281 EMBRYOTOXICITY ANALYSIS OF NANOCAPSULES AND THEIR EFFECTIVENESS IN RELEASING FSH ON THE IN VITRO MATURATION OF BOVINE OOCYTES

Abstract

Liposomes and nanocapsules (NC) are nanotechnologies that allow for controlled drug-release systems, providing slow release of the incorporated or adsorbed substance in the lipid or polymeric particle. Therefore, slow-release FSH-loaded nanocapsules could be an innovative tool for the improvement of production systems. We aimed to evaluate the embryotoxicity of the NC (the vehicle without any incorporation) and analyse the effectiveness of FSH release through the addition of NC to in vitro maturation (IVM). Cumulus-oocyte complexes (COC, grades I and II) from follicles ranging 3 to 8 mm were obtained from bovine ovaries from abattoir. Ten to 20 COC were washed in TCM199 hepes medium droplets and, subsequently, in droplets through the specific group to which they were allocated. In experiment 1 (E1), 6 groups (G1, G2, G3, G4, G5, and G6; 5 replicates, n = 76 oocytes/group) were defined: G1 = negative control [1 mL of TCM199 bicarbonate, 5 μL of amikacin (16.67 μg μL^–1), 2 μL of pyruvate (0.011 g mL^–1)], G2 = experimental control [5 mL of TCM199 bicarbonate, 0.030 g of BSA, 5 μL of FSH (0.1 mg mL^–1), 25 μL of amikacin (16.67 mg μL^–1), 10 μL of pyruvate (0.011 g mL^–1)], G3 = laboratory control [0.9 mL of G2, 100 mL of FCS, 10 μL of LH (50 μg mL^–1), and 1 μL of oestradiol] and groups G4, G5, and G6 contained 0.9 mL of G2 plus different concentrations of empty NC: 10% (~0.1 g), 1%, and 0.1% vol/vol, respectively. In the second experiment (E2), we used the same groups, but now groups G4 to G6 were supplemented with FSH derived from NC loaded with FSH (5 replicates, n = 98 oocytes/group). The NC was produced by the coacervation method containing grape seed oil, propylene glycol, isopropyl myristate, and Tween 20 in mixture to the aqueous phase with atelocollagen and xanthan gum. The NC were submitted to sonication and produced without any active compounds for the E1 and incorporated with FSH (10 μg mL^–1) for E2. There was a clear morphological difference (expansion) in cumulus cells after IVM (method according to Ali and Sirard 2002 Biol. Reprod. 66, 901–905). Data were analysed with ANOVA and post-hoc Tukey-Kramer. There was no expansion in G1, but cumulus in G2 and G3 expanded as expected (for both experiments). In both E1 and E2 there was partial expansion in G4 while G5 showed full expansion, similar to G2 and G3. Expansion of G6 was fair in E1, but in E2 the G6 expansion was similar to G1 (not expanded). In E1, cleavage (D3) rates of the highest tested concentration of NC [G4 (36.6%)] was different from G2 (72.1%), G3 (68.7%), G5 (59.3%), and G6 (69.5%; P < 0.001). Also in E2, cleavage rates of G4 (28.5%) differed from G2 (61.8%), G3 (77.2%), and G5 (64.9%). The blastocyst production did not differ between groups in E1 (P > 0.1). In E2, the group with the highest concentration of NC tested [G4 (9.5%)] and the control group [G2 (19.5%)] had different blastocyst rates (P < 0.05). Our results suggest a potential toxic effect for the pre-implantation embryo after using NC on the IVM of bovine oocytes.

Authors acknowledge funding from grants #12/50533-2, #13/05083-1, #12/24423-5, #13/07730-4, São Paulo Research Foundation (FAPESP).