Publication
Molecular and metabolic pathways for endocrine-disrupting chemicals action in male fertility: focus on atrazine
| Summary: | In the last half-century sperm quality has been declining globally. Although its causes remain elusive, it is suggested that alterations in environmental conditions and increased exposure to industrial and pharmaceutical chemicals play a key role due to their capacity as endocrine-disrupting chemicals (EDCs). EDCs are ubiquitous in our daily lives, being present in our environment, food, and consumer products. Atrazine (ATZ) is a pesticide listed as a potent EDC, capable of causing a variety of negative health outcomes, including decreased testosterone production, delayed puberty, and infertility. However, our understanding of the influence of ATZ on testicular metabolism remains limited. Within the male reproductive tract, somatic Sertoli cells (SCs) act as sentinels and regulate spermatogenesis. Thus, we hypothesized that ATZ may alter the function of SCs and consequently affect spermatogenesis. Herein we aimed to study the effects of environmentally relevant concentrations of ATZ on the metabolic and mitochondrial function of SCs. For this, mouse SCs (mSCs) were exposed to increasing concentrations of ATZ (0.3, 3, 30, 300 and 3000 μg/L) for 24h at 37°C. ATZ concentrations were selected based on concentrations found in human occupational exposure (0.3 μg/L), the legal limit of ATZ in drinking water (3 and 30 μg/L), and concentrations found in soils and rivers near agricultural crops where ATZ was used (300 and 3000 μg/L). After incubation, cytotoxicity was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assays. Cellular proliferation was assessed by sulforhodamine B (SRB) assay. Mitochondrial activity and endogenous reactive oxygen species (ROS) production were evaluated using JC-1 and CM-H2DCFDA probe, respectively. The ferric reducing antioxidant power (FRAP) assay was used to measure the antioxidant potential of culture media. Protein levels of LDH were analyzed using Western Blot and the mSCs glycolytic function using the Seahorse XF Glycolysis Stress Test Kit. Metabolites in the culture media were identified and quantified by the Nuclear Magnetic Resonance (1H-NMR) technique. Our findings provide compelling evidence that ATZ exposure led to a notable reduction in the metabolic activity of mSCs at all tested concentrations, without inducing cytotoxicity. Despite existing reports indicating that ATZ can induce oxidative stress, our study did not reveal any significant changes in mitochondrial function or ROS production in mSCs. ATZ exposure significantly impaired mSCs glycolysis and glycolytic capacity, at all tested concentrations. These findings were further corroborated by a decrease in the expression of LDH. Interestingly, glucose and pyruvate consumption, and acetate and lactate production were unaffected by ATZ exposure. In this study, we present, for the first time, evidence that ATZ exerts a detrimental effect on the metabolic activity of mSCs, without altering cell viability and proliferation. This discovery underscores the need for future studies to unravel the intricate mechanisms through which ATZ operates and to gain a deeper understanding of how it may disrupt the crucial nutritional support required for spermatogenesis. |
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| Main Authors: | Andrade, Daniela Patrícia Gomes de |
| Subject: | Atrazine Sertoli cell Spermatogenesis Cell metabolism Lactate Male fertility |
| Year: | 2023 |
| Country: | Portugal |
| Document type: | master thesis |
| Access type: | open access |
| Associated institution: | Universidade de Aveiro |
| Language: | English |
| Origin: | RIA - Repositório Institucional da Universidade de Aveiro |
| Summary: | In the last half-century sperm quality has been declining globally. Although its causes remain elusive, it is suggested that alterations in environmental conditions and increased exposure to industrial and pharmaceutical chemicals play a key role due to their capacity as endocrine-disrupting chemicals (EDCs). EDCs are ubiquitous in our daily lives, being present in our environment, food, and consumer products. Atrazine (ATZ) is a pesticide listed as a potent EDC, capable of causing a variety of negative health outcomes, including decreased testosterone production, delayed puberty, and infertility. However, our understanding of the influence of ATZ on testicular metabolism remains limited. Within the male reproductive tract, somatic Sertoli cells (SCs) act as sentinels and regulate spermatogenesis. Thus, we hypothesized that ATZ may alter the function of SCs and consequently affect spermatogenesis. Herein we aimed to study the effects of environmentally relevant concentrations of ATZ on the metabolic and mitochondrial function of SCs. For this, mouse SCs (mSCs) were exposed to increasing concentrations of ATZ (0.3, 3, 30, 300 and 3000 μg/L) for 24h at 37°C. ATZ concentrations were selected based on concentrations found in human occupational exposure (0.3 μg/L), the legal limit of ATZ in drinking water (3 and 30 μg/L), and concentrations found in soils and rivers near agricultural crops where ATZ was used (300 and 3000 μg/L). After incubation, cytotoxicity was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assays. Cellular proliferation was assessed by sulforhodamine B (SRB) assay. Mitochondrial activity and endogenous reactive oxygen species (ROS) production were evaluated using JC-1 and CM-H2DCFDA probe, respectively. The ferric reducing antioxidant power (FRAP) assay was used to measure the antioxidant potential of culture media. Protein levels of LDH were analyzed using Western Blot and the mSCs glycolytic function using the Seahorse XF Glycolysis Stress Test Kit. Metabolites in the culture media were identified and quantified by the Nuclear Magnetic Resonance (1H-NMR) technique. Our findings provide compelling evidence that ATZ exposure led to a notable reduction in the metabolic activity of mSCs at all tested concentrations, without inducing cytotoxicity. Despite existing reports indicating that ATZ can induce oxidative stress, our study did not reveal any significant changes in mitochondrial function or ROS production in mSCs. ATZ exposure significantly impaired mSCs glycolysis and glycolytic capacity, at all tested concentrations. These findings were further corroborated by a decrease in the expression of LDH. Interestingly, glucose and pyruvate consumption, and acetate and lactate production were unaffected by ATZ exposure. In this study, we present, for the first time, evidence that ATZ exerts a detrimental effect on the metabolic activity of mSCs, without altering cell viability and proliferation. This discovery underscores the need for future studies to unravel the intricate mechanisms through which ATZ operates and to gain a deeper understanding of how it may disrupt the crucial nutritional support required for spermatogenesis. |
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