Hypoxic mesenchymal stem cell-derived exosomal circDennd2a regulates granulosa cell glycolysis by interacting with LDHA

doi:10.21203/rs.3.rs-4635583/v1

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Hypoxic mesenchymal stem cell-derived exosomal circDennd2a regulates granulosa cell glycolysis by interacting with LDHA

https://doi.org/10.21203/rs.3.rs-4635583/v1

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Background

Premature ovarian insufficiency (POI) is an ovarian dysfunction disorder that significantly impacts female fertility. Ovarian granulosa cells (GCs) are crucial somatic components supporting oocyte development that rely on glycolysis for energy production, which is essential for follicular growth. Hypoxia-induced exosomal circRNAs regulate glycolysis, but their biological functions and molecular mechanisms in POI are largely unexplored. The present comprehensive investigation revealed a substantial reduction in ovarian glycolysis levels in POI rats. Notably, hypoxia-induced exosomes originating from mesenchymal stem cells (HM-Exs) exhibit a remarkable capacity to enhance ovarian glycolysis, mitigate GCs apoptosis, reinstate disrupted estrous cycles, modulate sex hormone levels, and curtail the presence of atretic follicles. These restorative actions collectively contribute to fostering fertility revival in POI-afflicted rats.

Methods

Cyclophosphamide was administered for 2 weeks to induce POI rat model, and POI rats were randomly divided into two groups and treated with NM-Exs and HM-Exs, respectively. Ovarian function and fertility were assessed at the end of the study and ovarian tissues were collected for analysis of energy metabolites. The relationship between circDennd2a and POI was explored in vitro by qRT-PCR, Western blotting, CCK-8 assay, EdU staining, TUNEL staining, extracellular acidification rate (ECAR) measurements, and ATP, lactate and pyruvate level assays.

Results

Our findings revealed depletion of circDennd2a in serum samples and GCs from individuals suffering from POI. The introduction of HM-Exs-derived circDennd2a (HM-Exs-circDennd2a) effectively counteracted GCs apoptosis by enhancing glycolytic processes and driving cellular proliferation. CircDennd2a interacted with lactate dehydrogenase A (LDHA), which served as a catalyst to increase LDHA enzymatic activity and facilitate the conversion of NADH to NAD+. This biochemical cascade worked synergistically to sustain glycolytic function within GCs.

Conclusion

This study revealed that HM-Exs-circDennd2a promoted LDHA activity and enhanced GCs glycolytic capacity, both of which support its use as a potential clinical diagnostic and therapeutic target for POI.

Premature ovarian insufficiency (POI)

Hypoxia

Exosomes

circDennd2a

glycolysis

Primary ovarian insufficiency (POI) presents a significant challenge. POI is characterized by the loss of ovarian function in women before age 40, which presents as amenorrhea and altered gonadotropin and estradiol levels ¹. This condition affects approximately 1% of women under reproductive age and may lead to severe consequences for fertility ^2,3. Abnormalities in metabolic parameters, such as lipid, glucose, and amino acid metabolism, have been linked to POI pathophysiology ^4–6. The demise of ovarian granulosa cells (GCs), which causes follicular atresia and a diminished follicle count, is a key factor in POI development ^7,8. Glycolysis is the main energy source for GCs during follicular growth ⁹. The lncRNA ZNF674-AS1 modulates GC glycolysis via interactions with ALDOA to promote GC proliferation ¹⁰. Reduced glycolysis accelerates GC apoptosis and impacts follicular health via ATP and lactate pathways ⁹. The identification of pivotal glycolysis regulators within GCs may offer novel avenues for the prevention and effective management of POI.

Although hormone replacement therapy (HRT) is the conventional approach for managing POI, its effectiveness is limited due to its incomplete restoration of ovarian function and associated side effects ¹¹. Mesenchymal stem cell-derived exosomes (MSC-Exs) therapy has emerged as a promising alternative in the treatment of POI, because MSCs exhibit tissue regeneration capabilities similar to MSCs ¹². Notably, MSC-Exs offer advantages compared to MSCs, such as non-tumorigenicity, low immunogenicity, enhanced clinical safety, and reduced ethical concerns ¹³. MSC-Exs enhance glycolysis and protect endothelial cells from injury under oxygen and glucose deprivation (OGD) conditions via the SIX1/HBO1 signaling pathway ¹⁴. These exosomes improved the local ovarian tissue microenvironment in POI models by modulating metabolic processes, such as androgen metabolism, glucocorticoid activity, and glucose metabolism ¹⁵, but the precise underlying mechanisms are not clear. Oxygen levels are crucial for MSC proliferation and differentiation ¹⁶, but most MSCs thrive in a hypoxic environment in vivo ¹⁷. Hypoxia-treated MSC-exosomes (HM-Exs) have shown promise in facilitating cardiac repair by reducing cardiomyocyte apoptosis post-ischemia ¹⁸ and enhancing angiogenesis, proliferation, and migration for fracture healing ¹⁹. These findings support the potential of HM-Exs to effectively enhance their biological functionalities.

Non-coding RNAs (ncRNAs) carried within MSC-derived exosomes, particularly miRNAs ²⁰ and circular RNAs (circRNAs) ²¹ play a role in primary ovarian insufficiency (POI). Circular RNAs (circRNAs) are a subset of ncRNAs with a closed-loop structurec ²² that have gained attention due to their stability against degradation by nucleases, which makes these molecules valuable as biomarkers and therapeutic targets ²³. High-throughput sequencing identified unique circRNA profiles in hypoxia-treated MSC-exosomes (HM-Exs) compared to normoxia-treated MSC-exosomes (NM-Exs), which suggested their potential in the treatment of various ailments ²⁴. Notably, the delivery of circ-Epc1, which influences microglial M1/M2 polarization, improved cognitive function in mouse models of Alzheimer's disease ²⁴. HM-Exs expedited wound healing in diabetic mice by delivering circ-Snhg11 and promoting M2 macrophage-like polarization ²⁵. However, whether circRNAs released by HM-Exs aid in the restoration of ovarian function in POI patients and whether this effect is mediated by enhancing GC glycolysis are not clear. Further investigation is needed to elucidate the potential role of these circRNAs in MSC-exosome therapy for POI and their impact on the recovery of ovarian function.

The present study established POI rat models using intraperitoneal injections of cyclophosphamide (CTX). These model mice were then treated with normoxia-treated MSC-exosomes (NM-Exs) or hypoxia-treated MSC-exosomes (HM-Exs) via tail vein injection. Targeted metabolomics analysis revealed significant alterations in energy metabolites after exosome transplantation. HM-Exs notably enhanced ovarian glycolytic function in POI rats. To elucidate the molecular mechanisms involved, hsa_circ_0002142 (circDennd2a), which originates from Dennd2a, was screened and characterized, and it was highly enriched in HM-Exs. CircDennd2a interacted with a glycolysis-associated enzyme, LDHA, which increased LDHA activity. This interaction regulated glycolysis in granulosa cells (GCs) in vitro and in vivo, which ultimately aided in the restoration of impaired ovarian function. Therefore, the present study provides novel insights into the pathophysiology of POI with promising implications for future therapeutic interventions.

Patients and samples

This study was approved by the Ethics Committee of The Fourth Affiliated Hospital of Jiangsu University (Zhenjiang Maternal and Child Health Hospital). Informed consents were obtained from the patients, GCs and serum samples (on an empty stomach in the early morning of the 2nd-3rd day of menstruation) were collected.

50 patients with POI (POI group) who were treated with in vitro fertilization or intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET) at the Reproductive Center of the Fourth Affiliated Hospital of Jiangsu University were selected from June 2021 to July 2023 and all POI patients were clearly diagnosed by attending physicians and above. 50 patients with normal ovarian reserve function (NC group) who underwent IVF/ICSI-ET due to male and/or tubal factors were selected as controls during the same period. There was no significant difference between the age and BMI of the two groups. Patient information is shown in Table S1.

Animals

All animal experiments and conducted procedures were in accordance with the law on animal experimentation and are approved by the regulatory authorities. The work has been reported in line with the ARRIVE guidelines 2.0. 6-week-old healthy female SD rats weighing 162 ± 5g were purchased and housed from the Experimental Animal Center of Jiangsu University with the required constant temperature and relative humidity.

Two weeks prior to the start of the experiment, vaginal smears were collected from rats at 9 a.m. each day to observe the estrous cycle. The regular estrous cycle of rats consisted of the following four consecutive phases: proestrus, estrus, metestrus, and diestrus, which were identified on the basis of the presence or absence of keratinised epithelial cells, nucleated epithelial cells and leucocytes. The normal estrous cycle in rats ranges from 4 to 5 days, and experiments included rats that experienced at least two consecutive normal estrous cycles.

Isolation and identification of exosomes

BMSCs were extracted from femoral bone marrow of 6-week-old healthy SD rats, and generation 3–8 cells were used for subsequent experiments. BMSCs were incubated under normoxic conditions, reaching 80% concentration and then continued to be cultured for 48 hours in exosome-free medium at 20% (normoxic) or 1% (hypoxic) O₂. Subsequently, the culture medium supernatant was collected and exosomes were separated using Total Exosome Separation Reagent (Umibio, UR52121). The resulting exosomes were preserved by resuspension in PBS at -80. Exosomes were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting analysis for size, morphology and concentration of markers, respectively.

POI rat models establishment and treatment

To establish the chemotherapy-induced POI models, 6-week-old female SD rats were administered cyclophosphamide (CTX, Sigma, USA) via intraperitoneal injection. The injection dose was 50mg/kg on the first day, followed by 8mg/kg daily for 14 days. Additionally, twelve 6-week-old SD rats were taken as normal controls and injected daily intraperitoneally with the same volume of PBS. Regular monitoring of body weight, serum sex hormone levels and oestrous cycle of the POI rat models to assess modelling efficacy.

After successful modelling, all POI rats were randomly divided into three groups of 12 rats each, and injected with PBS (100 µL), NM-Exs (150 µg/100 µL PBS) and HM-Exs (150 µg/100 µL PBS) by tail vein every 2 days for a fortnight. Regular monitoring of body weight, serum sex hormone levels and oestrous cycle of the POI rat models to assess treatment efficacy. After 2 weeks of treatment, 6 rats in each group were randomly selected to be executed by injection of an overdose of pentobarbital sodium (500 mg/kg; Sigma, USA), and ovarian tissues were removed for subsequent experiments. The remaining 6 rats were evaluated for fertility, including pregnancy rate and number of offspring, at 4 and 8 weeks after treatment, respectively. The total number of samples for the whole animal experiment was 48, 12 animals per group and 6 animals per cage.

Cell culture and transfection

KGNs and 293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). KGNs were cultured in DMEM/F12 medium (Gibco, USA), 293T cells were cultured in DMEM medium (Gibco, USA) and BMSCs were cultured in DMEM (Gibco, USA). All the culture medium contained 10% foetal bovine serum (FBS, Gibco, USA) and 1% penicillin sodium and streptomycin (Gibco, USA).

SiRNA (circDennd2a) was synthesized and purchased from GenePharma (Jiangsu, China). The oligonucleotide sequences were as follow: 5′-TCAAGAATGCAGGCTCAAC-3′. For the transient transfection, siRNA was mixed with Lipofectamine 2000 (Invitrogen, USA) in DMEM/F12 medium to form complexes, and then transfected into the BMSCs.

RNase R and Actinomycin D treatment

Total RNA (1 µg) of BMSCs was incubated with or without 4 U of RNase R (Lucigen, USA) for 30 min at 37°C and terminated for 10 min at 70°C. The expression of circDennd2a and linear Dennd2a mRNA were detected by qRT-PCR.

BMSCs were treated with Actinomycin D (ActD, Sigma, USA) to evaluate the stability of circDennd2a and linear Dennd2a mRNA. The stability of RNA was detected by qRT-PCR.

Nuclear-cytoplasmic fractionation

A Nuclear and Cytoplasm Extraction kit (Beyotime, Shanghai, China) was used to separate the nuclear and cytoplasm of KGNs. As directed by the manufacturer, RNA was isolated from the nuclear and cytoplasm, respectively. Finally, the results were standardized to GAPDH (cytoplasmic control) and U6 (nuclear control), and calculated using the 2-ΔΔCt method.

CCK-8 assay

Cell proliferation was determined by using a CCK-8 kit (Vazyme, Nanjing, China) assay. Briefly, KGNs (1×10⁴ cells/well) were cultured in a 96-well plate for overnight with three replicates. After 0h, 24h, 48h, 72h and 96h of incubation, the CCK-8 solution (10 µL/well) was added and incubated for 2h at 37°C. The optical density (OD) value at 450 nm was measured by using a micro-plate reader (Thermo, USA).

EdU proliferation assay and TUNEL apoptosis assay

KGNs were seeded into 96-well plates in preparation for the cell proliferation and apoptosis assay. Following the manufacturer's instructions, a EdU Cell Proliferation Assay Kit (EdU, Ribobio, China) was used to detect the proliferation. Red: EdU staining; blue: nuclear staining. Apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. A TUNEL staining kit (Beyotime, Shanghai, China) was used to detect the apoptosis. Green: TUNEL staining; blue: nuclear staining. Fluorescent images were obtained under fluorescence microscope (Leica Microsystems, Mannheim, Germany).

RNA immunoprecipitation (RIP)

RIP assay was performed using a RNA Immunoprecipitation (RIP) Kit (BersinBio, Guangzhou, China) according to the manufacturer’s instructions. RNase inhibitor and protease inhibitor cocktail were added to RIP lysis solution to facilitate the lysing of 293T cells cellular proteins. Subsequently, 293T cells lysates were incubated with anti-LDHA (Abcam, USA, 4µL) or anti-IgG (BersinBio, Guangzhou, China, 4µL) at 4°C overnight. Co-precipitated RNA was extracted with TRIzol reagent and quantified by qRT-PCR as described previously.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from cells and ovarian tissues was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA). Genomic DNA (gDNA) was extracted from cells by the Genomic DNA kit (Tiangen, Beijing, China). RNA was reverse transcribed into cDNA using HiScript II Q RT SuperMix (Vazyme, Nanjing, China). Quantitative reverse transcription polymerase chain reaction (PCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). β-action, GAPDH and U6 were used as internal controls for relevant mRNA expression. The relative expression of RNAs was calculated using the comparative Ct method. The primer sequences are shown in Table S2.

Western Blot

Protease inhibitor-containing RIPA lysis buffer (Solarbio, Beijing, China) was used to lyse cells and ovarian tissues (Solarbio, Beijing, China). After determining the samples' protein content, 5×Lodding buffer (Beyotime, Shanghai, China) was added, and boiled for 5 minutes in the water bath. Electrophoresis was performed using 8% or 10% sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE), which were accompanied and transferred to membranes using polyvinylidene fluoride (PVDF) membranes (Millikon, USA). After sealing the membranes with 5% skimmed milk for 2 h, the membranes were incubated with anti-FSHR (proteintech, USA, 1:1500), anti-PCNA (proteintech, USA, 1:10000), anti-Bcl-2 (Wanlei Biotechnology, China, 1:500), anti-Bax (proteintech, USA, 1:8000), anti-Casp-3 (Abcepta, China, 1:1000), anti-LDHA (proteintech, USA, 1:1500), anti-HK2 (Wanlei Biotechnology, China, 1:500), anti-PKM2 (Wanlei Biotechnology, China, 1:500) or anti-β-actin (Biosharp, China, 1:10,000) at 4°C overnight. Anti-IgG (Biosharp, China, 1:10,000) was incubated for 2 hours at room temperature. Exposure was performed using an enhanced chemiluminescence kit (ECL; Vazyme, Nanjing, China) and the signals were then identified and analysed using Image J software.

Determination of extracellular acidification rate (ECAR) and ATP levels

The XFp Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used for real-time analysis of the extracellular acidification rate (ECAR). The ECAR was measured according to the manufacturer’s guidelines. ATP levels were determined using the ATP Assay Kit (Beyotime, China) according to the manufacturer's instructions.

Determination of lactate and pyruvate levels

Treat cells and continue incubation for 36h, lactate and pyruvate accumulation in culture medium were determined by a lactate test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and a pyruvate determination kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively. The culture media from each group of cells were collected in 1.5 mL centrifuge tubes and photographed.

Measurement of hormone levels

Serum samples from rats were collected every 7 days, centrifuged (2000g) at 4°C to further isolate the serum, and serum hormone concentrations, including AMH, FSH, LH and E₂, were determined by radioimmunoassay (ImmunoWay, USA). Briefly, the serum samples were incubated with the corresponding labelled antibodies, and the radioactivity of the compounds was detected using an enzyme marker to measure the corresponding hormone levels.

Targeted energy metabolomics

Ovarian metabolites were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), courtesy of Shanghai Applied Protein Technology (Shanghai, China). Overall, 100mg of ovarian tissues were homogenated in 20 mL of ultrapure water and 800 mL of methanol/acetonitrile (1:1) solution, incubated for 1h at -20℃ using an MP homogenizer and ultrasonicator, followed by centrifugation at 2000g for 20 min at 4°C. The supernatant was collected and analysed by mass spectrometry using a 5500 QTRAP mass spectrometer (AB Sciex, Framingham, MA, USA). Chromatographic peak areas and retention times were obtained using Multiquant software and normalised to standard metabolite preparations.

Histopathological Examination

Ovarian tissues were dissected, fixed with 4% paraformaldehyde overnight at 4°C, embedded in paraffin, sectioned into 4µm thick sections, deparaffinised and stained with hematoxylin and eosin. Follicular morphology was observed and photographed under the Pathology Image Scanner (Pannoramic MIDI, Hungary) and follicles at every level were counted.

Immunohistochemistry

Immunohistochemistry was performed on formalin-fixed and paraffin-embedded specimens with primary antibodies including anti-FSHR (proteintech, USA, 1:200), anti-PCNA (proteintech, USA, 1:3000), anti-Bcl-2 (Wanlei Biotechnology, China, 1:100), anti-Bax (proteintech, USA, 1:2000), anti-Casp-3 (Abcepta, China, 1:200), anti-LDHA (proteintech, USA, 1:200), anti-HK2 (Wanlei Biotechnology, China, 1:100) and anti-PKM2 (Wanlei Biotechnology, China, 1:100), respectively. Finally, sections were observed and photographed with the Pathology Image Scanner (Pannoramic MIDI, Hungary).

Statistical analysis

Differences between multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. All data were shown as mean ± standard error of the mean (SEM). All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Prism, USA). P < 0.05 was considered statistically significant.

HM-Exs significantly restored ovarian function in POI rats

To determine the role of MSC-Exs in restoring the glycolytic capacity of GCs, BMSCs were isolated from rats. Changes in oxygen levels influence the unique characteristics of BMSCs, which allows these cells to convey biological information via internalization into neighboring or distant cells. The present study was conducted to determine whether the hypoxic state of BMSCs affects the secretion of Exs. We isolated and characterized Exs secreted by BMSCs under normoxic (N-BMSC-Exs, NM-Exs) and hypoxic (Hy-BMSC-Exs, HM-Exs) conditions. Analysis using nanoparticle tracking (NTA) and transmission electron microscopy (TEM) revealed no discernible morphological differences in size, shape, or electron density between the NM-Exs and HM-Exs (Fig. 1A, B). Western blot analysis confirmed the presence of the Exs surface markers CD63 and CD9 and the absence of β-actin. Notably, higher protein levels of CD63 and CD9 were detected in HM-Exs compared to NM-Exs (Fig. 1C).

Previous findings indicated that MSC-Exs targeted ovarian tissue, and the strongest fluorescence signal was detected 24 hours post-injection using an in vivo imaging system. Co-localization studies using FSHR-labeled GCs and DiR-labeled HM-Exs within rat ovaries indicated the targeted delivery of Exs to GCs. Notably, more intense fluorescence signals were detected in the HM-Ex group than the NM-Ex group, which highlighted the superior targeting ability of the former group (Fig. 1D).

This research supports the potential contribution of MSC-Exs in enhancing the glycolytic function of GCs and emphasizes the importance of BMSC-derived Exs in intercellular communication within the ovarian microenvironment. To investigate the biological functions of HM-Exs, we established POI rat models by administering cyclophosphamide (CTX) followed by transplantation with NM-Exs/HM-Exs (Fig. 1E). Throughout the study period, various specific physiological indices were evaluated. Notably, the POI rats exhibited significantly lower body weights (Supplementary Fig. 1A, B), ovary sizes (Fig. 1F and Supplementary Fig. 2A), ovary weights (Supplementary Fig. 2B), and ovarian indices (Supplementary Fig. 2C) compared to the control rats. The levels of AMH and E2 were notably reduced in the POI rats, and the FSH and LH levels were markedly elevated (Supplementary Fig. 1C-F), which disrupted the estrous cycle (Supplementary Fig. 1G, H). Pathological studies to assess ovarian reserve across all groups revealed substantial reductions in primordial, primary, and secondary follicles and a significant increase in atretic follicles in POI rats compared to controls (Fig. 1G and Supplementary Fig. 2D, E). NM-Ex and HM-Ex transplantation facilitated the restoration of ovarian function in POI rats to varying degrees. Notably, the HM-Exs significantly restored ovarian function compared to NM-Exs in all aspects (Fig. 1F, G and Supplementary Fig. 1A-H and Supplementary Fig. 2A-E).

To further evaluate HM-Exs-mediated fertility restoration, four groups of female rats were paired with proven fertile males. Fertility outcomes were assessed at 4 weeks and 8 weeks post-transplantation. Heightened pregnancy rates and offspring numbers were found in the NM-Exs and HM-Exs groups compared to the POI group. The HM-Exs group exhibited a more substantial increase, which supports the greater therapeutic potential of HM-Exs in ameliorating CTX-induced fertility loss (Fig. 1H and Supplementary Fig. 2F-H)).

HM-Exs promote proliferation and inhibit apoptosis of GCs in POI rats

Ki67 and TUNEL staining revealed changes in GC proliferation and apoptosis, which were indicated by a decrease in the number of Ki67-positive cells and an increase in the number of TUNEL-positive cells in the POI group. These alterations were reversed in the NM-Exs and HM-Exs groups, and the HM-Exs group showed superior restoration effects (Fig. 1I and Supplementary Fig. 2I). To assess the impact of HM-Exs on the proliferation and apoptosis of GCs, we evaluated the mRNA levels of FSHR, PCNA, Bcl-2, Bax, and caspase-3 using quantitative real-time polymerase chain reaction (qRT-PCR) and assessed the levels of the corresponding proteins using Western blotting and immunohistochemistry (IHC).

Immunohistochemical analysis of ovarian tissues demonstrated that these proteins were predominantly expressed in GCs, which is consistent with the Western blot findings (Fig. 1J and Supplementary Fig. 2J). FSHR, PCNA, and Bcl-2 mRNA levels were notably lower in the POI group compared to controls but were restored in the NM-Exs and HM-Exs groups. A more substantial recovery was observed in the HM-Exs group. Conversely, Bax and caspase-3 levels were elevated in the POI group but normalized in the NM-Exs group and mostly restored to baseline levels in the HM-Exs group (Supplementary Fig. 2K). Therefore, the POI group exhibited the lowest Bcl-2/Bax ratio, and the HM-Exs group exhibited a significantly higher ratio than the NM-Exs group. Western blot analysis verified the reductions in FSHR, PCNA, and Bcl-2 and the increases in Bax and caspase-3 in the POI group compared to the controls (Supplementary Fig. 2L). In summary, the NM-Exs and HM-Exs groups showed improvements, and the restorative effect was more pronounced in the HM-Exs treatment. These findings suggest that Exs enhance GC proliferation and inhibit apoptosis, and HM-Exs demonstrated enhanced therapeutic potential.

Effect of HM-Exs on ovarian energy metabolism

To evaluate the effect of HM-Exs on ovarian energy metabolism, a metabolomic study of rat ovaries was performed using liquid chromatography-tandem mass spectrometry. The analysis focused on key metabolites, such as lactate, pyruvate, ATP, D-fructose 1,6-bisphosphate, and D-glucose 6-phosphate, which are crucial for folliculogenesis. The heatmap demonstrated that HM-Exs significantly restored CTX-induced glycolytic abnormalities compared to the NM-Exs group (Fig. 2A). Statistical evaluation of ovarian glycolytic metabolites revealed that ATP, lactate, D-fructose 1,6-bisphosphate, and D-glucose 6-phosphate levels were notably lower in the POI group than the control group, and pyruvate concentrations exhibited the opposite trend (Fig. 2B). Following HM-Exs transplantation, the concentrations of these glycolytic metabolites normalized, but NM-Exs transplantation did not significantly improve these levels. These findings suggested that HM-Exs transplantation partially rectified CTX-induced glycolytic metabolic irregularities.

Down-regulation of SUVmax values in PET/CT scans of older women's ovaries

FDG is a tracer for the visualization of glucose metabolism. PET/CT scans using FDG are extensively used in cancer diagnosis due to the increased glycolysis observed in cancer cells. Similarly, active ovarian tissues exhibit increased glucose metabolism from glycolysis in proliferating KGNs. In this study, we analyzed PET/CT data from 30 young patients (< 35 years old) and 30 older patients (> 35 years old), all of whom were free from reproductive system-related diseases. We assessed an indicator of glycolytic metabolism, the SUVmax, using PET/CT scans and demonstrated that the ovaries of younger patients exhibited elevated levels of glucose metabolism, which is consistent with the anticipated outcomes (Fig. 2C).

HM-Exs promote proliferation and inhibit apoptosis in KGNs

The KGNs were treated with 250 µM CTX for 48 hours to establish an in vitro POI cell model (CTX-KGNs). PKH26-labeled Exs were co-cultured with KGNs to investigate their ability to be endocytosed into KGNs. Laser scanning confocal microscopy images revealed that PKH26-labeled Exs (in red) were localized in the perinuclear region of the KGNs, which confirmed the endocytosis process (Fig. 3A). PBS, NM-Exs, and HM-Exs were individually co-cultured with CTX-KGNs, and the impact of Exs on KGN proliferation was assessed using CCK-8 and EdU staining assays.

The results depicted in Figs. 3B and C indicate that NM-Exs and HM-Exs enhanced the viability and proliferative capacity of CTX-KGNs. HM-Exs produced a more pronounced pro-proliferative effect compared to NM-Exs. The influence of HM-Exs on KGN apoptosis was evaluated using TUNEL staining assays, which revealed a significant reduction in the level of CTX-KGN-induced apoptosis in the NM-Exs and HM-Exs groups compared to the PBS group. Notably, HM-Exs exhibited superior anti-apoptotic effects on CTX-KGNs (Fig. 3D).

The expression of various proliferation- and apoptosis-related genes was examined at the mRNA and protein levels. The results demonstrated that HM-Exs notably increased the levels of FSHR, PCNA, and Bcl-2 but decreased the levels of Bax and caspase-3 in KGNs (Fig. 3E, F). Overall, these findings validate the potential of HM-Exs in enhancing the biological functions of KGNs in vitro.

HM-Exs improve the glycolysis of KGNs

Glycolysis plays a pivotal role in folliculogenesis and follicular maturation in GCs. Therefore, we investigated the impact of HM-Exs on glycolysis in KGNs. HM-Exs significantly upregulated the levels of glycolysis-related enzymes, including HK2, PKM2, and LDHA, but NM-Exs did not have a notable effect on these enzymes (Fig. 3G). The influence of HM-Exs on glycolysis in KGNs was assessed using a Seahorse XF extracellular flux analyzer. HM-Exs notably increased the extracellular acidification rate (ECAR) of CTX-KGNs, but NM-Exs transplantation did not significantly change the ECAR of CTX-KGNs (Fig. 3H). HM-Exs markedly increased ATP and lactate levels but reduced pyruvate levels in KGNs (Fig. 3I-K). HM-Exs decreased the pH levels of KGNs compared to the NM-Exs group (Fig. 3L). These collective findings suggest that HM-Exs alleviate glycolytic activity in KGNs.

The primary function of LDHA is the conversion of pyruvate to lactate and transformation of NADH to NAD. Building on the aforementioned results, we evaluated LDHA catalytic activity and the NAD+/NADH ratio (Fig. 3M, N). HM-Exs enhanced LDHA catalytic activity and the NAD+/NADH ratio in KGNs, which indicated that the beneficial effects of HM-Exs on CTX-KGNs may be associated with LDHA catalytic activity.

CircDennd2a is enriched in HM-Exs and associated with POIs

To identify circRNAs that are specifically expressed in HM-Exs, a circRNA microarray analysis was performed on NM-Exs and HM-Exs. This analysis revealed a total of 4731 differentially expressed circRNAs, which were comprised of 3177 up-regulated and 1554 down-regulated circRNAs. After assessing human-mouse homology within the top 100 significantly up-regulated circRNAs in the 400-2000-bp length range, we selected 52 up-regulated circRNAs for subsequent validation.

We knocked down these circRNAs in N-BMSCs and H-BMSCs and obtained Exs 48 hours post-transfection for co-culture with CTX-KGNs. The levels of these circRNAs were confirmed using qRT-PCR analysis, and hsa-circ-0002142 (circDennd2a) was the most prominently altered circRNA based on its fold change value, which may be due to its enrichment in HM-Exs (Fig. 4A, B). Notably, circDennd2a levels were decreased in GCs and serum samples from POI patients, which suggests an association between circDennd2a and POI (Fig. 3C).

Identification and characterization of circDennd2a

CircDennd2a (chr7:140301202–140302342) originates from exon 3 (1140 bp) of the host gene Dennd2a via back-splicing. To characterize circDennd2a, specific convergent and divergent primers were designed for the amplification of linear Dennd2a mRNA and circDennd2a sequences. Sanger sequencing confirmed the anticipated back-splicing junction of circDennd2a (Fig. 4D), and qRT-PCR analysis indicated that circDennd2a was solely amplified from a cDNA template using divergent primers and not from genomic DNA (gDNA) in KGNs. Conversely, linear Dennd2a mRNA was detected from cDNA and gDNA templates with convergent primers (Fig. 4E).

Subsequent qRT-PCR analysis following ActD treatment of KGNs revealed that circDennd2a exhibited greater stability than linear Dennd2a Mrna (Fig. 4F). Notably, circDennd2a was resistant to RNase R digestion, and the linear Dennd2a mRNA level decreased significantly after RNase R treatment in KGNs (Fig. 4G). A nuclear-plasmid separation assay demonstrated that circDennd2a was predominantly localized in the cytoplasm (Fig. 4H). These combined outcomes suggested that circDennd2a was a stably expressed circRNA in KGNs.

HM-Exs-circDennd2a promotes proliferation and inhibits apoptosis in KGNs

The effect of circDennd2a on the proliferation of KGNs was assessed using CCK-8 and EdU staining assays. The proliferative restorative effects of HM-Exs on KGNs were nullified after knockdown of circDennd2a in HM-Exs (HM-Exs-si-circDennd2a) (Fig. 5A, B). TUNEL staining assays indicated that the anti-apoptotic effect of HM-Exs on KGNs was reversed following circDennd2a knockdown (Fig. 5C). The expression levels of proliferation-related genes (FSHR, PCNA, and Bcl-2) decreased, and the expression levels of apoptosis-related genes (Bax and caspase-3) increased in the HM-Exs-si-circDennd2a group (Fig. 5D, E). These observations confirmed that circDennd2a played a crucial role in enhancing proliferation and suppressing apoptosis in KGNs.

HM-Exs-circDennd2a promotes LDHA-mediated glycolysis in KGNs

LDHA is a crucial enzyme in glycolysis that converts pyruvate to lactic acid. Therefore, we used molecular docking analysis to investigate the interactions between circDennd2a and LDHA. The results of molecular docking suggested that LDHA was a target of circDennd2a (Fig. 6A). RIP analysis confirmed the binding of circDennd2a to LDHA, which prompted an examination of how circDennd2a levels impact LDHA catalytic activity (Fig. 6B).

Knockdown of circDennd2a significantly inhibited LDHA catalytic activity in KGNs (Fig. 6C), which decreased the NAD+/NADH ratio (Fig. 6D). Notably, circDennd2a knockdown had minimal effects on the protein levels of LDHA, HK2, and PKM2 (Fig. 6E). These findings support the interaction of circDennd2a and LDHA. The downregulation of circDennd2a reduced glycolytic capacity in KGNs, which was accompanied by decreased ATP and lactate production and an increase in pyruvate levels and PH (Fig. 6F-I). These findings suggest that circDennd2a enhanced LDHA-mediated glycolysis in KGNs to influence the progression of POI.

To further validate the impact of LDHA enzymatic activity on the glycolytic capacity of KGNs, we added (R)-GNE-140 (an LDHA inhibitor) to reduce the enzymatic activity of LDHA during the co-culture of HM-Exs with CTX-KGNs. Inhibition of LDHA enzymatic activity notably reduced the NAD+/NADH ratio, ECAR, ATP, and lactate levels in KGNs and increased pyruvate levels and neutral PH (Fig. 6J-O). These results suggested that boosting LDHA enzyme activity contributed to the restoration of glycolytic capacity of KGNs. Overall, these findings demonstrate that circDennd2a influences LDHA enzymatic activity via binding to LDHA to regulate glycolysis in KGNs.

Metabolic abnormalities play a significant role in POI, and lipid and glucose metabolism disorders are closely intertwined with its pathophysiological mechanisms ⁶. The targeting of FABP4 in elderly mice showed promise in addressing the metabolic issues related to aging by inhibiting gluconeogenesis and promoting fatty acid and cholesterol breakdown ²⁶. Interventions, such as fine lysis, removal of senescent cells and ABT263 administration, improve glucose metabolism and β-cell function and reduce senescence marker expression, which support the potential of cellular senescence correction to alleviate metabolic disorders ²⁷. Energy metabolism in folliculogenesis heavily relies on granulosa cells (GCs), and disruptions in GCs energy metabolism may adversely impact follicular development ^28,29. Notably, Human Umbilical Cord Mesenchymal Stem Cells (HucMSCs) have been found to restore the ovarian metabolome and address ovarian insufficiency in mice ³⁰. Moreover, Mesenchymal Stem Cell-Derived Exosomes (MSC-Exs) have exhibited regulatory roles in various aging-related conditions through their impact on glucose metabolism ^31,32. Hypoxic preconditioning of MSCs enhances the paracrine effects of MSC-exosomes ¹⁹, suggesting that HM-Exs with altered cargos would significantly boost their biological functions, which creates exciting prospects for further exploration in this field.

Study of the protective mechanism of HM-Exs-circDennd2a in POI rats elucidates the pivotal role of metabolic shifts. Investigation of MSC-Exs treatment using CTX-induced POI rat models revealed that HM-Exs were more effective at restoring CTX-induced POI than NM-Exs, which demonstrated their potential therapeutic benefits. The significant decrease in ATP levels and various metabolites in the ovaries of POI rats indicated the presence of CTX-induced metabolic disorders, which was partially reversed with MSC-Exs treatment. Notably, HM-Exs treatment substantially increased glycolysis-related products in the ovaries of POI rats, which suggests a marked restoration of glycolytic capacity in these rats. NM-Exs treatment did not notably improve glycolysis, which suggests that its therapeutic impact is linked to elevated levels of oxidative phosphorylation. The importance of the glycolytic pathway in GCs for energy during follicle maturation and development has been highlighted in previous studies ^33,34. Higher glycolytic activity in developing follicles and increased lactate production in follicular fluid with larger follicular diameters further emphasize the critical role of glycolysis in POI development ³⁵. PET-CT data comparison between younger (< 35 years old) and older (> 35 years old) patients revealed higher glycolytic levels in the ovaries of younger patients, which suggests a strong correlation between ovarian function and glycolytic capacity. These findings support the significance of metabolic processes, particularly glycolysis, in the mechanisms and treatment of POI.

This study demonstrated the pivotal role of circDennd2a in restoring glycolytic capacity in GCs and the potential reversal of impaired ovarian function. The down-regulation of circDennd2a observed in the serum and GCs of POI patients and CTX-KGNs, support its significance in the pathogenesis of POI. Notably, the restoration of circDennd2a expression after treatment with HM-Exs, but not NM-Exs, suggests a unique regulatory mechanism specific to HM-Exs-circDennd2a in managing POI. Knockdown experiments further supported this hypothesis and revealed that reduced circDennd2a levels decreased glycolytic capacity and ATP levels in CTX-KGNs, which ultimately inhibited cell proliferation and promoted apoptosis. By demonstrating the positive impact of HM-Exs-circDennd2a on glycolysis within KGNs, this study introduces an innovative approach for alleviating POI. These results highlight the therapeutic potential of circDennd2a in restoring the metabolic functionality of GCs and provide a promising avenue for further investigations and the development of treatments for POI.

This research supports the pivotal role of LDHA in glycolysis regulation, which orchestrates the conversion of pyruvate to lactate. Our investigation revealed LDHA as a prospective target of circDennd2a, and RIP assays confirmed its binding interaction. Previous studies indicated that diverse factors, such as LNC CRYBG3 ³⁶, GLTC ³⁷, and LINC00973 ³⁸, influence LDHA function and glycolysis in distinct scenarios. Notably, we observed that circDennd2a knockdown minimally affected LDHA protein expression but profoundly impaired LDHA catalytic activity. Phosphorylation ³⁹, acetylation ⁴⁰ and succinylation ³⁷. In addition, the coenzyme NAD binds to LDHA and acts as an electron carrier in catalytic redox reactions to modulate the catalytic activity of LDHA ⁴¹. Because circDennd2a binds to LDHA at NAD binding sites, we evaluated the NAD+/NADH ratio. The depletion of circDennd2a decreased the NAD+/NADH ratio in CTX-KGNs and decreased ATP and lactate levels, which demonstrated the impact of circDennd2a on LDHA-mediated glycolysis modulation via NAD redox equilibrium. Subsequent experiments of the co-culture of HM-Exs with CTX-KGNs and an LDHA inhibitor revealed reduced LDHA enzyme activity with a subsequent decrease in the NAD+/NADH ratio and ATP and lactate levels. These results highlight the potential of LDHA enzyme activity to regulate glycolysis in CTX-KGNs. This study highlights the complex interplay between circDennd2a, LDHA, and NAD in regulating glycolysis and offers critical insights into metabolic pathways in KGN cells.

In conclusion, we introduced a novel molecular mechanism model demonstrating the influence of circDennd2a on glycolysis regulation (Fig. 7). The interaction between circDennd2a and LDHA increased LDHA enzyme activity to increase glycolysis via the regulation of NADH/NAD oxidative reduction. This process increased ATP and lactate production to provide CTX-KGNs with energy, which stimulated their proliferation and potentially restored impaired ovarian function. HM-Exs-circDennd2a may emerge as a promising target for POI treatment and offers a new avenue for clinical interventions in POI patients. However, further studies are essential to elucidate the precise mechanism of the interplay between circDennd2a and LDHA.

POI

Premature ovarian insufficiency

BMSCs

Mesenchymal stem cells

NM-Exs

Normoxia-treated MSC-exosomes

HM-Exs

Hypoxia-treated MSC-exosomes

GCs

Granulosa cells

circRNAs

Circular RNAs

miRNAs

MicroRNAs

TEM

Transmission electron microscopy

NTA

Nanoparticle tracking analysis

ECAR

Extracellular Acidification Rate

Ethics approval and consent to participate

(1) Title of the approved project: Hypoxic mesenchymal stem cell-derived exosomal circDennd2a regulates granulosa cell glycolysis by interacting with LDHA. (2) Name of the institutional approval committee or unit: The Ethics Committee of The Fourth Affiliated Hospital of Jiangsu University (Zhenjiang Maternal and Child Health Hospital). (3) Approval number: 202217. (4) Date of approval: 2022.10.31.

Competing of interests

The authors declare no competing interests.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

This research was supported by grants from the National Natural Science Foundation of China (Grant No.82172838), 333 Project Excellent Young Talents Project of Jiangsu Province, Key Medical Research Projects of Jiangsu Provincial Health Commission (K2023078), and Social Development Project of Zhenjiang, Jiangsu Province (Grant No. SH2023057 and Grant No. SH2022060). Medical Education Collaborative Innovation Foundation of Jiangsu University (JDY2023010).

Author contributions

XZ designed this study. XZ, LL, YL and DZ obtained the funding. LL and YL performed the clinical studies. WL and ML performed the experiments. WL wrote the manuscript. XZ revised the manuscript. All authors read, revised, and approved the final manuscript.

Acknowledgements

The authors declare that they have not used Artificial Intelligence in this study.

Consent for publication

All authors confirm their consent for publication.

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Hypoxic mesenchymal stem cell-derived exosomal circDennd2a regulates granulosa cell glycolysis by interacting with LDHA

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Patients and samples

Animals

Isolation and identification of exosomes

POI rat models establishment and treatment

Cell culture and transfection

RNase R and Actinomycin D treatment

Nuclear-cytoplasmic fractionation

CCK-8 assay

EdU proliferation assay and TUNEL apoptosis assay

RNA immunoprecipitation (RIP)

Quantitative real-time polymerase chain reaction (qRT-PCR)

Western Blot

Determination of extracellular acidification rate (ECAR) and ATP levels

Determination of lactate and pyruvate levels

Measurement of hormone levels

Targeted energy metabolomics

Histopathological Examination

Immunohistochemistry

Statistical analysis

Results

HM-Exs significantly restored ovarian function in POI rats

HM-Exs promote proliferation and inhibit apoptosis of GCs in POI rats

Effect of HM-Exs on ovarian energy metabolism

Down-regulation of SUVmax values in PET/CT scans of older women's ovaries

HM-Exs promote proliferation and inhibit apoptosis in KGNs

HM-Exs improve the glycolysis of KGNs

CircDennd2a is enriched in HM-Exs and associated with POIs

Identification and characterization of circDennd2a

HM-Exs-circDennd2a promotes proliferation and inhibits apoptosis in KGNs

HM-Exs-circDennd2a promotes LDHA-mediated glycolysis in KGNs

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1