돼지난소에서 난포폐쇄와 큰포식세포에 관한 형태학적 연구

김원식(Won-Sik Kim),한승로(Seung-Ro Han),김수일(Soo-Il Kim),박창식(Chang-Sik Park) 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.1

난포폐쇄가 과립층세포의 세포자멸사에 의해 이루어지고 이 과정에 큰포식세포가 직, 간접적으로 관여한다는 사실은 널리 알려져 있다. 그러나 난포내에서 일어나는 세포자멸사가 과립층의 어디에서부터 개시되고 어떻게 파급되는지, 세포자멸사 산물과 퇴화된 난모세포의 제거 방법 그리고 난포폐쇄의 완성은 어떻게 이루어지는가에 대해서는 아직 확실히 밝혀져 있지 않다. 이에 저자들은 가임기 돼지(Yorkshire-breed)를 실험동물로 난소내 난포의 광학현미경적 및 투과전자현미경적 관찰과 돼지 대식세포 단크론항체 4E9를 이용한 면역조직화학적 방법으로 본 연구를 실시하였다. 광학현미경적 관찰 결과, 난포폐쇄는 과립층세포의 세포자멸사로부터 개시되고 이어 난모세포의 퇴화와 난포막 속층 세포들의 세포자멸사가 일어나면서 난포폐쇄가 완성되는 것으로 보인다. 과립층세포의 세포자멸사는 핵농축으로부터 시작되고 과립층의 깊이에 관계없이 짧은 시간안에 모든 층으로 파급되고 최종적으로 난모세포의 투명대를 둘러싸고 있는 과립층세포의 세포자멸사로 이어지는 것으로 보인다. 투과전자현미경 관찰에서는 세포자멸사중에 있는 과립층세포들이 인접한 정상적인 과립층세포들과 큰포식세포들에 의해 포식되는 것을 확인할 수 있었고, 난포동내에 산재한 세포자멸사 소체들의 제거에도 큰포식세포들이 관여하고 있음을 알 수 있었다. 난포폐쇄와 큰포식세포와의 관계를 알아보기 위해 실시한 면역조직화학적 관찰에서는, 과립층세포의 세포자멸사 초기에는 큰포식세포가 과립층내에서만 발견되었으나, 세포자멸사의 진행과 함께 난포동과 난모세포를 둘러싸고 있는 투명대 주위에서 각각 순서대로 관찰되었고, 마지막으로 난포막 속층에서도 발견되어, 이들이 과립층세포들의 세포자멸사 소체들을 제거하는 것은 물론 최종적으로는 난포막 속층 세포들의 세포자멸사 소체들을 제거하여 난소 실질화에 관여하는 것으로 보인다. 이러한 본 연구의 결과는 난포폐쇄와 관련된 큰포식세포의 구조, 기능적 연구에 유용한 자료로 생각된다. Apoptosis of granulosa cells leads follicular atresia and macrophages have an important role during the apoptotic process. However, the propagation of apoptosis within the follicle, the ways of elimination of apoptotic bodies and degenerated oocyte, and the completion of follicular atresia are still controversial and unidentified clearly. Using adult porcine (Yorkshire-breed) ovary, in this morphological study, transmission electron microscopic observation and immunohistochemical study with pig macrophage monoclonal antibody 4E9 were performed. In light microscopy, the follicular atresia initiated with apoptosis of granulosa cells, followed by degeneration of oocyte and apoptosis of theca interna cells. Apoptosis occured in random fashion among the granulosa cells and propagated multidirectionally, and finally to the granulosa cells surrounding zona pellucida of degenerating oocyte. Pyknosis of granulosa cells was the first sign of apoptosis. In immunohistochemistry, macrophages were found only in the granulosa layer at the stage of beginning of apoptosis. With progression of apoptosis, they were proliferated greatly in number enough to eliminate all the apoptotic bodies, and found within the follicular antrum. In advanced stage of atresia, macrophages surrounded the zona pellucida of degenerating oocyte, and found also in the theca interna. In transmission electron microscopy, phagocytic granulosa cells maintained characteristic gap junctions with neighboring granulosa cells and contained several apoptotic bodies and lipid droplets within their cytoplasm. Macrophages kept many apoptotic bodies, vacuoles and autophagosomes in their cytoplasm. Apoptotic granulosa cells were ingested by intact granulosa cells and macrophages initially, but lately, all the apoptotic granulosa cells and degenerated oocyte were eliminated by macrophages. Ovarian follicular atresia completed with phagocytosis of apoptotic theca interna cells by macrophages, and the remnants of the atretic follicle became ovarian stroma. It is well known that macrophages may play an important role during follicular atresia, such as elimination of apoptotic granulosa cells, theca interna cells and degenerated oocytes, but, the valid action mechanisms of macrophages on the initiation of granulosa cell apoptosis and on the completion of atresia through the secretion of paracrine factors and autocrine factors still unclear.

Effects of Steroid Hormone in Avian Follicles

Caicedo Rivas, R.E.,Nieto, M. Paz-Calderon,Kamiyoshi, M. Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.4

The aim of the present study was to examine the effects of testosterone (T) and estradiol-$17{\beta}$ ($E_2$) on the production of progesterone ($P_4$) by granulosa cells, and of the $E_2$ on the production of $P_4$ and T by theca internal cells. In the first experiment, granulosa cells isolated from the largest ($F_1$) and third largest ($F_3$) preovulatory follicle were incubated for 4 h in short-term culture system, $P_4$ production by granulosa cells of both $F_1$ and $F_3$ was increased in a dose-dependent manner by ovine luteinizing hormone (oLH), but not T or $E_2$. In the second experiment, $F_1$ and $F_3$ granulosa cells cultured for 48 h in the developed monolayer culture system were recultured for an additional 48 h with increasing doses of various physiological active substances existing in the ovary, including T and $E_2$. Basal $P_4$ production for 48 h during 48 to 96 h of the cultured was about nine fold greater by $F_1$ granulosa cells than by $F_3$ granulosa cells. In substances examined oLH, chicken vasoactive intestinal polypeptide (cVIP) and T, but not $E_2$, stimulated in a dose-dependent manner $P_4$ production in both $F_1$ and $F_3$ granulosa cells. In addition, when the time course of $P_4$ production by $F_1$ granulosa cells in response to oLH, cVIP, T and $E_2$ was examined for 48 h during 48 to 96 h of culture, although $E_2$ had no effect on $P_4$ production by granulosa cells of $F_1$ during the period from 48 to 96 h of culture, $P_4$ production with oLH was found to be increased at 4 h of the culture, with a maximal 9.14 fold level at 6 h. By contrast, $P_4$ production with cVIP and T increased significantly (p<0.05) from 8 and 12 h of the culture, respectively, with maximal 6.50 fold response at 12 h and 6, 48 fold responses at 36 h. Furthermore, when $F_1$ granulosa cells were precultured with $E_2$ for various times before 4 h culture with oLH at 96 h of culture, the increase in $P_4$ production in response to oLH with a dose-related manner was only found at a pretreatment time of more than 12 h. In the third experiment, theca internal cells of $F_1$, $F_2$ and the largest third to fifth preovulatory follicles ($F_{3-5}$) were incubated for 4 h in short-term culture system with increasing doses of $E_2$. The production of $P_4$ and T by theca internal cells were increased with the addition of $E_2$ of $10^{-6}M$. These increases were greater in smaller follicles. These results indicate that, in granulosa cells of the hen, T may have a direct stimulatory action in the long term on $P_4$ production, and on $E_2$ in long-term action which may enhance the sensitivity to LH for $P_4$ production, and thus, in theca internal cells, $E_2$ in short term action may stimulate the production of $P_4$ and T.

Induction of Fas-Mediated Apoptosis by Interferon-g is Dependent on Granulosa Cell Differentiation and Follicular Maturation in the Rat Ovary

Lee, Hye-Jeong,Kim, Ji Young,Park, Ji Eun,Yoon, Yong-Dal,Tsang, Benjamin K.,Kim, Jong-Min The Korean Society of Developmental Biology 2016 발생과 생식 Vol.20 No.4

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-${\gamma}$) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-${\gamma}$ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-${\gamma}$ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-${\gamma}$ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-${\gamma}$ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (${\geq}1,000U/mL$) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-${\gamma}$ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-${\gamma}$ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

Induction of Fas-Mediated Apoptosis by Interferon-g is Dependent on Granulosa Cell Differentiation and Follicular Maturation in the Rat Ovary

이혜정,김지영,박지은,윤용달,Benjamin K. Tsang,김종민 한국발생생물학회 2016 발생과 생식 Vol.20 No.4

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-g) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-g in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-g immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-g was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-g (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCGprimed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-g (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-g is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

난소과립막세포 스테로이드호르몬 생성 및 증식에 대한 Mullerian Inhibiting substance의 효과

김장흡(JH Kim),제동성(DS Jae),김태응(TE Kim),신재인(JI Shin),김은중(EJ Kim),이진우(JW Lee),나종구(JG Na),김수평(SP Kim) 대한산부인과학회 1996 Obstetrics & Gynecology Science Vol.39 No.12

Ovarian follicular growth is a consequence of granulosa cell proliferation, and steroid production by these cells appears to be a major determinant of the endocrine microenvironment of ovum maturation. The gonadotropins, FSH ad LH, regulate directly the growth and differentiation of the granulosa cells in the ovary, but there is evidence to suggest that the gonadotropins act partly through locally produced growth factors and that this interaction is complex. A number of growth factors, such as epidermal growth factor(EGF), insulin-like growth factor-I(IGF-I), and transforming growth factor-β(TGF-β) are produced in the ovarian follicle and might act in an autocrine and/or paracrine manner to directly control granulosa cell proliferation and differentiation. While it has been demonstrated that steroid hormones, gonadotropins and growth factors regulate proliferation and differentiation of ovarian follicles, little is known concerning the fators involved in the inhibition of ovarian function. Recently, Mullerian inhibiting substance(MIS), a non-steroidal testicular Sertoli cell product responsible for the regression of Mullerian duct in male embryo, has been shown to be produced by ovarian granulosa cells in adolescnet and ault females. Although the function of MIS in the ovary has not been fullydelineated, MIS appears to be a regulator of oocyte maturation and follicular development in the rat. In this study, in order to investigate the influence of MIS on steroidogenesis and proliferation of human granulosa cells, we performed culture of human granulosa cells. The cells were cultured for 2 to 12 days under two conditions, with and without MIS(20 ng/ml). Each condition was additionally defined by the presence and absence of EGF(20 ng/ml), FSH(10 ng/ml), or LH (10 ng/ml). Estradiol, progesterone, and testosterone were mearsured form the spent media by radioimmunoassay and the cell number was determined by trypsinizing the cells and counting them with a Coulter counter. The result were as follows: 1. There was about 6-fold increase in the final granulosa cell number when the culture were maintained for 12 days in Ham`s F-10 supplemented with 10% MIS-free female fetal calf serum(control). FSH and EGF caused a significant increase in granulosa cell number compared with the control but LH significantly suppressed cell number after 8 days in culture. 2. MIS caused a significant decrease in granulosa cell number compared with the control after 8 days in culture in the 20 ng/ml dose, and on day 12 in the 2 ng/ml dose(p

Induction of Fas-Mediated Apoptosis by Interferon-γ is Dependent on Granulosa Cell Differentiation and Follicular Maturation in the Rat Ovary

Hye-Jeong Lee,Ji Young Kim,Ji Eun Park,Yong-Dal Yoon,Benjamin K. Tsang,Jong-Min Kim 한국발생생물학회 2016 발생과 생식 Vol.20 No.4

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCGprimed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

소 과립막세포의 배양 상층액이 생쥐배의 체외발달에 미치는 영향

이상범(Sang Bum Lee),문신홍(Sin Hong Moon),김선구(Seon Ku Kim) 한국생명과학회 2009 생명과학회지 Vol.19 No.12

본 연구는 소의 과립막세포 배양상층액에서 스테로이드 호르몬의 농도와 생쥐 배의 체외 발달에 미치는 효과를 검토하기 위하여 수행되었다. 소의 성숙난포(직경 6~15 ㎜)와 미성숙 난포(직경 2~5 ㎜)로부터 과립막세포를 각각 분리하여 15% FCS가 첨가된 Ham's F-10 배양약에서 16일간 배양하였다. 과립막세포들은 쉽게 단층배엽을 형성 하였으며, 배양과정 중 비슷한 성장패턴을 나타내었다. 또한 성숙 과립막세포와 미성숙 과립막세포 간에 형태적인 차이가 없었다. 과립막세포 배양 중 progesterone과 estradiol 생산이 활발하게 이루어졌으며, progesterone은 배양후기에, estradiol은 배양초기에 분비량이 높았다. 성숙 과립막세포와 미성숙 과립막세포에서 내분비 양상은 비슷하였다. 생쥐배가 상실배, 배반포 및 부화배반포 단계로 체외발달된 비율은 Ham'F-10배양액(86.7%, 41.7%, 13.3%)에 비하여 성숙 과립막세포 배양상층액(92.7%, 78.1%, 34.5%)과 미성숙 과립막세포 배양상층액(96.4%, 78.5%, 26.8%)에서 유의적으로 높았다(p<0.05). 그러나 배의 발달에 대하여 성숙 과립막세포배양상층액과 미성숙 과립막세포 배양상층액 간에는 차이가 없었다. This study was carried out to examine a concentration of steroid hormones and in vitro development of mouse embryos in culture supernatant of bovine granulosa cells (GC). To obtain the culture supernatant, granulosa cells were retrieved from mature follicles (6~15 ㎜ diameter) and immature follicles (2~5 ㎜ diameter) of bovine ovary and were cultured, respectively, in media of Ham's F-10 with 15% FCS for 16 days. Mature and immature granulosa cells formed their monolayers easily and showed similar growth patterns in culturing. There was no morphological difference between mature and immature granulosa cells. High levels of both progesterone and estradiol were detected in the culture supernatant of mature granulosa cells and immature granulsa cells, and the endocrine profiles of the two types of cells were similar. Progesterone secretion of granulosa cells was high in the late stage of culturing and estradiol secretion was high in the early stage of culturing. In vitro development rates of mouse embryos to morula, blastocyst and hatched blastocyst were significantly (p<0.05) higher in culture supernatant of mature granulosa cells (92.7%, 78.1% and 34.5%) and in culture supernatant of immature granulosa cells (96.4%, 78.5% and 26.8%) than in Ham's F-10 (86.7%, 41,7% and 13.3%). However, there was no difference between the culture supernatant of mature granulosa cells and the culture supernatant of immature granulosa cells in the development of embryos.

Induction of Ski Protein Expression upon Luteinization in Rat Granulosa Cells

김현,Takashi Matsuwaki,Keitaro Yamanouchi,Masugi Nishihara,양보석,고응규,김성우 사단법인 한국동물생명공학회 2011 한국동물생명공학회지 Vol.26 No.4

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vitro models. RT-PCR and real time PCR analysis respectively revealed that LH had no effect on c-Ski mRNA expression in the cultured granulosa cells regardless of LH treatment. Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally. Moreover, expression of mRNA of Arkadia, an E3 ubiquitin ligases,in luteinizing granulosa cells in vivo was assessed by realtime-PCR. The levels of Arkadia mRNA expression were unchanged during follicular growth and postovulatory luteinization. These findings suggest that Ski protein level may be regulated during luteinization at translational and/or post-translational level but not by Arkadia

돼지 난소 과립막세포 배양액으로부터 발현된 GnSIF의 활성에 관한 연구

서수형(Soo Hyung Seo) 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.12

Objectives: It has been identified a novel protein in porcine and bovine follicular fluid termed Gonadotropin Surge Inhibiting Factor (GnSIF) which inhibits GnRH-stimulated LH secretion by pituitary cells in culture. The purpose of this study was to investigate the production of GnSIF by porcine granulosa cell in vitro. Materials and Methods: Granulosa cells were aspirated from different size of porcine follicles (1-3mm, 3-5mm, >6mm in diameter) and cultured in 24-well plates in Ham's F-12/DMEM at 500,000 cells/well for 24h. After plating, cells were washed and incubated in triplicate for 72h in DMEM/F-12 containing 2% FBS in the presence of 100ng/ml FSH and the media were collected. Granulosa cell-conditioned media were chromatographed on heparin-Sepharose to remove inhibin and follistatin, and assayed for GnSIF activity using a rat pituitary cell bioassay. Briefly, dispersed pituitary cells from adult female Sprague-Dawley rats were plated (500,000 cells/well) for 72h, washed, and incubated with granulosa cell-conditioned media in the presence of 10nM GnRH for 4h. The media were collected and assayed for LH by RIA. GnSIF activity is defined as the suppression of GnRH-stimulated LH secretion. Results: Granulosa cell-conditioned media significantly inhibited GnRH-stimulated LH secretion by rat pituitary cells in vitro (69% of control, p<0.001, n=2 experiments). Preliminary data suggest that granulosa cells from small follicles (1-3mm) produce greater amounts of GnSIF than those obtained from larger follicles (>6mm). Conclusions: GnSIF activity is produced by porcine granulosa cells in vitro. Granulosa cell production of GnSIF, along with other gonadal proteins such as inhibin, follistatin, and activin may be an important component of the ovarian control of pituitary gonadotropin secretion.

난소의 생식세포 Apoptosis에 대한 JNK1과 JNK2의 역할

김미란 ( Mee Ran Kim ),최윤경 ( Yoon Kyong Choi ),송재연 ( Jae Yen Song ),신현미 ( Hyun Mi Shin ),정재은 ( Jae Eun Chung ),임용택 ( Young Taik Lim ),김장흡 ( Jang Heub Kim ),김진홍 ( Jin Hong Kim ),유영옥 ( Young Oak Lew ) 대한폐경학회 2007 대한폐경학회지 Vol.13 No.3

연구목적: 난자와 난포 과립막세포가 외부 자극에 대하여 JNK의 발현에 어떠한 영향을 미치는지 알아보고, JNK 유전자 중 JNK1이나 JNK2가 결핍된 마우스를 이용하여 난자와 과립막세포의 apoptosis에 대한 JNK유전자의 역할을 규명하고자 하였다. 연구재료 및 방법: 생후 21∼24일의 C57BL/6 마우스를 이용하여 과배란 유도를 통해 난자를 채취하여 자외선 조사 후 phospho-JNK에 대한 면역세포화학법을 시행했다. 과립막세포를 배양하고 자외선 조사 후 단백질을 추출하여 웨스턴블롯을 시행했다. Hepa 1c1c7 cell도 위와 같은 방법으로 웨스턴블롯팅을 시행하였다. JNK1이나 JNK2 유전자 결핍 마우스에서 난자의 apoptosis의 차이를 알기 위해 난소 전체의 난자의 수를 비교하였다. 과립막세포를 자외선 조사 2시간, 4시간, 6시간 후 고정하고 Hoescht 33342로 염색 후 현광현미경으로 핵의 형태를 관찰하여 apoptosis 여부를 판별했다. 결과: 자외선 조사 후 난자에서 인산화 JNK에 대한 면역세포화학염색 반응이 대조군에 비하여 현저히 증가되었다. Hepa 1c1c7 cell에서는 자외선 조사 후 인산화 JNK의 발현이 나타나지 않았으나 과립막 세포에서는 자외선 조사 30분 후 각각 55 kD와 46 kD의 인산화 JNK의 단백이 발현되었다. JNK1 유전자 결핍 마우스의 난소에서 총 난자의 수와 원시난포의 수는 출생 후 제 4일과 제 42일 정상 마우스에서와 유의한 차이가 없었다. JNK2의 경우도 JNK2 유전자 결핍 마우스의 난소에서 총 난자의 수와 원시난포의 수가 정상 마우스에서와 유의한 차이를 보이지 않았다. 자외선 조사에 의한 과립막세포의 apoptosis는 대조군의 13.0±3.3%에서 자외선 조사 2시간 후에 22.2±12.8%, 4시간 후에 32.0±10.4%, 6시간 후에 42.9±11.3%로 관찰되었다. JNK1 유전자 결핍 마우스의 난포 과립막 세포는 대조군의 10.5±4.1%에서 2시간 후에 18.3±9.1%, 4시간 후에 24.8±9.1%, 6시간 후에 29.5±12.4%의 apoptosis를 보여 부분적으로 apoptosis에 대한 보호작용이 있는 것으로 보였지만, 통계적인 의미가 없었다. JNK2 유전자 결핍 마우스에서도 정상 마우스에서의 apoptosis와 유의한 차이가 없었다. 결론: JNK는 포유동물의 세포 생리작용에 중요한 매개체로서 세포의 증식, 생존, 사망, DNA의 대사에 다양하게 작용하며 향후 치료적 표적으로 중요한 역할을 할 것으로 기대된다. JNK는 마우스의 난자와 과립막 세포에서 자외선 조사에 의해 특이적으로 활성화 되었다. JNK1이나 JNK2 유전자결핍 마우스를 이용한 연구 결과를 통하여 마우스의 난소에서 JNK1과 JNK2는 상호보완적으로 작용함으로 특이한 표현형을 발견할 수 없었다. 이러한 연구 결과는 향후 폐경 전 여성에서 비정상적으로 가속화되는 난자 고갈에 대한 JNK의 역할을 규명하는데 도움을 줄 수 있을 것이며 노화로 인한 자연적인 난소기능의 퇴화기전을 밝히는데 도움이 될 것으로 생각한다. Objectives: Each female is endowed with a finite numbers of oocytes, which when depleted leads to ovarian senescence, the menopause. The objective of this study was to investigate the role of JNK1 and JNK2 in murine ovarian germ cell apoptosis. Methods: Murine oocytes were retrieved by superovulation of female C57BL/6 mice at the age of 21∼24 days. At 10 minutes after UV irradiation, immunocytochemistry was performed. Ovarian granulosa cells were cultured from antral follicles. At the 24 hours of the culture period, UV was irradiated on culture dishes. After 30 minutes, protein was extracted and western blotting done. Same procedures were performed with Hepa 1c1c7 cells. Serial ovarian sections (8μm) were stained with modified Lee`s picric methyl blue, and we estimated the numbers of oocyte-containing primordial, primary and small preantral follicles of female mice at day 4 and day 42 postpartum. Ovarian granulosa cells were cultured from female wild-type and JNK1-/-or JNK2-/-mice. The granulosa cells were fixed at 2, 4, 6 hours after UV irradiation and stained with Hoescht 33342, and then evaluated for characteristics of apoptosis under fluorescence microscopy. Results: After UV irradiation, oocytes showed intense signal of phospho-JNK. In ovarian granulosa cells, JNK was activated to phospho-JNK after UV irradiation, but not in Hepa 1c1c7 cells. There was no difference in oocyte counts between wild type and JNK1-/-or JNK2-/-at day 4 and day 42 postpartum. The incidence of apoptosis of ovarian granulosa cells from wild type were 13.0±3.3%, 22.2±12.8%, 32.0±10.4%, 42.9±11.3% after 0, 2, 4, 6 hour of UV irradiation, respectively. The incidences were 10.5±4.1%, 18.3±9.1%, 24.8±9.1%, 29.5±12.4%, respectively in granulosa cells from JNK1-/-mice. These results showed only a partial suppression of apoptosis in granulosa cells from JNK1-/-mice, however, it was not statistically significant. There was no significant difference in the rate of apoptosis of granulosa cells after UV irradiation between wild type and JNK2-/-mice. Conclusion: JNK1 and JNK2 are recognized as critical regulators of various aspects of mammalian physiology, including cell proliferation, cell survival and cell death. This study showed that UV irradiation caused activation of JNK in murine oocytes and granulosa cells of ovary. In murine ovary, JNK1 and JNK2 may be redundant or have compensatory role to each other in apoptosis. These findings may help to elucidate the one of mechanisms for acceleration of oocyte depletion in women with premature menopause.