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Ecotoxicol Environ Saf
2024 Jan 15;270:115876. doi: 10.1016/j.ecoenv.2023.115876.
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Impaired spermatogenesis and associated endocrine effects of azole fungicides in peripubertal Xenopus tropicalis.
Svanholm S
,
Brouard V
,
Roza M
,
Marini D
,
Karlsson O
,
Berg C
.
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Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3β-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.
Fig. 1. mRNA concentration in testis from Xenopus tropicalis males at the age of 4 (PROP) or 8 (IMZ) weeks post metamorphosis after two weeks exposure to 0 (PROPcontrol), 17 (PROPlow) or 178 (PROPhigh) µg propiconazole/L or 0 (IMZcontrol), 1 (IMZlow), 12 (IMZmid) or 154 (IMZhigh) µg imazalil/L. Each data point represents one individual, data is presented as mean 2-ddCt relative to respective control (SD). ddx4: DEAD-box helicase 4, id4: Inhibitor of DNA binding 4, 3β-hsd: 3β-hydroxysteroid dehydrogenase, cyp19: Cytochrome P450 19, aldh1a2: aldehyde dehydrogenase 1 family, member A2. In the PROP exposure, cyp19 was not detected in two males. In the IMZ exposure, ddx4 was not detected in one male and cyp19 was not detected in another. n = 3–8. *Statistically significant from respective control (Generalized linear model with Holm-Bonferroni post hoc test), p < 0.05. **Statistically significant from respective control (Generalized linear model with Holm-Bonferroni post hoc test), p < 0.01. ***Statistically significant from respective control (Generalized linear model with Holm-Bonferroni post hoc test), p < 0.001.
Fig. 2. mRNA concentration in ovaries from female Xenopus tropicalis at the age of 4 (PROP) or 8 (IMZ) weeks post metamorphosis after two weeks exposure to 0 (PROPcontrol), 17 (PROPlow) or 178 (PROPhigh) µg propiconazole/L or 0 (IMZcontrol), 1 (IMZlow), 12 (IMZmid) or 154 (IMZhigh) µg imazalil/L. Data is presented as mean 2-ddCt relative to respective control (SD). esr1: Estrogen receptor 1, rsbn1: Round spermatid basic protein 1. n = 4–7. *Statistically significant from respective control (Generalized linear model, with Holm-Bonferroni post hoc test), p < 0.05. ***Statistically significant from respective control (Generalized linear model with Holm-Bonferroni post hoc test), p < 0.001.
Fig. 3. Principle component analysis of mRNA concentration for selected genes and germ cell counts per testis area of Xenopus tropicalis males at 8 weeks post metamorphosis after two weeks exposure to 0 (C), 1 (L), 12 (M) or 154 (H) µg imazalil/L. The parameters have been shown to differ significantly between the treatment groups and control. DDX4: DEAD-box helicase 4, ID4: Inhibitor of DNA binding 4, 3βHSD: 3β-hydroxysteroid dehydrogenase, CYP19: Cytochrome P450 19. Dark SSC: dark spermatogonial stem cells, Sec. spg: secondary spermatogonia. n = 3–7.
Fig. 4. Suggested adverse outcome for decreased fertility as a result of juvenile imazalil exposure. Boxes encircled by whole lines represent effects observed in the present study. ER: estrogen receptor. AR: androgen receptor. FSH: Follicle-stimulating hormone. cyp19: Cytochrome P450 19. 3β-hsd: 3β-hydroxysteroid dehydrogenase. SSC: spermatogonial stem cell. Sec. spg: secondary spermatogonia. Boxes encircled by dashed lines represent events/outcomes based on data from the literature: Bar-Shira Maymon et al. (2003); Jorgensen et al. (2021); Kojima et al. (2004); Meehan et al. (2000); Orton et al., 2018, Orton et al., 2011; Roco et al. (2021); Steger et al. (1998).
Supplementary Fig. S1. Illustration of the gonadkidney complex. The dashed line illustrates how the complex was divided during sampling. The right part of the complex was used for histological analysis of gonads and Müllerian ducts. The gonad was separated from the left part and used for gene expression. G: gonad, K: kidney, MD: Müllerian duct. Redrawn after Jansson et al. (2016).
Supplementary Fig. S2. Histographs of testis from Xenopus tropicalis 4 weeks post metamorphosis after exposure to A) 0 µg imazalil/L, B) 1 µg imazalil/L, C) 12 µg imazalil/L or D) 154 µg imazalil/L. The arrows indicate spermatogonial stem cells.
Supplementary Fig. S3. Maturation of 4 and 8 week post metamorphic (PM) male Xenopus tropicalis testis. Proportion of animals with the most germ cell either pre- or post-meiotic. n=8 for each age. *Significantly different from 4 weeks PM males (Fisher’s exact test).
Supplementary Fig. S4. mRNA concentration in testis from Xenopus tropicalis males 4 (PROP) or 8 (IMZ) weeks post metamorphosis after juvenile exposure to 0 (PROPcontrol), 17 (PROPlow) or 178 (PROPhigh) µg propiconazole/L or 0 (IMZcontrol), 1 (IMZlow), 12 (IMZmid) or 154 (IMZhigh) µg imazalil/L for two weeks. Each data point represents one individual, data is presented as mean 2-ddCt relative to respective control (SD). amh: Anti-Müllerian hormone, amhr2: Anti-Müllerisan hormone Receptor 2, esr1: Estrogen receptor 1, sox9: SRY-Box Transcription Factor 9, cyp17: Cytochrome P450 17, cyp26: Cytochrome P450 26, rsbn1: Round spermatid basic protein 1. In the PROP exposure, cyp17 was not detected in one male. In the IMZ exposure, amh was not detected in three of the males. In one of these males, esr1 was not detected either. n=3-8.
Supplementary Fig. S5. mRNA concentration in ovaries from female Xenopus tropicalis, 4 (PROP) or 8 (IMZ) weeks post metamorphosis after juvenile exposure to 0 (PROPcontrol), 17 (PROPlow) or 178 (PROPhigh) µg propiconazole/L or 0 (IMZcontrol), 1 (IMZlow), 12 (IMZmid) or 154 (IMZhigh) µg imazalil/L for two weeks. Data is presented as mean 2-ddCt relative to respective control (SD). amh: Anti-Müllerian hormone, amhr2: Anti-Müllerian hormone Receptor 2, sox9: SRY-Box Transcription Factor 9, cyp17: Cytochrome P450 17, cyp19: Cytochrome P450 19, 3β-hsd: 3β-hydroxysteroid dehydrogenase, aldh1a2: Aldehyde dehydrogenase 1 family member A2, cyp26: Cytochrome P450 26, ddx4: DEAD-box helicase 4, id4: Inhibitor of DNA binding 4. In the PROP exposure, amh was not detected in two females, cyp19 in one female and cyp17 in one female. n=4-7.