Influence of phenolic flavonols (Kaempferol, Querectin and Myricetin) on the survival and growth of ovine preantral follicles and granulosa cells cultured in vitro

Abstract

Study was carried out to examine the influence of plant bioactive compounds [Kaempferol (KAE), Querectin (QUE) and Myricetin (MYR)] on the survival and growth parameters of cultured ovine preantral follicles (PFs) granulosa cells (GCs) and expression of some key developmental genes. Ovine PFs were isolated from slaughterhouse derived ovaries and KAE, QUE and MYR were supplemented to the standard culture medium of GCs and PFs at concentrations of 0, 5, 10, 25, 50 and 100 μM and cultured for 5 and 7 days respectively. PFs morphological and functional parameters [follicle and enclosed oocyte growth rate, viability of follicles, antrum formation rate, oocyte maturation rate, estradiol concentration, reactive oxygen species (ROS) production] and GC growth parameters (metabolic activity, viability rate, cell number increment, ROS production) were measured after culture. Significantly higher PF growth, viability rate and estradiol concentration was observed at 10 μM, 25 μM and 10 μM concentration of KAE, MYR and QUE respectively compared to the control. ROS production was significantly decreased in the PF culture media treated with 10 μM KAE or MYR 25 μM or 10 μM QUE compared to those observed in the control group. Likewise, metabolic activity of GCs, viability rate and cell number increment cultured with KAE, MYR and QUE was significantly higher at 10, 25 and 10 μM concentrations respectively compared to those observed in control group. ROS production was significantly lower in the GC cultured with KAE, MYR and QUE at 10, 25 and 10 μM concentrations respectively compared to the control. Based on the results of the growth parameters, gene expression of PFs and GCs were studied by qPCR at selected concentrations (KAE, MYR and QUE at 10, 25 and 10 μM concentrations respectively) in the cultured PFs and GCs. Gene expression of GDF9, FGF2, CYP19A1 was significantly higher and Bax, Bcl2 expression was significantly lower in the PFs and GCs cultured with the KAE or QUE at 10 μM concentration. KAE, MYR and QUE have dose dependant responses on PFs and GCs morphological and functional parameters; however, KAE is more potent amongst the three in augmenting the ovarian functions.