• Proceedings of the KSAR Conference
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• 2000.10a
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• pp.9-9
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• 2000

The mammalian ovary has a large number of primordial and preantral follicles, which are a potential source of oocytes for the in vitro mass production of embryos. Several in vitro culture systems have been developed to support the growth and development of oocytes from mouse preantral follicles. Under the appropriate condition, meiotically incompetent oocytes from preantral follicles can grow to final size and complete nuclear maturation in vitro. Furthermore, the successful production of live young from in vitro grown and matured oocytes demonstrates that oocytes from preantral follicles are able to acquire full developmental capacity in vitro. However, the efficiency of in vitro production of embryos from mouse preantral follicles is still low. In farm animals as well as human, the growth of oocyte from preantral follicle to the meiotic competence stage has yet to be demonstrate. Therefore, further studies to improve the culture condition or to develope new culture system should be needed in the future. In addition, the visible progress in the establishment of the in vitro culture system for preantral follicles of farm animals and human could help to enlarge the populations of valuable agricultural, phamaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that jeopardize oocytes.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.117-122
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• 2019

The objective of this study was to establish an in vitro culture system for ovarian preantral follicles of B6D2F1. First, we optimized the in vitro preantral-follicle culture by culture duration, follicle stimulating hormone (FSH) type, and activin A concentration. Duration of in vitro culture for 9, 11, and 13 days was sufficient for the normal development of preantral follicles to antral follicles. Formation of cumulus cell-oocyte complex (COC) was induced by treatment with human chorionic gonadotropin (hCG; 2.5 IU/mL) and epidermal growth factor (EGF; 5 ng/mL). In addition, metaphase II (MII) oocytes formed during this in vitro culture of preantral follicles. In vitro preantralfollicle culture for 9 days showed higher rates of growth and maturation, thus yielding a greater number of antral follicles, and there were significant differences (p < 0.05) in the number of MII oocytes (that formed from these preantral follicles via differentiation) between the 9-day culture and 11-day or 13-day culture. The follicles cultured for 9 days contained a tightly packed well-defined COC, whereas in follicles cultured for 11 days, the COC was not well defined (spreading was observed in the culture dish); the follicles cultured for 13 days disintegrated and released the oocyte. Second, we compared the growth of the preantral follicles in vitro in the presence of various FSH types. There were no significant differences in the growth and maturation rates and in differentiation into MII oocytes during in vitro culture between preantral follicles supplemented with FSH from Merck and those supplemented with FSH from Sigma. To increase the efficiency of MII oocyte formation, the preantral follicles were cultured at different activin A concentrations (0 to 200 ng/mL). The control follicles, which were not treated with activin A, showed the highest rate of differentiation into antral follicles and into MII oocytes among all the groups (0 to 200 ng/mL). Therefore, activin A (50 to 200 ng/mL) had a negative effect on oocyte maturation. Thus, in this study, we propose an in vitro system of preantral-follicle culture that can serve as a therapeutic strategy for fertility preservation of human oocytes for assisted reproductive medicine, for conservation of endangered species, and for creation of superior breeds.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.4
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• pp.152-165
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• 2019

Objective: This study aimed to examine the effect of vitrification on apoptosis and survival in human preantral follicles after thawing. Methods: This experimental study was conducted at an acute tertiary care hospital from March 2012 to April 2013. Ovaries were sliced into 5 × 5 × 1-mm pieces and divided into the following three groups: preantral follicle isolation, ovarian tissue vitrification-warming followed by follicle isolation, and immunohistochemistry of fresh ovarian tissue. For statistical analyses, the Student t-test, chi-square test, Kruskal-Wallis test, and Kaplan-Meier survival analysis were used. Results: A total of 161 preantral follicles (70% secondary) were collected from ovarian cortex tissue of six women between 30 and 37 years of age who underwent oophorectomy due to cervical cancer or breast cancer. There were no significant differences in the follicular morphology of fresh preantral follicles and vitrified follicles after thawing. The mean Fas ligand (FasL) mRNA expression level was 0.43 ± 0.20 (relative to β-actin) in fresh preantral follicles versus 0.51 ± 0.20 in vitrified follicles (p= 0.22). The mean caspase-3 mRNA expression level in fresh preantral follicles was 0.56 ± 0.49 vs. 0.27 ± 0.21 in vitrified follicles (p= 0.233). One vitrified-thawed secondary follicle grew and developed to an antral follicle within 6 days of culture. Conclusion: Vitrification did not affect preantral follicle morphology or mRNA expression of the apoptosis markers FasL and caspase-3. Further studies are required to establish whether vitrification affects the outcomes of in vitro culture and the maturation of preantral follicles.

• Clinical and Experimental Reproductive Medicine
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• v.48 no.3
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• pp.245-254
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• 2021

Objective: In humans, polycystic ovary syndrome (PCOS) is an androgen-dependent ovarian disorder. Aberrant gene expression in folliculogenesis can arrest the transition of preantral to antral follicles, leading to PCOS. We explored the possible role of altered gene expression in preantral follicles of estradiol valerate (EV) induced polycystic ovaries (PCO) in a mouse model. Methods: Twenty female balb/c mice (8 weeks, 20.0±1.5 g) were grouped into control and PCO groups. PCO was induced by intramuscular EV injection. After 8 weeks, the animals were killed by cervical dislocation. Blood serum (for hormonal assessments using the enzyme-linked immunosorbent assay technique) was aspirated, and ovaries (the right ovary for histological examinations and the left for quantitative real-time polymerase) were dissected. Results: Compared to the control group, the PCO group showed significantly lower values for the mean body weight, number of preantral and antral follicles, serum levels of estradiol, luteinizing hormone, testosterone, and follicle-stimulating hormone, and gene expression of TGFB1, GDF9 and BMPR2 (p<0.05). Serum progesterone levels were significantly higher in the PCO animals than in the control group (p<0.05). No significant between-group differences (p>0.05) were found in BMP6 or BMP15 expression. Conclusion: In animals with EV-induced PCO, the preantral follicles did not develop into antral follicles. In this mouse model, the gene expression of TGFB1, GDF9, and BMPR2 was lower in preantral follicles, which is probably related to the pathologic conditions of PCO. Hypoandrogenism was also detected in this EV-induced murine PCO model.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.387-392
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• 2000

Objectives: 1) To compare the efficacy of urofollitropin (Follimon) to that of recombinant human FSH (rhFSH) on the growth and maturation of mouse early preantral follicles in vitro, and 2) effect of vitrification on the growth and maturation of preantral follicles and oocytes . Methods: Isolated early preantra1 follicles (100-130 ${\mu}m$ diameter) were cultured for 12 days in 20 ${\mu}l$ ${\alpha}$-MEM media drop under the mineral oil. Follimon or rhFSH was added to the culture medium at various concentrations (0, 10, 100, and 1000 mIU/ml). Results: With Follimon, the dose of 10 mIU/ml showed the best follicle survival, growth, and MIl rate of oocyte than the other concentrations. Whereas the optimal dose of rhFSH was 100 mIU/ml. Despite the different optimal doses, the efficacy of two different FSHs on the follicle growth and maturation was similar. Isolated mouse preantral follicles were cryopreserved by vitrification and cultured in vitro for 12 days with 100 mIU/ml rhFSH. Despite the decreased follicular survival rate after thawing, the follicular growth and maturation rate of its oocyte were comparable to those of the fresh follicle. Conclusion: Results from the present study revealed that 1) the optimal doses of Follimon and rhFSH for in-vitro culture of mouse follicles are different, and 2) the frozen-thawed follicles develop normally after vitrification.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.532-537
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• 2008

In order to investigate the factors affecting the culture of mouse preantral follicles in vitro, we examined the effect of culture media, protein supplements, and culture period on their growth. The oocyte diameter (initial size: $55.6{\pm}2.5{\mu}m$) was progressively increased during culture, and the maximum size ($72.0{\pm}2.4{\mu}m$) was reached on day 10 of the in vitro culture. The chromatin configuration in the germinal vesicle (GV) oocyte progressively shifted from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN). On day 10 of the culture, most of the oocytes progressed to the SN pattern. The survival and metaphase II rates of the oocytes in alpha-minimal essential medium (alpha-MEM) were significantly higher (p<0.05) than those in Waymouth and tissue culture medium (TCM)-199. As a protein source, fetal bovine serum (FBS) was more suitable for the culture of mouse preantral follicles as compared to human follicular fluid (hFF) and bovine serum albumin (BSA); the optimal concentration of FBS was 5%. These results suggest that in a culture of mouse preantral follicles, alpha-MEM and 5% FBS are an optimal medium and a protein source, respectively; further, the 10 days of culture is required for the complete growth of oocytes in this culture system.

• Clinical and Experimental Reproductive Medicine
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• v.12 no.2
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• pp.71-79
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• 1985

Systemic extrogen therapy promotes multiple preantral follicular development in immature mice. Estrogen pretreated ovaries might therefore be a useful source of cells for in vitro studies of oocytes maturation. Silastic capsules (5.0 mm length; 3.18 mm outer diameter, 1.57 mm inner diameter) filled with diethylstilbesterol were implanted subcutaneously in experimental mice (ICR) for up to 6 days. Ovarian weight and histology in diethylstilbesterol pretreated and control animal were assessed before and after pregnant mare serum gonadotrophin treatment and after human chorionic gonadotrophin. The following results were obtained; 1. Ovarian weight was significantly increased by 6 days of diethylstilbesterol pretreatment. Subsequent ovarian weight gain in response to pregnant mare serum gonadotrophin and human chorionic gonadotrophin was increased. 2. Diethylstilnbesterol pretreatment stimulated the developed healthy preantral follicles. 3. Forty eight hours after pregnant mare serum gonadotrophin treatment, a larger number of the antral follicles which developed in diethylstilbesterol pretreated animals showed signs of atresia, whereas in the control ovaries there was a higher incidence of premature luteinization. 4. Forty eight hours after human chorionic gonadotrophin, numerous corpora lutea and occasional luteinized unruptured follicles were present in both control and diethylstilbesterol ovaries. 5. Ovulation rate, fertilization rate and subsequent preimplantation development in vitro were not adversely affected by diethylstilbesterol pretreatment. However, there was considerable variation in the ovulation rate the number of animals with more than 60 ovulations was greater in the diethylstilbesterol gorup (52.4%) as compared to the control (33.3%).

• 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.

• Korean Journal of Veterinary Research
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• v.37 no.1
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• pp.71-78
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• 1997

This study was designed to investigate the number of the growing and mature follicles following gonadotrophin treatments for superovulation in mature rats. Eighteen mature rats (Sprague-Duwely, initially 190~230gm) were randomly alloted into 3 groups. One group was control group, another FSH-treated group was injected intramuscularly with 0.5 units of follicular stimulating hormone (FSH) / rat, and third PMS and HCG-treated group was intramuscularly injected with 20~25IU of pregnant mare serum (PMS) / rat and then at the 48 hrs later, with 20~25IU of human chorionic gonadotrophin (HCG) / rat. The uteri and ovaries of rats were collected and then were observed grossly and serial sections of paraffin embedding ovaries were stained with H-E. Number of ovarian follicles by following 3 grades of large, middle and small follicles from secondary and tertiary follicles were investigated by LM photography of preparations. Small follicles were classified as secondary follicles of preantral follicles with more than 2 layers of granulosa cells surrounding the oocyte and middle follicles were classified as secondary follicles with early signs of antral cavity or with more than one small cavity on either side of the oocytes and large follicles were classified as tertiary follicles with a single medium sized antral cavity or large well-formed antral cavity. In gross findings, the uteri were slightly swelling in FSH-treated group and markedly swelling or filled with fluid in the uterine lumen in PMS and HCG-treated group. In histological findings, the shape and size of the follicles were diverse in middle and large follicles of FSH-treated group and PMS and HCG-treated group, and proportion of atretic follicles was increased in FSH-treated group and PMS and HCG-treated group than those in control group. The uteri of FSH-treated group and PMS and HCG-treated group were hypertropied or filled with fluid in the lumens and walls of uteri. The wall tissue layers were flattened and their blood and lymph vessels were dilated. The mean number of follicle per ovary in control group were appeared to be $17.1{\pm}5.6$($14.0%{\pm}4.6%$), $37.8{\pm}9.1$($30.9{\pm}7.4%$) and $67.6{\pm}30.1$($55.2{\pm}24.6%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $122.5{\pm}40.0$. The mean number of follicle per ovary in FSH-treated group were appeared to be $22.8{\pm}7.0$($17.4%{\pm}5.3%$), $43.4{\pm}6.6$($33.2{\pm}5.1%$) and $64.5{\pm}13.0$($49.3{\pm}9.9%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $130.7{\pm}16.6$. The mean number of follicle per ovary in PMS and HCG-treated group were appeared to be $29.7{\pm}11.0$($16.3%{\pm}6.0%$), $61.9{\pm}17.2$($33.9{\pm}9.4%$) and $91.1{\pm}28.2$($49.9{\pm}15.4%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $182.6{\pm}32.7$. The above findings reveal that large follicles were increased 29.8% in FSH-treated group and 73.7% in PMS and HCG-treated group than those in control group and in histologic findings, proportion of atretic follicles were more increased in ovaries with more number of more developing follicles.

• Development and Reproduction
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• v.7 no.1
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• pp.49-56
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• 2003

There have been reports that administrated high-dose gonadotropin-releasing hormone-agonist(GnRH-Ag) suppresses endogenous gonadotropin production and inhibits function of ovary. In human IVF-ET program, however, GnRH-Ag is employed in large amounts during superovulation induction resulting to luteal phase defects which must be supported with progesterone. To elucidate the reason of luteal phase defects by GnRH-Ag, the aim of this study was to investigate the apoptosis changes in the ovary and the hormonal changes in the serum after GnRH-Ag and PMSG administration in adult mice in a method similar to human superovualtion induction. GnRH-Ag(10 ${\mu}$g) or saline was injected every 12h beginning 48h prior to PMSG injection until 48h at)or PMSG injection when blood sampling and ovary collection was performed. In results, the ovary weight in the GnRH-Ag only injection group was significantly lower when compared with the other two groups, PMSG only or PMSC + GnRH-Ag injection. The ratio of preantral follicles in the ovary are increased in the GnRH-Ag only group, while the ratio of antral follicles are decreased and the corpus luteum ratio is increased in the PMSG + GnRH-Ag group. The proportion of all follicles showing apoptosis in the GnRH-Ag only in.iection group was seen to be more than twice the proportion seen in the PMSC only injection group, and such increased apoptosis is decreased after addition of PMSC. The serum levels of both estradiol and progesterone were significantly lower in the CnRH-hg only group compared to those in the other two groups. When the administration of GnRH-Ag were followed by PMSG in;ection, however, estradiol concentration was completely recovered compared to the serum level of PMSG group, but not progesterone level. In conclusion the use of GnRH-Ag in human IVF-ET program may induce the apoptosis and the suppression of hormone production by ovary leading to luteal phase defects, thus adequate progesterone support seems to be necessary against them.