Main Research Findings

1) Treatment with FOXO4-DRI was found to reduce age-related testosterone secretion insufficiency in aged animals by targeting Leydig cells.

2) FOXO4-DRI was shown to improve spermatogenesis in aged mice by decreasing the secretion of senescence-associated secretory phenotype.

 

Selected Data

1) The study conducted by the research team of Zhang et al employed a multi-faceted approach, combining human tissue analysis, in vitro cell culture experiments, and in vivo animal models, to investigate the role of FOXO4 and the therapeutic potential of FOXO4-DRI in age-related testosterone secretion insufficiency. First, the research utilized fourteen human testes specimens. These were sourced from donor patients and prostate cancer patients. Obtainment of these samples were critical for establishing baseline FOXO4 expression patterns and age-related changes in Leydig cells [1].

For both human testis tissues and cultured TM3 cells, immunofluorescence and immunohistochemical staining were performed to visualize protein expression and localization. Tissues were fixed and processed, then stained with specific primary antibodies. Key antibodies used included: anti-FOXO4 (two different clones, ab128908 and sc-373877), anti-StAR, anti-CYP11A1, anti-3β-HSD, and anti-Ser15-phospho-p53. StAR, steroidogenic acute regulatory protein, and CYP11A1, cytochrome P450 family 11 subfamily A member 1, are markers for Leydig cells, while 3β-HSD, 3-beta-hydroxysteroid dehydrogenase, is a key enzyme in testosterone synthesis. Ser15-phospho-p53 marks activated p53. Imaging was conducted using a Leica confocal microscope, allowing for detailed cellular and subcellular localization studies [1].

Protein expression levels were quantified using Western blot analysis. Total protein was extracted from tissues and cells using RIPA lysis buffer containing proteinase and phosphatase inhibitors. For specific analysis of cytoplasmic and nuclear proteins, the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit was employed for TM3 cells. A comprehensive panel of primary antibodies was used to detect various proteins: FOXO4, p53, Ser15-phospho-p53, p21 (cyclin-dependent kinase inhibitor 1A), p16 (cyclin-dependent kinase inhibitor 2A), 3β-HSD, CYP11A1, CYP17A1 (cytochrome P450 family 17 subfamily A member 1), and several senescence-associated secretory phenotype (SASP) factors including IL-1α, IL-1β, IL-6, IL-10, TNF-α, and TGF-β. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as a loading control for total protein, while Histone-H3 was used for nuclear extracts.

The in vitro experiments were performed using TM3 mouse Leydig cells that were cultured in DMEM/F-12 medium supplemented with 5% horse serum and 2.5% fetal bovine serum, and maintained at 37°C under a 5% CO2 atmosphere. This established Leydig cell model allowed for controlled induction of senescence and testing of FOXO4-DRI effects. Cellular senescence was assessed by measuring senescence-associated β-galactosidase (SA-β-gal) activity using a commercial staining kit. This assay was applied to both cultured cells and frozen tissue sections, with tissue sections counterstained with Nuclear Fast Red for better visualization. Increased blue staining indicated senescent cells [1].

To investigate the specific role of FOXO4, small interfering RNA (siRNA) was used for FOXO4 knockdown. The specific antisense sequences were 5′-GGCUCCUACACUUCUGUUATT-3′ for FoxO4 siRNA and 5′-UAACAGAAGUGUAGGAGCCTT-3′ for the negative control. Transfection was carried out using Lipofectamine 3000 Kit on cells at 50% confluence for 6 hours, followed by protein extraction 48 hours post-transfection. Cell viability was quantified using the Cell Counting Kit-8 (CCK8) assay. TM3 cells were seeded in 96-well plates at a density of 5×10^3 cells/well. After various treatments, 10 µl of CCK8 solution was added to each well and incubated for 2 hours at 37°C. Absorbance at 450 nm was then measured using a microplate reader, with lower absorbance indicating reduced viability. Apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection Kit. TM3 cells were harvested after treatment, stained with Annexin V-FITC and PI reagent, and incubated in the dark for 15 minutes. Flow cytometric analysis was then performed to quantify apoptotic cells. The apoptotic index was calculated as the sum of early apoptotic and late apoptotic cell populations [1].

The in vivo study utilized naturally aged male C57BL/6 mice between 20-24 months old and young adult male C57BL/6 mice that were 3 months old. Aged mice were intraperitoneally injected with 5 mg/kg FOXO4-DRI or PBS as a control every other day for three administrations, and effects were assessed 30 days post-treatment. Serum testosterone levels in mice were measured. Blood samples were collected 60 minutes after removal from room temperature, centrifuged to obtain serum, and analyzed using an electrochemiluminescence immunoassay.

2) The study performed by Li et al employed a multi-pronged experimental design, integrating human tissue analysis, in vitro cell culture, and in vivo animal models to comprehensively investigate the role of FOXO4 in age-related male fertility and the therapeutic potential of FOXO4-DRI. The methodological approach was designed to address cellular and systemic effects, utilizing a range of established biochemical and histological techniques. Initially, human testes samples were procured from young donor patients and aged prostate cancer patients. These samples assisted in identifying baseline FOXO4 expression patterns and age-related changes in Leydig cells, with all procedures strictly adhering to ethical guidelines and informed consent protocols [2].

For cell culture experiments mouse TM3 Leydig cells and GC-1 spg spermatogonia cells were utilized. Both cell lines were maintained under standard conditions in specific DMEM/F-12 media supplemented with serum. A critical component was the establishment of an in vitro co-culture system using 12-well plates and 0.4 µm pore size transwell inserts. TM3 cells were seeded in the plate wells, and GC-1 spg cells were placed in the transwell inserts, facilitating indirect interaction. This system allowed for the investigation of crosstalk between senescent Leydig cells and spermatogonia. Treatments in these co-cultures included PBS as a control, FOXO4-DRI at 50 µM, hydrogen peroxide (H2O2) at 50 µM to induce senescence, and a combination of H2O2 with FOXO4-DRI. For silencing FOXO4, specific small interfering RNA (siRNA) was employed, with a negative control siRNA, and transfection was conducted using Lipofectamine 3000 [2].

Animal studies involved naturally aged male C57BL/6 mice that were 20 months old and younger controls that were 3 and 13 months old. Aged mice were divided into control and treatment groups, with the latter receiving intraperitoneal injections of FOXO4-DRI at doses of 0.5 mg/mL and 5 mg/kg every other day for 30 days, while controls received PBS. Multiple assays were performed to assess cellular status and molecular changes. Senescence-associated β-galactosidase (SA-β-gal) activity was measured in both cells and frozen tissues using a commercial kit, providing a visual and quantitative indicator of senescent cells. Cell viability was assessed using the Cell Counting Kit-8 (CCK8) assay, measuring metabolic activity as an indicator of cell health. Cell apoptosis was quantified via Annexin V-FITC/PI staining followed by flow cytometric analysis, differentiating early and late apoptotic cells.

For molecular analysis, immunofluorescence, and immunohistochemistry techniques were extensively used on human testis, mouse testis, and TM3 cells to visualize the expression and subcellular localization of key proteins. Primary antibodies targeted FOXO4, Leydig cell markers (StAR, CYP11A1, 3β-HSD), spermatogenesis markers (PCNA, PLZF, UCHL1, SCP3), and senescence/apoptosis markers (Ser15-phospho-p53). Images were captured using confocal and inverted fluorescence microscopes. Western blot analysis quantified protein expression levels, utilizing total protein extracts as well as nuclear and cytoplasmic fractions. A broad panel of antibodies was employed, including those for FOXO4, p53, phospho-p53, p21, p16, cleaved-Caspase3, BAX, BCL-2, testosterone synthesis enzymes (3β-HSD, CYP11A1, CYP17A1), and various SASP factors (IL-1β, IL-6, TNF-α, TGF-β). GAPDH and Histone-H3 served as loading controls. Quantitative Real-Time PCR (qRT-PCR) was used to measure mRNA expression of SASP factors (IL-1β, IL-6, TNF-α, TGF-β) in TM3 cells, normalizing data to GAPDH. Additionally, ELISA was performed to quantify secreted IL-6 and TGF-β in co-culture media [2].

To evaluate spermatogenesis and male fertility in mice, Computer-Aided Sperm Motion Analysis (CASA) was conducted on epididymal sperm to determine motility parameters, including the percentage of motile and forward progressive sperm. Testicular histology was assessed using Hematoxylin-Eosin staining to observe seminiferous tubule morphology, and a Johnson’s score was applied to quantify spermatogenic function based on a 10-point scale. Serum testosterone levels were measured using an electrochemiluminescence immunoassay. Drug safety testing involved analyzing serum biochemical markers (CK, CKMB, ALT, AST, CREA, BUN) to evaluate potential adverse effects on myocardial, liver, and renal function in treated mice [2].

 

Discussion

1) The study completed by Zhang et al yielded several key findings regarding the role of FOXO4 in Leydig cell senescence and the therapeutic efficacy of FOXO4-DRI in alleviating age-related testosterone insufficiency. Immunofluorescent staining of human testes revealed that FOXO4 expression was primarily localized to the interstitial or peritubular cells, but not within the seminiferous tubules. Further analysis confirmed that FOXO4-positive cells co-expressed Leydig cell markers, specifically StAR and CYP11A1, indicating that FOXO4 is indeed expressed in human Leydig cells [1].

While total FOXO4 protein levels did not significantly differ between young men of <30 years old and elderly men ≥65 years old, its subcellular localization changed with age. In young men, FOXO4 was predominantly found in the cytoplasm of Leydig cells, whereas in elderly men, it was primarily localized in the nucleus. Given that FOXO4 is a transcription factor, this nuclear translocation suggests increased transcriptional activity. Importantly, nuclear-localized FOXO4-positive Leydig cells in elderly testes showed reduced expression of 3β-HSD, a rate-limiting enzyme in testosterone synthesis, implying a connection between nuclear FOXO4 and age-related decline in testosterone production [1].

To model Leydig cell senescence in vitro, TM3 mouse Leydig cells were treated with hydrogen peroxide (H2O2). This treatment successfully induced senescence, as evidenced by a significant increase in SA-β-gal activity. Consistent with the observations in human elderly Leydig cells, H2O2-induced senescent TM3 Leydig cells exhibited a predominant nuclear localization of FOXO4, in contrast to untreated controls where it remained mostly cytoplasmic. Western blot analysis further confirmed that H2O2 treatment led to increased levels of senescence-associated proteins p53, Ser15-phospho-p53, and p21. Concurrently, levels of testosterone synthesis-related proteins CYP11A1 and CYP17A1 were significantly decreased in these senescent cells. These in vitro results demonstrated that H2O2 induces Leydig cell senescence, accompanied by FOXO4 nuclear translocation and activation of senescence pathways.

To explore the functional role of FOXO4 in senescence, its expression was silenced using siRNA in TM3 Leydig cells prior to H2O2-induced senescence. FOXO4 knockdown resulted in increased levels of p53 and Ser15-phospho-p53, but paradoxically decreased the level of p21 . Crucially, FOXO4 knockdown significantly reduced the viability of senescent TM3 Leydig cells and substantially increased their rate of apoptosis. These findings suggest that while FOXO4 is involved in the senescence pathway, it also plays a role in maintaining the viability of senescent Leydig cells by suppressing their apoptotic response, which could contribute to the accumulation of senescent cells [1].

FOXO4-DRI, designed to disrupt the FOXO4-p53 interaction, was tested on senescent TM3 Leydig cells. Treatment with FOXO4-DRI led to the nuclear exclusion of active Ser15-phospho-p53 in senescent cells. Moreover, FOXO4-DRI significantly decreased the viability of senescent Leydig cells and dramatically increased their apoptosis rate from 10% to 27%. Importantly, FOXO4-DRI showed no significant toxicity or impact on viability and apoptosis in normal TM3 Leydig cells, where FOXO4 expression is typically low. This demonstrates that FOXO4-DRI selectively targets and eliminates senescent Leydig cells by causing p53 nuclear exclusion and subsequent apoptosis.

Translating these in vitro findings to an in vivo model, naturally aged male mice from 20-24 months old were treated with FOXO4-DRI. Aged mice exhibited lower serum testosterone levels compared to young adult mice, confirming age-related hypogonadism. Immunohistochemical staining in aged mice showed FOXO4 expression in Leydig cells, often with nuclear localization, whereas it was rarely expressed in young mice. Following FOXO4-DRI treatment, the aged mice showed a significant increase in serum testosterone levels. This improvement was correlated with increased protein levels of testosterone synthesis-related enzymes, 3β-HSD and CYP11A1, in the testes of treated aged mice. Notably, FOXO4-DRI treatment did not cause significant changes in body weight or testis weight, suggesting a targeted effect [1].

Further analysis of aged mice testes after FOXO4-DRI treatment revealed a reduction in interstitial SA-β-gal activity, along with decreased levels of senescence-associated proteins p53, p21, and p16. Additionally, the treatment led to decreased levels of several key SASP factors, including IL-1β, IL-6, and TGF-β, in testicular tissue. These improvements in senescence markers and the inflammatory microenvironment underscore FOXO4-DRI’s potential to rejuvenate testicular function by clearing senescent cells and mitigating their detrimental effects. In summary, the study demonstrates that FOXO4-DRI effectively eliminates senescent Leydig cells, improves the testicular microenvironment, and alleviates age-related testosterone secretion insufficiency, highlighting its therapeutic potential for male late-onset hypogonadism [1].

2) The study conducted by the research team of Li et al meticulously delineated the role of FOXO4 in Leydig cell senescence and its impact on male fertility, while also demonstrating the therapeutic efficacy of FOXO4-DRI in alleviating age-related testosterone and spermatogenesis insufficiencies. The findings spanned human tissue observations, in vitro cellular models, and in vivo animal studies, providing a holistic view of the peptide’s effects.

Initial human tissue analysis revealed that FOXO4 is specifically expressed within Leydig cells of the human testis, as confirmed by its co-expression with Leydig cell markers StAR and CYP11A1 through immunofluorescent staining. Notably, while total FOXO4 protein levels remained consistent between young and elderly men, a striking age-dependent shift in its subcellular localization was observed: FOXO4 predominantly resided in the cytoplasm in young Leydig cells but translocated to the nucleus in aged cells. This nuclear localization in elderly Leydig cells correlated with reduced expression of 3β-HSD, a crucial enzyme for testosterone synthesis, suggesting a link between nuclear FOXO4 and age-related decline in testosterone production [2].

To further investigate this, an in vitro senescence model was established using TM3 mouse Leydig cells treated with H2O2. This treatment successfully induced cellular senescence, marked by increased SA-β-gal activity. Consistent with human observations, H2O2 also promoted FOXO4 nuclear translocation in TM3 cells, along with an increase in senescence markers p53, phospho-p53, and p21, while reducing testosterone synthesis-related enzymes like CYP11A1 and CYP17A1. This validated the in vitro model as representative of age-related Leydig cell dysfunction. Interestingly, when FOXO4 expression was silenced in TM3 cells using siRNA prior to H2O2 treatment, cell viability decreased, and apoptosis increased, despite elevated p53 levels . This counterintuitive finding suggested that while FOXO4 is involved in the senescence program, it also helps maintain the viability of senescent cells, potentially contributing to their accumulation [2].

The therapeutic potential of FOXO4-DRI was then explored. This peptide was found to alleviate H2O2-induced senescence in TM3 Leydig cells. Crucially, FOXO4-DRI selectively induced apoptosis in senescent TM3 Leydig cells by causing nuclear exclusion of active Ser15-phospho-p53, leading to a significant increase in apoptosis rate and altering pro-apoptotic (BAX, cleaved-Caspase3 increase) and anti-apoptotic (BCL-2 decrease) protein levels. Importantly, FOXO4-DRI showed no significant toxicity towards normal TM3 Leydig cells, demonstrating its selective action against senescent cells.

Further investigation into the testicular microenvironment using a co-culture system of senescent TM3 Leydig cells and GC-1 spg spermatogonia revealed that H2O2-induced senescent TM3 cells secreted increased levels of SASP factors, particularly IL-6 and TGF-β. This secretion impaired the proliferation of GC-1 spg cells. However, FOXO4-DRI treatment significantly reduced the expression of these detrimental SASP factors and, critically, restored the proliferation of GC-1 spg cells in the co-culture system, as evidenced by CCK-8 assays and PCNA expression, without inducing apoptosis in the spermatogonia. This highlighted FOXO4-DRI’s ability to rejuvenate the supporting microenvironment for germ cells.

The most significant results came from the in vivo animal studies. Naturally aged mice exhibited significantly lower sperm concentration and motility compared to young mice , confirming age-related declines in sperm quality. FOXO4-DRI treatment in these aged mice led to a remarkable increase in both sperm concentration and motility. Histological analysis showed that FOXO4-DRI treatment improved the testicular microenvironment, leading to thicker seminiferous epithelia, increased spermatogenic cells, and more mature spermatozoa in the central lumen. The Johnson’s score, an indicator of spermatogenesis function, also significantly improved in the FOXO4-DRI-treated aged group. Moreover, the expression of key spermatogenesis markers, including UCHL1, PLZF, SCP3, and PCNA, which were reduced in aged testes, were significantly restored or increased following FOXO4-DRI treatment. Crucially, comprehensive drug safety testing in mice indicated no significant adverse effects on myocardial, liver, or renal function markers after 30 days of treatment, suggesting a favorable safety profile [2].

 

Figure 1: Changes in blood serum biochemical detection of mice in the aged group and in the group treated with FOXO4-DRI.

In conclusion, the study robustly demonstrated that FOXO4-DRI selectively eliminates senescent Leydig cells, reduces detrimental SASP factor secretion, improves the testicular microenvironment, and ultimately enhances sperm quality and spermatogenesis in aged mice, positioning it as a promising therapeutic candidate for age-related male infertility [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Zhang C, Xie Y, Chen H, et al. FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice. Aging (Albany NY). 2020;12(2):1272-1284. doi:10.18632/aging.102682

[2] Li Y, Zhang C, Cheng H, et al. FOXO4-DRI improves spermatogenesis in aged mice through reducing senescence-associated secretory phenotype secretion from Leydig cells. Exp Gerontol. 2024;195:112522. doi:10.1016/j.exger.2024.112522

 

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