Endothelin 2 (EDN2) induces follicular rupture by constricting periovulatory follicles. In this study, it was investigated the mechanisms of EDN2 action on follicular rupture with respect of receptor using the conditionally granulosa cell specific EDN2 receptor type A (ETa) KO mice (gcETaKO; ETaflox/-․Amhr2Cre). It was generated the gcETaKO mice by breeding with ETaflox/- mice after mono-alleic ETa knockout by ZP3Cre and Amhr2Cre mice. Fertility, ovulation and maturation rates of ovulated oocytes after super ovulation were investigated in the gcETaKO mice compared with wild-type mice (ETaflox/flox and ETaflox/-) as a control group. In the gcETaKO mice, normal fertility after breeding with male mice was shown compared with wild-type mice. And, there was no significant differences in ovulation rates after super ovulation, however its maturation rates was lower than that of wild type mice. These findings show that EDN2 in follicular rupture for ovulation is related with an other ETa not in granulosa cells. Further studies are needed to investigate how EDN2 is acted in ovarian follicular rupture for ovulation.

The aim of this study was to evaluate the changes of protein patterns in granulosa cells and corpus luteum during the estrus cycle in bovine ovary by proteomics ^techniques. Our study was devided into five steps for follicular, ovulatory, early-lteal, midluteal and late-luteal. The protein was extracted from glanulosa cell and corpus luteum proteins by using M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was 700 μg. Immobilized pH gradient (IPG) strip was used 18 cm and 3 11 NL. SDS-PAGE was used 10% acrylamide gel. The protein spots were visualized by Coomassie Brilliant Blue (CBB) staining, analyzed by MALDI mass spectrometry and searched on NCIBlnr. As the result, 61 spots of total 85 spots were repeated on follicular stage and 51 spots of total 114 spots were repeated on ovulatory stage. 40 spots of total 129 were repeated on early-luteal and 49 spots of total 104 spots were repeated on mid-luteal stage. Also 41 spots of total 60 spots were repeated on last-luteal stage. There were differences in the ovulation (follicular∼ovultory stage) in which the spots of follicular stage 19 was only and in ovulation stage was 10 spots. The difference between the luteinization (ovultory∼mid-luteal stage) was the spots counted in each stage. The spots of ovulatory stage was 1, early-luteal stage was 1 and in mid-luteal stage was 2. Eleven spots were found in mid-luteal stage and 2 spots were found in last-luteal stage. In conclusion, we confirmed that there were 7 spots in ovulation, 4 spots in luteinization and 2 spots in luteolysis. Spot No. 89-93 from ovulation were transferrin, and spot No.94 and 95 were HSP60. Spot No. 103 were Dusty PK, spot No. 135 were OGDC-E2, and spot No. 175, 176 were Rab GDI beta from luteinization. Spot No. 178 and 179 from luteolysis were vimentin.

Relaxin과 insulin이 돼지 난포 과립막세포의 스테로이드 호르몬 분비에 미치는 영향을 연구하기위하여 체외에서 황체화된 과립막세포에서 prosesterone과 의 생산을 조사하였다. 돼지난포 과립막세포를 혈청 존재하에 배양접시에 부착 후 48시간 동안 체외배양하고 무혈청 배지에서 24시간 배양하였다. Relaxin과 insulin의 용량의존성을 확인하기 위하여 다양한 농도 (10, 100, 1,000 ng/ml)를 각각 무혈청 배지에 첨가하였다.

The greater horseshoe bat (Rhinolophus ferrumequinum) is distributed throughout Europe, Africa, Australia, and South Asia. It habits mainly in the cave in small groups and forming communities in late spring. It has interesting reproductive behavior because it keeps sperm for a few months in female reproductive tracts and then those sperms attend in fertilization. This breeding pattern is a sperm storage type and belongs to Rhinolophidae or Hipposideridae. The greater horseshoe also habits in Korea. However, the reasons of reproductive behaviors has not much uncovered. In this study the characters of ovary and the levels of steroid hormones were investigated from September to November. The histological, ELISA, and immunohistochemical methods were employed. The pre-ovulatory follicle was detected only at October sample. On the other hand, the blood level of testosterone was not detectable but the levels of 17β-estradiol and progesterone were exist within the detectable range. E2 and P4 levels were peak in October. Besides, the key enzymes for estradiol synthesis, CYP17 and CYP19 were localized in the theca layer and granulosa cells, respectively. October is known as mating time in this species. However, progesterone receptors could not detect at this period. Put together, it is suggested that, the increase of estrogen and the absence of progesterone receptors on preovulatory follicle is the cause of the mating without ovulation. The understanding of the expression regulation in this system will be base of the understanding the anovulation in mammals.

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.

Early growth response 1 (Egr1) is an inducible zinc finger transcription factor. Egr1 binds specific GC-rich region. Egr1 mRNA is rapidly and transiently expressed in pre-ovulatory follicles by LH and expressed in decidual cell by estrogen. Progesterone receptor (PR) is a nuclear transcription factor that is induced in granulosa cells of pre-ovulatory follicles following the LH surge. PR regulates ADAMTS1, which downstream gene of PR. In previous study, we observed ADAMTS1 mRNA expression pattern changed in Egr1 KO mice. Therefore, we expected that progesterone receptor gene expression is directly regulated by early growth response 1 in mouse ovarian granulosa cell. We could found the ER binding domain, Egr1 binding domain and CAAT box in PR promoter using the web tool AliBaba 2.1. We construct the PR promoter vectors truncated ER binding domain, Egr1 binding domain, CAAT box, respectively. We also construct the Egr1 expression vector using pcDNA 3.1 (+) vector. Granulosa cells are isolated from female ICR mice after 12h PMSG injection. To confirm the Egr1 overexpression, we performed western blot. For reporter assay, we used Dual-Luciferase reporter assay system. In conclusion, Egr1 may regulate PR expression in granulosa cell.

Some mutations in FOXL2 are responsible for premature ovarian failure accompanied with blepharophimosis-ptosis-epicanthusinversus syndrome (BPES) typeI disease, and FOXL2-null mice exhibit developmental defects of granulosa cells. Recently, a new somatic mutation in FOXL2,c.402C>G, leading top. C134W change, has been identified in a vast majority of adult-type ovarian garnulosa cell tumors (GCTs). In the current study, we investigated possible mechanisms by which the C134W mutation could contribute to GCT development. The wild-type (WT) FOXL2 and its mutant form displayed differential apoptotic activities, in which WT induced a significant granulosa cell death while the mutant exhibited a minimal cell death effect. The FOXL2-induced apoptotic response was greatly dependent on caspase8, BID, or BAK since the depletion of either of them prevented FOXL2 to elicitits full apoptotic responses. Stimulated activation of caspase8, consequently resulting increased production of truncated BID (tBID), up-regulation and oligomerization of BAK, and release of cytochromec were all associated with the apoptosis followed by WT FOXL2 expression. In contrast, the mutant FOXL2 was deficient to elicit the full apoptotic signaling responses. In addition, we found the differential up-regulations of expression of death receptors including Fas and TNF-R1 between the WT and the mutant. Moreover, granulosa cells expressing either the WT FOXL2 or its variant form (C134W) exhibited distinct cell death sensitivities by the activation of death receptors. Thus, these differential activities of FOXL2 and it mutant may partly account for the pathophysiology of GCT development occurred by the somatic mutation (C134W) of FOXL2.

포유류 난포의 폐쇄 과정은 매우 정교한 내분비적 조절작용에 의해 일어나며, 이 과정중에 발생하는 난포 내 과립세포의 퇴화는 핵응축 현상을 동반하는 것으로 알려져 있다. 본 연구는 핵응축 현상과 관련하여 돼지 난소 내 폐쇄난포의 과립세포가 퇴화 시 동반되는 세포 사멸이 아포토시스의 과정에 의해서 일어나는지의 여부와 아포토시스 관련 주요 단백질 분해 효소인 캐스파제-3과 연관된 세포 사멸 기전과 관련이 있는지에 대해서 조사하고자 하였다. 돼지 난소로부터 정

본 연구에서는 자연세포사 (apoptosis)를 유발시키는 것으로 알려진 ceramide를 배양중인 생쥐 과립세포에 처리한 뒤 형광염색, in-situ 3'-end labeling(ISEL), 그리고 flow cytometry 기법을 이용하여, 자연세포사 및 세포주기에 미치는 ceramide의 영향을 조사하였다. Ceramide를 처리하지 않은 대조군에 비하여, ceramide를 처리한 실험군에서 세로의 생존율은 농도에 비레하여 유의하게 감소하였다. 또

To study the regulation of porcine follicular cell apostosis by gonadotropin, steroid, and nitric oxide, we analyzed DNA fragmentation, the hallmark of apoptosis, and nitrite production of porcine granulosa cells. Dissected indiidual follicles from ovary were separated in size (small, 2-3 mm; medium, 5-6 mm; large, 7-8 mm) and isolated granulosa cells were classified morpholocally as atretic or nonatretic. Nitrite concentration was measured by mixing follicular fluids with an equal volume of Griess reagent. Follicular nitric oxide (NO) concentration of healthy follicles was higher than that of atretic follicles. Apoptotic DNA fragmentation was suppressed in non-apoptotic granulosa cells. Follicular apoptosis was induced by androgen but prevented by gonadotropin in vitro. Apoptosis was confined to the granulosa cells. But it was not clear whether apoptosis of granulosa cells were isolated, incubated with or without gonadotropin, androgen and sodium nitroprusside (SNP), respectively at for 24 hrs. Cultured granulosa cells were used to extract genomic DNA and culture media was asssayed for nitrite concentration. Nitrite production of culture media was increased, while apoptotic DNA fragmentation was suppressed in PMSG, hCG, testosterone+SNP and SNP treated groups. Nitrite concentration in culture media was decreased, but apoptotic DNA fragmentation was induced in testosterone treated group. These data suggest that NO production and apoptosis may be involved of granulosa cell apoptosis induced by testosterone.