162 Supplementation of Fibroblast Growth Factor 2, Leukemia Inhibitory Factor, and Insulin-Like Growth Factor 1 (FLI) Enables Efficient Production of Competent Porcine Oocytes Without Gonadotropins in the Maturation Medium

Abstract

In vitro maturation (IVM) of oocytes is a critical step in assisted reproductive technologies carried out in species such as cattle and swine, for generating oocytes capable of being fertilized in vitro and providing healthy offspring useful for biomedical and agricultural purposes. Cumulus-oocyte complexes (COC) collected from abattoir ovaries for IVM often respond poorly to gonadotropins, resulting in compromised oocyte competence. Our previous work demonstrated that the combination of fibroblast growth factor 2, leukemia inhibitory factor, and insulin-like growth factor 1 (FGF2, LIF, and IGF1; FLI) significantly improved nuclear maturation of porcine oocytes and their developmental competence. However, it is unclear whether the benefits of FLI are mediated through increased gonadotropin sensitivity of COC or improved downstream signalling. Here we investigated the effect of FLI supplementation of IVM medium with and without gonadotropins. The COC, collected from 2- to 6-mm follicles from prepubertal ovaries, were matured in 5% CO₂/air at 38.5°C for 42 h in chemically defined TCM-199 medium (supplemented with 0.1% polyvinyl alcohol, 3.05 mM d-glucose, 0.91 mM pyruvate, 0.57 mM cysteine, 10 ng mL⁻¹ epidermal growth factor), with or without FLI (40 ng mL⁻¹ FGF2, 20 ng mL⁻¹ LIF, and 20 ng mL⁻¹ IGF1), and with or without gonadotropins (0.5 μg mL⁻¹ LH, 0.5 μg mL⁻¹ FSH), in a 2 × 2 factorial design experiment. After IVM, oocytes were fertilized in vitro and cultured under standard conditions until Day 6 when blastocyst formation was assessed. The experiment was replicated 4 times with a total of 792 oocytes. Percentage data were arcsin transformed and analysed by ANOVA to detect differences (significance, P < 0.05). When FLI was absent from the maturation medium, oocytes matured in presence of gonadotropins demonstrated improved nuclear maturation (67.0 ± 2.2% v. 52.7 ± 5.3%) and blastocyst formation (23.7 ± 3.3% v. 10.2 ± 2.3%) relative to oocytes matured without gonadotropins, respectively. However, when FLI was present in the medium, the extent of oocyte maturation and subsequent blastocyst development was unaffected by gonadotrophin addition (maturation: 84.2 ± 1.8% with and 79.2 ± 3.4% without; blastocyst formation: 29.5 ± 4.3% with and 24.9 ± 4.3% without). In summary, these results suggest that FLI, rather than enhancing COC sensitivity to gonadotrophins, may activate certain downstream signalling pathways that are normally controlled by gonadotropins during IVM, thereby enhancing oocyte quality. Therefore, FLI appears able to substitute for gonadotropins in the maturation medium and supports porcine oocyte competence in the absence of FSH and LH. These observations will help us better understand the mechanisms whereby FLI enhances oocyte nuclear maturation and improves developmental competency.

Supported by NIH R01HD69979, U42OD011140, and University of Missouri Food for the 21^st Century Program.