Introduction:Cell culture techniques of human mural granulosa cells (MGCs) aspirated during IVF serve as a majorin vitroresearch tool. However, the use of luteinized MGCs has major limitations as a model for assessing the effect of hCG stimulation due to their luteinized state. We recently characterized the molecular profile of these cells and established a model for FSH induction (MCE 384/1-2 (2014), pp. 165-174). However, the optimal protocol to explore the effect of hCG in cell culture is still unknown
Aim: Our aim was to establish a standardized protocol for MGCs culture model for exploring the efficiency of hCG stimulationin vitro.
Materials & Methods:We stimulated MGCs with hCG (1U/ml) for 24h on either the seeding day, days 1-3 in culture, or on day 6 in culture after 48h FSH stimulation (FSH model) and compared the induction of LH/hCG target genes and hormone known to be involved in steroidogenesis and ovulation. RT-PCR analysis and EIA were performed in order to evaluate gene expression and progesterone secretion.
Results: Our results showed that ovulation related genes were maximally upregulated by hCG stimulation on day 1-2 while steroidogenesis related gene were maximally elevated in response to hCG on day 6 after 48 h of FSH stimulation.
Conclusion: This novel model may provide a standardized research tool. For exploring the effect of hCG in culture, mimicking the molecular processes occurring following the LH/hCG surge during the latter stages of follicular development in the human ovary.
