Figure 1.
Tissue-specific melanocortin 2 receptor (MC2R) gene expression.
Expression of melanocortin 2 receptor (MC2R) and β-actin in adult zebrafish tissues as determined with RT-PCR. Products were amplified (MC2R-36 cycles, β-actin- 32 cycles) from total RNA extracts. Images are representative of the results observed from three to five fish. Water replaced cDNA in the negative (Neg.) control treatments.
Figure 2.
Temporal (3 or 8 h incubations) cortisol production by zebrafish ovarian follicles.
Cortisol (grey bars) is not detected in the culture media of control or ACTH treated follicles, but is detected in the human chorionic gonadotropin (hCG, 10 I.U./mL) and hCG plus adrenocorticotropic hormone (ACTH, 1.5 I.U./mL; hatched bars) treatments. Control (open bar) and hCG-stimulated (black bar) estradiol (E2) media concentrations after 3 h are provided as a reference. Values represent mean ± SEM (N = 3; each N is a pool of follicles from three fish). Treatments with different letters are significantly different, as determined by one-way ANOVA at each time point followed by Student-Newman-Keuls test for multiple comparisons (P<0.05). An asterisk (*) with the E2 measurements indicates a significant difference determined by a Student's t test (P<0.05).
Figure 3.
Effect of ACTH on estradiol (E2) production by zebrafish ovarian follicles.
E2 production by follicles after 1.5 h incubations under A) basal conditions (no hCG; open bars) with a range of ACTH concentrations (0 to 3.33 I.U./mL; hatched bars), and B) hCG treatment (10 I.U./mL; black bars) and a range of ACTH concentrations (hatched bars). Additionally, the effect of 500 nM (181 ng/mL; horizontal striped bar) cortisol (F) on hCG-induced E2 production was also tested. Values represent mean ± SEM (N = 4, with each N representing a pool of follicles from three different fish). Treatments with different letters are significantly different (one-way repeated measures ANOVA followed by Student-Newman-Keuls test for multiple comparisons; P<0.05). The effects of cortisol on E2 production was analyzed using a Student's t test. C) Expression of the glucocorticoid receptor (GR) and β-actin in adult zebrafish tissues as determined with RT-PCR (GR-36 cycles, β-actin- 32 cycles). Images are representative of the results observed from three fish.
Figure 4.
Temporal E2 production by zebrafish ovarian follicles.
Temporal E2 production (1.5 h, 3 h or 8 h incubations) by zebrafish ovarian follicles in control (open bars), human chorionic gonadotropin (hCG; 10 I.U./mL; black bars) and hCG plus adrenocorticotropic hormone (ACTH; 1 I.U./mL at 1.5 h, 1.5 I.U./mL at 3 h and 8 h; hatched bars) treatments. Each time point is a separate experiment using follicles from different fish. Values represent mean ± SEM (N = 3 or 4, where each N is a pool of follicles from three different fish). Time points with the same lower-case letter are not different, while treatments within a given time that are different are indicated by different upper-case letters (two-way ANOVA; P<0.05).
Figure 5.
Estradiol (E2) production by zebrafish ovarian follicles in response to 8-bromo-cAMP and forskolin.
Follicles were exposed for 1.5 h to basal conditions (open bar), hCG (10 I.U./mL; black bar), 8-bromo-cAMP (0.5 mM; grey bar), 8-bromo-cAMP and ACTH (1.0 I.U./mL; hatched bar), forskolin (10 µM; dark grey bar) or forskolin and ACTH (1.0 I.U./mL; hatched bar). Values represent mean ± SEM (N = 3 pools of follicles from three different fish). Treatments with different letters are significantly different (one-way ANOVA followed by Student-Newman-Keuls test for multiple comparisons; P<0.05).
Figure 6.
Overview of the impact of the stress axis (hypothalamus-pituitary-adrenal axis) on the reproductive axis (hypothalamus-pituitary-gonadal axis).
Human chorionic gonadotopin (hCG) was used as a luteinizing hormone receptor agonist to stimulate estradiol (E2) secretion from ovarian follicles. Corticotropic releasing factor (CRF, green arrows) inhibits GnRH within the hypothalamus [1] and steroidogenesis in the ovary [31]. Adrenocorticotropic hormone (ACTH, blue arrows) inhibits stimulated estradiol (E2) synthesis by ovarian follicles (present study; blue bold arrow) and stimulates lipolysis in adipocytes [22]. Cortisol (purple arrows) modulates reproductive process in the hypothalamus, pituitary and testis [5]. However, cortisol has no effect on E2 synthesis in zebrafish ovarian follicles (present study; purple dashed arrow). In fish, cortisol treatment decreases the expression of hepatic estrogen receptor (ER), vitellogenin (vtg) and vitelline envelope protein-β (vep) [10], [11]. See text for more examples.