Unraveling Temperature-Dependent Sex Determination in Leopard Geckos

Temperature-dependent sex determination (TSD) is a biological process in reptiles where the sex of an embryo is determined by the temperature it experiences during a specific developmental period. For instance, American alligators typically develop into females at approximately 30 °C and males near 33 °C, though extremely high temperatures can also yield females. Despite decades of study on TSD, its mechanisms in squamates, a diverse group including lizards and snakes, have been largely unexplored.

Leopard Gecko Study Sheds Light

To address this research gap, a team led by Professor Shinichi Miyagawa from the Department of Biological Science and Technology at Tokyo University of Science, Japan, investigated TSD in the leopard gecko (Eublepharis macularius). In this lizard species, incubation at around 26 °C typically produces females, while temperatures near 32 °C primarily result in males.

The findings from this study were published in Volume 533 of the journal Developmental Biology, marking a significant step in understanding squamate TSD.

Pinpointing the Mechanisms: Experimental Design and Findings

The research team incubated gecko eggs at either 26.5 °C (female-producing temperature) or 31.5 °C (male-producing temperature). To pinpoint the exact period when temperature influences sex, they conducted shift experiments, moving eggs between the two temperatures on different days post-laying.

Upon examination of gonads as the eggs approached hatching, it was observed that incubation at the cooler temperature produced 100% females, while the warmer temperature resulted in 91% males. Early in development, embryos from both temperature groups appeared similar without obvious external differences. Structural differences emerged later, with ovaries becoming spherical and testes elongating to form seminiferous tubules.

Molecular Divergence Precedes Physical Changes

However, gene expression analysis showed that male and female developmental pathways began to diverge molecularly before these visible changes occurred. Key testis-related genes, including AMH, DMRT1, and SOX9, were activated earlier at male-producing temperatures, while ovarian genes such as FOXL2 and CYP19A1 became more active at female-producing temperatures.

Identifying the Temperature-Sensitive Period and Unique Features

Researchers determined that the temperature-sensitive period concludes at embryonic stage 36. Before this stage, altering the incubation temperature could still change the sex of the embryo; however, after this point, the sex was established and immutable by temperature. The study also identified features unique to the leopard gecko. For example, the KDM6B gene, which plays a role in male determination in turtles, exhibited a different regulatory pattern in this species. Additionally, early temperature-responsive genes involved in RNA splicing and cell adhesion were identified, suggesting molecular changes precede any physical differentiation between sexes.

The temperature-sensitive period for sex determination in leopard geckos concludes at embryonic stage 36, after which sex is immutably established.

Broader Implications

The researchers indicated that factors such as the mother's body temperature before egg laying might influence early development and could vary between laboratory settings. The findings suggest that while the genes responsible for gonad formation are largely shared across reptiles, the precise mechanisms by which temperature controls these genes have evolved differently among various reptile groups. The study contributes to the understanding of evolutionary plasticity and the molecular complexity of how environmental cues direct biological outcomes.