Use of apolipoprotein e as a marker of testicular leydig cell aging
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI FIRST PEOPLES HOSPITAL
- Filing Date
- 2026-02-06
- Publication Date
- 2026-06-16
Smart Images
Figure CN122218243A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of clinical and molecular biology, and more specifically, to the application of apolipoprotein E as a marker of senescence in Leydig cells of the testis. Background Technology
[0002] Elderly men often experience reproductive endocrine disorders characterized by decreased serum testosterone levels, including systemic symptoms such as osteoporosis, muscle atrophy, and metabolic syndrome. These symptoms also lead to neurological and psychological issues such as decreased energy, memory loss, sleep disturbances, and depression, severely impacting their health and quality of life. Therefore, exploring the regulatory mechanisms of decreased testosterone levels in elderly men and finding ways to control and delay testicular aging are urgent national needs in addressing population aging and are crucial components of the Healthy China strategy.
[0003] Leydig cells are responsible for synthesizing and secreting androgens such as testosterone, which are crucial for maintaining male secondary sexual characteristics, muscle mass, bone mineral density, cognitive function, and overall metabolic homeostasis. However, current clinical assessments of Leydig cell function mainly rely on indirect indicators such as serum total testosterone, free testosterone, and luteinizing hormone (LH), lacking specific molecular markers that can directly reflect the aging state of Leydig cells themselves.
[0004] Apolipoprotein E (APOE) is a classic lipid transporter primarily expressed in the liver and central nervous system, and its gene polymorphisms are closely associated with diseases such as Alzheimer's disease and atherosclerosis. In recent years, sporadic studies have suggested that APOE may have novel functions in non-classical tissues, but its expression characteristics in the male reproductive system, particularly in Leydig cells, its relationship with aging, and its biological functions have not yet been reported. Summary of the Invention
[0005] The purpose of this invention is to provide a molecular marker indicating the senescence state of Leydig cells and to elucidate its role in regulating Leydig cell function, thereby providing a new theoretical basis and technical means for male reproductive health assessment and intervention.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] In a first aspect, the present invention provides the application of apolipoprotein E (APOE) as a marker of senescence in Leydig cells of the testis.
[0008] Secondly, the present invention provides the use of apolipoprotein E (APOE) or a substance for detecting the expression level of apolipoprotein E (APOE) in identifying or assisting in the identification of the senescence level of testicular Leydig cells or in the preparation of products for identifying or assisting in the identification of the senescence level of testicular Leydig cells.
[0009] Thirdly, the present invention provides a product for identifying or assisting in the identification of the senescence level of testicular Leydig cells, comprising a substance for detecting the expression level of apolipoprotein E (APOE).
[0010] Fourthly, the present invention provides the use of apolipoprotein E (APOE) or a substance regulating the expression level and / or activity of apolipoprotein E (APOE) in any of the following A1)-A3):
[0011] A1) To prepare products that improve or regulate the function of Leydig cells in the testes;
[0012] A2) To prepare products that improve or regulate the synthesis or secretion of male hormones;
[0013] A3) Prepare products for the treatment or prevention of androgen deficiency caused by the decline of Leydig cell function.
[0014] Fifthly, the present invention provides a product whose active ingredient is apolipoprotein E (APOE) or a substance that regulates the expression level and / or activity of apolipoprotein E (APOE); the function of the product is any one of the following B1)-B3):
[0015] B1) Improve or regulate the function of Leydig cells in the testes;
[0016] B2) Improves or regulates the synthesis or secretion of androgens;
[0017] B3) Treatment or prevention of androgen deficiency caused by the decline of Leydig cell function.
[0018] This invention is the first to discover and confirm that APOE is specifically expressed in Leydig cells of human and mouse testes, and its expression level is significantly upregulated with age, positively correlated with the degree of senescence of Leydig cells. More importantly, although APOE does not induce typical cellular senescence phenotypes (such as increased SA-β-gal activity), it can significantly affect lipid droplet accumulation in Leydig cells and positively regulate testosterone synthesis. Clinical data show that serum APOE concentration is significantly negatively correlated with the levels of multiple sex hormones. Furthermore, in the aging stage, APOE gene knockout (APOE-KO) mice exhibit spermatogenesis disorders and reduced lipid droplets in Leydig cells, the mechanism of which is closely related to cholesterol metabolism pathway disorders.
[0019] Therefore, this invention breaks through the limitations of traditional methods that rely on indirect hormone indicators to assess Leydig cell function. It proposes for the first time APOE as a specific molecular marker of Leydig cell aging and reveals its key role in maintaining Leydig cell function, providing a new approach and tool for the precise assessment and targeted intervention of male reproductive aging. Attached Figure Description
[0020] Figure 1 The single-cell transcriptional atlas of human testes throughout their lifespan reveals a significant positive correlation between APOE expression and age. A: Dimensionality reduction display of 37 human testicular single-cell databases using UMAP. B: Dimensionality reduction display of testicular cells in different age groups using UMAP. C: Bar chart of age-related genes in Leydig cells. D: Heatmap of age-related genes in Leydig cells. E: Scatter plot of APOE expression increasing with age. F: Scatter plot of APOE and HUSI scores.
[0021] Figure 2 The spatiotemporal expression characteristics of APOE in human testes are shown. A: Immunofluorescence staining confirmed that APOE protein expression in human testicular tissue co-localized with CYP11A1, scale bar = 20 μm. B: Immunofluorescence staining confirmed that APOE expression increased during human testicular aging, scale bar = 20 μm. C: Immunofluorescence staining confirmed that APOE expression increased during mouse testicular aging, scale bar = 20 μm. D, E: Western blot analysis of APOE protein expression levels in young and aged humans and mice.
[0022] Figure 3 The following statements illustrate the relationship between serum APOE levels and sex hormones: A: Serum testosterone concentration decreases with age. B: Serum APOE concentration is negatively correlated with testosterone levels. C: APOE is negatively correlated with sex hormone-binding protein. D: APOE levels are negatively correlated with dihydrotestosterone. E: APOE levels are negatively correlated with LH. F: APOE levels are negatively correlated with FSH.
[0023] Figure 4 show APOE Expression is associated with aging and Leydig cell function. A: Construction APOE Overexpression of human Leydig cell line. B: Construction APOE Knock down the human Leydig cell line. C: APOE The expression is related to the number of Leydig lipid droplets. D: APOE Expression was not correlated with SA-β-gal staining.
[0024] Figure 5 show APOE Identification of reproductive phenotype in knockout mice. A, B: Normal aging and... APOE HE staining of testicular tissue from knockout aging mice. C: Young and aging wild-type mice. APOE Oil Red staining of testicular tissue from knockout mice. D: Youth and aging. APOE Transmission electron microscopy image of Leydig cells in mice.
[0025] Figure 6 This chart displays the results of primary Leydig cell isolation and transcriptomic / proteomic analysis. Multi-omics sequencing data suggests that APOE may regulate Leydig cell function through the autophagy-lysosomal pathway. A: Heatmap of differentially expressed genes in the transcriptome of APOE knockout Leydig cells relative to the WT group. B: Bar chart of differentially expressed genes in GO enrichment analysis. C: Bubble chart of differentially expressed genes in the KEGG pathway. D: Volcano plot of differentially expressed proteins in the proteome of APOE knockout Leydig cells relative to the WT group. C: Bar chart of differentially expressed proteins in the KEGG pathway. D: Bar chart of differentially expressed proteins in the GO pathway. Detailed Implementation
[0026] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0027] Example 1: Establishing a single-cell RNA-seq database of the entire human testis life cycle and screening for genes upregulated by Leydig cell senescence. APOE
[0028] 1.1 Sample and data sources, inclusion rules
[0029] 1. We included publicly available single-cell transcriptome data from human testes aged 17–76 years (37 samples in total) and combined them with single-cell testes data from donors aged 0–81 years from our research group (17 samples in total) to form a dataset covering the entire life cycle.
[0030] 2. Exclusion criteria: cells with too low gene count (indicating empty droplet / low quality), abnormally high mitochondrial gene ratio (indicating death / stress), and cells with a high probability of doublet. Specific thresholds can be set according to the distribution in different batches of data (e.g., gene count 200–6000, mitochondrial ratio <15%, etc. are commonly used ranges), and the thresholds should be recorded for reproduction.
[0031] 1.2 Data Preprocessing and Batch Integration
[0032] 1. Compare and count the raw sequencing data (e.g., FASTQ → gene expression matrix) to obtain the gene × cell expression matrix for each sample.
[0033] 2. Perform the analysis using standard single-cell analysis procedures:
[0034] • Normalization: Scale and log transform based on total reads / UMI per cell;
[0035] • Selection of highly variable genes: Selecting a set of highly variable genes for dimensionality reduction;
[0036] • Dimensionality reduction: UMAP / t-SNE visualization is performed after PCA;
[0037] • Batch effect correction: Methods such as anchors or nearest neighbors (MNN) are used to integrate different cohorts / batches to ensure that samples from different age groups and different sources can be compared in the same embedding space.
[0038] 1.3 Cell type annotation and Leydig cell extraction
[0039] 1. Annotate major cell populations (such as spermatogenic cells, Sertoli cells, Leydig cells, immune cells, endothelial cells, etc.) based on classical marker genes.
[0040] 2. After extracting the Leydig cell subsets, a second clustering was performed to remove potential contaminants (such as cells expressing immune cells or cells with significant endothelial markers).
[0041] 1.4 Aging-related analysis and candidate gene screening
[0042] 1. Age-related analysis:
[0043] • Treat age as a continuous variable and perform correlation analysis between the expression of each gene in Leydig cells and age (either Spearman or linear model is acceptable), and perform multiple test correction (FDR).
[0044] 2. Correlation with aging index:
[0045] • Calculate the senescence score for each Leydig cell (e.g., based on a known set of senescence genes or a universal senescence index hUSI), and analyze the correlation between candidate genes and the senescence score. Your original manuscript clearly shows a significant positive correlation between APOE and hUSI.
[0046] 3. Example of screening criteria:
[0047] • Significantly correlated with age (FDR < 0.05) and with a relatively large effect size;
[0048] • It is expressed with relative specificity or significant enrichment in Leydig cells;
[0049] • Significantly positively correlated with the aging score (hUSI).
[0050] 4. Candidate gene APOE was obtained: its transcription level was significantly upregulated with age in Leydig cells and was positively correlated with hUSI.
[0051] 1.5 Visualization and Results Output
[0052] 1. Output UMAP cluster plots, UMAPs grouped by different age groups, bar charts / heatmaps of age-related genes in Leydig cells, scatter plots of APOE versus age, and scatter plots of APOE versus hUSI (corresponding to...) Figure 1 (A–F).
[0053] 2. Statistics: Spearman's correlation was used for scatter plots, and ρ and P values were given; t-tests or non-parametric tests were used for comparisons of multiple groups, and FDR / Bonferroni correction was performed.
[0054] The results showed that APOE was one of the top 10 most relevant genes, and its expression was almost entirely confined to the Leydig cell subset (see [link to study]). Figure 1 ).
[0055] Example 2:
[0056] Validating the spatiotemporal expression characteristics of APOE in human / mouse testes (immunofluorescence / Western blot)
[0057] 2.1 Processing of human testicular tissue samples and preparation of paraffin sections
[0058] 1. Take testicular tissue from normal individuals (the source can be donated or clinical surplus tissue, which must meet ethical approval and informed consent requirements), and record age stratification.
[0059] 2. Fixation: Fix with 4% paraformaldehyde (usually 4–24 hours, adjust according to the size of the tissue block).
[0060] 3. Dehydration and clearing: Gradient ethanol dehydration → xylene clearing → paraffin embedding.
[0061] 4. Sectioning: Paraffin sections are generally 3–5 μm thick and are baked before use.
[0062] 2.2 Immunofluorescence staining (co-localization of APOE and Leydig marker CYP11A1)
[0063] 1. Dewaxing and rehydration: Dewaxing with xylene → rehydration with graded ethanol → rinsing with PBS.
[0064] 2. Antigen retrieval: Heat retrieval using citrate buffer or EDTA buffer (microwave / pressure cooker, conditions optimized according to antibody instructions).
[0065] 3. Blocking: Block with 5% BSA or normal serum for 30–60 min.
[0066] 4. Primary antibody incubation: Add anti-APOE and anti-CYP11A1 primary antibodies and incubate overnight at 4°C.
[0067] 5. Secondary antibody incubation: Add fluorescent secondary antibody and incubate at room temperature for 1 h under light-protected conditions; counterstain cell nuclei with DAPI.
[0068] 6. Mounting and Imaging: Mount the slide with anti-quenching mounting medium, and acquire images using a confocal or fluorescence microscope; adjust the scale according to your preference. Figure 2 Set to 20 μm.
[0069] 7. Quantitative:
[0070] • Using the Leydig cell region as the ROI, calculate the mean fluorescence intensity or the percentage of positive area for APOE;
[0071] • Colocalization analysis can use Pearson correlation coefficient or Manders coefficient (to compare different age groups under the same imaging parameters).
[0072] The results showed that APOE signaling and CYP11A1 (Leydig cell marker) signaling were highly colocalized in the cytoplasm of Leydig cells (see [link to study]). Figure 2 A).
[0073] 2.3 Immunofluorescence of mouse testicular tissue (APOE upregulation during aging)
[0074] 1. Grouping: Young mice and aged mice (e.g., young mice aged 8–12 weeks, aged mice aged ≥18 months; specific groups were determined based on animal resources and design).
[0075] 2. The procedures for sample collection, fixation, sectioning, and immunofluorescence are the same as in 2.1–2.2.
[0076] 3. Output Correspondence Figure 2 B–C: APOE expression increases during human / mouse aging.
[0077] 2.4 Western blot detection of APOE protein levels (human / mouse, young / aged controls)
[0078] 1. Lysis: Take testicular tissue or isolated Leydig cells, add RIPA lysis buffer (containing protease inhibitor) to homogenize, centrifuge at 4°C and collect the supernatant.
[0079] 2. Quantitative analysis: Protein concentration was determined using the BCA method.
[0080] 3. Electrophoresis and membrane transfer: After separation by SDS-PAGE, the membrane is transferred to a PVDF membrane.
[0081] 4. Sealing: Sealing with 5% skim milk powder or BSA for 1 hour.
[0082] 5. Antibody incubation: Primary antibody (anti-APOE, internal control β-actin / GAPDH) incubated overnight at 4°C; secondary antibody incubated at room temperature for 1 h.
[0083] 6. Colorimetric Development and Analysis: ECL colorimetry was performed, and grayscale quantification was conducted using software such as ImageJ. APOE / internal control normalization was followed by inter-group comparisons (corresponding to...). Figure 2 D–E).
[0084] Example 3:
[0085] Correlation analysis between serum APOE and sex hormones (clinical testing procedures and statistics)
[0086] 3.1 Subjects and Sample Collection
[0087] 1. Inclusion: 962 male subjects of different ages (2-88 years) were included. Age, BMI, comorbidities, and medication history (especially those affecting lipid metabolism / sex hormones) were recorded.
[0088] 2. Blood collection: Collect venous blood into a procoagulant tube or an EDTA anticoagulant tube (as required by the kit).
[0089] 3. Separate serum / plasma: After coagulation at room temperature, centrifuge (3000 g, 10 min), collect the supernatant, aliquot, and store at -80℃, avoiding repeated freeze-thaw cycles.
[0090] 3.2 APOE and Sex Hormone Testing
[0091] 1. APOE quantification: Use ELISA or chemiluminescence method; it is recommended to set up replicates (≥2 wells) for each sample, and provide a standard curve and quality control materials.
[0092] 2. Sex hormones: Total testosterone, SHBG, DHT, LH, FSH, etc., should be tested using the hospital's testing platform or commercial reagent kits; the testing units should be consistent and the batch number of the testing platform should be recorded.
[0093] 3. Data quality control:
[0094] Standard curve R 2 Meets the standards;
[0095] • The CV of the duplicate holes is within an acceptable range (e.g., <10–15%).
[0096] • Samples that exceed the linear range should be appropriately diluted and retested.
[0097] 3.3 Statistical Analysis and Output
[0098] 1. Stratification: Compare the differences in testosterone and APOE by age group; plot trend graphs (corresponding to...) Figure 3 A).
[0099] 2. Correlation: Correlation analysis was performed between APOE and testosterone, SHBG, DHT, LH, and FSH, and the correlation coefficients and p-values were output (corresponding to...). Figure 3 B–F).
[0100] 3. Optional enhancement (suggested to be included in the patent to enhance persuasiveness): After adjusting for confounding factors such as age and BMI using multiple linear regression / partial correlation, it was verified that the negative correlation between APOE and testosterone remains significant.
[0101] The results showed that serum APOE concentration was significantly negatively correlated with testosterone, DHT, LH, and FSH (see [link to study]). Figure 3 ).
[0102] Example 4:
[0103] Constructing an APOE overexpression / knockdown Leydig cell model and evaluating lipid droplet, SA-β-gal, and testosterone synthesis.
[0104] 4.1 Cell source and culture
[0105] 1. Cells: Primary Leydig cells derived from human testes were used in this experiment; as a control, primary Leydig cell lines derived from mouse testes could also be used in parallel to verify consistency.
[0106] 2. Culture conditions: Use the corresponding basal medium DMEM / F12 + 10% FBS + 1% antibiotics according to the cell instructions; culture at 37℃ and 5% CO2.
[0107] 4.2 Construction of APOE overexpression model (lentiviral transfection was used in this study)
[0108] Route A: Stable Lentiviral Strains
[0109] 1. Vector construction: Human APOE CDS is cloned into an expression vector (e.g., containing CMV / EF1α promoter) with a selection marker (puromycin / neomycin).
[0110] 2. Virus packaging and transduction: Viral supernatant was collected by co-transfection with packaging plasmid in 293T cells; Leydig cells were infected with an appropriate MOI, and polybrene was added if necessary.
[0111] 3. Screening: 48–72 h after infection, add the corresponding antibiotic for screening until the cells in the control wells are basically dead; expand to obtain a stable overexpressing cell line.
[0112] 4. Validation: qPCR was used to detect the upregulation of APOE mRNA; Western blot was used to detect the upregulation of APOE protein (same procedure as in Example 2.4). Validation results are shown in [link to documentation]. Figure 4 A.
[0113] 4.2.1 Vector Construction and Sequence Confirmation
[0114] 1. Target gene preparation: Obtain human APOE CDS, and design a tag (FLAG) at the 5' or 3' end for subsequent detection.
[0115] 2. Vector backbone selection: The third-generation lentiviral expression vector pLVX with promoter EF1α was selected.
[0116] 3. Selection marker: The vector carries the puro resistance gene.
[0117] 4. Cloning steps: Use restriction endonuclease ligation or Gibson assembly to insert APOE CDS into the multiple cloning site to ensure correct reading frame.
[0118] 5. Quality control: After screening positive clones by colony PCR, plasmids were extracted and Sanger sequencing was performed to cover the full length of the inserted fragment and the linker region to confirm that there were no mutations and the orientation was correct.
[0119] 4.2.2 Lentiviral Packaging (293T Cells)
[0120] 1. 293T plate seeding: Seed 293T cells into 10 cm dishes or 6-well plates one day before transfection, so that the cell confluence reaches 70%–90% at the time of transfection.
[0121] 2. Packaging system: Each 10 cm dish can be prepared in the following proportions: transfer vector (APOE expression vector), packaging plasmid (psPAX2), and coating plasmid (pMD2.G).
[0122] 3. Transfection: Use Lipo transfection reagent; mix DNA and transfection reagent, incubate at room temperature to form a complex, and then add dropwise to 293T medium. Replace with fresh complete medium 6–12 h after transfection to reduce toxicity.
[0123] 4. Collect viral supernatant: Collect supernatant at 48 h and 72 h after transfection; centrifuge at 300–500×g for 5–10 min to remove cell debris, and filter the supernatant through a 0.45 μm filter membrane.
[0124] 5. Storage: Aliquot the virus and store at -80℃, avoiding repeated freeze-thaw cycles.
[0125] 4.2.3 Leydig cell transduction (determining MOI and infection conditions)
[0126] 1. Cell preparation: Human Leydig cells (primary) are plated one day before infection to achieve a confluence of approximately 30%–60% at the time of infection.
[0127] 2. MOI gradient preliminary experiment: It is recommended to first perform MOI gradient (MOI=1, 5, 10, 20) to evaluate infection efficiency with GFP control virus and select the most suitable MOI (taking into account both efficiency and cell status).
[0128] 3. Infection enhancer: Polybrene (final concentration 5 μg / mL) can be added to enhance infection.
[0129] 4. Infection procedure: Remove the old culture medium and add culture medium containing viral supernatant (virus volume should account for 60% of the total volume, with the remainder added to complete culture medium). Gently shake well and incubate in an incubator for 12 hours; after infection, replace with fresh complete culture medium and continue incubation for 24 hours.
[0130] 4.2.4 Establishing stable strains (including killing curves) through antibiotic screening
[0131] 1. Killing curve: Before formal screening, antibiotic gradient tests were performed on uninfected Leydig cells to determine the minimum complete killing concentration: puromycin test concentration 5 μg / mL; G418 test concentration 500 μg / mL; the lowest concentration that resulted in the near-total death of control well cells within 7 days was used as the screening concentration.
[0132] 2. Start screening: 48 hours after infection, start adding screening antibiotics (at the concentration determined by the killing curve), change the medium every 2-3 days, and continue for 5-14 days (adjust according to cell tolerance and death rate).
[0133] 3. Obtaining a stable cell population: After the control wells die, the surviving cells are retained as a stable overexpression cell population (polyclonal).
[0134] 4. Cryopreservation backup: After successful screening, cryopreserve a multi-tube cell bank (P3–P5) as soon as possible to avoid drift caused by long-term culture.
[0135] 4.2.5 Overexpression validation (mRNA + protein + function)
[0136] 1. qPCR validation (mRNA): Total RNA extraction → reverse transcription → qPCR detection of APOE relative expression level; GAPDH / ACTB can be used as internal control. Results are expressed as ΔΔCt / Delta / DeltaCtΔΔCt. Compared with the stable control with empty vector, APOE mRNA should be significantly upregulated.
[0137] 2. Western blot verification (protein).
[0138] 3. Infection efficiency / expression uniformity (optional): If the vector is fluorescent, the positive rate can be detected by flow cytometry; or the intracellular localization and expression ratio can be observed by immunofluorescence / immunocytochemistry.
[0139] 4. Positive and negative controls: Negative: uninfected cells, stable empty vector strains; Positive: 293T or known APOE-overexpressing cells / exogenous recombinant protein control (used for antibody band location confirmation).
[0140] 4.3 Lipid droplet detection and quantification (Oil Red O or BODIPY)
[0141] 1. Oil Red O:
[0142] • Cell fixation (4% paraformaldehyde for 15 min) → washing with PBS → staining with Oil Red O working solution for 15 min → washing → microscopic photography;
[0143] • After elution with isopropanol, semi-quantitative analysis can be achieved using spectrophotometry.
[0144] 2. Body:
[0145] • After fixation, incubate with BODIPY lipid droplet dye in the dark, and counterstain the nucleus with DAPI; collect via confocal microscopy.
[0146] • Calculate the number / area / fluorescence intensity of lipid droplets per cell using image processing software.
[0147] See results Figure 4 C showed that the number of lipid droplets in the APOE overexpression group was significantly higher than that in the control group, while the number in the shAPOE group was significantly lower.
[0148] 4.4 SA-β-gal staining (for assessing aging phenotype)
[0149] Perform the standard SA-β-gal staining procedure for cells as follows: remove culture medium → wash with PBS → fix with fixative at room temperature for about 15 min → wash 3 times with PBS → add staining working solution → incubate at 37℃ in the dark for 12 h → observe the positive rate under a light microscope.
[0150] Output: The proportion of SA-β-gal positive cells was statistically analyzed, and the results were compared among the control, APOE overexpression, and APOE knockdown groups. See [link to results]. Figure 4 D. No significant difference was observed in the proportion of senescent positive cells among the groups.
[0151] 4.6 Testosterone synthesis function test (basal secretion and stimulation secretion)
[0152] 1. Stimulation: Leydig cells were stimulated with LH (concentration 1 IU / L, time 6 h).
[0153] 2. Obtain the supernatant: Centrifuge to remove cell debris, then aliquot and store.
[0154] 3. ELISA: A standard curve was established using a total testosterone ELISA kit; replicates were set for each sample and the mean was taken; results were normalized by cell number or total protein.
[0155] 4. Output: Differences in testosterone secretion between control group vs. APOE overexpression vs. APOE knockdown.
[0156] See results Figure 4 E showed that the APOE overexpression group had significantly higher testosterone secretion than the control group, while the shAPOE group had significantly lower levels.
[0157] Example 5:
[0158] Germ phenotype identification of ApoE knockout mice and isolation and transcriptomic / proteomic analysis of primary Leydig cells
[0159] 5.1 Animal grouping and genotyping
[0160] 1. Animals: ApoE knockout mice (ApoE- / -) and wild-type controls with the same background (WT).
[0161] 2. Grouping:
[0162] • Youth group: 8–12 weeks old;
[0163] • Aging Group: The natural aging age group (18 months) was used to verify that "the differences in aging stages are more obvious".
[0164] 3. Genotype: DNA was extracted from tail tip samples and PCR amplification was used to identify ApoE gene deletion / insertion bands.
[0165] 5.2 Fertility and General Phenotype
[0166] 1. Mating experiment: Each male mouse was paired with a female mouse of the same background (1:2), and the number of conceptions and litters were recorded within 2–8 weeks.
[0167] 2. Body weight and organ coefficient: Measure body weight and testicular weight, and calculate the testis / body weight ratio (T / W), see Table 1 and Table 2;
[0168] Table 1. Body weight and organ coefficients of ApoE knockout mice
[0169]
[0170] Table 2. Body weight and organ coefficient of WT mice
[0171]
[0172] 5.3 Testicular histology and lipid droplet detection (HE + Oil Red O)
[0173] 1. HE:
[0174] Fixation, embedding, and sectioning (same as in Example 2.1) → HE staining → observation of seminiferous tubule structure;
[0175] • Output: Aging ApoE- / - shows “focal spermatogenic insufficiency lumen”.
[0176] 2. Oil Red O (frozen slices):
[0177] • OCT embedding → frozen sectioning (8–10 μm) → fixation → Oil Red O staining → hematoxylin counterstaining → mounting;
[0178] • Quantitative: Area / intensity of lipid droplet positivity in the interstitial region or Leydig cell-rich region.
[0179] The results showed that only aging APOE-KO mice exhibited spermatogenesis disorders (semiolecular tubule atrophy and reduced sperm cells) and a significant decrease in Leydig cell lipid droplets (see [link to study]). Figure 5 AC).
[0180] 5.4 Transmission electron microscopy observation of the ultrastructure of lipid droplets in Leydig cells
[0181] 1. Small tissue fragments enriched in the testicular interstitium were obtained, fixed with 2.5% glutaraldehyde, then post-fixed with osmium tetroxide, dehydrated in a gradient manner, embedded in epoxy resin, and then sectioned ultrathinly.
[0182] 2. Electron microscopy imaging: Record the number, size, and membrane structure of lipid droplets in Leydig cells.
[0183] The results showed that lipid droplets were significantly downregulated in aging APOE-KO mice compared to the control group (see [link]). Figure 5 D).
[0184] 5.5 Primary Leydig cell isolation (for omics and functional validation)
[0185] 1. Tissue digestion:
[0186] Remove the testicles, remove the white membrane, and cut them into pieces;
[0187] • Digest with collagenase IV at 37°C with shaking to release mesenchymal cells;
[0188] • Filter to remove large pieces of tissue, and centrifuge to collect cells.
[0189] 2. Enrichment and purification: Leydig cell layers were separated using a density gradient (Percoll gradient);
[0190] 3. Purity verification: Detect the positive rate of Leydig markers (CYP11A1, HSD3B, etc.) by qPCR or immunostaining, and purify further if necessary.
[0191] 5.6 Transcriptome sequencing (RNA-seq)
[0192] 1. RNA extraction and quality control: Total RNA was extracted using TRIzol or column method, and purity and integrity (RIN met) were assessed using Nanodrop and Bioanalyzer.
[0193] 2. Library preparation and sequencing: After mRNA enrichment or rRNA removal, a library is prepared and then sequenced.
[0194] 3. Analysis:
[0195] • Aligned to a reference genome;
[0196] • Counting and normalization;
[0197] • Differential expression: Compare WT vs ApoE- / -, and analyze separately for young and aged individuals.
[0198] 5.7 Proteomics (LC–MS / MS)
[0199] 1. Protein extraction: Protein was obtained by lysing primary Leydig cells, quantified, and then digested with trypsin.
[0200] 2. On-machine operation: LC–MS / MS data acquisition;
[0201] 3. Analysis: Identify and quantify differentially expressed proteins, and perform pathway enrichment.
[0202] 5.8 Joint enrichment and mechanism orientation
[0203] Joint enrichment analysis was performed on the differential sets of the transcriptome and proteome. (See results below) Figure 6 A: Heatmap of differentially expressed genes in APOE knockout Leydig cells relative to the WT group (transcriptome sequencing). B: Bar chart of differentially expressed genes in GO enrichment analysis. C: Bubble chart of differentially expressed genes in KEGG pathway analysis. D: Volcano plot of differentially expressed proteins in APOE knockout Leydig cells relative to the WT group (proteomics sequencing). C: Bar chart of differentially expressed proteins in KEGG pathway analysis. D: Bar chart of differentially expressed proteins in GO analysis.
[0204] Leydig primary cells from the testes of APOE knockout mice were isolated in vitro and subjected to transcriptomic and proteomic sequencing. Differentially expressed genes and proteins were obtained by comparison with age-matched WT mice. GO and KEGG pathway enrichment analyses were performed. GO enrichment analysis showed that differentially expressed genes and proteins were mainly enriched in biological processes such as phagocytosis, receptor-mediated endocytosis, high-density lipoprotein assembly, and cholesterol transport; cellular components such as extracellular components and lipoprotein particles; and molecular functional items such as lipoprotein receptor binding. KEGG pathway enrichment was mainly concentrated in pathways such as autophagy, phagocytosis, and cholesterol metabolism. All these results suggest that APOE may participate in the regulation of Leydig cell senescence by affecting the autophagy-lysosome pathway and lipid metabolism.
Claims
1. Application of apolipoprotein E as a marker of senescence in testicular Leydig cells.
2. Application of apolipoprotein E or substances for detecting apolipoprotein E expression levels in identifying or assisting in the identification of testicular Leydig cell senescence levels or in the preparation of products for identifying or assisting in the identification of testicular Leydig cell senescence levels.
3. A product for identifying or assisting in the identification of the senescence level of Leydig cells in the testes, characterized in that, This includes substances that detect the expression level of apolipoprotein E.
4. The product according to claim 3, characterized in that: The substance used to detect the expression level of apolipoprotein E is either a substance used to detect the expression level of APOE protein or a substance used to detect the expression level of APOE mRNA.
5. The product according to claim 4, characterized in that: The substance used to detect APOE protein expression level is an antibody that specifically binds to APOE protein; the substance used to detect APOE mRNA expression level is a primer that specifically amplifies the APOE gene or a probe that specifically recognizes the APOE gene.
6. The application of apolipoprotein E or substances that regulate the expression and / or activity of apolipoprotein E in any of the following A1)-A3): A1) Preparation of products that improve or regulate the function of Leydig cells in the testes; A2) Preparation of products that improve or regulate the synthesis or secretion of androgens; A3) Preparation of products that treat or prevent androgen deficiency caused by the decline of Leydig cell function.
7. A product characterized in that, Its active ingredient is apolipoprotein E or a substance that regulates the expression and / or activity of apolipoprotein E; the function of the product is any one of the following B1)-B3): B1) improving or regulating the function of Leydig cells in the testes; B2) improving or regulating the synthesis or secretion of androgens; B3) treating or preventing androgen deficiency caused by the decline of Leydig cell function.
8. The product according to claim 7, characterized in that: The substance that regulates the expression level and / or activity of apolipoprotein E is a substance that increases the expression level and / or activity of APOE; the substance that increases the expression level and / or activity of APOE is a nucleic acid molecule encoding APOE protein, an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant microorganism, or a recombinant cell line.
9. The product according to claim 7, characterized in that: The male hormone in question is testosterone.
10. The product according to claim 7, characterized in that: The symptoms of androgen deficiency are selected from one or more of osteoporosis, muscle atrophy, metabolic syndrome, decreased energy, memory loss, sleep disorders, or depression.