绵羊羔羊卵巢皮质组织在序贯培养液中的体外培养。
In vitro culture of sheep lamb ovarian cortical tissue in a sequential culture medium.
机构信息
Department of Animal Science, ShiHeZi University, ShiHeZi, Xinjiang Uygur Autonomous Region, China.
出版信息
J Assist Reprod Genet. 2010 May;27(5):247-57. doi: 10.1007/s10815-010-9415-6.
PURPOSE
This study evaluates the effect of a sequential culture system on the follicular development of sheep lamb ovaries, aiming to establish an available in vitro culture system for ovarian culture.
METHODS
Lamb ovarian cortical fragments were cultured on a steel mesh with a nitrocellulose membrane pre-coated by type 1 collagen. Several culture media were used for the determinations, specifically, a control medium (alpha-MEM), a constant medium (control medium supplemented with 75 ng/mL human recombinant EGF, 200 mIU/mL sheep FSH, 100 ng/mL human recombinant GDF-9, and 100 ng/mL human recombinant bFGF), and a sequential medium (control medium supplemented with sequential growth factors added on different days). Ovarian tissues, both fresh and cultured, were processed for histological and apoptotic assays, while spent culture media were processed for hormone assays.
RESULTS
It was found that the growth of lamb primordial follicles can be initiated during culture in vitro. Compared to the control medium, sequential culture medium significantly increased the percentage of secondary follicles in cultures, while the follicle and oocyte diameters of primary and secondary follicles were also observed to increase in this medium. The constant medium was found to increase the number and diameter of secondary follicles only 18 days after culture. After this same period of time, some normal antral follicles were found in the sequential medium, while a few abnormal antral-like follicles were found in the control medium. Moreover, sequential medium appeared to significantly increase estradiol and inhibin production, especially 10-18 days after culture. The highest percentage of normal follicles and the lowest apoptotic cell rates were observed in the sequential medium, suggesting that a sequential addition style of culture can improve follicle and tissue viability.
CONCLUSIONS
The sequential addition of FSH, EGF, GDF-9,and bFGF can stimulate primordial follicle transmittal into the later development stages, even as far as the antral stage, improve the survival rate of follicles, and maintain follicular viability.
目的
本研究评估了序贯培养系统对绵羊羔羊卵巢卵泡发育的影响，旨在建立一种可行的卵巢体外培养系统。
方法
将羔羊卵巢皮质碎片置于涂有 1 型胶原的硝化纤维素膜的钢网上进行培养。使用几种培养基进行测定，具体为对照培养基（α-MEM）、恒化培养基（对照培养基中添加 75ng/mL 人重组 EGF、200mIU/mL 绵羊 FSH、100ng/mL 人重组 GDF-9 和 100ng/mL 人重组 bFGF）和序贯培养基（对照培养基中添加在不同天添加序贯生长因子）。新鲜和培养的卵巢组织进行组织学和凋亡检测，而用过的培养物用于激素检测。
结果
发现羔羊原始卵泡的生长可以在体外培养中启动。与对照培养基相比，序贯培养基显著增加了培养物中次级卵泡的比例，同时初级和次级卵泡的卵泡和卵母细胞直径也观察到在该培养基中增加。恒化培养基仅在培养 18 天后才发现次级卵泡数量和直径增加。经过同样的时间，在序贯培养基中发现了一些正常的腔前卵泡，而在对照培养基中发现了一些异常的腔前样卵泡。此外，序贯培养基似乎显著增加了雌二醇和抑制素的产生，特别是在培养 10-18 天后。在序贯培养基中观察到正常卵泡的比例最高，凋亡细胞率最低，表明序贯添加培养方式可以提高卵泡和组织的活力。
结论
FSH、EGF、GDF-9 和 bFGF 的序贯添加可以刺激原始卵泡进入后期发育阶段，甚至是腔前阶段，提高卵泡的存活率，并维持卵泡的活力。
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