• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.113-113
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• 2002

The stimulatory effect of EGF and FSH on oocyte maturation have been reported in various mammalian species. And some reports presented FSH enhanced the effect of EGF on oocyte maturation. But, the interaction between EGF and FSH on nuclear maturation of mammalian oocytes is not fully understood. We observed the effect of EGF and FSH on nuclear maturation during in vitro maturation of mouse oocytes. Also, we examined the interaction between EGF and FSH on nuclear maturation of mouse oocytes using the EGFR inhibitor or FSH inhibitor. Germinal vesicle (GV) stage oocytes were obtained from 3-4weeks PMSG primed BCFI hybrid mice and cultured in TCM-199 medium with 0.4%PVP supplemented with/without EGF (1ng/ml), FSH (1ug/ml), EGFR specific tyrosine kinase inhibitors: Tyrphostin AG 1478 (500nM), MAP kinase kinase inhibitor : U0126 (20uM) or PD 98059 (100uM) for 14-l5hr. Rapid staining method were used for the assessment of nuclear maturation. Nuclear maturation rates of EGF indjor FSH-treated group were significantly higher than those of control group. Treatment of EGFR inhibitor significantly block the nuclear maturation of GV oocyte in EGF-treated group, but it did not block those of GV oocyte in FSH-treated or FSH and EGF-treated group. Treatment of FSH inhibitor(U0126, PD98059) significantly block the nuclear maturation of EGF-treated group, FSH-treated and FSH and EGF-treated group. These results show that EGF has a stimulatory effect as well as different action pathway with FSH on in-vitro maturation of mouse oocyte in vitro. Therefore, further studies will be needed to find the signaling pathway of EGF associated with nuclear maturation.

• 대한수의학회지
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• 제36권4호
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• pp.929-939
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• 1996

The present study was undertaken to establish a relationship between bovine follicle size and oocyte diameter, compare the nuclear maturation competence of oocytes of different diameter groups and the nuclear maturation changes in Korean Native Cattle according to in vitro maturation period. To compare the relationship between follicle size and oocyte diameter, follicles were dissected, measured, and assigned to one of the following size categories($4{\geq}mm$, 3-4mm, 2-3mm, 1-2mm, and < 1mm), investigate the maturation competence in the different-sized oocytes, which were divided into three groups( < $110{\mu}m$, 110 - < $120{\mu}m$, and ${\geq}120{\mu}m$). Oocytes were cultured in the culture medium during 0, 6, 12, 18, and 24hrs, respectively, stained, and measured the nuclear maturation degree according to period. When compared the relationship between follicle size and intrafollicular oocyte diameter, oocyte diameters of three groups of ${\geq}3mm$ follicle-sized were significantly higher than < 3mm (p<0.01). After in vitro maturation, the rates reached to MI stage of < $110{\mu}m$ oocyte groups(25%) was higher than $110-120{\mu}m$ and ${\geq}120{\mu}m$ oocyte groups(11 and 10%) reached to the same stage(p<0.01), and the rates throughout MII stage of $110-120{\mu}m$ and ${\geq}120{\mu}m$ and < $110{\mu}m$(70 and 76%) groups were higher than < $110{\mu}m$(35%)(p<0.01). When nuclear maturation rates were measured according to period, < 6hr groups(7 and 10%) showed lower rates reached to MI than ${\geq}12hr$ groups(100%), 24hr groups(76%) revealed higher rates throughout MII than 18hr groups(40%). These results indicate that the preparation of oocyte for the production of in vitro fertilization embryos and nuclear transplantation ones could be adapted, as follicle increased up to appointed size there was a corresponding increase in oocyte diameter, and differences of nuclear maturation rate revealed according to oocyte diameter and maturation period.

• 한국수정란이식학회지
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• 제13권2호
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• pp.139-145
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• 1998

In the present study, effects of interleukin-2 (IL-2), a differentiator and proliferator of T-cells, on nuclear maturation and sperm penetration of bovine oocytes was examined in a serum-free or serum-containing medium. Basic medium was used TCM-199 supplemented with 2.2g / ι sodium bicarbonate, 100 i.u. /rnl penicillin. 100$\mu$g /ml streptomycin, 0.25$\mu$g/ml Fungizone, this medium treated with FCS and IL-2. In experiment 1, we examined the effect of the addition of 0, 1, 5, 10 or 15nM /ml IL-2 to tissue culture medium (TCM-199) on nuclear maturation of oocytes Development of oocytes to the Metaphase II (M II) stage (%) was significantly (P<0.05) higher at 1, 5,10 and 15 nM /ml IL-2(54.2, 73.5, 80.0 and 69.6%, respectively) than at 0 nM /ml IL-2(35.7%). In experiment 2, we examined the effect of the addition of l0nM /ml IL-2 or 5% FCS in oocyte maturation. Nuclear maturation rates were significantly(P<0.05) higher l0nM /ml IL-2(80%) than non-treatment(35.7%) and 5% FCS(63.6%) treatment. On the other hand, there were no significant difference in the proportion of oocytes developed to the 2-cell stage after addition of IL-2 and/or FCS. These results suggest that IL-2 supports nuclear maturation of bovine immature oocytes in vitro. Serum-free maturation system using IL-2 might be useful for evaluation of various factors on oocyte maturation.

• Journal of Magnetics
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• 제19권3호
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• pp.241-247
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• 2014

The objective of this study was to study the effect of magnetized water on porcine cumulus cell-oocyte complexes (COCs). Oocytes obtained from female pig were cultured in a medium magnetized at 0, 2000, 4000, and 6000 Gauss (G) for 5 minutes using the neodymium magnet. Subsequently, intracellular hydrogen peroxide ($H_2O_2$) concentration, glutathione (GSH) activity, oocyte membrane integrity, anti-apoptosis factor Bcl-xL expression, and nuclear maturation were analyzed. The intracellular $H_2O_2$ levels in COCs cultured for 44 hours were not significantly different among the variously magnetized samples. However, GSH activity were significantly higher in the magnetized samples compared to the 0 G sample. The Bcl-xL mRNA expression in COCs cultured for 44 hours was higher in the 4000 G sample than other treatment groups. Membrane damage in COCs cultured for 22 and 44 hours was significantly lower in 4000 G group than control group. On the other hand, nuclear stages as maturation indicator significantly increased in 2000, 4000, and 6000 G groups compared to 0 G group. These results indicate that incubation of porcine oocytes and cumulus cells in magnetized medium improves intracellular GSH levels, membrane integrity and nuclear maturation, and inhibits apoptosis in vitro.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.147-152
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• 2008

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at $56^{\circ}C$ and $97^{\circ}C$, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and $56^{\circ}C$ group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

• 한국수정란이식학회지
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• 제27권3호
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• pp.163-169
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• 2012

The 3-isobutyl-1-methylxanthine (IBMX) is non-selective phosphodiesterase and is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte. The present study was conducted to analyze: (1) nuclear maturation (examined by the Hoechst staining), (2) whether cytoplasmic maturation (examined by the intracellular glutathione (GSH) concentration) of porcine oocytes is improved during meiotic arrest after prematuration (22 h) with IBMX. Before in vitro maturation (IVM), oocytes were treated with 1 mM IBMX for 22 h. After 22 h of pre-maturation, the higher rate of IBMX treated group oocytes were arrested at the germinal vesicle (GV) stage (42.3%) than control IVM oocytes (10.1%). It appears that the effect of IBMX on the resumption of meiosis has shown clearly. In the end of IVM, the reversibility of the IBMX effect on the nuclear maturation has been corroborated in this study by the high proportions of MII stage oocytes (72.5%) reached after 44 h of IVM following the 22 h of inhibition. However, intracellular GSH concentrations were lower in the oocytes treated with IBMX than the control oocytes (6.78 and 12.94 pmol/oocyte, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pre-treated with IBMX for 22 h did not equal that of control oocytes in the current IVM system. These results indicate that pre-maturation with IBMX for 22 h may not be beneficial in porcine IVM system.

• 한국발생생물학회지:발생과생식
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• 제22권4호
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• pp.297-307
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• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• 한국가축번식학회지
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• 제21권2호
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• pp.177-183
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• 1997

The present study examined the effects of epidermal growth factor(EGF) and transforming growth factor-$\alpha$(TGF-$\alpha$) on in vitro maturation of porcine follicular oocytes. Basic medium used TCM-HEPES, and oocytes cultured for 42 hours in vitro. The results obtained are as follows; 1. The nuclear maturation rates of EGF-treated groups(10ng/ml, 75.9% ; 30ng/ml, 69.2% ; 50ng/ml, 67.2% ; 100ng/ml, 71.0%) on the porcine oocytes cultured in medium without pFF in vitro were significantly(P<0.01) higher than those of non-treated group(57.1%). When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation rates of 30ng EGF/ml(77.1%) treated group were significantly(P<0.01) higher than those of non-(59.2%) and EGF-treated groups(10ng/ml, 65.4% ; 50ng/ml, 65.5% ; 100ng/ml, 70.4%). 2. The nuclear maturation rates of 30ng TGF-$\alpha$/ml treated group(71.9%) in media without pFF in vitro were significatnly(P<0.01) higher than those of non-(57.1%) and TGF-$\alpha$ treated groups(10ng/ml, 60.4% ; 50ng/ml, 65.4% ; 100ng/ml, 60.0%). When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation rates of 30 and 50ng TGF-$\alpha$/ml(77.4% and 79.6%) treated groups(10ng/ml, 64.2% ; 100ng/ml, 61.6%). 3. On the effect of EGF(30ng/ml) and/or TGF-$\alpha$(30ng/ml) treated groups in medium without pFF in vitro, the nuclear maturation rates indicated 57.3, 60.4, 75.9 and 79.7% in media with no EGF & TFG-$\alpha$, TGF-$\alpha$ only, EGF only nad EGF＋TGF-$\alpha$ treated groups, respectively. The nuclear maturation rates in medium with EGF only or EGF＋TGF-$\alpha$ were significantly(P<0.01) higher than those non- and TGF-$\alpha$ treated groups. When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation ratesof EGF＋TGF-$\alpha$ treated group(75.9%) were significantly(P<0.01) higher than those of non-(59.2%), TGF-$\alpha$ only (64.2%) and EGF only(69.4%) treated groups.

• Journal of Veterinary Science
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• 제23권2호
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• pp.31.1-31.13
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• 2022

Background: Compared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes. Objectives: This study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes. Methods: Pig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM. Results: Regardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference. Conclusions: IVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.

• Reproductive and Developmental Biology
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• 제39권2호
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• pp.37-41
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• 2015

The oocyte undergoes various events during maturation and requires many substances for the maturation process. Various intracellular organelles are also involved in maturation of the oocyte. During the process glucose is essential for nuclear and cytoplasmic maturation, and adenosine triphosphate is needed for reorganization of the organelles and cytoskeleton. If mitochondrial function is lost, several developmental defects in meiotic chromosome segregation and maturation cause fertilization failure. The endoplasmic reticulum, a store for $Ca^{2+}$, releases $Ca^{2+}$ into the cytoplasm in response to various cellular signaling molecules. This event stimulates secretion of hormones, growth factors and antioxidants in oocyte during maturation. Also, oocyte nuclear maturation is stimulated by growth factors such as epidermal growth factor. This review summarizes roles of organelles with focus on the Golgi apparatus during maturation in oocyte.