Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR - National Dairy Research Institute, Karnal, Haryana, India.
Mol Reprod Dev. 2021 Apr;88(4):287-301. doi: 10.1002/mrd.23465. Epub 2021 Mar 18.
Hanging drop (HD) three-dimensional (3D) culture model for buffalo granulosa cells (GC) was reported to mimic the preovulatory stage of ovarian follicles in our previous study. To further verify its reliability, the present study attempted a comparative transcriptome profile of buffalo GC freshly isolated from ovarian follicles (<8 mm diameter) (FC) and their cultures in normal culture dish (ND or 2D), polyHEMA coated dish (PH) and HD culture systems (3D). Out of 223 significantly (-log2 fold change: >3; p < .0005; false discovery rate [FDR]: <0.1) differentially expressed genes (SDEGs) among different culture systems, 137 were found unannotated, and 94, 29, and 66 were exclusively expressed in FC, PH, and HD, respectively. However, on eliminating the fixed points of p values and FDR from the entire raw data, only 11 genes related to long noncoding RNA, 12 genes related to luteinization, and 3 genes related to follicular maturation were exclusively expressed in FC, PH, and HD culture systems, respectively. The quantitative real time-PCR validation and the next generation sequencing data had more than 90% correlation. Bioinformatics analyses of the exclusively expressed SDEG revealed that the freshly aspirated GCs were a true representative of GCs from small follicles (<8 mm diameter), the GC spheroids under PH maintained mitochondrial function, and those cultured in HD system for 6 days simulated the inflammatory milieu required for ovulation. Therefore, the comparative transcriptome profile also reinforced that HD culture system is better in vitro culture method than the other methods analyzed in this study for buffalo GC.
在我们之前的研究中，曾报道过一种水牛颗粒细胞（GC）的悬滴（HD）三维（3D）培养模型，该模型可模拟卵巢卵泡的排卵前阶段。为了进一步验证其可靠性，本研究尝试比较了从卵巢卵泡（<8mm 直径）（FC）中新鲜分离的水牛 GC 及其在普通培养皿（ND 或 2D）、聚己内酯（PH）和 HD 培养系统（3D）中的培养物的转录组图谱。在不同培养系统之间的 223 个显著差异表达基因（SDEGs）中（-log2 倍变化：>3；p<0.0005；错误发现率[FDR]：<0.1），有 137 个未注释，94、29 和 66 个分别仅在 FC、PH 和 HD 中表达。然而，在从整个原始数据中消除 p 值和 FDR 的固定点后，只有 11 个与长非编码 RNA 相关、12 个与黄体化相关和 3 个与卵泡成熟相关的基因分别仅在 FC、PH 和 HD 培养系统中表达。定量实时 PCR 验证和下一代测序数据的相关性超过 90%。对 SDEG 的专门表达进行的生物信息学分析表明，刚从卵泡中吸出的 GC 是小卵泡（<8mm 直径）中 GC 的真实代表，PH 下的 GC 球体保持了线粒体功能，而在 HD 系统中培养 6 天则模拟了排卵所需的炎症环境。因此，比较转录组图谱也强化了 HD 培养系统是比本研究中分析的其他方法更好的水牛 GC 体外培养方法。
