5 THE EFFECT OF ELEVATED NONESTERIFIED FATTY ACID CONCENTRATIONS ON MURINE IN VITRO FOLLICULOGENESIS AND SUBSEQUENT OOCYTE DEVELOPMENTAL COMPETENCE

Abstract

Metabolic disorders, like negative energy balance and obesity, are characterized by elevated serum non-esterified fatty acid (NEFA) concentrations, due to upregulated lipolysis. We previously showed that short-term (24-h) NEFA exposure of bovine oocytes during their final maturation is detrimental to oocyte and embryo quality (Van Hoeck et al. 2011 PLoS ONE 6, e23183). However, the typical chronic exposure as present in diseased females has never been studied before. Therefore, we aimed to use a murine preantral follicle culture model to study the effect of long-term NEFA exposure on folliculogenesis, antrum formation, and the developmental competence of the enclosed oocyte. Early secondary murine follicles (n = 629) from 13-day-old B6CBAF1 mice were individually cultured for 12 days. Follicles were exposed to 4 treatments containing physiological or pathological concentrations of stearic acid (SA), palmitic acid (PA), or oleic acid (OA) for 12 days: BASAL (72 µM NEFA mix, n = 154), high SA (280 µM SA, n = 160), high OA (210 µM OA, n = 159), and NEFA (720 µM NEFA mix, n = 156). Oocytes were routinely fertilized 20 h after a final maturation stimulus [hCG, epidermal growth factor (EGF)]. Presumptive zygotes were denuded and cultured until the blastocyst stage (Day 5 pre-implantation). Data were analyzed with a mixed-model ANOVA or binary logistic regression. The SA-exposed follicles displayed a reduced Day-12 antrum formation, compared to BASAL and NEFA follicles (68 v. 78 and 83%, P = 0.03 and P < 0.01), which was similar for OA follicles compared with the NEFA follicles (72 v. 83%, P = 0.04). Follicles cultured in SA conditions were, on average, 10, 13, and 11% smaller in diameter compared with BASAL, OA, and NEFA follicles (P = 0.05, P = 0.01, and P = 0.04, respectively). Likewise, the follicular growth during the 12-day culture was significantly reduced for SA follicles compared with BASAL and NEFA follicles (P < 0.01). Neither Day-12 oocyte diameter nor oocyte growth were affected by treatment. After fertilization, the oocytes from NEFA-exposed follicles showed a decreased cleavage rate on Day 1 pre-implantation, compared with BASAL oocytes (53 v. 69%; P < 0.01). Accordingly, the percentage of Day-5 blastocysts was reduced for oocytes from NEFA follicles compared with BASAL follicles (42 v. 63%; P < 0.01). Also, oocytes from SA and OA follicles showed reduced blastocyst rates compared with BASAL oocytes (32 and 33 v. 63%; P < 0.01). Exposure of follicles to SA or OA seems to slightly impair antrum formation and follicle growth. Furthermore, oocytes originating from NEFA-, OA-, and SA-exposed follicles had a dramatically reduced developmental competence compared with oocytes from follicles cultured in physiological BASAL conditions, an effect most apparent at blastocyst formation. In conclusion, we show for the first time that elevated NEFA concentrations affect murine follicular development and that this negative effect is carried through to the oocyte and pre-implantation embryo development. Ongoing research focuses on the pathways involved.