Temporal and spatial SOX9 expression patterns in the course of gonad development of the caudate amphibian Pleurodeles waltl.

The SOX family of transcription factors is thought to regulate gene expression in a wide variety of developmental processes. Namely, SOX9 expression is conserved in vertebrate sex determination or differentiation. Nevertheless, information about caudate amphibians is lacking. In this study, we provide data on Pleurodeles waltl, a species that displays a ZZ/ZW genetic mode of sex determination and a temperature-dependent mechanism of female-to-male sex reversal. Phylogenetic analysis of SOX9 P. waltl ortholog reveals that the deduced protein segregates from the group of anuran and could be more closely related to amniote than to anamniote. However, SOX9 lacks the PQA-rich domain present in amniotes. In larvae, SOX9 is expressed in both sexes in gonad-mesonephros complexes as soon as stage 42, before gonad differentiation. At stage 54(60d) at which testis differentiation is already in progress, analyses of isolated gonads reveal a male-enriched expression of SOX9, which was quantified by real-time PCR. At the end of metamorphosis (stage 56), SOX9 shows a nuclear localization only in the testis. In adults, SOX9 is still expressed in testes and ovaries. In the ovary, SOX9 is found in oocytes from stage I to stage VI but it is never detected in the nucleus. Our results suggest that in P. waltl, like in non mammalian vertebrates, SOX9 could play a role during the late phase of gonad differentiation rather than in sex determination. Its role in germ cells of the adult ovary has still to be elucidated.

Müllerian inhibiting substance in the caudate amphibian Pleurodeles waltl.

Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-ß family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.