The GC-IGF1 axis-mediated testicular dysplasia caused by prenatal caffeine exposure

in Journal of Endocrinology
Correspondence should be addressed to H Wang: wanghui19@whu.edu.cn

*(L Pei and Q Zhang contributed equally to this work)

This paper is part of a thematic section on 30 Years of the Developmental Origins of Health and Disease. The guest editors for this section were Sean Limesand, Kent Thornburg and Jane Harding

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Prenatal caffeine exposure (PCE) can induce testicular developmental toxicity. Here, we aimed to explore the underlying mechanism of this process in reference to its intrauterine origin. Pregnant rats were intragastrically administrated caffeine (30 and 120 mg/kg/day) from gestational days 9 to 20. The results showed that the male fetuses exposed to high dose of caffeine (120 mg/kg/day) had a decreased bodyweight and inhibited testosterone synthetic function. Meanwhile, their serum corticosterone concentration was elevated and their testicular insulin-like growth factor 1 (Igf1) expression was decreased. Moreover, the histone 3 lysine 14 acetylation (H3K14ac) level in the Igf1 promoter region was reduced. Low-dose (30 mg/kg/day) caffeine exposure, however, increased steroidogenic enzymes expression in male fetuses. After birth, the serum corticosterone concentration gradually decreased in the PCE (120 mg/kg/day) offspring rats, whereas the expression and H3K14ac level of Igf1 gradually increased, with obvious catch-up growth and testicular development compensation. Intriguingly, when we subjected the offspring to 2 weeks of chronic stress to elevate the serum corticosterone concentration, the expression of Igf1 and testosterone synthesis were inhibited again in the PCE (120 mg/kg/day) group, accompanied by a decrease in the H3K14ac level in the Igf1 promoter region. In vitro, corticosterone (rather than caffeine) was proved to inhibit testosterone production in Leydig cells by altering the H3K14ac level and the expression of Igf1. These observations suggested that PCE-induced testicular developmental toxicity is related to the negative regulation of corticosterone on H3K14ac levels and the expression of Igf1.

Downloadable materials

  • Fig. S1 The schematic illustration of animal treatment. From gestational day (GD) 9 to 20, pregnant Wistar rats were administrated intragastrically with caffeine (30 and 120 mg/kg/d). At postnatal week (PW) 10, one male rat was randomly selected from each litter to exposure to chronic stress for two weeks, and the other one was selected from each litter to mate with normal females (avoid inbreeding) at PW12.
  • Table S1. Antibodies
  • Table S2. Oligonucleotide primers and PCR conditions for real-time quantitative PCR.

 

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    Effects of prenatal caffeine exposure (PCE) on testicular morphology in male fetal rats. Pregnant rats were intragastrically administered with low-dose (30 mg/kg/day) and high-dose (120 mg/kg/day) caffeine once per day from gestational day (GD) 9 to 20, which were assigned to PCE(L) and PCE(H) groups, respectively. At GD20, the fetal rats were taken out. The bodyweight, intrauterine growth retardation (IUGR) rate, testicular diameter and area of male fetal rats were recorded (A, n = 12) (B, n = 3, 100×). The testicular morphology was observed by hematoxylin-eosin dying (C, 200×, 400×), and Ki67 protein expression (red arrows) was detected by immunohistochemistry (D, n = 3, 200×, 400×). The Leydig cells structure (red arrows represent injured mitochondria) was observed by transmission electron microscopy (E, 5000×). Mean ± s.d., *P < 0.05, **P < 0.01 vs control by one-way ANOVA. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0684.

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    Effects of prenatal caffeine exposure (PCE) on testicular steroidogenesis in male fetal rats. Pregnant rats were intragastrically administered with low-dose (30 mg/kg/day) and high-dose (120 mg/kg/day) caffeine once per day from gestational day (GD) 9 to 20, which were assigned to PCE(L) and PCE(H) groups, respectively. At GD20, the fetal rats were taken out. Serum testosterone concentration and intra-testicular testosterone content were measured by ELISA (A, n = 8–12). The mRNA expression of steroidogenic acute regulatory protein (Star), cytochrome P450 cholesterol side chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (Hsd3b) were detected by RT-qPCR (B, n = 8–12). HSD3B protein level was detected by immunohistochemistry (C, n = 3, 400×). Leydig cells (stained with HSD3B, a cytoplasmic steroidogenic enzyme marker) counts were detected by immunofluorescence staining (D, n = 3, 200×). Mean ± s.d., *P < 0.05, **P < 0.01 vs control by one-way ANOVA. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0684.

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    Effects of prenatal caffeine exposure (PCE) on testicular morphology in male offspring rats after birth. Pregnant rats in the PCE(H) group were allowed to deliver spontaneously at term. Bodyweight/bodyweight gain rate, testis weight/index and seminiferous tubule diameter/epithelium thickness were recorded and calculated at different time points after birth (A, B, n = 8–12) (C, n = 3, 400×). Mean ± s.d., *P < 0.05, **P < 0.01 vs control by the Student’s t-test. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0684.

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    Effects of prenatal caffeine exposure (PCE) on testicular function in male offspring rats after birth. Pregnant rats in the PCE(H) group were allowed to deliver spontaneously at term. Serum testosterone concentration and intra-testicular testosterone content were measured by radioimmunoassay (A, n = 8–12). The mRNA expression of steroidogenic acute regulatory protein (Star), cytochrome P450 cholesterol side chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (Hsd3b) were detected by RT-qPCR (B, n = 8–12). HSD3B protein level was detected by Western blot (C, n = 3). Leydig cells (stained with HSD3B, a cytoplasmic steroidogenic enzyme marker) counts were detected by immunofluorescence staining (D, n = 3, 400×). Sperm counts and sperm motility were measured (E, n = 5). The rate of pregnancy and bodyweight of F2 generation male rats were recorded (F, n = 8–12). Representative photomicrographs of sperm smear collected from epididymal cauda in PCE(H) group were shown (head abnormalities (thin arrows) and tail abnormalities (thick arrows)) (G). The frequency of spermatozoa with abnormal morphology was observed (G, n = 5). Mean ± s.d., *P < 0.05, **P < 0.01 vs control by the Student’s t-test. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0684.

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    Effects of prenatal caffeine exposure (PCE) on corticosterone concentration and insulin-growth factor 1 (IGF1) expression in male offspring rats before and after birth. Pregnant rats were intragastrically administered with low-dose (30 mg/kg/day) and high-dose (120 mg/kg/day) caffeine once per day from gestational day (GD) 9 to 20, which were assigned to PCE(L) and PCE(H) groups, respectively. And pregnant rats in the PCE(H) group were allowed to deliver spontaneously at term. Serum corticosterone concentration was measured by ELISA (A, F, n = 8–12). The mRNA expression of glucocorticoid receptor (Gr) and Igf1 were detected by RT-qPCR (B, C, G, n = 8–12). The levels of histone 3 lysine 14 acetylation (H3K14ac) in the Igf1 promoter were measured by ChIP-PCR (E, I, n = 3). IGF1 protein level was detected by immunohistochemistry (D, n = 3, 400×) or Western blot (H, n = 3). Mean ± s.d., *P < 0.05, **P < 0.01 vs control by one-way ANOVA or Student’s t-test. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0684.

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    Effects of chronic stress on testicular insulin-growth factor 1 (IGF1) expression and steroidogenesis in male offspring rats followed prenatal caffeine exposure (PCE). Pregnant rats in the PCE(H) group were allowed to deliver spontaneously at term. Male pups were randomly selected from each dam and subjected to two weeks chronic stress or not from postnatal week 10 to 12. Serum corticosterone concentration was measured by ELISA (A, n = 8–12). The mRNA expression of Igf1, steroidogenic acute regulatory protein (Star), cytochrome P450 cholesterol side chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (Hsd3b) were detected by RT-qPCR (B, D, n = 8–12). The levels of histone 3 lysine 14 acetylation (H3K14ac) in the Igf1 promoter were measured by ChIP-PCR (C, n = 3). Serum testosterone concentration and intra-testicular testosterone content were measured by radioimmunoassay (E, F, n = 8–12). Mean ± s.d., *P < 0.05, **P < 0.01 vs control by independent t-test; # P < 0.05, ## P < 0.01 vs no stress by paired t-test.

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    Direct effects of caffeine and corticosterone on steroidogenesis in mouse Leydig TM3 cells. The TM3 cells were treated with different concentrations of caffeine and corticosterone for 3 days. Above-mentioned treatment of the cells showed no cytotoxicity. Then, TM3 was treated in the presence of corticosterone (750 nM) with insulin-growth factor 1 (IGF1) (100 ng/mL) or RU486 (2.5 μM). The mRNA expression of Igf1, steroidogenic acute regulatory protein (Star), cytochrome P450 cholesterol side-chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (Hsd3b) were determined by RT-qPCR (A, B, D, E, G, I, n = 5). The testosterone concentration in the cell culture supernatant was detected by radioimmunoassay (C, F, H, n = 5). The levels of histone 3 lysine 14 acetylation (H3K14ac) in the Igf1 promoter were measured by ChIP-PCR (J, n = 3). Mean ± s.d., *P < 0.05, **P < 0.01 vs control; # P < 0.05, ## P < 0.01 vs 750 nM corticosterone by one-way ANOVA or Student’s t-test.

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    The intrauterine programming alteration of GC-IGF1 axis caused by prenatal caffeine exposure is involved in testicular dysplasia and dysfunction in male offspring rats. GC, glucocorticoid; H3K14ac, histone 3 lysine 14 acetylation; HPA, hypothalamic–pituitary–adrenal; IGF1, insulin-like growth factor 1. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0684.

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