Nanos3 of the frog Rana rugosa: Molecular cloning and characterization

Abstract

Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3-deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT-PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3-knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild-type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.

Participation of androgen and its receptor in sex determination of an amphibian species

“…Previously we have shown that the W - AR is hardly expressed in R . rugosa embryos compared with Z-AR [10]. We then hypothesized that Z - AR came to play a role, perhaps a critical one given its sex hormone-signaling function, in male sex determination in ZZ/ZW R .…”

“…Tg and pTg embryos and tadpoles were reared as previously described [3]. To confirm Z-AR integration into genomic DNA, we extracted DNA from the tail tip of all Tg tadpoles at St. V, using the AllPrep DNA/RNA Micro Kit (QIAGEN, Tokyo, Japan) [10]. The PCR primers used were: forward, and reverse, .…”

“…rugosa frog the AR gene is located on the sex (X, Y, W and Z) chromosomes, reportedly on the inverted region of the Y and W chromosomes [8]. Structural rearrangements such as inversions, translocations and deletions are known to cause degradation of native genes by accumulation of deleterious mutations [9, 10]. Thus, it is likely that the AR gene is undergoing a process of evolutional degradation due to the lack of recombination between the inverted and non-inverted regions of the sex chromosomes (X vs. Y, and Z vs. W).…”

“…It is known that the AR gene on the W chromosome ( W - AR ) is hardly expressed in R . rugosa embryos, perhaps due to variations in promoter element and cognate transcription factor interaction between the W- and Z-AR genes [10]. However, when W - AR is transgenically expressed in Xenopus A6 cells, W-AR proteins can trans-activate androgen-dependent transcription in reporter assays [10], indicating that W-AR degradation is incomplete in R .…”

“…Interestingly, up-regulation of the AR expression is observed in the male gonad of R . rugosa tadpoles prior to sex determination [10]. Moreover, male gonads of R .…”