Isolation and characterization of a novel member of the relaxin/insulin family from the testis of the frog Rana esculenta

A complementary DNA (cDNA) encoding a frog relaxin/insulin member family (f RLX) from testis cDNA library was isolated and characterized. The fRLX cDNA predicted a 155-amino acid protein with a low homology to mammalian RLF an d relaxin. Northern blot analysis revealed a single transcript expressed in the interstitial compartment, RT-PCR, evidenced that fRLX is expressed at low levels in the oviduct and ovary too. The predicted mature fRLX protein, composed of the signal peptide, B, C, and A domains, has conserved amino a cid sequences in the characteristic functional domains. A different express ion of the transcript was found during the frog reproductive cycle, with a peak in Spring. After administration of ethane dimethane sulfonate, by in s itu hybridization, fRLX messenger RNA disappeared from the interstitial com partment and reappeared again at the time of generating of a new population of Leydig cells (LC), strongly indicating that LC are the interstitial cel l type expressing fRLX. Preliminary results obtained by in situ hybridizati on, performed on testis of hypophysectomized frogs evidenced a pituitary co ntrol of fRLX expression. This study is the first cloning of a relaxin/insu lin family member in a nonmammalian vertebrate. In addition, because fRLX e xpression changes during the annual cycle suggesting its involvement in spe rmatogenesis, fRLX may be considered a new marker for the study of spermato genesis in the Rana esculenta.