Yadav Monica, Agrawal Himanshu, Pandey Mamta, Singh Dheer, Onteru Suneel K
Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, India.
J Cell Physiol. 2018 Mar;233(3):1959-1970. doi: 10.1002/jcp.25909. Epub 2017 May 3.
Granulosa cell (GC) culture models mimicking the intrafollicular environment are limited. Such models have a great potential in reproductive toxicity studies. The buffalo, a monovulatory species like humans, could be a better model than polyovulatory rodents. Therefore, we targeted the development and characterization of three-dimensional (3D) culture systems for buffalo GCs. The GCs from small ovarian follicles (SF) maintained the CYP19 gene expression for 144 hr in a 2D culture system. Hence, GCs from SF were cultured directly in 3D using hanging drop and Poly-([2-hydroxyethyl methacrylate]) (polyHEMA) methods in the DMEM media containing 1 ng/ml FSH and 10 ng/ml IGF-1 for 144 hr. The expression profile of nine GC-specific transcripts; CYP19, TNFAIP6, AMH, PTI, NR4A1, FSHR, RUNX, LHR, and COX2/PTGS2; revealed that 3D-spheroids developed in hanging drop method maintained the GC phenotype of preovulatory follicles. Therefore, hanging drop method is a best method for culturing GCs to mimic the intrafollicular environment.
模拟卵泡内环境的颗粒细胞（GC）培养模型有限。此类模型在生殖毒性研究中具有巨大潜力。水牛与人类一样是单排卵物种，可能比多排卵啮齿动物更适合作为模型。因此，我们致力于水牛颗粒细胞三维（3D）培养系统的开发与特性研究。来自小卵泡（SF）的颗粒细胞在二维培养系统中可维持CYP19基因表达144小时。因此，将来自小卵泡的颗粒细胞在含有1 ng/ml促卵泡激素（FSH）和10 ng/ml胰岛素样生长因子-1（IGF-1）的杜氏改良 Eagle 培养基（DMEM）中，使用悬滴法和聚（甲基丙烯酸 - 2 - 羟乙酯）（聚HEMA）方法直接进行三维培养144小时。九种颗粒细胞特异性转录本（CYP19、TNFAIP6、抗苗勒管激素（AMH）、妊娠特异性β1糖蛋白（PTI）、核受体亚家族4A成员1（NR4A1）、促卵泡激素受体（FSHR）、 Runt相关转录因子1（RUNX）、促黄体生成素受体（LHR）和环氧化酶2/前列腺素内过氧化物合酶2（COX2/PTGS2））的表达谱显示，悬滴法培养出的三维球体维持了排卵前卵泡的颗粒细胞表型。因此，悬滴法是培养颗粒细胞以模拟卵泡内环境的最佳方法。
