• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.79-86
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• 2001

Objective: Mouse pre-antral follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and/or phygiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Methods: Pre-antral follicles ($91{\sim}120{\mu}m$) were isolated mechanically by fine 30G needles not using enzymes from ovaries of 3-6 week-old female ICR mice. Isolated pre-antral follicles were cultured in $20{\mu}l$ droplets of TCM (n=17; follicles: $107.8{\pm}1.58{\mu}m$; oocytes: $57.9{\pm}1.2{\mu}m$) or MEM (n=12; follicles: $109.3{\pm}2.53{\mu}m$; oocytes: $55.4{\pm}1.6{\mu}m$) under mineral oil on the 60 mm culture dish. All experimental media was supplemented with 10% FBS without Gns and/or physiological factors. Pre antral follicles were individually cultured for 8 days. Antram formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using precalibrated ocular micrometer at X200 magnifications during in vitro culture. Results were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when p<0.05. Results: Antrum formation had started in two culture media on day 2. On day 8, antrum formation had occurred in 58.3% of pre-antral follicles cultured in DMEM, but only in 23.5% of those cultured in TCM (p=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day 4 and 8. On day 4, follicular diameter was similar (p=0.1338) in TCM ($119.4{\pm}2.58{\mu}m$) and MEM ($125.4{\pm}4.52{\mu}m$). However, on day 8, diameters of pre-antral follicles cultured in MEM ($168.9{\pm}17.29{\mu}m$) were significantly bigger (p=0.0248) than that in TCM ($126.7{\pm}4.28{\mu}m$). On day 4 and 8, diameters of intra-follicular oocytes were similar in TCM ($67.1{\pm}1.3$ and $72.4{\pm}0.9{\mu}m$) and MEM ($65.2{\pm}1.7$ and $73.3{\pm}1.5{\mu}m$), respectively. Conclusion: We can conform that medium without Gns and/or physiological factors can be used for in vitro antrum formation and growth of pre-antral follicles and intra-follicular oocytes in mouse. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.3
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• pp.107-113
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• 2012

Objective: To determine whether animal age impacts in vitro preantral follicle growth. Effects of hCG, stem cell factor (SCF), and/or insulin-like growth factor (IGF) supplementation in growth medium were also investigated. Methods: Intact preantral follicles were mechanically isolated from fresh ovaries of BDF1 mice and cultured in growth medium for 9 to 11 days. Surviving follicles with antrum formation were transferred to maturation medium for 14 to 18 hours. Follicle survival, antrum formation, and retrieval of metaphase II (MII) oocytes were compared among three age categories (4-5, 7-8, and 10-11 week-old). By using 7- to 8-week-old mice, preantral follicles were cultured in growth medium supplemented with hCG (0, 5, or 10 mIU/mL), SCF (50 ng/mL), IGF-1 (50 ng/mL), and SCF+IGF-1. Results: Seven- to eight-week-old mice showed a higher follicle survival and antrum formation and produced more MII oocytes compared to other groups. In the 7- to 8-week-old mice, supplementation of 5 mIU/mL hCG significantly enhanced the antrum formation but the percentage of MII oocytes was similar to that of the control. Supplementation of SCF+IGF-1 did not enhance follicle survival or antrum formation but the percentage of MII oocytes increased modestly (39.1%) than in the control (28.6%, p>0.05, statistically not significant). Conclusion: Seven- to eight-week-old mice showed better outcomes in growth of preantral follicles in vitro than 4- to 5- or 10- to 11-week-old mice. Supplementation of hCG enhanced antrum formation and supplementation of SCF+IGF-1 yielded more mature oocytes; hence, these should be considered in the growth of preantral follicles in vitro.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.8-8
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• 2001

Mouse follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation of follicles in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and physiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Pre-antral follicles (91-120${\mu}{\textrm}{m}$) were isolated mechanically by fine 30G needles not using enzymes from ovary of 3-6 weeks old female ICR mice. Isolated pre-antral follicles were cultured in 20 ${mu}ell$ droplets of TCM (n=17; follicles: 107.8 $\pm$ 1.58 ${\mu}{\textrm}{m}$; oocytes: 59.9$\pm$1.2 ${\mu}{\textrm}{m}$) or MEM (n=12; follicles: 109.3$\pm$2.53 ${\mu}{\textrm}{m}$; oocytes: 55.4 $\pm$1.6${\mu}{\textrm}{m}$) under mineral oil on the 60mm culture dish. All experimental media was supplemented with 10% FBS but without Gns and/or physiological factors. Pre-antral follicles were individually cultured in drops for 8 days. Antrum formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using a precalibrated ocular micrometer at $\times$200 magnifications during in vitro culture. Results between different groups were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when P<0.05. Antrum formation of pre-antral follicles had started in two culture media on day-2. On day-8, antrum formation had occurred in 58.3%(7/12) of pre-antral follicles cultured in MEM, but only in 23.5% (4/17) of those cultured in TCM (P=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day-4 and -8. On day-4, follicular diameters was similar (P=0.1338) in TCM (119.4$\pm$2.58 ${\mu}{\textrm}{m}$) and MEM (125.4$\pm$4.52 ${\mu}{\textrm}{m}$). However, on day-8, diameters of pre-antral follicles cultured in MEM (168.9$\pm$17.29 ${\mu}{\textrm}{m}$) was significantly (P=0.0248) bigger than that in TCM (126.7$\pm$4.28 ${\mu}{\textrm}{m}$). On day-4 and -8, diameters of intra-follicular oocytes were similar TCM (67.1$\pm$1.3 and 72.4$\pm$0.9${\mu}{\textrm}{m}$) and MEM (65.2$\pm$1.7 and 73.3$\pm$1.5 ${\mu}{\textrm}{m}$), respectively. We can conform that medium not supplemented with Gns and/or physiological factors can be used for in vitro antrum formation and growth of mouse pre-antral follicles and intra-follicular oocytes. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.207-211
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• 2010

Current developments in IVF and animal cloning have resulted in increasing demand for large quantities of oocytes and ovarian follicles at specific stages of development. These medical and scientific needs may be met by developing an optimal culture system for preantral follicles. In this study, we investigated the growth of porcine preantral follicle cultures in different media and in the presence and absence of serum. Follicles were manually dissected from ovaries obtained from prepubertal gilts at a local slaughterhouse, and cultured for 3 days in M199 or NCSU23 medium supplemented with porcine FSH, transferrin, L-ascorbic acid and insulin. Follicle diameters were measured on day 1 and 3 of culture. In Experiment 1, the effect of supplementing culture medium with fetal calf serum (FCS) on porcine preantral follicle growth was examined. In the group of cultures supplemented with FCS, follicle diameter after 3 days of culture, survival rate and antrum formation rate in the FCS group were significantly higher than those of the control group. In Experiment 2, the effects of culture medium (M199 and NCSU23) on follicle growth were compared. Follicle diameters were increased in the M199 group, compared with those in NCSU23 (p<0.05), but we observed no significant differences in survival and antrum formation rates between cultures grown in the two media. In conclusion, supplementation of the culture medium with serum enhances preantral follicle growth and antrum formation, and M199 is superior to NUSU23 for porcine preantral follicle culture in vitro.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.3
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• pp.95-106
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• 2012

It has been revealed that multiple cohorts of tertiary follicles develop during some animal estrous cycle and the human menstrual cycle. To reach developmental competence, oocytes need the support of somatic cells. During embryogenesis, the primordial germ cells appear, travel to the gonadal rudiments, and form follicles. The female germ cells develop within the somatic cells of the ovary, granulosa cells, and theca cells. How the oocyte and follicle cells support each other has been seriously studied. The latest technologies in genes and proteins and genetic engineering have allowed us to collect a great deal of information about folliculogenesis. For example, a few web pages (http://www.ncbi.nlm. nih.gov; http://mrg.genetics.washington.edu) provide access to databases of genomes, sequences of transcriptomes, and various tools for analyzing and discovering genes important in ovarian development. Formation of the antrum (tertiary follicle) is the final phase of folliculogenesis and the transition from intraovarian to extraovian regulation. This final step coordinates with the hypothalamic-pituitary-ovarian axis. On the other hand, currently, follicle physiology is under intense investigation, as little is known about how to overcome women's ovarian problems or how to develop competent oocytes from in vitro follicle culture or transplantation. In this review, some of the known roles of hormones and some of the genes involved in tertiary follicle growth and the general characteristics of tertiary follicles are summarized. In addition, in vitro culture of tertiary follicles is also discussed as a study model and an assisted reproductive technology model.

• Clinical and Experimental Reproductive Medicine
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• v.48 no.4
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• pp.347-351
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• 2021

Objective: We investigated the impact of vitamin D₃ (VD3) supplementation during mouse preantral follicle culture in vitro and the mRNA expression of 25-hydroxylase (CYP2R1), 1-alpha-hydroxylase (CYP27B1), and vitamin D receptor (VDR) in mouse ovarian follicles at different stages. Methods: Preantral follicles were retrieved from 39 BDF1 mice (7-8 weeks old) and then cultured in vitro for 12 days under VD3 supplementation (0, 25, and 50 pg/mL). Follicular development and the final oocyte acquisition were assessed. Preantral follicles were retrieved from 15 other BDF1 mice (7-8 weeks old) and cultured without VD3 supplementation. Three stages of mouse ovarian follicles were obtained (preantral, antral, and ruptured follicles). Total RNA was extracted from the pooled cells (from 20 follicles at each stage), and then reverse transcriptase-polymerase chain reaction was performed to identify mRNA for CYP2R1, CYP27B1, and VDR. Results: The survival of preantral follicles, rates of antrum formation and ruptured follicles (per initiated follicle) and the number of total or mature oocytes were all comparable among the three groups. Both CYP2R1 and CYP27B1 were expressed in antral and ruptured follicles, but not in preantral follicles. VDR was expressed in all three follicular stages. Conclusion: VD3 supplementation in vitro (25 or 50 pg/mL) did not enhance mouse follicular development or final oocyte acquisition. Follicular stage-specific expression of CYP2R1, CYP27B1, and VDR was observed.

• Journal of Gastric Cancer
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• v.6 no.1
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• pp.52-56
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• 2006

A 69-year old man presented with severe epigastric pain for 1 day. He had early gastric cancer at the antrum and underwent a distal subtotal gastrectomy and Billorth II gastrojejunostomy one month later without any post-operative complications. Radiologic examination revealed a large amount of retroperitoneal free air formation. Because of unremitting pain and unstable vital sign, exploratory laparotomy was followed. During the operation, a perforated duodenal diverticulum at the posterior wall of the 2nd portion of the duodenum was identified. He underwent diverticulectomy and primary closure. He was discharged on the 18th post operative day and has been followed up without any evidence of comlpication for several months.