303PARTHENOGENETIC DEVELOPMENT OF PIG OOCYTES BY CHEMICAL 
ACTIVATION

Efficient activation is essential for the success of animal cloning by nuclear transfer. The aim of this study was to investigate the effects of chemical activation agents on parthenogenetic development of pig oocytes matured in vitro. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were selected for activation. Oocytes were activated as follows. First, all oocytes were activated with 25mM HEPES buffered NCSU-23 medium containing 8% ethanol for 10min. After that, in treatment 1, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 3h. In treatment 2, oocytes were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 3h. In treatment 3, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 1.5h, and then were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 1.5h. In treatment 4, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B plus 10μgmL−1 cycloheximide for 3h. Following activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture for 20 and 144h. Activated oocytes were fixed and stained for evaluation of activation rate, cleaved oocytes, blastocyst formation rate and cell numbers per blastocyst. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of oocyte activation was higher in treatment 4 (62.1%) than in treatment 1, 2 and 3 (52.0, 49.6 and 58.0%, respectively). The percentage of cleaved oocytes was lower in treatment 1 and 2 (56.9 and 55.2%) than in treatment 3 and 4 (68.8 and 68.5%). The rate of blastocyst formation from the cleaved oocytes was higher in treatment 3 and 4 (19.8 and 22.0%) than in treatment 1 and 2 (12.1 and 11.7%). Mean cells per blastocyst were lowest in treatment 2 (21.2±0.9) compared to treatment 1, 3 and 4 (27.3±2.2, 30.4±3.8 and 30.9±3.4, respectively). In conclusion, cytochalasin B combined with cycloheximide was more efficient for parthenogenetic development of pig oocytes matured in vitro.

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315 EFFICIENCY OF CHEMICAL OR ELECTRICAL ACTIVATION OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab315 ◽

2006 ◽

Vol 18 (2) ◽

pp. 265

Author(s):

M. P. Milazzotto ◽

W. B. Feitosa ◽

R. Simões ◽

C. M. Mendes ◽

M. E. O. A. Assumpção ◽

...

Keyword(s):

Nuclear Transfer ◽

Embryo Quality ◽

Calcium Influx ◽

Polar Body ◽

Chemical Activation ◽

Electrical Activation ◽

Activation Process ◽

Oocyte Activation ◽

Blastocyst Formation ◽

First Polar Body

Activation of in vitro matured oocytes is essential for the success of nuclear transfer embryo production. Oocyte activation is promoted by the release of intracellular calcium and influx of extracellular ions, and can be chemically induced by calcium ionophores such as A23187 (CA) or ionomycin (IO). Electrical stimulation (EL) is an essential stage in nuclear transfer protocols for the fusion of enucleated oocytes with the donor's cell nucleus. Moreover, EL can be used as an alternative method to induce calcium influx through the formation of pores in the plasma membrane. This work aimed to evaluate the effect of electrical pulse vs the use of different calcium ionophores (A23187 or ionomycin) as primary agents of bovine oocyte activation, with or without the addition of BSA, on the rate of blastocyst formation and blastocyst quality. BSA was used to quench the activation process after a 5-min exposure to CA or IO. Cumulus-oocyte complexes were matured in TCM-199 medium with FCS and hormones for 18 h at 38.5�C and 5% CO2 in air. After removal of cumulus cells, oocytes presenting the first polar body were selected and maintained in SOFaa medium to complete 24 h of maturation. They were then divided into five treatments groups 1-CA (CA 5 mM, 5 min); 2-CAB (CA 5 mM, 5 min; BSA, 5 min); 3-IO (IO 5 mM, 5 min); 4-IOB (IO 5 mM, 5 min; BSA, 5 min); and 5-EL (EL 1.5 kV/cm, 20 �s, 2 pulses). After treatments, oocytes were kept in 6-dimethylaminopurine for 3 h and cultured in SOFaa medium for 7 days at 38.5�C and 5% CO2 in air. Rates of cleavage and blastocyst were evaluated respectively on Days 2 and 7 of culture. To evaluate embryo quality, Hoechst 33342/propidium iodide staining was used. Data were evaluated by ANOVA and submitted to LSD test for embryo rates and t-test for embryo quality. Four replicates were carried out with a total of 89 oocytes per treatment. There was a difference (P < 0.05) in rate of development to blastocyst between treatments 1-CA (54.4%a), 3-IO (51.4%a), and 5-EL (54.5%a) compared with 4-IOB (18.3%b). Treatment 2-CAB (39.8%ab) did not show any difference from the others. There was no difference (P > 0.05) among treatments in total number of cells: 1-CA (63.1a), 2-CAB (57.2a), 3-IO (60.9a), 4-IOB (72.4a), and 5-EL (58.4a). However, there was a difference (P < 0.01) in the percentage of viable cells between treatments 1-CA (49.9%a), 2-CAB (45.8%a), 3-IO (64.9%a), and 4-IOB (50.9%a) in comparison to 5-EL (82.7%b). In conclusion, BSA, when associated with IO, had a negative effect on embryonic developmental rates. The different calcium ionophores used and the BSA did not improve embryo quality. Although there were no significant differences between electrical and chemical activation on the rate of blastocyst formation, it is important to point out that higher quality embryos were achieved by using electrical activation. This work was supported by FAPESP 03/00156-9.

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

Reproduction ◽

10.1530/rep.1.01216 ◽

2006 ◽

Vol 132 (4) ◽

pp. 559-570 ◽

Cited By ~ 17

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.

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304INFLUENCE OF ELECTRICAL STIMULUS ON ACTIVATION OF PIG OOCYTES MATURED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab304 ◽

2004 ◽

Vol 16 (2) ◽

pp. 271

Author(s):

Chang Sik Park ◽

Dong Il Jin ◽

Young June Chang ◽

Moon Young Kim ◽

Young Joo Yi

Keyword(s):

Acetic Acid ◽

Polar Body ◽

Stimulus Frequency ◽

Electrical Stimulus ◽

Electrical Activation ◽

Penicillin G ◽

Oocyte Activation ◽

Activation Rate ◽

First Polar Body

Electrically induced activation of pig oocytes deserves particular attention for research on parthenogenesis. The aim of this study was to improve electrical activation of in vitro matured pig oocyte. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes involving first polar body were selected for activation. For electrical activation, oocytes were rinsed twice in 0.3M mannitol solution supplemented with 0.1mM CaCl2, 0.2mMMgCl2, 0.5mM HEPES and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1mm apart which were overlaid with the same activation solution. Experiment 1 was conducted to investigate the effect of electrical pulse on oocyte activation. Oocytes were activated with DC pulses of 1.0, 1.5, 2.0 and 2.5kVcm−1 for 30, 60 and 90μs, respectively. Experiment 2 was carried out to investigate the effect of electrical stimulus frequency on oocyte activation. Oocytes were activated one, two and three times, with a DC pulse of 1.0kVcm−1 for 60μs. After activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA and cultured for 20h. Activated oocytes were fixed for 48h in 25% acetic acid (v:v) in ethanol at room temperature, and stained with 1% orcein (w:v) in 45% acetic acid (v:v) to examine pronucleus formation. Data were analyzed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of activation was highest in the DC pulse of 1.0kVcm−1 for 60μs (75.1%) compared with the other durations and strengths (62.5–63.1%). Activation rate by electrical stimulus frequency was highest (76.0%) when oocytes were activated by a one-time pulse. In conclusion, the results suggested that electrical stimulus with a single DC pulse of 1.0kVcm−1 for 60μs might be more efficient than other strengths and durations for activation of pig oocytes.

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312 CHEMICAL ACTIVATION OF IN VITRO-MATURED PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab312 ◽

2006 ◽

Vol 18 (2) ◽

pp. 263

Author(s):

A. Bali Papp ◽

E. Varga

Keyword(s):

Cytochalasin B ◽

Chemical Activation ◽

Basic Medium ◽

Ionophore A23187 ◽

Strontium Chloride ◽

Oocyte Activation ◽

Parthenogenetic Development ◽

Activation Rate ◽

Significant Difference

Parthenogenetic oocyte activation is important for nuclear transfer and for the understanding of cell cycle regulation of oocytes. Several chemical agents, including ethanol, cycloheximide, strontium, cytochalasin B, 6 dimethylaminopurine, CaCl2 and ionophore A23187 can induce mammalian oocyte activation in vitro. The objectives of the present study were: (1) to assess the ability of strontium chloride (S), cytochalasin B (CB), cycloheximide (CX), and 6-dimethylaminopurine (D) to induce activation and parthenogenetic development in porcine oocytes; and (2) to verify whether the combinations of treatments (SB group = strontium combined with cytochalasin; SX group = strontium combined with cycloheximide, and SD group = strontium combined with 6-dimethylaminopurine) improves activation and parthenogenetic development rates. Oocytes from slaughterhouse ovaries were matured in vitro for 42 h at 39�C, in 5% CO2 in air. The basic medium used for oocyte maturation was TCM-199 supplemented with 10% pig follicular fluid, 1.25 mM L-glutamine, 0.9 mM Na-pyruvate, 100 �M cysteamine, 0.1 mg/mL streptomycin sulfate, 10 IU/mL pregnant mare serum gonadotropin (PMSG), and 10 IU/mL hCG (Werfft-Chemie GmbH, Vienna, Austria). Denuded MII oocytes were cultured in activation solution for 5 h. Thereafter the oocytes were cultured in NCSU37 for 6 days. At 48 h and 6 days after activation, oocytes, zygotes were fixed in acetic acid:alcohol (1/3 w/v), then stained with 0.1% (w/v) orcein in 45% (v/v) acetic acid, and evaluated under a phase contrast microscope. Each experiment was repeated four times. All data were analyzed by ANOVA, followed by Duncan's multiple range test (P < 0.05). A total of 2243 oocytes were activated in the different groups. In all groups, more than 45% of the oocytes were activated. No significant difference was observed in activation rate among SD (346/170, 49.13%), SX (302/164, 54.3%), and SB (318/182, 57.23%) groups. The activation rate for CB was significantly higher (P < 0.05) than for D or S (323/192, 59.44 � 6.84%; 366/176, 48.09 � 3.43%; and 319/183, 53.29 � 5.39%, respectively). The blastocyst rate for SX was significantly higher (P < 0.05) than that for D, SD, or SB (8.64 � 8.07%; and 0 � 0%; 0 � 0%; and 1.27 � 2.41%, respectively). In conclusion, this study suggests that chemical activation procedure is the most effective in strontium chloride combined with cycloheximide. The lowest oocyte fragmentation rates were in SX (28.40 � 1.26%) and CX (21.05 � 1.12%). This work was supported by the the Hungarian Scientific T 43131 Research Foundation and the Hungarian Science on Technology Foundation E 14/04.

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63 PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES RECONSTRUCTED BY CENTRIFUGATION AND ELECTROFUSION AFTER METAPHASE-II CHROMOSOME TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab63 ◽

2007 ◽

Vol 19 (1) ◽

pp. 149

Author(s):

N. Maedomari ◽

K. Kikuchi ◽

M. Fahrudin ◽

M. Nakai ◽

M. Ozawa ◽

...

Keyword(s):

Treatment Group ◽

Cytochalasin B ◽

Polar Body ◽

Formation Rate ◽

Cell Number ◽

Control Group ◽

Metaphase Chromosomes ◽

Chromosome Transfer ◽

First Polar Body

Metaphase-II chromosome transfer (M-II transfer) is considered to be a useful technique for studying nucleus–cytoplasm relationships, or for generating oocytes with good developmental ability after transfer of the nucleus to the cytoplasm. The reconstructed oocytes carry the original genomic information within the metaphase chromosomes from the donor oocytes. The objective of the present study was to evaluate the parthenogenetic developmental ability of porcine M-II transferred oocytes. In vitro maturation was carried out as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). After culture for 44 h, cumulus cells were removed by hyaluronidase treatment and gentle pipetting. Oocytes that had extruded the first polar body were selected and centrifuged at 13 000g for 9 min to stratify the cytoplasm. The zonae pellucidae were removed after exposure to pronase, and zona-free oocytes were layered on a 300 �L discontinuous gradient (100 �L each of 45%, 30%, and 7.5%) of Percoll in TCM-HEPES supplemented with 5 �g mL-1 cytochalasin B. After centrifugation of the oocytes on the gradient in microcentrifuge tubes at 6000g for 20 s, fragmented cytoplasm with an equal volume was obtained, stained with Hoechst 33342, and classified as cytoplasm with or without chromosomes by observation with a fluorescence microscope. One fragmented cytoplasm with chromosomes and 2 fragmented cytoplasms without chromosomes were fused by electric stimulation with a single DC pulse (1.5 kV cm-1, 20 �s) and cultured temporarily for 1 h. The reconstructed oocytes were then stimulated again to induce parthenogenetic activation (0.8 kV cm-1, 30 �s, 2 DC pulses) (treatment group). Zona-free mature oocytes that had not been subjected to reconstruction were activated as a control group. The oocytes in both groups were treated with 5 �g mL-1 cytochalasin B for 2 h, and then cultured for 6 days in media (Kikuchi et al. 2002) using the WOW system (Gabor et al. 2000 Mol. Reprod. Dev.). The blastocyst formation rate in the control group (22.9 � 5.5%) was significantly higher (P < 0.05; ANOVA and PLSD-test) than that in the treatment group (7.6 � 1.8%). The total cell number per blastocyst in the control group (28.7 � 4.6) was significantly higher (P < 0.05) than that in the treatment group (16.7 � 1.0). These results suggest that reconstructed porcine oocytes following M-II transfer by centrifugation and electrofusion can develop to the blastocyst stage in vitro. This technique enables transfer of the nucleus to cytoplasm with good developmental ability without the use of a micro-manipulation system.

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294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab294 ◽

2007 ◽

Vol 19 (1) ◽

pp. 262

Author(s):

W. Fujii ◽

H. Funahashi

Keyword(s):

Chemical Activation ◽

Formation Rate ◽

Cumulus Cell ◽

Cumulus Cells ◽

Female Rats ◽

Blastocyst Formation ◽

In Vitro Development ◽

Pronuclear Formation ◽

Vitro Development

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.

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301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab301 ◽

2004 ◽

Vol 16 (2) ◽

pp. 270

Author(s):

I. Lagutina ◽

G. Lazzari ◽

C. Galli

Keyword(s):

Cytochalasin B ◽

Average Rate ◽

Polar Body ◽

Cell Number ◽

Developmental Competence ◽

Blastocyst Formation ◽

Average Cell ◽

Electric Impulse ◽

Polar Body Extrusion

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

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Luteolin Orchestrates Porcine Oocyte Meiotic Progression by Maintaining Organelle Dynamics Under Oxidative Stress

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.689826 ◽

2021 ◽

Vol 9 ◽

Author(s):

Soo-Hyun Park ◽

Pil-Soo Jeong ◽

Ye Eun Joo ◽

Hyo-Gu Kang ◽

Min Ju Kim ◽

...

Keyword(s):

Oxidative Stress ◽

Oocyte Maturation ◽

Polar Body ◽

Blastocyst Formation ◽

Cortical Granules ◽

Expression Levels ◽

Meiotic Progression ◽

First Polar Body ◽

Underlying Mechanisms

Increasing evidence has demonstrated that oxidative stress impairs oocyte maturation, but the underlying mechanisms remain largely unknown. Here, for the first time, we examined the antioxidant role of luteolin in meiotic progression and the underlying mechanisms. Supplementation of 5 μM luteolin increased the rates of first polar body extrusion and blastocyst formation after parthenogenetic activation, and the expression levels of oocyte competence (BMP15 and GDF9)-, mitogen-activated protein kinase (MOS)-, and maturation promoting factor (CDK1 and Cyclin B)-related genes were also improved. Luteolin supplementation decreased intracellular reactive oxygen species levels and increased the expression levels of oxidative stress-related genes (SOD1, SOD2, and CAT). Interestingly, luteolin alleviated defects in cell organelles, including actin filaments, the spindle, mitochondria, the endoplasmic reticulum, and cortical granules, caused by H2O2 exposure. Moreover, luteolin significantly improved the developmental competence of in vitro-fertilized embryos in terms of the cleavage rate, blastocyst formation rate, cell number, cellular survival rate, and gene expression and markedly restored the competencies decreased by H2O2 treatment. These findings revealed that luteolin supplementation during in vitro maturation improves porcine meiotic progression and subsequent embryonic development by protecting various organelle dynamics against oxidative stress, potentially increasing our understanding of the underlying mechanisms governing the relationship between oxidative stress and the meiotic events required for successful oocyte maturation.

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236 PARTHENOGENETIC ACTIVATION OF IN VITRO-MATURED OVINE OOCYTES WITH STRONTIUM

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab236 ◽

2008 ◽

Vol 20 (1) ◽

pp. 197

Author(s):

J. Zhu ◽

K. H. S. Campbell

Keyword(s):

Nuclear Transfer ◽

Cytochalasin B ◽

Blastocyst Stage ◽

Oocyte Activation ◽

Parthenogenetic Development ◽

Activation Rate ◽

Electrical Pulses ◽

Blastocyst Rate ◽

Activation Rates

The objective of the present experiments was to examine whether strontium could activate in vitro-matured ovine oocytes. Oocytes were collected and matured as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). Briefly, selected cumulus–oocyte complexes were cultured in modified TCM-199 medium supplemented with 20% sheep serum and hormones for 22–23 h, at 39°C, 5% CO2 in air. Matured oocytes were randomly divided into four groups and treated as follows: (1) cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (2) electrically activated in Ca2+-containing medium, then cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (3) electrically activated in Ca2+-containing medium and then cultured in SOF medium containing 5 μg mL–1 cytochalasin B for 4–5 h; and (4) electrically activated in Ca2+-free medium and then transferred into SOF medium + 5 μg mL–1 cytochalasin B for 4–5 h. This experiment was repeated three times. Activation rates based on the number of pronuclear formations/the number of oocytes cultured were 96.7% (147/152), 95.9% (116/121), 75.9% (101/133), and 43.0% (56/107) in Groups 1–4, respectively. After 7 days of culture in SOF medium, 26.8%, 33.3%, 19.6%, and 0% of oocytes in Groups 1, 2, 3, and 4 developed to the blastocyst stage, respectively. Significant differences in blastocyst rate were observed across these groups except between groups 1 and 2 (P < 0.01). However, there were no significant differences in mean number of nuclei/blastocyst across Groups 1, 2, and 3 (P > 0.05). Our results demonstrated that in vitro-matured ovine oocytes can be effectively activated with strontium alone, resulting in an activation rate of 96.7% and a blastocyst rate of 26.8% (blastocysts/oocytes). Also, a combination of strontium and electrical pulses could benefit sheep oocyte activation and embryo development to the blastocyst stage (95.9% and 33.3%, respectively). We conclude that strontium is an effective activator for sheep oocyte activation and it could be used for sheep nuclear transfer. Table 1. Parthenogenetic development of oocytes activated by SrCl2+ and electrical pulses

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216 PRONUCLEAR FORMATION AND DEVELOPMENTAL COMPETENCE OF MATURE PORCINE OOCYTES DERIVED FROM SMALL- AND MEDIUM-SIZED FOLLICLES

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab216 ◽

2011 ◽

Vol 23 (1) ◽

pp. 207

Author(s):

C. Kohata ◽

H. Funahashi

Keyword(s):

Polar Body ◽

Formation Rate ◽

Cell Number ◽

Developmental Competence ◽

Blastocyst Formation ◽

Dibutyryl Camp ◽

First Polar Body ◽

Porcine Oocyte ◽

Sperm Penetration ◽

Pronuclear Formation

The maturation rate of oocytes derived from small follicles (SF) is known to be lower than that of oocytes from medium follicles (MF). The objective of this study was to assess the fertilizability and developmental competence of mature SF oocytes that were selected by the presence of the first polar body. Cumulus–oocyte complexes (COC) were aspirated from SF (1 to 2 mm in diameter) or MF (3 to 6 mm in diameter) of prepuberal ovaries. The COC were cultured in modified porcine oocyte medium supplemented with gonadotropins and dibutyryl cAMP for the first 20-h period and then in gonadotropin-free and dibutyryl cAMP-free porcine oocyte medium for another 24 h. Following IVM culture, mature oocytes with the first polar body were selected under a stereomicroscope, co-incubated with spermatozoa in a drop of modified TCM-199 containing 0.4% BSA and 5 mM caffeine for 6 h, and then incubated in porcine zygote medium-5 for 7 days. Sperm penetration, cleavage, and early development of the oocytes were examined before culture in porcine zygote medium-5 on Days 2 and 7 of culture. To analyse the fertilizability and developmental competence of oocytes from the SF and MF groups, sperm penetration, pronuclear formation, cleavage, blastocyst formation, and mean cell number in a blastocyst (as determined by fluorescence observation following Hoechst 33342 staining) were examined. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post-hoc test (P < 0.05). The percentages of oocytes in which the first polar body could be observed were 51.0 ± 4.5% and 78.5 ± 2.8% for SF- and MF-oocytes, respectively, whereas the maturation rates were 83.8 ± 4.0% and 62.8 ± 4.4% following fixation and staining. When only mature oocytes were co-cultured with sperm for 6 and 9 h, sperm penetration, monospermic penetration, and pronuclear formation were not different (P > 0.33) between mature SF- and MF-oocytes. Although there was no difference in cleavage rates between the mature SF- and MF-oocyte groups, blastocyst formation rate and mean cell number in the blastocyst were higher in mature MF-oocytes (31.0 ± 3.6% and 38.7 ± 1.9 cells, respectively) than in mature SF-oocytes (14.7 ± 3.2% and 31.2 ± 2.0 cells). From these results, we conclude that mature oocytes derived from SF have a similar fertilizability when compared with mature MF-oocytes, but the developmental competence to the blastocyst stage following IVF is significantly lower in mature SF-oocytes than in mature MF-oocytes.

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