Temporal difference between testis and ovary determinations with possible involvement of testosterone and aromatase in gonadal differentiation in TSD lacking lizard, Calotes versicolor

In the garden lizard, Calotes versicolor, which lacks identifiable sex chromosomes, incubation temperature also does not have a deterministic effect on the gender. However, the embryos reared at high temperature (33-35 degrees C) have a shorter duration of incubation as well as gonadal differentiation. In contrast, exogenous application of the male hormone testosterone to embryos at ambient temperature (28 degrees C) results in almost all individuals with only testis. Thus the testosterone treatment reverts genic females to males and accelerates the differentiation of testis, a feature similar to the high-temperature treatment. Treatment of eggs with estradiol shows no difference from that seen in the untreated eggs. The present series of experiments was done to establish the "window" of testosterone sensitivity and to understand the interaction between sex hormones and high temperature on gonadal differentiation. The period between day 5 and 15 of embryonic development was the window period of testosterone sensitivity for sex reversal. This period coincided with the formation of the genital ridge and its differentiation into cortex and medulla. Treatment of the 33 degrees C-reared embryos with testosterone resulted in hatchlings of both the sexes, in contrast to only males at the ambient temperature. In contrast, at the same temperature (33 degrees C), all the dihydrotestosterone (nonaromatisable testosterone)-treated embryos hatched into males. However, those given estradiol showed no sex bias regardless of the day of application and the concentration of drug. Eggs were also treated with aromatase inhibitor, CGS 16949 A, at ambient temperature and at 33 degrees C. All the 33 degrees C eggs to which the drug was given on day 25 hatched into males. These results suggest that though high temperature has no direct effect on sex determination in this species, it may have a stimulatory effect on aromatase activity, leading to the conversion of the exogenously applied testosterone into estradiol and permitting ovarian differentiation in the genic females. It also follows from the present report that the pathway of testis formation in Calotes versicolor is triggered much earlier, and irreversibly, than that for the ovary.     

Cell aggregation precedes the onset of Sox9-expressing preSertoli cells in the genital ridge of mouse

SOX9 is expressed at the onset of the genital ridge formation in both sexes. It is assumed that SRY, the testis determining gene, turns SOX9 on in male embryos because it is turned off in female embryos. Spatial expression of SRY follows a cranio-caudal pattern. Here, we asked if SOX9 is expressed in the same cell lineage and with a similar pattern as SRY. A correlative study between the structural changes in the genital ridge and the immunocytochemical localization of SOX9-positive cells was undertaken. We used a transgenic strain expressing the green fluorescent protein (GFP) that considerably enhanced the cell context where the first SOX9-positive cells appear. Although SOX9-positive cells are located among loose mesenchymal cells by stages of 8-14 tail somites (ts) in both sexes, they are absent in the thickening coelomic epithelium of females. At 15 ts the first SOX9-positive cells appear within the core of the condensed cells only in male genital ridges. At 17 ts, a gradient of SOX9-positive cells in males is apparent, closely following the cranio-caudal pattern of cell aggregation seen in genital ridges of both sexes. Hence, our results suggest that SOX9 is expressed only in loose mesenchymal cells in both sexes and that expression of SOX9 in males requires the prior aggregation of cells in the genital ridges. The correspondence of SOX9 and SRY pattern of expression supports that both genes are expressed in the preSertoli cell lineage in the core of the genital ridges.     

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.     

Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development.

Sex-determining mechanisms are highly variable between phyla. Only one example has been found in which structurally and functionally related genes control sex determination in different phyla: the sexual regulators mab-3 of Caenorhabditis elegans and doublesex of Drosophila both encode proteins containing the DM domain, a novel DNA-binding motif. These two genes control similar aspects of sexual development, and the male isoform of DSX can substitute for MAB-3 in vivo, suggesting that the two proteins are functionally related. DM domain proteins may also play a role in sexual development of vertebrates. A human gene encoding a DM domain protein, DMRT1, is expressed only in the testis in adults and maps to distal 9p24.3, a short interval that is required for testis development. Earlier in development we find that murine Dmrt1 mRNA is expressed exclusively in the genital ridge of early XX and XY embryos. Thus Dmrt1 and Sry are the only regulatory genes known to be expressed exclusively in the mammalian genital ridge prior to sexual differentiation. Expression becomes XY-specific and restricted to the seminiferous tubules of the testis as gonadogenesis proceeds, and both Sertoli cells and germ cells express Dmrt1. Dmrt1 may also play a role in avian sexual development. In birds the heterogametic sex is female (ZW), and the homogametic sex is male (ZZ). Dmrt1 is Z-linked in the chicken. We find that chicken Dmrt1 is expressed in the genital ridge and Wolffian duct prior to sexual differentiation and is expressed at higher levels in ZZ than in ZW embryos. Based on sequence, map position, and expression patterns, we suggest that Dmrt1 is likely to play a role in vertebrate sexual development and therefore that DM domain genes may play a role in sexual development in a wide range of phyla.     

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.     

Analysis of sex differences in EGC imprinting

Sex-specific differences are apparent in the methylation patterns of H19 and Igf2 imprinted genes in embryonic germ cells (EGCs) derived from 11.5 or 12.5 days post coitum (dpc) primordial germ cells (PGCs). Here we studied whether these differences are associated either with the sex chromosome constitution of the EGCs or with the sex of the genital ridge (testis versus ovary) from which the PGCs were isolated. For this purpose we derived pluripotent EGC lines from sex-reversed embryos, either XY embryos deleted for Sry (XY(Tdym1)) or XX embryos carrying an Sry transgene. Southern blotting of the EGC DNA was used to analyze the differentially methylated regions of Igf2 and H19. The analysis revealed that both genes were more methylated in EGCs with an XY sex chromosome constitution than in those with an XX sex chromosome constitution, irrespective of the phenotypic sex of the genital ridge from which the EGCs had been derived. We conclude that the sex-specific methylation is intrinsic and cell-autonomous, and is not due to any influence of the genital ridge somatic cells upon the PGCs.     

Temperature regulates SOX9 expression in cultured gonads of Lepidochelys olivacea, a species with temperature sex determination

Although sex determination starts in the gonads, this may not be the case for species with temperature sex determination (TSD). Since temperature affects the whole embryo, extragonadal thermosensitive cells may produce factors that induce gonadal sex determination as a secondary event. To establish if gonads of a species with TSD respond directly to temperature, pairs of gonads were cultured, one at female-promoting temperature (FPT) and the contralateral at male-promoting temperature (MPT). Histological and immunohistochemical detection of SOX9 revealed that the response to temperature of isolated gonads was similar to that of the gonads of whole embryos. While gonads cultured at MPT maintained SOX9 expression, it was downregulated in gonads at FPT. Downregulation of SOX9 took longer in gonads cultured at stage 23 than in gonads cultured at stage 24, suggesting that a developmental clock was already established at the onset of culture. To find out if sex commitment occurs in vitro, gonads were switched from FPT to MPT at different days. Results showed that the ovarian pathway was established after 4 days of culture. The present demonstration that gonads have an autonomous temperature detector that regulates SOX9 expression provides a useful starting point from which the molecular pathways underlying TSD can be elucidated.