在恒河猴的三维包被培养中，次级卵泡生长和卵母细胞成熟：促性腺激素、氧和胎球蛋白的影响。
Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin.
机构信息
Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR 97006, USA.
出版信息
Hum Reprod. 2011 May;26(5):1061-72. doi: 10.1093/humrep/der049. Epub 2011 Feb 28.
BACKGROUND
An alginate-based matrix supports the three-dimensional (3D) architecture of non-human primate follicles and, in the presence of FSH, permits the in vitro development of pre-antral follicles to the small antral stage, including the production of ovarian steroids and paracrine factors. The current study investigated the ability of gonadotrophins, fetuin and oxygen (O₂) to improve primate follicle growth and oocyte maturation in vitro.
METHODS
Macaque secondary follicles were isolated from the early follicular phase ovaries, encapsulated in a sodium alginate matrix and cultured individually for 40 days in supplemented medium. The effects of recombinant human (rh) FSH (15, 3 and 0.3 ng/ml for high, medium and low FSH, respectively), bovine fetuin (1 or 0 mg/ml) and O₂ (5 or 20% v/v) were examined. Half of the follicles in each culture condition received rhLH on Day 30-40. Follicles that reached antral stage were treated with rh chorionic gonadotrophin for 34 h to initiate oocyte meiotic maturation. Media were analyzed for ovarian steroids and anti-müllerian hormone (AMH).
RESULTS
Improved culture conditions supported non-human primate, secondary follicle growth to the antral stage and, for the first time, promoted oocyte maturation to the MII stage. In the presence of fetuin at 5% O₂, follicles had the highest survival rate if cultured with high or medium FSH, whereas follicles grew to larger diameters at Week 5 in low FSH. Oocyte health and maturation were promoted under 5% O₂. High FSH stimulated steroid production by growing follicles, and steroidogenesis by follicles cultured with low FSH was promoted by LH. AMH biosynthesis was elevated with high compared with low FSH and for longer under 5% O₂ than under 20% O₂.
CONCLUSIONS
This encapsulated 3D culture model permits further studies on the endocrine and local factors that influence primate follicle growth and oocyte maturation, with relevance to enhancing fertility preservation options in women.
背景
基于海藻酸盐的基质支持非人类灵长类动物卵泡的三维（3D）结构，并且在 FSH 的存在下，允许体外将原始卵泡发育到小窦卵泡阶段，包括卵巢类固醇和旁分泌因子的产生。本研究探讨了促性腺激素、胎球蛋白和氧气（O₂）改善灵长类卵泡体外生长和卵母细胞成熟的能力。
方法
从小卵泡期卵巢中分离出猕猴次级卵泡，包裹在海藻酸钠基质中，并在补充培养基中单独培养 40 天。研究了重组人（rh）FSH（高、中、低 FSH 分别为 15、3 和 0.3ng/ml）、牛胎球蛋白（1 或 0mg/ml）和 O₂（5 或 20% v/v）的作用。在每个培养条件下，一半的卵泡在第 30-40 天接受 rhLH。达到窦卵泡阶段的卵泡用 rh 绒毛膜促性腺激素处理 34 小时以启动卵母细胞减数分裂成熟。分析培养基中的卵巢类固醇和抗缪勒管激素（AMH）。
结果
改进的培养条件支持非人类灵长类动物次级卵泡生长到窦卵泡阶段，并且首次促进卵母细胞成熟到 MII 阶段。在 5% O₂存在下，胎球蛋白的情况下，如果在高或中 FSH 存在下培养，卵泡的存活率最高，而在低 FSH 下，卵泡在第 5 周时生长到更大的直径。在 5% O₂下促进卵母细胞健康和成熟。高 FSH 刺激生长卵泡的类固醇生成，LH 促进低 FSH 培养的卵泡的类固醇生成。与低 FSH 相比，高 FSH 增加了 AMH 的生物合成，并且在 5% O₂下比在 20% O₂下延长了 AMH 的生物合成。
结论
这种包封的 3D 培养模型允许进一步研究影响灵长类卵泡生长和卵母细胞成熟的内分泌和局部因素，对增强女性的生育力保存选择具有重要意义。
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