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Sex hormones are essential for sexual differentiation and play a key role in the development of gonads in amphibians. The goal of this study was to evaluate the influence of exogenous sex steroids, testosterone, and 17β-estradiol (E₂) on development of gonads in five anuran species differing in their evolutionary positions, sex determination, and mode of gonadogenesis. We found that in two closely related species of fire-bellied toad, Bombina bombina and Bombina variegata, testosterone and E₂ exposure results in sex reversal as well as intersex and undifferentiated gonads. Similarly, sex reversal was observed in Hyla arborea after exposure to male or female sex steroids. Xenopus laevis was sensitive to E₂ but only moderately to testosterone. In Bufo viridis, treatment with either sex hormone provoked a developmental delay in gonads and Bidder's organs. Therefore, susceptibility to hormonal sex reversal appeared species dependent but unrelated to genetic sex determination and the type of gonadogenesis. We also found that the onset of sex steroid exposure influences gonad differentiation and the meiotic status of the germ cells depends on their location within the gonad. Our findings reveal differential sensitivity of amphibians to testosterone and E₂, establishing a hierarchy of sensitivity to these hormones among different anuran species.

The normal course of gonad development is critical for the sexual development and reproductive capacity of the individual. During development, an incipient bipotential gonad which consists of unorganized aggregate of cells, must differentiate into highly structured testis or ovary. Cell adhesion molecules (CAMs) are a group of proteins crucial for segregation and aggregation of different cell types to form different tissues. E-cadherin (Cdh1) is one of the CAMs expressed in the developing gonads. We used tissue-specific knockout of Cdh1 gene in OCT4⁺ germ cells and, separately, in SF1⁺ somatic cells of developing gonads. The knockout of E-cadherin in somatic cells caused decrease in the number of germ cells, while the knockout in the germ cells caused their almost complete loss. Thus, the presence of E-cadherin in both the germ and somatic cells is necessary for the survival of germ cells. Although the lack of E-cadherin did not impair cell proliferation, it enhanced apoptosis, which was a possible cause of germ cell loss. However, the somatic cells of the gonad differentiated normally into Sertoli cells in the testis cords, and into follicular cells in the ovaries. The testis and ovigerous cords maintained their integrity; they were covered by continuous basement membranes. The testicular interstitium with steroidogenic fetal Leydig cells did not show any noticeable changes. However, in the female gonads, because of the lack of germ cells, the ovarian follicles were absent. The sex determination and sexual differentiation of the gonad were not impaired. These results underscore an important role of E-cadherin in germ cell survival and gonad development.

Cell to cell interactions are crucial for morphogenesis and tissue formation. Desmoplakin (encoded by the Dsp gene) is a component of desmosomes and anchors the transmembrane adhesion proteins to the cytoskeleton. Its role in gonad development remains vague. To study the role of desmoplakin in gonad development, we used a tissue-specific knockout of the Dsp gene in the NR5A1⁺ somatic cells of the gonads. We show here that desmoplakin is necessary for the survival of germ cells in fetal testes and ovaries. The Dspknockout in NR5A1⁺ somatic cells in testes decreased the number of germ cells, and thus the size of the testes, but did not affect the Sertoli cells or the structure of testis cords and interstitium. The Dspknockout in NR5A1⁺ somatic cells in ovaries decreased the number of female germ cells and drastically reduced the formation of ovarian follicles. Dsp knockout in NR5A1⁺ somatic cells did not affect the sex determination and sexual differentiation of the gonads, as judged from an unchanged expression of genes essential for these processes. We conclude that mediation by desmoplakin cell adhesion between the gonadal cells is necessary for germ cell survival.