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

AI Article Synopsis

  • Researchers aimed to create a successful method for transfecting ovine secondary follicles to study the effects of reducing aquaporin 3 (AQP3) on follicle growth and antrum formation.
  • They tested various volumes of Lipofectamine® and concentrations of fluorescent oligonucleotides, finding that specific combinations effectively allowed transfection to occur.
  • Ultimately, the study concluded that transfecting ovine secondary follicles successfully can lead to reduced antrum formation when AQP3 is inhibited, highlighting the potential for further research in ovarian follicle development.

Article Abstract

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.

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Source
http://dx.doi.org/10.1017/S096719941800031XDOI Listing

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