GnRH-II presence in rat brain and pituitary.

MONGIAT LA; FERNANDEZ MO; SOMOZA GM; LUX-LANTOS VA; LIBERTUN C

San Diego, USA

Congreso; Endocrine Society, 87th Annual Meeting; 2005

Endocrine Society

The second GnRH form originally identified in chicken (cGnRH-II or GnRH-II), is the most ubiquitous peptide of this family, being present from jawed fish to human beings. However, the presence of GnRH-II in such an important model as the rat is still object of discussion since it has not been possible to elucidate the GnRH-II gene sequence in rat. Here we present chromatographic, immunologic and activity evidences supporting the expression of GnRH-II in the rat. Olfactory bulb, hypothalamus, remnant brain and anterior pituitary from a pool of 50 female adult rats were extracted and subjected to RP-HPLC on a Supelcosil LC-18 column. Fractions were collected and evaluated by two different RIA systems, specific for GnRH-I (antibody HU60, gift from Dr. H. Urbanski) and GnRH-II respectively (antibody acII6, gift from Dr. K. Okuzawa). Under these conditions the GnRH-I standard eluted in fraction 21 (f21) and was only detected with the GnRH-I RIA system, whereas GnRH-II standard was only detected in the fraction 27 (f27) by GnRH-II RIA system. The HPLC column was rigorously washed after each use and a blank run was performed each time previous to sample injection. In the olfactory bulbs extract, the fractions analyzed by GnRH-I RIA systems showed a single peak in f21, whereas by using GnRH-II RIA system a single peak at f27 was observed. In the hypothalamus GnRH-I was detected in f21 whereas GnRH-II could not be detected. When the remnant brain and pituitary were analyzed, both GnRHs forms were detected. Furthermore, the immunological properties of f27 were tested in displacement experiments in comparison to GnRH-II, and no differences were found. Regarding biological activity, we reported that GnRH-II is able to release LH and FSH from rat pituitary cells, being this effect mediated by the classical GnRH type-I receptor (1). In this work, we demonstrated that after 60 minutes stimulation, both f27 and GnRH-II posses LH and FSH releasing activity in 12 day-old rat pituitary primary cell cultures (FSH in ng/ml, control: 3.6±0.5, GnRH-II 10-9M: 19.9±1.4, f27 10-9M: 13.9±1.1, n=3, p<0.01 vs control; LH in ng/ml, control: 12.2±0.8, GnRH-II 10-9M: 45.0±6.5, f27 10-9M: 27.8±2.4, n=3, p<0.05 vs control). These results are in agreement with a previous publication suggesting presence of GnRH-II peptide in rat whole brain (2), and provide strong evidence for the expression of GnRH-II in rat with some regional distribution.   With the support of UBA, CONICET, ANPCYT.   1) Mongiat LA, Lux-Lantos VA and Libertun C. (2004) Biol. Reprod.; 71: 464-9. 2) Chen A, Yahalom D, Ben-Aroya N, Kaganovsky E, OkonE and Koch Y. (1998) FEBS Lett. 435: 199-203.