Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo

in Reproduction
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  • 1 Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Chungcheongbuk-do, Republic of Korea
  • 2 Department of Biotechnology, College of Engineering, Daegu University, Jillyang, Gyeongsan, Gyeongsangbuk-do, Republic of Korea
  • 3 Department of Animal Science and Biotechnology, College of Agriculture and Life Science, Chungnam National University, Daejeon, Republic of Korea
  • 4 Department of Animal Science, College of Natural Resources & Life Science, Pusan National University, Samrangjin-ro, Samrangjin-eup, Miryang, Gyeongsangnam-do, Republic of Korea
  • 5 Primate Resource Center, Korea Research Institute of Bioscience and Biotechnology, Jeollabuk-do, Republic of Korea
  • 6 Department of Functional Genomics, University of Science and Technology, Daejeon, Republic of Korea

Correspondence should be addressed to D-B Koo or S-U Kim; Email: dbkoo@daegu.ac.kr or sunuk@kribb.re.kr

*(M-H Lee, P-S Jeong and B-W Sim contributed equally to this work)

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In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0–2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2–4 days) and late phases (4–6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

Supplementary Materials

    • Supplementary Figure 1. Effect of various hypoxia treatment periods on the size of blastocysts derived from porcine parthenogenetically activated (PA) embryos. (A) Representative photographs of blastocysts 150, 200, and 250 μm in diameter. Scale bar, 50 μm. (B) Proportion of blastocysts 100-150, 150-200, and 200-250 μm in diameter after cultivation under 1% oxygen tension during different culture periods. Data are means ± standard error of the mean (SEM; *P < 0.05).
    • Supplementary Figure 2. Effects of autophagy induction on developmental competence of porcine in vitro-fertilized (IVF) embryos under extreme hypoxic conditions during early-phase in vitro culture (IVC). (A) Representative photographs of blastocysts (white asterisks) developed in the presence or absence of 10 nM rapamycin (RM) under 1% oxygen tension. Scale bar, 100 μm. (B) Representative fluorescence images of blastocysts stained with 4′,6-diamidino-2-phenylindole (DAPI) at 1% oxygen tension with and without RM, compared with control. Scale bar, 100 μm. Cleavage at (C) 24 h, and (D) 48 h; (E) blastocyst formation rates; (F) total cell numbers within blastocysts at 1% oxygen tension with and without RM, compared with control. (G) Representative merged images of DAPI [blue, inner cell mass (ICM)], CDX2 [green, trophectoderm (TE)], or terminal deoxynucleotidyl transferase-mediated dUTP-digoxygenin nick end-labeling (TUNEL; green) signals after immunostaining of porcine IVF blastocysts in different treatment combinations. Scale bar, 50 μm. (H) ICM, TE, and total cell numbers; (I) ICM/TE ratios; (J) apoptotic cell numbers; and (K) apoptosis rates for 1% oxygen tension with and without RM, compared with control. Data are means ± SEM (*P < 0.05).
    • Supplementary Figure 3. Effects of 3-methyladenine (3-MA) treatment on early development of porcine PA embryos cultivated under normoxia conditions during early-phase IVC. (A) Representative photographs of blastocysts (white asterisks) developed at various doses of 3-MA under 20% oxygen tension during early-phase IVC. Scale bar, 100 μm. (B) Cleavage (left panel) and blastocyst development (right panel) rates for different treatment combinations. Data are means ± SEM (*P < 0.05).
    • Supplementary Figure 4. Effects of RM treatment on mitochondrial function in porcine PA embryos cultivated under extreme hypoxic conditions during early-phase IVC. (A) Representative fluorescence images of PA embryos stained with MitoTracker (red) and DAPI (blue) after cultivation under 1% or 20% oxygen tension for 10, 24, and 48 h post-PA. Scale bar, 50 μm. (B) Relative fluorescence intensity of MitoTracker signals and (C) representative fluorescence images of porcine PA embryos stained with JC-1 after cultivation under 1% or 20% oxygen tension with and without RM for 10, 24, and 48 h post-PA. Scale bar, 50 μm. (D) Mitochondrial membrane potential of porcine PA embryos determined by densitometric analysis of JC-1 fluorescence after cultivation under 1% or 20% oxygen tension with and without RM. Data are means ± SEM (*P < 0.05).
    • Supplementary table S1. Primer sequences for RT-PCR
    • Supplementary Table S2. Effects of various oxygen concentrations on early development of porcine PA embryos
    • Supplementary Table S3. Effects of various extreme hypoxia treatment periods on cleavage and blastocyst formation rates in porcine PA embryos
    • Supplementary Table S4. Effects of various extreme hypoxia treatment periods on the size of blastocysts derived from porcine PA embryos
    • Supplementary Table S5. Effects of various extreme hypoxia treatment periods on the total cell numbers of blastocysts derived from porcine PA embryos
    • Supplementary Table S6. Effects of rapamycin (RM) treatment on cleavage and blastocyst formation rates in porcine PA embryos cultivated under extreme hypoxic conditions during early-phase in vitro cultivation (IVC)
    • Supplementary Table S7. Effects of rapamycin treatment on cleavage and blastocyst formation rates of porcine IVF embryos cultivated under extreme hypoxic conditions during early-phase IVC
    • Supplementary Table S8. Effects of 3-methyladenine (3-MA) treatment on the early development of porcine PA embryos cultivated under normoxia conditions during early-phase IVC
    • Supplementary Table S9. Effects of rapamycin treatment on ICM/TE ratios of porcine PA embryos cultivated under extreme hypoxic conditions during early-phase IVC
    • Supplementary Table S10. Effects of rapamycin treatment on ICM/TE ratios of porcine IVF embryos cultivated under extreme hypoxic conditions during early-phase IVC
    • Supplementary Table S11. Effects of rapamycin treatment on apoptosis of porcine PA blastocysts developed under extreme hypoxic conditions during early-phase IVC
    • Supplementary Table S12. Effects of rapamycin treatment on apoptosis of porcine IVF blastocysts developed under extreme hypoxic conditions during early-phase IVC

 

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