• 한국발생생물학회지:발생과생식
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• 제1권1호
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• pp.45-56
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• 1997

Befor fertilization, mammalian oocytes undergo meiotic maturation, which consists of nuclear and cytoplasmic differentiation. In this study, changes of $Ca^{2+}$ stores in mouse oocytes were examined during meiotic maturation and the role of $Ca^{2+}$ in the regulation of the maturation was investigated by using monoclonal antibodies against smooth endoplasmic reticulum $Ca^{2+}$-ATPase(SERCA-ATPase) and calreticulin. Observations were made under epifluorescence microscope and/or confocal laser scanning microscope. In immature oocytes which did not resume meiotic maturation, SERCA-ATPases were mostly localized in the vicinity of the germinal vesicle and calreticulins were distributed evenly throughout the cytoplasm. In mature oocytes, SERCA-ATPases were observed throughout the cytoplasm, butwere absent from the nuclear region. In contrast, calreticulins were localized mostl in the cortex of the oocyte and were absent from the cytoplasm. However, bright fluoresence stainings were wbserved in the perimeiotic spindle region of mature oocyte when labeled with antibodies against calreticulin. These results indicate that mouse oocytes undergo distinct rearrangement of the localization of $Ca^{2+}$-ATPases and calreticulins during meiotic maturation. Thus it can be suggested that redistribution of the $Ca^{2+}$ stores, as revealed by differential fluorescence stainings, is deeply involved in the regulatory mechanism of mammalian oocyte maturation.

• Reproductive and Developmental Biology
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• 제30권4호
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• pp.301-305
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• 2006

The aim of this study was to investigate whether addition of porcine epididymal fluid (pEF) into culture medium during in vitro maturation influences the nuclear maturation of porcine germinal vesicle (GV) oocytes. Porcine cumulus-oocyte complexes (COCs) from follicles were cultured in tissue culture medium 199 (TCM 199) containing pEF. After 48hr of culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II (M II) stage was significantly (p<0.05) increased in oocytes cultured in the media supplemented with 10% pEF during in vitro maturation than in those without pEF regardless of cumulus presence or absence (54.6% vs 22.5%, 51.7% vs 24.2%). The supplementation of pEF during maturation of oocyte enhanced oocytes maturation in a dose-dependent manner in vitro. Also significant differences (p<0.05) in the percentage of MII oocytes were observed according to exposure period in pEF. Present study suggests that pEF contains a enhancing component(s) for nuclear maturation of porcine immature oocytes in vitro.

• 한국동물생명공학회지
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• 제35권1호
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• pp.2-11
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• 2020

Assisted reproductive technologies (ART) merely depend on improving the oocyte maturation and their developmental competence to produce good quality embryos. Oocyte maturation passes through long and complex molecular steps starts from the early embryonic life and ends with sperm fertilization. Oocyte developmental competence can be attained by improving the nuclear and cytoplasmic mechanisms together with some epigenetic maturation. In this review, we highlight the cornerstones of oocyte maturation on both nuclear and cytoplasmic levels. Interfering or supporting these molecular mechanisms would help in the development of novel regulating agents for reproductive performance of humans and livestock species.

• 한국수정란이식학회지
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• 제31권3호
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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.

• 한국가축번식학회지
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• 제21권2호
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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.

• Reproductive and Developmental Biology
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• 제29권2호
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• pp.121-125
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• 2005

The present study was performed to determine the ability of canine oocytes to achieve nuclear maturation according to oocyte diameter and different culture environments. All of the collected oocytes were classified by grade 1 to 3 and by their diameters such as $<100{\mu}m,\;<100{\mu}m\;to\;<110{\mu}m,\;<110{\mu}m,\;to\;<120{\mu}m,\;>120{\mu}m,$. Oocytes were cultured in culture medium supplemented with $10\%\;FBS,\;0.4\%\;BSA,\;10\%$ porcine follicular fluid (pFF), $10\%$ canine serum (CS), or $10\%$ canine estrus serum (CES). The mean number of oocytes recovered from estrus status ovaries was significantly higher than that of anestrus status ovaries (p<0.01). The maturation rate of grade 1 oocytes $(>120{\mu}m)$ was significantly higher than that of the other groups (p<0.05). Nuclear maturation to MI to MII in diameter of $>110{\mu}m$ groups was significantly higher than that in $<100{\mu}m$ group (p<0.05). The oocytes cultured in $10\%$ FBS­supplemented group were significantly higher rate of GVBD compared to the other supplemented groups (p<0.05), and oocytes maturation to MI to MII in $10\%$ FBS-, $0.4\%$ BSA-, and $10\%$ pFF-supplemented groups were significantly higher than those in $10\%$ CS-supplemented group (p<0.05). Based on these results, the estrus status and the size of oocyte affect positively to improve nuclear maturation of canine immature oocytes in vitro. Among several protein sources, porcine follicular fluid was the most effective supplementation to culture medium to achieve higher in vitro maturation rate.

• Clinical and Experimental Reproductive Medicine
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• 제23권1호
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• pp.33-39
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• 1996

본 연구는 돼지 미성숙난포란의 체외배양시 EGF가 핵성숙의 GVBD와 M II 에 미치는 효과를 조사하였다. 실험 1에서는 EGF를 첨가하였을 때, 난포란의 배양경과 시간 (0, 16, 24, 42시간)에 따른 핵성숙도를 조사하였던바, EGF 10ng/ml이 첨가된 군이 무첨가된 군에 비해서 24시간 이후에 GVBD가 유의하게 높았다 (p < 0.001). 실험 2에서는 난포란의 배양시 EGF 노출시간에 따른 난포란의 핵성숙의 효과를 조사하였던바, 배양 초기 (0-24시간)와 배양 전시간 (0-42시간) 동안 EGF를 배양액내에 첨가한 군의 경우 최종 성숙단계인 M II까지의 핵성숙율은 72.8과 84.8%로써 배양 후기 (24-42시간)에 EGF가 첨가된 군의 53.5%와 배양 전시간 (0-42시간) 동안 무첨가한 군의 26.1%보다 유의하게 높았다 (p < 0.001). 그리고 실험 3에서는 난구세포 부착 난포란과 난구세포가 제거된 난포란에 있어서 EGF의 효과를 조사하였던바, EGF가 첨가된 난구세포 부착 난포란 군의 경우 핵성숙율 (M II)은 84.6%로써 EGF가 첨가된 난구세포 제거군의 53.0%와 무첨가 난구세포 부착군의 27.6%, 난구세포 제거군의 44.2%보다 유의하게 높았다 (p < 0.001). 따라서 이상의 결과로 미루어보아 EGF 단독만으로도 돼지 미성숙난포란의 체외성숙의 핵성숙에 있어서 GVBD와 M II 을 유도할 수 있다고 사료된다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.87-88
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• 2005

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.87-88
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• 2005

• Asian-Australasian Journal of Animal Sciences
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• 제14권6호
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• pp.777-784
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• 2001

Evaluation of the granulosa cell (GC) monolayers developed from small (<5 mm) and large (>5 mm) follicles on the meiotic competence of caprine oocytes during in vitro maturation was done in this study in comparison to the granulosa cell coculture. Ovaries were collected from the local abattoir and follicular contents were aspirated for the monolayer culture. For IVM the oocytes were collected by puncturing the nonatretic follicles (>4 mm). Results revealed that at the same seeding rate, small follicular granulosa cell monolayer achieved confluence 24-48 h earlier than large follicular granulosa cell monolayer. GC monolayers significantly p (<0.05) improved the rate of meiotic resumption and nuclear maturation (84.76% vs 74.74%) after 27 h of culture in comparison to GC coculture. Statistically there was no significant difference in the maturation rate between the caprine oocytes matured over small or large follicular GC monolayers. It is concluded from the present study that GC monolayers support better nuclear and cytoplasmic maturation of growing caprine oocytes which is evident by better maturation rate over GC monolayer as compared to the oocytes matured with GC coculture. Granulosa cells from small and large follicles can be used for IVM with more or less in the same efficiency after conditioning them with maturation media in 18-24 h before the onset of culture.