Role of steroidogenic factor 1 and aromatase in temperature-dependent sex determination in the red-eared slider turtle.

Red-eared slider turtles are genetically bipotential for sex determination. In this species, as in many other reptiles, incubation temperature of the egg determines gonadal sex. At higher incubation temperatures females are produced and increasing temperature appears to increase estrogen production in the embryonic brain. Treatment of eggs incubating at a male-producing temperature with exogenous estrogen causes ovaries to form. At a female-biased incubation temperature, prevention of estrogen biosynthesis or administration of nonaromatizable androgens results in the development of testes. In mammals, steroidogenic factor 1 (SF-1) regulates most genes required for estrogen biosynthesis, including aromatase. In both mammals and red-eared sliders, SF-1 is differentially expressed in males and females during gonadogenesis. We have examined both SF-1 gene expression and aromatase activity in embryos incubating at different temperatures and after manipulation to change the course of gonadal development. Our findings indicate a central role for SF-1 in enacting the effect of estrogen. Estrogen treatment directly or indirectly downregulates SF-1 and, ultimately, causes development of females. The inhibition of estrogen results in upregulation of SF-1 and male hatchlings. Thus, SF-1 may lie at the center of one molecular crossroad in male versus female differentiation of the red-eared slider.     

Temperature-dependent sex ratio in a bird

To our knowledge, there is, so far, no evidence that incubation temperature can affect sex ratios in birds, although this is common in reptiles. Here, we show that incubation temperature does affect sex ratios in megapodes, which are exceptional among birds because they use environmental heat sources for incubation. In the Australian brush-turkey Alectura lathami, a mound-building megapode, more males hatch at low incubation temperatures and more females hatch at high temperatures, whereas the proportion is 1:1 at the average temperature found in natural mounds. Chicks from lower temperatures weigh less, which probably affects offspring survival, but are not smaller. Megapodes possess heteromorphic sex chromosomes like other birds, which eliminates temperature-dependent sex determination, as described for reptiles, as the mechanism behind the skewed sex ratios at high and low temperatures. Instead, our data suggest a sex-biased temperature-sensitive embryo mortality because mortality was greater at the lower and higher temperatures, and minimal at the middle temperature where the sex ratio was 1:1.     

High temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by increasing cortisol levels during gonadal sex differentiation in Japanese flounder

The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. XX flounder can be induced to develop into phenotypic females or males, by rearing them at 18°C or 27°C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that cortisol, the major glucocorticoid produced by the interrenal cells in teleosts, causes female-to-male sex reversal by directly suppressing mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, an inhibitor of cortisol synthesis prevented masculinization of XX flounder at 27°C, suggesting that masculinization by high temperature is due to the suppression of cyp19a1 mRNA expression by elevated cortisol levels during gonadal sex differentiation in the flounder. In the present study, we found that exposure to high temperature during gonadal sex differentiation upregulates the mRNA expression of retinoid-degrading enzyme (cyp26b1) concomitantly with masculinization of XX gonads and delays meiotic initiation of germ cells. We also found that cortisol induces cyp26b1 mRNA expression and suppresses specific meiotic marker synaptonemal complex protein 3 (sycp3) mRNA expression in gonads during the sexual differentiation. In conclusion, these results suggest that exposure to high temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by elevating cortisol levels during gonadal sex differentiation in Japanese flounder.     

Temperature sex-reversal in amphibians and reptiles

The sexual differentiation of gonads has been shown to be temperature-sensitive in many species of amphibians and reptiles. In two close species of salamanders, Pleurodeles poireti and P. waltl, both displaying a ZZ/ZW mechanism of genotypic sex determination (GSD), the rearing of larvae at high temperatures (30 degrees-32 degrees C) produces opposite effects: ZZ genotypic males of Pleurodeles poireti become phenotypic females whereas ZW genotypic females of P. waltl become phenotypic males. Sex-reversal of these individuals has been irrefutably demonstrated through genetic, cytogenetic, enzymatic and immunological studies. In many turtles, both sexes differentiate only within a critical range of temperature: above this range, all the individuals become phenotypic females, whereas below it, 100% become phenotypic males. The inverse occurs in some crocodiles and lizards. In many species of these three orders of reptiles, females are obtained at low and high temperatures, and males at intermediate ones. Preliminary studies in turtles (Emys orbicularis) indicate that within the critical range of temperature, sexual phenotype conforms with GSD, but that above and below this range, GSD is overriden. Temperature shifts during larval development in salamanders and during embryonic development in reptiles allowed the determination of thermosensitive stages for gonadal differentiation. Estrogens synthesized in the gonads at these stages appear to be involved in their sexual differentiation, higher levels being produced at feminizing temperatures than at masculinizing ones. The phenomenon of temperature sensitivity of gonadal differentiation occurs in species showing a very early stage in the evolution of sex chromosomes. Its adaptive value, chiefly in reptiles, remains an open question.     

Expression of P450(arom) in Malaclemys terrapin and Chelydra serpentina: a tale of two sites.

The formation of estrogens from androgens in all vertebrates is catalyzed by the "aromatase" complex, which consists of a membrane bound P(450) enzyme, P(450) aromatase (which binds the androgen substrate and inserts an oxygen into the molecule), and a flavoprotein (NADPH-cytochrome P450 reductase). Among vertebrates, the two major sites of aromatase expression are the brain and gonads. Given the importance of estrogen in reptile sex determination, we set out to examine whether P450arom was involved in the initiation and/or stabilization of sex determination in turtles. We examined the expression of aromatase activity in the brain and gonads of two turtle species exhibiting temperature dependent sex determination (TSD), the diamondback terrapin (Malaclemys terrapin), and the common snapping turtle (Chelydra serpentina). Estradiol when applied at stage 14 of the terrapin induces expression of aromatase in the gonad of embryos incubated at male temperatures (26.5 degrees C). The level of expression is similar to that of a normal embryonic ovary. When applied at stage 22, estradiol does not induce aromatase expression in the terrapin. The xenoestrogen, nonylphenol, sex reverses terrapin embryos at 26.5 degrees C. Letrazole, a nonsteroidal aromatase inhibitor, suppresses aromatase activity in the brain at either incubation temperature. Ovotestes are produced by letrazole administration in the terrapin when incubated at 30.5 degrees C. In the snapping turtle at stage 23, gonadal and brain aromatase activity in embryos incubated at female temperatures (30.5 degrees C) is nearly half that exhibited in terrapin embryos at the same temperature. Moreover, letrazole administration suppresses aromatase expression to nearly basal levels. At male incubation temperatures (26.5 degrees ), brain aromatase expression is nearly three times higher than at female temperatures, while gonadal expression levels are nearly one third lower. However, the gonadal expression levels at male temperatures in the snapping turtle are nearly 25 times higher than that found in the terrapin. Estradiol administration elevates this level nearly three fold. These data suggest that is not merely the expression of aromatase that is important for ovarian development, but that the level of expression may be more important.     

Epigenetic control of cyp19a1a expression is critical for high temperature induced Nile tilapia masculinization

In fish species with temperature-dependent sex determination (TSD) or genotypic sex determination plus temperature effects (GSD + TE), temperature can either affect sex differentiation or determine the sex. However, it is unknown if epigenetic control of cyp19a1a expression is critical for high temperature induced masculinization in the freshwater fish Nile tilapia. We analyzed the cyp19a1a DNA methylation levels in three age groups and found that they were lower in females than in males. At 8 months of age, males had DNA methylation levels of the cyp19a1a promoter that were almost twice as high as those of females. Exposure to high temperatures increased the cyp19a1a promoter DNA methylation levels from 30.87 ± 4.56% to 48.34 ± 0.92% (P = 0.035) in females and from 50.33 ± 7.38% to 51.66 ± 4.75% in males (P = 0.867). The increases in the cyp19a1a promoter DNA methylation levels were associated with the mRNA expression levels and might play a role in promoting gonadal differentiation in high temperature induced group females toward the male pathway. Western blot analysis revealed that the cyp19a1a protein expression levels in females significantly declined after high temperature treatment; only a slight decline was recorded in male fish. These results reveal that epigenetic control of cyp19a1a mRNA and protein expression is related to the environmental temperature and sex ratios in fish with TSD or GSD + TE.     

Environmental regulation of sex determination in reptiles

The various patterns of environmental sex determination in squamates, chelonians and crocodilians are described. High temperatures produce males in lizards and crocodiles but females in chelonians. Original experiments on the effects of incubation at 30 degrees C (100% females) or 33 degrees C (100% males) on development in Alligator mississippiensis are described. These include an investigation of the effect of exposing embryos briefly to a different incubation temperature on the sex ratio at hatching, and a study of the effects of 30 degrees C and 33 degrees C on growth and development of alligator embryos and gonads. A 7-day pulse of one temperature on the background of another was insufficient to alter the sex ratio dramatically. Incubation at 33 degrees C increased the rate of growth and development of alligator embryos. In particular, differentiation of the gonad at 33 degrees C was enhanced compared with 30 degrees C. A hypothesis is developed to explain the mechanism of temperature-dependent sex determination (TSD) in crocodilians. The processes of primary sex differentiation are considered to involve exposure to a dose of some male-determining factor during a specific quantum of developmental time during early incubation. The gene that encodes for the male-determining factor is considered to have an optimum temperature (33 degrees C). Any change in the temperature affects the expression of this gene and affects the dose or quantum embryos are exposed to. In these cases there is production of females by default. The phylogenetic implications of TSD for crocodilians, and reptiles in particular, are related to the life history of the animal from conception to sexual maturity. Those animals that develop under optimal conditions grow fastest and largest and become male. A general association between the size of an animal and its sex is proposed for several types of vertebrate.     

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.     

Temperature-dependent sex-biased embryo mortality in a bird

Sex ratios have important evolutionary consequences and are often biased by environmental factors. The effect of developmental temperature on offspring sex ratios has been widely documented across a diverse range of taxa but has rarely been investigated in birds and mammals. However, recent field observations and artificial incubation experiments have demonstrated that the hatching sex ratio of a megapode, the Australian brush-turkey (Alectura lathami), varied with incubation temperature; more females hatched at high incubation temperatures and more males hatched at low temperatures. Here, we investigated the causes of this temperature-dependent sex-biasing system. Molecular sexing of chicks and embryos confirmed that male embryo mortality was greater at high temperatures while female embryo mortality is greater at low temperatures, with mortality in both sexes similar at intermediate incubation temperatures. Temperature-dependent sex-biased embryo mortality represents a novel mechanism of altering sex ratios in birds. This novel mechanism, coupled with the unique breeding biology of the brush-turkey, offers a potentially unparalleled opportunity in which to investigate sex allocation theory in birds.     

Incubation temperatures, sex ratios and sex determination in a population of Nile crocodiles (Crocodylus niloticus)

A demographic study of the Nile crocodile Crocodylus niloticus at Lake Ngezi, Zimbabwe, revealed that females predominated in all size classes and among embryos. The sex of C. niloticus was shown to be determined by the temperature of egg incubation in constant temperature laboratory experiments. At 31 °C and below only females were produced. The threshold temperature for maleness was between 31 ° and 34 °C, but appeared to vary between clutches. The duration of the incubation period varied with temperature and was 110 days at 28 °C, falling to 85 days at 34 °C. Incubation temperature affected hatchling length, but not mass. Hatchlings from incubation at 34 °C were shorter on average than those from incubation at 28 °C and 31 °C, but by three months had outgrown them. There was no sex-related difference in length in a random sample of 200 two-year-old C. niloticus on a crocodile farm. Mean temperatures in wild nests were consistently lower than 31 °C and therefore the male threshold as determined in the laboratory. Embryonic development was slow and hatching success poor. The shallowest eggs in a nest had higher mean temperatures and more advanced embryos than the deepest eggs. They also experienced daily temperature fluctuations of up to 10 °C during which the maximum occasionally rose to 35 °C. Constant temperature incubation was not a good model of field conditions, but the correlation between nest temperatures and embryonic sex is consistent with temperature-dependent sex determination in the wild.