Park, Sung-Rok;Lee, Byung-Seok;Yang, Woo-Ick;Kim, Jong-Hwa;Park, Byung-Joo;Park, Ki-Hyun;Cho, Dong-Jae;Song, Chan-Ho
Clinical and Experimental Reproductive Medicine
/
v.29
no.3
/
pp.195-200
/
2002
Objective : To investigate the expression of apoptosis related proteins and apoptotic cells on the human ovarian follicles. Materials and Methods: Thirty five Formalin-fixed paraffin-embedded human ovarian tissue blocks were selected from the surgical pathology files of the department of pathology, College of Medicine, Yonsei University, for the period from 1996 to 1998. All specimen were from premenopausal women aged from $32{\sim}45$. Ovarian tissues were collected from the patients performing hysterectomy for benign uterine diseases. Immunohistochemical staining was performed for the detection of DNA fragmented cell, Bcl-2, Bax, Fas and Fas-ligand. Results: Bcl-2 and bax were not expressed on the surrounding cells and oocyte of the primary, primordial and preantral follicles. Fas and Fas-ligand (Fas-L) were not expressed on the surrounding cells on the primordial and primary follicles. But expressed on the surrounding granulosa cells and oocyte in the primordial and primary follicles. In the healthy follicles, Bcl-2 was expressed on the granulosa cells, however, Bax was not expressed. DNA fragmented cells were expressed on the inner granulosa cell layer of atretic follicles. Conclusion: Fas, Fas-ligand, and Bax may be responsible for the follicular atresia and Bcl-2 may be involved in the follicular survival in the human ovary.
The aims of this study were to detect spontaneously occurring apoptosis in cultured porcine ovarian cells, to examine the role of growth hormone (GH), tyrosine kinase (TK), protein kinase G (PKG) and cyclin-dependent kinase (CDK) in the control of this process, and to determine whether the effect of GH on apoptosis is mediated by TK-, PKG- and cdc2-dependent intracellular mechanisms. We studied the action of pGH (10 ng/ml), blockers of TK (genistein, lavendustin, both 100 ng/ml), PKG (Rp-Br-PET-cGMPS, 50 nM; KT5823, 100 ng/ml) and CDK (olomoucine, $1{\mu}g/ml$), as well as combinations of GH with these blockers, on the onset of apoptosis in cultured granulosa cells isolated from antral (3-6 mm) porcine follicles. The functional characteristics of an early apoptotic event, DNA fragmentation, were determined using terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL), whilst morphological signs of advanced apoptosis such as pyknosis, chromatin marginalization, shrinkage and fragmentation of nucleus, were detected using routine light microscopy. After culture, some ovarian granulosa cells exhibited DNA fragmentation, which in some cases was associated with morphological apoptosis-related changes (pyknosis, shrinkage and fragmentation of the nucleus). GH significantly reduced the proportion of TUNEL-positive cells. Neither TK nor CDK blockers when given alone, significantly affected the percentage of TUNEL-positive cells although both PKG blockers significantly increased this index. Furthermore, TK and PKG blockers given together with GH, prevented or reversed the inhibitory effect of GH on apoptosis, whilst the CDK blocker olomoucine promoted it. These observations demonstrate apoptosis in porcine ovaries and suggest the involvement of GH, TK, PKG and CDK in the control of this process. They also suggest that the effect of GH on ovarian apoptosis is mediated or regulated by multiple signalling pathways including TK-, PKG- and CDK-dependent intracellular mechanisms.
There have been many reports to date regarding the role of GnRH as a local regulatory factor of ovarian function as studies of human and rat ovaries revealed GnRH and its receptor. In recent studies it has been shown that GnRH directly causes apoptosis in the granulosa cells of the rat ovary, and such results leads to the suggestion that the use of GnRH agonist for more stable long term ovarian hyperstimulation in human IVF-ET programs causes granulosa cell apoptosis which may lead to follicular atresia. Therefore this study attempts to determine if granulosa-luteal cell apoptosis occurs in patients during IVF-ET programs in which GnRH agonist is employed for ovarian hyperstimulation. The quality of oocyte-cumulus complexes obtained during ovum pickup procedures were assessed morphologically and then the fertilization rate and developmental rate was determined. Apoptotic cells among the granulosa-luteal cells obtained during the same procedure were observed after staining with Hematoxylin-eosin. The fragmentation degree of DNA extracted from granulosa-luteal cells was determined and comparatively analyzed. There was no difference in the average age of the patients, the number of oocytes retrieved, and fertilization and developmental rates between the FSH/hMG group and GnRH-long group. There was also no difference in the apoptosis rate and pyknosis rate in the granulosa-luteal cells between the two groups. However, when the oocyte-cumulus complexes were morphoogically divided into the healthy group and atretic group without regard for the method of hyperstimulation, the results showed that the number of oocytes obtained averaged $11.09{\pm}8.75\;and\;10.33{\pm}4.53$ per cycle, respectively, showing no significant difference, but the fertilization rate (77.05%, 56.99%, respectively, p<0.01) and developmental rate (65.96%, 41.51%, respectively, p<0.01) was significantly increased in the healthy group when compared to the atretic group. The degree of apoptosis in the granulosa-luteal cells showed that in the healthy group it was 2.25% which was not significantly different from the atretic group (2.77%), but the pyknosis rate in the atretic group (27.81%) was significantly higher compared to the healthy group (11.35%, p<0.01). The quantity of DNA fragmentation in the FSH/hMG group was 32.22%, while in the GnRH-long group it was 34.27%, showing no significant difference. On the other hand the degree of DNA fragmentation was 39.05% and 11.83% in the healthy group and atretic group, respectively, showing significantly higher increase in the atretic group (p<0.01). The above results suggest that death of granulosa-luteal cells according to the state of the oocyte-cumulus complex is more related to pyknosis rather than apoptosis. Also, the GnRH agonist used in ovarian hyperstimulation does not seem to directly affect the apoptosis of retrieved oocytes and granulosa-luteal cells, and which is thought to be due to the suppression of the apoptogenic effect of GnRH agonist as a result of the high doses of FSH administered.
Kim, Kyeoung-Hwa;Park, Chang-Eun;Yoon, Se-Jin;Lee, Kyung-Ah
Clinical and Experimental Reproductive Medicine
/
v.32
no.3
/
pp.269-277
/
2005
Objectives: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. Methods: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (${\underline{c}}onnective$ tissue growth factor/${\underline{c}}ysteine$-rich 61/${\underline{n}}ephroblastoma$-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. Results: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. Conclusions: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.
Objectives: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the mouse ovary during the follicle development. Methods: The animals were implanted subcutaneously with Silastic brand capsules containing the synthetic estrogen, DES at $21{\sim}23$ days of age. Ovaries were collected $1{\sim}3$ days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for $1{\sim}2$ days to induce ovarian follicle apoptosis. Ovaries were also collected from 26 day-old immature mice at various times after treatment with 10 IU PMSG. Some mice received a single intraperitoneal injection of 10 IU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analysis, respectively. Results: Treatment of immature mice with diethylstilbestrol (DES) for $24{\sim}48$ h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for $24{\sim}48$ h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature mice with PMSG for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. Interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSGprimed mice with hCG stimulated strongly ovarian Bok mRNA expression at $6{\sim}9$ h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. Conclusion: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.
Yan Liang;Huimin Wang;Jin Chen;Lingyan Chen;Xiaoyong Chen
The Korean Journal of Physiology and Pharmacology
/
v.27
no.2
/
pp.167-176
/
2023
This study aims to explore the impact of Rehmannioside D (RD) on ovarian functions of rats with diminished ovarian reserve (DOR) and its underlying mechanisms of action. A single injection of cyclophosphamide was performed to establish a DOR rat model, and fourteen days after the injection, the rats were intragastrically administrated with RD for two weeks. Rat estrus cycles were tested using vaginal smears. Ovarian tissues were histologically evaluated, the number of primordial, mature, and atretic follicles was calculated, and the apoptotic rate of granulosa cells. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels were determined by ELISA assays. Protein levels of Forkhead Box O1 (FOXO1), KLOTHO, Bcl-2, and Bax were investigated in ovarian tissues of DOR rats. The binding between FOXO1 and KLOTHO was verified by ChIP assay. High-dose administration of RD into DOR rats improved their estrus cycles, increased ovarian index, enhanced the number of primordial and mature follicles, reduced the number of atretic follicle number, and ovarian granulosa cell apoptosis in addition to inhibiting FSH and LH levels and upregulating E2 expression. FOXO1 and KLOTHO were significantly suppressed in DOR rats. FOXO1 knockdown partially suppressed the protective effects of RD on DOR rats, and KLOTHO overexpression could restore RD-induced blockade of DOR development despite knocking down FOXO1. FOXO1 antibody enriched KLOTHO promoter, and the binding between them was reduced in DOR group compared to that in sham group. RD improved ovarian functions in DOR rats and diminished granulosa cell apoptosis via the FOXO1/KLOTHO axis.
This experiment has been done in order to study the correlation between the ultrastructure changes and the atresia phenomenon of the follicles in porcine ovary. The ovaries were assorted according to the presence or absence of corpus luteum. Thereafter, the follicles were classified into normal, pyknotic, necrotic and cystic groups by atretic characteristics on the histological observation, and then their ultrastructures were examined with an electron microscope. The results were as followings. 1. In normal group, granulosa cells represented the ultrastructural characteristics of protein-synthesizing cells. Since the initiation of atresia, the line structure of granulosa cells showed many of the characteristic features of steroid-secreting cells, followed by gradual pyknosis. 2. In necrotic group of the ovary without corpus luteum, the theca interns became hypertrophic and displayed the ultrastructural features of active steroidsecreting cells. But this phenomenon was not seen in the follicles of the ovary with corpus luteum. 3. Degenerative changes of cumulus cells were similar to those of granulosa cells, and the degenerating oocytes showed the degeneration of cellular organelles, cytoplasmic vacuolization and disappearance of microvilli on the surface. The degeneration of granulosa cells tended to procede that of oocytecumulus complex in the follicles of the ovary having no corpus luteum, but this tendency was reversed in the case of presence of corpus luteum. In conclusion, it may be unable to identi(y the initiation of follicular atresia
Vitamin D3 is a fat-soluble secosteroid responsible for enhancing intestinal absorption of calcium, iron, and other materials. Vitamin D3 deficiency, therefore, can cause health problems such as metabolic diseases, and bone disorder. Female sex hormones including estrogen and progesterone are biosynthesized mainly in the granulosa cells of ovary. In this study, we isolated granulosa cells from porcine ovary and cultured for the experiments. In order to examine the effect of vitamin D3 on the ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by real-time PCR and Western blot assay. The production of estrogen from the granulosa cells was also measured by the ELISA assay. Genes associated with follicle growth were not significantly altered by vitamin D3. However, it increases expression of genes involved in the estrogen-biosynthesis. Further, estrogen concentrations in porcine granulosa cell-cultured media increased in response to vitamin D3. These results showed that vitamin D3 is a powerful regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.
Kim, Chang-Woon;Choi, Eun-Ju;Kim, Eun-Jin;Siregar, Adrian S.;Han, Jaehee;Kang, Dawon
Journal of Animal Reproduction and Biotechnology
/
v.35
no.4
/
pp.315-322
/
2020
Aquaporin channels (AQPs) are known to play an important role in the development of ovarian follicles through their function in water transport pathways. Compared to other AQPs, research on the role of AQP4 in female reproductive physiology, particularly in cattle, remains limited. In our previous study, gene chip microarray data showed a downregulation of AQP4 in bovine cystic follicles. This study was performed to validate the AQP4 expression level at the protein level in bovine follicles using immunohistochemistry, Western blotting, and immunoprecipitation assays. Immunostaining data showed that AQP4 was expressed in granulosa and theca cells of bovine ovarian follicles. The ovarian follicles were classified according to size as small (< 10 mm) or large (> 25 mm) in diameter. Consistent with earlier microarray data, semi-quantitative PCR data showed a decrease in AQP4 mRNA expression in large follicles. Western blot analysis showed a downregulation of the AQP4 protein in large follicles. In addition, AQP4 was immunoprecipitated and blotted with anti-AQP4 antibody in small and large follicles. Accordingly, AQP4 exhibited a low expression in large follicles. These results show that AQP4 is downregulated in bovine ovarian large follicles, suggesting that the downregulation of AQP4 expression may interfere with follicular water transport, leading to bovine follicular cysts.
Wee1 is a kinase regulator of the M-phase promoting factor (MPF; a complex of cdc2 and cyclin B1). The present study was undertaken to determine the role(s) of wee1 in the early stages of mouse ovarian follicles. The expression of wee1 and the correlated cell-cycle components, namely cdc2, cyclin B1, and cdc25C, were evaluated by immunohistochemistry. In addition, the expression of Tyr15-phosphorylated cdc2 (cdc2-p) was also examined to determine whether wee1 kinase phosphorylates cdc2 existed. Each component except cdc25C was found cytoplasmic in the oocytes at all stages of follicles, while cdc25C was not detected in primordial follicles. It was found primarily in ovarian somatic cells and to a small extent in granulosa cells of the growing follicles. To further confirm the expression of cell-cycle components in the primordial follicular oocytes, day1 ovaries were enzymatically and mechanically dissociated, then oocytes were isolated from somatic including pre-granulosa cells, and we confirmed that cdc2-p was expressed in oocytes of primordial follicles. From the results of the present study, we concluded wee1, without the counteracting cdc25C, would cause meiotic arrest of oocytes by the inhibitory phosphorylation of cdc2. The expression of all these proteins in the granulosa cells of growing follicles may regulate their mitosis concurrently with the growth of oocytes and follicles.
본 웹사이트에 게시된 이메일 주소가 전자우편 수집 프로그램이나
그 밖의 기술적 장치를 이용하여 무단으로 수집되는 것을 거부하며,
이를 위반시 정보통신망법에 의해 형사 처벌됨을 유념하시기 바랍니다.
[게시일 2004년 10월 1일]
이용약관
제 1 장 총칙
제 1 조 (목적)
이 이용약관은 KoreaScience 홈페이지(이하 “당 사이트”)에서 제공하는 인터넷 서비스(이하 '서비스')의 가입조건 및 이용에 관한 제반 사항과 기타 필요한 사항을 구체적으로 규정함을 목적으로 합니다.
제 2 조 (용어의 정의)
① "이용자"라 함은 당 사이트에 접속하여 이 약관에 따라 당 사이트가 제공하는 서비스를 받는 회원 및 비회원을
말합니다.
② "회원"이라 함은 서비스를 이용하기 위하여 당 사이트에 개인정보를 제공하여 아이디(ID)와 비밀번호를 부여
받은 자를 말합니다.
③ "회원 아이디(ID)"라 함은 회원의 식별 및 서비스 이용을 위하여 자신이 선정한 문자 및 숫자의 조합을
말합니다.
④ "비밀번호(패스워드)"라 함은 회원이 자신의 비밀보호를 위하여 선정한 문자 및 숫자의 조합을 말합니다.
제 3 조 (이용약관의 효력 및 변경)
① 이 약관은 당 사이트에 게시하거나 기타의 방법으로 회원에게 공지함으로써 효력이 발생합니다.
② 당 사이트는 이 약관을 개정할 경우에 적용일자 및 개정사유를 명시하여 현행 약관과 함께 당 사이트의
초기화면에 그 적용일자 7일 이전부터 적용일자 전일까지 공지합니다. 다만, 회원에게 불리하게 약관내용을
변경하는 경우에는 최소한 30일 이상의 사전 유예기간을 두고 공지합니다. 이 경우 당 사이트는 개정 전
내용과 개정 후 내용을 명확하게 비교하여 이용자가 알기 쉽도록 표시합니다.
제 4 조(약관 외 준칙)
① 이 약관은 당 사이트가 제공하는 서비스에 관한 이용안내와 함께 적용됩니다.
② 이 약관에 명시되지 아니한 사항은 관계법령의 규정이 적용됩니다.
제 2 장 이용계약의 체결
제 5 조 (이용계약의 성립 등)
① 이용계약은 이용고객이 당 사이트가 정한 약관에 「동의합니다」를 선택하고, 당 사이트가 정한
온라인신청양식을 작성하여 서비스 이용을 신청한 후, 당 사이트가 이를 승낙함으로써 성립합니다.
② 제1항의 승낙은 당 사이트가 제공하는 과학기술정보검색, 맞춤정보, 서지정보 등 다른 서비스의 이용승낙을
포함합니다.
제 6 조 (회원가입)
서비스를 이용하고자 하는 고객은 당 사이트에서 정한 회원가입양식에 개인정보를 기재하여 가입을 하여야 합니다.
제 7 조 (개인정보의 보호 및 사용)
당 사이트는 관계법령이 정하는 바에 따라 회원 등록정보를 포함한 회원의 개인정보를 보호하기 위해 노력합니다. 회원 개인정보의 보호 및 사용에 대해서는 관련법령 및 당 사이트의 개인정보 보호정책이 적용됩니다.
제 8 조 (이용 신청의 승낙과 제한)
① 당 사이트는 제6조의 규정에 의한 이용신청고객에 대하여 서비스 이용을 승낙합니다.
② 당 사이트는 아래사항에 해당하는 경우에 대해서 승낙하지 아니 합니다.
- 이용계약 신청서의 내용을 허위로 기재한 경우
- 기타 규정한 제반사항을 위반하며 신청하는 경우
제 9 조 (회원 ID 부여 및 변경 등)
① 당 사이트는 이용고객에 대하여 약관에 정하는 바에 따라 자신이 선정한 회원 ID를 부여합니다.
② 회원 ID는 원칙적으로 변경이 불가하며 부득이한 사유로 인하여 변경 하고자 하는 경우에는 해당 ID를
해지하고 재가입해야 합니다.
③ 기타 회원 개인정보 관리 및 변경 등에 관한 사항은 서비스별 안내에 정하는 바에 의합니다.
제 3 장 계약 당사자의 의무
제 10 조 (KISTI의 의무)
① 당 사이트는 이용고객이 희망한 서비스 제공 개시일에 특별한 사정이 없는 한 서비스를 이용할 수 있도록
하여야 합니다.
② 당 사이트는 개인정보 보호를 위해 보안시스템을 구축하며 개인정보 보호정책을 공시하고 준수합니다.
③ 당 사이트는 회원으로부터 제기되는 의견이나 불만이 정당하다고 객관적으로 인정될 경우에는 적절한 절차를
거쳐 즉시 처리하여야 합니다. 다만, 즉시 처리가 곤란한 경우는 회원에게 그 사유와 처리일정을 통보하여야
합니다.
제 11 조 (회원의 의무)
① 이용자는 회원가입 신청 또는 회원정보 변경 시 실명으로 모든 사항을 사실에 근거하여 작성하여야 하며,
허위 또는 타인의 정보를 등록할 경우 일체의 권리를 주장할 수 없습니다.
② 당 사이트가 관계법령 및 개인정보 보호정책에 의거하여 그 책임을 지는 경우를 제외하고 회원에게 부여된
ID의 비밀번호 관리소홀, 부정사용에 의하여 발생하는 모든 결과에 대한 책임은 회원에게 있습니다.
③ 회원은 당 사이트 및 제 3자의 지적 재산권을 침해해서는 안 됩니다.
제 4 장 서비스의 이용
제 12 조 (서비스 이용 시간)
① 서비스 이용은 당 사이트의 업무상 또는 기술상 특별한 지장이 없는 한 연중무휴, 1일 24시간 운영을
원칙으로 합니다. 단, 당 사이트는 시스템 정기점검, 증설 및 교체를 위해 당 사이트가 정한 날이나 시간에
서비스를 일시 중단할 수 있으며, 예정되어 있는 작업으로 인한 서비스 일시중단은 당 사이트 홈페이지를
통해 사전에 공지합니다.
② 당 사이트는 서비스를 특정범위로 분할하여 각 범위별로 이용가능시간을 별도로 지정할 수 있습니다. 다만
이 경우 그 내용을 공지합니다.
제 13 조 (홈페이지 저작권)
① NDSL에서 제공하는 모든 저작물의 저작권은 원저작자에게 있으며, KISTI는 복제/배포/전송권을 확보하고
있습니다.
② NDSL에서 제공하는 콘텐츠를 상업적 및 기타 영리목적으로 복제/배포/전송할 경우 사전에 KISTI의 허락을
받아야 합니다.
③ NDSL에서 제공하는 콘텐츠를 보도, 비평, 교육, 연구 등을 위하여 정당한 범위 안에서 공정한 관행에
합치되게 인용할 수 있습니다.
④ NDSL에서 제공하는 콘텐츠를 무단 복제, 전송, 배포 기타 저작권법에 위반되는 방법으로 이용할 경우
저작권법 제136조에 따라 5년 이하의 징역 또는 5천만 원 이하의 벌금에 처해질 수 있습니다.
제 14 조 (유료서비스)
① 당 사이트 및 협력기관이 정한 유료서비스(원문복사 등)는 별도로 정해진 바에 따르며, 변경사항은 시행 전에
당 사이트 홈페이지를 통하여 회원에게 공지합니다.
② 유료서비스를 이용하려는 회원은 정해진 요금체계에 따라 요금을 납부해야 합니다.
제 5 장 계약 해지 및 이용 제한
제 15 조 (계약 해지)
회원이 이용계약을 해지하고자 하는 때에는 [가입해지] 메뉴를 이용해 직접 해지해야 합니다.
제 16 조 (서비스 이용제한)
① 당 사이트는 회원이 서비스 이용내용에 있어서 본 약관 제 11조 내용을 위반하거나, 다음 각 호에 해당하는
경우 서비스 이용을 제한할 수 있습니다.
- 2년 이상 서비스를 이용한 적이 없는 경우
- 기타 정상적인 서비스 운영에 방해가 될 경우
② 상기 이용제한 규정에 따라 서비스를 이용하는 회원에게 서비스 이용에 대하여 별도 공지 없이 서비스 이용의
일시정지, 이용계약 해지 할 수 있습니다.
제 17 조 (전자우편주소 수집 금지)
회원은 전자우편주소 추출기 등을 이용하여 전자우편주소를 수집 또는 제3자에게 제공할 수 없습니다.
제 6 장 손해배상 및 기타사항
제 18 조 (손해배상)
당 사이트는 무료로 제공되는 서비스와 관련하여 회원에게 어떠한 손해가 발생하더라도 당 사이트가 고의 또는 과실로 인한 손해발생을 제외하고는 이에 대하여 책임을 부담하지 아니합니다.
제 19 조 (관할 법원)
서비스 이용으로 발생한 분쟁에 대해 소송이 제기되는 경우 민사 소송법상의 관할 법원에 제기합니다.
[부 칙]
1. (시행일) 이 약관은 2016년 9월 5일부터 적용되며, 종전 약관은 본 약관으로 대체되며, 개정된 약관의 적용일 이전 가입자도 개정된 약관의 적용을 받습니다.