Effect of aquaporin 3 knockdown by RNA interference on antrum formation in sheep secondary follicles cultured in vitro

• Published in: Zygote (Cambridge) Vol. 26; no. 5; p. 350
• Main Authors: Paz, Marcela Pinheiro, de Sousa, Francisca Geovania Canafístula, Alves, Benner Geraldo, Lobo, Carlos Henrique, Sales, Antonia Débora, Tavares, Kaio Cesar Simiano, de Sá, Naíza Arcângela Ribeiro, Guerreiro, Denise Damasceno, Maside, Carolina, Rocha, Rebeca Magalhães Pedrosa, Bertolini, Luciana Relly, Bordignon, Vilceu, de Figueiredo, José Ricardo, Rodrigues, Ana Paula Ribeiro
• Format: Journal Article
• Language: English
• Published: England 01-10-2018
• Subjects: Animals; Aquaporin 3 - genetics; Aquaporin 3 - metabolism; Cell Culture Techniques; Female; Gene Knockdown Techniques; Lipids; Ovarian Follicle - growth & development; Ovarian Follicle - physiology; RNA Interference; Sheep; Transfection - methods; GC granulosa cells; t0/3D6 transfected on day 0 and day 3; t0D3 transfected on day 0 and cultured for 3 days; t3D6 transfected on day 3 and cultured for 3 more days; Small interfering RNA; Ovine; siRNA small interfering RNA; AQP aquaporin; siRNA-AQP3 small interfering against aquaporin 3; t0D6 transfected on day 0 and cultured for 6 days; Transfection; and cultured until day 6; CD3 cultured in vitro for 3 days; Secondary follicle; AF/t0 antral follicle transfected on day 0; CD6 cultured in vitro for 6 days; IVC in vitro culture; AF in vivo antral follicle in vivo; tNEG Negative Control Transfected; Aquaporin 3
• ISSN: 1469-8730
• DOI: 10.1017/S096719941800031X

SummaryThe 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.