Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre la Sal, Consejo Superior de Investigaciones Científicas (CSIC), Torre la Sal, Ribera de Cabanes s/n, 12595, Castellón, Spain.
Cytotechnology. 2013 Mar;65(2):273-86. doi: 10.1007/s10616-012-9484-8. Epub 2012 Jul 4.
The layers of follicular cells surrounding the oocyte and the interactions among them and the germ cells are critical for the successful maintenance of the ovarian functions. We have set up the isolation procedure and culture conditions of sea bass ovarian follicular cells. Their behaviour at three different physiological temperatures (25, 18 and 15 °C) was evaluated by verifying their steroidogenic capacity along time together with the expression of follicular specific genes (cyp19a1, fshr, lhr and star). These characteristics revealed this culture as a good in vitro alternative to short term in vivo studies at the level of the ovarian follicle. Moreover, to evaluate the suitability of this system for gene function studies conditions for transient transfection of plasmid DNA were optimized. Finally, the characteristics of the follicular culture were not affected by freezing and thawing cycles what facilitates the performance of experiments independently of the reproductive season. In conclusion, we have developed an in vitro homologous system that enables functional and gene expression studies and resembles the in vivo situation in the ovarian follicle.
围绕卵母细胞的滤泡细胞层及其与生殖细胞之间的相互作用对于成功维持卵巢功能至关重要。我们已经建立了鲈鱼卵巢滤泡细胞的分离程序和培养条件。通过随着时间的推移验证它们的甾体生成能力以及滤泡特异性基因（cyp19a1、fshr、lhr 和 star）的表达，评估了它们在三种不同生理温度（25、18 和 15°C）下的行为。这些特征表明，这种培养物是卵巢滤泡水平短期体内研究的良好体外替代物。此外，为了评估该系统用于基因功能研究的适用性，优化了质粒 DNA 的瞬时转染条件。最后，滤泡培养的特征不受冷冻和解冻循环的影响，这使得实验可以在不受生殖季节影响的情况下独立进行。总之，我们已经开发出一种能够进行功能和基因表达研究的同源体外系统，并且类似于卵巢滤泡中的体内情况。
