Developmental asynchrony and antagonism of sex determination pathways in a lizard with temperature-induced sex reversal

Abstract

Vertebrate sex differentiation follows a conserved suite of developmental events: the bipotential gonads differentiate and shortly thereafter sex specific traits become dimorphic. However, this may not apply to squamates, a diverse vertebrate lineage comprising of many species with thermosensitive sexual development. Of the three species with data on the relative timing of gonad differentiation and genital dimorphism, the females of two (Niveoscincus ocellatus and Barisia imbricata) exhibit a phase of temporary pseudohermaphroditism or TPH (gonads have differentiated well before genital dimorphism). We report a third example of TPH in Pogona vitticeps, an agamid with temperature-induced male to female sex reversal. These findings suggest that for female squamates, genital and gonad development may not be closely synchronised, so that TPH may be common. We further observed a high frequency of ovotestes, a usually rare gonadal phenotype characterised by a mix of male and female structures, exclusively associated with temperature-induced sex reversal. We propose that ovotestes are evidence of a period of antagonism between male and female sex-determining pathways during sex reversal. Female sexual development in squamates is considerably more complex than has been appreciated, providing numerous avenues for future exploration of the genetic and hormonal cues that govern sexual development.

Figure 1

The timing and duration of temporary pseudohermaphroditism (TPH) in squamates. White bars indicate indeterminate sex, dark grey shows the TPH phase, and light grey indicates dimorphic sexes. The asterisk symbol for Anolis carolinensis indicates that there is a short delay (half a stage) between ovarian differentiation and genital dimorphism but is functionally lacking TPH. The dagger symbol on the dotted section of developmental stage axis indicates the approximate post-hatch timing of hemipenis regression in Barisia imbricata. The timing of events are approximations standardised to the staging system described for Pogona vitticeps. The reproductive mode (O/V) and sex determination mode (SDM) is reported for each species (O = oviparity, V = viviparity, G + T = genetic with thermal influence, GSD = genetic sex determination). The dashed vertical line at stage 18 denotes approximate time of hatching/birth. Phylogeny adapted from, branch length is for illustrative purposes only.

Figure 2

Histological sections of embryonic Pogona vitticeps urogenital systems stained with haematoxylin and eosin (H & E). (a) Bipotential gonads with developing cortex and medullary regions during migration towards the anterior mesonephros. (b) Ovotestes from an embryo incubated at 36 °C undergoing sex reversal showing a proliferating cortex with oogonia, a medulla with numerous rudimentary seminiferous tubules. (c) Differentiated ovary with a reducing medulla, cortex proliferating with oogonia. (d) Differentiated testes with a reducing cortex, and medulla with developing seminiferous tubules. B. P = bipotential gonad, Mes. = mesonephros, C = cortex, M = medulla, black arrows = seminiferous tubules.

Figure 3

Timing of gonad (a,b) and genital (c,d) development for Pogona vitticeps at normal (28 °C; a,c) and sex-reversing (36 °C; b,d) incubation temperatures. Sexual phenotype is indicated by colour as per the legend, and sexual genotype is indicated by shape (squares = ZZ specimens, triangles = ZW specimens, circles = unknown). The grey shading defines the period of temporary pseudohermaphroditism during female development, persisting for approximately 9 stages. The black asterisks denote approximate time of hatching (stage 18, ~73 dpo at 28 °C and ~47 dpo at 36 °C; Holleley et al.). Genital development data was re-analysed from Whiteley et al..

Figure 4

Homologous hemipenal structures in male and female Pogona vitticeps. Scanning electron micrographs of stage 14 (a–f) and stage 17 (g–i) embryonic genitalia. (a) Reduced hemipenes of a ZW female showing trilobes. (b) ZW female from the same clutch as specimen in with well-developed bilobed hemipenes each with a sulcus spermaticus. (c) Enhanced view of right hemipenis of specimen in (b) showing the beginnings of the hemipenal lattice. (d) Bilobed hemipenes of a ZZ male with a sulcus spermaticus. (e) Bilobed hemipenes with sulcus spermaticus of a ZZ embryo incubated at 36 °C that did not undergo sex reversal. (f) Bilobed hemipenes with sulcus spermaticus of a sex reversed female. (g) Well developed bilobed hemipenes of a ZZ male showing the sulcus spermaticus and hemipenal lattice. (h) Enhanced view of left hemipenis of specimen shown in (g). (i) Enhanced view of the hemipenal lattice from specimen shown in (g) and (h). C = cloaca, P = pedicel, blue arrows = sulcus spermaticus. The specimens presented in E and F were validated histologically to have testes and ovaries respectively.