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. 2021 Sep 1:2021:1764929.
doi: 10.1155/2021/1764929. eCollection 2021.

HIF-1 α Activation Promotes Luteolysis by Enhancing ROS Levels in the Corpus Luteum of Pseudopregnant Rats

Affiliations

HIF-1 α Activation Promotes Luteolysis by Enhancing ROS Levels in the Corpus Luteum of Pseudopregnant Rats

Zonghao Tang et al. Oxid Med Cell Longev. .

Abstract

The increase of oxidative stress is one of the important characteristics of mammalian luteal regression. Previous investigations have revealed the essential role of reactive oxygen species (ROS) in luteal cell death during luteolysis, while it is unknown how ROS is regulated in this process. Considering the decrease of blood flow and increase of PGF2α during luteolysis, we hypothesized that the HIF-1α pathway may be involved in the regulation of ROS in the luteal cell of the late corpus luteum (CL). Here, by using a pseudopregnant rat model, we showed that the level of both HIF-1α and its downstream BNIP3 was increased during luteal regression. Consistently, we observed the increase of autophagy level during luteolysis, which is regulated in a Beclin1-independent manner. Comparing with early (Day 7 of pseudopregnancy) and middle CL (Day 14), the level of ROS was significantly increased in late CL, indicating the contribution of oxidative stress in luteolysis. Inhibition of HIF-1α by echinomycin (Ech), a potent HIF-1α inhibitor, ameliorated the upregulation of BNIP3 and NIX, as well as the induction of autophagy and the accumulation of ROS in luteal cells on Day 21 of pseudopregnancy. Morphologically, Ech treatment delayed the atrophy of the luteal structure at the late-luteal stage. An in vitro study indicated that inhibition of HIF-1α can also attenuate PGF2α -induced ROS and luteal cell apoptosis. Furthermore, the decrease of cell apoptosis can also be observed by ROS inhibition under PGF2α treatment. Taken together, our results indicated that HIF-1α signaling is involved in the regression of CL by modulating ROS production via orchestrating autophagy. Inhibition of HIF-1α could obviously hamper the apoptosis of luteal cells and the process of luteal regression.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Immunohistochemistry (IHC) of LC-3I/II and p62 on the adjacent section of corpus luteum (CL) from pseudopregnant rats on Days 7, 14, and 21. Negative control (Ctrl) used serum instead of primary antibody. Bar = 100 μm.
Figure 2
Figure 2
Expressions of LC-3I/II, p62, and Beclin1 in the corpus luteum (CL) from pseudopregnant rats on Days 7, 14, and 21. (a) Immunohistochemistry (IHC) of Beclin1 on the adjacent section of corpus luteum (CL) from pseudopregnant rats on Days 7, 14, and 21. (b) Representative immunoblotting of LC-3I/II, p62, and Beclin1. (c) Densitometric qualification of LC-3I/II, p62, and Beclin1. P < 0.05 was considered to indicate a statistically significant difference. #P < 0.05, vs. Day 7. &P < 0.05, vs. Day 14.
Figure 3
Figure 3
Expressions of cleaved caspase-3, Bcl-2, and Bax in the corpus luteum (CL) from pseudopregnant rats on Days 7, 14, and 21. (a) Representative immunoblotting of cleaved caspase-3, Bcl-2, and Bax. (b) Densitometric qualification of cleaved caspase-3, Bcl-2, and Bax. (c) Relative ROS level in CLs of pseudopregnant rats. P < 0.05 was considered to indicate a statistically significant difference. #P < 0.05, vs. Day 7. &P < 0.05, vs. Day 14.
Figure 4
Figure 4
Expressions of VDAC1 and PINK1 in the corpus luteum (CL) from pseudopregnant rats on Days 7, 14, and 21. (a) Representative immunoblotting of VDAC1 and PINK1. (b) Densitometric qualification of VDAC1 and PINK1. Data are presented as the mean ± SE. The significant differences within or between groups were evaluated by one-way analysis of variance, followed by Tukey's multiple range test. P < 0.05 was considered to indicate a statistically significant difference. #P < 0.05, vs. Day 7. &P < 0.05, vs. Day 14.
Figure 5
Figure 5
Expressions of HIF-1α, BNIP3, and NIX in the corpus luteum (CL) from pseudopregnant rats on Days 7, 14, and 21. (a) Representative immunoblotting of HIF-1α, BNIP3, and NIX. (b) Densitometric qualification of HIF-1α, BNIP3, and NIX. (c) BNIP3 and NIX mRNA expressions in the corpus luteum (CL) from pseudopregnant rats on Days 7, 14, and 21. (d) Morphology of the ovary on Day 21 from pseudopregnant rats treated with vehicle (Veh) or echinomycin (Ech). P < 0.05 was considered to indicate a statistically significant difference. #P < 0.05, vs. Day 7. &P < 0.05, vs. Day 14.
Figure 6
Figure 6
Expressions of cleaved caspase-3, Bcl-2, and Bax in the corpus luteum (CL) on Days 7 and 21 from pseudopregnant rats treated with vehicle (Veh) or echinomycin (Ech). (a) Representative immunoblotting of cleaved caspase-3, Bcl-2, and Bax. (b) Densitometric qualification of cleaved caspase-3, Bcl-2, and Bax. (c) Relative ROS level of CLs with or without Ech treatment on Days 7 and 21 of pseudopregnancy. P < 0.05 was considered to indicate a statistically significant difference. #P < 0.05, vs. Day 21 without Ech.
Figure 7
Figure 7
Expressions of LC-3I/II, BNIP3, and NIX in the corpus luteum (CL) on Days 7 and 21 from pseudopregnant rats treated with vehicle (Veh) or echinomycin (Ech). (a) Representative immunoblotting of LC-3I/II, BNIP3, and NIX. (b) Densitometric qualification of LC-3I/II, BNIP3, and NIX. P < 0.05 was considered to indicate a statistically significant difference. #P < 0.05, vs. Day 21 without Ech.
Figure 8
Figure 8
Expressions of p62 and VDAC1 in the corpus luteum (CL) on Days 7 and 21 from pseudopregnant rats treated with vehicle (Veh) or echinomycin (Ech). (a) Representative immunoblotting of p62 and VDAC1. (b) Densitometric qualification of p62 and VDAC1. P < 0.05 was considered to indicate a statistically significant difference. #P < 0.05, vs. Day 21 without Ech.
Figure 9
Figure 9
Inhibition of autophagy attenuates PGF2α-induced ROS production and cell apoptosis. (a) Inhibition of autophagy attenuates ROS production in luteal cells. Cells were treated with PGF2α for 24 h with or without 3-MA. (b) Representative immunoblotting and (c) densitometric qualification for the effect of autophagy inhibition on caspase-3 activation. Cells were treated with PGF2α for 24 h with or without 3-MA. #P < 0.05, vs. PGF2α.

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