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 (fRLX) from testis cDNA library was isolated and characterized. The fRLX cDNA predicted a 155-amino acid protein with a low homology to mammalian RLF and 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 acid sequences in the characteristic functional domains. A different expression of the transcript was found during the frog reproductive cycle, with a peak in Spring. After administration of ethane dimethane sulfonate, by in situ hybridization, fRLX messenger RNA disappeared from the interstitial compartment and reappeared again at the time of generating of a new population of Leydig cells (LC), strongly indicating that LC are the interstitial cell type expressing fRLX. Preliminary results obtained by in situ hybridization, performed on testis of hypophysectomized frogs evidenced a pituitary control of fRLX expression. This study is the first cloning of a relaxin/insulin family member in a nonmammalian vertebrate. In addition, because fRLX expression changes during the annual cycle suggesting its involvement in spermatogenesis, fRLX may be considered a new marker for the study of spermatogenesis in the Rana esculenta.

Effects of multiple injections of ethane 1,2-dimethane sulphonate (EDS) on the frog, Rana esculenta, testicular activity.

Ethane 1,2-dimethane sulphonate (EDS) is an alkylating agent, which has a selective cytotoxic effect on Leydig cells in some mammalian species. Similarly, in the frog, Rana esculenta, Leydig cells are destroyed after a single EDS injection and regenerate after 28 days. Regeneration of Leydig cells in frogs appears to be independent of the pituitary. The present experiments in R. esculenta were carried out: a) to investigate Leydig cell responsiveness to gonadotropin stimulation during 58 days after a single EDS injection; and b) to assess whether four consecutive EDS injections induce additional effects on the testicular cell population. Our results show that androgen stimulation after gonadotropin injections is restored after 44 days from a single EDS injection. Since the interstitial compartment appears to be normal at least 28 days after EDS treatment, it is likely that new Leydig cells lack gonadotropin receptors. With respect to multiple-EDS injections, Leydig cells completely disappear in several areas and the adjacent germinal compartment is disorganised. In some cases damaged germinal compartment is still surrounded by intact Leydig cells. Surprisingly, testicular and plasma androgens strongly increase in EDS-treated animals. Therefore, Sertoli cells may produce substances inhibiting androgen production in Leydig cells. J. Exp. Zool. 287:384-393, 2000.

Morphology of the Harderian gland of the Gecko, Tarentola mauritanica.

The Harderian gland of the gecko, Tarentola mauritanica, was studied at the histological, histochemical, and ultrastructural levels. It is a nonlobate compound acinar gland surrounded by a thin capsule of connective tissue. Numerous connective tissue-type mast cells, ultrastructurally similar to those described in other higher vertebrates, were identified in the interstitial tissue between the acini. Pyramidal or columnar-shaped secretory glandular cells were observed in the acini. In the glandular cells, two types of structures could be distinguished on the basis of their high or low electron density. Lipid droplets were found in the cytoplasm of the Harderian gland of both sexes. Histochemical tests showed that the Harderian gland of the gecko is a seromucous gland. The secretion is essentially merocrine, although an apocrine type of secretion is sometimes observed.