Estradiol production of granulosa cells is unaffected by the physiological mix of non-esterified fatty acids in follicular fluid.

The ovarian cycle is controlled by circulating levels of the steroid hormone 17-β-estradiol, which is predominantly synthesized by the granulosa cells (GCs) of ovarian follicles. Our earlier studies showed that unsaturated fatty acids (USFs) downregulate and saturated fatty acids (SFAs) upregulate estradiol production in GCs. However, it was unclear whether pituitary gonadotropins induce accumulation of free fatty acids (FFA) in the follicular fluid since follicle-stimulating hormone (FSH) induces and luteinizing hormone (LH) inhibits estradiol production in the mammalian ovary. Interestingly, we show here gas chromatography analysis of follicular fluid revealed no differential accumulation of FFA between pre- and post-LH follicles. We therefore wondered how estradiol production is regulated in the physiological context, as USFs and SFAs are mutually present in the follicular fluid. We thus performed in vitro primary GC cultures with palmitate, palmitoleate, stearate, oleate, linoleate, and alpha-linolenate, representing >80% of the FFA fraction in the follicular fluid, and analyzed 62 different cell culture conditions to understand the regulation of estradiol biosynthesis under diverse FFA combinations. Our analyses showed co-supplementation of SFAs with USFs rescued estradiol production by restoring gonadotropin receptors and aromatase, antagonizing the inhibitory effects of USFs. Furthermore, transcriptome data of oleic acid-treated GCs indicated USFs induce the ERK and Akt signaling pathways. We show SFAs inhibit USF-induced ERK1/2 and Akt activation, wherein ERK1/2 acts as a negative regulator of estradiol synthesis. We propose SFAs are vital components of the follicular fluid, without which gonadotropin signaling and the ovarian cycle would probably be shattered by USFs.