All animal experiments were approved by the Institutional Animal Care and Use Committee of South China Agricultural University. Adult CD1 mice (6–8 weeks old) were maintained in a temperature- and light-regulated environment with a 14 h light/10 h dark photoperiod. Pregnant and pseudopregnant female mice were obtained by mating with fertile or vasectomized male mice, respectively. The day when the vaginal plug was detected was defined as day 1 of pregnancy (D1) or pseudopregnancy.

The P₄ doses used in this experiment were based on our previous study (Liang et al., 2018). To investigate effects of excessive P₄ on early pregnancy, pregnant mice were subcutaneously injected with 2, 4, or 8 mg of P₄ (P0130, Sigma-Aldrich, St. Louis, MO) in 100 μL of sesame oil (S9057, Macklin, Shanghai, China) at 9:00 AM daily from days 3–7. Control mice received 100 μL of sesame oil. On day 8, the mice were sacrificed to collect uteri for further analysis.

To further examine effects of P₄, ovariectomized mice rested for 2 weeks were subcutaneously injected with 2, 4, or 8 mg of P₄ in 100 μL of sesame oil for 1, 3, or 7 consecutive days. Control mice received 100 μL of sesame oil. Mice were sacrificed 24 h after the last injection to collect uteri for further analysis.

Artificial decidualization was induced as previously described (Liang et al., 2018). Briefly, on day 4 of pseudopregnancy, 10 μL of sesame oil was injected into one uterine horn to induce decidualization, and the contralateral horn served as a control. Female mice undergoing artificial decidualization were subcutaneously injected with 4 mg P₄ daily from days 5–7, while controls received 100 μL of sesame oil. On day 8 of pseudopregnancy, mice were sacrificed to collect uteri for further analysis.

Mouse endometrial stromal cells were isolated and cultured as previously described (Li et al., 2023a). Briefly, the uteri of day 4 pseudopregnant mice were longitudinally incised and digested with HBSS (PB180321, Procell, Wuhan, China) containing 1% trypsin (0,458, VWR, Radnor, PA) and 6 mg/mL dispase (82,003,500, Sigma-Aldrich, St. Louis, MO). After the uteri were rinsed in HBSS to remove luminal epithelial cells, the remaining tissue was further digested with 0.15 mg/mL collagenase I (2,691,550, Gibco, Grand Island, NY). The collected stromal cells were cultured in DMEM/F12 medium (D2906, Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (164,210, Procell, Wuhan, China).

Mouse stromal cells were induced for in vitro decidualization using 10 nM E₂ (HY-B0141, MedChemExpress, NJ, USA) and 1 μM P₄ as previously described (Chen et al., 2023). The P₄ doses for the in vitro experiments were based on previous studies (Liang et al., 2018; Suthaporn et al., 2021). To investigate the effects of excessive P₄ on decidualization, stromal cells under in vitro decidualization were treated with different doses of P₄ and analyzed the mRNA levels of Prl8a2 and Prl3c1, markers of mouse in vitro decidualization. To examine effects of Kyn on decidualization, stromal cells under in vitro decidualization were treated with different concentrations of L-kynurenine (HY-104026; MedChemExpress, NJ, USA).

Kynurenine amount was measured as previously described (Chen et al., 2024a). Briefly, the cultured medium was collected from cultured stromal cells and centrifuged at 5,000×g for 10 min to remove cellular debris. Total 360 μL supernatant was mixed with 180 μL of 30% trichloroacetic acid (TCA; T6399, Sigma-Aldrich, St. Louis, MO) and incubated at 50 °C for 30 min. After the mixture was centrifuged at 3,000 × g for 10 min, the supernatant was thoroughly mixed with an equal volume of Ehrlich reagent (2% p-dimethylaminobenzaldehyde, D109644, Aladdin, Shanghai, China) and incubated for 12–30 min. The absorbance was measured at 492 nm to calculate the concentration using a standard curve of L-kynurenine.

qPCR was performed as previously described (Li et al., 2024). Total RNAs were extracted from mouse uterine tissue or mouse stromal cells using TRIzol (AG21101, Accurate Biology, Changsha, China). cDNA was synthesized from RNA using the HiScript II Q RT SuperMix kit (R222-01-AB, Vazyme, Nanjing, China). qPCR was performed using the SYBR Premix (Q311-02-AA, Vazyme, Nanjing, China). The data were analyzed using the 2^−ΔΔCt method and normalized to mouse Rpl7. The primer sequences were listed in Table 1.

Western blot was performed as previously described (Chen et al., 2024b). After tissues or cultured cells were lysed with RIPA (R0010, Solarbio, Beijing, China), the protein concentration was determined by the BCA method (23,225, Thermo Fisher Scientific, Waltham, MA). The samples were separated via SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Immobilon®-P, IPVH00010, Millipore, Billerica, MA). After blocked with 5% nonfat milk (A600669, Sangon Biotech, Shanghai, China), the PVDF membranes were incubated with each primary antibody and secondary antibody (1:5,000). The signal was detected using the ECL chemiluminescence kit (Millipore). The primary antibodies utilized in this study include IDO1 (51,851, Cell Signaling Technology, Danvers, MA), TDO (ab259359, Abcam, Cambridge, United Kingdom), BMP2 (A0231, ABclonal, Wuhan, China), SNAIL (3879T, Cell Signaling Technology, Danvers, MA), AhR (A00225-4, Boster, Wuhan, China), CYP1A1 (GTX55582, GeneTex), CYP1B1 (GTX104424, GeneTex), and α-TUBULIN (2144S, Cell Signaling Technology, Danvers, MA), GAPDH (SC-32233, Santa Cruz Biotechnology, Dallas, TX), Histone H3 (ab176842, Abcam, Cambridge, United Kingdom).

Immunofluorescence was performed as previously described (Li et al., 2023b). Briefly, paraffin sections were dewaxed and rehydrated. Antigen retrieval was achieved with citrate buffer (pH 6.0) or Tris/EDTA buffer (pH 9.0). Cell membranes were permeabilized with 0.1% Triton X-100 (T0694, Sangon Biotech, Shanghai, China) in PBS. After non-specific binding was blocked with horse serum (ZLI-9024, ZSGB-BIO, Beijing, China) for 1 h, sections were incubated with each primary antibody overnight at 4 °C and Alexa 488-conjugated secondary antibody (169,549, Jackson ImmunoResearch, West Grove, PA) at 37 °C for 30 min. Nuclei were counterstained with propidium iodide (PI, P4170, Sigma-Aldrich, St. Louis, MO) or 4′,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich, St. Louis, MO). Fluorescence signals were captured using a Nikon C2 confocal microscope. The primary antibodies used in this study include IDO1 (66,528-1, Proteintech, Wuhan, China), Phospho-AhR (PA5-36025, Invitrogen, Carlsbad, CA), AhR (A00225-4, Boster, Wuhan, China) and Nucleolin (14,574, Cell Signaling Technology, Danvers, MA).

The nuclear and cytoplasmic extractions were conducted as previously described (Deng et al., 2014). Cultured cells were washed twice with pre-chilled PBS, incubated with Buffer B (5 mM EDTA in PBS) on ice for 5 min and scraped off from culture plates. After centrifuged at 1,000 g for 5 min at 4 °C, the pellet was resuspended in Buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA with fresh added dithiothreitol and phenylmethylsulfonyl fluoride) and shaked at 4 °C for 20 min, mixed with 2.5% Nonidet P-40 and vortexed for 10 s. Following centrifugation at 15,000 g for 5 min at 4 °C, the supernatant was collected as cytoplasmic protein. The remaining pellet was resuspended in Buffer C (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, freshly added DTT and PMSF), vortexed, and centrifuged at 18,000 g for 5 min at 4 °C, and collected the supernatant as nuclear protein.

Data are presented as mean ± standard deviation. The two-tailed Student’s t-test was used to compare two groups. For more than two groups, one-way ANOVA was conducted with post hoc tests: LSD (if equal variances were assumed based on Levene’s test) or Games-Howell (if variances were unequal). Statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001.

To examine effects of excessive P₄ on decidualization, pregnant mice were subcutaneously injected with 4 mg or 8 mg of P₄ in 100 μL sesame oil daily from days 3–7 of pregnancy. Compared with controls, the decidual weight of implantation site on day 8 was significantly reduced by 4 mg or 8 mg P₄ treatments (Figure 1A). Alkaline phosphatase is a marker of mouse decidualization (Yee and Kennedy, 1988). The staining density of alkaline phosphatase activity in day 8 pregnant uterus was also significantly decreased by 4 mg or 8 mg P₄ (Figure 1B). Under artificial decidualization, the decidual weight on day 8 pseudopregnant mice was significantly reduced by 4 mg P₄ treatments from days 5–7 (Figure 1C). Prl8a2 and Prl3c1 serve as markers for mouse in vitro decidualization (Rasmussen et al., 1997). Under in vitro decidualization, Prl8a2 mRNA was significantly downregulated by 20 μM P₄, while no significant changes were observed by 0.16, 0.8, or 4 μM P₄ treatment for 2 days (Figure 1D). Meanwhile, Prl3c1 mRNA levels were significantly reduced by 10 μM or 20 μM P₄ (Figure 1D).

Trp is crucial during pregnancy and mainly metabolized via Kyn pathway (Badawy, 2015; Badawy et al., 2016; Hoang et al., 2023; Xue et al., 2023). TDO, IDO1 and IDO2 are the key rate-limiting enzymes in Kyn pathway and essential for pregnancy (Munn et al., 1998). Because excess P₄ is detrimental for pregnancy, we wondered whether Kyn pathway was affected by excess P₄. When ovariectomized mice were treated with 4 mg or 8 mg P₄ for 7 days, uterine Kyn levels were significantly increased (Figure 2A). IDO1 immunofluorescence signals in the uterine luminal epithelium were clearly increased after ovariectomized mice were treated with 4 mg or 8 mg P₄ for 24 h, while 2 mg P₄ had no obvious effect (Figure 2C). Uterine TDO protein levels were also upregulated by 2 mg or 4 mg P₄, but not by 8 mg P₄ (Figure 2D).

After stromal cells were treated with 0.16, 0.8, 4, or 20 μM P₄ for 2 days, Kyn secretion was significantly increased, which was abrogated by RU486, an antagonist of progesterone receptor (Figure 2B). IDO1 and TDO protein levels were also significantly increased after stromal cells were treated with 0.16, 0.8, or 4 μM P₄ for 3 days (Figure 2E). P₄-induced increases in IDO1 and TDO protein levels were blocked by RU486 treatments (Figure 2F). Epacadostat, a selective inhibitor of IDO1, effectively suppressed P₄-induced increases in IDO1 protein levels and Kyn secretion (Figures 2G,H).

Because high-dose P₄ increases Kyn levels, we explored whether Kyn had any effects on decidualization. Under in vitro decidualization, Prl8a2 mRNA levels were significantly downregulated in a dose-dependent manner by 0.25, 0.5, or 1 mM Kyn (Figure 3A). Meanwhile, Prl3c1 mRNA levels were upregulated by 0.5 mM Kyn, but downregulated by 1 mM Kyn (Figure 3A). BMP2 is essential for decidualization (Wang and Dey, 2006). BMP2 protein levels were downregulated after stromal cells were treated with 0.2, or 1 mM Kyn, whereas 0.04 mM Kyn had no detectable change on BMP2 protein levels for 2 days (Figure 3B). SNAIL, a key player during the epithelial-mesenchymal transition, is decreased during decidualization (Zhang et al., 2013; Serrano-Gomez et al., 2016). SNAIL protein levels were significantly upregulated after stromal cells were treated with 0.2, or 1 mM Kyn rather than 0.04 mM Kyn for 2 days (Figure 3B).

Kyn is an effective AhR agonist (DiNatale et al., 2010). Treatment of stromal cells with 1 mM Kyn increased the fluorescence intensity of nuclear AhR, which was abrogated by CH-223191, a specific AhR antagonist (Figure 3C). CYP1A1 and CYP1B1 are downstream targets of AhR (Denison and Whitlock, 1995; Nebert and Dalton, 2006; MacPherson et al., 2013). Under in vitro decidualization, Cyp1a1 and Cyp1b1 mRNA levels were significantly downregulated, but upregulated in a dose-dependent manner by 0.25, 0.5, or 1 mM Kyn (Figure 3D).

We further explored whether excessive P₄ could directly activate the AhR pathway. When ovariectomized mice were treated with 2 or 4 mg P₄, p-AhR immunofluorescence in stromal cells was enhanced (Figure 4A). The mRNA levels of Cyp1a1 and Cyp1b1 were significantly increased after ovariectomized mice were treated with 2, 4, or 8 mg P₄ for 7 days (Figure 4B). Furthermore, CYP1A1 and CYP1B1 protein levels in uterine tissues of ovariectomized mice significantly increased after 4 mg or 8 mg P₄ treatment (Figure 4C). After stromal cells were treated with 2.5, 5, 10, or 20 μM P₄ for 2 days, nuclear AhR protein levels were clearly elevated (Figure 4D). In addition, nuclear AhR fluorescence in stromal cells was enhanced after treatment with 0.8, 4, or 20 μM P₄ for 48 h (Figure 4E).

Under in vitro decidualization, Kyn significantly suppressed Prl8a2 mRNA levels, but upregulated Cyp1a1 and Cyp1b1 mRNA levels, which were reversed by CH-223191, a specific AhR antagonist (Figures 5A,B). CYP1A1 and CYP1B1 are cytochrome P450 enzymes that catalyze the formation of non-toxic 2-OH-E₂ and genotoxic 4-OH-E₂ from E₂ (Mao et al., 2023). Under in vitro decidualization, Prl8a2 and Prl3c1 mRNA levels were downregulated by 10 μM 2-OH-E₂ and 10 μM 4-OH-E₂, respectively (Figures 5C,D).

The nucleolus plays a crucial role in ribosome biogenesis. The morphology, size, and activity of nucleolus are closely linked, exhibiting diverse reorganization patterns under stress (Yang et al., 2018). AhR modulates nucleolar activity and enhances protein synthesis (Lafita-Navarro et al., 2018). Given that excess P₄ was detrimental to pregnancy and could activate the AhR pathway, we investigated excess P₄ effects on nucleoleus. Nucleolin (NCL), constituting approximately 10% of total nucleolar protein, serves as a nucleolar marker (Lo et al., 2006). After ovariectomized mice were subcutaneously injected with 2 mg or 8 mg P₄ for 7 days, the size and NCL intensity of nucleolus in the uterine stromal cells were obviously increased, while there were no clear changes for NCL immunofluorescence in luminal and glandular epithelium (Figure 6A). When stromal cells were treated with 0.5, 5, or 20 μM P₄ for 24 h, the size of nucleolar NCL immunofluorescence was also increased (Figure 6B).