Nonmammalian gonadotropin-releasing hormone molecules in the brain of promoter transgenic rats

Abstract

Mammalian gonadotropin-releasing hormone (GnRH1) and nonmammalian immunoreactive GnRH subtypes were examined in transgenic rats carrying an enhanced GFP (EGFP) reporter gene driven by a rat GnRH1 promoter. Double-label immunocytochemistry was performed on EGFP(+)/GnRH1 brain sections by using antisera against GnRH1, GnRH2 (chicken II), GnRH3 (salmon), or seabream GnRH. EGFP(+)/GnRH1 neurons were in the septal-preoptic hypothalamus but not in the midbrain, consistent with GnRH1-immunopositive neurons in WT rats. Apparent coexpression of EGFP(+)/GnRH1 with other GnRH subtypes was observed. All EGFP(+) neurons in the septal-preoptic hypothalamus were GnRH1-immunopositive. However, only approximately 80% of GnRH1-immunopositive neurons were EGFP(+), which awaits further elucidation. GnRH subtypes-immunopositive fibers and EGFP(+)/GnRH1 fibers were conspicuous in the organum vasculosum of the lamina terminalis, median eminence, and surrounding the ependymal walls of the third ventricle and the aqueduct in the midbrain. These results demonstrate that the expression of the EGFP-GnRH1 transgene is restricted to the bona fide GnRH1 population and provide clear morphological evidence supporting the existence of GnRH1 neuronal subpopulations in the septal-preoptic hypothalamus, which might be driven by different segments of the GnRH promoter. This genetic construct permits analyses of promoter usage in GnRH neurons, and our histochemical approaches open questions about functional relations among isoforms of this peptide, which regulates reproductive physiology in its behavioral and endocrine aspects.

Fig. 1.

Gel showing expression of EGFP amplicons. EGFP⁺/GnRH1 rats show a 1,057-bp PCR amplicon in lanes 1, 3, 5, 8, 9, and 10, and EGFP^– littermates in lanes 2, 4, 6, and 7. DNA size marker (M) in base pairs, is given.

Fig. 2.

Distribution of GnRH soma and fibers in the brain of EGFP⁺/GnRH1 rats is shown. (A) TT. (B and C) Soma (B) and fibers (C) in the HB. (D and E) Soma (D) and fibers (E) in the MM. (F and G) EGFP⁺/GnRH1 fibers (F) and Cy3-labeled GnRH2 fibers (G) surrounding the Aq in the midbrain. (Scale bars: A and D–G, 100 μm; B and C, 50 μm.)

Fig. 3.

Coronal sections through the caudal region of the septal–preoptic area of EGFP⁺/GnRH1 rats immunoreactive to GnRH subtypes are shown. Cy3-labeled soma and fibers (red) reveal GnRH1 (LRH13) (A), GnRH3 (lot 2) (B), and GnRH2 (aCII6) (C). Note the typical fusiform shape of GnRH neurons and the abundance of EGFP⁺ cells immunoreactive to GnRH1 in A; EGFP⁺ soma and fibers coexpressing each GnRH subtype appear in yellow. Cy3-labeled nonmammalian GnRH soma and fibers (red) intermingled with EGFP⁺/GnRH1 cells (green). (Scale bars, 50 μm.)

Fig. 4.

Coronal sections through the medial region of the ME of EGFP⁺/GnRH1 rats immunoreactive to GnRH subtypes are shown. (A and B) Cy3-labeled (red) fibers immunoreactive to GnRH1 (LRH 13) (A) or GnRH2 (aCII6) (B). EGFP⁺ fibers coexpressing either nonmammalian GnRH appear in yellow. (C) Cy3-labeled GnRH2 (aCII6) fibers (red) in close contact with EGFP⁺/GnRH1 soma (green) in the septal–preoptic area are shown. (Scale bars: A and B, 100 μm; C, 50 μm.)