• 한국가축번식학회지
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• 제19권3호
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• pp.217-226
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• 1995

본 연구는 돼지 미성숙난포란의 체외배양시 핵성숙에 미치는 EGF의 효과를 검토하고자 실시하였다. 기초 배양액으로는 TCM-199에 0.2mM pyruvate, 1$\mu\textrm{g}$/ml estradiol-17$\beta$, 25$\mu\textrm{g}$/ml gentamycin을 첨가하였으며, 이 배양액에 EGF, FSH와 FBS을 처리하였다. 실험 1에서는 난포란 성숙에 있어서 FSH와 0, 10, 100 ng EGF/ml의 농도에 따른 효과를 조사하였던 바, 1, 10, 100 ng EGF/ml가 처리된 군의 핵성숙율은 83.0, 86.7, 87.5%로서 무처리군 27.3%와 FSH 처리군 60.3%보다 유의하게 높았다(p<0.001). 실험 2에서는 EGF, FSH와 FBS의 상호작용에 대해서 조사하였다. EGF 단독, EGF에 FSH 첨가, EGF에 FBS 첨가, FSH에 FBS 첨가와 EGF와 FSH에 FBS가 첨가된 군의 핵성숙율은 86.7, 90.2, 87.1, 89.6, 92.6%로서 무처리 군 22.3%, FSH와 FBS가 각각 단독 처리된 군의 52.2, 42.3%보다 유의하게 높았다(p<0.001). 또한, 정상적인 난구세포의 팽창은 FSH에 FBS, FSH에 EGF 혹은 FSH, EGF와 FBS가 첨가된 군에서 나타났으며, EGF가 첨가된 군에서는 대부분의 난구세포가 난포란으로부터 분리되었지만, 일부는 덩어리로 난포란에 부착되어 있었다. 따라서, EGF는 돼지 미성숙난포란에 있어서 핵성숙을 유도할 수 있다고 사료된다.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.117-117
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• 2002

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.

• 한국수정란이식학회지
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• 제29권4호
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• pp.361-368
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• 2014

This study was designed to evaluate the effect of bovine serum albumin (BSA) in a maturation medium on oocyte maturation and embryonic development in pigs. Immature pig oocytes were matured for 44 h in a medium supplemented with 0.4% (w/v) BSA, 0.1% (w/v) polyvinyl alcohol (PVA), or 10% (v/v) pig follicular fluid (PFF). After IVM, oocytes reached metaphase II stage were activated for parthenogenesis (PA) or used as cytoplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation (89.5%, 90.7% and 91.3% for BSA, PVA and PFF, respectively) and intraoocyte glutathione contents (1.20, 1.16 and 1.00 pixels/oocyte for BSA, PVA and PFF, respectively) were not altered by the macromolecules added to maturation medium. IVM of oocytes in a medium containing BSA (21.4%) and PVA (20.7%) showed significantly lower blastocyst formation after PA than culture in medium with PFF (39.2%). After SCNT, oocytes matured in medium with BSA showed decreased embryonic development to the blastocyst stage (9.2%) compared to those matured in medium with PFF (28.9%), while 23.6% of SCNT oocytes matured in medium with PVA developed to the blastocyst stage. When the effect of BSA in a maturation medium during the first 22 h and the second 22 h of IVM in combination with PFF or PVA was examined, PVA-BSA showed a higher nuclear maturation (94.1%) than BSA-PFF (84.5%). However, there was no significant difference in the blastocyst formation among tested combinations (47.3, 52.2, 50.0, 44.4 and 49.0% for PFF-PFF, PFF-BSA, PVA-BSA, BSA-PVA and BSA-PFF, respectively). Our results demonstrate that BSA and PVA added to maturation medium can support oocyte maturation comparable to PFF-supplemented medium. However, maturation of oocytes in a BSA-containing medium decreases embryonic development after PA and SCNT when compared with the medium supplemented with PFF.

• International Journal of Oral Biology
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• 제30권4호
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• pp.131-134
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• 2005

In the present study, performances of several in vitro maturation (IVM) systems for porcine follicular oocytes were evaluated, and an efficient chemically defined IVM system for porcine oocytes was proposed. The proposed one-step culture system supplemented with polyvinylalcohol (PVA) gave competitive efficiencies in terms of oocyte maturation and blastocyst development after parthenogenetic activation and in vitro culture, compared with the conventional two-step culture system by a supplementation of porcine follicular fluid (pFF). Additionally, it is identified that the proposed chemically defined one-step culture system yielded the comparable level of blastocyst production to the conventional maturation system in porcine somatic cell nuclear transfer (SCNT). Therefore, one can eliminate un-expected effects accompanied by supplementation of pFF. No medium replacement during whole maturation period is an additional benefit by applying this new system. Thus, these data support that the developed PVA supplemented chemically defined one-step IVM system for porcine follicular oocyte might be used in porcine SCNT program.

• Asian-Australasian Journal of Animal Sciences
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• 제23권11호
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• pp.1421-1426
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• 2010

We investigated the effects of various concentrations of ${\beta}$-hydroxybutyrate (BHB, 0, 0.1, 1 and 10 mM), a ketone body, added to chemically-defined maturation medium with or without energy substrates (glucose, pyruvate and lactate) on nuclear maturation rates up to the metaphase stage of the second meiotic division (M-II stage). In addition, we also assessed the influence of BHB on glutathione content, sperm penetration rate and embryonic development up to the blastocyst stage of oocytes matured under the presence of these energy substrates. Nuclear maturation rates up to the M-II stage of oocytes matured with BHB in each concentration group did not show a significant increase compared with the control (0 mM) groups in both the presence and absence of energy substrates. Although glutathione contents were not significantly different in each BHB concentration group, the sperm penetration rate in the 1 mM BHB group was significantly higher (p<0.05) and the embryonic development rate of oocytes up to the blastocyst stage was significantly lower (p<0.05) than the respective values of the control groups. These results suggest that BHB added to a chemically-defined maturation medium may stimulate sperm penetration while inhibiting embryonic development of porcine oocytes.

• 한국발생생물학회지:발생과생식
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• 제21권2호
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• pp.131-138
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• 2017

This study was conducted to investigate stimulatory effect of epidermal growth factor (EGF) on nuclear maturation and the expression level of EGF-receptor (EGFR), GM-130 (a marker of Golgi apparatus), transport protein Sec61 subunit beta ($Sec61{\beta}$), and coatomer protein complex subunit gamma 2 (COPG2) in porcine oocytes. The cumulus-oocyte complexes were collected from follicle with 3-6 mm in diameter. They were incubated in medium with/without EGF for 22 h (IVM I) and subsequently incubated hormone-free medium with/without EGF for 22 h (IVM II). Nuclear maturation state was checked by aceto-orcein stain. Protein expression of EGFR, GM-130, $Sec61{\beta}$, and COPG2 were measured by immunofluorescence. In results, nuclear maturation of oocytes in EGF non-treated oocytes were significantly lower than EGF-treated groups at IVM I or IVM II stage (P<0.05), whereas maturational rate in EGF treatment groups at both of IVM stage was higher in among the all treatment groups (P<0.05). EGFR, GM-130, $Sec61{\beta}$ and COPG2 were expressed in the cytoplasm of oocytes. Especially, GM-130 and EGFR were strongly expressed, but $Sec61{\beta}$ and COPG2 were weakly expressed in cortical area of cytoplasm. The protein level of GM-130, $Sec61{\beta}$, and COPG2 were significantly higher in the EGF-treated groups (P<0.05). However EGFR was no difference between non EGF-treated groups and control. In conclusion, EGF plays an important role in the systems for oocyte maturation with endoplasmic reticulum and Golgi apparatus. In addition, the protein levels of $Sec61{\beta}$ and COPG2 could be changed by EGF in the porcine oocytes during maturation.

• Reproductive and Developmental Biology
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• 제30권1호
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• pp.7-11
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• 2006

본 연구는 호르몬과 Na-pyruvate의 첨가가 개 미성숙난자의 체외성숙에 미치는 영향을 검토하였다. 개의 발정주기와 관계없이 암캐의 난소에서 회수한 난자를 NCSU-37 배양액에서 72시간 체외 배양하였다. 호르몬의 영향을 검토하기 위하여 배양액 중에 다양한 호르몬을 $24{\sim}72$시간 첨가하여 성숙율을 검사하였으며, Na-pyruvate 첨가 농도의 영향을 검토하였다. $E_2$ 단독 첨가구에서는 제2감수분열 중기(MII) 단계까지 성숙이 관찰되었으나(5.7%), 타 처리구에서는 MII기까지의 성숙이 이루어지지 않았다(P<0.05), $E_2$를 $6{\sim}24$시간만 처리하였을 때는 MII기로 성숙되는 난자가 없었으나 72시간 처리하였을 때는 6.0%로 유의적으로 높게 나타났다(P<0.05). Na-pyruvate의 농도를 0.25 mM 첨가한 구보다 2.5 mM 첨가한 구에서 제1감수분열 중기(MI) 단계까지의 성숙율이 증가하는 경향을 보였으나, MII 단계까지의 성숙율은 차이가 없었다. 본 실험의 결과는 개 난자의 체외성숙시 $E_2$가 첨가된 배양액에서 72시간 배양시에 성숙율을 증진시킬 수 있으나, Na-pyruvate의 농도는 개 체외배양에 있어 큰 영향을 미치지 않는 것을 나타낸다.

• 한국수정란이식학회지
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• 제24권2호
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.

• Asian-Australasian Journal of Animal Sciences
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• 제31권6호
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• pp.827-834
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• 2018

Objective: The aim of this study was to compare the effects of $36.5^{\circ}C$ and $38.5^{\circ}C$ incubation temperatures on the maturation of bovine oocytes and developmental competence of embryos. Methods: In experiment 1, oocytes were maturated in bicarbonate-buffered TCM-199 for 22 hours in a humidified atmosphere of 5% $CO_2$ in the air at either $36.5^{\circ}C$ or $38.5^{\circ}C$ and nuclear maturation status were determined. In experiment 2, in vitro fertilized oocytes were allocated randomly into synthetic oviductal fluid medium with or without a mixture of 1 mM L-glutathione reduced and 1,500 IU superoxide dismutase and cultured in a humidified atmosphere of 5% $CO_2$, 5% $O_2$, and 90% $N_2$ in the air at $38.5^{\circ}C$ for 8 days. Results: There were no significant differences between incubation temperatures in terms of oocyte maturation parameters such as cumulus expansion, first polar body extrusion and nuclear maturation. Incubation temperatures during in vitro maturation had no effects on developmental competence of embryos, but supplementation of antioxidants increased (p<0.05) developmental competence of the embryos. Blastocysts from oocytes matured at $38.5^{\circ}C$ had comparatively higher inner cell mass, but low overall and trophectoderm cell numbers (p<0.05). Conclusion: The results of present study showed that maturation of bovine oocytes at $36.5^{\circ}C$ may provide a suitable thermal environment for nuclear maturation and subsequent embryo development.

• 한국가축번식학회지
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• 제17권4호
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• pp.331-337
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• 1994

처녀발생은 난자의 세포질성숙을 투명대경화나 체외수정에 있어 난자 외적요소의 문제점을 배제하고 측정할 수 있는 지표이다. 본 실험에서는 체외성숙시간에 따른 소 난자의 처녀발생활성을 조사하였다. 도살장 난소로부터 회수한 미성숙난포란을 15% 소 태아혈청이 첨가된 TCM 199에서 6시간 간격으로 24~48시간까지 성숙시킨 후 7% ethanol로 7분간 활성화시켰다. 핵성숙과 세포질성숙은 rapid staining에 의해 핵형태와 전핵의 형성 유무로 판정하였다 핵성숙율은 24~48시간 사이 각각 81, 89, 72, 60 및 60%로 체외성숙 36시간에 성숙율이 최고였으나, 반면 감수분열 중기 II 염색체이상은 36시간부터 증가(0~30%)하였다. 에탄올처리에 의한 전핵형성율은 체외성숙 24~48시간에 각각 67, 68, 73, 84 및 87%였고, 그 중 이배체율은 각각 4, 5, 10, 16 및 20%로 성숙시간이 증가함에 따라 증가하였다. 위 실험의 결과 난자의 체외성숙 연장에 따라 전핵형성과 이배체수가 증가되는 것으로 나타났으며, 정상적인 핵성숙에 비해 세포질성숙은 더 많은 성숙시간이 필요한 것으로 나타났다. 이러한 결과들은 소 초기배 체외생산시와 핵치환용 핵수용란 생산시 적정 성숙시간 결정에 유용하게 이용될 수 있을 것이다.