In mice and humans, the androgen receptor (AR) gene, located on the X chromosome, is not known to be involved in sex determination. In the Japanese frog Rana rugosa the AR is located on the sex chromosomes (X, Y, Z and W). Phylogenetic analysis shows that the AR on the X chromosome (X-AR) of the Korean R. rugosa is basal and segregates into two clusters: one containing W-AR of Japanese R. rugosa, the other containing Y-AR. AR expression is twice as high in ZZ (male) compared to ZW (female) embryos in which the W-AR is barely expressed. Higher AR-expression may be associated with male sex determination in this species. To examine whether the Z-AR is involved in sex determination in R. rugosa, we produced transgenic (Tg) frogs carrying an exogenous Z-AR. Analysis of ZW Tg frogs revealed development of masculinized gonads or ‘ovotestes’. Expression of CYP17 and Dmrt1, genes known to be activated during normal male gonadal development, were up-regulated in the ZW ovotestis. Testosterone, supplied to the rearing water, completed the female-to-male sex-reversal in the AR-Tg ZW frogs. Here we report that Z-AR is involved in male sex-determination in an amphibian species.
The first cell fate commitment during mammalian development is the specification of the inner cell mass and trophectoderm. This irreversible cell fate commitment should be epigenetically regulated, but the precise mechanism is largely unknown in humans. Here, we show that naïve human embryonic stem (hES) cells can transdifferentiate into trophoblast stem (hTS) cells, but primed hES cells cannot. Our transcriptome and methylome analyses reveal that a primate-specific miRNA cluster on chromosome 19 (C19MC) is active in naïve hES cells but epigenetically silenced in primed ones. Moreover, genome and epigenome editing using CRISPR/Cas systems demonstrate that C19MC is essential for hTS cell maintenance and C19MC-reactivated primed hES cells can give rise to hTS cells. Thus, we reveal that C19MC activation confers differentiation potential into trophoblast lineages on hES cells. Our findings are fundamental to understanding the epigenetic regulation of human early development and pluripotency.
Androgens play a critical role in testicular differentiation in many species of vertebrates. While female-to-male sex reversal can be induced by testosterone (T) in some species of amphibians, the mechanism still remains largely unknown even at the histological level. In this study, we determined a threshold dosage of T to induce female-to-male sex reversal in the Japanese frog Rana (R.) rugosa. Tadpoles were allowed to metamorphose into frogs with T present in the rearing water. At 0.2 ng/mL T, female frogs formed tissue comprising a mixture of ovary and testis, the so-called ovotestis, the size of which was significantly smaller than the wild-type ovary. Histological changes occurring in the oocytes of T-treated ovaries induced oocyte degeneration in the masculinizing ovaries leading to their final disappearance. In parallel, many germ cells emerged in the cortex of the ovotestis and, later, in the medulla as well. RT-PCR analysis revealed upregulated expression of CYP17 and Dmrt1 but not 17βHSD in the ovotestis, and downregulation of Pat1a expression. Furthermore, immunohistology revealed CYP17-positive signals in the cortex of the masculinizing ovary, spreading throughout the whole area as the testis developed. These results indicate that oocytes are sensitive to T in the ovary of R. rugosa and that male-type germ cells expand in the masculinizing gonad (testis) contemporaneous with oocyte disappearance.
Molluscan gastropods have long been used for studying the cellular and molecular mechanisms underlying learning and memory. One such gastropod, the pond snail Lymnaea stagnalis , exhibits long-term memory (LTM) following both classical and operant conditioning. Using Lymnaea , we have successfully elucidated cellular mechanisms of learning and memory utilizing an aversive classical conditioning procedure, conditioned taste aversion (CTA). Here, we present the behavioral changes following CTA training and show that the memory score depends on the duration of food deprivation. Then, we describe the relationship between the memory scores and the monoamine contents of the central nervous system (CNS). A comparison of learning capability in two different strains of Lymnaea , as well as the filial 1 (F 1 ) cross from the two strains, presents how the memory scores are correlated in these populations with monoamine contents. Overall, when the memory scores are better, the monoamine contents of the CNS are lower. We also found that as the insulin content of the CNS decreases so does the monoamine contents which are correlated with higher memory scores. The present review deepens the relationship between monoamine and insulin contents with the memory score.
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.
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