Viability of feline preantral follicles in vitro cultured with Insulin Growth Factor and Epidermal Growth Factor supplemented medium

In vitro culture of ovarian preantral follicles (PF) has emerged as a potential reproductive technology for the production of mature oocytes capable of fertilization [1]. Advances concerning the role of several growth factors (GF) on in vitro activation of primordial follicles have been described. The addition of EGF (Epidermal Growth Factor) and IGF (Insulin-like Growth Factor) in the in vitro culture of PF of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells (GC) [2]. However, there are only few reports regarding the use of these factors on feline PF in vitro culture. We previously demonstrated that IGF-1 exerts a positive influence on follicular development [3] and has been recently shown that EGF sustains in vitro primordial follicle viability in the prepubertal cat ovary [4]. Thus, the aim of this study was to investigate the effect of a combination of IGF and EGF on in vitro growth and GC viability of PF collected from domestic cats. A total of 64 PF characterized by multi-layer granulosa cells and theca cells with 150–200 μm of diameter were isolated and individually cultured for 6 days (T6) in minimum essential medium (MEM; Sigma-Aldrich Co., St. Louis, LO, USA) supplemented with IGF+EGF (100 ng/ml each; Sigma) or without GF (control). Data from follicular and oocyte diameters were submitted to Kolmogorov–Smirnov. Mean values of the increase in follicular size were analysed by Student–Newman–Keuls test, and GC viability was analysed by chi-square test (P<0.05). After culture, PF showed a significant increase in size when IGF+EGF were added to the medium (170.0 ± 32.4 μm (T0) vs 201.0 ± 22.3 μm (T6); P<0.05). An increase of the diameter was also observed in PF cultured without GF (166.0 ± 16.1 μm (T0) vs 181.0 ± 10.4 μm (T6); P<0.05), but the increase was only 8.3% compared to 15.4% of those cultured with GF (P<0.05). No differences were found in the diameter of oocytes contained in follicles cultured in the non-supplemented or supplemented media (15.2% and 16.2%; P> 0.05; Sigma). The GC of PF cultured with GF maintained a greater viability (fluorescein diacetate staining; Sigma) than those of PF cultured without (84.3% and 68.7% respectively; P<0.05). These data suggest that the addiction of IGF and EGF to the culture medium promotes better conditions to the in vitro development of preantral follicles of cats. It remains to investigate the precise role of the single growth factor to establish optimal culture conditions. [1] Demeestere I, Centner J, Gervy Y, et al. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction 2005; 130:147-56. [2] Silva J R V, Van Den Hurk R, Figueiredo J R. Ovarian follicle development in vitro and oocyte competence: advances and challenges for farm animals. Domest Anim Endocrinol 2016, 55:123-5. [3] Alves EA, Padilha L, Savi PA, et al. In vitro survival of follicles recovered from queens at different stages of estrous cycle and cultured with IGF-1. Reprod Domest Anim 2012; 47 (Suppl. 6):109-12. [4] Fujihara M, Comizzoli P, Keefer CL, et al. Epidermal growth factor (EGF) sustains in vitro primordial follicle viability by enhancing stromal cell proliferation via MAPK and PI3K pathways in the prepubertal, but not adult, cat ovary. Biol Reprod 2014; 90:86.