Confocal imagery of tg(gnrh2:eGFP) pituitaries in normally fed (a), 7-day fasted (b) and 14-day fasted (c) conditions. Antibody staining of the specific GAP2 region of Gnrh2 in pituitary cryosections of fed (d), 7-day fasted (e), and 14-day fasted zebrafish (d) with the quantification of Gnrh2 neuronal fiber lengths confirming increased Gnrh2 in 14-day fasted conditions (e). Confocal imagery of tg(gnrh3:tdTomato) pituitaries in normally fed (f) and 14-day fasted (h) conditions. Immunohistochemistry antibody staining of the specific GAP3 region of Gnrh3 in pituitary cryosections of fed (g) and 14-day fasted zebrafish (i) with the quantification of Gnrh3 neuronal fiber lengths confirming decreased Gnrh3 in 14-day fasted conditions (j). Pictoral representation of the pituitary orientation of images shown (k). NH, neurohypophysis; PPD, proximal pars distalis; RPD; rostral pars distalis. All data expressed as means ± S.E.M, n = 6 pituitaries per group, **P < 0.01, *P < 0.05.

Confocal imagery of whole tg(gnrh2:eGFP;lh:mCherry) pituitaries in normally fed (a, b) and 14-day fasted zebrafish (c, d). B’ and d’ are magnifications of the boxed regions shown in b and d. Quantification of Lh cells with close Gnrh2 neuronal contact confirms increased interactions after 14-day fasting (E). Immunohistochemistry antibody staining of Gnrh2 and Fsh on pituitary cryosections of fish in normally fed (f, g) and 14-day fasted (h, i) conditions. G’ and i’ are magnifications of the squared regions shown in g and i. Quantification of Fsh cells with close Gnrh2 contact confirms increased interactions after 14-day fasting (j). ∆ indicate close contact of Gnrh2 neurons with gonadotropes. All data expressed as means ± S.E.M, n = 6 pituitaries per group, *P < 0.05.

Expression profiles of the Gnrh system in fed and fasted conditions of wild-type zebrafish. Relative mRNA levels of gnrh2 (a) and gnrh3 (b) from brain tissues. Protein content of Gnrh2 in pituitaries from fed and 14-day fasted male and female wild-type zebrafish (c). Relative mRNA levels of gnrhr1 (d), gnrhr3 (e), and gnrhr4 (f) in pituitaries from fed and fasted male and female wild-type zebrafish. All data expressed as means ± S.E.M, n = 8 pituitaries or brains per group, *P < 0.05.

Expression differences of Lh and Fsh in wild-type and gnrh2^−/− zebrafish. Relative mRNA levels of lhb (a) and fshb (c) from pituitaries of wild-type ● and gnrh2^−/− ∆ zebrafish undergoing normally fed, 7-day fasting, or 14-day fasting conditions. Protein content of Lh (b) and Fsh (d) from pituitaries of wild-type ● and gnrh2^−/− ∆ zebrafish undergoing normally fed, 7-day fasting, or 14-day fasting conditions. All data expressed as means ± S.E.M, n = 8 pituitaries per group, *P < 0.05.

Gametogenesis analysis of knockout and WT zebrafish undergoing fed and fasted conditions. Percentage of oocytes at pre-vitellogenic, early-mid vitellogenic, and late/fully mature stages of WT and gnrh2^−/− ovaries after fed and fasted conditions demonstrate significantly fewer late/fully mature oocytes in gnrh2^−/− in fasted conditions (a). Histological sections of ovaries with labelled germinal vesicles (GV), cortical alveoli (ca), pre-vitellogenic oocytes (PV), and primary-growth follicles (PG), demonstrating fully mature oocytes (yellow star *) present in fed and fasted WT ovaries, but only present in fed gnrh2^−/− ovaries (b). GSI % quantification indicates reduced GSI in gnrh2^−/− undergoing fed and fasted conditions (c). Histological sections of testes demonstrate mature spermatozoa ▲ in fed and fasted WT and gnrh2^−/− males (d) with GSI % quantification indicating no difference in genotypes undergoing fed and fasted conditions (e). All data expressed as means ± S.E.M, n = 10 gonads per group, *P < 0.05, **P < 0.01, different letters indicate significant differences (P < 0.05) from each other (statistical comparisons made only between similarly-staged oocytes which are characterized by quote marks: no mark = pre-vitellogenic, ‘ = early-mid vitellogenic, ‘’ = late/fully mature).

Reproductive outputs of male (n = 8–10 per group) and female (n = 5–8 per group) WT, gnrh2, and gnrh3 fish undergoing 0, 7, and 14-day fasting, including WT and gnrh2^−/− comparisons of male spawning percentages (a), female spawning percentages (b), quantification of male courtship attempts with female partners (c), and female fecundities (d). WT and gnrh3^−/− comparisons of male spawning percentages (e), female spawning percentages (f), quantification of male courtship attempts with female partners (g), and female fecundities (h). Still image from video of spawning tanks demonstrating typical behavior of WT males chasing females and gnrh2^−/− males less frequently chasing female partners (i) All data expressed as means ± S.E.M, *P < 0.05.