Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006, USA.
Reproduction. 2010 Nov;140(5):685-97. doi: 10.1530/REP-10-0284. Epub 2010 Aug 20.
A three-dimensional culture system supports the development of primate preantral follicles to the antral stage with appreciable steroid production. This study assessed i) whether in vitro developmental competence of follicles is age dependent, ii) the role of gonadotropins and insulin in supporting folliculogenesis, and iii) anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) production by growing follicles. Ovaries were obtained from prepubertal, young, and older adult rhesus macaques. Secondary follicles were encapsulated into alginate beads and cultured individually for 40 days in media containing 0.05 or 5 μg/ml insulin, with or without recombinant human (rh) FSH (500 mIU/ml). No follicles survived in the culture without rhFSH. In the presence of rhFSH, survival was lower for follicles from older animals, whereas growth, i.e. follicle diameter, was less by day 40 for follicles from prepubertal animals. The surviving follicles were categorized as no-grow (NG; ≤ 250 μm), slow-grow (SG; 250-500 μm), and fast-grow (FG; ≥ 500 μm) according to their diameters. SG follicles cultured with 5 μg/ml insulin produced more ovarian steroids than those cultured with 0.05 μg/ml insulin by week 5. SG and FG follicles produced more AMH and VEGF than the NG, and levels peaked at weeks 2 and 5 respectively. After 100 ng/ml rh chorionic gonadotropin treatment for 34 h, more healthy oocytes were retrieved from young adults whose follicles were cultured with 5 μg/ml insulin. This culture system offers an opportunity to characterize the endocrine and paracrine function of primate follicles that influence follicle growth and oocyte maturation.
三维培养体系支持灵长类动物原始腔前卵泡发育到腔卵泡阶段，并能产生可观的类固醇。本研究评估了：i）卵泡体外发育能力是否与年龄有关，ii）促性腺激素和胰岛素在支持卵泡发生中的作用，以及 iii）生长卵泡产生抗苗勒管激素（AMH）和血管内皮生长因子（VEGF）的情况。从未成年、年轻和老年恒河猴中获取卵巢。将次级卵泡包被到藻酸盐珠中，并在含有 0.05 或 5μg/ml 胰岛素的培养基中进行个体培养，有或没有重组人（rh）FSH（500mIU/ml）。没有 rhFSH 的培养物中没有卵泡存活。在 rhFSH 的存在下，来自老年动物的卵泡存活率较低，而来自未成年动物的卵泡在第 40 天的生长（即卵泡直径）较小。根据卵泡直径，将存活的卵泡分为无生长（NG；≤250μm）、缓慢生长（SG；250-500μm）和快速生长（FG；≥500μm）卵泡。用 5μg/ml 胰岛素培养的 SG 卵泡比用 0.05μg/ml 胰岛素培养的卵泡在第 5 周时产生更多的卵巢类固醇。SG 和 FG 卵泡产生的 AMH 和 VEGF 比 NG 多，且分别在第 2 和第 5 周达到峰值。经过 34 小时 100ng/ml rh 绒毛膜促性腺激素处理后，用 5μg/ml 胰岛素培养的年轻成年猴的卵泡中可回收更多健康卵母细胞。该培养体系为研究影响卵泡生长和卵母细胞成熟的灵长类动物卵泡的内分泌和旁分泌功能提供了机会。
