Diabetes mellitus is the most common chronic disease worldwide that causes numerous complications, including male infertility. The prevalence of DM is 451 million people and estimated that would increase to 693 million in 2045. Fluorosis caused by drinking water contaminated with inorganic fluoride is a public health problem in many areas around the world. Previous studies have shown that fluoride exposure damages the male reproductive function. This study aimed to evaluate the fluoride sub-chronic exposure on the spermatozoa function in streptozotocin (STZ)-induced diabetic mice. After confirming diabetes by measuring blood glucose levels, the male mice received 45.2 ppm of fluoride added or deionized water. We evaluated several parameters in diabetic mice exposed to fluoride: standard quality analysis, the mitochondrial transmembrane potential (
Fluoride is an abundant environmental pollutant widely existing in rocks, soil, water, food, and others [
The world prevalence of Diabetes mellitus (DM) in adults reached 451 million people in 2017, and it estimated that would increase to 693 million people in 2045 [
Male CD1 mice were obtained from the Institute of Health Sciences at the Autonomous University of Hidalgo (Hidalgo, Mexico). The animals were maintained according to the norms of the Institutional Ethics Animal Care and Use Committee (CIECUAL), under standard conditions with a 12 h/12 h light/dark cycle, constant temperature (22 ± 2°C) and humidity (50%). Food (Lab Diet® 5013) and water freely available in their home cages. The experimental procedures approved by CIECUAL of the Autonomous University of Hidalgo.
Twenty-four animals were randomly issued into four experimental groups as follows: group I (control), group II (STZ-induced diabetes), group III (fluoride-exposure), and group IV (STZ-induced diabetes plus fluoride exposure). Mice (45-day-old) were made diabetic by applying a single dose intraperitoneal injection of 150 mg/kg STZ dissolved in citrate buffer (pH 4.5). The tail vein blood glucose was measured after seven days of injection, and those animals with a blood glucose concentration ≥250 mg/dl were included in this study. At 60 days, the mean blood glucose in mice-STZ-treated was 460 mg/dl. Co-exposure with fluoride started once diabetes was confirmed in the animals. Control group mice received with deionized water, and the mice from groups III and IV received water-containing fluoride at 45.2 ppm during 60 days. The animals were kept for 60 days to assure more than one spermatogenic cycle in mice (approximately 40 days) [
After 60 days of treatment, the mice were euthanized by cervical dislocation, the testis-epididymis-vas deferens complexes were dissected, and the spermatozoa were isolated by flushing the vas deferens and cauda epididymis lumens with one ml of phosphate-buffered saline (PBS, pH 7.4) at 37°C. Sperm parameters, including motility, viability, and concentration, were evaluated according to the method previously described under a light microscope at a magnification of ×400 [
The mitochondrial membrane potential (
The detection of active Caspase 3 and 7 was carried out by Cell Event caspase 3/7 Green detection reagent, which is a four-amino acid peptide (DEVD), conjugated with a nucleic acid binding dye. After the activation of caspase-3/7 in apoptotic cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright, fluorogenic response. For each spermatozoa sample, 1 ml of sperm suspension in PBS (10 × 106 spermatozoa/ml) was incubated during 30 min at 37°C with caspase 3/7 reagent (2
During the exposure period to fluoride, urine was collected three times at 0, 30, and 60 days of the study in a nonfasted state. For the urine collection, the animals remained in metabolic cages, and urine was collected over 24 h to quantify the fluoride concentration by a potentiometric method using an ion-selective electrode (Orion 9609). For its analysis, the urine was mixed with TISAB II (1 : 1). The electrode was calibrated at the standard range of 0.01–10 ppm [
The weight of testes was recordered to calculate the gonadosomatic index using the following formula: [gonada weight × body weight−1] × 100. The control testes samples and the diabetic fluoride-exposed mice were fixed with to 10% of formalin in phosphate-buffered saline (pH 7.4) and processed with conventional paraffin-embedding methods. Afterward, the paraffinized-tissue blocks were sectioned at 5
Moreover, Johnsen’s criteria evaluated spermatogenesis was performed by analyzing twenty-five seminiferous tubules per animal. In this evaluation, a score is assigned to describe the spermatogenesis process quantitatively. Considering that the progressive degeneration of the seminiferous tubules presents the loss of mature spermatozoa and continues until the loss of spermatogonia, then to Sertoli cells. According to these criteria, a score of 9 or 10 indicates normal testicular histology, a score of 8 signifies hypo-spermatogenesis. A score of 3–7 represents maturation arrest, a score of 2 indicates Sertoli cells aplasia and, a score of 1 indicates tubular fibrosis. The samples were examined with phase-contrast microscopy at 40× magnification [
The program Sigma Stat Statistics Analysis System version 4.0 was used to analyze the data statistics. The one-way ANOVA followed by a Bonferroni correction to evaluate pairwise differences. A
In general, at 60 days, the mean body weight in the fluoride plus STZ group decreased significantly 1.40-fold and 1.16-fold, compared with the control and fluoride-exposed groups respectively. Also, there was no significant difference in body weight compared with the STZ group (Table
Assessment of spermatozoa parameters.
Sperm parameters | Control | STZ | Fluoride | Fluoride + STZ |
---|---|---|---|---|
Motility (%) | 88.66 ± 3.82 | 55.83 ± 13.57a | 57.83 ± 7.75a | 23.33 ± 22.50a,b,d |
Sperm concentration (106/ml) | 62.12 ± 11.43 | 40.43 ± 10.82a | 46 ± 8.02a | 15.81 ± 13.24a,b,d |
Viability (%) | 86 ± 1.09 | 57.33 ± 8.93a | 48.5 ± 6.74a | 33 ± 23.84ab |
GSI (%) | 0.592 ± 0.041 | 0.406 ± 0.018a | 0.476 ± 0.065a | 0.375 ± 0.072a,d |
Body weight (g) | 49.17 ± 1.74 | 35.06 ± 3.82a | 30.56 ± 3.90a | 42.25 ± 1.92a,d |
Number of seminiferous tubules | 290 ± 13.3 | 259 ± 8.2a | 268 ± 7.2a | 251 ± 9.1a,b |
Seminiferous tubule diameter (mm) | 0.23 ± 0.020 | 0.19 ± 0.017a | 0.20 ± 0.019a | 0.15 ± 0.015a,b,d |
Histology (Johnsen’s score) | 9.14 ± 1.81 | 7.33 ± 2.27a | 8.01 ± 1.61a,d | 6.41 ± 2.17a,b |
Values are means ± SD. a(
The mitochondrial membrane potential (Δ
Effect of subchronic fluoride exposure in STZ-induced on Δ
Effect of subchronic fluoride exposure in STZ-induced on caspase 3/7 activity. Detection of spermatozoa with activated caspases by cytometry flow using invitrogen cell event caspase 3/7. Subchronic fluoride exposure in STZ-induced diabetes leads to a greater increase in caspase 3/7 activated. Values are means ± SD of 6 mice per group (
Figure
Urinary fluoride concentration during treatment for all groups. Control group, STZ group, fluoride group, fluoride in STZ-induced diabetes. Values are means ± SD of 6 mice per group. (
This study aimed to assess the effect of a fluoride subchronic-exposure on impairments caused in spermatozoa due to the exposure of fluoride in STZ-induced diabetes mice. Several studies have shown that fluoride exposure has a negative effect on male reproduction, altering sperm quality, epidydimal maturation, capacitation, acrosome reaction, damage to DNA integrity, and fertilization [
On the other hand, both induced diabetes and diabetes disease have demonstrated adverse effects on sperm quality and sperm DNA integrity [
In the same study, a subchronic fluoride exposure under diabetic conditions negatively aggravated the spermatozoa quality (motility, viability, and concentration) compared to the diabetic and fluoride groups. Sperm motility is dependent upon the availability of energy obtained through ATP hydrolysis produced by oxidative phosphorylation [
Apoptosis is a regulated cell death program that is a trigger via the extrinsic pathway, which implicates the activation of the cell surface death receptors, or the intrinsic pathway, which involves mitochondrial outer membrane permeabilization, or the apoptotic signaling caused by endoplasmic reticulum stress [
Likewise, several alterations caused by the exposure to fluoride in male reproduction have been corroborated by microarray analysis and real-time RT-PCR, 63 down-regulated genes, which are involved in several sperm biological processes including signal transduction, oxidative stress, apoptosis, electronic transport chain, glycolysis, chemotaxis, spermatogenesis, and spermatozoa capacitation [
Additionally, fluoride causes degeneration and necrosis of the tubular cells, renal tubular hyaline casts, and glomeruli swelling, accompanied by alterations of renal function caused by renal oxidative damage [
On the other hand, DM is a disease that affects the duration and quality of life due to various intrinsic complications. DM can affect spermatogenesis process in multiple ways [
Recently it has been shown that co-exposures with fluoride with sulfur dioxide worsen on brain and kidney toxicity [
Results of this study suggest that the exposure of mice in diabetic conditions to fluoride aggravates the spermatozoa function, decreasing the mitochondrial membrane potential, increasing apoptosis and worsening histological changes of testicular cells. Besides, the diminution found in male quality parameters could be a reflection of the impairment in spermatogenesis by the co-exposure to fluoride and DM. Further studies is required, to characterize the mechanism by which the combined effect of fluoride in diabetic conditions exert a great toxic effect.
The data used to support the findings of this study are available from the corresponding author upon request.
Authors declare no conflicts of interest.
The authors wish to acknowledge Alvaro Rubén Hernández Cruz for technical support in histology technique performed. This study was partially supported by PROFOCIE 2018.