Materials and methods Directory UMM :Data Elmu:jurnal:A:Animal Reproduction Science:Vol59.Issue1-2.Apr2000:

2. Materials and methods

2.1. Experimental animals Ž . Prepubertal female mice ICR strain aged 3 weeks were used to avoid the interven- ing results, which could come from the reproductive cycles. Female mice were obtained from Toxicology Research Center, Korea Research Institute of Chemical Technology, Taejon, Republic of Korea. The mice were maintained in a 238C-controlled animal care Ž . room with lightrdark 12r12 h . The animals had free access to tap water and commercial chow during the experiments. 2.2. Irradiation Five mice per group were whole-body irradiated with g-radiation from 60 Co isotopic Ž source dose rate: 6.94 cGyrmin, source strength: approximately 150 TBq, Panoramic . Irradiator, Atomic Energy of Canada, Limited at Korea Atomic Energy Research Ž . Ž . Institute KAERI as previously reported by Kim et al. 1999 . The radiation dose was 8.3 Gy, which was LD for the mice. The irradiation was done for 2 h. As a control, 80Ž30. the sham-irradiation was carried out by placing the mice in the preparation room during the irradiation period. Groups of mice were killed by cervical dislocation before irradiation, and at 0, 3, 6 and 12 h after the end of the 2-h irradiation. 2.3. Histological preparation After irradiation, mice of each group were kept in a 238C-controlled animal care room at KAERI. Each group of mice was killed by cervical dislocation at 0, 3, 6 and 12 h after irradiation. The ovaries were collected and fixed to observe the changes in the architecture of primordial and primary follicles. Ž . Postfixation using 1 of osmium tetraoxide Sigma, MO was conducted for 2 h at Ž . 48C after prefixation with 2.5 glutaraldehyder0.1 M phosphate buffer pH 7.3 . Embedding of specimens after an alcoholic dehydration and displacement by propylene w Ž . oxide was carried out in epon mixture PolyrBed 812 resin Epon 812 :dodecenylsuc- Ž . Ž cinic anhydride:nadic methyl anhydride:2,4,6-tri dimethylaminomethyl phenol DMP- . x Ž . 30 s 19.3:12.3:9.4:0.6 ml, Polysciences . Using ultramicrotome Leica , semithin sec- tions were prepared by 1 mm in thickness and stained with 1 toluidine blue O in 1 borax solution. The largest cross-sections were used in this study. Observation of Ž . morphological changes was done under a light microscope Olympus BX50 . 2.4. Identification of oÕarian status Normal primordial follicles were identified by the presence of normal granulosa cells surrounding the healthy oocyte. Follicles with shrunk oocytes, with one or more C.J. Lee et al. r Animal Reproduction Science 59 2000 109 – 117 112 Table 1 Criteria for the identification of normal and atretic primordial follicles in the present experiment Primordial follicle Primary follicle Normal Atretic Normal Atretic Granulosa cells Flattened or round, Irregular or amorphous, Round, no pyknotic, Pyknosis of one or no pyknotic apoptotic some cells mitotic more cells Germinal vesicle Round and clear Irregular, absent Round and clear Irregular, absent Ooplasmic membrane Clear, regular Unclear, irregular Clear, regular Unclear, irregular Ooplasm Even, clear Uneven, unclear, dark Even, clear Uneven, unclear, dark Basement membrane Clear, thin, regular Unclear, relatively thick, Clear, regular Unclear, irregular irregular pyknotic granulosa cells, andror with apoptotic oocyte or without oocyte were classified into atretic ones based on the criteria listed in Table 1. The criteria for classification of the primary follicles were also described in Table 1. Under a microscope, the number of normal and atretic follicles was counted and their ratio was calculated. Data were expressed as mean SEM and the statistical differences between the experimental groups were considered when p value was smaller than 0.05. Fig. 1. Microphotographs of the primordial and the primary follicles in normal and irradiated mouse ovaries. The identification of normal and atretic primordial and primary follicles based on the criteria depicted in Table 1. Panel A shows morphologically the normal primordial follicles in the sham-irradiated mouse ovary. Panel B shows the atretic primordial follicles. Four degenerating primordial follicles were shown. In the right-upper primordial follicle, the apoptotic nucleus of oocyte was identified. In the left-upper and middle primordial follicles, the remnants of degenerating oocyte nuclei were shown. In the left-lower primordial follicle, the oocyte was no longer observable, and the granulosa cells, instead of the oocyte, were filled in a basement membrane. These primordial follicular changes were observed in the irradiated mouse ovaries at 6 h after irradiation. In panel C, in a basement membrane, some of the granulosa cells were recognized pyknotic. The peculiar changes in the primordial follicular shapes were shown at 12 h after irradiation, and also shown in the sham-irradiated normal mouse ovaries. In panel D, a normal primary follicle with a mitotic granulosa cell was shown. The upper primary follicle in panel E was shown with a pyknotic or apoptotic nucleus, and the lower primary follicle had a degenerated remnant of oocyte nucleus. These follicles were shown 6 h after irradiation. In panel F, the primary follicle with apoptotic granulosa cells and without oocyte was shown at 12 h after irradiation. Bars: 10 mm. Thin arrows, basement membranes; thick arrows, oocytes; arrow heads, granulosa cells; white thick arrow, follicles with pyknotic or apoptotic granulosa cells; white thin arrow, mitotic granulosa cell.

3. Results