• 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.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.31-31
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• 2001

This study was to establish in uitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, This study was to establish in vitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, $\geq$morula: 4.8%) and 7 hrs ($\geq$2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.morula: 4.8%) and 7 hrs (2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.255-260
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• 2012

The purpose of this study was to assess follicular viability and competence through in vitro culture of preantral follicles isolated from vitrified mouse whole ovaries. Mouse preantral follicles were enzymatically isolated from vitrified- warmed and fresh ovaries and cultured for 10 days followed by in vitro oocyte maturation. In vitro matured oocytes were fertilized and cultured to the blastocyst stage. Five minutes pre-exposure to vitrification solution of whole ovaries had significantly higher (p<0.05) oocyte survival and maturation rates than between 10 min exposure groups. Oocyte diameter was significantly smaller (p<0.05) in the 5 and 10 min exposure groups ($69.4{\pm}2.8$ and $67.8{\pm}3.1$) when compared to that of control group ($71.7{\pm}2.1$). There was no statistical significant difference in blastocyst development rates between vitrification group (8.6%) and the fresh control group (12.0%). The mean number of cells per blastocyst was significantly lower (p<0.05) in the vitrification group ($41.9{\pm}20.2$) than in the fresh control group ($55.1{\pm}22.5$). The results show that mouse oocytes within preantral follicles isolated from the vitrified whole ovaries can achieve full maturation, normal fertilization and embryo development.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.53-61
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• 1999

The present study was designed to investigate the effects of gonadotrophins on in vitro growth and maturation in mouse preantral follicles. Ovaries were removed from 12-day-old ICR mice. Follicles were dissociated enzymetically in Leibovitz L-15 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I. The follicles were cultured on Transwell-COL membrane inserts in six well cluster dishes for 10 days. The culture medium was $\alpha$MEM medium supplemented with 5% fetal bovine serum and FSH or HMG. After 10 days of growth in vitro, follicles were allowed to mature for 18~20 hr in medium supplemented with 1.5 IU/$m\ell$ hCG. The oocytes were then denuded of their cumulus cells and assessed maturation status. Concentrations of oestradiol and progesterone were measured with a radioimmunoassay. Oocyte diameter was determined with an ocular micrometer. The survival and Metaphase II rates of oocytes were significantly higher in FSH treatment groups than in control group (P＜0.001), but there were no differences among the groups of treated FSH concentration. The survival and Metaphase II rates of oocytes in HMG treatment group (60.9 and 40.6%) were higher than in FSH treatment group (76.6 and 48.2%) and control group (49.2 and 7.1%). The survival and Metaphase II rates of oocytes on both FSH and LH treatment groups were no differences among the ratios of FSH and LH. Diameter of oocyte was no differences among the treatment groups, but smaller than compared to in vivo grown oocyte. Through the entire culture period, secretions of oestradiol and progesterone were significantly less in control group than in HMG and FSH treatment groups. These results suggest that gonadotrophins playa key role in in vitro culture of mouse preantral follicles. Especially, addition of FSH and LH should be more effective than FSH alone.

• Development and Reproduction
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• v.17 no.3
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• pp.269-274
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• 2013

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12-14 day old mice and these were cultured individually in ${\alpha}$-minimal essential medium (${\alpha}$-MEM) supplemented with 5% fetal bovine serum (FBS), $100mIU/m{\ell}$ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, $100{\mu}g/ml$ penicillin and $50{\mu}g/m{\ell}$ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.35-41
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• 2009

This study was undertaken to evaluate how ${\beta}$-mercaptoethanol (bME), an exogenous antioxidant, interacts with preantral follicles cultured in vitro. Mouse primary or secondary follicles were cultured in glutathione (GSH)-free or GSH-containing medium supplemented with bME of various concentrations, and the growth of preantral follicles, the maturation of intrafollicular oocytes and preimplantation development after parthenogenesis were monitored. In experiment 1, 0, 25, 50 or 100 ${\mu}M$ bME was added to culture medium supplemented with 100 ${\mu}M$ GSH or not. When secondary follicles were cultured in GSH-free medium, no significant change in follicle growth was detected after bME addition. However, exposure to bME in the presence of GSH significantly inhibited both follicle growth and oocyte maturation. Such detrimental effect became prominent in primary follicles and bME strongly inhibited follicle growth in the absence of GSH. In conclusion, there are stage-dependent effects of bME on follicle growth and oocyte maturation, and selective use of antioxidants contributes to establishing an efficient follicle culture system.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.235-243
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• 2000

Objective: Our present studies were conducted to examine more effective isolating method of preantral follicles from mouse ovaries. Methods: ICR mice (3-6 weeks old) were sacrificed through cervical dislocation and their ovaries were removed and put into watch glasses containing Hams F-10 supplemented with 10% fetal bovine serum (FBS). Preantral follicles were isolated by three different methods; 1) enzymatical method and 2) mincing method, and 3) scraping method. Enzymatical method was carried out as following. Ovaries were bisected with a pair of fine 30G needles. Bisected ovaries were incubated at $37^{\circ}C$ and 5% $CO_2$ incubator in 2-well dish containing Hams F-10 supplemented with collagenase 600 lU/ml and DNAse 20 lU/ml. After 20 min., follicles were isolated by repeated pipetting. Isolated preantral follicles were collected, and the remnant of tissues was placed in incubator and previous procedure was repeated. Mincing method was carried out with a pair of fine 30G needles attached to 1 ml syringes and minced ovary. Scraping method was carried out with a pair of fine 30G needles and scratched to surface of ovary. The differences between isolating methods were analyzed using Student's t-test and Chi-square. Results were considered statistically significant when ${\rho}$ value was less than 0.05. Results: In handling time, mincing or scraping method ($28{\pm}3.42$ min or $16{\pm}1.58$ min) were significantly (p<0.00001) shorter than enzymatical method ($72{\pm}1.69$ min), and scraping method was significantly (p<0.01) shorter than mincing method. Total number of isolated follicles was significantly (p<0.0001) higher in enzymatical method ($49.8{\pm}3.91$) than in mincing or scraping method ($25.3{\pm}2.33$ or $20.5{\pm}1.75$). Isolated follicles in ${\leq}$90${\mu}m$ were significantly (p<0.005) higher in enzymatical method ($15{\pm}1.71$) than in mincing or scraping method ($7.8{\pm}0.98$ or $8.1{\pm}1.31$). In 91~130 ${\mu}m$, isolated follicles were significantly (p<0.0005) higher in enzymatical method ($33{\pm}3.27$) than in mincing or scraping method ($16.3{\pm}1.82$ or $10.7{\pm}1.38$). In ${\geq}$ 131 ${\mu}m$, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated follicles in ${\leq}$ 90 ${\mu}m$ was highest in scraping method (39.6% vs. enzymatical method: 30.1%, p<0.05; mincing method: 30.9%, p=0.11719, NS). Rate of follicles in $91{\sim}130$ ${\mu}m$ was significantly (p<0.05) lower in scraping method (52.7%) than in enzymatical or mincing method (66.3% or 64.5%). Rate of follicles in ${\geq}$131 ${\mu}m$ was highest in scraping method (8.3% vs. enzymatical or scraping method: 3.6%, p<0.05 or 4.6%, p=0.19053, NS). Conclusions: This study suggests that scraping method is simple and useful for isolation of preantral follicles, because this method reduced handling time and recovered enough follicles. The recovered rate of isolated follicles in diameter of 91 ~ 130 ${\mu}m$ was highest in all methods.

• Nuclear Engineering and Technology
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• v.33 no.3
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• pp.255-260
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• 2001

In mammals, most of the follicles can not be ovulated, and instead, are degenerated throughout the entire reproductive period. However, the precise mechanism of follicle atresia is unknown. Three weeks old female mice (ICR strain) were ${\gamma}$-irradiated with a dose of LD$^{50}$ . Before irradiation (day 0) and at day 1, 2, and 3 after irradiation, the normal and atretic preantral and antral follicles of the left ovaries were morphologically observed. Atretic follicles at 2 days after irradiation had numerous cell debris, apoptotic cells and bodies, and polymorphonuclear leukocytes in the antral cavity. In severely atretic follicles, numerous polymorphonuclear leukocytes infiltrated into the follicle. The frequencies of atretic antral (58.0 $\pm$8.6) and preantral follicles (27.3$\pm$11.2) induced by ${\gamma}$-radiation increased to 94.0$\pm$3.4 and 86.9$\pm$7.6, respectively at 2 days after irradiation (p<0.05). The number of follicles with one or more neutrophils in the largest cross sections at 2 and 3 days after irradiation significantly increased (p<0.05). It can be concluded that ${\gamma}$-radiation triggers the recruitment of neutrophils into the follicles during degeneration. The ovarian follicles can make a role of informative biological end-point useful for disaster-prevention.

• 대한생식의학회:학술대회논문집
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• 2001.06a
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• pp.54-55
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• 2001

• 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.