Expression of GnRH variants and their receptors in the zebrafish (Danio rerio) testis: A preliminary study.

JULIANO BUTZGE; J. M. RICCI; L. DORETTO; E.R. MARTÍNEZ; GABRIELA C. LÓPEZ; GUSTAVO M. SOMOZA; RAFAEL H. NÓBREGA

Águas de Lindóia

Congreso; 59 Congresso Brasileiro de Genética; 2013

Sociedade Brasileira de Genética

Hypothalamic-GnRH (Gonadotropin-Releasing Hormone) is a key hormone in vertebrate reproduction by stimulating gonadotropin (Lh and Fsh) release from pituitary. Most vertebrates also express more than one variant of GnRH, which can be detected in several extra-hypothalamic brain tissue and peripheral organs as well. The anatomic distribution of the extra-hypothalamic GnRH, its regulation and function in different tissues remain poorly known in fish. The existence of the multiple extra-hypothalamic variants suggests that besides its primary function on reproduction,GnRH might be involved in other unknown physiological processes. In this context, the aim of this work is to evaluate the expression of GnRH variants (zfgnrh2, zfgnrh3) and their receptors (zfgnrhr1, zfgnrhr2, zfgnrhr3, zfgnrhr4) in the zebrafish D. rerio (Cyprinidae, Cypriniformes) testis and verify by immunofluorescence the testicular cell types that express GnRH. Forward and reverse specific primers were designed based on the full-length cDNA sequences deposited at NCBI public database. To evaluate the gonadal gene expression, total RNA from testes and ovaries were extracted, followed by cDNA synthesis, and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) using different annealing temperatures (52, 54, 56, 58, 60 and 62°C). For immunofluorescence, a mammalian monoclonal antibody raised against GnRH1 that also cross-reacts with GnRH3 was used. For the different forms of GnRH, we found double bands for zfgnrh3 in testis and single band in the ovary. zfgnrh2 was no expressed at all in testes and ovaries. For the receptors, all were expressed; zfgnrhr2, zfgnrhr4 showed single bands for both males and females, while zfgnrhr1 and zfgnrhr3 double bands in females and in males. We still do not know the significance of the double bands which persisted in all temperatures studied. Different GnRH splicing variants may be present in the gonads but this will be further confirmed by sequencing studies of these bands. Preliminary results of the immunofluorescence, showed that GnRH is expressed in Sertoli cells and Leydig cells. The presence of GnRH and its receptors in the gonads may be related to paracrine/autocrine functions of GnRH on gametogenesis and gonad functions. The interpretation of the possible role of GnRH and its receptors will be assess by real-time qPCR in different gonadal stages (recrudescence versus differentiation) in order to understand the mechanism of action of GnRH in the gonads of zebrafish. Financial Support: FAPESP (2012/00423-6); CNPq (482946/2012-1).