| Lipopolysaccharide (LPS) is a reproductive toxicant, which induces the apoptosis of germ cells, damages the function of Sertoli cells, inhibits the synthesis of steroid hormone, and affects spermatogenesis. On the other hand, many studies have showed that LPS is associated with adverse developmental outcomes, including intra-uterine fetal death, intra-uterine growth retardation and fetal neurological injury in animals. In this study, we aimed to investigate the effects of maternal LPS exposure on reproductive development in male offspring and to explore the role of tumor necrosis factor-α(TNF-α) on LPS-induced damage in reproductive development in male offspring.1. Effects of maternal LPS exposure on reproductive development in male offspring.To investigate the effects of maternal LPS exposure on reproductive development in male offspring, the pregnant mice were randomly divided into two groups. The pregnant mice in LPS group received an intraperitoneal (i.p.) injection of LPS (8μg/kg) daily from gestational day 8 to 18. Control mice received saline. Twelve dams each group were sacrificed on gestational day 18. Fetuses were dissected under a binocular microscope. Sex of fetuses was determined by the morphology and fetal testes were aseptically removed. Fetal testes were immersed in modified davidson's fluid for testicular histology. Serum testosterone (T) was measured by radioimmunoassay (RIA). Steady state mRNA level of StAR and T synthetic enzymes in fetal testes was determined by RT-PCR. Leydig cells in fetal testes were identified by immunohistochemistry. For the detection of apoptosis, paraffin-embedded sections were stained with the TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique. The remaining pregnant mice were born by natural birth. All male pups were sacrificed at postnatal day 70. The testes were collected and weighed as quickly as possible. Histopathological changes in testes were observed. Serum testosterone (T) and testicular T were measured by RIA. The apoptosis of germ cells was measure by TUNEL. Results showed in fetal mice that maternal LPS exposure during pregnancy significantly downregulated expression of StAR and P450scc mRNA in fetal testes, whereas there was no significant difference in T of fetal serum and in testicular histopathological changes between LPS group and the controls. However, results showed in adult that testicular weight of male offspring significantly was significantly decreased in LPS group as compared with the control group. The number of epididymal spermatozoa was significantly decreased in LPS group. Histological examination showed that maternal LPS exposure increased the inside diameter of seminiferous tubules, decreased the counts of spermatogenic cells, and disturbed the array of spermatogenic cells in adult male offspring. RIA showed that the levels of serum T and testicular T were significantly decreased in adult offspring of LPS-treated mice. TUNEL showed that maternal LPS exposure increased the number of TUNEL-positive cells in testes of adult male offspring.2. Effect of TNF-αon LPS-induced damage in reproductive development in male offspring.To investigate the role of TNF-αin LPS-induced damage in reproductive development in male offspring, the pregnant mice were randomly divided into six groups. All pregnant mice except controls received an intraperitoneal (i.p.) injection of LPS (500μg /kg) on gestational day 17. Some pregnant mice were pretreated with a low dose of LPS (10μg/kg, i.p.) at different time point (4, 12, 24 or 48 h) before LPS (500μg/kg, i.p.). Control mice received saline. All pregnant mice were sacrificed at 1.5 h after LPS (500μg/kg, i.p.). TNF-αin maternal serum, amniotic fluid, fetal liver and fetal brain was measured by enzyme linked immunosorbent assay (ELISA). TNF-αmRNA in fetal liver and fetal brain was determined by RT-PCR. The levels of TNF-αin maternal serum, amniotic fluid, fetal liver and fetal brain were significantly increased in LPS-treated mice, whereas there was no significant difference in TNF-αmRNA in fetal liver and brain among different groups. Interestingly, a low dose of LPS pretreatment significantly attenuated LPS-evoked elevation of TNF-αin maternal serum, amniotic fluid, fetal liver and fetal brain, while a low dose of LPS pretreatment had little effect on TNF-αmRNA expression in fetal liver and brain.In summary, the present results allow us to reach the following conclusions. First, maternal LPS exposure induces a long-term effect on reproductive development in male offspring. Second, TNF-αmay be involved in LPS-induced damage in reproductive development in male offspring.
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