난자-난구 복합체의 전자현미경적 및 fibronectin, tenascin, laminin분포에 대한 면역세포화학적 연구

이여일(Yu Il Lee),조주은(Ju Eun Cho),박현정(Hyun Jeong Park),권영숙(Young Sook Kwon),이재혁(Jae Hyuk Lee) 대한산부인과학회 2000 Obstetrics & Gynecology Science Vol.43 No.2

목적: 난자의 성숙 및 수정에서 초기 난할에 이르는 과정과 배란된 난자와 난관 내부환경사이의 복잡한 생화학적 기전을 이해하는데 중요한 fibronectin, tenascin 및 laminin의 생성을 난자-난구 복합체에서 알아보고자 면역형광염색후 공초점 레이져 주사현미경 검색을 시행하였다. 방법: 생쥐의 난소로 부터 얻은 난자-난구 복합체를 hCG가 첨가된 배양액에서 24시간, 48시간 배양한 후 각각의 복합체를 3.7% formaldehyde에 고정한 다음 3종류의 부착단백질에 대한 단클론성 일차항체의 혼합용액과 형광염료가 부착된 이차항체 혼합용액을 이용하여 3중 면역형광염색을 시행하였다. 염색된 슬라이드는 각각의 형광신호를 동시에 또는 분리하여 관찰이 가능한 공초점 레이져 주사현미경으로 관찰하였으며 동일한 조건의 난자-난구 복합체에서 투사전자현미경을 이용하여 세포내 미세구조물들의 변화를 관찰하였다. 결과: 면역형광염색 결과 적색의 형광신호로 관찰되는 fibronectin과 청색의 형광신호로 출현하는 tenascin이 많은 난구-관상세포(cumulus-corona cell)들에서 관찰되었으며 특히 fibronectin은 난자에 인접한 관상세포(corona cell)들의 세포질내와 세포막뿐만 아니라 세포외 기질에서 강한 염색반응이 관찰되었다. 그리고 fibronectin과 tenascin의 발현부위는 서로 일치하는 경향을 보였다. 녹색의 형광신호로 나타나는 laminin은 난구세포의 세포막을 따라서 그리고 세포외 기질에서 출현하였으며 fibronectin이나 tenascin과의 발현부위가 대부분에서 일치하였지만 드물게는 발현부위의 차이를 보이기도 하였다. 전자현미경 검색결과 난구세포의 성장 및 확장은 배양시간의 경과에 따라 증가하였다. 난자에서 멀어질수록 난구세포의 크기가 커지고 세포의 밀집도가 증가하였으며 세포사이의 공간이 적어져 세포들 사이에 접촉면이 많아졌다. 세포사이의 공간내로 돌출되는 가늘고 긴 미세융모(microvilli)가 다수 관찰되었으며 세포들 사이에 접촉면을 따라 길고 짧은 선모양의 간극결합(gap junction)을 흔히 볼 수 있었다. 특히 난구세포체 주변에 위치한 세포들의 세포질내에는 스테로이드 호르몬의 생성과 관계가 깊은 미세구조물들, 즉 관상의 사립체, 잘 발달된 활면 내형질망, 전자치밀성 지방적(fat droplet)과 이들과 연관된 세사다발(microfilaments)와 세관구조물(microtubules) 등이 보다 풍부히 관찰되었다. 그러나 단절된 기저막의 흔적 이외에 세포외 기질과 관계되는 미세구조물들은 관찰하기 어려웠다. 결론: 따라서 난자-난구 복합체내에서 이러한 부착단백들의 발현양상과 분포의 차이뿐만 아니라 전자현미경적으로 관찰되는 스테로이드 호르몬생성과 관계가 있는 미세구조물들의 형태학적 변화를 고려하여 볼 때 난자-난구 복합체를 구성하는 주변부 난구세포와 중심부 관상세포의 기능이 서로 다를 것으로 생각되었다. Objective: Immunofluorescence microscopy including confocal laser scanning microscopy and electron microscopy were used to study the production of fibronectin, tenascin, and laminin in the cumulus-corona (CC) cells surrounding mature, unfertilized oocytes after ovulation in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm-egg interaction, and the complex biochemical mechanism between the ovum and the oviduct. Methods: Mature oocyte-cumulus complex (OCC) was cultured for 24 and 48 hour and fixed in 3.7% formaldehyde. Specimens were incubated with a mixture of primary monoclonal antibodies recognizing different epitopes of fibronectin, tenascin, and laminin, and then with a mixture of secondary antibodies containing FITC, TRITC, and Cy-5 conjugated antibodies. Observation was made by confocal laser scanning microscope equipped with epifluorescece optics. Transmission electron microscopy were used to observe the OCC at 24 and 48 hours after cultrue. Results: The immunocytochemical date demonstrated that CC masses are capable of producing fibronectin and tenascin but their production is heterogeneous in the CC population. Immunoreactivity to fibronectin and tenascin was shown mostly by inner corona cells, and the intensity of immunofluorescence decreased from the central corona cells to the peripheral cumulus cells. Colocalization of fibronectin and tenascin was evident in most CC cells. Moreover, fibronectin and tenascin immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. Whereas, laminin immunofluorescence was found around plasma membrane and extracellular area, but a intracytoplasmic reaction was rarely observed. The distribution of laminin immunofluorescence was similar to that of fibronectin and tenascin, but in some cumulus cells, colocalization between them was not found. Ultrastructurally, cumulus cells projected numerous long, thin microvilli into the intercellular area and some micovilli penetrated into zona pellucida. The inner layer of the cumulus mass was loose arrangement of relatively uniform, small cells with widened intercellular spaces, whereas in the outer layer, cumulus cells are rather larger in size and compact arrangement by narrow, irregular spaces. A small and large linear gap junctions were easily found at cell contacts. The cytoplasm of most cells had abundant organelles typical of steroidogenesis: numerous mitochondrias, a well-developed smooth endoplasmic reticulum, electron dense lipid droplets, and bundles of microtubules and microfilaments. Rudimentary disrupted basal lamina along the cytoplasmic border was rarely seen in a few inner conora cells. Conclusion: Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process. Cumulus cells appears to be a heterogeneous and dynamic system for suitable microenviroment of fertilization. And functional differences between corona and cumulus cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins and steroidogenesis-related organelles.

인간 조기 황체화 과립막세포에서 황체화호르몬에 의한 steroid thyroid hormone receptor 3 ( TR 3 ) mRNA 의 발현 조절

이여일(Yu Il Lee),박현정(Hyun Jeong Park),김병룡(Byung Ryeong Kim),전상영(Sang Young Chun) 대한산부인과학회 2002 Obstetrics & Gynecology Science Vol.45 No.10

목적 : TR3는 생쥐에서는 nur77, 흰쥐에서는 NGFI-B로도 불리우는 인간 상동단백으로 핵 고아수용체 (nuclear orphan receptor)를 암호화하는 immediate-early 유전자이다. 최근 TR3 유전자는 흰쥐 난소에서 성선자극 호르몬에 의해 유도되는 것으로 보이기 때문에 본 연구에서도 배양중인 인간 과립막세포에서 TR3 유전자 발현이 황체화호르몬에 의해 조절되는지 조사해 보았다. 연구 방법 : TR3 mRNA의 수준은 체외수정 환자로부터 얻어 배양중인 조기 황체화 과립막세포에서 competitive RT-PCR 방법으로 측정하였다. 결과 : TR3 mRNA 발현은 LH에 의해 일시적으로 유도되었으며 LH 자극 후 1시간째 최고치에 도달하였고, 농도 의존적이었다. LH에 의해 유도된 TR3 발현은 actinomycin-D에 의해 소실되었으나, cycloheximide에 의해서는 과잉 유도되었다. Protein kinase A의 저해제인 Rp-cAMP 뿐만 아니라 protein kinase C의 저해제인 chelerythrin과 함께 처리한 과립막세포에서는 LH에 의해 자극된 TR3 mRNA 수준이 억제되었으나 forskolin과 TPA는 TR3유전자 유도에 대한 LH 활성과 유사하였다. 결론 : 본 연구에서 TR3 유전자는 인간의 조기 황체화 과립막세포에서 LH에 의해 빠르게 일시적으로 유도되었다. 이 결과는 TR3가 LH에 반응하는 배란 특이 유전자의 cascade를 개시하여 배란을 조절할 것이라 사료된다. Objective : The present study examined the gonadotropin regulation of TR3 gene expression by luteinizing hormone (LH) in cultured human luteinized granulosa cells. Methods : TR3 mRNA levels were detected by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) method in cultured human luteinized granulosa cells collected from patients undergoing in vitro fertilization. Results : TR3 transcript was transiently induced by LH, reaching maximum levels 1 hr after stimulation, in a dose-dependent manner. LH-stimulated TR3 expression was abolished by actinomycin D, but was superinduced by cycloheximide. Treatment of luteinized granulosa cells with Rp-cAMP, an inhibitor of protein kinase A, as well as, chelerythrin, an inhibitor of protein kinase C, suppressed LH-stimulated TR3 mRNA levels. In addition, forskolin and TPA mimicked the LH action on the induction of TR3 gene, implying the role of protein kinase A and C activation. Conclusion : Taken together, the present study demonstrates that TR3 gene was rapidly and transiently induced by LH in human luteinized granulosa cells. The results imply that TR3 may play a role in ovulation by initiating a cascade of ovulation-specific gene expression in response to LH.

폐경 호르몬 대체 요법에 따른 산화질소와 항산화능의 변이

이여일 ( Yu Il Lee ),강우대 ( Woo Dae Kang ),조명숙 ( Myung Sook Cho ),이기영 ( Kee Young Lee ) 대한폐경학회 2004 대한폐경학회지 Vol.10 No.2

생쥐배포의 유리화 동결보존에 관한 연구

이유진 ( Yu Jin Lee ),김미영 ( Mi Young Kim ),이석원 ( Seok Won Lee ),이여일 ( Yu Il Lee ) 대한산부인과학회 2004 Obstetrics & Gynecology Science Vol.47 No.7

생쥐배아의 동결보존에 관한 실험적 연구

이여일,권영숙,박현정,Lee, Yu-Il,Kwon, Young-Sook,Park, Hyun-Jeong 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.1

Objectives: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Materials and Methods: Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. Results: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1 %,79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The developmen1 rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. Conclusions: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

정자 세정후 Swim-up 처치가 정자의 운동성에 미치는 영향

이여일,Lee, Yu-Il 대한생식의학회 1991 Clinical and Experimental Reproductive Medicine Vol.18 No.1

Thirty five couples were treated by intrauterine insemination with sperm prepared by a washing and swim-up method. Fifteen women conceived(42.9%). Sperm washing and swim-up was found to significantly improve sperm motility for men of infertile couples and the increment of percent sperm motility after sperm preparation allowed significant differentiation of pregnant and nonpregnant patients in asthenozoospermia(submotile) group (p<0.01). The author suggest that the increment of percent sperm motility after sperm washing and swim-up could be a useful screening tool for in vitro procedure proposed to improve fertility in the intrauterine insemination of asthenozoospermia.

체외수정 실패 정자에 대한 전자현미경적 연구

이여일,나재형,이재혁,정상우,Lee, Yu-Il,Na, Jae-Hyung,Lee, Jae-Hyuk,Juhng, Sang-Woo 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.2

Failure of in vitro fertilization may occur even though oocyte and semen parameters seem satisfactory. Quantified ultrastructural study of spermatozoa was performed in three cases of failed in vitro fertilization. The results were compared to those of four fertile men. Quantification was achieved by cataloguing cell defects of the spermatozoon heads and mid/principal pieces of the flagella. Using the data from each specimen, the percentages of total cellular abnormalities in the head/mid/principal pieces were established. The percentages of anomalies of the midpiece and of the principal piece were not significantly different between failed cases and controls. The percentage of cell alterations of the head (96-100 vs 75${\pm}$3,4%), the percentage of combined anomalies of the head (80-86 vs 52.5${\pm}$1.9%), and the percentages of nuclear shape deformation (68-86 vs 47.5${\pm}$6.3%), acrosomal defects (86-96 vs 50${\pm}$4.3%), and postacrosomal sheath defects (78-88 vs 44.5${\pm}$7.2%) of the head were significantly different between failed cases and controls. Due to the cost and time involved in processing semen samples for electron microscopy, the widespread application of this technique to all couples presenting for IVF certainly is not warranted. However, in selected instances electron microscopy may play a crucial role in identifying an occult male factor.

신생아제대혈청이 난자성숙과 난구세포 분산에 미치는 영향

이여일,박현정,권영숙,Lee, Yu-Il,Park, Hyun-Jeong,Kwon, Young-Suk 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1

This study was performed to investigate the stimulating effect on oocyte maturation and cumulus cell expansion in TC199 media by human cord serum (HCS) supplementation. Immature mouse oocyte cumulus complexes (OCCs) were cultured in TC199 media supplemented with bovine serum albumin (BSA), HCS and human chorionic gonadotropin (hCG) instead of luteinizing hormone (LH) respectively, and the expression of cumulus expansion and oocyte maturation were observed. After 4hr and 24hr culture with or without OCCs, media containing 0.4% BSA, 10% HCS and 10 IV hCG respectively were collected and analyzed for changing concentrations of estradiol $(E_2)$, progesterone $(P_4)$, testosterone (T), and $PGF_{2\alpha}$. There were no elevation of $E_2$, T, and $PGF_{2\alpha}$ by OCCs culture, but minute elevation of $P_4$ level by 24hr OCCs culture in hCG supplementation (p=0.048). The stimulating pattern of cumulus expansion of OCCs by HCS and hCG supplementation was similar to our previously report using Ham's F-10 media, however oocyte maturation rates after 24hr OCCs culture in all media were increased by $20\sim30%$ compared to Ham's F-10 media. These results suggest that LH in HCS induce cumulus expansion probably by $P_4$ secretion of OCCs, and TC199 is efficient media for immature mouse oocyte maturation.

Gonadotropin Regulation of Regulator of G Protein Signaling 2 (RGS-2) Expression in the Rat Ovary

이여일,이은숙,김선애,김미영,조문경,전상영,Lee, Yu-Il,Lee, Eun-Suk,Kim, Sun-Ae,Kim, Mi-Young,Cho, Moon-Kyoung,Chun, Sang-Young The Korean Society for Reproductive Medicine 2008 Clinical and Experimental Reproductive Medicine Vol.35 No.2

연구방법: 미성숙 백서 난소의 과배란 유도를 위해 PMSG를 주사하고, 배란을 위해서 hCG를 주입하였다. RGS-2의 유전자 발현양상을 조사하기 위하여는 Northern blot 분석과 in situ hybridization 분석을 시행하였다. 결 과: 미성숙 백서에 성선자극호르몬인 PMSG를 복강내 주사했을 때 RGS-2 mRNA 발현에 영향을 미치지 않음을 Northern blot analysis로 확인할 수 있었으나, hCG를 주입했을 때는 1시간에서 3시간 내에 발현이 증가됨을 알 수 있었다. In situ hybridization으로 살펴본 RGS-2 mRNA의 발현세포는 난포의 크기에 관계없이 난자였으나, hCG로 처리한 후에는 배란 전 난포와 성장중인 난포의 과립막 세포이었다. 그러나, RGS-2 단백의 발현은 hCG 처치와 관계없이 난포막 세포이었다. 상기 생체 실험과 마찬가지로 시험관에서도 배란 전 난포의 과립막 세포에 대한 LH 처리는 RGS-2 유전자 발현을 1시간 내에 촉진하였다. 또한, 성선자극호르몬 분비호르몬 2 길항제도 이러한 LH의 촉진작용을 증진시켰다. 결 론: 본 연구로 배란 전 과립막 세포에서 성선자극호르몬인 LH/hCG와 성선자극호르몬 분비호르몬 길항제에 의해 RGS-2의 발현이 증진되는 양상으로 보아 RGS-2가 배란과정 동안에 Gq protein 신호전달을 조절할 것으로 추정된다. Objective: The purpose of the present study was to examine the hormonal regulation of RGS-2 in the rat ovary. Methods: Immature rats were injected with 10 IU of PMSG to induce multiple growth of preovulatory follicles and 10 IU of hCG to induce ovulation. Northern blot analysis performed for gene expression and in situ hybridization performed for mRNA localization. Results: Northern blot analysis revealed that pregnant mare's serum gonadotropin (PMSG) treatment did not affect RGS-2 mRNA levels. In contrast, human chorionic gonadotropin (hCG) treatment of PMSG-primed rats resulted in an increase in RGS-2 expression within $1{\sim}3\;h$. The major cell-types expressing RGS-2 mRNA were oocytes regardless of follicle size. Interestingly, hCG treatment caused the stimulation of RGS-2 gene expression in granulosa cells of preovulatory and growing follicles. In contrast, cell types expressing RGS-2 protein were theca cells regardless of hCG treatment. Like in vivo, treatment of preovulatory granulosa cells with LH in vitro stimulated RGS-2 levels within 1 h. Interestingly, GnRH antagonist II enhanced the stimulatory action of LH. Conclusion: The present study demonstrates the LH/hCG induction of RGS-2 in preovulatory granulosa cells and suggests a role of RGS-2 in Gq protein signaling pathway during ovulation.

Control Mechanisms of Ovarian Follicle Development by Follicle Stimulating Hormone and Pituitary Adenylate Cyclase-activating Polypeptide

이여일,신진옥,김미영,전상영,Lee, Yu-Il,Shin, Jin-Ok,Kim, Mi-Young,Chun, Sang-Young The Korean Society for Reproductive Medicine 2006 Clinical and Experimental Reproductive Medicine Vol.33 No.1

목 적: 본 연구는 흰쥐 난소를 실험모델로 하여 미성숙 전동 난포의 성장에 대한 pituitary adenylate cyclase-activating polypeptide (PACAP)의 영향을 얄아보고자 하였다. 연구방법: 미성숙 전동 난포를 생후 21일된 흰쥐로부터 분리하여 PACAP을 첨가하거나 첨가하지 않은 무혈청 배양액에서 3일 동안 배양하고, 푸로게스테론 호르몬의 생성, 난포의 성장, 과립막세포의 증식 및 유전자의 동태 등을 관찰하였다. 증식의 정도는 thymidine incorporation 방법으로 검색하고 유전자의 변동은 Northern 분석을 이용하였다. 결 과: PACAP으로 처리한 군은 난포의 직경이 75% 증가한 반면 난포자극호르몬인 FSH로 처리한 군은 65% 증가하였고, PACAP 처리는 과립막 세포의 증식을 강화시켰다. FSH와 PACAP 공히 배양된 흰쥐 난포의 과립막 세포와 FSH에 반응하는 세포주인 GFSHR-17에서의 프로게스테론 생성을 촉진시켰고, PACAP이 FSH의 작용을 증진시켜 SF-1과 아로마타제 유전자 발현을 촉진시켰다. 결 론: 본 연구는 PACAP이 과립막증식과 스테로이드합성을 통하여 전동 난포의 성장을 촉진함을 시사하였고, 또한, SF-1, 아로마타제 등에 대한 FSH의 작용을 도와주는 역할을 PACAP이 담당하므로 PACAP은 초기 난포성장에 필요한 난소국소인자임을 유추할 수 있었다. Objective: Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel hypothalamic neuropeptide, has been suggested to play a role in ovarian folliculogenesis. The present study evaluated the effect of PACAP on the growth of preantral follicles. Methods: Preantral follicles were mechanically isolated from ovaries of 21-day-old rats and cultured in groups for 3 days in serum-free medium in the absence or presence of PACAP-38 ($10^{-6}M$). Results: Treatment with PACAP-38 resulted in an increase in follicle diameter by 75% whereas treatment with follicle stimulating hormone (FSH) increased follicle diameter by 65%. PACAP-38 treatment enhanced the granulosa cell proliferation as measured by thymidine incorporation analysis. Furthermore, the production of progesterone by cultured granulosa cells and GFSHR-17 cell line was stimulated by PACAP-38. Interestingly, PACAP enhanced FSH action on stimulation of SF-1 and aromatase gene expression. Conclusion: The present results demonstrate that PACAP stimulated preantral follicle growth by potentiating proliferation and by stimulating steroidogenesis.