**3. Effects of Cd on proliferation of spermatogenic cells from** *M. nipponense in vitro*

The toxic effects of Cd on male reproductive system is obvious, it can significantly damage the testicles and the testicular parenchyma cells, leading to pathological testicular alterations and morphological abnormalities of spermatozoa, directly affected the reproductive capacity (Luo et al., 1993; Mohan et al., 1992; Saygi et al., 1991).

Culture of spermatogenic cells *in vitro* is important in development. Establishing the model of culture of spermatogenic cells in vitro is helpful for studying the regulate mechanism of spermatogenesis. In addition, the environment factor and presence of a poisonous substance can have grave effect on idioplasm, and thus restrict the development of marine species. Studying the effects of poisonous substances on reproduction and differentiation of spermatogenic cells has theoretical significance on clarifying the mechanism of poisonous substance, and has practical significance on idioplasm profect and health breed aquatics.

Juvenile male *M. nipponense* (20 to 25 mm body length) for the experiments were purchased from Baiyangdian Lake, Hebei Province, China. Spermatogenic cells of *M. nipponense* were isolated and sublimated with the method of trypsinization and differential speed adherence. Cell suspensions were seeded into M199 medium (pH 7.2, supplemented with 10% fetal bovine serum (FBS), 1 g/L glucose, 0.3 g/L glutamine, 0.11 g/L sodium pyruvate, 0.01% 2 mercaptoethanol, 100 IU/mL penicillin, 100 IU/mL streptomycin, 20 μg/mL gentamicin) and kept under 5% CO2 at 26°C for 12 h before being incubated with various concentrations of Cd (5, 50, 500, 1 000 ng/mL). Equal volumes of culture medium containing no Cd were added to the control groups. Subsequently, MTT [3-(4, 5-dimethylthia- zole-2-yl)-2,5 diphenyl-tetrazoliumbromide] assay (Mosmann, 1983) was used to evaluate the proliferation of spermatogenic cells after 0 h, 24 h, 48 h, 72 h, 96 h exposure.

MTT assay is widespread method to assess cell viability. In living cells, MTT is deoxidized by mitochondrial dehydrogenases to a blue formazan product. The results can be read on a multi-well scanning spectrophotometer (ELISA reader) and the absorption of dissolved formazan correlates with the number of alive cells (Mosmann, 1983). Cytotoxic compounds (e.g. heavy metals) are able to damage and destroy cells, and thus decrease the reduction of MTT to formazan, the absorbance value therefore will decline.

**3. Effects of Cd on proliferation of spermatogenic cells from** *M. nipponense* 

**1µm** 

The toxic effects of Cd on male reproductive system is obvious, it can significantly damage the testicles and the testicular parenchyma cells, leading to pathological testicular alterations and morphological abnormalities of spermatozoa, directly affected the reproductive

Culture of spermatogenic cells *in vitro* is important in development. Establishing the model of culture of spermatogenic cells in vitro is helpful for studying the regulate mechanism of spermatogenesis. In addition, the environment factor and presence of a poisonous substance can have grave effect on idioplasm, and thus restrict the development of marine species. Studying the effects of poisonous substances on reproduction and differentiation of spermatogenic cells has theoretical significance on clarifying the mechanism of poisonous substance, and has practical significance on

Juvenile male *M. nipponense* (20 to 25 mm body length) for the experiments were purchased from Baiyangdian Lake, Hebei Province, China. Spermatogenic cells of *M. nipponense* were isolated and sublimated with the method of trypsinization and differential speed adherence. Cell suspensions were seeded into M199 medium (pH 7.2, supplemented with 10% fetal bovine serum (FBS), 1 g/L glucose, 0.3 g/L glutamine, 0.11 g/L sodium pyruvate, 0.01% 2 mercaptoethanol, 100 IU/mL penicillin, 100 IU/mL streptomycin, 20 μg/mL gentamicin) and kept under 5% CO2 at 26°C for 12 h before being incubated with various concentrations of Cd (5, 50, 500, 1 000 ng/mL). Equal volumes of culture medium containing no Cd were added to the control groups. Subsequently, MTT [3-(4, 5-dimethylthia- zole-2-yl)-2,5 diphenyl-tetrazoliumbromide] assay (Mosmann, 1983) was used to evaluate the

MTT assay is widespread method to assess cell viability. In living cells, MTT is deoxidized by mitochondrial dehydrogenases to a blue formazan product. The results can be read on a multi-well scanning spectrophotometer (ELISA reader) and the absorption of dissolved formazan correlates with the number of alive cells (Mosmann, 1983). Cytotoxic compounds (e.g. heavy metals) are able to damage and destroy cells, and thus decrease the reduction of

proliferation of spermatogenic cells after 0 h, 24 h, 48 h, 72 h, 96 h exposure.

MTT to formazan, the absorbance value therefore will decline.

Fig. 8. Endoplamic reticulum vesicles exposed to 0.50 mg/L Cd

capacity (Luo et al., 1993; Mohan et al., 1992; Saygi et al., 1991).

idioplasm profect and health breed aquatics.

*in vitro* 

A concentration-response curve for Cd obtained with the MTT assay is shown in Fig. 9. Before 24 h, the absorbance curve of each group showed no regularity; downward trend of the curve was not obvious. 24 h later, the absorbance of groups exposed to Cd at dose of 50 ng/mL, 500 ng/mL, 1 000 ng/mL, but not 5 ng/mL, were significantly lower than those of the controls (*P* < 0.01). The cell proliferation rate was found to decrease with increasing Cd concentration, and after 24 h exposure the absorbance of each concentration was significantly different from the absorbance at the start of the experiment (*P* < 0.01). In brief, rate of cell proliferation showed negative correlation with dose and exposure time at 50 ng/mL, 500 ng/mL, 1 000 ng/mL after 24 h.

Fig. 9. Absorbency of spermatogenic cells disposed with Cd

There is growing evidence that suggests the mechanism of cytotoxicity of Cd may be mitochondrial dysfunction (Sokolova, 2004; Sokolova et al., 2004; Ivanina et al., 2010). In terrestrial plants and mammals, Cd is known as a powerful modulator of mitochondrial function, inhibiting electron transport chain, increasing generation of reactive oxygen species (Sokolova, 2004, as cited in Miccadei & Floridi, 1993 and Wallace & Starkov, 2000), and stimulating proton leak through the inner mitochondrial membrane (Sokolova, 2004, as cited in Belyaeva et al., 2001). In marine mollusks, such as oysters, Cd also affects mitochondrial function (Sokolova, 2004; Sokolova et al., 2004; Ivanina et al., 2010). These data strongly suggest that mitochondria are key intracellular targets for Cd (Sokolova, 2004). Heavy metals, such as Cd, are known to induce apoptosis and necrosis in invertebrates and vertebrates and result in increased cellular mortality (Benoff et al., 2004; Sokolova et al., 2004, as cited in Li et al., 2000 and Sung et al., 2003). In vertebrates, undergoing Cd stress, cells activate the classical intrinsic death pathway, in which mitochondria have a central role (Sokolova et al., 2004, as cited in Shih et al., 2004 and Hüttenbrenner et al., 2003). Cd exposure induces apoptosis in oyster immune cells and does so through a mitochondria/caspase-independent pathway (Sokolova et al., 2004). These results suggest that the mechanism of apoptosis induced by Cd exposure is very complex.

In our study, the results of MTT assay showed that Cd restrained the proliferation of isolated spermatogenic cells from *M. nipponense*. According to other investigations, it is due to cells apoptosis or necrosis induced by Cd exposure. The cause is unclear and further research will be needed.

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