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The Ultrastructural Characteristics of Preimplantation Embryonic Envelope in the Rat
홍순갑,이준영 한국발생생물학회 1998 발생과 생식 Vol.2 No.2
흰쥐의 배아발생 동안에 피질반응 후 배아 외부에 새롭게 형성된 피질과립막 (cortical granule envelope, CGE)이 존속하는지 여부와 투명대와 배아표면의 미세구조 변화를 조사하였다. 흰쥐배아의 투명대와 배아표면의 미세구조는 주사전자현미경으로 관찰하였으며, 피질과립막 형성과 분포는 Ulex europaeus agglutinin I lectin을 표지하여 형광현미경으로 관찰하였다. 배아표면은 미수정란 과는 다르게 배아표면의 미세융모가 단축 Preimplantation embryos of the rat was examined by the morphological changes in the cortical granule envelope (CGE), blastomere surface, and zona pellucida (ZP) of embryo after cortical reaction. The ultrastructural characteristics and CGE of embryos were observed with the scanning electron microscope and fluorescence microscope. In the ultrastructural characteristic of embryo surface, surface microvilli were shortened and the CGE-like structure existed above microvilli in eight-cell embryo. Rough spongy surface and decreased network numbers were key characters of embryonic ZP compared to unfertilized oocyte. The CGE formed by cortical reaction existed in perivitelline space during embryo development but it was thin and locally distributed ill contrast to fertilized oocyte. The present results indicate that cortical reaction forms cortical granule envelope in perivitelline space and causes not only zona hardening, but also ultrastructural changes in ZP and cell membrane of preimplantation embryos.
홍순갑,이준영,Hong, Soon-Gab,Lee, Joon-Yeong 대한생식의학회 1996 Clinical and Experimental Reproductive Medicine Vol.23 No.3
The present experiments aimed to investigate the metabolism of calcium during oocyte maturation in rat. The concentration of free calcium and calmodulin in oocytes was measured respectively by using of fluo-3/AM and FITC with microscope fluorescence spectrometer. The ultrastructural localization of calcium precipitates in oocytes was observed with the transmission electron microscope. Cumulus-free immature oocytes(GV-oocyte) were cultured in vitro through 15 hours. The free calcium concentration in GV oocyte was $55.9{\pm}3.5nM$. In calcium-containing medium, the free calcium concentration was increased in germinal vesicle breakdown(GVBD) oocyte($64.2{\pm}7.3nM$). In normal medium after calcium chelator treatment ($10{\mu}M$ BAPTA/AM), the free calcium contents were slightly lower than those in control group. In calcium-free medium, the free calcium content was drastically increased in GVBD($72.7{\pm}3.4nM$) and metaphase I - anaphase I ($88.0{\pm}3.4nM$) oocyte. In maturation rate of oocytes, GVBD rate was high in control group($82.9{\pm}6.55%$) and calcium chelator treatment group($91.2{\pm}4.4%$), but in calcium-free medium group, it was low and then the oocyte was degenerated without polar body formation. Relative content of calmodulin in oocyte was significantly(P<0.001) increased in metaphase I - anaphase I than in GV and GVBD oocyte. The calcium precipitates were observed in mitochondria and cytoplasm of GV oocyte but that were not observed in mitochondria of GVBD and metaphase I - anaphase I oocyte. And then the calcium precipitates reappeared in mitochondria of metaphase II oocyte. The above results indicate that changes in free calcium and calmodulin concentration of oocyte occur according to the maturational stages and the extracellular calcium is required during oocyte maturation. Also change of calcium localization in oocyte occurs according to the maturational stages.
Effects of Calcium Chelator on in vitro Fertilization of Rat Oocyte
홍순갑,김해권,김성례,이영기,이준영 한국발생생물학회 1999 발생과 생식 Vol.3 No.1
흰쥐 난자의 체외수정에 있어서 calcium chelator처리에 따른 피질반응 및 피질과립막의 형성 여부와 피질과립막의 전자현미경적 미세구조를 관찰하고, 수정율 및 단정자수정과 다정자수정 빈도에 미치는 영향을 조사하여 수정기간중 calcium의 역할을 조사하였다. Calcium chelator로는 BAPTA/AM을 사용하였으며, 난자의 미세구조와 피질과립막은 주사전자현미경으로 관찰하였다. 투명대가 제거된 난자의 체외수정 과정에서는 피질반응에 의해 형성 In order to examine the effect of -chelation on in vitro fertilization of rat zona-free oocyte, the formation of cortical granule envelope (CGE) and the rate of fertilization related to monospermy and polyspermy were determined. The ultrastructural characteristics of oocytes were observed with the scanning electron microscope and BAPTA/AM was used for calcium-chelation. The CGE formed by cortical reaction was observed in zona-free oocyte inseminated in vitro and it was also observed in the calcium chelator (1, 5, 10M BAPTA/AM) treated zona-free oocytes inseminated in vitro. The CGE developed according to incubation time. The fertilization rate was decreased in the calcium chelator-treated group (59.8, 38.1, 37.0%) compared to the control group (60.6%) but monospermy rate was increased in the calcium chelator-treated group (45.0, 47.3, 50.9%) compared to control group (37.5%). The above results demonstrate that the CGE is formed during fertilization in rat and the extracellular calcium is used in cortical reaction. Also the results suggest that proper concentration of free calcium in oocyte acts as important factor in fertilization.n.
ɑ-Tocopherol이 인간지방조직-유래 중간엽 줄기세포에 미치는 영향
김동윤, 홍순갑, 이준영 忠北大學校部設基礎科學硏究所 2011 自然科學硏究 Vol.25 No.-
본 실험은 ɑ-tocopherol이 체외 배양에서 인간지방 조직 유래 중간엽 줄기세포(ATMSCs)에 미치는 항 산화 효과와 골분화에 미치는 영향을 알아보고자 수행하였다. 세포의 생존률과 증식률은 각각 MTT assay와 hemocytometer를 사용하여 측정하였다. ATMSCs의 골 분화에 미치는 ɑ-tocopherol의 영향을 알아보기 위하여 alkaline phosphatase(ALP) 활성은 p-nitrophenol method로, glycosaminoglycan(GAG) 합성 은 DMMB method로 측정하였다. 활성산소에 대한 ATMSC의 생존률은 150μM ɑ-tocopherol과 600μM H2O2를 동시에 처리한 군에서 높게 나타났다. ATMSCs의 세포증식률은 ɑ-tocopherol이 첨가된 군에서 대 조군보다 높게 나타났다. 배양 기간동안 ɑ-tocopherol이 첨가된 osteogenic induction(OI)배양액군이 기본 OI배양액보다 낮은 ALP활성을 나타내었다. GAG 함량은 대조군과 실험군간에 유의한 차이가 없었다. 본 실 험의 결과들을 종합해 볼 때, ɑ-tocopherol의 항산화 효과에 의해 ATMSC의 생존률과 증식률이 증가된 것 으로 생각된다. 또한 ɑ-tocopherol이 ATMSC의 osteoblast로의 분화를 지연시키는 것으로 생각된다.