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. 2018 Aug 15;16(1):79.
doi: 10.1186/s12958-018-0396-0.

Peroxiredoxin I Maintains Luteal Function by Regulating Unfolded Protein Response

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Free PMC article

Peroxiredoxin I Maintains Luteal Function by Regulating Unfolded Protein Response

Hyo-Jin Park et al. Reprod Biol Endocrinol. .
Free PMC article

Abstract

Background: Mounting evidence shows that ROS regulation by various antioxidants is essential for the expression of enzymes involved in steroidogenesis and maintenance of progesterone production by the corpus luteum (CL). However, the underlying mechanisms of peroxiredoxin 1 (PRDX1), an antioxidant enzyme, in luteal function for progesterone production in mice have not been reported. The aim of this study was to evaluate the functional link between PRDX1 and progesterone production in the CL of Prdx1 knockout (K/O) mice in the functional stage of CL.

Methods: The expression pattern of the unfolded protein response (UPR) signaling pathways, endoplasmic reticulum (ER) stress-induced apoptosis related genes and peroxiredoxins 1 (PRDX1) were investigated by western blotting analysis in CL tissue of 10 weeks mice during functional stage of CL. The protein levels of these genes after ER-stress inducer tunicamycin (Tm), ER-stress inhibitor tauroursodeoxycholic acid (TUDCA) and ROS scavenger, N-acetylcysteine (NAC) stimulation by intraperitoneal (i.p) injection were also investigated in CL tissue of wild type (WT) mice. Finally, we examined progesterone production and UPR signaling related gene expression in CL tissue of Prdx1 K/O mice.

Results: We demonstrated that PRDX1 deficiency in the functional stage activates the UPR signaling pathways in response to ER stress-induced apoptosis. Interestingly, CL number, serum progesterone levels, and steroidogenic enzyme expression in Prdx1 K/O mice decreased significantly, compared to those in wild type mice. Levels of UPR signaling pathway markers (GRP78/BIP, P50ATF6, and phosphorylated (p)-eIF2) and ER-stress associated apoptotic factors (CHOP, p-JNK, and cleaved caspase-3) were dramatically increased in the CL tissue of Prdx1 K/O mice. In addition, administration of the NAC, reduced progesterone production and activated ER-stress-induced UPR signaling in the CL tissue obtained from the ovary of Prdx1 K/O mice. Taken together, these results indicated that reduction in serum progesterone levels and activation of ER-stress-induced UPR signaling are restored by NAC injection in the CL of Prdx1 K/O mice.

Conclusion: These observations provide the first evidence regarding the basic mechanisms connecting PRDX1 and progesterone production in the functional stage of CL.

Keywords: Apoptosis; Corpus luteum; Peroxiredoxin 1; Unfolded protein response.

Conflict of interest statement

Ethics approval

All animal care and experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of KRIBB (permit number: KRIBB-AEC-17092).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
The number of CL, serum progesterone and 3β-HSD expression were increased 48 h to peak levels of progesterone production after PMSG/hCG injection. a Schematic diagram of ovary tissue selected 16, 24, 48, 72, and 96 h after PMSG/hCG injection. CL tissues were carefully collected from the ovaries after ovulation. b Luteal phase samples were collected at different time points based on CL morphology. c Serum progesterone levels were determined by progesterone ELISA kit (ALPCO) for 4–5 animals at each time point of the luteal phase during the estrous cycle. d Western blotting analysis of 3β-HSD, a marker of progesterone synthesis in the CL. The 3β-HSD level was normalized to that of the β-actin control. The histogram represents values of densitometry analysis obtained using the Image J software (NIH). The data are representative of at least three independent experiments conducted in triplicate. Values represent means ± SEM (4–5 mice/group) * P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 2
Fig. 2
ER-stress-induced reduction in progesterone production at the functional stage of CL is recovered by ROS scavenger NAC. a Schematic diagram showing the injection and collection times of the ER stress-regulator and ROS scavenger injection. Tm (an ER stress inducer; 0.5 μg/g body weight), TUDCA, (an ER stress inhibitor; 0.5 μg/g body weight), or NAC (a ROS scavenger; 1.0 μg/g body weight) were injected into mice 45 h after the PMSG/hCG injection. b Peak production of serum progesterone was measured (48 h after the PMSG/hCG injection) in the luteal phase. c Protein level of the UPR signaling activation marker CHOP in selected CL tissues was measured by western blot analysis after ER stress regulator and ROS scavenger stimulation. The levels of 3β-HSD, P450scc, and CHOP were normalized to that of β-actin. The histogram represents values of densitometry analysis obtained using the Image J software (NIH). The bar graphs represent the least-squares means ± SEM (4–5 mice/group) of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; Dunnett’s multiple comparison test compared to 48 h after PMSG/hCG injection
Fig. 3
Fig. 3
Increased PRDX1 expression by injection of ER-stress inducer (Tm) in the CL tissue was recovered by ER-stress inhibitor (TUDCA) or ROS scavenger (NAC). a Schematic diagram showing the time taken to reach peak progesterone levels in CL 48 h after PMSG/hCG injection. b Protein levels of PRDX1 and 3β-HSD were measured by western blot analysis in different sampling times of the luteal phase. c Schematic diagram showing the timing of ER stress-regulator and ROS scavenger injection and sample collection. d PRDX1 levels in selected CL tissues were measured using western blot analysis in the presence of the ER stress inducer (Tm) (0.5 μg/g body weight) or ER stress inhibitor (TUDCA) (0.5 μg/g body weight). And ER stress inhibitor (TUDCA) (0.5 μg/g body weight) or ROS scavenger (NAC) (1.0 μg/g body weight) after pre-treatment with the ER stress inducer (0.5 μg/g Tm), respectively. 3β-HSD and PRDX1 levels were normalized to that of β-actin. The histogram represents values densitometry analysis was performed using the Image J software (NIH). The bar graphs represent the least-squares means ± SEM (4–5 mice/group) of three independent experiments. * P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 4
Fig. 4
The CL number, serum progesterone, 3β-HSD, and P450ssc expression were decreased in Prdx1 K/O mice. a We performed genotyping of PRDX1 by PCR in the tail of Prdx1 K/O mice. b CL number in the ovary of Prdx1 K/O mice was observed by hematoxylin and eosin (H & E) staining. c Serum progesterone levels were measured using a progesterone kit in Prdx1 K/O mice. d The levels of the steroidogenic enzymes 3β-HSD and P450SCC were determined in CL tissue by western blot analysis. The relative levels of these proteins were obtained after normalization with β-actin levels. The histogram values of densitometry analysis were obtained using the Image J software. The bars represent the mean of two independent experiments means ± SEM (4–5 mice/group) * P < 0.05, **P < 0.01, and ***P < 0.001; two-tailed Student’s t-test compared to WT mice
Fig. 5
Fig. 5
Protein levels of UPR signal activation and ER stress-mediated apoptosis in CL functional stage were induced in the CL tissue of Prdx1 K/O mice. a Western blot analysis to evaluate the levels of PRDX1 and UPR signaling-related proteins (GRP78/BIP, p-EIF2α, EIF2α, p50ATF5, and p-IRE1) and (b) ER stress-mediated apoptotic factors (CHOP, p-JNK, JNK, and caspase-3) in the CL. The relative levels of these proteins were obtained after normalization with β-actin levels. The histogram values of densitometry analysis were obtained using the Image J software. The bars represent the means of two independent experiments ± SEM (4–5 mice/per group) * P < 0.05, **P < 0.01, and ***P < 0.001; two-tailed Student’s t-test compared to WT mice. c IHC staining for UPR markers (p-EIF2α and ATF6) and ER stress apoptosis marker (cleaved caspase-3) in CL tissue from ovary of Prdx1 K/O mice. Scale bar = 200 μm
Fig. 6
Fig. 6
Deceased serum progesterone, steroidogenesis enzymes, and increased activation of UPR signaling in response to ER stress in Prdx1 K/O mice are restored by injecting NAC, a ROS scavenger. a Serum progesterone levels of PRDX1 were measured using a progesterone kit after NAC (1.0 μg/g) i.p injection. b RT-PCR analysis of Prdx1 and three major genes encoding steroidogenesis enzymes (3β-HSD, StAR, and P450scc). c The levels of PRDX1, UPR signaling-related proteins (GRP78/BIP and p50ATF5), and ER stress-mediated apoptotic factors (CHOP) were determined in the CL tissue of Prdx1 K/O mice by western blot analysis after NAC (1.0 μg/g) i.p injection. The relative mRNA/protein levels were normalized to those of GAPDH/β-actin controls. The histogram values of densitometry analysis were obtained using Image J software. The data are representative of at least three independent experiments conducted in triplicate and shown as means ± SEM (3–5 mice/group). * P < 0.05, **P < 0.01, and ***P < 0.001; Dunnett’s multiple comparison test, compared to 48 h after PMSG/hCG injection
Fig. 7
Fig. 7
Roles of PRDX1 on progesterone production, and UPR signaling activation in CL tissue of mice. Graphical summary. Right panel; ROS reduction by PRDX1 during the luteal functional stage (48 h after PMSG/hCG injection) in WT mice decreased activated UPR signaling in response ER stress and increased steroidogenesis enzymes expression in the CL to promote progesterone production. Left panel; Increasing ROS by PRDX1 deficiency during the luteal functional stage of Prdx1 K/O mice increased activated UPR signaling in response ER stress mediated apoptosis and decreased progesterone production related steroidogenesis enzymes expression in CL tissue and serum progesterone levels

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