• 한국가축번식학회지
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• 제22권4호
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• pp.395-403
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• 1998

본 연구에서는 돼지난자내 원형정자 주입후 수정율과 체외 배 발달을 조사하였다. 원형정자 주입 2시간 전에 인위적 전기자극을 주고 원형정자를 주입했을 때 난자들의 수정율이 주입 직후에 전기자극을 준 것들과 전기 자극을 주지 않은 것들보다 수정율이 높았다. 원형정자와 원형 정자핵을 각각 주입 한 후 전핵 형성율과 전핵 이동율을 조사하였으나 유의차를 발견할 수 없었다. 전핵의 형성과 이동중 미세소관의 움직임을 간접형광면역법 및 공춧점 현미경을 사용하여 조사해본 결과, 난활성 직후 난자의 표층에서 미세소관이 발생해서 이것에 의해 웅성 및 자성전핵이 난자 중심부로 이전됨을 볼 수 있었다. 원형정자와 원형정자핵 주입후 배양 6일째에 각각 25% 와 27%의 배반포로 형성되었고, 8 일째 형성된 배반포를 염색하여 관찰한 철과 세포수가 각각 평균 87에서 99개가 형성되었음을 알 수 있었다. 이러한 결과는 체외 성숙된 돼지 난자 내에 원형정자 혹은 원형정자 핵을 미세 주입하는 방법에 의해 정상척인 배발달을 하는 돼지 수정란 생산이 가능한 것을 보여주는 것이다.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.169-173
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• 2010

Correlations between cumulus cells and germinal vesicle (GV) chromatin configuration were examined in porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from 2~6 mm follicles and divided into three categories according to cumulus cell morphology. "A" group was compacted COCs with more than three cumulus cell layers. "B" group was COCs with less cumulus cell layers than "A" group. "C" group was COCs with one or less layer of cumulus cells. Cumulus cells were removed 0.1% hyaluronidase, and denuded oocytes were stained with Hoechst 33342. GV chromatin configuration was classified into GV-Con and GV-Dis. GV-Con meant that a nucleus was surrounded by condensed chromatin in a ring. GV-Dis meant that filamentous chromatin clumps were distributed in nucleus. The proportion (80.2%) of GV-Con in "A" group was significantly higher than "B" (62.0%) or "C" (44.9%). The proportion (55.1%) of GV-Dis in "C" group was significantly higher than "A" (19.8%) or "B" (38.0%). The meiotic competence of COCs was examined after 44 h culture. The proportion (90.0%) of oocytes reaching to metaphase II (M-II) in "A" group was significantly higher than "B" (76.5%) or "C" (45.5%). In conclusion, oocytes with good quality cumulus cell layers are synchronized early GV stage, and early GV stage is important for meiotic competence in pigs.

• Reproductive and Developmental Biology
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• 제40권2호
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• pp.23-26
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• 2016

Transcription factor called activating enhancer binding protein 2C (AP2-gamma) is found in a variety of species and expressed from oocyte stage onwards, particularly restricted to the trophectoderm. Recent studies demonstrated that ablation of Tfap2c led to failure of tight junction biogenesis, particularly the knock-down embryos of Tfap2c did not form cavity from morula to blastocyst in mouse and pig. We speculated that the Tfa2pc may also be involved in desmosome biogenesis because blastocoel formation is coincident with the establishment of desmosome. To determine this, we depleted Tfap2c injecting siRNA into one-cell zygote and analysed the expression levels of genes that are required for desmosome complex such as PkP2, Pkp3, Dsc2, and Dsg2. We found only Pkp3 was up-regulated in the knockdowned morula embryos. Interestingly, upstream region of Pkp3 had putative Tfap2c binding sites. In conclusion, our results suggest that Tfap2c is not a crucial factor but somehow it might be involved in desmosome biogenesis directly or indirectly via Pkp3.

• Mycobiology
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• 제44권1호
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• pp.54-57
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• 2016

Davallialactone (DAVA) is a hispidin analogue derived from the medicinal fungus Phellinus baumii. We examined the effect of DAVA on in vitro fertilization (IVF) of pigs. Boar spermatozoa were incubated in fertilization medium with varying concentrations of DAVA, then sperm motility and reactive oxygen species (ROS) level were evaluated. Higher sperm motility was found following the addition of 0.5 or $1{\mu}M$ DAVA after incubation than addition of other concentrations or controls. ROS level decreased significantly with the addition of DAVA. The rate of normal fertilization was higher in the presence of $1{\mu}M$ DAVA (65.1%) than were those of other concentrations or controls (45.4~59.4%), and the highest total fertilization rate (mono- and polyspermic oocytes) was observed at $1{\mu}M$ DAVA (83%). In conclusion, addition of DAVA to fertilization medium improved sperm motility, and reduced ROS level so as to potentially improve sperm-oocyte binding in IVF, suggesting the potential of a compound isolated from mushrooms in assisted reproductive technology for humans and animals.

• 대한수의학회지
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• 제32권1호
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• pp.135-142
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• 1992

This experiment was carried out to establish an effective technique of in vitro maturation of porcine follicular oocytes. Porcine ovaries were collected from an abbatoir and delivered to the laboratory in phosphate buffered saline in an hour. Immatured follicular oocytes were collected from the ovaries and divided into groups by the size of follicles and by the attachment of granulosa cells. The follicular oocytes were cultured in m-KRB solution supplemented with FCS(10%), follicular fluid(10%) or hormones of PMSG(10IU/ml), hCG(10IU/ml ) and $estradiol-17{\beta}(1{\mu}g/ml)$ for 48 hours at $39^{\circ}C$ under an atmosphere of 5% $CO_2$ in air. The results are as follows ; 1. The mean recoveration rate of follicular oocytes was 61.8%. 2. The maturation rate was significantly(p<0.05) higher when the oocytes were collected from large-sized follicles and under good state of granulosa cell attachment. 3. The maturation rate was significantly(p<0.01) promoted when the follicular oocytes were cultured in m-KRB solution supplemented with follicular fluid(74.8%) or hormones and fetal calf serum(70.6%).

• 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.

• 한국수정란이식학회지
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• 제26권1호
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• pp.79-84
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• 2011

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.

• Clinical and Experimental Reproductive Medicine
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• 제29권3호
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• pp.179-186
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• 2002

Objective: Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the zona pellucida (ZP) of several mammalian species including pigs. Treatment of oocytes with various lectins blocks sperm binding to the ZP in various mammalian species. This study was undertaken to examine the distribution of sugar residues in the ZP of pig oocytes matured in vitro and the ability of spermatozoa to bind to ZP and in vitro penetration in oocytes treated with fluorescein isothiocyanate (FITC)-labelled lectins. Materials and Methods: The lectins of Banderiaea simplicifolia (BS-II, bind to $\beta$-D-N-acetylglucosamine), Canavalin ensiformis (Con A, bind to $\alpha$-D-Mannose), Lens culinaris (LCA, bind to a-D-Mannose), Ricinus communis (RCA-I, bind to $\beta$-D-Galactose) and Ulex europaeus (UEA-I, bind to $\alpha$-L-Fucose) were examined for spermatozoa penetration, binding capacity to ZP and distribution of lectins. Results: The penetration rates were significantry (p<0.05) higher in control oocytes (63%) than those treated with all lectins, but penetration rates ($40{\sim}49%$) were simililar in group treated with lectins. The incidence of monospermy was similar in oocytes untreated and UEA-I, but it was higher in oocytes treated with BS-II, Con A, RCA-I and LCA. The porcine oocytes cultured for 48 h in TC-199 medium were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescein illumination, higher (p<0.001) proportions of oocytes showed fluorescein of zona pellucida after treatment with Con A (93%), LCA (93%) and RCA-I (100%) than BS-II (37%) and UEA-I (50%). All of the oocytes treated with RCA-I exhibited strong fluorescein in the outer region of the zona pellucida while those treated with LCA exhibited strong fluorescein throughout the zona pellucida. BS-II bounded mainly to the outer region and UEA-I bounded mainly to the inner region of the zona pellucida, with either strong or weak fluorescein. At 120 min after insemination in vitro, fewer spermatozoa were bound to the zona pellucida of the oocytes treated with BS-II, Con-A and RCA-I. Of the lectins, Con A most inhibited sperm binding. Conclusions: These results suggest that $\beta$-D-Galactose residues in the porcine zona pellucida may act as primary sperm receptors and inducers of the sperm acrosome reaction and these sugar residues may be involved in the block to polyspermy.

• 한국수정란이식학회지
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• 제20권3호
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• pp.289-295
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• 2005

본 연구는 돼지에서 스트레스 관련 HSP70 유전자의 다형성과 IVF 수정란의 배발달간의 관련성을 조사하기 위하여 수행되었으며 그 결과는 다음과 같다. HSP70-K1, -K3 및 -K4의 PCR 산물로부터 SSCP 다형성은 각각 다르게 확인되었다. 체외수정란의 분할율 시험에서 HSP70 K1-AA($73.1\%$) 및 K1-AB($62.3\%$) 난할율은 HSP70 K1-BB($49.3\%$)보다 유의적으로 높게 나타났다(p<0.05). 또한 HSP70 K3-AA($72.4\%$) and K3-AB($62.2\%$)형도 HSP70 K3-BB($49.1\%$)형보다 유의적으로 높았다(p<0.05). 돼지품종들과 정자의 HSP70 유전자형에 따른 2-cell 단계까지의 체외수정란 배발달은 유의적 차이를 보였다. Landrace(28.8)와 Duroc(29.8)에서 2세포기까지 발달된 수정란의 수는 Yo.kshire(10.9) 보다 유의적으로 높았다(p<0.05). 또한 HSP70 K4-AB(29.6)형의 2세포기 단계까지의 배발달 수정란 수는 HSP70 K4-AA(10.6)형보다 유의적으로 높았다(p<0.05). 그러나 돼지 품종 및 HSP70 유전자형에 따른 배반포기까지 수정란 배발달 정도는 유의적 차이는 없었다. 이러한 결과들은 돼지 체외수정란의 초기 배발달은 HSP70 유전자형과 품종에 따라서 영향을 받는 것으로 확인되었다.

• 한국가축번식학회지
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• 제27권3호
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• pp.215-223
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• 2003

본 연구는 체외성숙된 돼지난포란을 액상정액으로 수정시 수정시간과 배양배지가 난포란의 발달에 미치는 영향을 조사하기 위하여 실시하였다. 정자농후정액 (30∼60 ml)을 채취하여 실온에서 2시간 정도 서서히 냉각시킨 후, 정액을 15 ml 튜브에 담아 800${\times}$g로 10분간 원심분리하였다. 상층액은 버리고 하부의 정자는 5 ml LEN 희석액으로 1${\times}$$10^{9}$ 전자/ml가 되도록 재희석하였다. 희석된 정액은 4$^{\circ}C$ 냉장고에 보존하였다. 미성숙 난모세포의 성숙에 사용된 배지는 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml PMSG, 10 IU/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate그리고 10% pFF를 첨가한 TCM-199 배지였다. 22시간 성숙 배양한 후 난모세포는 cysteamine과 hormone들을 배제한 후 38.5$^{\circ}C$, 5% $CO_2$ incubator에서 22시간 더 성숙시켰다. 성숙된 난모세포는 채취 후 2일간 4$^{\circ}C$에 보존된 액상정액으로 수정되었다. 난모세포는 500 $\mu$l mTBM 수정 배지에서 1${\times}$$10^{6}$ 정자/ml의 농도로 1, 3, 6 그리고 9시간 동안 수정시켰다. 그 후 난모세포는 500 $\mu$l NCSU-23, Hopes buffered NCSU-23, PZM-3 그리고 PZM-4 배양배지에 옮겨서 6, 48 그리고 144시간을 더 배양하였다. 정자침투율, 웅성전핵형성율 그리고 난모세포의 난할율은 6 및 9시간 수정시간에서 1 및 3시간 수정시간 보다 높았다. 6시간 수정시 배반포형성율 (33.6%)은 1, 3 그리고 9시간 수정시 배반포형성율 (11.4, 23.0 그리고 29.6%) 보다 높았다. 배반포의 평균세포수는 6, 9, 3 그리고 1시간 수정시 각각 32.9, 27.6, 26.3 그리고 24.4개 였다. 분할된 난모세포의 배반포형성율 그리고 배반포의 평균세포수는 NCSU-23, PZM-3 그리고 PZM-4 배양배지보다 HEPES buffered NCSU-23 배양배지가 우수하였다. 결론적으로 4$^{\circ}C$ 보존 돼지액상정액은 체외성숙된 돼지 난모세포의 체외수정에 사용될 수 있음이 입증되었다. 또한 체외성숙된 돼지 난모세포는 500 $\mu$l mTBM 수정배지에서 1${\times}$$10^{6}$ 정자/ml로 6시간 공배양시키는 것이 바람직하며, HEPES buffered NCSU-23 배양배지에서 배양하는 것이 좋다는 결과를 얻었다.