Xian-Feng Zhao

Melatonin alleviates cadmium‐induced cellular stress and germ cell apoptosis in testes

Abstract:  Increasing evidence demonstrates that melatonin has an anti‐apoptotic effect in somatic cells. However, whether melatonin can protect against germ cell apoptosis remains obscure. Cadmium (Cd) is a testicular toxicant and induces germ cell apoptosis. In this study, we investigated the effects of melatonin on Cd‐evoked germ cell apoptosis in testes. Male ICR mice were intraperitoneally (i.p.) injected with melatonin (5 mg/kg) every 8 hr, beginning at 8 hr before CdCl2 (2.0 mg/kg, i.p.). As expected, acute Cd exposure resulted in germ cell apoptosis in testes, as determined by terminal dUTP nick‐end labeling (TUNEL) staining. Melatonin significantly alleviated Cd‐induced testicular germ cell apoptosis. An 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 endoplasmic reticulum (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 eIF2α and JNK phosphorylation, indicating that the unfolded protein response (UPR) pathway was activated by CdCl2. Interestingly, melatonin almost completely inhibited Cd‐induced ER stress and the UPR in testes. In addition, melatonin obviously attenuated Cd‐induced heme oxygenase (HO)‐1 expression and protein nitration in testes. Taken together, these results suggest that melatonin alleviates Cd‐induced cellular stress and germ cell apoptosis in testes. Melatonin may be useful as pharmacological agents to protect against Cd‐induced testicular toxicity.

Mitochondrial signaling pathway is also involved in bisphenol A induced germ cell apoptosis in testes

Bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. An earlier study showed that BPA-induced germ cell apoptosis through the Fas/FasL apoptotic pathway. In the present study, we aimed to investigate whether the mitochondrial pathway is also involved in the process of BPA-mediated germ cell apoptosis in testes. Male mice were administered with BPA (160 or 480 mg/kg) by gavage daily from postnatal day 35 (PND35) to PND49. Germ cell apoptosis in testes was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). As expected, the number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with BPA during puberty. TUNEL+ germ cells were observed mainly in stages VII-VIII seminiferous tubules in testes. An increase in the level of Fas and FasL was observed in testes of mice exposed to BPA during puberty. In addition, pubertal BPA exposure evoked the activation of caspase-8 and caspase-3 in testes. Interestingly, pubertal BPA exposure also caused the translocation of cytochrome c from mitochondria into cytosol. In addition, pubertal BPA exposure upregulated the level of Bax and active caspase-9 in testes. Taken together, these results suggest that pubertal BPA exposure induces germ cell apoptosis in testes through not only the Fas/FasL signaling pathway but also the mitochondrial apoptotic pathway.

Pubertal cadmium exposure impairs testicular development and spermatogenesis via disrupting testicular testosterone synthesis in adult mice

Cadmium (Cd) is a well-known testicular toxicant. However, the effects of pubertal Cd exposure on testicular development and spermatogenesis remained to be elucidated. The present study investigated the effects of pubertal Cd exposure on testicular development and spermatogenesis. Male CD-1 mice were intraperitoneally injected with CdCl(2) (1mg/kg) daily from postnatal day 35 (PND35) to PND70. As expected, 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 testosterone (T) was observed in mice exposed to Cd during puberty. Moreover, pubertal Cd exposure markedly reduced mRNA and protein levels of testicular StAR, P450scc, P450(17alpha) and 17beta-HSD. Taken together, these results suggest that the decreased testicular T synthesis might partially contribute to pubertal Cd-induced impairment on testicular development and spermatogenesis in mice.

Effects of maternal cadmium exposure during late pregnant period on testicular steroidogenesis in male offspring

Cadmium (Cd) is a testicular toxicant and endocrine disruptor. In the present study, we investigated the effects of maternal Cd exposure during the late pregnant period on testicular development and steroidogenesis in male offspring. Pregnant mice were injected intraperitoneally with CdCl(2) (0.5mg/kg) daily from gestational day (gd) 13 to gd 17. As expected, fetal weight and crown length were significantly decreased in pups whose mothers were exposed to Cd. Importantly, absolute and relative weights of testes were significantly decreased in male fetuses. In addition, maternal Cd exposure during pregnancy markedly reduced serum T level and downregulated the expression of steroidogenic acute regulatory (StAR) protein, P450scc, P45017α and 17β-HSD in testes of male fetuses. Interestingly, the level of serum and testicular T at adulthood remained decreased in male offspring of Cd-exposed mice. Correspondingly, the expression of testicular P450scc was downregulated in male adult offspring whose mothers were exposed to Cd during pregnancy. Fertility analysis found that the number of live fetuses per litter in F2 generation was significantly decreased in Cd-treated group. Additional experiment showed that placental Cd level was increased about 750 folds in dams injected with Cd. However, only traces of blood Cd was measured in fetuses whose mothers were exposed to Cd during the late pregnant period. Taken together, these results suggest that 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.

Age- and gender-dependent impairments of neurobehaviors in mice whose mothers were exposed to lipopolysaccharide during pregnancy.

Lipopolysaccharide (LPS)-induced intrauterine infection has been associated with neurodevelopmental injury in rodents. The purpose of the present study was to analyze the dynamic changes of neurobehaviors in mice whose mothers were exposed to LPS during pregnancy. The pregnant mice were intraperitoneally (i.p.) injected with LPS (8 microg/kg) daily from gestational day (gd) 8 to gd 15. A battery of neurobehavioral tasks was performed in mice at postnatal day (PND) 70, 200, 400 and 600. Results showed that the spatial learning and memory ability, determined by radial six-arm water maze (RAWM), were obviously impaired in two hundred-day-old female mice and four hundred-day-old male mice whose mothers were exposed to LPS during pregnancy. Open field test showed that the number of squares crossed and peripheral time, a marker of anxiety and exploration activity, were markedly increased in two hundred-day-old female mice following prenatal LPS exposure. In addition, prenatal LPS exposure significantly shortened the latency to the first grid crossing in six hundred-day-old female offspring. Moreover, sensorimotor impairment in the beam walking was observed in two hundred-day-old female mice whose mothers were exposed to LPS during pregnancy. Species-typical behavior examination showed that prenatal LPS exposure markedly increased weight burrowed in seventy-day-old male offspring and six hundred-day-old female offspring. Correspondingly, prenatal LPS exposure significantly reduced weight hoarded in two hundred-day-old female offspring. Taken together, these results suggest that prenatal LPS exposure induces neurobehavioral impairments at adulthood in an age- and gender-dependent manner.

Crosstalk Between Endoplasmic Reticulum Stress and Mitochondrial Pathway Mediates Cadmium-Induced Germ Cell Apoptosis in Testes

Cadmium (Cd) is associated with male infertility and poor semen quality in humans. Increasing evidence demonstrates that Cd induces testicular germ cell apoptosis in rodent animals. However, the molecular mechanisms of Cd-induced testicular germ cell apoptosis remain poorly understood. In the present study, we investigated the role of endoplasmic reticulum (ER) stress on Cd-evoked germ cell apoptosis in testes. We show that spliced form of XBP-1, the target of the IRE1 pathway, was significantly increased in testes of mice injected with CdCl(2). 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 caused eIF2α and JNK phosphorylation in testes, indicating that the unfolded protein response pathway in testes was activated by Cd. Interestingly, phenylbutyric acid (PBA), an ER chemical chaperone, attenuated Cd-induced ER stress and protected against germ cell apoptosis in testes. In addition, PBA significantly attenuated Cd-evoked release of cytochrome c from mitochondria to cytoplasm in testes. Taken together, these results suggest that crosstalk between ER stress signaling and mitochondrial pathway mediates Cd-induced testicular germ cell apoptosis.

Maternal cypermethrin exposure during lactation impairs testicular development and spermatogenesis in male mouse offspring

Within the last decade, numerous epidemiological studies have demonstrated that endocrine disruptors are a possible cause for a decline in semen quality. Cypermethrin is a widely used pyrethroid insecticide, but little is known about its potentially adverse effects on male reproduction. In the present study, we investigated the effects of maternal cypermethrin exposure during lactation on testicular development and spermatogenesis in male offspring. Maternal mice were administered with cypermethrin (25 mg/kg) by gavage daily from postnatal day 0 (PND0) to PND21. Results showed that the weight of testes at PND21 was significantly decreased in pups whose mothers were exposed to cypermethrin during lactation. Maternal cypermethrin exposure during lactation markedly decreased the layers of spermatogenic cells, increased the inside diameter of seminiferous tubules, and disturbed the array of spermatogenic cells in testes of pups at PND21. In addition, maternal cypermethrin exposure during lactation markedly reduced mRNA and protein levels of testicular P450scc, a testosterone (T) synthetic enzyme. Correspondingly, the level of serum and testicular T at weaning was significantly decreased in pups whose mothers were exposed to cypermethrin during lactation. Although the expression of testicular T synthetic enzymes and serum and testicular T in adulthood had restored to control level, the decreased testicular weight and histological changes were irreversible. Importantly, the number of spermatozoa was significantly decreased in adult male offspring whose mothers were exposed to cypermethrin during lactation. In conclusion, maternal cypermethrin exposure during lactation permanently impairs testicular development and spermatogenesis in male offspring, whereas cypermethrin‐induced endocrine disruption is reversible.

Pubertal and early adult exposure to fenvalerate disrupts steroidogenesis and spermatogenesis in mice at adulthood

Fenvalerate, a pyrethroid insecticide used worldwide, has been shown to have a potentially adverse effect on male reproduction. Our earlier study showed that maternal fenvalerate exposure during lactation impaired testicular development in male offspring. In this study, we investigated the effects of pubertal and early adult exposure to fenvalerate on steroidogenesis and spermatogenesis in mice. Male mice were administered fenvalerate (60 mg/kg) by gavage daily from postnatal day 35 (PND35) to PND63. Results showed that sperm count was significantly decreased in fenvalerate‐treated mice. In addition, fenvalerate markedly decreased the layers of spermatogenic cells, disturbed the array of spermatogenic cells and increased the number of apoptotic cells in testes. The adverse effects of fenvalerate on male reproduction seemed to be associated with a decrease in serum and testicular testosterone (T). Although pubertal and early adult exposure to fenvalerate had little effect on the number of Leydig cells in testes, mRNA and protein levels of testicular T biosynthetic enzymes including P45017α and P450scc were significantly downregulated in fenvalerate‐treated mice. In conclusion, pubertal and early adult fenvalerate exposure induces a deleterious effect on steroidogenesis and spermatogenesis in adulthood. The decreased testicular T synthesis partially contributes to fenvalerate‐induced impairment on spermatogenesis. Copyright

Maternal fenvalerate exposure during pregnancy persistently impairs testicular development and spermatogenesis in male offspring.

Fenvalerate, a widely used pyrethroid insecticide, has been associated with poor semen quality. As yet, little is known about the effects of prenatal fenvalerate exposure on testicular development. The present study investigated the effects of prenatal fenvalerate exposure on testicular development and spermatogenesis. The pregnant mice were administered fenvalerate (30 mg/kg) by gavage daily from gestational day (gd) 13 to gd 18. The weights of testes and epididymides were significantly decreased in mice whose mothers were exposed to fenvalerate during pregnancy. Importantly, maternal fenvalerate exposure during pregnancy markedly decreased the number of mature seminiferous tubules (stages VII and VIII) in testes of adult male offspring. In addition, maternal fenvalerate exposure during pregnancy significantly reduced the number of epididymal spermatozoa in adult male offspring. Additional experiments showed that the level of serum testosterone (T) was significantly decreased in male fetuses whose mothers were exposed to fenvalerate during pregnancy. Correspondingly, mRNA and protein levels of P450(17alpha), a T synthetic enzyme, were significantly decreased in fetal testes. Moreover, the disruptive effect of prenatal fenvalerate exposure on testicular T synthesis was irreversible. In conclusion, prenatal fenvalerate exposure irreversibly impairs testicular development and spermatogenesis at least into early adulthood.

Lactational fenvalerate exposure permanently impairs testicular development and spermatogenesis in mice.

Fenvalerate, a widely used synthetic pyrethroid insecticide, has been associated with poor semen quality in human being. However, little is known about the effects of lactational fenvalerate exposure on testicular development and spermatogenesis. The purpose of the present study was to investigate the effects of maternal fenvalerate exposure during lactation on testicular development and spermatogenesis in male offspring. Maternal mice were administered with fenvalerate (60 mg/kg) by gavage daily from postnatal day (PND) 0 to PND21. Lactational fenvalerate exposure markedly decreased the absolute and relative weights of testes and increased the number of apoptotic cells in testes of pups at weaning. Histological examinations showed abnormal seminiferous tubules with large vacuoles or complete spermatogenic failure in testes of fenvalerate-treated mice at weaning. Additional experiment showed that lactational fenvalerate exposure markedly reduced mRNA and protein levels of testicular P450scc, a testosterone (T) synthesis enzyme. Consistent with down-regulation of testicular P450scc, the level of serum and testicular T at weaning was significantly decreased in pups whose mothers were exposed to fenvalerate during lactation. Although the expression of testicular P450scc and serum and testicular T in adulthood restored to control level, the decreased weight of testes and histological changes were irreversible. Importantly, the percentage of mature seminiferous tubules (stages VII and VIII) and the number of spermatozoa were obviously decreased in adult male mice whose mothers were exposed to fenvalerate during lactation. Taken together, these results suggest that lactational fenvalerate exposure permanently impairs testicular development and spermatogenesis.