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제안 방법
- The vitrified oocytes were stored in LN2 for 2 weeks. After 2 weeks, they were warmed with sucrose solutions of decreasing concentrations (0.5, 0.25, 0.125, and 0 M) and 20% FBS for 2.5 minutes each. The vitrified-warmed oocytes were then cultured for 1 to 2 hours in medium with HEPES containing 20% FBS for recovery.
- Atg7f/f female mice were intraperitoneally injected with 7.5 IU of pregnant mare’s serum gonadotropin (Sigma-Aldrich, St. Louis, MO, USA) to induce folliculogenesis, followed by injection of 7.5 IU of human chorionic gonadotropin (Sigma-Aldrich) 48 hours later to induce superovulation.
- Rabbit IgG was used as a mock control. Immunofluorescence images were obtained using an LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany), and analyzed using the Zen 2009 Light Edition (Carl Zeiss), a platform associated with the confocal microscope. For quantification of the LC3 puncta, each oocyte was imaged at four planes (3 μm intervals) using an LSM710 confocal microscope.
- Thus, it remains to be determined whether a complete absence of autophagy has any effect on the survival, fertilization, or development of vitrified-warmed oocytes. In this work, we aim to address two aspects of autophagy in mouse oocytes: first, the consequences of a complete absence of Atg7, a seminal gene in autophagy, in vitrified-warmed oocytes, and second, assessment of LC3 immunofluorescence staining as a reliable method of monitoring autophagy in oocytes.
대상 데이터
- KU14099). Atg7 floxed mice (Atg7f/f mice, C57BL/6) [7] were obtained from the RIKEN BioResource Center (Ibaraki, Japan). Zp3-Cre transgenic mice (C57BL/6) were purchased from Jackson Laboratory (Bar Harbor, ME, USA).
- Atg7 floxed mice (Atg7f/f mice, C57BL/6) [7] were obtained from the RIKEN BioResource Center (Ibaraki, Japan). Zp3-Cre transgenic mice (C57BL/6) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). To produce the Atg7f/f;Zp3-Cre mice, Atg7f/f female mice were mated with Atg7f/+;Zp3-Cre male mice, and the litters were selected as either experimental or control depending on the genotype (Table 1).
데이터처리
- 5 (GraphPad, La Jolla, CA, USA). Statistical analyses were performed using the Student t-test (one-tailed). The criteria for statistical significance were p< 0.
- The values represent the mean ± standard deviation (n = 4). Statistical significance was assessed by the Student t-test. (D) Fertilization and developmental rates of vitrified-warmed Atg7d/d and Atg7f/f oocytes.
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