Kiss2 neurons (red) and Gnrh3 fibers (green) in the zebrafish hypothalamus. (A) Kiss2 neurons are located in the dorsal zone of the periventricular hypothalamus (Hd). Kiss2 fibers project downward to the anterior tuberal nucleus (ATN) and lateral hypothalamic nucleus (LHN), while few fibers extend to the ventral zone of the periventricular hypothalamus (Hv). (B) Gnrh3 nerve fibers extend to the bottom of the ventral hypothalamus where nerve fibers converge into the pituitary stalk. Few Kiss2 fibers were detected at the bottom. PTN, posterior tuberal nucleus.

Kiss2 neurons (red) and Gnrh3 fibers (green) in the zebrafish pituitary. (A) The distribution of Gnrh3 nerve fibers in the female pituitary. A very large number of Gnrh3 axonal projections penetrate the proximal pars distalis (PPD). (B) Kiss2 nerve structures distribute though the female pituitary. (C) Kiss2 fibers show a similar distribution pattern to that of Gnrh3 fibers in the PPD regions. (D) Gnrh3 fiber bundles invade through the pituitary stalk (rectangular frame), but no Kiss2 fibers are detected in the pituitary stalk. The Kiss2 cells (arrow, high-resolution image) and fibers arise near the pituitary stalk. (E) Kiss2 neurons (arrow, high-resolution image) and fibers (asterisk) reside side by side with Gnrh3 nerve bundles penetrating the neurohypophysis (NH). (F) Gnrh3 fibers are contacted by Kiss2 fibers in the PPD. Kiss2 cells (arrow, high-resolution image) are detected in this area. (G) Gnrh3 fibers are detected in the male pituitary. (H) Kiss2 fibers are rarely found in the male pituitary. (I) Gnrh3 fibers are not in contact with Kiss2 fibers in the male pituitary.

Transcriptional expression of kiss2 and kiss1r in the zebrafish pituitary. (A) kiss2 and kiss1r mRNAs are examined by reverse transcription-polymerase chain reaction (RT-PCR). kiss2 mRNA is expressed in the female but not in the male pituitary. kiss1ra mRNA is not detected in the zebrafish pituitary. kiss1rb mRNA is expressed in both the female and male pituitary. (B) In situ hybridization analysis shows kiss1rb-positive signals in the proximal pars distalis (PPD) and pars intermedia (PI) in the female pituitary. (C) kiss1rb-positive signals are also detected in the male pituitary.

Histological analysis of 3~6 months sexual mature zebrafish gonads. (A) The oocytes of all stage of development are present in sexual mature female. Most of the oocytes are in full-grown stage. (B) Spermatogenesis is undergoing in sexual mature male.

In situ hybridization with sense probes of kiss1rb mRNA shows no signaling in the zebrafish pituitary.