Detection of mitomycin C‐induced testicular toxicity by micronucleus assay in mice

Abstract

Background, Aims and Methods:  Mitomycin C is a promising cancer agent that has been shown to inhibit DNA synthesis. Our previous study showed that mitomycin C induces spermatogenic cell apoptosis in the mouse testis. By using TdT‐mediated dUTP‐biotin nick‐end labeling in the study, we confirmed that apoptotic cell death was most commonly found in the spermatogonia and less frequently found in spermatocytes in mitomycin C‐treated mice. We therefore hypothesized that the spermatogenic cell apoptosis induced by mitomycin C occurred as the result of a mechanism to eliminate male germ cells with DNA damage or chromosomal aberrations. To test our hypothesis, we used a micronucleus assay for the detection of chromosomal damage induced in the spermatogonia or spermatocyte stages.

Results and Conclusions:  The frequency of micronuclei was clearly increased in the mitomycin C‐treated animals, and the number of micronuclei was greater at the spermatogonia or early spermatocyte stage than at the secondary spermatocyte stage. These results revealed that apoptosis and chromosomal aberration in the mouse testis after mitomycin C treatment occurred in the same cell types, that is, spermatogonia and spermatocytes. These findings indicate that chromosomal aberration of the spermatogenic cells induced by mitomycin C may have caused the spermatogenic cell apoptosis. (Reprod Med Biol 2003; 2: 69–73)

INTRODUCTION

IT IS WELL known that germ cell degeneration plays an important role in normal spermatogenesis. ¹ , ² , ³ However, the mechanism of this degeneration remains to be clarified. Previous studies have also indicated that male germ cell degeneration in rodents occurs spontaneously, as well as in response to endocrine disruption, various environmental agents (e.g. heat, irradiation, and ischemia), and exposure to chemicals. ² , ⁴ , ⁵ , ⁶ , ⁷ , ⁸ Recently, some studies have provided evidence that degeneration of the male germ cells involves apoptosis. ⁴ , ⁵ , ⁶ , ^{8 , 9} In a study using TdT‐mediated dUTP‐biotin nick‐end labeling (TUNEL), transmission electron microscopy and molecular methods to detect DNA ladders, we also revealed that apoptosis was induced in mouse testes after injection with mitomycin C (MC), and we confirmed that MC induces apoptosis with fragmentation of nuclear DNA in mouse spermatogenic cells. ¹⁰

Mitomycin C is a promising cancer agent that has been shown to inhibit DNA synthesis. Previous studies have shown that MC induces chromosomal aberration, dominant‐lethal mutations, and DNA damage in the spermatogonia. ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ Therefore, we hypothesized that the spermatogenic cell apoptosis induced by MC occurred as the result of a mechanism to eliminate male germ cells with DNA damage or chromosomal aberrations. To test our hypothesis, we used a micronucleus assay for the detection of chromosomal damage induced in the spermatogonia or in the spermatocyte stages. The chromosomal aberration induced by mutagenic treatments causes fragmentation of chromosomes during anaphase. During telophase, this material is surrounded by a nuclear envelope giving rise to micronuclei in the daughter cells. Such genetic damage was originally quantified by counting the number of polychromatic erythroblasts in micronuclei, but the spermatid micronucleus assay has since been developed as a simple and sensitive method for measuring genetic damage. ¹⁶ , ¹⁷ In this paper, we investigated the relationship of apoptosis and chromosomal aberration in mouse testis after MC treatment.

MATERIALS AND METHODS

Chemicals

MITOMYCIN C, TRYPSIN inhibitor, collagenase and phosphate buffered solution (PBS) were purchased from Wako Pure Chemical Industry Co., Ltd. (Osaka, Japan). 2‐[4‐(2‐Hydroxyethyl)‐1‐piperadinyl]ethansulfonic acid (HEPES) was purchased from Nakalai Tesque Co. Ltd. (Kyoto, Japan) and both CaCl₂ and NaHCO₃ were purchased from Hayashi Pure Chemical Inc. Ltd. (Osaka, Japan).

Animals and treatment

Mature male Institute of Cancer Research (ICR) mice were obtained from Japan Clea Co. Ltd. (Tokyo, Japan). Male mice aged between 10 and 11 weeks and weighing 40–42 g were used in the experiments. Animals were allowed free access to food and water. The room in which the animals were kept was maintained at approximately 22 ± 2°C and 55 ± 5% humidity with a 12‐h light–dark cycle. Mitomycin C was dissolved in physiological saline, and intraperitoneally injected in a single dose of 1.25 or 2.5 mg/kg. Control animals were given a single intraperitoneal injection of physiological saline. The animals were killed on days 3, 10 and 19 after MC treatment. These sacrifice days were set to detect the relationship between micronuclei occurrence and the stage of spermatogenic cells exposed to MC. In the specimens prepared on days 3, 10 and 19 after MC treatment, the early spermatids that were exposed to MC at the stage of spermatogonia, primary and secondary spermatocyte could be observed (Fig. 1). Each experimental group consisted of six treated and four control animals. The dosing solutions were injected at a volume of 0.01 mL/g bodyweight.

Figure 1.

Scheme of spermatogenic cell development and test schedule. Arrows indicate the dosing of mitomycin C, and the days represent the period from dosing to observation. () spermatogonia, () spermatocyte, () spermatid.

Slide preparation

The testis of each animal was minced in 1 mL of Testes Isolation Solution (TIS), which was modified from Testes Isolation Medium. ¹⁷ Testes Isolation Solution was mixed in a 1‐L batch that contained the following: 10 mmol/L HEPES, 5 mmol/L CaCl₂, 4.2 mmol/L NaHCO₃, 0.05 g trypsin inhibitor, and 0.5 g collagenase (pH 7.2). Nine milliliters of TIS was added to the 1 mL of TIS in which the testis was minced, and this solution was shaken for 60 min in a water bath at 37°C. After incubation, the germ cell suspension was centrifuged at 1500 g for 10 min. The supernatant was removed, and the cells were washed twice with 10 mL of fresh PBS. The pellet was unfastened with a pipette and suspended after adding 1 mL of 4% paraformaldehyde. After fixation for 24 h, 40 µL of the germ cell suspension and 0.006% acridine orange solution were mixed well and added to the glass slide. The slides were then cover‐slipped for observation under a fluorescent microscope. The micronuclei assay with the early spermatids was performed according to the method of Tates. ¹⁷ The number of micronucleated spermatids in 1000 spermatids was determined and the incidence of micronucleated spermatids (%) for each slide was calculated.

Statistical analysis

The Chi‐squared test was used to analyze the number of micronuclei in the early spermatids. It was performed at P < 0.05 and P < 0.01.

RESULTS

Application of the micronucleus assay for polychromatic erythroblasts to spermatids in testis

FIRST, WE ASSAYED for the presence of micronuclei in spermatids after chemical treatment. Micronuclei were clearly detected in the early spermatids stained with acridine orange after MC treatment (days 3, 10 and 19). In Fig. 2, the presence of nuclei or micronuclei is indicated by yellow fluorescence, whereas the red fluorescence indicates cytoplasm.

Figure 2.

Acridine orange staining of the early spermatids. The nuclei and micronuclei (arrows) emitted yellow fluorescence.

Micronucleus assay in spermatids after mitomycin C treatment

The high incidence of micronuclei by the micronucleus assay on days 3, 10 and 19 after MC treatment indicates testicular toxicity at the secondary spermatocyte stage, early spermatocyte stage and spermatogonia stage, respectively. The results of this experiment are presented in Fig. 3. In the spermatids of the control group, the percentage of spontaneously derived micronuclei was 0.03–0.08%. The percentage of spermatids containing micronuclei in the MC treated mice was significantly higher than that of the control group on every sacrifice day after MC treatment. In addition, when the data on days 3, 10 and 19 were compared, the values on days 10 and 19 were higher than that on day 3 in each dosing group (Fig. 3). However, the values on day 10 and 19 were almost the same. Our results indicated that MC has higher testicular toxicity at the spermatogonia stage or early spermatocyte stage.

Figure 3.

Incidence of early spermatids with micronuclei after mitomycin C (MC) treatment. The incidence was calculated as (No. early spermatids with micronuclei × 100)/(No. observed 1000 early spermatids). Each column represents the mean ± SE. *P < 0.05 versus controls; **P < 0.01 versus controls. () Control, () 1.25 mg/kg, () 2.5 mg/kg.

DISCUSSION

MITOMYCIN C IS well known as a DNA inhibitor. Previous studies have indicated that MC induces chromosomal aberration and DNA damage in the spermatogonia. ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ In the present study, we examined the induction of chromosomal aberration and DNA damage by MC by using a micronuclei assay. The micronuclei assay with the early spermatids was performed according to the method of Tates et al., ¹⁷ with the exception that the cells were stained with acridine orange. Hayashi et al. reported the advantages of staining with acridine orange, a fluorescent dye that combines with a nucleic acid base, in a micronuclei assay. ¹⁸ When the compound formed by nucleic acid and acridine orange is excited by blue light with a wavelength of 490 nm, it emits a yellow–green or orange fluorescence. Therefore, it is very easy to identify the micronuclei becaues only the nuclei and micronuclei are stained yellow–green. The micronuclei assay with acridine orange staining has been reported to be very accurate. ¹⁹

Hayashi et al. reported that micronuclei are derived from structural chromosomal aberrations induced by chemicals. ²⁰ In the present study, the number of micronuclei in the MC treatment grew higher with each observation day. This finding proved that MC induced chromosomal aberration in the mouse spermatogenic cells.

To investigate the frequency of chromosome aberration in the spermatogonia, early spermatocytes and secondary spermatocytes, we prepared samples on days 3, 10 and 19 after MC treatment. The spermatogonia and spermatocytes stages generally last 7 and 13 days, respectively. Therefore, the spermatids with micronuclei on days 3, 10 and 19 after MC treatment reflected the cells that were exposed to MC at the spermatogonia, early spermatocyte and secondary spermatocyte stages, respectively. When the frequency of micronuclei among sampling days for each dosing group were compared, it was found that the frequency of micronuclei on days 10 and 19 was apparently higher than that on day 3. This result showed that MC has higher toxicity at the spermatogonia or early spermatocyte stage than at the secondary spermatocyte stage. Oakberg and Clermont reported that the degeneration of the male germ cells might be a mechanism for eliminating cells with abnormal chromosomes. ²¹ , ²² Our previous study demonstrated that MC induces apoptosis in mouse spermatogonia and spermatocytes. ¹⁰ The present study showed that MC also induces chromosomal aberration in same‐cell types. The finding that apoptosis and chromosomal aberration occurred in spermatogonia and spermatocytes implies that MC induced chromosome aberration in these cells, and that these cells were subsequently eliminated by apoptosis. As just described, the combination of TdT‐mediated dUTP biotin nick‐end labeling (TUNEL) methods and micronucleus assay demonstrated the degree of cell toxicity (apoptosis), and indicated the cause of apoptosis in the mouse testis after MC treatment.

In conclusion, our study findings suggested that spermatogenic cell apoptosis induced by MC occurred as the result of a mechanism to eliminate male germ cells with DNA damage or chromosomal aberration in mice. These results also indicate that TUNEL and a micronuclei assay are useful for detecting the induction of spermatogenic cell apoptosis and chromosomal aberration.