Acute restraint stress triggers progesterone withdrawal and endometrial breakdown and shedding through corticosterone stimulation in mouse menstrual-like model

in Reproduction
Correspondence should be addressed to X-B Xu; Email:
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Stress impacts the reproductive axis at the level of the hypothalamus and the pituitary gland, which exert an effect on the ovary. Menstruation is regulated by the hypothalamic–pituitary–ovary (HPO) axis. However, the role of stress in menstruation remains unclear. The objective of this study was to explore the role of stress in endometrial breakdown and shedding, using the pseudopregnant mouse menstrual-like model. Female mice were mated with vasectomized males and labeled day 0.5, upon observation of a vaginal seminal plug. On day 3.5, decidualization was induced in pseudopregnant mice using arachis oil. On day 5.5, pseudopregnant mice with artificial decidualization were placed in restraint tubes for 3 h. The findings indicated that acute restraint stress resulted in the disintegration of the endometrium. While corticosterone concentration in the serum increased significantly due to restraint stress, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and progesterone (P4) levels in the serum decreased significantly. An endometrial histology examination indicated that progesterone implants may rescue P4 decline caused by acute stress and block endometrium breakdown and shedding. In addition, mice were treated with metyrapone, an inhibitor of corticosterone synthesis, 1 h prior to being subjected to restraint stress. Interestingly, metyrapone not only inhibited stress-induced endometrium breakdown and shedding, but also prevented stress-induced reduction of P4, LH and FSH. Furthermore, real-time PCR and western blot showed that mRNA and protein expression of CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and steroidogenic acute regulatory protein (StAR), the two rate-limiting enzymes for progesterone synthesis in the ovary, decreased following acute stress. But metyrapone prevented the reduction of StAR expression induced by restraint stress. Overall, this study revealed that acute stress results in an increase in corticosterone, which may inhibit LH and FSH release in the serum and CYP11A1 and StAR expression in the ovary, which finally leads to the breakdown and shedding of the endometrium. These experimental findings, based on the mouse model, may enable further understanding of the effects of stress on menstruation regulation and determine the potential factors affecting stress-associated menstrual disorders.


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    Schematic representation of the experimental design and timeline for restraint stress. Female mice were mated with vasectomized male mice. Pseudopregnancy in female mice was confirmed by observing vaginal plug (0.5 day). On day 3.5 at 15:30, 20 μL of arachis oil was injected into the bilateral uterine lumen of each mouse to induce decidualization. (A) Control group: on day 5.5, 19:30 was designated as 0 h. (B) Restraint stress group: on day 5.5, 19:30 was designated as 0 h. On day 5.5 from 16:30 to 19:30, pseudopregnant mice were individually put into transparent 50 mL plastic centrifuge tubes for 3 h. The end of the stress period at 19:30 was recorded as 0 h. (C) Metyrapone group and vehicle group (sterile saline): on day 5.5 at 19:30 was designated as 0 h. (D) P4 implant group: on day 5.5 at 15:30, P4 implants were inserted, and from 16:30 to 19:30, pseudopregnant mice were stressed for 3 h. Sham-operated mice underwent the same procedures except for the implantation of P4.

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    Macroscopic and histological examination of mouse uterus tissues 24 h after restraint stress. (A), (B) and (C) represent control group. (D), (E) and (F) represent restraint stress group. (A) and (D) represent the macroscopic observation. The uterine horns in control group are light pink in color, whereas those in restraint stress group were dark red. (B), (C), (E) and (F) represent morphological changes in the two groups. The whole endometrium was structurally intact in the control group, whereas the functional layer was sloughed off into the uterine lumen in the restraint stress group. (C) and (F) represent higher magnification of the areas indicated by an asterisk in (B) and (E).

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    Immunocytochemistry for MMP-2, MMP-9 and MMP-13 expression patterns 16 h after restraint stress. (A): (a) and (b) represent morphological changes in the control and stress groups, respectively, 16 h after restraint stress. (a1) and (b1) are higher magnification of the areas indicated by an asterisk in (a) and (b). Pyknosis and karyorrhexis are indicated in (b1) by a red arrow and black arrow, respectively. (c and d) MMP-2 (e and f) MMP-9 and (g and h) MMP-13 staining was observed by immunohistochemistry. MMP-2-, MMP-9- and MMP-13-positive signals were stronger in decidual stromal cells in the stress group than that in the control group. Insert images are parts of cross-section fields of uterine tissues. (B) Quantification and comparison of MMP-2, -9, -13 expression in endometrium after restraint stress. **P < 0.01.

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    Effect of stress on the concentrations of corticosterone (ng/mL), FSH (ng/mL) and LH (ng/mL). (A), (B) and (C) represent the serum levels of corticosterone, FSH and LH, respectively. Data are represented as mean ± s.d. of hormone concentration at the different time points. *P < 0.05, **P < 0.01.

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    Expression of glucocorticoid receptor (GR) in endometrium after restraint stress. (A) Immunohistochemical staining: There was no significant difference in the intensity of staining of GR between the pre-stress and stress (0, 16, 24 h) groups. Insert images are parts of cross-section fields of uterine tissues; magnified fields are indicated by the red square. (B) Western blot analysis: No significant expression of GR was observed in the endometrium of mice before stress as compared to expressions at 0, 16, 24 h after stress. S, stress group. Data are represented as mean ± s.d. β-actin was used as a loading control.

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    Effect of stress on the serum levels of E2 (pmol/L) and progesterone (nmol/L) in serum. Data were represented as mean ± s.d. of hormone concentration at the different time points. (A) E2 concentrations between the pre-stress and stress groups at different time points were not significant (P > 0.05). (B) The serum P4 levels in stress group were significantly lower than those in pre-stress group and control group at the same point of time. **P < 0.01.

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    Effect of metyrapone on the endometrial breakdown and shedding during the stress period. Metyrapone (150 mg per kg) dissolved in sterile saline and vehicle were administered intraperitoneally (IP) 1 h prior to the restraint stress (at 15:30 on day 5.5). (A) Representative macroscopic and morphologic changes of the bilateral uterine horns at 24 h after treatment with metyrapone show that uterine horns treated with vehicle are dark red in color, whereas those treated with metyrapone are light pink. Morphological analysis showed that endometrial breakdown was inhibited by metyrapone. (a2) and (b2) represent higher magnifications of the areas indicated by the red square in (a1) and (b1). (B) At 0 h, the serum level of corticosterone in metyrapone group was significant lower than vehicle group. (C) Serum levels of P4 (nmol/L) in the metyrapone group were significantly higher than those in the vehicle group after restraint stress. (D) At 0 h, the serum levels of FSH in the metyrapone group were significantly higher than those in the vehicle group. In the vehicle group, serum levels of FSH were not changed before and after stress. (E) At 0 h, there was a significant increase in serum concentrations of LH in the metyrapone group as compared to that in the vehicle group. Data are represented as mean ± s.d. *P < 0.05, **P < 0.01.

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    Effect of P4 implant on the endometrium breakdown and shedding during the stress period. (A) Cross-sections of uterine horns with P4 implant 24 h after restraint stress. (B) Cross-sections of stress uterine horns without P4 implant 24 h after restraint stress: (a) and (b) represent higher magnifications of the areas indicated by an asterisk in (A) and (B). (C) Serum concentrations of P4 (nmol/L) in the sham-operated group, P4 implant group and stress group before stress and at 0, 16 and 24 h after restraint stress (the sham-operated group was not subjected to stress). (D) Serum corticosterone level in the sham-operated group, progesterone implant group, and stress group before stress and 0, 16 and 24 h after restraint stress. Data were represented as mean ± s.d. **P < 0.01.

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    Expression patterns of Cyp11a1 and Star mRNA and protein after restraint stress. (A) Quantitative real-time PCR analysis demonstrates that the expression of Cyp11a1 and Star mRNA levels decreased after restraint stress. (B and C) Western blot analysis shows that CYP11A1 protein levels decreased from 8 to 16 h after restraint stress. And StAR protein levels sharply decreased from 0 to 16 h. (D) Western blot analysis of CYP11A1 and StAR in the ovary at 0 and 24 h. β-actin was used as a loading control. C, control group; M, metyrapone group; S, stress group; V, vehicle group. Data are represented as mean ± s.d. *P < 0.05, **P < 0.01.


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