Calcium fluoride

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Remarks:

Mechanistic assessment of effects on spermatogenesis

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Non-standard published study / Non-standard parameters

Data source

Reference

Reference Type:

publication

Title:

Fluoride exposure changed the structure and the expressions of Y chromosome related genes in testes of mice

Author:

Jinling Cao, Yan Chen, Jianjie Chen, Hanghang Yan, Meiyan Li, Jundong Wang

Year:

2016

Bibliographic source:

Chemosphere 161 (2016) 292-299

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
In the present study, the effects of fluoride on histological structure, sex testosterone contents, sperm quality, and the mRNA expression and protein level profiles of Y chromosome microdeletion genes associated with spermatogenesis were examined in testes of male mice treated with the different concentrations of sodium fluoride (NaF) in drinking water to investigate the effects of fluoride on spermatogenesis and the underlying toxic mechanisms caused by fluoride
- Short description of test conditions:
One hundred and twenty healthy male Kunming mice (8-week old, the mean body weight of 30 ± 0.16 g) were obtained from the Experimental Animal Center of Shanxi Medical University. Prior to the experiment, they were kept in the plastic cage containing shavings as bedding material and maintained under standard laboratory conditions, room temperature of 18-22°C, good ventilation and a certain humidity and hygienic conditions. The mice were fed commercial rodent pellets and distilled water ad libitum. After acclimatization for 1 week, the male Kunming mice were randomly divided into four groups of thirty mice each, the control group and three NaF treatment groups. The control group received distilled water. Three NaF-treated groups have ad libitum access to distilled water containing 25, 50, 100 mg/L NaF, respectively, for 11 weeks. Each group was studied in triplicate (n = 10 for each group). The duration and doses of NaF exposure used were based on our previous study (Han et al., 2014). The everyday water-drinking amount for each mouse in our study was 7.2 ± 0.3 mL/day. All animal work was authorized by the Institutional Animal Care and Use Committee of Shanxi Agricultural University.
- Parameters analysed / observed:
During the treatment period, the body weights of all the mice were recorded each week. After 11 weeks, all male mice were executed by a cervical vertebrae luxation according to animal ethical standards. The femur was collected to determine fluoride content by fluoride ion selective electrode method (Inkielewicz et al., 2003 ). The epididymis were taken and used for the organ coefficient, sperm quality evaluation. The testes were taken and used for the histological determination, organ coefficient, testosterone assay, and the analysis of mRNA expressions and protein levels. Blood was obtained from the eyeballs of mice and used for serum testosterone assay.

GLP compliance:

not specified

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Sodium fluoride

EC Number:

231-667-8

EC Name:

Sodium fluoride

Cas Number:

7681-49-4

IUPAC Name:

sodium fluoride

Details on test material:

Studies were performed with sodium fluoride

Test animals

Species:

mouse

Strain:

other: Kunming

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Animal Center of Shanxi Medical University
- Age at study initiation: (P) 8 wks;
- Weight at study initiation: (P) Males: mean body weight of 30 ± 0.16 g
- Housing: Prior to the experiment, they were kept in the plastic cage containing shavings as bedding material and maintained under standard laboratory conditions
- Diet (e.g. ad libitum): The mice were fed commercial rodent pellets ad libitum.
- Water (e.g. ad libitum): The mice were fed distilled water ad libitum.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22°C
- Humidity (%): a certain humidity
- Air changes (per hr): good ventilation

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

other: distilled water

Details on exposure:

VEHICLE
- Concentration in vehicle: 25, 50, 100 mg/L NaF in drinking water
The everyday water-drinking amount for each mouse in our study was 7.2 ± 0.3 mL/day.

Duration of treatment / exposure:

11 weeks

Frequency of treatment:

continuous via drinking water

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L drinking water

Doses / concentrations 2

Dose / conc.:

25 mg/L drinking water

Remarks:

In the absence of mean average body weight at study termination it is not possible to calculated actual received doses.

Doses / concentrations 3

Dose / conc.:

50 mg/L drinking water

Remarks:

In the absence of mean average body weight at study termination it is not possible to calculated actual received doses.

Doses / concentrations 4

Dose / conc.:

100 mg/L drinking water

Remarks:

In the absence of mean average body weight at study termination it is not possible to calculated actual received doses.

No. of animals per sex per dose:

30 (Each dose group was studied in triplicate (n = 10 for each group))

Control animals:

yes, concurrent vehicle

Details on study design:

1. Animals and treatments
One hundred and twenty healthy male Kunming mice (8-week old, the mean body weight of 30 ± 0.16 g) were obtained from the Experimental Animal Center of Shanxi Medical University. Prior to the experiment, they were kept in the plastic cage containing shavings as bedding material and maintained under standard laboratory conditions, room temperature of 18-22°C, good ventilation and a certain humidity and hygienic conditions. The mice were fed commercial rodent pellets and distilled water ad libitum.
After acclimatization for 1 week, the male Kunming mice were randomly divided into four groups of thirty mice each, the control group and three NaF treatment groups. The control group received distilled water. Three NaF-treated groups have ad libitum access to distilled water containing 25, 50, 100 mg/L NaF, respectively, for 11 weeks. Each group was studied in triplicate (n = 10 for each group).
The duration and doses of NaF exposure used were based on our previous study (Han et al., 2014). The everyday water-drinking amount for each mouse in our study was 7.2 ± 0.3 mL/day. All animal work was authorized by the Institutional Animal Care and Use Committee of Shanxi Agricultural University.
During the treatment period, the body weights of all the mice were recorded each week. After 11 weeks, all male mice were executed by a cervical vertebrae luxation according to animal ethical standards. The femur was collected to determine fluoride content by fluoride ion selective electrode method (Inkielewicz et al., 2003). The epididymis were taken and used for the organ coefficient, sperm quality evaluation. The testes were taken and used for the histological determination, organ coefficient, testosterone assay, and the analysis of mRNA expressions and protein levels. Blood was obtained from the eyeballs of mice and used for serum testosterone assay.

2. Organ coefficient
The testes or epididymis were weighed and the organ coefficient was calculated according to the formulae: Organ coefficient = Organ wet weight (g)/Body weight (g) x 100%.

3. Sperm quality evaluation
Sperm were obtained from the cauda epididymis and rinsed through 0.9% sodium chloride. Sperm quality was evaluated by sperm count, abnormality rate of sperm and sperm head. Sperm count was calculated by counting the number of sperm per milliliter sperm suspension under the micro scope with the method of erythrometry. The sperm morphology was observed and the abnormal rates of sperm and sperm head were calculated according to the shapes of teratospermia described in Fig. 1 (Supplement material). As shown in Fig. 1 (Supplement material), A is healthy sperm, and B to L indicates the morphologies of abnormal sperm. Sperm could be induced a variety of malformations. When the abnormality of spermatozoon occurred in head, we would record it as sperm head abnormality.

4. Histological examination
The testes were immediately fixed in Bouins' solution for 24 h and rinsed by running water. Then testes samples were dehydrated in graded alcohol, cleared in xylene, and embedded in paraffin.
Next, they were cut into 4-µm sections with a rotary microtome (Paraffin machine, Leica RM 2245, Germany) and stained with Delafield's hematoxylin and alcoholic Eosin. Finally, the histological changes were observed under the optical microscope (Olympus, Japan).

5. Determination of testosterone concentration
The testosterone concentrations in the blood and testes were measured using γ Radiation immunization register (USTC Zonkia Scientific Instruments Co. Ltd, Anhui, China) based on the instruction of testosterone radioimnunoassay kit (HTA Co., Ltd. Beijing, China). Units for results were in ng/dL.

6. Total RNA extraction and quantitative real-time PCR (QRT-PCR)
Total RNA was extracted from mouse testes using Trizol Reagent, (TaKaRa Biological Engineering Company, Dalian, China) based on the manufacturer's instructions and digested by RNase-free DNase I (Promega Madison, WI, USA) to remove genomic DNA contamination.
After that, NanoDrop ND1000 Spectrophotometer (NanoDrop, USA) and agarose gel electrophoresis were used for the examination of RNA quality. The reverse transcription (RT) assays were performed with 500 ng total RNA in a 10 µL reaction mixture according to the protocol of PrimeScript® RT Master Mix (Takara Biological Engineering Company, Dalian, China). The specific primers of spermiogenesis specific transcript on the Y 2 (Ssty2), DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), sycp3 like Y-linked (Sly) and heat shock factor 2 (HSF2), β-actin for QRT-PCR were designed using Primer 3.0 software based on the corresponding mouse sequences in Genbank (Table 1) and synthesized by Biosynthesis Biotechnology CO., LTD (Beijing, China).
Before quantitative analysis, the amplification efficiencies between reference genes and target genes were determined, and the results showed that the efficiency differences were less than 5%. Thus, mRNA expressions by QRT-PCR were calculated by the comparative Ct method in the study (Livak and Schmittgen, 2001). Afterwards, the QRT-PCR was performed in a total reaction volume of 10 µL containing 5 µL of SYBR Premix Ex TaqTM (2x), 0.4 µL of forward primer and reverse primer, 0.2 µL of ROX Reference Dyell(50x), 1 µL of cDNA, and 3 µL of RNase free dH2O by using SYBR Premix Ex TaqTM 11 QRT-PCR kit (TaKaRa Co., Dalian, China) and the Mx3000PTM QRT-PCR system (Stratagene, USA). The reaction conditions were as follows: an initial denaturation at 95°C for 10 s, followed by 40 cycles of 95°C for 10 s, 61°C for 15 s and 72°C for 6 s. Negative control without template was used to eliminate contamination. All the reactions were performed in triplicate. The target gene expression level was normalized to β-actin mRNA level.
To verify each amplification's specificity, a melt curve analysis was performed after QRT-PCR and the PCR product was examined on a 1.5% agarose gel. The data indicated that only 1 PCR product was obtained for each individual primer set, indicating the specificity of amplification. The relative expression levels of genes were calculated using 2 -ΔΔCt method (Livak and Schmittgen, 2001).

7. Enzyme-linked immunosorbent assay
Testes were extracted by pre-cooling RIPA Tissue Protein Extraction Reagent (Beyotime Biotechnology, China), mixed with 1% proteinase inhibitor. Then testicular homogenate was treated with ice-water bath for 30 min. The precipitate was removed by centrifugation at 12,000 x g for 5 min at 4°C, and the clear supernatant was used for measuring the protein concentration by BCA Protein Assay Kit (Beyotime Biotechnology, China). The protein contents of Ssty2 (genecopoeia, America), DDX2Y (cusabio, America), Sly (cusabio, America), and HSF2 (cusabio, America) were determined following the instructions of the corresponding ELISA kits.

Statistics:

Experimental data were expressed as mean ± SD and analyzed with GraphPad Prism 5 software (GraphPad Software Inc., San Diego, USA). Prior to the statistical analysis, all data were checked for homogeneity of variances (Levene's F test) and all date met the assumptions of equal variances. Differences among the treatment groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's test for Multiple Comparison, at 95% confidence limits. The differences were considered statistically significant when p < 0.05.

Results and discussion

Effect levels

1

Dose descriptor:

conc. level:

Remarks:

sperm count

Effect level:

50 mg/L drinking water

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: significant decrease in sperm counts

Remarks on result:

other: Compared with the control group, sperm counts in 50 and 100 mg/L NaF groups were significantly decreased by 25.51% and 30.82%,

2

Dose descriptor:

conc. level:

Remarks:

deformity ratio of sperm and sperm head

Effect level:

25 mg/L drinking water

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

The deformity ratio of sperm and sperm head were significantly increased in all NaF-treated groups.

3

Dose descriptor:

conc. level: serum testosterone level

Effect level:

100 other: mg/L NaF

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

clinical biochemistry

Remarks on result:

other: The serum testosterone was significantly decreased by 61.22% in 100 mg/L NaF groups.

4

Dose descriptor:

conc. level: testosterone content testis

Effect level:

50 other: mg/L NaF

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: testosterone content in testis

Remarks on result:

other: Testis testosterone was remarkably reduced by 43.95% in 100 mg/L NaF groups compared with the control group.

5

Dose descriptor:

conc. level: expression of HSF2 mRNA

Effect level:

100 mg/L drinking water

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: Expression of HSF2 mRNA

Remarks on result:

other: The expression of HSF2 mRNA was significantly decreased by 28.06% in 100 mg/L NaF group

6

Dose descriptor:

conc. level: expression of Sly mRNA

Effect level:

100 mg/L drinking water

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: Expression of Sly mRNA

Remarks on result:

other: he expression Sly mRNA was significantly decreased by 51.34% in 100 mg/L NaF group

7

Dose descriptor:

conc. level: Ssty2 mRNA levels

Effect level:

50 mg/L drinking water

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: Ssty2 mRNA levels

Remarks on result:

other: The Ssty2 mRNA levels were significantly reduced by 64.90% in 100 mg/L NaF group (p < 0.05).

8

Dose descriptor:

conc. level: Ssty2 protein levels

Effect level:

50 mg/L drinking water

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: Ssty2 protein levels

Remarks on result:

other: The Ssty2 protein levels were significantly decreased by 38.46% in 100 mg/L NaF group treatment groups (p < 0.05).

9

Dose descriptor:

conc. level: protein levels of Sly

Effect level:

50 mg/L drinking water

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: Protein levels of Sly

Remarks on result:

other: The protein levels of Sly were significantly reduced by 35.42% in 100 mg/L NaF group (p < 0.05).

10

Dose descriptor:

conc. level: HSF2 protein levels

Effect level:

100 mg/L drinking water

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: HSF2 protein levels

Remarks on result:

other: The HSF2 protein levels showed a decreasing trend and were significantly suppressed by 24.61 % in the highest concentration of fluoride (100 mg/L NaF)

Observed effects

1. Effects of NaF on growth in male mice
The male mice in the control group were in good nutritional status with well-developed figures, soft and silky coats, and health teeth. The mice in NaF-treated groups showed poor development, rough coats and even rough teeth with dark brown stains. The mice in each group gained weight during the NaF treatment, but no differences were found between the control and the experimental groups (Fig. 1 ).
The organ coefficients of testes in treatment groups were less than that in the control group, but the differences were not significant (p > 0.05). There was a decreasing tendency in the epididymal viscera coefficients, and the reduction was significant in the highest fluoride group (100 mg/L of NaF) (p < 0.01) compared with the control group. These results indicated that the effects of NaF on epididymis were greater than on testis.

2. Fluoride concentration of bone in NaF-treated male mice
After being treated with NaF for 11 weeks, the fluoride concentration in femur of all NaF-treated male mice were increased significantly compared with the control group.

3. Effects of NaF on the sperm quality
The sperm count, the abnormal ratio of sperm and sperm head in NaF-treated male mice were shown in Fig. 2. Compared with the control group, sperm counts in 50 and 100 mg/L NaF groups were significantly decreased by 25.51% and 30.82%, while the deformity ratio of sperm and sperm head were significantly increased in all NaF-treated groups, the maximum increase by 72.89% and 72.73% in 100 mg/L NaF group, respectively.

4. Effects of NaF on testes tissue in male mice
Testis histological changes were shown in Fig. 3. In the control testes, testicular interstitial was normal and 3-5 interstitial cells constituted a group. The structure of seminiferous tubules was normal in which a large number of sperm were found. The Sertoli cells and spermatogenic cells were morphologically normal and orderly arranged. After treatment with NaF, compared with the control group, HE staining found a few vacuoles in seminiferous tubules, irregular arrangement and decreased layers of spermatogenic cells in mice treated with 25 and 50 mg/L NaF. The most obvious damage of testicular structure was found in 100 mg/L NaF, including the abnormal arrangement and morphological malformations of spermatogenic cells, and decreased number of sperm in the lumen. All above indicated that the histological damages of testicular tissues in all treatment groups were aggravated with the increase of NaF concentration.

5. Testosterone content in serum and testis of NaF-treated male mice
As shown in Fig. 2, the testosterone contents in serum and testis were lower than that of the control group. The serum testosterone was significantly decreased by 61.22% in 100 mg/L NaF groups and the testis testosterone was remarkably reduced by 44.72% and 43.95% in 50 and 100 mg/L NaF groups compared with the control group, respectively.

6. Effects of NaF on the mRNA expressions of Y chromosome related genes
After treatment for 11 weeks, the mRNA expressions of the Y chromosome microdeletion genes related spermatogenesis were changed markedly.
Compared with the control group, the DDX3Y mRNA level increased with the NaF concentration, but the differences were not significant (p > 0.05). The expressions of HSF2 and Sly mRNA were significantly decreased by 28.06% and 51.34% in 100 mg/L NaF group, and the Ssty2 mRNA levels were significantly reduced by 66.98% and 64.90% in 50 and 100 mg/L NaF group (p < 0.05), respectively.

7. Effects of NaF on Y chromosome related protein levels
According of the mRNA expression results, ELISA was performed to determine and analyze the corresponding protein levels. The results indicated that compared with the control group, the DDX3Y protein levels were relatively unchanged in all NaF groups.
The Ssty2 protein levels were significantly decreased by 36% and 38.46% in 50 and 100 mg/L NaF group treatment groups (p < 0.05), respectively. The protein levels of Sly were significantly reduced by 18.37% and 35.42% in 50 and 100 mg/L NaF group (p < 0.05), respectively. The HSF2 protein levels showed a decreasing trend and were significantly suppressed by 24.61 % in the highest concentration of fluoride (100 mg/L NaF).

Any other information on results incl. tables

Discussion

Reproduction is a biological process, and it can be easily affected by toxicants. The testosterone is an important androgen produced in the testis and is involved in the initiation of spermatogenesis (Pushpalatha et al., 2005). Many studies have reported the male reproductive defects induced by fluoride, such as the deleterious effects of NaF on testosterone level, spermatogenesis, sperm motility and morphology, and fertilizing ability (Long et al., 2009; Sun et al., 2014; Zhang et al., 2006). When the male mice were exposed to 500 ppm and 1000 ppm of fluoride for 3 months, the spermatogenesis failed (Kaminsky et al., 1990). When male mice were treated with 100 and 500 mg/L of NaF in drinking water, there was a lack of maturation and differentiation of spermatogenesis in male mice (Kour and Singh, 1980). After sexually mature male Wistar rats were exposed to 2, 4, and 6 ppm NaF in their drinking water for 6 months ad libitum, fluoride significantly decreased the weight of testis, epididymis, and ventral prostate, and the sperm motility and density were significantly reduced (Gupta et al., 2007).

In present study, the testosterone of serum and testis, and sperm count in all treatment groups were decreased, while the abnormality ratio of sperm and sperm head were increased in all NaF treated groups. These results indicated that NaF damaged the male reproductive system and led to the impairment of spermatogenesis by decreasing the testosterone level and sperm quality, which was supported by the previous studies (Lubkowska et al., 2006). We speculated that this might be due to the changes of the testicular structure as evidenced by our histological observation.

People have been only focused on the mammalian Y chromosome on male-specific functions until Tiepolo L found the long arm of Y chromosome of azoospermia patients was deleted and concluded that the Y chromosome was important in the spermatogenesis in 1976 (Tiepolo and Zuffardi, 1976). Since then, more and more studies on spermatogenesis were performed based on the Y chromosome deletions in infertile men and male mice (Campbell et al., 2013: Foresta et al., 2001 ). A large number of epidemiological investigations showed the decreased male sperm number and the semen quality, and found that constant environmental destruction resulted in more and more serious male infertility. The second most frequent genetic defect was found in the pathogenesis of infertility of mouse (Campbell et al., 2013 ). Previous studies found that when some genes were deleted in Y chromosome, male spermatogenesis would fail (Fu et al., 2012; Kim et al., 2012). Deletions of the male-specific region of the mouse Y chromosome long arm (MSYq), harboring multiple copies of a few genes, led to sperm head defects, anomalies and even infertility (Ellis et al., 2007; Riel et al., 2013 ).

DDX3Y gene, located in the Yq11.23 region, belongs to no sperm factor (AZF) and is the main candidate gene of AZFa (Vergnaud et al., 1986). The deletion of DDX3Y is frequently found in infertile men, which leads to the failure of spermatogenesis, so DDX3Y is regarded as an important gene associated with spermatogenesis and mainly exists in the sperm cell. Studies showed that the deletion of DDX3Y resulted in the failure of spermatogenesis (Foresta et al., 2000; Lardone et al., 2007). In this study, the mRNA expressions and protein levels of DDX3Y were not significantly different between the treatment groups and the control group, which indicated that NaF did not affect the DDX3Y expressions.

Sly and Ssty2, which belong to the genes of Y chromosome microdeletion, are located at the male-specific region of MSYq and specifically express during spermatogenesis (Calenda et al., 1994; Reynard et al.. 2007). Sly-deletion mice have seriously damaged sperm differentiation and even infertility (Burgoyne et al., 1992; Riel et al., 2013 ). In the present study, Sly mRNA expression was significantly decreased in 100 mg/L NaF group and Sly protein levels were significantly decreased in 50 and 100 mg/L NaF groups. Large deletions of MSYq are associated with sperm head defects for which Ssty deficiency has been presumed to be responsible (Touré et al., 2005). A previous study indicated that the loss of Ssty expression in the mouse Y Chromosome Long Arm effected the development and sterility of sperm (Touré et al., 2004). In this study, Ssty2 mRNA expressions were significantly decreased in 50 mg/L and 100 mg/L of NaF groups. Similar results were obtained in the Ssty2 protein levels.

HSF2 is expressed in embryogenesis (Pirkkala et al., 2001 ). For adult mice, HSF2 is mainly expressed in the nuclei of meiotic and differentiated male germ cells (Bettegowda and Wilkinson, 2010). Kallio et al. found that the size of testicles and epididymides were reduced, while apoptosis of pachytene spermatocytes was increased in HSF2-mutant male mice (Kallio et al., 2002). The target genes of HSF2 include Ssty2 and Sly (Åkerfeit et al., 2010; Bettegowda and Wilkinson, 2010; Björk et al., 2010). The levels of Ssty2 and Sly mRNA expression were reduced in knockout Hsf2 mice (Abane and Mezger, 2010 ). In this study, the HSF2 mRNA and protein levels were significantly down-regulated in the 100 mg/L of NaF group, which was correlated with the decreased expressions of Ssty2 and Sly. The deficiency of Ssty2, Sly and Hsf2 resulted in a remarkably increase in sperm head abnormality ratio (Bettegowda and Wilkinson, 2010; Cocquet et al., 2009; Liu et al., 2011; Ward and Burgoyne, 2006; Zhang et al., 2009). This study indicated that the abnormality ratio of sperm head was increased when the male mice was exposed to fluoride, especially in 100 mg/L of NaF group.

In summary, NaF did have toxic effects on male reproductive system, which reduced the testosterone content and sperm number, and increased the abnormality ratio of sperm and sperm head, supported by the damages of the testicular structure, as a consequence of depressed HSF2 level, which resulted in the downregulation of Ssty2 and Sly mRNA and protein.

Applicant's summary and conclusion

Conclusions:

In summary, NaF did have toxic effects on male reproductive system, which reduced the testosterone content and sperm number, and increased the abnormality ratio of sperm and sperm head, supported by the damages of the testicular structure, as a consequence of depressed HSF2 level, which resulted in the downregulation of Ssty2 and Sly mRNA and protein.
Effects started to occur as off the lowest dose concentration of 25 mg/L NaF in the drinking water (sperm and sperm head abnormalities).
In the absence of mean average body weight at study termination it is not possible to calculated actual received doses.

Executive summary:

In this study, the effects of fluoride on the body weight, fluoride content in femur, testosterone levels in serum and testis, sperm quality, and the expressions of Y chromosome microdeletion genes and protein levels were examined in testes of Kunming male mice treated with different concentrations of 0, 25, 50, 100 mg/L of NaF in drinking water for 11 weeks, respectively. The results showed that compared with the control group, fluoride contents in three treatment groups were significantly increased and the structure of testes was seriously injured. The testosterone contents and the sperm count were decreased. Sperm malformation ratio was distinctly elevated. The expressions of Sly and HSF2 mRNA were markedly reduced in 100 mg/L NaF group and Ssty2 mRNA expression was dramatically decreased in 50 and 100 mg/L NaF groups. Meanwhile, the protein levels of Ssty2 and Sly were significantly reduced in 50 and 100 mg/L NaF groups and HSF2 protein levels were significantly decreased in 100 mg/L NaF group. These studies indicated that fluoride had toxic effects on male reproductive system by reducing the testosterone and sperm count, and increasing the sperm malformation ratio, supported by the damage of testicular structure, as a consequence of depressed HSF2 level, which resulted in the down-regulation of Ssty2 and Sly mRNA and protein.

In the absence of mean average body weight at study termination it is not possible to calculated actual received doses.