Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development
Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development
复制标题
DOI：
10.1095/biolreprod.106.054833
复制
发表时间：
2006-12-01
影响因子：
3.6
通讯作者：
Woodruff, Teresa K.
中科院分区：
文献类型：
--
作者：
Xu, Min;West, Erin;Woodruff, Teresa K.
可用卵母细胞的可获得性是新生殖技术发展的限制因素。近几十年来，在体外培养未成熟卵母细胞方面进行了许多尝试。最近，一种改良的基于藻酸盐的三维培养系统被设计出来支持多层次级卵泡的生长和成熟。该系统能够生产出成功完成减数分裂、受精和发育到囊胚期的卵母细胞。在原始条件下，随后的培养双层次级毛囊的尝试都失败了。在此，我们通过将卵泡包裹在不同浓度的海藻酸盐支架中，研究了海藻酸盐浓度对双层卵泡生长和卵母细胞发育能力的影响。培养8d后，0.25%和0.5%的藻酸盐对卵泡的生长和腔的形成较1.5%和1.0%的藻酸盐有更大的促进作用，但成活率没有显著差异。藻酸盐支架浓度也影响体细胞(膜细胞和颗粒细胞)的增殖和分化，根据形态变化、类固醇(雄烯二酮、雌二醇和孕酮)和特定分子标记(FSHR、Lhcgr和Gja1)进行测量。在1.5%海藻酸盐中培养的卵泡膜细胞的增殖和类固醇的产生受到抑制。体外受精和胚胎培养表明，0.25%海藻酸盐的卵母细胞具有最高的发育能力。总体而言，本研究表明，藻酸盐支架的浓度影响卵泡发生和卵母细胞的体外发育，藻酸盐培养系统可以而且应该根据选定培养的卵泡的大小和阶段而定制以最大限度地支持卵泡的生长。
The availability of viable oocytes is the limiting factor in the development of new reproductive techniques. Many attempts have been made to grow immature oocytes in vitro during recent decades. Recently, a modified alginate-based three-dimensional culture system was designed to support the growth and maturation of multilayered secondary follicles. This system was able to produce oocytes that successfully completed meiosis, fertilization, and development to the blastocyst stage. Subsequent attempts to culture two-layered secondary follicles were unsuccessful under the original conditions. Herein, we investigated the effect of alginate consistency on two-layered follicle growth and oocyte developmental competence by encapsulating follicles into alginate scaffolds of various concentrations. Although there were no significant differences in survival rates, 0.25% and 0.5% alginate supported more rapid growth of follicles and antrum formation compared with 1.5% and 1.0% alginate after 8 days of culture. Alginate scaffold concentration also affected the proliferation and differentiation of somatic cells (theca and granulosa cells), measured in terms of morphological changes, steroid profiles (androstenedione, estradiol, and progesterone), and specific molecular markers (Fshr, Lhcgr, and Gja1). Theca cell proliferation and steroid production were hindered in follicles cultured in 1.5% alginate. In vitro fertilization and embryo culture revealed that oocytes obtained from 0.25% alginate retained the highest developmental competence. Overall, the present study showed that the alginate scaffold consistency affects folliculogenesis and oocyte development in vitro and that the alginate culture system can and should be tailored to maximally support follicle growth depending on the size and stage of the follicles selected for culture.