Exposure To Inflammation At Different Life Stages Persistently Impairs Male Reproduction And Endocrine Function In Male Mice

Objective The present study was to investigate the persistent impairment of inflammation exposure at different life stages on male reproduction and endocrine function in male mice. Two experiments were conducted to explore the lasting effects of prenatal LPS exposure on testicular development, testosterone biosynthesis, spermatogenesis in male mice and acute LPS exposure on testosterone biosynthesis, testicular morphology in adult male mice, respectively.Methods Two experiments were conducted in this study.(1) To explore the long-term effects of prenatal LPS exposure on male reproduction and endocrine function in male mice, adult CD-1 male mice were mated with female mice and the pregnant mice were randomly divided into two groups. In LPS group, the pregnant mice were intraperitoneally injected with LPS(50 mg/kg) daily from gestational day(GD) 13 to GD 17. The normal saline-treated pregnant mice served as controls. Twelve pregnant mice in each group were sacrificed on GD 18. Fetuses were dissected, sexed and weighed. Sera from male fetuses were collected and serum testosterone was measured by RIA. Fetal testes were immersed in modified Davidson’s fluid(MDF) for 6 hr. Subsequently, testicular histology and immunohistochemistry(IHC) were detected. The remaining pregnant mice delivered their fetuses naturally. At postnatal day(PND) 21, pups were weaned and housed five to a cage. Anogenital distance(AGD) in males and females were evaluated at PND 26. At PND 35 and 63, twelve male mice from two groups were sacrificed by exsanguination. Testes and prostates plus seminal vesicles were excised and weighed. Sera from male pups were collected and kept for T and luteinizing hormone(LH) measurement. The left testes were immersed in MDF. HEstaining was conducted to observe histopathological changes in testis and 3β-HSD was detected by IHC to estimate the number of Leydig cells. The number of spermatozoa in cauda epididymis was counted at PND 63.(2) To investigate the effects of acute LPS exposure on reproductive endocrine function in adult male mice, the CD-1 male mice were randomly divided into control group and LPS group. Mice in LPS group were intraperitoneally(i.p.) injected with LPS(1 mg/kg), while mice in control group were i.p. injected with normal saline. At different time points(3, 6, 12 and 24 h) after LPS treatments, mice were killed in batches. The weight of testes was measured, and then the organ coefficient of testis was calculated. HE staining was conducted to observe histopathological damage in testis. Serum testosterone was measured by RIA. 3β-HSD was detected by immunohistochemistry to estimate the number of Leydig cells. Testis was collected and total cellular RNA was extracted. The m RNA levels of T biosynthetic enzymes in testis were detected using RT-PCR. Related proteins, including St AR, were detected by immunoblotting.Results At fetal period, a significant decrease in body weight and abnormal Leydig cell aggregations were observed in males whose mothers were exposed to LPS daily from GD 13 to GD 17. At postnatal day(PND) 26, anogenital distance(AGD), a sensitive index of altered androgen action, was markedly reduced in male pups with LPS exposure. At PND 35, the weight of testes, prostates and seminal vesicles, and serum testosterone(T) level were significantly decreased in LPS-treated male pups. Prenatal LPS exposure obviously diminished the percent of seminiferous tubules in stages I–VI, increased the percent of seminiferous tubules in stages IX–XII, and caused massive sloughing of germ cells in seminiferous tubules in testes. Moreover, prenatal LPS exposure significantly reduced serum T level and the number of sperm in male mice at PND 63. When adult male mice were exposed to LPS(1 mg/kg), testicular weight and coefficient were significantly increased at 12 and 24 h after LPS. Moreover,histopathological change, as determined by a decrease in the number of germ cells and structural damages in seminiferous epithelium, was observed in seminiferous tubules at 12 and 24 h after LPS. In addition, the levels of serum testosterone were significantly decreased at 3, 6 and 12 h after LPS. No significant difference was observed in the number of testicular Leydig cells between LPS-treated mice and controls. Futher study showed that LPS treatments could down-regulate the m RNA levels of T biosynthetic enzymes in testis, such as St AR, 17β-HSD, CYP17A1, CYP11A1 and so on. After detecting the expression of proteins involved using immunoblotting, only testicular St AR was found to be significantly reduced within 24 hours after LPS.Conclusion Taken together, these results suggest that exposure to inflammation at different life stages could persistently impair male reproduction and endocrine function in male mice. More specifically, prenatal LPS exposure disrupts T production, impairs testicular development and leads to spermatogenesis dysfunction in male mice. Acute exposure to LPS in adulthood causes damages in testicular morphology and might eventually decrease serum T level through inhibiting T biosynthetic enzymes in male mice.