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

Published online by Cambridge University Press:  05 October 2018

Marcela Pinheiro Paz
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
Francisca Geovania Canafístula de Sousa
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
Benner Geraldo Alves
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
Carlos Henrique Lobo
Affiliation:
Laboratory of Reproductive Biology, Animal Science, Federal University of Ceará, Fortaleza, CE, Brazil
Antonia Débora Sales
Affiliation:
Estacio Fic, College & University. Fortaleza, Brazil
Kaio Cesar Simiano Tavares
Affiliation:
Molecular and Developmental Biology Lab, School of Medicine, University of Fortaleza, Fortaleza, CE, Brazil
Naíza Arcângela Ribeiro de Sá
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
Denise Damasceno Guerreiro
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
Carolina Maside
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
Rebeca Magalhães Pedrosa Rocha
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
Luciana Relly Bertolini
Affiliation:
Pontifical Catholic University of Rio Grande do Sul, Brazil
Vilceu Bordignon
Affiliation:
Department of Animal Science, McGill University, Ste-Anne-de-Bellevue, QC, Canada
José Ricardo de Figueiredo
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
Ana Paula Ribeiro Rodrigues*
Affiliation:
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil
*
Author for correspondence: Ana Paula Ribeiro Rodrigues. Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil. Av. Dr. Silas Munguba, 1700, Campus do Itaperi. Fortaleza, CE, Brasil. CEP: 60.714.903. Tel: +55 85 3101 9852. Fax: +55 85 3101 9840. E-mail: [email protected]

Summary

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.

Type
Research Article
Copyright
© Cambridge University Press 2018 

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