JBRA Assist. Reprod. 2025;29(1):53-60
ORIGINAL ARTICLE
doi: 10.5935/1518-0557.20240089
Protective Effects of Adipose Mesenchymal Stem Cell Secretome On Oxidative Stress-Induced Bisphenol-A in Isolated Rat Testes Mitochondria and Sperm Quality
1Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
2Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
3Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
4Department of Anatomy, School of Medicine, Arak University of Medical Sciences, Arak, Iran
5Department of Anatomy, School of Medicine, Fasa University of Medical Sciences, Fasa, Iran
CONFLICT OF INTEREST
The authors have no conflict of interest.
ABSTRACT
Objective: This study aimed to explore the potential protective effects of adipose-derived mesenchymal stem cell secretome (ASE) on oxidative stress triggered by Bisphenol-A (BisA) exposure in testicular mitochondria and sperm quality of rats.
Methods: Testicular tissue mitochondria and sperms were exposed to BisA (8 μM) and ASE (50 or 100 μg). ΔΨm (mitochondrial membrane potential), reactive oxygen species (ROS) levels, antioxidant biomarkers, and sperm parameters were measured.
Results: BisA elevated biomarkers of oxidative stress in mitochondria, while the levels of antioxidant activity and ΔΨm decreased significantly. BisA harmed the morphology, survival rate, and mobility of the spermatozoids. ASE lowered malondialdehyde contents and ROS generation in the mitochondria, increased ΔΨm, and reversed sperm quality.
Conclusions: These data indicated that ASE effectively reduced BisA-induced damage to mitochondria and enhanced sperm quality by averting oxidative stress.
Keywords: adipose-derived mesenchymal stem cell, secretome, Bisphenol-A, sperm, mitochondria
INTRODUCTION
Infertility affects approximately 12% of the global population. The increased prevalence of infertility has been attributed to various factors such as a sedentary lifestyle, nutritional behavior, and environmental pollution (Bader et al., 2019). Bisphenol-A (BisA) is a prominent plasticizer known for its exceptional cross-linking properties. Human exposure to BisA can occur through inhalation, dermal contact, and consumption of water and foods stored in plastic packaging (Liu et al., 2022). Exposure to BisA has been associated with a range of health issues, particularly disorders affecting reproductive function.
BisA causes male reproductive toxicity, resulting in decreased sperm quality, reduced testosterone synthesis, and impaired supporting cell functions (Singh et al., 2015; Tao et al., 2019; Li et al., 2021; Qi et al., 2024). Different doses of BisA reduce sperm motility in fish, bovines, mice, and chickens (Hulak et al., 2013; Lukacova et al., 2015; Singh et al., 2015). Chronic exposure to BisA decreased mouse sperm motility and impaired germ cell proliferation (Liu et al., 2020). Furthermore, paternal exposure to BisA in CD-1 mice decreased total sperm counts and sperm motility among the offspring (Rahman et al., 2015).
BisA induces cellular toxicity by interfering with the function and structure of mitochondria. Exposure to BisA results in oxidative stress and alterations in mitochondrial biogenesis, mitochondrial membrane potential (ΔΨm), and mitochondrial DNA (Wang et al., 2019; Nayak et al., 2022; Meng et al., 2024; Qi et al., 2024). BisA impairs the function of mitochondria by reducing ATP and lowering mitochondrial mass (Kaur et al., 2018), which harms sperm motility. BisA reduces ΔΨm in chicken sperms (Singh et al., 2015) and elevates reactive oxygen species (ROS) levels in bovine and fish spermatozoa (Hulak et al., 2013; Lukacova et al., 2015). Accumulation of ROS may be responsible for causing BisA-induced germ cell toxicity (Wang et al., 2019; Nayak et al., 2022; Qi et al., 2024). The physiological amount of ROS regulates normal spermatogenesis, while disruption of the oxidant-antioxidant system damages spermatogenesis and induces male infertility (Zhang et al., 2023a). In the study of Khazaeel et al. (2022), BisA significantly declined normal sperm morphology, sperm count, motility, count, well as testosterone levels, CAT (catalase), and SOD (superoxide dismutase) activity. Also, BisA significantly increased the sperm anomalies, and MDA amount (Khazaeel et al., 2022).
Mesenchymal stem cells (MSCs) for treating reproductive disorders have attracted the attention of recent researchers (Ahmed et al., 2021; Zhang et al., 2023b). MSCs derived from human amniotic membranes, bone marrow, and umbilical cords can improve chemical-induced testicular injuries (Qian et al., 2020). Nafchi et al. (2024) reported that adipose-derived MSCs (ASCs) conditioned media improves human sperm count and motility. Adipose tissue, commonly known as fat, is abundant in the human body and can be easily harvested through minimally invasive procedures such as liposuction. This makes it a convenient and accessible source of stem cells for regenerative medicine and tissue engineering applications (Schneider et al., 2017).
ASCs release hormones and growth factors into the surrounding environment, known as secretome (SE). SE derived from ASCs (ASE) has been reported to repair some degenerative disorders (Mehrabani et al., 2015). SE has various advantages compared to cell-based therapies, such as being easily obtained, freeze-dried, packed, and transported (Khodayar et al., 2022). ASE improved dog sperm quality after freezing and thawing (Qamar et al., 2020). The co-culture of ASE improved human sperm vacuolization and DNA fragmentation (Bader et al., 2019). Moreover, the antioxidant properties of ASCs and ASE have been reported (Kim et al., 2008; Tofiño-Vian et al., 2018; Alonso-Alonso et al., 2020). Microvesicles from ASCs suppress oxidative stress in Osteoarthritic chondrocytes (Tofiño-Vian et al., 2018). Another study reported the antioxidant property of ASCs in damaged alveolar epithelial cells (Peñuelas et al., 2013).
Since the accumulation of ROS is the main mechanism of BisA-mediated mitochondrial dysfunction (Gassman, 2017; Nayak et al., 2022), targeting the oxidative stress pathway may reverse the toxic effects of BisA on male germ cells. Due to the antioxidant and other beneficial impacts of ASE, this study aimed to investigate the protective effect of ASE against BSA-induced mitochondrial oxidative stress and sperm quality in rats.
MATERIALS AND METHODS
Study design (Figure 1)
Fifteen male Wistar rats (200-250 g) contributed their sperm and mitochondria to the study. The current study was approved by the Animal Research Ethics Committee (IR.AJUMS.ABHC.REC.1399.018).
Figure 1. Schematic illustration detailing the experimental design of the study.
The epididymis sperm were separated and classified into the groups listed below:
Control. Given only Ham’s F-10 media for four hours.
BisA. Received BisA (0.8 μM) for two hours
ASE50+ BisA. Received 50 μg of ASE, respectively, for two hours before BisA.
ASE100+ BisA. Received 100 μg of ASE, respectively, for two hours before BisA.
ASE100. Received 100 μg ASE for four hours
ASE or BisA treatment was applied to 5×106 sperm/mL within each group. Because the untreated sperm expired after 4 hours, treatment with ASE or BisA was limited to a total of 4 hours. BisA (Sigma) was dissolved in 0.1% DMSO and mixed with Ham’s F-10 media. Concentration levels of BisA were determined using the results from the MTT test (Table 1).
Table 1. The impact of BisA on spermatozoa viability (Mean±SD; n=6).
Secretome Preparation
The characterized ASCs were obtained from the Royan Institute in Tehran, Iran. In short, the ASCs (1 × 105 cells/well) were seeded onto 6-well plates and incubated in complete media. When reaching 80% confluence, the cells were washed with Hanks’ solution and cultured in serum-free media containing low glucose DMEM (Gibco, USA), 100 U/ml penicillin/streptomycin (Sigma, USA), 2mM L-glutamine (Sigma, USA), and 10% AdvanceSTEM Stem Cell Growth (GE Life Sciences, USA) for three days. After collection, the conditioned media was subjected to centrifugation at 800. g for ten minutes to remove cell debris. The conditioned medium underwent a second round of centrifugation using an Amicon Ultra-15 centrifugal filter (Sigma, USA) for two hours. The protein concentrations in the supernatants were quantified using a Bradford assay and stored at -70°C.
Mitochondria Isolation
The testicular tissues were chopped up and placed in a liquid mixture containing fat-free BSA (0.1%), sucrose (250mM), HEPES-KOH (5mM), EGTA (0.2mM), and EDTA (0.1mM). The sample, after the homogenization process, underwent four rounds of centrifugation in an isolating buffer containing 1.0 M sucrose, 0.5 M MgCl2, 0.1 M KCl, 0.1 M EGTA, 0.1 M K2HPO4, 1.0 M mannitol, 0.1 M MOPS, 0.5 M HEPES, 10% BSA, 0.5 M glutamate, and 1.0 M succinate at a temperature of 4°C to separate the mitochondrial fractions: once at 3,000· g (for 10 min) and three times at 10,000·g (for 7 min each). The protein concentrations in the supernatants were quantified using a Bradford assay and stored at -70°C.
Sperm Parameters
The epididymis was dissected and placed in a 1% trisodium citrate solution (1 mL) for eight minutes. Eight mL of the trisodium citrate solution were added and mixed for one minute. The spermatozoa suspension was diluted in 10% formalin at a 1:1 ratio. Spermatozoa were counted using a Neubauer hemocytometer. A 100 µL sperm suspension was placed on a glass slide for morphological observation. Morphological evaluation was performed on 100 spermatozoa in each mouse. Sperm mobility was assessed according to the protocols established by the World Health Organization. Suspended sperm (10 μL) was poured into the semen analysis chamber. Six fields were evaluated for the motility rate of at least 200 sperm for each specimen.
Determining antioxidant levels, MDA content, and ROS formation
The mitochondria were incubated in DCFH-DA (10 μM; Sigma) along with Hanks buffered salt solution (100 μL) for 25 minutes. The ROS levels were determined using a spectrofluorometer with an excitation wavelength of 490 nm and an emission wavelength of 570 nm. Levels of superoxide dismutase (SOD), catalase (CAT), as well as malondialdehyde (MDA) were examined using commercially available kits from ZellBio Company.
Mitochondrial membrane potential (ΔΨm) evaluation
The mixture of Rhodamine 123 (10 μM) and mitochondrial fractions was prepared. The fluorescence was recorded using a spectrophotometer (LS50B, USA) with an excitation wavelength of 490 nm and an emission wavelength of 535 nm.
Statistical Analysis
In this study, the data were analyzed using a one-way analysis of variance in SPSS (version 21.0), with post-hoc pairwise comparisons. Statistical significance was determined for the p-values less than 0.05.
RESULTS
Sperm Quality
ASE (100 µg) had no significant impact on the number, motility, and typical morphology of the sperms. In the treatment with BisA, a noticeable reduction in sperm motility and numbers (p<0.01) was observed compared to the control. Bis-A significantly increased the percentage of immotile sperms compared to the control (p<0.01). ASE pretreatment, concentration-dependently, caused a significant reduction in the number of immotile sperm while enhancing overall sperm motility in the BisA treatment (Figure 2).
Figure 2. Total rat sperm motility (%), abnormality (%), and count in the various groups (mean±SD, n=5). * and # indicate comparisons against control and BisA-treated groups, respectively.
The level of abnormality in sperm exposed to BisA was significantly more than the control (p<0.01). Pre-exposure to ASE resulted in a dose-dependent decrease in sperm abnormalities compared to the BisA group. ASE at the concentration of 100 µg had a more positive impact on sperm quality than the 50 µg (Figure 2).
Antioxidant levels, MDA content, and ROS formation
No significantly lower levels of MDA and ROS were observed in the groups that received ASE (100 µg) compared to the control. Levels of ROS and MDA showed a significant increase in the BisA group compared to the control (p<0.01). ASE concentration-dependently reduced MDA and ROS levels in the BisA-exposed mitochondria (Figure 3). The mitochondria exposed to only ASE (100 µg) exhibited no significantly higher levels of CAT and SOD activity than the untreated groups. Treatment with BisA resulted in decreased activity of CAT and SOD enzymes in the isolated mitochondria compared to the control (p<0.001). In a concentration-dependent way, ASE reversed the CAT and SOD activity of the mitochondria. ASE at the concentration of 100 µg had more effect on the reducing MDA and ROS levels and increased activity of CAT and SOD enzymes compared to the 50 µg (Figure 4).
Figure 3. ROS and MDA levels in the mitochondrial fractions (mean±SD, n=5). * and # indicate comparisons with untreated control and BisA-treated groups, respectively.
Figure 4. CAT and SOD levels in the mitochondrial fractions (mean±SD, n=5). * and # indicate comparisons with untreated control and BisA-treated groups, respectively.
ΔΨm Assay
ΔΨm was not significantly changed in ASE (100 µg)-treated cells compared to the control. There was a significant decrease in ??m within the BisA group compared to the control group (p<0.01) (Figure 5). ASE could dose-dependently reverse the ΔΨm of BisA-exposed mitochondria. 100 µg of ASE showed more impact on the ΔΨm than 50 µg (p<0.01).
Figure 5. ΔΨm measurement in different groups (mean±SD, n=5). * and # indicate comparisons against untreated control and BisA-treated groups, respectively.
DISCUSSION
In this study, ASE could improve sperm quality, attenuate oxidative stress, and increase ΔΨm of BisA-exposed sperm and testicular tissue mitochondria. BisA significantly decreased sperm motility, normal morphology, and sperm count. In parallel with our results, previous studies conducted in humans and animals revealed the negative impacts of BisA on sperm parameters (Knez et al., 2014; Kotwicka et al., 2016; Cariati et al., 2019; Liu et al., 2020; Khazaeel et al., 2022; Lü et al., 2024). An increased number of abnormal spermatozoa after 4 hours of BisA exposure was reported by Nguyen et al. (2022). A meta-analysis study revealed that BisA impairs sperm quality (Lü et al., 2024).
In our study, the decreased sperm quality caused by Bisphenol A (BisA) was associated with increased MDA and ROS levels within the isolated testicular tissue mitochondria. The overproduction of ROS hurts spermatozoa, resulting in elevated MDA production and subsequent lipid peroxidation (Hsieh et al., 2006; Wagner et al., 2017).
Due to low cytoplasm, spermatozoa lack mechanisms for relieving oxidative damage (Agarwal et al., 2014). Additionally, the rich polyunsaturated fatty acids of sperm membranes make them susceptible to oxidative damage through lipid peroxidation (de Lamirande & Gagnon, 1995; Alahmar, 2019; Rahman & Pang, 2019). Other studies have also demonstrated enhanced lipid peroxidation and ROS levels following exposure to BisA in spermatozoa (Rahman et al., 2016; Gassman, 2017).
The ROS elevation levels were along with a decrease in ΔΨm in the BisA group. Exposure to BisA can cause a reduction in ΔΨm for human sperm, even at very low doses (Grami et al., 2020). The regulation role of the ΔΨm is crucial for maintaining mitochondrial function, structure, and metabolism. Additionally, ΔΨm regulates ROS generation and removes damaged mitochondria (Zorov et al., 2014). BisA alters ΔΨm to promote mitochondrial dysfunction and negatively affects ATP levels in mouse spermatozoa (Kaur et al., 2014; Jiang et al., 2015; Rahman et al., 2016; Shirani et al., 2019).
The decreased sperm quality in the BisA group was along with a reduction in ΔΨm. ΔΨm has a direct positive impact on both the mobility and quantity of sperm cells (Madeja et al., 2021). BisA-mediated mitochondrial dysfunction may stimulate the mitophagy pathway (Meng et al., 2024), thus interfering with mitochondrial mass.
Moreover, Bisphenol A (BisA) may induce germ cell apoptosis leading to decreased sperm quality. The decreased ΔΨm by BisA supports this hypothesis (Zhang et al., 2019). The apoptotic impact of BisA on testicular cells has been reported in many studies (Zhang et al., 2017, 2023a; Chen et al., 2022). Increased germ cell apoptosis was observed during sperm development following postnatal exposure to BisA (Shaha et al., 2010; Xie et al., 2016).
Our research revealed that ASE improved sperm count, normal morphology rate, and motility of the rats treated with BisA, corroborating findings from previous studies (Bader et al., 2019; Qamar et al., 2020). Bader et al. (2019) demonstrated that ASE improves sperm motility and viability. ASE may enhance sperm quality by suppressing apoptosis. In addition, ASE might reduce levels of ROS by enhancing ΔΨm, which could subsequently decrease germ cell apoptosis. The antiapoptotic impact of ASE has been reported in previous studies (Jiao et al., 2021; Seo et al., 2022). ASCs inhibited apoptosis caused by ionizing radiation in the testicular tissue (Cetinkaya-Un et al., 2022). Jiao et al. (2021) reported that ASE reversed the ischemia-reperfusion-induced by decreasing the P53, Bax, Fas, FasL, and Bcl-2 expression.
In our study, the enhanced SOD and CAT levels indicate the antioxidant properties of ASE against BisA. The antioxidant effects of ASCs have also been demonstrated in dermal fibroblasts, degenerative diseases of the retina, and osteoarthritic chondrocytes (Kim et al., 2008; Tofiño-Vian et al., 2018; Alonso-Alonso et al., 2020). Based on our findings, ASE exhibited a concentration-dependent reversal of ΔΨm and ROS production. Therefore, ASE may protect testicular mitochondria by minimizing oxidative stress. This data aligns with existing research that shows the positive effects of ASE on mitochondrial function (Ma et al., 2022).
The secretome derived from other sources of the MSCs has also been shown to have positive impacts on sperm parameters. Cai et al. (2021) reported that bone marrow-derived secretome improves spermatogenesis in mice with busulfan-induced azoospermia. Canine amniotic membrane-derived MSCs protected dog sperm in the freezing and thawing processes (Mahiddine et al., 2020).
CONCLUSION
In summary, ASE improved ΔΨm and declined mitochondrial oxidative damages. ASE also enhanced rat sperm quality. It is suggested that ASE might ameliorate Bis-A-induced mitochondrial injury and rat sperm quality impairment by inhibiting oxidative stress. Future studies are required to clarify the mechanism of ASE on spermatozoa and mitochondrial-related pathways such as mitophagy and apoptosis. This primary study stimulates the research to explore ASE’s benefits on male infertility disorders.
ACKNOWLEDGMENT
Student Research Committee of Ahvaz Jundishapur University granted this study (Grant No: 99s33).
Ethical Approval
The Animal Research Ethics Committee (IR.AJUMS.ABHC.REC.1399.018) approved the current study of Ahvaz Jundishapur University of Medical Sciences.
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