Reduced testicular steroidogenesis in rat offspring by prenatal nicotine exposure: Epigenetic programming and heritability via nAChR/HDAC4
Introduction
Although there is increasing awareness that smoking seriously endangers human health, the prevalence of tobacco consumption among pregnant women is estimated to be 10%–25% worldwide, even up to 59.3% in some areas (Tong et al., 2013; Cui et al., 2014). In addition, 25%–29% of women smoke throughout the duration of their pregnancy despite intentions to refrain from smoking, and 50% of non-smoking mothers are exposed to an environment polluted by tobacco (Contal et al., 2005). Tobacco consumption is known to be associated with adverse pregnancy outcomes, including spontaneous abortion, premature birth, stillbirth and lower birthweight (George et al., 2006; Ion and Bernal, 2015; Marufu et al., 2015; Talati et al., 2017). It also provokes, the decline in fertility and disorders of spermatogenesis in adult male offspring (Ramlau-Hansen et al., 2007; Sobinoff et al., 2014). Nicotine, a very toxic alkaloid extracted from the tobacco plant, is widely consumed in the world through cigarettes. Because of its high lipid solubility, nicotine can accumulate in breast milk and amniotic fluid and readily crosses the placental tissue into the bloodstream of the fetus (Dahlstrom et al., 1990). Studies have shown that prenatal nicotine exposure (PNE) has a long-term effect on reproductive health in male offspring (Paccola et al., 2014; Paccola and Miraglia, 2016). This effect is likely ascribable to alterations in the synthetic function of sexual hormone (Dempsey and Benowitz, 2001).
Besides its fundamental role in sexual behavior, testosterone also plays a key role in either the development of the male genital system or the maintenance of spermatogenesis. A reduction in testosterone level may lead to testicular injury, gonadal dysfunction, and male infertility (Macleod et al., 2010). Testosterone belongs to the steroid hormone, and its biosynthesis is catalyzed by a series of steroidogenic enzymes. Among them, the steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are the rate-limiting factors, and their expression is susceptible to external environments (e.g., xenobiotics) (Simard et al., 2005; Scott et al., 2009; Selvaraj et al., 2018). Studies have shown that maternal intake of di(n-butyl) phthalate during pregnancy inhibits testosterone synthesis in fetal rats, which is caused by the low expression of StAR and 3β-HSD (Barlow et al., 2003; Lehmann et al., 2004). All of these findings suggest that PNE-induced testicular development toxicity might be related to the low expression of StAR and 3β-HSD.
It has been shown that environmental toxins can promote epigenetic intergenerational inheritance of reproductive disorder. The exposure of gestating female rats to endocrine disruptors induced an adult phenotype of decreased spermatogenic capacity in the F1 to F4 generation, which was related to epigenetic alteration in the germ line (Anway et al., 2005). However, similar studies on male endocrine function (mainly testosterone synthesis) are slightly inadequate. Despite evidence that adverse events of early life program lower testosterone levels in contemporary adult males (Drake et al., 2009; Eisenberg et al., 2012), the intergenerational transmission and mechanism through which adverse events exert their effect on testicular steroidogenesis remain unclear. Prenatal bisphenol A exposure has been shown to alter the histone code of StAR promoter to disrupt testicular steroidogenesis in mice (Hong et al., 2016). Arsenic exposure induced 3β-HSD up-regulation by suppressing H3K9me2/3 status in Leydig cells (LCs), and caused male reproductive dysfunction (Alamdar et al., 2017). Evidence from our laboratory revealed that histone acetylation status and associated phenotype can be inherited in F2 generations in response to PNE (Xie et al., 2018). Hence, we speculated that StAR/3β-HSD may be involved in PNE-induced low steroidogenesis and intergenerational effect through epigenetic mechanism.
In this study, pregnant rats were treated with nicotine during middle and late pregnancy, as described in our previous study (Xu et al., 2013). To better understand the intergenerational effects of nicotine on testicular development and its intrauterine origin, we detected the changes of testicular morphology and testosterone synthetic function in both F1 and F2 generations. Combining the LC experiment in vitro, we confirmed whether the epigenetic modifications of StAR and 3β-HSD mediated the low testosterone synthesis in the fetal testis. This study will clarify the intergenerational effect and intrauterine programming mechanism of testicular dysplasia in male offspring caused by nicotine exposure during pregnancy.
Section snippets
Chemicals and reagents
Nicotine (CAS no. 54-11-5) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Isoflurane was purchased from Baxter Healthcare Co. (Deerfield, IL, USA). The enzyme-linked immunosorbent assay (ELISA) kit for testosterone was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). A rat/mouse Iodine [125I] testosterone radioimmunoassay kit was purchased from North Institute of Biological Technology (Beijing, China). The antibodies of StAR (8449s) and cytochrome P450 cholesterol side chain
Effects of PNE on body weight and testicular morphology in F1 male fetal rats
First, we observed the effects of PNE on testicular morphological development in F1 fetal rats. A significant reduction of body weight and testicular area occurred in the PNE group at GD20 (P < 0.05, P < 0.01, Fig. 2A, Fig. 2B). The interstitial region among seminiferous tubules appeared slightly enlarged (Fig. 2C, red arrowhead), but there were no other significant pathological changes. In addition, immunolocalization for Ki67 demonstrated that the cell proliferation was reduced (P < 0.01,
PNE induced testicular dysplasia and its heritable effect on low testosterone synthetic function
Several studies have confirmed that nicotine exposure during the embryonic period induces male reproductive damage in adulthood (Lagunov et al., 2011; Miranda-Spooner et al., 2016). In the present study, we found that PNE reduced testicular weight and index, seminiferous tubule diameter, LCs number and testosterone production from puberty to adulthood in the F1 generation. What do these postnatal changes have to do with early testicular development? The abnormal environment in early life could
Conclusion
In conclusion, PNE induced testicular dysplasia and lower testosterone synthetic function, which can be inherited from the F1 generation to the F2 generation. The programming mechanism could be that nicotine increased the HDAC4 expression via nAChR, which further contributed to the decreased H3K9ac and expression levels of StAR and 3β-HSD (Fig. 8). Our study provides novel experimental evidence for exploring the intrauterine origin, programming alteration, intergenerational effect, and
Author contribution statement
Qi Zhang: Methodology, Investigation, Writing- Original draft preparation. Lin-guo Pei: Methodology, Investigation, Writing- Original draft preparation. Min Liu: Resources, Investigation, Formal analysis. Feng Lv: Investigation, Formal analysis. Guanghui Chen: Resources, Formal analysis. Hui Wang: Conceptualization, Methodology, Writing- Reviewing and Editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by grants from the National Key Research and Development Program of China (2017YFC1001300), the National Natural Science Foundation of China (Nos. 81430089, 81673524), and Hubei Province Health and Family Planning Scientific Research Project (No. WJ2017C0003).
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2022, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :This is the deficiency of the model and the limitation of the conclusion. However, based on the low birth weight and multiple organs dysplasia in the PNE offspring in clinical and animal studies [26,27,37–43], the conclusions of the PNE rat model can still provide new insights and basis for adrenal dysplasia in offspring. More and more studies reported that PNE could cause adverse pregnancy outcomes and even long-term harm [44–49].
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Qi Zhang and Lin-guo Pei have contributed equally to this work.