  
Jitka Danadova ◽  
Natalie Matijescukova ◽  
Anna Mac Gillavry Danylevska ◽  
Martin Anger
Keyword(s):  
Developmental Disorders ◽  
Incubation Temperature ◽  
Small Deviation ◽  
Polar Body ◽  
Culture Conditions ◽  
Mouse Oocytes ◽  
Chromosome Congression ◽  
Lower Temperature ◽  
Polar Body Extrusion

Optimal culture conditions are essential for successful IVM of mammalian oocytes and for their further development into an embryo. In the present study we used live cell imaging microscopy to assess the effects of suboptimal culture temperature on various aspects of IVM, including duration of meiosis I, dynamics of polar body extrusion, chromosome congression, anaphase-promoting complex/cyclosome (APC/C) activation and aneuploidy. The data showed that even a small deviation from the optimal incubation temperature causes marked changes in the duration and synchronicity of meiosis, APC/C activity and the frequency of chromosome congression and segregation errors. In vitro manipulation and maturation of germ cells is widely used in both human and animal artificial reproduction techniques. Mammalian oocytes are naturally prone to chromosomal segregation errors, which are responsible for severe mental and developmental disorders. The data presented herein demonstrate that exposure of mouse oocytes to suboptimal temperature during manipulation and maturation could further increase the frequency of chromosome segregation defects in these cells.

130 PREDICTION OF PARTHENOGENETIC DEVELOPMENTAL POTENTIAL BY POLAR BODY EXTRUSION AND FIRST CLEAVAGE ON IN VITRO MATURATION AND DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 145
Author(s):  
H. J. Kim ◽  
S. R. Cho ◽  
C. Y. Choe ◽  
S. H. Choi ◽  
D. S. Son ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Non Invasive ◽  
First Polar Body ◽  
Polar Body Extrusion

The objective of this study was to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as a non-invasive marker to know the developmental competence in advance. Porcine oocytes matured for 48 h and then examined for polar body extrusion. The examined oocytes were matured for an additional 16–18 h, activated with 7% ethanol, and cultured in 5 µg mL–1 cytochalasin B for 5 h for diploid formation. The treated oocytes were examined for cleavage after 48 h and continued culturing for 5 days. Each treatment was replicated by 3–4 times. Oocytes of 21.9% (70/320) were discarded in morphological selection, and 32.1% (167/520) oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated, and after 48 h, the cleavage rate was examined. In morphologically selected oocytes, 15.8% (30/190) were not cleaved, 52.6% (100/190) were normally cleaved (consisted of 2–7 cells), and 31.6% (60/190) were hyper-cleaved (consisted of 8 cells or more) at 48 h after activation. However, in the first polar body extruded oocytes, 7.1% (18/253) were not cleaved, 73.1% (185/253) were normally cleaved, and 19.8% (50/253) were hyper-cleaved. From the morphologically selected oocytes, 16.7% (10/60) were developed up to blastocyst stage from those in which cleavage selection was not performed and 31.7% (19/60) from those in which cleavage selection was performed. From the polar body extruded oocytes, 39.0% (39/100) were developed up to blastocyst stage from those in which cleavage selection was not performed and 49.0% (49/100) from those in which cleavage selection was performed. Cleavage was examined within 12 h interval after activation (0 = time of activation) up to 48 h. At 0–12, 12–24, 24–36, and 36–48 h intervals, 4.1% (9/220), 68.6% (151/220), 19.1% (42/220), and 2.3% (5/220) oocytes were cleaved, respectively, and 5.9% (13/220) oocytes were not cleaved at 48 h after activation. The cleaved embryos in each interval were cultured and developed up to blastocyst with 0 (0/9), 39.1 (59/151), 9.5 (4/42), and 0% (0/5), respectively. This result suggests that the polar body extruded and cleaved at 12–36 h embryo has higher developmental potential than the others.

P–134 Zygote morphokinetic parameters (ZMP) differs between fertilized and non-fertilized (in vitro maturation) oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Zhao ◽  
H Li ◽  
S Wang ◽  
O Alqawasmeh ◽  
M Xu ◽  
...  
Keyword(s):  
Neural Network ◽  
Hong Kong ◽  
Convolutional Neural Network ◽  
Zona Pellucida ◽  
Dynamic Change ◽  
Polar Body ◽  
First Polar Body ◽  
The Difference ◽  
Polar Body Extrusion

Abstract Study question Is there any difference on the ZMP between the fertilized and the non-fertilized oocytes in an IVM cohort? Summary answer The zona pellucida (zp_g) , cytoplasm greyscale（cm_g),the cytoplasm size (cm_size), radius (cm_r) and deviation (cm_d) showed different patterns from the two groups. What is known already We developed a convolutional neural network (CNN) based algorithm that provides instant and bias-free analytical outcomes of oocyte morphological segmentation. The mature but not-fertilized oocytes tend to be static while the fertilized oocytes are more dynamic for preparing its following biological events. Study design, size, duration This was a case-control study on oocytes including 631 normal fertilized oocytes and 100 IVM oocytes from 01/08/2017 to 31/12/2019 conducted in the Prince of Wales Hospital, The Chinese University of Hong Kong. Participants/materials, setting, methods We used the convolutional neural network (CNN) algorithm to segment the ZMPs of the cytoplasm and zona pellucida of the oocytes. The ZMPs include cm_g, cm_size, cm_r, cm_d, zp_g, thickness of zona pellucida and the area of perivitelline space. For the ZMPs that did not change with time, we used t-test to test the significance and for the parameters changed with time we used dynamic warp timing and similarity test to find the difference. Main results and the role of chance The IVM group had a higher intensity of zp_g of 142.03 (128.52–158.70) compared with the fertilized group of 137.04 (121.69–154.37). The cm_g of IVM group was higher than fertilized group [122.55 (114.87–137.62) vs 119.37(108.88–132.87)]. The cm_size, cm_r and cm_d as parameters changed with time and showed a different pattern in two groups. The IVM group decreased the cm_size faster than the fertilized group but the fertilized group had a more dynamic change in the shape of cytoplasm (cm_d) during the development. The cm_r changed with the same pattern of cm_size provided evidence supporting the finding above. Limitations, reasons for caution The ZMPs in the IVM group was captured for 21 hours from the first polar body extrusion. Though the duration was similar to the one from fertilized to the first cleavage. The morphology change during that period may not represent the holistic one of IVM oocytes. Wider implications of the findings: The IVM oocytes have different morphokinetic performance from fertilized oocytes. We used a novel method based on CNN to confirm the differences between the two groups showing that our algorithm was able to describe the morphokinetic changes in a quantitative way and corresponded with embryologist’s experience. Trial registration number The Hong Kong Obstetrical & Gynaecological Trust Fund

scholarly journals Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes

Reproduction ◽  
2005 ◽  
Vol 130 (6) ◽  
pp. 829-843 ◽  
Author(s):  
Hayden A Homer ◽  
Alex McDougall ◽  
Mark Levasseur ◽  
Alison P Murdoch ◽  
Mary Herbert
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Polar Body ◽  
Cyclin B ◽  
Mutant Form ◽  
Anaphase Promoting Complex ◽  
Mouse Oocytes ◽  
First Polar Body ◽  
Polar Body Extrusion ◽  
Meiosis I

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.

scholarly journals 313IN VITRO MATURATION AND FERTILIZATION OF OOCYTES COLLECTED FROM UNSTIMULATED MACACA NEMISTRINA OVARIES

2004 ◽  
Vol 16 (2) ◽  
pp. 276
Author(s):  
E.S. Hayes ◽  
E.C. Curnow
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Pantothenic Acid ◽  
Macaca Nemestrina ◽  
Chi Square ◽  
Maturation In Vitro ◽  
First Polar Body ◽  
Chi Square Analysis ◽  
Polar Body Extrusion

Reports describing the IVM of Macaca nemestrina (Mn) oocytes are limited (Cranfield MR et al. 1989 Zoo. Biol. (Supp. 1), 33). The use of gonadotrophins (Gnt) for IVM of non-human primate (NHP) oocytes is common but the concentrations used are often high (8–40IUmL−1) and the species of origin and biological activity of Gnt varies (Schramm RD and Paprocki AM, 2000 Hum. Reprod. 15, 2411). We have compared two different IVM systems with human Gnt on maturation and fertilization of oocytes collected from unstimulated Mn ovaries (n=6–10 animals). Oocytes were subjected to IVM in modified (minus PVA and pantothenic acid, plus 20 amino acids) HECM−10+15% FCS (Zheng P et al., 2001 Mol. Reprod. Dev. 58, 348) for a) 36h in the presence (mHECM+36, n=322) or absence (mHECM−36, n=99) of FSH and LH applied sequentially (FSH 1IUmL−1 0–24h; 10IUmL−1 FSH and LH 24–36h) or b) 24h in the presence (mHECM+24, n=119) or absence (mHECM−24, n=56) of static concentrations of Gnt (FSH and LH 1IUmL−1 0–24h; no Gnt 24–30h). Oocytes exhibiting first polar body extrusion at 36 and 30h were recorded as mature (MII) and subjected to IVF in HTF+BSA (3mg mL−1) with Mn sperm pretreated with 1.0mM caffeine and 0.1mM dbcAMP. Fertilized oocytes (pronuclei and/or 2nd polar body extrusion) were cultured in sequential culture medium for 48h, assessed for cleavage and either fixed or frozen. Proportional data (mature/total, fertilized/mature or cleaved/fertilized) were compared by chi-square analysis and are reported as percentages. Oocytes cultured in mHECM+36 and mHECM−36 exhibited similar rates of GVBD (58.7% v. 53.5%) but the percentage of MII oocytes was significantly higher (P=0.0244) in mHECM+36 (41.3%) v. mHECM−36 (28.3%). Fertilization rates were comparable between mHECM+36 (61.5%) and mHECM−36 (60.9%), whereas cleavage rates were significantly higher (P=0.0004) in mHECM+36 (74.6%) v. HECM−36 (21.4%). Oocytes cultured in mHECM+24 and mHECM−24 exhibited similar rates of GVBD (76.5% v. 62.5%) but the proportion of MII oocytes was significantly higher (P=0.0159) in mHECM+24 (55.5%) v. mHECM−24 (35.7%). Fertilization and cleavage rates were comparable between mHECM+24 (58.8% v. 63.3%) and mHECM−24 (50.0% v. 42.8%). A comparison between mHECM+36 and mHECM+24 indicated a significantly lower (P=0.0005) percentage of GV oocytes and a significantly higher (P=0.0096) percentage of MII oocytes in mHECM+24 (23.5% v. 55.5%) compared to mHECM+36 (41.3% v. 41.3%). Fertilization and cleavage rates were not significantly different between mHECM+36 and mHECM+24. Oocyte maturation and fertilization and embryo cleavage were not different for mHECM−36 and mHECM−24 (P=0.3138–0.8202). Mn oocytes exhibit high rates of Gnt-independent GVBD (52.5%–53.5%) and maturation (28.3%–35.7%) in vitro, and maturation rates were improved in Gnt supplemented maturation medium. However, reduced exposure to lower concentrations of FSH and increased exposure to lower concentrations of LH was associated with higher rates of oocyte maturation in vitro. The use of lower concentrations of FSH and LH for reduced periods may improve IVM of NHP oocytes. This work was supported by the Tissue Distribution Program of the WaNPRC (NIH grant # R00166).

318 INHIBITION OF FIRST POLAR BODY EXTRUSION BY CYTOCHALASIN B DURING IN VITRO MATURATION OF PORCINE OOCYTES LEADS TO RE-ARRANGEMENT OF THE SEGREGATED CHROMOSOMES AND IMPROVEMENT IN THE QUALITY OF THE PARTHENOGENETIC BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 266
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Cytochalasin B ◽  
Polar Body ◽  
Metaphase Plate ◽  
Live Cells ◽  
Lower Probability ◽  
Control Group ◽  
First Polar Body ◽  
Polar Body Extrusion

Diploid parthenotes are usually obtained by the inhibition of second polar body (PB2) extrusion after activation of metaphase II (MII) oocytes. However, diploid embryos can be generated by the inhibition of the first polar body (PB1) extrusion as well, using cytochalasin B (CB) during in vitro maturation prior the activation procedure. A higher percentage of mouse embryos generated by the activation of MII oocytes and the inhibition of PB2 extrusion were proven to be homozygous than for parthenotes obtained by the latter method (Kubiak et al. 1991 Development 111, 763-769). The aim of the present study was to examine if such difference has any effect on the development of parthenogenetic embryos in vitro. Nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to CB during in vitro maturation (IVM) was investigated in the present study. The tendency of nuclear maturation was similar in oocytes matured in the presence of 1 �g/mL CB (IVM-CB group) and control oocytes matured without CB after 37 h of IVM; at this time the frequency of oocytes that had reached/or passed through anaphase-I stage did not differ significantly (P < 0.05) between the IVM-CB and the control groups (61.3% and 69.9%, respectively), however, no polar body extrusion was observed in the IVM-CB group and the two lumps of homologue chromosomes remained in the oocyte and turned into two irregular sets of condensed chromosomes. By 41 h of IVM, the double sets of chromosomes re-united in 89.5% of IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached metaphase-II stage (MII) by this time. When IVM-CB oocytes were electrically (1.5 kV/cm for 100 �s) activated and subsequently cultured without CB, 39% of the oocytes extruded a polar body (PB) and 82.9% of them had a female pronucleus. When those oocytes with PB were cultured, the blastocyst rate of the cleaved embryos did not differ (P < 0.05) from those of the control that were stimulated at MII and subsequently treated with CB (43.3% and 48.2%, respectively). The number of blastomeres in Day 6 blastocysts was significantly higher (P < 0.05) in the IVM-CB derived embryos than in those in the control group (47.8 and 40.7, respectively); moreover, the ratio of dead blastomeres (dead cells : live cells) was higher (P < 0.05) in the control than in the IVM-CB blastocysts (0.047 and 0.031, respectively). A possible explanation for this result might be a lower frequency of homozygous genes in IVM-CB parthenotes, in which segregation of sister chromatids were promoted instead of segregation of homologous chromosomes to obtain diploid embryos. In such embryos the expression of recessive lethal, sublethal and subvital genes might have a lower probability. This work was supported by the Japanese-Hungarian bilateral scientific and technological cooperation (TET JAP-11/02).

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