• Rana esculenta complex
• hybridogenesis
• gametogenesis
• expression pattern
• vasa

In metazoan species, sexual reproduction occur through the fusion of male and female gametes. Mature haploid gametes generate by the differentiation of the germ cells via meiotic cell division, the gametogenesis process. Both germline cells development than gametes formation from germ cells represent evolutionary controlled crucial steps in the maintenance of species identity. The study of germline development and gametogenesis have been greatly improved after the analysis of the genetic network involved in these processes in model animal organisms. Several genes are presently known to retain a conserved role in groups like amphibian anurans, insects and nematodes, which germline cells are specified by the inheritance of maternal determinants, than taxa such as mammals and amphibian urodeles, which germ cells arise after interactions between tissues during gastrulation.
Although limited by important evolutionary constrains, gametogenesis can undergo complex changes in some cases, for example after the crossbreeding of related but distinct species. Whenever pre-meiotic isolation mechanisms fail to avoid interspecific crosses, the consequent hybrids are usually non-fertile if not viable due to post-meiotic isolation processes. Among the few exceptions of this rule in vertebrates, the western Palaearctic group of water frogs, also know as ‘Rana esculenta complex’, offer a unique example. This complex is formed by several pure species and at least three different interspecific hybrid lineages characterized by the extremely rare reproductive mode named hybridogenesis. During the larval development of hybridogenetic forms, one parental genome is eliminated and the second chromosome set is duplicated only in the germline, before the beginning of the first meiosis division, and adult hybrids produce haploid gametes containing one unrecombined genome of the second parental species. The hybrids constitution is recovered by their backcrossing with the parental species whose genome has been eliminated.
Rana esculenta, the first green frog species classified by Linneus, represents instead the hybridogenetic hybrids of the two species Rana lessonae and Rana ridibunda, but it usually forms mixed populations only with R. lessonae in the most widespread and best characterized system belonging to the ‘Rana esculenta complex’, named L-E. Several investigations carried out on the L-E system demonstrated that in the vast majority of cases the R. lessonae genome is eliminated, whereas the R. ridibunda chromosomes are duplicated before the first meiosis in R. esculenta germ cells. However, a molecular approach to the study of the modified gametogenesis of these anuran hybrids have not been performed yet.
In this perspective, several genes known to be conservatively involved in the germline determination and maintenance, as well as in gametogenesis, have been isolated in R. lessonae, and their expression pattern has been determined in R. esculenta, R. lessonae, R. ridibunda, in the present work, in order to improve our knowledge of the gametogenesis in hybridogenetic forms.
Selected genes belong to the three different gene families of the DEAD box ATP-dependent RNA helicases, Pumilio and Y-box, which members encode translational regulators of messenger RNA.
Full-length cDNAs of the five genes RlVlg and RlPl10 (DEAD box ATP-dependent RNA helicases family), Rlpum1, Rlpum2 (Pumilio family) and Rlybox2 (Y-box family) were cloned from R. lessonae testis and ovary cDNA libraries, and both RT-PCR on adult tissues than in situ hybridization experiments on testis and ovary showed that these genes are expressed during early stages of both oogenesis and gametogenesis in adult gonads. RlVlg represents the homologue of Drosophila vasa gene, a specific marker of germ line in metazoans, and is active only in germ cells. RlPl10 is homologue to the DEAD box genes An3 in Xenopus and Pl10 in mouse and is expressed in both somatic than germline tissues, as well as the two pumilio genes Rlpum1 and Rlpum2. Rlybox2 instead constitutes the germline specifically-expressed member of the Y-box family in vertebrates and, as expected, its transcript has been detected only in testis and ovary of adult frogs.
During oogenesis of R. lessonae, R. ridibunda and R. esculenta, the five genes showed a decreasing expression in the early stages I-III (RlPl10, Rlpum1, Rlpum2 and Rlybox2), or I-IV (RlVlg), whereas no activity was observed in the late stages V-VI. On the basis of in situ hybridization experiments on testis sections, the genes RlVlg, RlPl10 and Rlpum1 showed a differential expression in spermatogenesis stages. RlVlg mRNA was detected in spermatogonia and primary spermatocytes, whereas RlPl10 is expressed in spermatogonia and all the meiotic stages. Immunohistochemical analysis of the expression of the specific G2 and M cell phases marker histone H3 phosphorylated at Serine ten (Ser-10-P-H3) allowed to determine that RlVlg expression in male germ cells occurred before and after the mitotic and first meiotic divisions, respectively. On the contrary, RlPl10 activity is extended to the whole cell cycle. Finally, Rlpum1 transcript was found in both interphase and dividing spermatogonia, and in primary spermatocytes before and after the first meiotic division.