抗坏血酸、表皮生长因子和促卵泡素对绵羊卵巢皮质体外培养的影响

Effect of ascorbic acid, epidermal growth factor and follicle stimulating hormone on in vitro culture of sheep ovarian cortical tissue

摘要:

本研究旨在评估抗坏血酸 (VC)、表皮生长因子 (EGF)、促卵泡素 (FSH) 对绵羊原始卵泡体外培养的影响以及它们之间的相互关系。实验按照2×2×2因子试验设计分为8组，分别为：MEM(对照组)，MEM+VC (50 μg/mL)，MEM+EGF (100 ng/mL)，MEM+FSH (50 ng/mL)，MEM+VC+EGF，MEM+VC+FSH，MEM+EGF+FSH，MEM+VC+ EGF+FSH。在培养0 (未培养对照组)、2、6、12 d后，对培养的卵巢皮质薄片进行组织学和增殖细胞核抗原 (PCNA) 检测以及透射电镜 (TEM) 观察。结果表明，与未培养组 (发育卵泡比例15.4％±1.9％，正常卵泡比例88.2％±4.6％) 比较，所有培养组中发育卵泡比例显著增加(P＜0.05)，正常卵泡比例下降(P＜0.05)。培养12 d后，与对照组 (卵泡直径 (34.5±3.3) μm，卵泡存活比例 (38.9％±3.9％)) 比较，MEM+VC+FSH和MEM+EGF+FSH组中卵泡直径 (分别为(39.7±3.4) μm和 (42.5±5.1) μm) 和卵泡存活比例 (分别为58.5％±4.3％和59.3％±3.7％) 都显著提高 (P＜0.05)；各处理组中，培养12 d后，MEM+VC+EGF组中发育卵泡比例 (49.3％±3.2％) 和卵泡直径 ((32.3±2.3) μm) 最低，颗粒细胞PCNA阳性卵泡比例 (26.4％±1.2％) 也最少，而MEM+VC+EGF+FSH组中卵泡存活率 (59.7％±6.1％) 和卵泡直径 ((42.5±5.1) μm) 都显著增加 (P＜0.05)，颗粒细胞PCNA (43.5％±4.1％，P＜0.05) 表达增加。电镜结果表明，VC+EGF+FSH组能够维持与正常卵泡类似的超微结构，而在MEM 和MEM+VC+EGF组却显示不同程度的退化特征。本研究结果提示在培养中联合添加VC与EGF抑制卵泡的发育和生长，而联合添加VC、EGF和FSH可能是促进绵羊原始卵泡体外激活和生长，维持卵泡存活以及结构完整的最有效的处理手段之一。

Abstract:

In this study, we evaluated the effects of ascorbic acid (VC), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) on in vitro culture of sheep ovarian cortical tissue. Using 2×2×2 factor experimental design, we cultured sheep ovarian cortex fragments in 8 media with MEM (control), MEM+VC (50 μg/mL), MEM +EGF (100 ng/mL), MEM+FSH (50 ng/mL), MEM+VC+EGF, MEM+VC+FSH, MEM+EGF+FSH, MEM+VC+EGF+FSH. After 0 (non-cultured control), 2, 6, 12 days of culture, the pieces of ovarian cortex were proceed to histological and proliferating cell nuclear antigen (PCNA) examination, or observed by transmission electron microscopy (TEM). The percentages of developing follicles were increased (P<0.05) and the percentages of healthy follicles were reduced (P<0.05). When compared to the MEM group, the addition of FSH with VC or EGF promoted a significant increase of follicles diameter and follicles survival rate (P<0.05), and stimulated the proliferation of granulosa cells. After 12 days of culture, medium supplemented with MEM+VC+EGF resulted the lowest proportion of developing follicles (49.3%±3.2%), follicles diameter((32.3±2.3) μm), follicles survival rate (41.6%±3.1%) and the proportion of PCNA stained follicles (26.4%±1.2%, P<0.05). In contrast, MEM+VC+EGF+FSH resulted the highest follicles diameter ((42.5±5.1) μm), follicles survival rate (59.7%±6.1%) and proportion of PCNA stained follicles (43.5%±4.1%, P<0.05). Ultrastructural analysis confirmed the integrity of follicles cultured in VC+EGF+FSH group, while follicles cultured in MEM+VC+EGF groups showed more degeneration characters. In conclusion, the addition of VC and EGF to culture medium inhibited follicular development, VC+EGF+FSH was the most effective treatment to maintain follicular integrity and promote sheep primordial follicular activation and growth during in vitro culture.