Adverse Effects of Formaldehyde Vapor on Mouse Sperm Parameters and Testicular Tissue

In this experimental study, mice were placed in a vitreous Plexiglass quadrangular chamber (30×25×29 cm) that contained two holes for in and out flows of air. Air circulation in the chamber was at a fixed flow rate (12 times per hour) maintained by air pumps. The exposure chamber temperature was 22 ± 2°C. FA gas was generated by thermal depolymerization of Paraformaldehyde (Merck AG, Darmstadt, Germany) at 70-90°C according to a method described by Chang et al. (14). FA concentration in the exposure chamber reached a desirable range by changing the airflow, using a micro valve, and control by a rotameter. FA concentration was measured and monitored four times each hour by a photo ionization detector (Photocheck +5000, Ionscience Co., UK). The preciseness of the measurement was controlled according to the 3500 method recommended by National Institute of Occupational Health and Safety (NIOSH) with a sensitivity of less than 0.1% ppm (5, 15).

This experimental study was performed on 36 normal, eight-week old male NMRI mice (25-35 g). The animals were purchased from Iran Pasteur Institute and maintained in the animal house at Tarbiat Modares University according to standard laboratory conditions in terms of temperature (22- 24ºC), light (12 hours light:12 hours dark), ventilation and free access to food and drinking water. The experiments were approved by the Institutional Animal Care and Use Committee of Tarbiat Modares University (Tehran, Iran).

The mice were randomly assigned to three groups (n=12) based on the study design. The control group (C) was maintained under experimental conditions but mice were not exposed to FA. Experimental group F1 was exposed to FA (10 ppm) eight hours per day (8:00 am- 4:00 pm) for ten consecutive days. Experimental group F2 was exposed to FA (20 ppm) eight hours per day (8:00 am- 4:00 pm) for ten consecutive days.

The population in each group (n=12) was determined based on the results from previous studies and preliminary experiments (16, 17). Other researchers reported that exposure to inhaled FA at 10 and 20 ppm which led to liver toxicity (16).

For epididymal sperm preparation, mice were sacrificed by cervical dislocation either 24 hours or 35 days after the end of the exposure period. The cauda epididymides were dissected and placed in 1 ml pre-warmed Ham's F-10 (nutrient mixture- Ham-X1, Gibco, UK) culture medium with 10% fetal bovine serum (FBS, Gibco, UK). Gentle agitation along with tearing of the tissue was applied to enable spermatozoa to swim into the medium in a Falcon culture dish (18,19). Semen samples were incubated at 37°C for 30 minutes before analysis of sperm parameters.

Sperm count and motility were analyzed with a Computer-assisted Sperm Analyzer (CASA) as described by Krause (18). The CASA system consisted of a phase contrast microscope (Eclipse E-200, Nikon Co., Japan) with a heat plate equipped with Sperm Class Analyzer® software (SCA, full research version 5.1, Microptic Co., Barcelona, Spain). Images were captured by a video camera (Basler Vision, A312FC at 50 fps, Tecnologie Co., Ahrensburg, Germany) at 100x magnification. For this purpose, 4 µl sperm samples were placed in a standard count analysis chamber (Leja, Nieuw Vennep Co., Netherlands). The loaded chamber was placed on the warm plate of the microscope (37°C) for 3 minutes before analysis. Then, specimens were observed with a Nikon microscope 10x/0.25 negative phase contrast field Ph1 BM, with an intermediate magnification of 0.7 and a green filter. Several fields of view were captured and at least 400 spermatozoa counted for each analysis.

performed eosin-Y staining by mixing 10 µl of the sperm sample with 10 µl of dye (0.5% wt/vol, Merck Chemical Co., Germany) on a microscope slide which was then covered with a coverslip. A total of 200 sperm cells were counted within a few minutes after the addition of the dye (19). Evaluation of live (unstained) and dead (red stained) spermatozoa was performed with a phase contrast microscope at x400 magnification.

For the assessment percentage of the normal sperm morphology, we used SpermBlue® fixative and stain. Briefly, once the smear air dried at room temperature it was fixed for 10 minutes by placing the slide with the dried smear in a staining tray that contained SpermBlue® fixative. The fixed smear was stained for 10 minutes by immersing the slide in a staining tray that contained SpermBlue® stain (20). Morphologic assessment was performed at x1000 magnification with a phase contrast microscope, and at least 200 spermatozoa were counted on each slide.

For histological examination, testicular tissues were dissected and samples fixed in Bouin’s fixative for 24 hours, then processed by a graded ethanol series and embedded in paraffin. The paraffin sections were cut in 5 µm thick slices and stained with hematoxylin and eosin for light microscopic examination (21, 22).

Sections were viewed and photographed by a microscope (Magnum-3, Ceti, England) with an attached camera (Sony-DSC-H50). We compared the integrity of seminiferous tubules and interstitial cells, including Leydig cells, with the control group. An estimate of STD was performed by examining 20 fields in 5 histological sections from each testis (22), using digitalized microscopic images (x10) with the software Image Tools 3.0 (Obtain from http://compdent.uthscsa.edu/dig/itdesc.html).

Serum was separated by centrifugation at 3000×g for 15 minutes, then collected and stored at -20°C until analysis. Serum testosterone level was evaluated following the standard protocol supplied by a kit (Monobind Inc., USA, Product code: 3775-300) and read by a fully automated ELISA reader (23).

All statistical analyses were performed using SPSS statistical software version 11.5 (SPSS, Inc., Chicago, IL). All data were expressed as mean ± SD. Comparisons between control and FA-exposed groups were performed using one-way analysis of variance (ANOVA), followed by the Scheffe test. The level of statistic significance was set at p<0.05.

The mean FA concentrations in the exposure chamber were 10.89 ± 0.76 ppm for group F1 and 19.79 ± 1.56 ppm for group F2. During all exposure sessions we registered minor, insignificant fluctuations in the FA vapor concentration.

The mean of testes weights in experimental groups F1 and F2 were 94.72 ± 8.88 and 98.58 ± 12.34 (mgs), respectively during short-term analysis. The mean testes weights in experimental groups F1 and F2 were 96.85 ± 7.15 and 106.03 ± 14.77(mgs), respectively during long-term analysis. No statistical decrease was observed in all experimental groups compared to the control group (p<0.05) in mice sacrificed at 24 hours and 35 days after exposure (Fig 1).

Sperm parameters in mice assessed 24 hours after FA exposure are shown in table 1. We observed no significant changes in sperm count, motility and percentage of normal morphology among the experimental groups compared to the control group (p>0.05) for short-term analysis (Table 1). FA exposure at 20 ppm was associated with a decreased percentage of progressive motility. Also in this concentration there was a significant decrease (p<0.05) in percentage of sperm viability compared with the control group (Table 1).

Sperm parameters in mice sacrificed 35 days after FA exposure are summarized in table 1. The progressive motility of sperm cells in both experimental groups compared to the control group decreased significantly (p<0.001). Regarding sperm count and normal morphology, a significant decrease was observed in all exposure groups compared to the control group (p<0.001) in long-term analysis. A comparison of exposure groups with one another showed no statistical difference (p>0.05) in terms of these two parameters (Table 1).

Histological examination of the testis showed numerous structural changes in the experimental groups (long-term analysis) compared to the control group. The main pathological changes included seminiferous tubule atrophy, increase in the spaces between germ cells, degeneration of Leydig cells, disintegration of seminiferous epithelial cells and degeneration of a number of seminiferous tubules. No histological changes were seen in the control specimens (Fig 2 a-d).

The morphometric findings indicated that the mean STD decreased in both short and long-term analysis in the experimental groups compared with the control groups,but it was significant (p<0.05) only in long-term analysis (Table 2).

Short-term analysis of testosterone levels showed a significant difference (p<0.001) between the F1 (2.58 ± 0.16 ng/ml) and F2 (1.95 ± 0.18 ng/ml) groups compared to the control (3.82 ± 0.35 ng/ ml). The comparison between the F1 and F2 groups was statistically significant (p<0.05; Table 3).