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Maternal metabolic stress conditions are of growing importance in both human and dairy cattle settings as they can have significant repercussions on fertility. Upregulated lipolysis is a common trait associated with metabolic disorders and results in systemically elevated concentrations of non-esterified fatty acids (NEFAs). The effects of high NEFA concentrations on the follicular environment, oocyte and embryo development is well documented. However, knowledge on the effects of NEFAs within the oviduct, representing the initial embryonic growth environment, is currently lacking. Therefore, the experiments outlined here were designed to obtain fundamental insights into both the direct and indirect interactions between NEFAs, bovine oviductal cells and developing zygotes. Hence, zygotes were co-cultured with NEFA-pre-exposed bovine oviductal cells or subjected to simultaneous NEFA exposure during the co-culture period. The outcome parameters assessed were embryo development with cleavage (48h post insemination (pi)), morula (120-126h pi) and blastocyst (192h pi) rates, as well as morula intracellular lipid content and blastocyst quality using Bodipy and differential staining respectively. Our data suggest a direct embryotoxicity of NEFAs as well as impaired embryo development through a reduced oviductal ability to support and protect early embryo development.

Maternal metabolic disorders linked to lipolysis are major risk factors for reproductive failure. A notable feature of such disorders is increased non-esterified fatty acid (NEFA) concentrations in the blood, which are reflected in the ovarian follicular fluid. Elevated NEFA concentrations impact on the maturing oocyte and even alter subsequent embryo physiology. The aetiological mechanisms have not been fully elucidated. Therefore, in the present study, bovine in vitro maturing cumulus-oocyte complexes were exposed (24 h) to three different maturation treatments containing: (1) physiological (72 µM) NEFA concentrations (= control); (2) elevated (75 µM) stearic acid (SA) concentrations (= HIGH SA); and (3) elevated (425 µM) NEFA concentrations (= HIGH COMBI). Zygotes were fertilised and cultured following standard procedures. Transcriptomic analyses in resulting Day 7.5 blastocysts revealed that the major pathways affected are related to lipid and carbohydrate metabolism in HIGH COMBI embryos and to lipid metabolism and cell death in HIGH SA embryos. Furthermore, lower glutathione content and a reduced number of lipid droplets per cell were observed in HIGH SA-exposed oocytes and resulting morulae, respectively, compared with their HIGH COMBI-exposed counterparts. Vitrified embryos originating from HIGH SA-exposed oocytes tended to exhibit lower survival rates compared with controls. These data suggest possible mechanisms explaining why females across species suffering lipolytic disorders experience difficulties in conceiving. Metabolic disorders are risk factors for reproductive failure and elevated non-esterified fatty acid (NEFA) concentrations have been proposed as key contributors by affecting oocyte quality. The aim of this study was to unravel the underlying molecular mechanisms through which NEFAs impact on bovine maturing oocytes and subsequent embryo development. Transcriptome and functional data point to metabolic dysregulation in the resulting preimplantation embryos, findings that might explain the reduced conception rates in females across species suffering lipolytic disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]