Low-Intensity pulsed ultrasound reduces ovarian cryopreservation injury through the Notch signaling pathway.

To evaluate the impact of cryopreservation on ovarian tissue, we assessed oxidative stress markers (3-NT, 4-HNE, and 8-OHdG) using IHC. The staining intensity and area were significantly greater in the cryopreserved group than in the control group ( p  < 0.05; Fig.  2 A). Moreover, we performed TUNEL staining, which is a marker of apoptosis. The cryopreserved group exhibited a significantly greater number of TUNEL-positive cells ( p  < 0.05; Fig.  2 E). We analyzed the expression of oxidative stress-related proteins via WB. As shown in Fig.  2 G, compared with those in fresh ovarian tissue, the expression levels of Nrf2 and HO-1 in cryopreserved tissue were significantly lower ( p  < 0.05), suggesting that cryopreservation may have compromised the antioxidant defense system, leading to oxidative stress. Collectively, these findings indicate that cryopreservation induces oxidative damage in ovarian tissue. Fig. 2 Cryopreservation causes oxidative damage to ovarian tissue: A - D : The immunohistochemical results and statistical bar charts of 3-NIT ( A , B ), 4-HNE ( A , C ), and 8-OHdG ( A , D ) in the control group and the cryopreservation (CP) group. The brown area indicates the positive area (50 μm). ( n  = 3 ovaries from different mice); E , F : TUNEL staining ( E ) and statistical bar chart of the control group and the CP group ( F ). Green indicates positive staining areas (50 μm). ( n  = 3 ovaries from different mice); G - I : Western blot and statistical bar charts of Nrf2 ( G , H ) and Ho-1 ( G , I ) expression in the control group and the CP group ((* P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001)). ( n  = 3 mice) Cryopreservation causes oxidative damage to ovarian tissue: A - D : The immunohistochemical results and statistical bar charts of 3-NIT ( A , B ), 4-HNE ( A , C ), and 8-OHdG ( A , D ) in the control group and the cryopreservation (CP) group. The brown area indicates the positive area (50 μm). ( n  = 3 ovaries from different mice); E , F : TUNEL staining ( E ) and statistical bar chart of the control group and the CP group ( F ). Green indicates positive staining areas (50 μm). ( n  = 3 ovaries from different mice); G - I : Western blot and statistical bar charts of Nrf2 ( G , H ) and Ho-1 ( G , I ) expression in the control group and the CP group ((* P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001)). ( n  = 3 mice) To assess the protective efficacy of LIPUS on cryopreserved ovarian tissue, we performed WB analysis to measure the expression of HO-1, a critical antioxidant enzyme regulated by the Nrf2 pathway [ 13 ]. As illustrated in Fig. 3 A-F, we systematically evaluated three key LIPUS parameters—treatment duration, duty cycle, and intensity—to identify the optimal conditions. We evaluated the effects of varying LIPUS exposure durations (1, 5, and 10 min) on HO-1 expression. Notably, HO-1 levels significantly increased at the 5-minute time point ( p < 0.05) (Fig. 3 A and B). HO-1 expression was significantly higher in the 20% duty cycle group than in the other groups ( p < 0.05; Fig. 3 C). Furthermore, analysis of different ultrasound intensities revealed that HO-1 expression peaked at 0.5 W/cm², with levels significantly exceeding those at 0.1 W/cm² and 1.0 W/cm² ( p < 0.05; Fig. 3 E). These results indicate that LIPUS upregulates HO-1 expression in a parameter-dependent manner, with optimal induction achieved at 5 min, a 20% duty cycle, and 0.5 W/cm². Fig. 3 LIPUS can ameliorate oxidative damage to mouse ovarian tissue caused by cryopreservation, and the optimal parameters for LIPUS can be verified. A , B : Western blot ( A ) and statistical bar charts ( B ) of HO-1 expression in the CP group and different ultrasound exposure times in the CP + LIPUS group. ( n  = 3 mice); C - D Western blot ( C ) and statistical bar charts ( D ) of HO-1 in the CP and different ultrasound duty cycles of the CP + LIPUS group. ( n  = 3 mice); E , F : Western blot ( E ) and statistical bar charts ( F ) of HO-1 expression in the CP group and different ultrasound emission intensity groups treated with CP + LIPUS. ( n  = 3 mice); G - J : Immunohistochemical and statistical bar charts of 3-NIT (G, H), 4-HNE (G, I), and 8-OHdG (G, J) in the CP group and the CP + LIPUS group. The brown area indicates the positive area (50 μm). ( n  = 3 ovaries from different mice); K , L : The TUNEL fluorescence staining ( K ) and statistical bar chart ( L ) of the cryopreservation group and the cryopreservation + LIPUS group. Green indicates positive staining areas (50 μm); * P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001; n  = 3 ovaries from different mice LIPUS can ameliorate oxidative damage to mouse ovarian tissue caused by cryopreservation, and the optimal parameters for LIPUS can be verified. A , B : Western blot ( A ) and statistical bar charts ( B ) of HO-1 expression in the CP group and different ultrasound exposure times in the CP + LIPUS group. ( n  = 3 mice); C - D Western blot ( C ) and statistical bar charts ( D ) of HO-1 in the CP and different ultrasound duty cycles of the CP + LIPUS group. ( n  = 3 mice); E , F : Western blot ( E ) and statistical bar charts ( F ) of HO-1 expression in the CP group and different ultrasound emission intensity groups treated with CP + LIPUS. ( n  = 3 mice); G - J : Immunohistochemical and statistical bar charts of 3-NIT (G, H), 4-HNE (G, I), and 8-OHdG (G, J) in the CP group and the CP + LIPUS group. The brown area indicates the positive area (50 μm). ( n  = 3 ovaries from different mice); K , L : The TUNEL fluorescence staining ( K ) and statistical bar chart ( L ) of the cryopreservation group and the cryopreservation + LIPUS group. Green indicates positive staining areas (50 μm); * P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001; n  = 3 ovaries from different mice To investigate the protective effect of LIPUS on cryopreserved ovarian tissue, we analyzed the expression of key antioxidant-related proteins and apoptosis markers. WB analysis revealed that compared with cryopreserved treatment, LIPUS treatment significantly upregulated the protein levels of HO-1, a critical regulator of antioxidant stress ( p  < 0.05). These results suggest that LIPUS enhances the activity of the antioxidant defense system, potentially mitigating oxidative damage in ovarian tissue. Additionally, we assessed oxidative stress markers (3-NT, 4-HNE, and 8-OHdG) via IHC. Compared with the cryopreserved group, the LIPUS group exhibited reduced staining intensity and area for these markers ( p  < 0.05; Fig.  3 G). To further validate the protective role of LIPUS, we performed TUNEL staining to evaluate apoptosis. LIPUS treatment significantly decreased the number of TUNEL-positive cells compared with that in the cryopreserved group ( p  < 0.05; Fig.  3 K). Collectively, these findings demonstrate that LIPUS attenuates oxidative damage and reduces apoptosis in cryopreserved ovarian tissue. To better understand the molecular mechanisms by which LIPUS improves oxidative damage, we conducted proteomic and phosphoproteomic sequencing and functionally enriched the differentially expressed proteins. A total of 346 differential proteins ( p value < 0.05, | log2(FC)| ≥ 1) were identified between the cryopreserved group and the control group, among which 142 were upregulated and 204 were downregulated. The upregulated genes were enriched mainly in the functions of the CAMP signaling pathway, osteoclast differentiation, the PPAR signaling pathway and ovarian steroidogenesis. The downregulated genes were enriched mainly in the terms “chemical carcinogenesis-reactive oxygen species”, “Parkinson’s disease”, and “glycolysis”. There were a total of 101 differentially expressed proteins in the cryopreserved + LIPUS group and the cryopreserved group, among which 61 were upregulated and 40 were downregulated. The upregulated differentially expressed proteins were enriched mainly in the terms “cytokine–cytokine receptor interaction”, “chemokine signaling pathway”, and “human papillomavirus infection”. The downregulated genes were enriched mainly in “osteoclast differentiation”, “cGMP-PKG signaling pathway” and “calcium signaling pathway”. A total of 3,061 differential sites ( p value < 0.05, | log2(FC)| ≥ 1) were identified between the cryopreserved group and the control group, among which 803 were upregulated and 2,258 were downregulated. The upregulated differentially phosphorylated proteins were enriched mainly in the terms “autophagy”, “transcriptional misregulation in cancer”, “thyroid hormone signaling pathway”, and “Notch signaling pathway”. The downregulated differentially phosphorylated proteins were enriched mainly in the terms “spliceosome”, “proteoglycans in cancer”, and “nucleocytoplasmic transport”. There were a total of 945 differences between the cryopreserved + LIPUS group and the cryopreserved group, among which 535 were upregulated and 410 were downregulated. The upregulated differentially phosphorylated proteins were enriched mainly in the terms “tight junction”, “cell cycle” and “necroptosis”. The downregulated differentially phosphorylated proteins were enriched mainly in the terms “motor proteins”, “apoptosis”, “microRNAs in cancer”, and “Notch signaling pathways”. Among these, the Notch pathway was upregulated in the cryopreserved group compared with that in the control group; however, LIPUS treatment inhibited this increase. These results indicate that LIPUS may ameliorate oxidative damage to ovarian tissue by regulating the Notch pathway (Figs.  4 and 5 ). Fig. 4 Proteomics detection and differential protein analysis. A : PCA plots of reliable protein analysis in the control group and the CP group; B : Volcano maps of differential proteins in the control group and the CP group. C : Comparison chart of upregulated and downregulated items in the KEGG enrichment analysis between the control group and the CP group; D : Top 15 bubble charts of the GO enrichment analysis in the control group and the CP group; E : PCA plots of the reliable protein analysis in the CP group and the CP + LIPUS group; F : Volcano maps of the differentially expressed proteins in the CP group and the CP + LIPUS group. G : Comparison chart of upregulated and downregulated genes according to the results of the KEGG enrichment analysis between the CP group and the CP + LIPUS group; H: Top 15 genes according to the results of the GO enrichment analysis in the CP group and the CP + LIPUS group. (CP_L: CP + LIPUS group); Ctr ( n  = 3 samples from more than 9 mice), CP ( n  = 3 samples from more than 9 mice), CP + LIPUS ( n  = 4 samples from more than 12 mice) Proteomics detection and differential protein analysis. A : PCA plots of reliable protein analysis in the control group and the CP group; B : Volcano maps of differential proteins in the control group and the CP group. C : Comparison chart of upregulated and downregulated items in the KEGG enrichment analysis between the control group and the CP group; D : Top 15 bubble charts of the GO enrichment analysis in the control group and the CP group; E : PCA plots of the reliable protein analysis in the CP group and the CP + LIPUS group; F : Volcano maps of the differentially expressed proteins in the CP group and the CP + LIPUS group. G : Comparison chart of upregulated and downregulated genes according to the results of the KEGG enrichment analysis between the CP group and the CP + LIPUS group; H: Top 15 genes according to the results of the GO enrichment analysis in the CP group and the CP + LIPUS group. (CP_L: CP + LIPUS group); Ctr ( n  = 3 samples from more than 9 mice), CP ( n  = 3 samples from more than 9 mice), CP + LIPUS ( n  = 4 samples from more than 12 mice) Fig. 5 Phosphorylated proteomics detection and differential site analysis. A : PCA plots of reliable site analysis in the control group and the CP group; B : Volcanic maps of differential phosphorylation sites in the control group and the CP group; C : Comparison chart of upregulated and downregulated items in KEGG enrichment analysis between the control group and the CP group; D : Top 15 bubble charts of GO enrichment analysis in the control group and the CP group; E : PCA plots of reliable site analysis in the CP group and the CP + LIPUS group. F : Volcanic maps of differential phosphorylation sites in the CP group and the CP + LIPUS group; G: Comparison chart of upregulated and downregulated genes according to the results of the KEGG enrichment analysis between the CP group and the CP + LIPUS group; H: Top 15 genes according to the results of the GO enrichment analysis in the CP group and the CP + LIPUS group. Ctr ( n  = 3 samples from more than 9 mice), CP ( n  = 3 samples from more than 9 mice), CP + LIPUS ( n  = 4 samples from more than 12 mice) Phosphorylated proteomics detection and differential site analysis. A : PCA plots of reliable site analysis in the control group and the CP group; B : Volcanic maps of differential phosphorylation sites in the control group and the CP group; C : Comparison chart of upregulated and downregulated items in KEGG enrichment analysis between the control group and the CP group; D : Top 15 bubble charts of GO enrichment analysis in the control group and the CP group; E : PCA plots of reliable site analysis in the CP group and the CP + LIPUS group. F : Volcanic maps of differential phosphorylation sites in the CP group and the CP + LIPUS group; G: Comparison chart of upregulated and downregulated genes according to the results of the KEGG enrichment analysis between the CP group and the CP + LIPUS group; H: Top 15 genes according to the results of the GO enrichment analysis in the CP group and the CP + LIPUS group. Ctr ( n  = 3 samples from more than 9 mice), CP ( n  = 3 samples from more than 9 mice), CP + LIPUS ( n  = 4 samples from more than 12 mice) To further validate whether LIPUS ameliorates oxidative damage associated with the Notch pathway in cryopreserved mouse ovarian tissue, we used the Notch pathway inhibitor CB103 to assess HO-1 expression and oxidative stress levels. CB103 at a concentration of 200 µM resulted in optimal inhibition of Notch and maximal upregulation of HO-1 protein expression ( p  < 0.05; Fig.  6 A). Cryopreserved ovarian tissues were subsequently treated with 200 µM CB103. Western blot analysis revealed that HO-1 expression was significantly higher in the LIPUS group than in both the cryopreserved-only group and the cryopreserved + CB103 group ( p  < 0.05; Fig.  6 C). TUNEL and immunohistochemical (IHC) analyses of oxidative damage markers revealed no significant difference between the LIPUS and cryopreserved + CB103 groups ( p  ≥ 0.05), although both groups exhibited markedly less oxidative damage than the cryopreserved control group did ( p  < 0.05; Fig.  6 E–I). These results confirm that LIPUS mitigates cryopreservation-induced oxidative stress associated with the Notch pathway in mouse ovarian tissue. Fig. 6 LIPUS ameliorates oxidative damage to mouse ovarian tissue through the NOTCH pathway: A , B : Western blot ( A ) and statistical bar charts ( B ) of the effects of different concentrations of inhibitors (CB103) on the ovarian tissue of the CP group ( n  = 3 mice); C , D : Western blot ( C ) and statistical bar charts ( D ) of the Ho-1 expression in the CP group, the CP + CB103 group, and the CP + CB103 + LIPUS group. ( n  = 3 mice); E - H : Immunohistochemistry and statistical bar charts of 3-NIT ( E , F ), 4-HNE ( E , G ), and 8-OHdG ( E , H ) in the CP group, CP + CB103 group, and CP + CB103 + LIPUS group. The brown area indicates the positive area (50 μm) ( n  = 3 ovaries from different mice). I , J : TUNEL staining ( I ) and statistical bar charts ( J ) of the cryopreservation group, the CP + CB103 group, and the CP + CB103 + LIPUS group. The green area indicates the positive area (50 μm). (* P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001) ( n  = 3 ovaries from different mice) LIPUS ameliorates oxidative damage to mouse ovarian tissue through the NOTCH pathway: A , B : Western blot ( A ) and statistical bar charts ( B ) of the effects of different concentrations of inhibitors (CB103) on the ovarian tissue of the CP group ( n  = 3 mice); C , D : Western blot ( C ) and statistical bar charts ( D ) of the Ho-1 expression in the CP group, the CP + CB103 group, and the CP + CB103 + LIPUS group. ( n  = 3 mice); E - H : Immunohistochemistry and statistical bar charts of 3-NIT ( E , F ), 4-HNE ( E , G ), and 8-OHdG ( E , H ) in the CP group, CP + CB103 group, and CP + CB103 + LIPUS group. The brown area indicates the positive area (50 μm) ( n  = 3 ovaries from different mice). I , J : TUNEL staining ( I ) and statistical bar charts ( J ) of the cryopreservation group, the CP + CB103 group, and the CP + CB103 + LIPUS group. The green area indicates the positive area (50 μm). (* P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001) ( n  = 3 ovaries from different mice) Next, we further investigated the mechanism by which LIPUS mitigates oxidative damage. Phosphorylated proteome profiling revealed a marked reduction in the S284 residue of TLE1 in both the cryopreservation + LIPUS group and the control group relative to that in the cryopreservation group (Fig. 7 A-B). Previous research has indicated that TLE1 forms a complex with HES1 to repress gene expression [ 14 ]. Fig. 7 Verification of the mechanism by which LIPUS improves oxidative damage to mouse ovarian tissue caused by cryopreservation. A , B : Volcano plots of differential phosphorylation sites enriched in the NOTCH pathway were constructed and analyzed by phosphorylation proteomics. C : Graph of the point mutation sequencing results for TLE1. D : Co-IP of HES1-WT, TLE1-S284A, and TLE1-WT; E , F : Western blot ( E ) and statistical bar charts ( F ) of Ho-1 in HES1-WT, TLE1-S284A, and TLE1-WT cells (* P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001) Verification of the mechanism by which LIPUS improves oxidative damage to mouse ovarian tissue caused by cryopreservation. A , B : Volcano plots of differential phosphorylation sites enriched in the NOTCH pathway were constructed and analyzed by phosphorylation proteomics. C : Graph of the point mutation sequencing results for TLE1. D : Co-IP of HES1-WT, TLE1-S284A, and TLE1-WT; E , F : Western blot ( E ) and statistical bar charts ( F ) of Ho-1 in HES1-WT, TLE1-S284A, and TLE1-WT cells (* P  < 0.05, ** P  < 0.01, *** P  < 0.001, **** P  < 0.0001) To further explore the functional relationship between HES1 and TLE1, we generated a phosphorylation-deficient TLE1 mutant by substituting the serine residue at position 284 with alanine (S284A) (Fig.  7 C). This mutation enabled us to examine the interaction between hypophosphorylated TLE1 and HES1. Co-IP assays confirmed that attenuated TLE1 phosphorylation decreased its binding affinity for HES1, resulting in an increase in HO-1 expression (Fig.  7 D-F). In summary, our findings demonstrate that LIPUS attenuates Notch signaling, leading to reduced phosphorylation of TLE1. This posttranslational modification weakens TLE1-HES1 binding, disrupting the formation of the HES1-TLE1 transcriptional repressor complex. Consequently, downstream HO-1 expression is upregulated, resulting in decreased oxidative damage.

A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit (KTA2010; Abbkine, Wuhan, China) was used. Primary antibodies against 3-nitrotyrosine (3-NT; catalog no. bs-8551R; Bioss, Beijing, China), 4-hydroxynonenal (4-HNE; bs-6313R; Bioss), 8-hydroxy-2-deoxyguanosine (8-OHdG; bs-1278R; Bioss), nuclear factor erythroid 2-related factor 2 (Nrf2; 16396-1-AP; Proteintech, Rosemont, USA), heme oxygenase-1 (HO-1; R24541 ; Zen-Bio, Chapel Hill, USA), TLE1 (CY8779; Abways, Shanghai, China) and HES1 (CY5649; Abways) were used. Matrigel: 40183ES08 (Yeasen, Shanghai, China). The protein A/G immunomagnetic beads C0104B (Targetmol, Boston, USA) and Limantrafin (CB-103, #HY-135145) were purchased from MCE (New Jersey, USA). A total of 120 SPF female ICR mice (3 weeks old) were used for this study and were obtained from the Anhui Provincial Laboratory Animal Center. All the animals were housed in a relatively stable environment, with lights turned on at 8 a.m. and off at 8 p.m., and maintained at room temperature (21–25 °C) with water and food ad libitum. The mice were acclimatized for one week. All of the experimental procedures were approved by the Ethics Committee and the Institutional Animal Care and Use Committee of Anhui Medical University (20232078). The specific number of mice in each experimental group is shown in Supplemental Material 1. The mice were randomly divided into 3 groups: control, cryopreservation and cryopreservation + LIPUS. The tissues in the cryopreservation group underwent vitrification and subsequent thawing following established standard protocols (Fig.  1 ). Fig. 1 Flow chart Flow chart A LIPUS device was used in this study (Chongqing Haifu Medical Technology Co., Ltd., Chongqing, China, RH2022D08). The transducer used in this study had an area of 5 mm², a frequency of 0.5 MHz, a duty cycle of 20%, and an average spatial time intensity (ISATA) of 0.5 mW/cm2. Each ovary was irradiated for 5 min (Fig.  1 ). Ovarian tissue sections were deparaffinized, washed once, treated with PBS, and then treated with 20 µg/ml proteinase K, and incubated for 10 min at room temperature. Proteinase K was washed away with PBS, and a TUNEL assay was performed. Fifty microlitres of the prepared solution was added, incubated at 37 °C for 60 min in the dark, and then washed 1 time with PBS. The sections were then sealed with anti-fluorescence quenching sealing solution and placed under a fluorescence microscope. At least 3 biological replicates were performed. The samples were deoxygenated in citrate buffer solution and boiled for 10 min for antigen retrieval. After they cooled to room temperature, the sections were incubated with primary polyclones for 4-HNE, 8-OHdG or 3-NIT. Each antibody was used at a 1:200 dilution and incubated for 2 h at room temperature. Immunostaining is accomplished by using diaminobenzene, tolbenzenyldiphenyl (DAB) as the chromogen. The slides were visualized using a light microscope. In each group, at least three pieces of unilateral ovarian tissue from different mice were randomly selected to repeat the experiment. After the tissues were rapidly excised and washed in 0.9% NaCl solution, the proteins were extracted and evaluated by 12% sodium lauryl sulfate–polyacrylamide. The product was subjected to gel electrophoresis and transferred to a blocked PDF membrane with nonfat powdered milk for 1 h and then incubated overnight at 4 °C with primary antibodies. After the primary antibody was discarded, the cells were washed with Tris-buffered saline for 10 min and incubated with the secondary antibody for 1 h at room temperature. The frequency bands were evaluated using an imaging system, and the gray values of the target bands were analyzed by ImageJ software for statistical analysis. Proteomics: Total protein was extracted from the sample. A portion was aliquoted for protein concentration determination and SDS–PAGE analysis, while another portion was subjected to tryptic digestion. After the enzymatically digested peptide segments were desalted, the samples were analyzed by LC–MS/MS. The LC–MS/MS analysis used data-independent acquisition (DIA) to collect mass spectrometry data for each sample, followed by spectral matching, quantitative information extraction, and subsequent statistical analysis. Differential proteins were screened out based on a p -value < 0.05 and a | log2(FC)| ≥ 1. Subsequently, functional enrichment and pathway analyses were conducted to elucidate the biological roles of these proteins. Phosphoproteomics: Total protein was extracted from the sample, and a portion was removed for protein concentration determination and SDS–PAGE separation. Another portion was used for trypsin enzymatic hydrolysis, peptide desalination, and enrichment and purification of phosphorylated peptides. For LC–MS/MS identification, DIA technology was used to collect mass spectrometry data for each sample, and spectrum matching, quantitative information extraction, and subsequent statistical analysis were performed. The differential phosphate sites were screened out based on p -value < 0.05 and a | log2(FC)| ≥ 1, and expression pattern clustering heatmap analysis was conducted for the difference comparison group data. Site motif analysis was also performed. Finally, multiple common databases were used to conduct functional annotation analysis on the proteins corresponding to the significantly different phosphorylation sites obtained through screening. The HES1 and TLE1 expression plasmids were synthesized by General Biotechnology Co., Ltd. and Qingke Biotechnology Co., Ltd., respectively, and were subsequently cloned and inserted into the pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro and pCDNA3.1(+) vectors (the cloning sites were XbaI-BamHI and BamHI-XhoI). The sequence accuracy was further confirmed by gene sequencing verification. Using molecular cloning techniques, the full-length mouse TLE1 cDNA sequence was inserted into the pCDNA3.1(+) plasmid vector to construct a fusion protein expression system. We used the Mut Express II Rapid Mutagenesis Kit V2 (Vazyme) to perform precise site-directed mutations on this plasmid. The 284th acidic amino acid, serine, located on TLE1 was specifically mutated to the neutral amino acid alanine to achieve targeted dephosphorylation. The mutated plasmid was named pCDNA3.1-TLE1-S284A. The mutant was confirmed to have been successfully constructed through sequencing and was then transfected into HEK293T cells for subsequent experiments (Table  1 ). Table 1 Mutants of TLE1 were generated by point Mutaiton using the following primers (5’-3’) Primer Primer sequence of the mutation site positive-sense strand 5’-TTCAGGTGCCCCGGCATCCACAGCCTCCTCTG-3’ Anti-sense strand 5’-ATGCCGGGGCACCTGAAGCATCTTTCTTCAGCAGACGG-3’ Mutants of TLE1 were generated by point Mutaiton using the following primers (5’-3’) HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Before transfection, the cells were seeded in 10 cm culture dishes at a density of 1 × 10⁶ cells per dish. When the degree of fusion reached 80%, the plasmid was transfected into Opti-MEM using Yisen Hieff Trans™ transfection reagent (catalog no. #40808ES03). The wild-type group used TLE1-WT + HES1-WT and the mutant group used TLE1-S284A + HES1-WT. The DNA-reagent complex was incubated at room temperature for 5 min and then added to the cell culture system. The complete medium was replaced 6 h after transfection, and the cells were collected 48 h later for coimmunoprecipitation analysis. Cells were collected 48 h after transfection and lysed in ice-precooled buffer containing protease inhibitors for 15 min. The lysate was centrifuged at 14,000×g at 4 °C for 10 min. The supernatant was used for subsequent experiments. For the input group, 10 µL of the supernatant was added to 25 µL of 5×SDS buffer for storage. For the precleaning group, the remaining supernatant was incubated with prewashed bare magnetic beads at 4 °C overnight. Simultaneously, antibody–magnetic bead complexes were prepared. Protein A/G magnetic beads (30 µL × 4 tubes) were bound to rabbit IgG or anti-HES1 antibody (Abways) at 4 °C overnight. The precleared lysate was transferred to the antibody-magnetic bead complex and incubated overnight at 4 °C with rotation. After the samples were washed, they were boiled in a 100 °C metal bath for 10 min, after which WB analysis was performed. At least 3 biological replicates were used to calculate the mean and SD. To compare significant differences between groups, one-way ANOVA was performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA), and P  < 0.05 was considered to indicate statistical significance.

In conclusion, our study revealed that cryopreservation triggers substantial oxidative stress in ovarian tissue, resulting in significant apoptosis. Through systematic optimization, we demonstrated that LIPUS application at defined parameters (5-minute duration, 20% duty cycle, 0.5 W/cm² intensity) effectively counteracts these adverse effects by bolstering endogenous antioxidant defense mechanisms. Mechanistically, we revealed that LIPUS-mediated protection is associated with the Notch pathway and subsequent upregulation of HO-1 expression. These findings position LIPUS as a promising adjuvant therapy for ovarian tissue cryopreservation, offering significant potential to improve fertility preservation protocols. Future investigations should focus on longitudinal assessments of LIPUS efficacy and translational studies to evaluate its clinical implementation.

As cancer survival rates continue to improve, fertility preservation has become an increasing concern for women undergoing chemotherapy [ 15 , 16 ]. Ovarian tissue cryopreservation, first demonstrated in sheep models more than three decades ago [ 17 , 18 ], remains a valuable fertility preservation option, especially for children and adolescent cancer patients in terms of fertility preservation [ 19 ]. While ovarian tissue cryopreservation has proven effective for patients with congenital ovarian anomalies, adult recipients often experience suboptimal outcomes following tissue transplantation, including elevated miscarriage rates during subsequent IVF treatments [ 20 , 21 ]. These clinical observations suggest that cryopreservation may compromise ovarian tissue functionality. Our study investigated this phenomenon by exploring cryopreservation-induced oxidative damage in ovarian tissue, thereby providing mechanistic insights into the poor transplantation outcomes associated with ovarian tissue cryopreservation. Nrf2 serves as a master regulator of cellular oxidative stress responses and plays a pivotal role in suppressing inflammation and maintaining redox homeostasis [ 22 ]. This transcription factor orchestrates cellular defense mechanisms by inducing the expression of numerous antioxidant and detoxification enzymes. Importantly, Nrf2 protects against oxidative stress by modulating the transcription of genes involved in inflammatory responses [ 23 ]. Among its downstream targets, HO-1 represents a crucial effector molecule regulated by this redox-sensitive transcription factor [ 24 ]. HO-1 induction constitutes a fundamental cellular stress adaptation mechanism, generating biologically active molecules, including carbon monoxide, iron, and biliverdin [ 25 ]. Our investigation revealed significant downregulation of key antioxidant proteins (Nrf2 and HO-1) in cryopreserved ovarian tissues. Concurrently, we observed marked increases in oxidative damage markers (3-NT, 4-HNE, and 8-OHdG), indicating that cryopreservation induces oxidative stress through impairment of the endogenous antioxidant defense system. These findings were further corroborated by the TUNEL staining results, which revealed significantly increased apoptosis in cryopreserved tissues. LIPUS is a type of ultrasound, with an energy intensity of less than 3 W/cm², which is much lower than that of traditional ultrasound energy. Its output is a pulse wave mode and is typically used in rehabilitation medicine. It has a good safety profile. Currently, LIPUS has received approval for clinical use in bone healing in the United States [ 26 ]. (Parameters: 1.5 MHz; 30 mW/cm², duty cycle 20%, pulse frequency 1 kHz). Applications in soft tissue repair have also advanced to the preclinical stage (parameters: <3 MHz, < 1 W/cm²) [ 27 ]. Clinical translation of research on male prostatitis and chronic pelvic pain syndrome has also been conducted [ 28 ]. (Parameters: 0.3 W/cm², pulse duration: 200 µs, pulse ratio: 1:4; each treatment lasted 30 min. Few studies have investigated the effects on the ovaries, and these studies have only been performed in animals. It has demonstrated efficacy in reducing cisplatin-induced primordial follicular depletion and suppressing granulosa apoptosis, offering a promising noninvasive therapeutic approach for female infertility [ 29 , 30 ]. In our study, we further revealed that LIPUS upregulates HO-1 protein expression, thereby mitigating oxidative damage. HO-1 plays a critical role in maintaining cellular redox homeostasis. Moreover, we determined the optimal LIPUS treatment parameters: a 5-minute exposure duration, 20% duty cycle, and an intensity of 0.5 W/cm² [ 31 – 33 ]. Apoptosis was evaluated using TUNEL staining, which revealed that LIPUS treatment significantly decreased the number of TUNEL-positive cells, further supporting its protective role against cryopreservation-induced apoptosis [ 34 , 35 ]. Additionally, proteomic analysis and subsequent validation demonstrated that LIPUS mitigated oxidative damage through inhibition of the Notch signaling pathway, accompanied by differential expression of HES1 and TLE1. Some studies have indicated that oxidative damage can affect the Notch pathway. Upon pathway activation, the Notch intracellular domain (NICD) is translocated to the nucleus, where it interacts with DNA-binding proteins, including CBF1/RBP-Jκ, to form a transcriptional activation complex. This complex subsequently upregulates the expression of the downstream effector HES1, a transcriptional repressor that inhibits target gene expression. Notably, the Tle protein exhibits intrinsic transcriptional repression activity. The formation of a HES1-TLE1 heterodimeric complex further enhances the transcriptional inhibition of downstream targets. Our findings demonstrate that LIPUS treatment effectively influenced Notch pathway activation, leading to decreased TLE1 expression. This reduction in TLE1 expression diminishes the formation of the HES1-TLE1 transcriptional repressor complex, ultimately resulting in upregulated HO-1 expression. These results provide novel mechanistic insights and potential technical advancements for optimizing ovarian tissue cryopreservation outcomes and provide a theoretical basis for subsequent clinical translation.

Ovarian cryopreservation has emerged as a valuable assisted reproductive technology for preserving fertility in women facing ovarian impairment because of medical treatments (e.g., chemotherapy) or age-related decline [ 1 , 2 ]. An overview of the current standard of slow freezing has been described by Anderson et al., and Sänger et al. recently reported the first successful delivery after retransplantation of vitrified ovarian tissue in Europe, demonstrating the clinical potential of this emerging technique [ 3 , 4 ]. However, current cryopreservation techniques may induce significant damage to both follicles and interstitial tissue during the freezing process, ultimately compromising fertility outcomes [ 5 ]. Therefore, finding effective protective measures to alleviate ovarian damage during cryopreservation has become an important research topic. Oxidative stress is a key factor that cannot be ignored in the process of ovarian cryopreservation. During the cryopreservation process, the level of reactive oxygen species (ROS) in ovarian tissue increases significantly because of temperature changes and changes in the composition of extracellular fluid, which in turn triggers a series of chain reactions, such as cell membrane damage, protein denaturation, and DNA damage [ 6 , 7 ]. Oxidative stress mediates dual pathogenic effects in ovarian tissue via direct structural damage coupled with oocyte depletion through apoptosis activation [ 8 ]. Low-intensity pulsed ultrasound (LIPUS), a noninvasive physiotherapeutic modality, has demonstrated significant therapeutic efficacy across multiple biomedical applications [ 9 ]. Recent studies have demonstrated that LIPUS exerts multiple tissue-protective effects through enhanced microcirculation, stimulation of cellular metabolism, and improved regenerative capacity, with documented efficacy in fracture healing, soft tissue regeneration, and neural repair [ 10 ]. LIPUS has been shown to alleviate oxidative stress by modulating nuclear factor erythroid 2-related factor (Nrf2) signaling [ 11 ]. Moreover, our previous study revealed that LIPUS can alleviate oxidative damage to ovarian tissue in chemotherapy-treated mice [ 12 ]. In summary, we hypothesized that LIPUS can ameliorated oxidative damage caused by ovarian tissue cryopreservation. In this study, we investigated this hypothesis and explored the related mechanisms.

Supplementary Material 1. Supplementary Material 1. Supplementary Material 2. Supplementary Material 2.