| A Simple Isolating Method of Preantral Follicles from Mouse Ovaries. |
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Ju Hwan Kim, Kee Sang Park, Hai Bum Song, Sang Sik Chun |
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| Abstract |
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 37degrees C and 5% CO2 incubator in 2-well dish containing Hams F-10 supplemented with collagenase 600 IU/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 mined ovary. Scraping method was carried out wit 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 P value was less than 0.05. RESULTS:In handling time, mincing or scraping method (28+/-3.42 min or 16+/-1.58 min) were significantly (p<0.00001) shorter than enzymatical method (72+/-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+/-3.91) than in mincing or scraping method (25.3+/-2.33 or 20.5+/-1.75). Isolated follicles in < or =90 micrometer were significantly (p<0.005) higher in enzymatical method (15+/-1.71) than in mincing or scraping method (7.8+/-0.98 or 8.1+/-1.31). In 91~130 micrometer, isolated follicles were significantly (p<0.0005) higher in enzymatical method (33+/-3.27) than in mincing or scraping method (16.3+/-1.82 or 10.7+/-1.38). In > or =131 micrometer, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated follicles in < or =90 micrometer 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 91~130 micrometer was significantly (p<0.05) lower in scraping method (52.7%) than in emzymatical or mincing method (66.3% or 64.5%). Rate of follicles in > or =131 micrometer 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 micrometer was highest in all methods. |
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