• Journal of Embryo Transfer
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• v.31 no.3
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• pp.153-159
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• 2016

(-)-Epicatechin gallate (ECG) is a polyphenol compound of green tea exhibiting biological activities, such as antioxidant and anticancer effects. To examine the effect of ECG on porcine oocytes during in vitro maturation (IVM), oocytes were treated with 0-, 5-, 15-, and $25{\mu}M$ ECG. After maturation, we investigated nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 42 hours of IVM, the $5{\mu}M$ group exhibited significantly increased (p< 0.05) nuclear maturation (89.8%) compared with the control group (86.1%). However, the $25{\mu}M$ group observed significantly decreased (p< 0.05) nuclear maturation (83.5%). In intracellular maturation assessment the 5-, 15-, and $25{\mu}M$ groups had significantly increased (p< 0.05) GSH levels and decreased ROS levels compared with the controls. The 5- and $15{\mu}M$ group showed significantly increased (p< 0.05) embryo formation rates and total cell number of blastocysts after PA (18% and 68.9, 15% and 85.1 vs. 12% and 59.5, respectively) compared with controls. Although the $25{\mu}M$ group observed significantly lower blastocyst formation rates after PA (27.6% vs. 23.2%) than control group, the $5{\mu}M$ group showed significantly increased blastocyst formation rates after PA (37.2% vs. 23.2%) compared to the control group. Furthermore, the $5{\mu}M$ group measured significantly increased blastocyst formation rates (20.7% vs. 8.6%) and total cell number after IVF ($88.3{\pm}1.5$ vs. $58.0{\pm}3.6$) compared to the control group. The treatment of $5{\mu}M$ ECG during IVM affectively improved the porcine embryonic developmental competence by regulating intracellular oxidative stress during IVM.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.123-130
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• 1997

Normal maturation of the mammalian oocytes is prerequisite for the fertilization and the early embryonic development. We have been tested the effects of purine and its de novo synthetic inhibitor, azaserine(Aza) on the maturation of germinal vesicle(GV) and germinal vesicle breakdown(GVBD) mouse oocytes. Denude-immature oocytes were cultivated in the media containing adenosine, guanosine, and/or azaserine, and checked the matruation stage by monitoring the prominent morphological changes. In GV stage oocytes, GV was arrested temporarily by the adenosine(1.0%) and protractedly by the guanosine(65.9%, P<0.001). The regression was increased significantly at the adenosine(90%, P<0.001) but decreased at the guanosine(1.6%, P<0.05). Inhibiting the de novo synthesis of purine, nuclear maturation rate was increase(90.4% : 96.7%), but GV arrest was significantly increased by cotreatment with guanosine(P<0.001). Polar body extraction significantly was increased at the Aza(P<0.05), but not in others. In GVBD oocytes, adenosine itself did not affect GVBD arrest. Guanosine, on the other hand, elevated GVBD arrest rate(P<0.001), but co-treated with Aza, decreased GVBD arrest(P<0.001). Aza increased GVBD arrest rate(20.2%, P<0.05) compared with control. From those results, we know that guanosine shows more prominent effect on the inhibition of nuclear maturation at the GV stage, and of the 1st polar body extrusion at the GVBD stage. Adenosine showed the cytoplasmic toxicity at GV stage oocyte. Our data speculate that cytoplasmic cAMP level is auto-regulated by endogenous adenylate cyclase while GVBD is inhibited by guanosine, since purine toxicity is not observed in the GVBD stage. And it is showed that purine metabolism is concerned with nuclear maturation, that the amounts of purine metabolism is not even during the oocyte maturation.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.28-28
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• 2001

The objective of this study was to the effect on subsequent development of EGF present in defined medium during bovine 1)oocyte maturation or 2)embryo culture. The presence of EGF during IVM, irrespective of concentration(1, 10, 100ng/$m\ell$), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphaseII, the rate of cleavage, and develop to blastocyst. 1. In the group of EGF-added medium(1, 10, 100ng/$m\ell$), nuclear maturation rate for in vitro maturation was 91% to 92% but was not significantly higher than control group(87%). 2. For in vitro maturation, in the group of EGF-added medium(1, 10, 100ng/$m\ell$)the rate of cumulus cell expansion degree 2 ranged from 81% to 87%, which was significantly higher than the control group(medium with EGF not added). The rate of in vitro fertilization, developing to 2-to 4- cell stage, was 76% to 80%, which was also significantly higher(p<0.05)than control group(62%). 3. For in vitro maturation, in the group of EGF added in medium(1, 10, 100ng/$m\ell$)the development rate to blastocyst was 24.3% to 27%, which was significantly higher than control group(13.7%). The total cleavage rate in the group of EGF-added medium was 77% to 82%, which was higher than control group. 4. The development rate to blastocyst for 6 days of cultivation and the hatching blastocyst were 30.6% and 59.1%, respectively, in the group of 100ng/$m\ell$ of EGF, which were significantly higher(p<0.05)than control group(14.0% and 24%, respectively), The numbers of cells in blastocyst were 140.2 and 148, respectively, in 10ng/$m\ell$ and 100ng/$m\ell$ of EGF-added medium, which were higher than 108.5 in control group. 5. The development rate of in vitro fertilized embryos to blastocyst in 10ng/$m\ell$ of EGF-added medium co-cultured with somatic cell was 28%, which was significantly higher(p<0.05)than control group(11.8%). The numbers of cells in blastocyst were 141.6 for EGF-added medium and 145 for EGF+co-culture group, which were higher than control(101.6)and medium co-cultured with somatic cells(110.6). These results showed that in vitro maturation and fertilization, EGF was found a significant effect of increase of development rate to blastocyst and cell number.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.71-80
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• 2012

Biotechnologies for cloning animals and in vitro embryo production have the potential to produce biomedical models for various researches. Especially, pigs are a suitable model for xenotransplantation, transgenic production and various areas of reproductive research due to its physiological similarities to human. However, utilization of in vitro-produced embryos for transfer remains limited. Despite improvement over past few decades, obstacles associated with the production of good quality embryos in vitro still exist which limit the efficiency of cloning. One of major problems includes improper in vitro maturation (IVM) and culture (IVC). Oxidative stress caused from in vitro culture conditions contributes to inadequate IVM and IVC which leads to poor developmental competence of oocytes, failure of fertilization and embryo development. To reduce the oxidative stress, various antioxidants have been used to IVM and IVC system. However, limited information is available on the effects of resveratrol on livestock reproductions. Resveratrol is a polyphenolic natural product and well known as an antioxidant in foods and beverages (e.g. in grapes and red wine). Resveratrol is known to be cardioprotective, anticarcinogenic, anti-inflammatory, antioxidant and antiapoptotic. This paper will review the effects of resveratrol on in vitro maturation of oocytes and embryo development.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.29-29
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• 2001

This study was carried out to examine, by the reverse transcription chain reaction(RT-PCR)and Immunostain assays, epidermal growth factor mRNA expression in bovine ova during oocyte maturation in vitro(0-2lh)and after fertilization in vitro(6-144hr: zygotes to blastocysts). In this study, the transcripts of EGF was detected in oocytes using primers for EGF. Transcripts for EGF mRNA was not detected in oocytes through in vitro maturation. But EGF mRNA were present after fertilization up to the 2-cell stage and the blastocyst stage. The highest mRNA levels in 4-cell stage embryos were decreased at 8cell stage and then reincreased upto morulae and blastocysts. The results of this study showed EGF mRNA are present in embryo after fertilization and this factors are involved in the regulation of bovine embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.293-301
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• 1996

In the present study, mouse follicular oocytes and 2-cell embryos(late -zygote stage embryos included) were cultured on the electric pad for electric stimulation in the culture incubator. In addition, follicular oocytes and embryos were tested for maturation and development under higher temperature condition($39^{\circ}C$).Mouse follicular oocyte maturation were not affected by anion electric stimulation and there is no significant difference in GBVD and MI between the control and experiment group after 4hr culture. In the embryo culture, it was found that more morula and blastocyst were found in the electric stimulation group rather than the control(96hr). This may seem to be caused with cytoplasmic $Ca^{2+}$ transient rise by electric stimulation(anion pad). On the other hand higher temperature incubation ($39^{\circ}C$) on the anion pad caused all the embryos degenerated within $12h{\sim}24hr$ culture. This was quite different from large animal embryos(bovine, pig, sheep), in which beneficial effect of high temperature incubation for oocyte maturation and embryo development were found.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.115-118
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• 2006

Cyclin B1 is known to reflect the M-phase promoting factor (MPF), a universal regulator of G2/M-phase transition, activity during the process of oocytes maturation. To investigate whether culture condition affects the maturation rate and the expression of cyclin B1 protein, bovine immature oocytes are stimulated and cultured according to the following protocols: Experiment 1: denuded oocytes (denude) only, COC only, denuded oocytes+granulosa cells (denude+GCs) and COC+GCs; Experiment 2: no-activation (control), 7% ethanol for 5 min and $10{\mu}l/ml$ ionomycin for 5 min at immediately before maturation. The maturation rates of denude and no-activation group were significantly lower in both experiments (P<0.05), respectively. Co-culture or stimulation method in bovine immature oocytes culture increases the cyclin B1 expression significantly in both experiments (P<0.05). Based on these results, culture condition affects the maturation rate and the expression of cyclin B1 protein during the first meiotic maturation in bovine immature oocytes.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.77-82
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• 2007

This study was conducted to investigate the effects of oocyte maturational age and activation condition on in vitro development of porcine parthenogenetic embryos (parthenotes). Porcine follicular oocytes were matured in vitro for 30 to 44 hr. Maturation rate was examined during in vitro maturation (IVM) every 2 hr interval. The cdc2 kinase activity was measured at 36 and 44 hr of IVM. Some oocytes were activated at 36 or 44 hr of IVM by three different conditions; 1) single electric stimulation (1.5 kV/cm for $30{\mu}sec$; ES), 2) double electric stimulations (1.5 kV/cm for $30{\mu}sec$, followed by 1.0 kV/cm for $50{\mu}sec$ after 1 hr; ES+ES) or 3) ES+ES followed by culture in 6-dimethlyaminopurine (6-DMAP) for 4 hr (ES+ES+D), and cultured for 6-7 days. Maturation rate was significantly increased as culture period was increased to 36 hr (66.9%, p<0.05), and then gradually increased to 87.1% at 44 hr of IVM. The cdc2 kinase activity was decreased (p<0.05) with culture period prolonged from 36 hr to 44 hr. Lower blastocyst formation rate (4.3%, p<0.05) were obtained by ES in 36 hr-matured oocytes compared to other treatments (16.5 and 20.5%) in the same age and the same treatment in 44 hr-matured oocytes (15.0%). High blastocyst formation rate (23.6%) was obtained by ES+ES+D in 44 hr-matured oocytes (p<0.05). These results demonstrate that porcine oocyte activation and in vitro development of parthenotes can be affected by interactions between oocyte maturational age and activation condition.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.754-758
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• 2001

Bovine oocytes with compact and complete cumulus cells were cultured for up to 24h in TCM199 buffered with 25mmol/l hepes and supplemented with 10% FBS (fetal bovine serum), 1mg/ml $17{\beta}$-estradiol, 20IU/ml hCG(human chorionic gonadotropin). All of the oocytes were divided into at 6 groups depending upon incubation times (control, 0 hour, 6 hours, 12 hours, 16 hours, 18 hours). To all experimental media, $200{\mu}g/ml$ puromycin was added at different incubation times mentioned above. Following these culture times, in vitro insemination was conducted with frozen-thawed bovine spermatozoa in medium BO (Brackett and Oliphant medium for in vitro insemination) with $10{\mu}g/ml$ BSA(bovine serum albumin) and 10 mg/ml heparin added. After 22h culture, the oocytes were fixed with acetic alcohol solution and stained with orcein acetic solution to evaluate sperm nuclear progression. Addition of puromycin after 0, 6 and 12 h of culture resulted in near of oocyte maturation at the M1 stage. Contrariwise, puromycin addition after 12 h of culture led to restoration of nuclear progression to M2 stage. On the one hand, puromycin affected the synthesis of Cyclin B protein that may be involved in the oocyte maturation and sperm capacitation for in vitro fertilization. The present study suggests the participation of protein synthesis, cyclin B, in the oocyte development from M1 to M2 stages in vitro.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.4
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• pp.483-488
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• 2015

We studied oocyte steroidogenesis in the blackfin flounder Glyptocephalus stelleri as a region-specific species, in the East Sea of Korea during the spawning season. Maturing oocytes (0.76, 0.82, 0.88, and 0.91 mm in oocyte diameter) were incubated in vitro in the presence of [$^3H$] $17{\alpha}$-hydroxyprogesterone ($[^3H]17{\alpha}$-OHP) as a precursor. Steroid metabolites were extracted from the incubated medium and oocytes, and the extracts were separated and identified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and gas chromatographymass spectrometry (GC/MS). The major metabolites produced from $[^3H]17{\alpha}$-OHP were androgens [androstenedione (A4) and testosterone (T)] and estrogens [$17{\beta}$-estradiol (E2) and estrone (E1)] and progestins [$17{\alpha},20{\alpha}$-dihydroxy-4-pregen-3-one ($17{\alpha}20{\alpha}P$) and $17{\alpha}20{\beta}$-dihydroxy-4-pregnen-3-one ($17{\alpha}20{\beta}P$)] in maturing oocytes. The metabolic rate of $17{\alpha}20{\beta}$ was elevated (29.04%) in oocytes measuring 0.88 mm (nucleus migration stage following the induction of germinal vesicle breakdown), but was very low in oocytes measuring 0.76, 0.82, and 0.91 mm (0.42, 0.67, and 2.62%, respectively). From these results, we suggest that $17{\alpha}20{\beta}P$ acts as a maturation-inducing steroid in the blackfin flounder.