Differential expression of SOX9 in gonads of the sea turtle Lepidochelys olivacea at male- or female-promoting temperatures.

In mouse and chick embryos, the SOX9 gene is down-regulated in genetic females whereas in genetic males it remains in the Sertoli cells. We studied the distribution of SOX9 protein in developing genital ridges of embryos of the sea turtle Lepidochelys olivacea incubated at male- or female-promoting temperatures, using the antibody for detection. At stages 22-24, cells in medullary cords show SOX9 positive nuclei, while coelomic epithelial cells appear negative. At stage 25 however, most medullary cells are SOX9 negative and at the female-promoting temperature, and from stage 26 onwards, SOX9 protein is not detected. At the male-promoting temperature, medullary cords remain SOX9-positive at all stages. These results suggest that SOX9 is up-regulated in Sertoli cells irrespective of primary sex-determining switch. Sex is irreversibly determined at stage 24 or 26 at the male- or female-promoting temperature, respectively (Merchant-Larios et al.,'97). The present results suggest that there is a correlation between SOX9 expression and sex determination in the olive ridley. At the male-promoting temperature, Sertoli cells expressing SOX9 become committed at stage 24 and male sex is determined, whereas at the female-promoting temperature, SOX9 is down-regulated at stage 26 and female sex is determined. J. Exp. Zool. 284:705-710, 1999.     

Turning on the male--SRY, SOX9 and sex determination in mammals

The decision of the bi-potential gonad to develop into either a testis or ovary is determined by the presence or absence of the Sex-determining Region gene on the Y chromosome (SRY). Since its discovery, almost 13 years ago, the molecular role that SRY plays in initiating the male sexual development cascade has proven difficult to ascertain. While biochemical studies of clinical mutants and mouse genetic models have helped in our understanding of SRY function, no direct downstream targets of SRY have yet been identified. There are, however, a number of other genes of equal importance in determining sexual phenotype, expressed before and after expression of SRY. Of these, one has proven of central importance to mammals and vertebrates, SOX9. This review describes our current knowledge of SRY and SOX9 structure and function in the light of recent key developments.     

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination

Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000.     

Formation of the genital ridges is preceded by a domain of ectopic Sox9-expressing cells in Lepidochelys olivacea

Bipotential gonads represent the structural framework from which alternative molecular sex determination networks have evolved. Maintenance of Sox9 expression in Sertoli cells is required for the structural and functional integrity of male gonads in mammals and probably in most amniote vertebrates. However, spatial and temporal patterns of Sox9 expression have diversified along evolution. Species with temperature sex determination are an interesting predictive model since one of two alternative developmental outcomes, either ovary or testis occurs under controlled laboratory conditions. In the sea turtle Lepidochelys olivacea, Sox9 is expressed in the medullary cords of bipotential gonads when incubated at both female- or male-promoting temperature (FT or MT). Sox9 is then turned off in presumptive ovaries, while it remains turned on in testes. In the current study, Sox9 was used as a marker of the medullary cell lineage to investigate if the medullary cords originate from mesothelial cells at the genital ridges where Sox9 is upregulated, or, if they derive from a cell population specified at an earlier developmental stage, which maintains Sox9 expression. Using immunofluorescence and in situ hybridization, embryos were analyzed prior to, during and after gonadal sex determination. A T-shaped domain (T-Dom) formed by cytokeratin (CK), N-cadherin (Ncad) and SOX9-expressing cells was found at the upper part of the hindgut dorsal mesentery. The arms of the T-Dom were extended to both sides towards the ventromedial mesonephric ridge before the thickening of the genital ridges, indicating that they contained gonadal epithelial cell precursors. Thereafter, expression of Sox9 was maintained in medullary cords while it was downregulated at the surface epithelium of bipotential gonads in both FT and MT. This result contrasts with observations in mammals and birds, in which Sox9 upregulation starts at a later stage in the inner cells underlying the Sox9-negative surface epithelium, suggesting that the establishment of a self-regulatory Sox9 loop required for Sertoli cell determination has evolved. The T-shaped domain at the upper part of the hindgut dorsal mesentery found in the current study may represent the earliest precursor of the genital ridges, previously unnoticed in amniote vertebrates.     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.     

Lymphoid aggregates in gonads of embryos, hatchlings, and young of turtles with temperature-dependent sex determination

Cellular infiltrations forming lymphoid-like aggregates were previously observed in gonads of two turtle species exhibiting temperature-dependent sex determination (TSD): at hatching in Chelydra serpentina; at and after hatching in Emys orbicularis. We show here that such aggregates are also present in gonads of Testudo graeca by the end of embryonic development, suggesting that their occurrence is general in turtles. Since in C. serpentina, infiltrations were observed mainly in testes exhibiting remnants of the germinal epithelium, it was assumed that their occurrence was an expression of maleness leading to rejection of this epithelium. The generality of this hypothesis was tested in E. orbicularis by looking for lymphoid-like aggregates in three types of gonads (testes, ovotestes, and ovaries) and for the stages at which they occur. Gonads were from embryos, hatchlings, and young incubated at various temperatures. Ovotestes obtained by treatment with an aromatase inhibitor of eggs incubated at female-producing temperature were also examined. In these gonads, the differentiation of Sertoli cells in testicular cords/tubes was ascertained by expression of SOX9. Moreover, the cell composition of aggregates was determined on electron micrographs. Aggregates appear in ovaries and ovotestes by the end of embryonic development and are present in the majority of these gonads at hatching, and at least up to one year after hatching. They are composed mainly of lymphocytes and fibroblasts. Aggregates are not present in typical testes. Since they occur in most ovaries, they cannot be seen as an expression of maleness. Rather, lymphocytic infiltration and formation of lymphoid aggregates in turtle gonads can be seen as components of the immune system, and can be under the control of gonadal endogenous sex steroids.     

Sex-specific DNA in reptiles with temperature sex determination

Banded krait minor ("Bkm") satellite DNA, originating in the W-chromosome of the snake Bungarus fasciatus, has been found in the genome of diverse eukaryotic species including fruit fly, quail, and horse. Concentrations of Bkm have been found in the presumptive W-chromosome of snakes with isomorphic sex chromosomes and in the male-determining region of the Y-chromosome in mouse and man. We therefore asked whether Bkm-related DNA might be present in quantitative excess in DNA from males or females in two related species of sea turtle, Chelonia mydas, in which sex is determined by the temperature of the incubating egg, and Lepidochelys kempi, in which the critical sex-determining temperature has recently been described. Filter hybridization with the Bkm 2(8) probe revealed male-specific fragments in both species; female-specific fragments were also revealed in C. mydas. Sex-specific DNA sequences in temperature-sex-determined species such as Kemp's ridley and the green turtle were unexpected, but could be explained if there were an underlying genetic mode of sex determination in these animals, or alternatively, if temperature-influenced sex determination involved structural modifications in DNA adjacent to, or directly concerned with, the sex-determining genes. If these results are confirmed across a broader sample of sea turtles, the techniques described in this paper might be used routinely to identify gener in the young of these endangered animals, in which male and female are grossly indistinguishable.     

First record of the Olive Ridley Turtle,Lepidochelys olivacea,in Iranian coastal waters (Testudines,Cheloniidae)

Abstract

Three specimens of the Olive Ridley Turtle (Lepidochelys olivacea) were collected in Guader (Gwatar) Bay in the Sea of Oman on 6 January, 1997. Two of them have been preserved for the Zoological Museum of Gorgan University. This is the first report of the Olive Ridley Turtle in Iranian coastal waters.     

Nest-site selection in two eublepharid gecko species with temperature-dependent sex determination and one with genotypic sex determination

At present, most turtles, all crocodilians, and several lizards are known to have temperature-dependent sex determination (TSD). Due to the dependence of sex determination on incubation temperature, the long-term survival of TSD species may be jeopardized by global climate changes. The current study was designed to assess the degree to which this concern is justified by examining nest-site selection in two species of Pattern II TSD geckos (Eublepharis macularius and Hemitheconyx caudicinctus) and comparing these preferences with those of a species with genotypic sex determination (GSD) (Coleonyx mitratus). Temperature preferences for nest sites were found to be both species-specific and female-specific. While H. caudicinctus females selected a mean nest-site temperature (32.4°) very close to the upper pivotal temperature (32°C) for the species, E. macularius females selected a mean nest-site temperature (28.7°C) well below this species' lower pivotal temperature (30.5°C). Thus, the resultant sex ratios are expected to differ between these two TSD species. Additionally, nest-site temperatures for the GSD species were significantly more variable (SE=+0.37) than were temperatures for either of the TSD species (E. macularius SE=±0.10; H. caudicinctus SE =+ 0.17), diereby further demonstrating temperature preferences within the TSD species.     

Relic thermosensitive gene expression in a turtle with genotypic sex determination

The evolution of sex determination remains one of the most fascinating enigmas in biology. Transitions between genotypic sex determination (GSD) and temperature‐dependent sex determination (TSD) have occurred multiple times during vertebrate evolution, however, the molecular basis and consequences of these transitions in closely related taxa remain unresolved. Here I address a critical question: Do species with GSD derived from ancestors possessing TSD retain any ancestral thermal sensitivity in the developmental pathways underlying gonadal differentiation? Results from an expression study of a gene involved in early gonadogenesis in GSD (Apalone mutica) and TSD (Chrysemys picta) turtles, support the hypothesis that Wt1 in A. mutica displays such a relic thermal sensitivity. This retention is likely enabled by Sf1, a gene immediately downstream from Wt1 whose expression is independent of temperature in this species. My results constitute the first empirical evidence of a GSD vertebrate exhibiting thermal sensitivity in the expression of a gene regulating gonadogenesis. This novel finding reveals an undocumented source of raw material for future evolutionary change that may exist in other GSD taxa, and one that enhances the evolutionary potential of the gene networks underlying sexual differentiation and contributes to the astonishing ability of sex‐determining mechanisms.