Vasotocin and mesotocin stimulate the biosynthesis of neurosteroids in the frog brain

Abstract

The neurohypophysial nonapeptides vasopressin (VP) and oxytocin (OT) modulate a broad range of cognitive and social activities. Notably, in amphibians, vasotocin (VT), the ortholog of mammalian VP, plays a crucial role in the control of sexual behaviors. Because several neurosteroids also regulate reproduction-related behaviors, we investigated the possible effect of VT and the OT ortholog mesotocin (MT) in the control of neurosteroid production. Double immunohistochemical labeling of frog brain sections revealed the presence of VT/MT-positive fibers in close proximity of neurons expressing the steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) and cytochrome P450 17alpha-hydroxylase/c17, 20-lyase (P450(C17)). High concentrations of VT and MT receptor mRNAs were observed in diencephalic nuclei containing the 3beta-HSD and P450(C17) neuronal populations. Exposure of frog hypothalamic explants to graded concentrations of VT or MT produced a dose-dependent increase in the formation of progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, and dehydroepiandrosterone. The stimulatory effect of VT and MT on neurosteroid biosynthesis was mimicked by VP and OT, as well as by a selective V1b receptor agonist, whereas V2 and OT receptor agonists had no effect. VT-induced neurosteroid production was completely suppressed by selective V1a receptor antagonists and was not affected by V2 and OT receptor antagonists. Concurrently, the effect of MT on neurosteroidogenesis was markedly attenuated by selective OT and V1a receptor antagonists but not by a V2 antagonist. The present study provides the first evidence for a regulatory effect of VT and MT on neurosteroid biosynthesis. These data suggest that neurosteroids may mediate some of the behavioral actions of VT and MT.

Figure 1.

Dual-channel confocal laser scanning microscope photomicrographs comparing the distribution of 3β-HSD- and VP-like immunoreactivity in the anterior preoptic area (Poa; A–C) and in the posterior tuberculum (PT; D–F). Frontal brain sections were labeled with the rabbit antiserum against 3β-HSD revealed with GAR/Alexa-594 (A, D) and the monoclonal antibody against VP revealed with GAM/Alexa-488 (B, E). Combination of the two images acquired in A and B and in D and E revealed that the 3β-HSD-positive cells in the anterior preoptic area and the posterior tuberculum are contacted by VT-immunoreactive fibers (C, F). The insets show higher-magnification views of VT-immunoreactive nerve fibers in the close vicinity of 3β-HSD-positive neurons (C, F). Scale bars, 10 μm.

Figure 2.

Dual-channel confocal laser scanning microscope photomicrographs comparing the distribution of P450_C17- and VP-like immunoreactivity in the anterior preoptic area (Poa; A–C) and in the medial amygdala (MA; D–F). Frontal brain sections were labeled with the rabbit antiserum against P450_C17 revealed with GAR/Alexa-594 (A, D) and the monoclonal antibody against VP revealed with GAM/Alexa-488 (B, E). Combination of the two images acquired in A and B and in D and E revealed that the P450_C17-positive cells in the anterior preoptic area and the medial amygdala are contacted by VT-immunoreactive fibers (C, F). The insets show higher-magnification views of VT-immunoreactive nerve fibers in the close vicinity of P450_C17-positive neurons (C, F). Scale bars, 10 μm.

Figure 3.

Labeling of consecutive frontal sections through the anterior preoptic area (Poa) showing the specificity control of the immunoreaction. Adjacent sections were incubated with the VP antibody (A, C) or with the antibody preincubated with 10⁻⁷ m synthetic VT (B) or 10⁻⁵ m MT (D). Scale bars, 100 μm.

Figure 4.

In situ hybridization analysis showing the distribution of VTR mRNA (A–C) and MTR mRNA (D–F) in the frog diencephalon. Frontal brain sections were hybridized with the antisense VTR riboprobe (A, B) or the antisense MTR riboprobe (D, E). Consecutive sections to those shown in A and E were hybridized with the sense VTR riboprobe (C) or the sense MTR riboprobe (F). The anatomical distributions of 3β-HSD-positive cell bodies (blue asterisks) and P450_C17-positive cell bodies (red asterisks) as well as the designation of the anatomical structures are indicated on the right hemisections. For abbreviations, see Table 1. Scale bar, 500 μm.

Figure 5.

HPLC analysis of radioactive steroids extracted from frog hypothalamic explants after a 2 h incubation with [³H]Δ⁵P in control conditions (A) or in the presence of 10⁻⁷ m VT (B). The ordinate axis indicates the radioactivity measured in the HPLC eluent. The dashed lines represent the gradient of secondary solvent (percentage solution B). The arrows indicate the elution positions of standard steroids.

Figure 6.

Effects of graded concentrations of VT (•), MT (▪), VP (▵), and OT (▾) on the conversion of tritiated pregnenolone into P, 17OH-Δ⁵P, 17OH-P, and DHEA by frog hypothalamic explants. The values were calculated from the areas under the peaks in chromatograms similar to those presented in Figure 5. Results are expressed as percentages of the amount of each steroid formed in the absence of peptides. Values are the mean ± SEM of four independent experiments.

Figure 7.

Time course of the conversion of [³H]Δ⁵P into radioactive P, 17OH-Δ⁵P, 17OH-P, and DHEA by frog hypothalamic explants in the absence (○) or presence (•) of 10⁻⁷ m vasotocin or 10⁻⁷ m mesotocin (▪). The values were calculated from the areas under the peaks in chromatograms similar to those presented in Figure 5. Results are expressed as percentages of the amount of each steroid formed compared with the total amount of radiolabeled compounds resolved by HPLC analysis, including [³H]Δ⁵P. Values are the mean ± SEM of four independent experiments.**p < 0.01; ***p < 0.001 compared with respective control values (one-way ANOVA followed by a post hoc Dunnett’s test).

Figure 8.

Effects of VT (10⁻⁷ m) and MT (10⁻⁷ m) in the absence or presence of the cytochrome P450scc inhibitor aminoglutethimide (10⁻⁴ m), and the 3β-HSD inhibitor trilostane (10⁻⁴ m) and cytochrome P450_C17 inhibitor ketoconazole (10⁻⁴ m) on the conversion of tritiated pregnenolone into P and 17OH-Δ⁵P by frog hypothalamic explants. The values were calculated from the areas under the peaks in chromatograms similar to those presented in Figure 5. Results are expressed as percentages of the amount of each steroid formed in the absence of drugs (control). Each value is the mean ± SEM of four independent experiments. ***p < 0.001 versus control; ^##p < 0.01, ^###p < 0.001 versus VT- or MT-stimulated levels; NS, not statistically different from control; ns, not statistically different from VT- or MT-stimulated levels (one-way ANOVA followed by a post hoc Bonferroni’s test).

Figure 9.

Effects of VT and various agonists (10⁻⁷ m each) on the conversion of tritiated pregnenolone into P and 17OH-Δ⁵P by frog hypothalamic explants. 1, [Phe², Orn⁸]OT, V1aR/V1bR agonist; 2, [deamino-Cys¹, β-(3-pyridyl)-d-Ala², Arg⁸]VP, selective V1bR agonist; 3, [deamino-Cys¹, Val⁴, d-Arg⁸]VP, V2R agonist. The values were calculated from the areas under the peaks in chromatograms similar to those presented in Figure 5. Results are expressed as percentages of the amount of each steroid formed in the absence of peptide (control). Values are the mean ± SEM of four independent experiments. **p < 0.01; ***p < 0.001; NS, not statistically different from control (one-way ANOVA followed by a post hoc Dunnett’s test).

Figure 10.

Effects of VT (10⁻⁷ m) in the absence or presence of various antagonists (10⁻⁶ m each) on the conversion of tritiated pregnenolone into P and 17OH-Δ⁵P by frog hypothalamic explants. 4, [d(CH₂)₅¹, Tyr(Me)², Arg⁸]VP, V1aR antagonist; 5, [Phe-acetyl¹, O-Me-d-Tyr², Arg^6,8, Lys⁹]VP-NH₂, V1aR antagonist; 6, [d(CH₂)₅¹, d-Ile², Ile⁴, Arg⁸]VP, V2R antagonist; 7, [d(CH₂)₅¹, Tyr(Me)², Thr⁴, Orn⁸, des-Gly-NH₂⁹]VT, OTR antagonist. The values were calculated from the areas under the peaks in chromatograms similar to those presented in Figure 5. Results are expressed as percentages of the amount of each steroid formed in the absence of peptide (control). Values are the mean ± SEM of four independent experiments. *p < 0.05, ***p < 0.001 versus control; ^##p < 0.01, ^###p < 0.001 versus VT-stimulated levels; NS, not statistically different from control; ns, not statistically different from VT-stimulated levels (one-way ANOVA followed by a post hoc Bonferroni’s test).

Figure 11.

Effects of MT (10⁻⁷ m) in the absence or presence of various antagonists (10⁻⁶ m each) and an OTR agonist (10⁻⁷ m) on the conversion of tritiated pregnenolone into P and 17OH-Δ⁵P by frog hypothalamic explants. 4, [d(CH₂)₅¹, Tyr(Me)², Arg⁸]VP, V1aR antagonist; 6, [d(CH₂)₅¹, d-Ile², Ile⁴, Arg⁸]VP, V2R antagonist; 7, [d(CH₂)₅¹, Tyr(Me)², Thr⁴, Orn⁸, des-Gly-NH₂⁹]VT, OTR antagonist; 8, [Thr⁴, Gly⁷]OT, OTR agonist. The values were calculated from the areas under the peaks in chromatograms similar to those presented in Figure 5. Results are expressed as percentages of the amount of each steroid formed in the absence of peptide (control). Values are the mean ± SEM of four independent experiments. ***p < 0.001 versus control; ^#p < 0.05, ^###p < 0.001 vs MT-stimulated levels; NS, not statistically different from control; ns, not statistically different from MT-stimulated levels (one-way ANOVA followed by a post hoc Bonferroni’s test).