The objectives were to develop an effective protocol for transfection of ovine secondary follicles and to assess the effect of attenuating aquaporin 3 (AQP3) using a small interfering RNA (siRNA-AQP3) on antrum formation and follicular growth in vitro. Various combinations of Lipofectamine® volumes (0.5, 0.75 or 1.0 µl), fluorescent oligonucleotide (BLOCK-iT ™) concentrations (3.18, 27.12 or 36.16 nM) and exposure times (12, 14, 16, 18 or 20 h) were tested. The BLOCK-iT™ was replaced by siRNA-AQP3 in the transfection complex. Ovine secondary follicles were isolated and cultured in vitro for 6 days using standard protocols. Follicles were transfected on day 0 or 3 or on both days (0 and 3) and then cultured for an additional 3 or 6 days. As revealed by the fluorescence signal, the Lipofectamine®/BLOCK-iT™ complex (0.75 µl + 27.12 nM by 12 h of incubation) crossed the basement membrane and granulosa cell and reached the oocytes. In general, the rate of intact follicles was higher and the rate of antrum formation was lower in transfected follicles compared with control follicles. In conclusion, ovine secondary follicles can be successfully transfected during in vitro culture, and siRNA-mediated attenuation of AQP3 gene reduced antrum formation of secondary follicles.

This study aimed to investigate whether aquaporin 3 (Aqp3) mRNAs are expressed in immature oocytes and altered during in vitro maturation process. Five- to 6-week-old female ICR mice were primed by gonadotropin for 24 and 48 h. Immature oocytes obtained 48 h after priming were also matured in vitro for 17 to 18 h. In vivo matured oocytes were obtained after 48 h priming followed by hCG injection. Total RNAs were extracted from 80 to 150 oocytes in each experimental group, and the levels of Aqp3 mRNA were quantified by real-time reverse transcriptase polymerase chain reaction. The experiments were repeated twice using different oocytes. The Aqp3 mRNA was expressed in immature oocytes, as well as in in vitro and in vivo matured oocytes. The expression level was higher in immature oocytes obtained 48 h after priming (17.2 ± 8.6, mean ± SD) than those with no priming (5.7 ± 0.8) or obtained 24 h after priming (2.5 ± 0.8). The expression of Aqp3 mRNA decreased after in vitro maturation (1.2 ± 0.5), which was similar to in vivo matured oocytes (1.0 ± 0.0). Our work demonstrated that Aqp3 mRNA expression increased during the development of immature oocyte but decreased after completion of in vitro maturation. The results indicate that AQP3 is certainly needed for the acquisition of immature oocytes’ full growing potential within antral follicles.