Effects Of Cadmium Exposure During Different Stages On Reproductive Development In Male Mice

Cadmium (Cd) is a toxic metal and a potent environmental pollutant. The general population is exposed to Cd via drinking water, food and cigarette smoking. Cd is a reproductive toxicant in human. Increasing evidence demonstrated that environmental exposure to Cd is associated with male infertility and the poor semen quality in humans. Even a low level of Cd accumulation in semen might contribute to male infertility by reducing sperm quality. Cd was also a testicular toxicant in rodent animals. Exposure to Cd causes damage in the testes and results in infertility in adult rodent animals. The level of serum testosterone (T) was also significantly decreased in Cd-treated adult rodent animals. A link between Cd exposure and germ cell apoptosis in testes has been demonstrated in rodent animals. Nevertheless, the molecular mechanisms of Cd-evoked germ cell apoptosis remain poorly understood. As yet, little is known about the effects of gestational and lactational Cd exposure on reproduction and endocrine in male offspring. In addition, the effects of pubertal Cd exposure on testicular development and spermatogenesis remained to be elucidated. In the present study, we investigated the effects of Cd exposure during different stages on reproductive development in male mice. Moreover, we also investigated the role of reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress on Cd-induced germ cell apoptosis in testes.1. Effects of maternal cadmium exposure during late pregnant period on testicular steroidogenesis in male offspringObjective The effects of maternal Cd exposure during the late pregnant period on testicular development and steroidogenesis in male offspring. Methods The pregnant mice were randomly divided into two groups. Mice in Cd group were intraperitoneally injected with CdCl₂ (0.5 mg/kg) daily from gestational day (GD) 13 to GD 17. Mice in control group were treated with normal saline (NS). Male pups were randomly sacrificed on GD18 and postnatal day(PND)70. The testes were collected and weighed. Histopathological changes in testes were observed. The cauda epididymides of male adult offspring were removed for spermatozoa count. The leydig cells in testes were counted and identified by staining for 3β-hydroxysteroid dehydrogenase (3β-HSD). The apoptotic germ cells in testes were measured by TdT-mediated dUTP-biotin nick end labeling (TUNEL). Serum and testicular T were measured by radioimmunoassay (RIA). The expression of testicular steroidogenic acute regulatory (StAR) protein and enzymes for T biosynthesis in male offspring was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Results Fetal weight and crown length were significantly decreased in mice whose mothers were exposed to Cd during the late pregnant period. Importantly, absolute and relative weights of testes were significantly decreased in male fetuses whose mothers were exposed to Cd during the late pregnant period. In addition, maternal Cd exposure during the late pregnant period markedly downregulated the expression of StAR protein, P450scc, P45017αand 17β-HSD in testes of male fetuses. Interestingly, the effects of maternal Cd exposure during the late pregnant period on testicular P450scc expression were irreversible. Additional experiment showed that placental Cd level was increased about 750 folds in the pregnant mice injected with Cd. However, only traces of blood Cd was measured in fetuses whose mothers were exposed during the late pregnant period. Conclusion Maternal Cd exposure during the late pregnant period impairs testicular development and steroidogenesis in male offspring. Placenta could deter most of Cd from passing from dams to fetuses. The impairments on testicular steroidogenesis in male offspring could not be attributed to a direct action of Cd on fetal testes.2. Effects of lactational Cd exposure on reproductive development in male mice Objective The present study was to investigate the effects of maternal Cd exposure during lactation on testicular development and spermatogenesis in male offspring. Methods Maternal mice were intraperitoneally injected with CdCl₂ (1.0mg/kg) daily from PND 0 to PND21. Male offspring were randomly sacrificed on PND22 and PND70. Sera were collected for the measurement of T. The testes were collected and weighed. Histopathological changes in testes were observed. The cauda epididymides of adult offspring were removed for spermatozoa count. The leydig cells in testes were counted and identified by 3β-HSD. The apoptotic germ cells in testes were measured by TUNEL. Serum and testicular T were measured by RIA. The expressions of testicular StAR protein and enzymes for T biosynthesis in male offspring were measured by RT-PCR and Western blotting. Results Cd concentation in maternal blood was significantly increased in Cd-treated mice. No significant differences on Cd concentration in serum, liver, kidney and testes of male offspring were observed between Cd-treated mice and controls. Moreover, there were no significant differences on the absolute and relative testis weight. In addition, no obvious pathohistological damage and germ cell apoptosis were observed in testes of Cd-treated mice. Interestingly, no significant differences on the levels of serum and testicular T were observed between controls and mice whose mothers were exposed to Cd during lactation. Importantly, lactational Cd exposure did not affect the mRNA and protein levels of testicular StAR and T synthetic enzymes in male offspring. In addition, lactational Cd exposure did not impair spermatogenesis in male adult offspring. Conclusion Maternal Cd exposure during lactation does not affect testicular development and spermatogenesis of male offspring.3. Effects of pubertal Cd exposure on testicular development and spermatogenesis in male miceObjective The present study was to investigate the effects of pubertal Cd exposure on testicular development and spermatogenesis. Methods Male mice were intraperitoneally injected with CdCl₂ (1.0 mg/kg) daily from PND35 to PND70. All mice were sacrificed at 24 h after the last Cd administration. Blood sera were collected. The testes were collected and weighed. Histopathological changes in testes were observed. The caudu epididymides were removed for spermatozoa count. The leydig cells in testes were counted and identified by staining for 3β-HSD. The apoptotic germ cells in testes were measured by TUNEL. Serum and testicular T were measured by RIA. The expression of testicular StAR protein and enzymes for T biosynthesis was measured by RT-PCR and Western blotting. The determination of Cd concentration in serum, testis, kidney and liver was performed by graphite furnace atomic absorption spectrometry (GFAAS). Results Pubertal Cd exposure significantly decreased the number of spermatozoa in epididymides. In addition, pubertal Cd exposure markedly reduced the weights of testes, epididymides and prostate and seminal vesicle in adult mice. A significant decrease in serum and testicular T was observed in mice exposed to Cd during puberty. Moreover, pubertal Cd exposure markedly reduced mRNA and protein levels of testicular StAR, P450scc, P45017αand 17β-HSD. Conclusion Pubertal Cd exposure impairs steroidogenesis and spermatogenesis in male mice. The decreased testicular T synthesis might partially contribute to Cd-induced impairment on testicular development and spermatogenesis in mice.4. Crosstalk between ER stress and mitochondrial pathway mediates Cd-induced germ cell apoptosis in testesObjective The present study was to investigate the role of ER stress on Cd-evoked germ cell apoptosis in testes and to explore the protective effects of antioxidants on Cd-induced germ cell apoptosis in testes. Methods Mice were injected with different doses of CdCl₂ (0.5, 1 or 2.0 mg/kg). Testes were collected at different time (0, 12, 24 and 48 h) after Cd. To investigate the protective effects of phenylbutyric acid (PBA) on Cd-induced testicular germ cell apoptosis, mice were injected with CdCl₂ (2.0 mg/kg). Some mice were injected with PBA (100 mg/kg). Testes were collected at 24 h after Cd treatment. To investigate the protective effects of antioxidants on Cd-induced testicular germ cell apoptosis, mice were injected with CdCl₂ (2.0 mg/kg). Some mice were injected with N-acetylcysteine (NAC, 100 mg/kg) or melatonin (5 mg/kg). Testes were collected at 24 h after Cd. Testicular germ cell apoptosis was determined by TUNEL. Testicular ER stress was measured using RT-PCR and Western blotting. Results Acute Cd exposure significantly increased testicular germ cell apoptosis, as determined by TUNEL staining. Additional experiment showed that spliced form of XBP-1, the target of the IRE-1 pathway, was significantly increased in testes of mice injected with CdCl₂. GRP78, an ER chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly increased testicular eIF2αand JNK phosphorylation, indicating that the unfolded protein response (UPR) pathway was activated by Cd. PBA, an ER chemical chaperone, attenuated Cd-induced ER stress and protected mice from germ cell apoptosis in testes. In addition, PBA significantly attenuated Cd-evoked release of cytochrome c from mitochondria to cytoplasm in testes. Moreover, Cd-evoked testicular ER stress and germ cell apoptosis were almost completely inhibited by NAC or melatonin, two famous antioxidants. Conclusion Crosstalk between ER stress signaling and mitochondrial pathway mediates Cd-induced testicular germ cell apoptosis. Reactive oxygen species contribute, at least partially, to Cd-evoked ER stress and subsequent germ cell apoptosis in testes.The present results allow us to reach the following conclusions. First, maternal Cd exposure during the late pregnant period impairs testicular development and steroidogenesis in male offspring. Second, maternal Cd exposure during lactation does not affect testicular development and spermatogenesis of male offspring. Third, pubertal Cd exposure impairs steroidogenesis and spermatogenesis in male mice. The decreased testicular T synthesis might partially contribute to Cd-induced impairment on testicular development and spermatogenesis in mice. Fourth, adult Cd exposure results testicular germ cell apoptosis. Crosstalk between ER stress signaling and mitochondrial pathway mediates Cd-induced testicular germ cell apoptosis. Reactive oxygen species contribute, at least partially, to Cd-evoked ER stress and subsequent germ cell apoptosis in testes.