Molecular characterization and expression of GDNFA and GDNFB and their putative receptors, GFRA1A, GFRA1B and ret during testicular development of the european seabass (Dicentrarchus Labrax L.)

Trabajo presentado en el 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022.
Spermatogenesis starts when undifferentiated type A spermatogonia (und-SpgA), also known as spermatogonial stem cells, become differentiated SpgA (diff-SpgA). In the mammalian testis, it is well established that Sertoli cells produce GDNF (glial cell line-derived neurotrophic factor) that binds the GFRA1 (GDNF family receptor alpha-1), which is expressed by und-SpgA promoting its self-renewal and maintenance. The Gdnf-Gfra1 complex interacts with the Ret receptor tyrosine-kinase activating the intracellular signaling pathway. A few recent studies have addressed the potential involvement of the Gdnf-Gfra1 system in the regulation of gametogenesis in fish but little is known about Ret receptor. As a first step to determine the Gdnf, Gfra1and Ret involvement in the self-renewal and maintenance of the und-SpgA in the European seabass, we have characterized and studied their expression at different stages of testicular development. By BLAST analysis against the NCBI whole-genome shotgun contigs (wgs) database for D. labrax (sequences CBXY010000001 to CBXY010037781), and BLAT analysis against the European seabass genome (http://seabass.mpipz.mpg.de) we found the sequences of the paralogs gdnfa and gdnfb, the paralogs gfra1a and gfra1b, and ret. The sequences were confirmed by PCR using testis cDNA and specific primers designed to the respective genomic sequences, showing open reading frames of 762, 807, 1419, 1425 and 3333 bp coding for 254, 269, 473, 475 and 1111 amino acid residues, respectively. Expression analysis by qrtPCR in adult testis during the reproductive cycle showed the highest levels of gdnfa, gdnfb, gfra1a, gfra1b and ret mRNA expression in testes containing only SpgA. The expression of all genes decreased sharply to low levels coinciding with the appearance of type B-spermatogonia, except for gfra1a, whose expression levels barely decrease during later stages of development. These results suggest that Gdnfa/b-Gfra1a/b-Ret regulatory pathway can have key roles during the early stages of testicular development. However, gfra1a could also have important roles throughout spermatogenesis.
Funded by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) y EU-FEDER (Grant RTI2018-094667-B-C2-1)