Widespread endocrine disruption and reproductive impairment in an estuarine fish population exposed to seasonal hypoxia

Abstract

The long-term effects on marine fish populations of the recent increase worldwide in the incidence of coastal hypoxia are unknown. Here we show that chronic environmental exposure of Atlantic croaker (Micropogonias undulatus) to hypoxia in a Florida estuary caused marked suppression of ovarian and testicular growth which was accompanied by endocrine disruption. Laboratory hypoxia studies showed that the endocrine disruption was associated with impairment of reproductive neuroendocrine function and decreases in hypothalamic serotonin (5-HT) content and the activity of the 5-HT biosynthetic enzyme, tryptophan hydroxylase. Pharmacological restoration of hypothalamic 5-HT levels also restored neuroendocrine function, indicating that the stimulatory serotonergic neuroendocrine pathway is a major site of hypoxia-induced inhibition. Inhibition of tryptophan hydroxylase activity to downregulate reproductive activity could have evolved as an adaptive mechanism to survive periodic hypoxia, but in view of the recent increased incidence of coastal hypoxia could become maladaptive and potentially affect fish population abundance and threaten valuable fishery resources.

Figure 1

Hypoxic sites (H1, H2, H3 and H4) in East Bay, normoxic sites (N1 and N2) in Pensacola Bay, Florida and transition site (TR) between the two bays where Atlantic croaker were collected in October and November 2003. Inset bar graphs: DO levels (mg l⁻¹, clear bars) at the bottom of the water column (y-axis on left), and ovarian expression of HIF-1α (H-1α) and HIF-2α (H-2α) mRNAs (solid bars: relative units, y-axis on right) in croaker at the time of the October sampling (N=6). A nested ANOVA analysis indicates that HIF-1α and -2α mRNA levels in croaker from the normoxic sites are significantly different from those in fish from the hypoxic sites (p<0.001; results not shown). Individual site differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters (a, b for HIF-1α and a′, b′ for HIF-2α, p<0.05).

Figure 2

Ovarian development and endocrine function in female croaker collected from hypoxic sites in (a,c–g) October and (b) November 2003. (a) GSI (ovarian growth) in October. (b) GSI in November. (c) Histological appearance of representative ovaries, (i) hypoxic site and (ii) normoxic site. (d) Percentage of oocytes at each development stage. (e) Plasma E₂ levels. (f) Hepatic ER mRNA levels. (g) Plasma Vg levels. PNS, peri-nucleolus; CA, cortical alveoli; PYS, primary yolk; SYS, secondary yolk; TYS, tertiary yolk. Scale bar, 200 μm (N=9–22). Asterisks indicate significant differences between normoxic and hypoxic sites (nested ANOVA, ^***p<0.001). Individual site differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters (p<0.05).

Figure 3

Testicular development and endocrine function in male croaker collected from hypoxic sites in (a–d) October and (e) November 2003. (a) GSI (testicular growth). (b) Relative sperm production. (c) Histological appearance of representative testes, (i) (ii) hypoxic sites and (iii) normoxic site. (d,e) Plasma 11-KT levels. SP, spermatozoa; SC, spermatocytes. Scale bar, 200 μm (N=15–31). Asterisks indicate significant differences between normoxic and hypoxic sites (nested ANOVA, ^*p<0.05 and ^***p<0.001). Individual site differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters (p<0.05).

Figure 4

Effects of laboratory hypoxia exposure on ovarian development and endocrine function in female croaker. (a) GSI (ovarian growth). (b) Oocyte development. (c) Histological appearance of representative ovaries, (i) (ii) hypoxia exposure and (iii) normoxic conditions. (d) Fecundity. (e) Plasma E₂. (f) Hepatic ER mRNA. (g) Plasma Vg. See figure 2 for key to abbreviations. Scale bar, 200 μm (N=20–23). Significant differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters (p<0.05). Preliminary results presented at Pollution Responses in Marine Organisms Symposium (Thomas et al. 2006b).

Figure 5

Effects of laboratory hypoxia exposure on testicular development and endocrine function in male croaker. (a) GSI (testicular growth). (b) Relative sperm production. (c) Histological appearance of representative testes, (i) (ii) hypoxia exposure and (iii) normoxic conditions. (d) Plasma 11-KT. (e) Plasma T. See figure 3 for key to abbreviations. Scale bar, 200 μm (N=8–24). Significant differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters (p<0.05).

Figure 6

Effects of hypoxia on neuroendocrine functions in croaker. Results from both sexes were combined because they were not significantly different. Mean±s.e.m. values and N for each sex in the experimental groups are shown in electronic supplementary material, table 6. (a) Plasma LH in response to GnRHa injection (N=8–11). LH values (N=7) for all saline-injected groups were below the detection limit of the assay and are not included in the figure. (b) GnRH mRNA expression in POAH (N=7). (c) 5-HT and (d) 5-HIAA levels in hypothalamus (N=19). (e) Hypothalamic TPH activity (N=6–7). (f,g) Effects of 5-HTP injection on (f) 5-HT (N=10) and (g) GnRH I mRNA levels in POAH (N=7–11). (a,f,g) Significant differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters (p<0.05). (b–e) Asterisk indicates significant difference from controls (Student's t-test, p<0.05).