Santos S S D, Biondi F C, Cordeiro M S, Miranda M S, Dantas J K, Figueiredo J R, Ohashi O M
Departamento de Histologia e Embriologia, Centro de Ciencias Biologicas, Universidade Federal do Pará, Belém, PA 66 075-000, Brazil.
Anim Reprod Sci. 2006 Sep;95(1-2):1-15. doi: 10.1016/j.anireprosci.2005.08.012. Epub 2006 May 2.
The aim of the present study was to determine the most desirable ovarian tissue section thickness to isolate preantral follicles (Experiment I), determine follicular density (follicles/mm(2) of cortex) of ovaries of fetal buffalo of different ages (Experiment II), and cultivate preantral follicles of buffalo fetuses (Experiment III). In Experiment I, ovary sections with different thicknesses (25, 50, 75, and 100 microm) had 415.0+/-285.2, 457.5+/-341.9, 585.0+/-309.3, and 685.0+/-278.8 isolated preantral follicles, respectively. In Experiment II, the follicular density of 46 buffalo fetuses with ages between 3 and 8 months was estimated to be between 0 and 7220, with means of 0.0, 2070.7+/-2190.3, 2570.8+/-1796.6, 2298.1+/-2286.5, 1277.5+/-1074.9, and 643.6+/-543.9 throughout the age range studied. The follicular density of 5-month-old fetuses was greatest, coinciding with the largest number of follicles isolated at this age. In Experiment III, preantral follicles isolated from the ovaries of buffalo fetuses aged from 5 to 9 months old were cultivated individually for 7 days in four different media: basic medium (Minimal Essential Medium (MEM), 10% SFB, kanamycin, pyruvate, glutamine, hypoxanthine) with additional ITS and FSH 0.5mg/ml (treatment 1); basic medium with FSH and EGF 100 ng/ml (treatment 2); basic medium with additional ITS, FSH, and EGF (treatment 3); basic medium supplemented with ITS and EGF (treatment 4). Integrity and morphological features, viability, and increase in diameter of follicles cultured in vitro were evaluated individually with an inverted microscope and an ocular micrometer. The results showed that follicle structure and form were maintained during culture. Growth and survival rates of treatments 1, 2, and 3 over 7 day culture were 23.25+/-17.06, 33.75+/-26.19, and 43.75+/-31.73 microm, and 31.3+/-22.7, 22.06+/-8.13, and 28.92+/-21.32%, respectively. However, neither growth nor survival was observed in treatment 4. In conclusion, this study showed that preantral follicles of buffalo fetuses can be cultured in vitro, and that FSH is essential for follicle survival.
本研究的目的是确定分离窦前卵泡时最理想的卵巢组织切片厚度（实验一），测定不同年龄胎水牛卵巢的卵泡密度（每平方毫米皮质中的卵泡数，实验二），以及培养胎水牛的窦前卵泡（实验三）。在实验一中，不同厚度（25、50、75和100微米）的卵巢切片分别分离出415.0±285.2、457.5±341.9、585.0±309.3和685.0±278.8个窦前卵泡。在实验二中，对46头年龄在3至8个月的胎水牛的卵泡密度进行了估算，其范围在0至7220之间，在所研究的整个年龄范围内，平均值分别为0.0、2070.7±2190.3、2570.8±1796.6、2298.1±2286.5、1277.5±1074.9和643.6±543.9。5月龄胎儿的卵泡密度最大，与该年龄分离出的卵泡数量最多相吻合。在实验三中，从5至9月龄胎水牛卵巢中分离出的窦前卵泡分别在四种不同培养基中单独培养7天：基础培养基（最低必需培养基（MEM）、10%胎牛血清、卡那霉素、丙酮酸、谷氨酰胺、次黄嘌呤）添加ITS和0.5毫克/毫升促卵泡素（处理1）；基础培养基添加促卵泡素和100纳克/毫升表皮生长因子（处理2）；基础培养基添加ITS、促卵泡素和表皮生长因子（处理3）；基础培养基添加ITS和表皮生长因子（处理4）。使用倒置显微镜和目镜测微计分别评估体外培养卵泡的完整性和形态特征、活力以及直径增加情况。结果表明，卵泡在培养过程中结构和形态得以维持。处理1、2和3在7天培养期内的生长率和存活率分别为23.25±17.06、33.75±26.19和43.75±31.73微米，以及31.3±22.7、22.06±8.13和28.92±21.32%。然而，处理4既未观察到生长也未观察到存活。总之，本研究表明胎水牛的窦前卵泡能够在体外培养，且促卵泡素对卵泡存活至关重要。
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