Chris Calamita

Temperature-dependent Sex Determination in Turtles

While sex determination in most species is genetically determined, in many species of egg-laying amniotes sex is determined by incubation temperature. Typically, lower incubation temperatures result in male hatchlings and higher incubation temperatures result in female-hatchlings. This phenomenon has been studied extensively in alligators and several species of turtles and is important for conservation purposes as well as for a thorough understanding of the process of sex determination itself. During spring/summer 2002, Chris Calamita will be working jointly with Dr. Russell Burke and I to complete a project designed to determine the temperature-sensitive period in Malaclemys terrapin, a temperature-sensitive species found locally. As a developmental biologist I now what to understand the mechanism that underlies this process of temperature-dependent sex determination.

In species exhibiting genetic sex determination (GSD), a cascade of differential gene expression early in development determines the gonadal sex of individuals. The switch that begins this cascade is the expression of a gene located on either the X or the Y chromosome. In GSD species, the sex hormones are not present in the gonads until sexual differentiation is well advanced. In species exhibiting temperature-dependent sex determination (TSD), on the other hand, the sex hormones, estrogen and dihydroxytestosterone (DHT), are present in the yolk and embryos throughout development starting from very early stages. Both sex determination and the normal differentiation of ovaries and testes are dependent on the presence of these hormones. Moreover, in species with TSD, the gonadal sex of individuals can be reversed after differentiation has begun by a change in incubation temperature or by the administration of hormones or hormone inhibitors. (Bergeron et al., 1999, Wibbels et al., 1998).

Because the presence of the sex hormones over an extended period of time is key to the normal differentiation of the gonads in TSD species, it is commonly assumed that the temperature-sensitive switch involves the regulation of either the enzymes that synthesize the different sex hormones from a common cholesterol precursor or the receptors for the respective hormones (Spotilla et al., 1994). The key enzymes involved in the production of DHT and estrogen from a common precursor are 5 a-reductase and aromatase respectively. Aromatase is pivotal in the process since it can convert most forms of the male sex hormones to estrogen. Estrogen also triggers a positive feedback mechanism whereby the presence of small amounts of estrogen up-regulates all of the genes that are important for the production of more estrogen and estrogen receptors. The same positive feedback mechanism does not seem to operate for testosterone, at least early in development (Rhen, 1999).

Most of the recent work on TSD has been based on the hypothesis that the underlying molecular mechanism is the differential activation of genes that are upstream in a regulatory cascade to the enzymes that directly metabolize testosterone to either DHT (the active male hormone) or estrogen. Several pieces of empirical evidence argue against this hypothesis. First of all, both 5 a-reductase and aromatase are expressed well before the beginning of the temperature-sensitive period of sex determination. Moreover, many of the genes that regulate the expression of the hormone-specific enzymes are themselves expressed in equal amounts at male and female-producing temperatures during the temperature-sensitive period (Jeyasuri et al, 1998). Finally, as mentioned above, it is known that even after the gonads have begun to differentiate, it is possible to reverse the sex of a hatchling by temperature change or steroid administration. Therefore, the period of sex determination must overlap the period of differentiation. The sex of the mature animal is determined and the switch is inactivated only after the gonads have begun to produce differentiated gametes. These three experimental results suggest that the temperature-dependent switch acts directly on the hormones or on the enzymes responsible for their synthesis and not on some up-stream regulatory molecule.

Temperature can directly affect the function of enzymes by changing their physical structure thereby changing their catalytic efficiency or binding capacity. It has been known for some time that mammals express two isoforms of 5 a-reductase (the enzyme that converts a common precursor to DHT instead of estrogen) and that one of the isoforms has a very sharp pH dependence (Wilson, 1992). It is also known that within the cytosol of poikilotherms (animals whose internal temperature is regulated by ambient temperature), pH is affected by alterations in temperature with a decrease in pH of about 0.017 pH units for every degree increase in temperature (Hochachka and Somero, 1984)). Thus even a small shift in pH could alter 5 a- reductase activity significantly.  Remarkably, this pH dependence of 5 a-reductase has not been studied in a TSD species. Higher temperatures can presumably decrease pH inside the egg by increasing metabolic rate and thus the production of CO2. Increased levels of CO2 raise carbonic acid levels; an increase in acid levels would decrease pH.

If changes in pH can affect 5 a-reductase activity in reptiles as it does in mammals, a simple mechanism for TSD can be proposed. It is conceivable that higher incubation temperatures result in higher CO2 levels in yolk and consequently in lower pH and lower 5 a-reductase activity. Lower 5 a-reductase activity then results in more female hatchlings. Likewise, lower incubation temperatures would result in lower yolk CO2 higher pH and high 5 a-reductase activity and consequently more male hatchlings. This simple mechanism is feasible since it is known that egg yolk contains not only cholesterol, the precursor for steroid hormones, but also stored transcript for enzymes and active steroid-producing enzymes (Bowden et al., 2001). In addition, both aromatase and 5 a-reductase are known to be present at low levels in egg yolk at male and female-producing temperatures and in embryos during the temperature-sensitive period. Chris will test this simple hypothesis by directly measuring the CO2 content and the pH of yolk at male and female-producing temperatures and by analyzing the pH dependence of both aromatase and 5 a-reductase in turtle eggs.

 

Literature Cited

Bergeron, JM. Willinghm, E, Osborn, CT, Rhen, T, Crews, D (1999). Developmental  synergism of steroid estrogens in sex determination. Environ. Health Perspec. 107: 93-97

Bowden, RM, Ewert, MA, Lipar, JL, Nelson, CE (2001). Concentrations of steroid hormones in layers and biopsies of chelonian egg yolks. Gen, Comp. Endocrinol. 121:95-103.

Etcberger, CR,Ewert, MA, Phillips, JB, Nelson, CE, Prange, HD (1992). Physiological responses to carbon dioxide in embryonic red-eared slider turtles, Trachemys scripta.J. Exp. Zool. 264: 1-10

Hochachka, PW, Somero, GN (1984). Biochemical Adaptation. Princeton University Press, Princeton, NJ.

Jeyasuria, P, Place, Ar (1998). Embryonic brain-gonadal axis in termperature-dependent sex determination of reptiles: a role for P450 aromatase (CYP19). J. Exp. Zool. 281: 428-449.

Rhen, T, Willingham, E, Sakta, JT, Crews, D (1999). Incubation temperature influences sex-steroid levels in juvenile red-eared slider turtles, Trachemys scripta, a species with temperature-dependent sex determination. Biol. Reprod. 61:1275-1280.

Spotilla, JR. Spotilla, LD, Kaufer, NE (1994). Molecular mechanisms of TSD in reptiles: a search for the magic bullet. J. Exp. Zool. 270:117-127.

Wibbels, T, Cowan, J, LeBoeuf, R (1998). Temperature-dependent  sex determination in the red-eared slider turtle, Trachemys scripta. J. Exp. Zool. 281:409-416.