• Proceedings of the Korean Society of Food Hygiene and Safety Conference
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• 2001.10a
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• pp.49-53
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• 2001

• Development and Reproduction
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• v.1 no.1
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• pp.79-89
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• 1997

Recent studies have demonstrated that apoptotic cell death plays an important role in the mechanism underlying follicular atresia and luteolysis. However, the mechanisms responsible for initiating these processes have not been elucidated. In in vitro fertilization (IVF) programs, it is highly possible that continuous and repeated administration of FSH/hMG and GnRH agonists for the usage of ovarian hyperstimulation may induce apoptotic death of granulosa cells leading to atresia in the human ovarian follicles. The present study was performed to investigate whether FSH/hMG and GnRh agonists used for a longer period in controlled ovarian hyperstimulation has any effect on the apoptosis of granulosa-luteal (GL) cells obtained from hyperstimulated ovaries. To examine apoptotic cell death in the GL cells, cells were stained with acridie orange followed by observed in some of GL cells. Similar but distinct staining of apoptotic GL cells was observed when the cells were examined by using in situ TUNEL method. The healthy-looking cells with normal nuclear morphology were not stained, whereas cells with pyknotic nuclei or with apoptotic nuclei were intensively stained. After examining the ultrastructural features of GL cells by TEM, it was confirmed that the majority of cells seemed to have normal nuclei while GL cells undergoing apoptotic cel death were rarely found. The DNA extracted from GL cells showed a typical pattern of fragmentation following DNA electrophoretic analysis. We have confirmed that the apoptosis occurs in granulosa-luteal cells obtained from hyperstimulated ovaries. Technically, in situ apoptosis detection method is simple and reproducible and is well suited to identify the quality of oocytes retrieved from hyperstimulated ovaries.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.93-102
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• 1998

Normal and abnormal follicular growth and steroidogenesis depend on gonadotropins as well as intraovarian peptides, which may mediate or potentiate gonadotropin action. Inhibin also affect follicular development and steroidogenesis and may play a role in dominant follicle selection and follicular atresia. Therefore, we studied the differences of serum inhibin, gonadotropin and androgen levels in the women with only the ultrasound findings and no disorder, and polycystic ovary (PCO) with ovulatory disturbance. We prospectively analysed forty-three women with PCO. The diagnosis of PCO was based on typical appearance of the ovaries on TVS. Twelve women with regular menstrual cycle and normal ovarian morphology were selected as control. Basal levels of inhibin, luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol $(E_2)$, testosterone (T), androstenedione (ADD), dehydroepiandrosterone-sulfate (DS), prolactin and TSH in serum were determined. There were significant differences in basal LH levels and LH/FSH ratio between the control and the women with PCO. The basal levels of inhibin and $E_2$ in the oligo-amenorrheic PCO (N=34) were significantly higher than those in the control. There was higher negative correlation between the inhibin and T levels in the oligo-amenorrheic PCO, but, not in the regular cycling PCO. Also, there was higher positive correlation between the LH and T levels in the oligo-amenorrheic PCO, but not in the regular cycling PCO. These data presume that the initial event of PCO is elevated pituitary LH secretion. Elevated levels of LH may down-regulate LH receptors on granulosa cells and also cause hypertrophy of the thecal layer. High level of androgen secreted by the hypertrophied thecal layer may stimulate inhibin secretion from granulosa cells and can be converted to estrogen by extraovarian tissues and could serve to augment pituitary sensitivity to GnRH with a resultant secretion of more LH than FSH. Inhibin may inhibit FSH action on granulosa cell in the PCO follicle, impairing follicular development and dominant follicle selection resulted in ovulatory disturbance.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.195-200
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• 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.

• International Journal of Molecular Medicine
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• v.46 no.5
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• pp.1923-1937
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• 2020

It has been suggested that oxidative stress involving reactive oxygen species (ROS) induces granulosa cell apoptosis, leading to follicular atresia, and that T-lymphokine-activated killer cell-originated protein kinase (TOPK) suppresses cancer cell apoptosis induced by several stimuli. However, it remains to be determined whether TOPK affects oxidative stress-induced granulosa cell apoptosis. The present study demonstrates that TOPK inhibition increases human granulosa COV434 cell apoptosis induced by hydrogen peroxide (H₂O₂). Co-treatment with the TOPK inhibitor, OTS514, in combination with H₂O₂ increased p53 acetylation and its expression, whereas it decreased Sirtuin 1 (SIRT1) expression, contributing to the promotion of apoptosis. In addition, the SIRT1 activator, resveratrol, or the SIRT1 inhibitor, Ex527, reduced or elevated H₂O₂-induced COV434 cell apoptosis, respectively. Furthermore, the p53 inhibitor, Pifithrin-μ, diminished the augmentation in poly(ADP-ribose) polymerase (PARP) cleavage induced by OTS514 plus H₂O₂, while the Mdm2 antagonist, Nutlin 3, increased PARP cleavage. Moreover, OTS514 further decreased the SIRT1 transcriptional activity decreased by H₂O₂, but promoted the H₂O₂-induced p53 or p21 transcriptional activity. Notably, the expression of exogenous p53 reduced SIRT1 transcriptional activity. Taken together, the findings of the present study demonstrate that TOPK inhibition promotes p53-mediated granulosa cell apoptosis through SIRT1 downregulation in response to H₂O₂. Therefore, it can be concluded that TOPK suppresses H₂O₂-induced apoptosis through the modulation of the p53/SIRT1 axis, suggesting a potential role of TOPK in the regulation of human granulosa cell apoptosis, leading to the promotion of abnormal follicular development.

• Clinical and Experimental Reproductive Medicine
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• v.18 no.2
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• pp.123-131
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• 1991

In order to study the growth and maturation of ovarian follicle, the localization and activity of alkaline phosphatase(ALPase) and adenosine triphosphatase(ATPase) of the granulosa cells and theca layer were examined according to the follicle size, the follicle state and the ovarian cyclic phase in pig. Theca interna of the small follicles was more heavyly localized with reaction product by the activites of ALPase and ATPase than that of the large follicles. It is assumed that, as the follicles proceed to growth and maturation, antrum formation is the result of the follicular fluid accumulation by means of active transport by the activities of ALPase and ATPase in theca interna. The activities of ALPase and ATPase in atretic follicles were higher than those of normal follicles. These results imply that the mechanisms of follicle maturation and atresia are different according to the phase of ovarian cycle.

• Korean Journal of Veterinary Research
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• v.16 no.1
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• pp.77-96
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• 1976

In order to investigate the effects and acting mechanism on ovaries, thyroid glands and hypophyses of rabbits in short term administration of sulfadimethoxine (SDM) as medical dose, a total of 90 virgin albino rabbits (mean body weight, 1,362g) were selected at random and alloted to two groups. Rabbits in one group served as controls and the others were administered SDM of 50 mg/kg/day for 5 weeks, and then reared without medication for 4 weeks. Pathological changes of the three organs were observed each week for 9 weeks and the results obtained were summarized as follows: 1. Mean body weights of both groups manifested slow increasing tendency but mean hypophysis weights fluctuated throughout the experimental term. Mean ovary weights of experiments were decreased significantly from the 3rd to 6th week but mean thyroid weights of experiments were increased significantly from the 1st to 6th week compared with those of controls. 2. Many ovarian follicles of each developing stage showed follicular atresia accompanying atrophy or necrosis of oocytes and of disintegrated follicular cells. Theca interna cells and sudanophilic interstitial cells showed atrophy and diminished sudanophilic granules and also liquor folliculi were diminished. These changes icreased from the 1st week, remaining so for 5 weeks and returned to normal status in the 8th or 9th week. 3. The thyroid gland showed a typical hyperplastic goiter. Hypertrophic and hyperplastic epithelia follicular manifested cuboidal or columnar form showing tiny or small vacuoles in cytoplasm. The follicles showed atrophy and decreasing colloidal materials. Necrotic and regenerative changes were also present. The interfollicular vessels showed congestion and hemorrhage. These changes increased from the 1st week, remaining so for 5 weeks and returned to normal status in the 9th week. 4. The rates of differential cell counts of hypophyses revealed increase of basophils (gonadotrophs and thyrotrophs) and decrease of chromophobes. Basophils which had diminished granules stainable with HE, PAS and AF revealed hypertrophy, hyperplasia, and increasing of tiny or small vacuoles in cytoplasm. These changes increased from the 1st week, remaining so for 5 weeks and returned to normal status in the 8th or 9th week. As summarized above histologically, administration of SDM led thyrotrophs and gonadotrophs of pituitary glands to hyperactivity but revealed retrogressive and compensatory changes with functional disturbance in ovaries and thyroid glands. These changes were transitional and attributed to direct actions of the drugs on the ovaries and thyroid glands.

• Journal of Radiation Protection and Research
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• v.37 no.4
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• pp.208-212
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• 2012

In previous studies, ovarian follicle in rat has been used a higher radiation dose than that for cancer radiotherapy in clinical practice. The aim of this study was to evaluate the effect of radiation dose used for cancer radiotherapy on ovarian follicle atresia in rat. Mice of 4-week-old female were whole body irradiated with 2 cGy or 2 Gy (Mevatron 67, Siemens, Germany) and sacrificed by cervical dislocation. Ovaries were collected at 24 hours after irradiation to observe the degree of follicular atresia. Ovaries were fixed in neutral formaldehyde solution for 24 hours and embedded with paraffin. Cutted in $5{\mu}m$ thickness with microtome and stained with hematoxylin and eosin (H&E) and TUNEL immunohistochemical stain, and examined histologically under a light microscope. All data were presented as mean ${\pm}SD$, calculating the ratio of normal or atretic follicles to total ovarian follicles. Statistical analysis was performed by the Mann Whitney test using the SPSS ver 19.0. Ratio of atretic to total follicles of 2 Gy group was significantly higher than control or 2 cGy groups (p<0.05). Ratio of normal to total follicles of 2 Gy group was significantly lower than control group in preantral follicle (64.0 vs. 87.7, p=0.027). Ratio of normal to total follicles of 2 cGy group was significantly increased more than control or 2 Gy groups in antral follicle, and there were no significant difference between control and 2 Gy groups (p=0.522). Radiation dose of 2 Gy for cancer radiotherapy have a significant effect on ovarian follicle atresia in rat.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.329-337
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• 2020

Cytochrome P450 1A2 (CYP1A2) is a member of the cytochrome P450 superfamily enzymes in mammals and plays a major role in metabolizing endogenous hormones in the liver. In recent days, CYP1A2 expression has been found in not only the liver but also other tissues including the pancreas and lung. However, little information is available regarding the expression of CYP1A2 in the ovary, in spite of the facts that the ovarian follicle growth and atresia are tightly associated with controls of endocrine hormonal networks. Therefore, the expression of CYP1A2 in the ovaries of prepubertal and pubertal rats was investigated to assess its expression pattern and puberty-related alteration. It was demonstrated that the expression level of CYP1A2 was significantly (p < 0.01) higher in the pubertal ovaries than prepubertal counterparts. At the ovarian follicle level in both groups, whereas CYP1A2 expression was less detectable in the primordial, primary and secondary follicles, the strongly positive expression of CYP1A2 was localized in the granulosa cell layers in the antral and pre-ovulatory follicles. However, the ratio of CYP1A2-positive ovarian follicle was significantly (p < 0.01) higher in the ovary of pubertal group (73.1 ± 3.1%) than prepubertal one (41.0 ± 10.5%). During the Immunofluorescence, expression of CYP1A2 was mainly localized in Fas-positive follicles, indicating the atretic follicles. In conclusion, these results suggested that CYP1A2 expression was mainly localized at the atretic follicular cells and affected by the onset of puberty. Further study is still necessary but we hypothesize that CYP1A2 expresses in the atretic follicles to metabolize residue of the reproductive hormones. These findings may have important implications for the fields of reproductive biology of animals.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.1
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• pp.69-79
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• 1989

Steroid hormone profiles during luteal phase of clomiphene citrate(CC)/human menopausal gonadotropin(hMG)/human chorionic gonadotropin(hCG)-stimulated in vitro fertilization (IVF) cycles and of follicle-stimulating hormone(FSH)/hMG/hCG-stimulated IVF cycles were compared. In seventy three cycles stimulated with CC/hMG/hCG regimen, follicles were aspirated during exploratory laparotomy and yielded 7 pregnancies, and in 83 cycles stimulated with FSH/hMG/hCG regimen, follicles were aspirated by laparoscope and made 13 pregnancies. Serum estradiol($E_2$) and progesterone($P_4$) levels were determined on days 2, 5, 7, and 9 after follicle aspiration. The FSH/hMG/hCG regimen was more effective than the CC/hMG/hCG regimen in folliculogenesis, ie, ovarian stimulation, follicular phase $E_2$ peak levels, oocyte maturation, and the number of retrieved oocytes. There was no significant difference between luteal serum $P_4/E_2$ ratio of the two regimens, suggesting that secretory endometrial build-up ability for implantation may not differ each other. Several significant correlations were observed between follicular phase seum $E_2$ peak levels and luteal phase serum $E_2$ and $P_4$ levels in the FSH/hMG/hCG-stimulated cycles but any correlation was not significant in the CC/hMG/hCG-stimulated cycles, suggesting that somewhat more follicles may eventually fall in atresia even after attaining dominant stage in the CC/hMG/hCG-stimulated cycles than the FSH/hMG/hCG-stimulated cycles.