Steroid signaling and temperature-dependent sex determination-Reviewing the evidence for early action of estrogen during ovarian determination in turtles

Abstract

The developmental processes underlying gonadal differentiation are conserved across vertebrates, but the triggers initiating these trajectories are extremely variable. The red-eared slider turtle (Trachemys scripta elegans) exhibits temperature-dependent sex determination (TSD), a system where incubation temperature during a temperature-sensitive period of development determines offspring sex. However, gonadal sex is sensitive to both temperature and hormones during this period-particularly estrogen. We present a model for temperature-based differences in aromatase expression as a critical step in ovarian determination. Localized estrogen production facilitates ovarian development while inhibiting male-specific gene expression. At male-producing temperatures aromatase is not upregulated, thereby allowing testis development.

Figure 1

Trajectory of gonadal developmental in the red-eared slider turtle (Trachemys scripta). The temperature-sensitive period is highlighted in grey. The bipotential gonad contains primitive sex cords in the medullary region, and germ cells in the outer germinal epithelium. At a female-producing incubation temperature, the primitive sex cords degenerate, while the germ and somatic cells in the cortical region proliferate. At a male-producing incubation temperature, the germ cells migrate into the medullary region where they are enclosed within the developing seminiferous tubules. Arrows indicate the two time periods where estrogen may be acting in ovarian development: during medullary regression and cortical proliferation. E₂ = 17β-estradiol.

Figure 2

A model for temperature and steroid hormone action during temperature-dependent sex determination in the red-eared slider turtle (Trachemys scripta). Temperature directly or indirectly causes an increase in aromatase expression at female-producing temperatures, which in concert with a spike in expression of ERα and AR and other female-specific genes (i.e., FoxL2, Rspo1), leads to ovarian determination. Increased estrogen levels combined with ERα expression inhibit male-specific gene expression in the developing ovary. ERβ expression increases later in ovarian development, and is associated with ovarian differentiation. In contrast, at male-producing temperatures aromatase expression is not increased, and so estrogen-related inhibition of male development does not occur. Testis determination is associated with increased expression of male-specific genes such as Sox9, Mis, Dmrt1 and Sf1.

Figure 3

Expression of components of the steroid signaling network is differential by temperature across development in the red-eared slider turtle (Trachemys scripta). Steroidogenic factor 1 (Sf1), aromatase, estrogen receptors (ERα and ERβ), and androgen receptor (AR) expression levels within the gonad were measured with real-time qPCR, and the normalized expression levels for a male-producing temperature (26° C) were subtracted from female-producing temperature (31° C) values. For each gene, expression was calibrated to female expression levels at Stage 17, and then male expression values were subtracted from the female expression values for each stage. Gene expression levels higher at the female-producing temperature are therefore positive values, while levels higher at male-producing temperature are negative. Aromatase expression is dramatically higher than the other genes at FPT, and the Y-axis is scaled to reflect this (calibrated aromatase value given in parentheses at the top of the bar).

Figure 4

Aromatase expression during the temperature-sensitive period in the red-eared slider turtle (Trachemys scripta). (A,B) Aromatase expression patterns were compared between isolated gonad and adrenal-kidney-gonad complex (AKG) tissues. Levels were normalized relative to PP1 housekeeping gene and then calibrated across temperatures to the normalized Stage 17 FPT. Therefore, Stage 17 FPT=1, and other values reflect the ratio of aromatase expression level/temp and stage to Stage 17 FPT. (C-H) Aromatase expression patterns were also measured using DIG-labeled whole mount in situ hybridization experiments. Expression patterns at a female-producing temperature (FPT) at Stages 15, 17 and 21 are depicted in panels C,D,E, while expression at a male-producing temperature (MPT) for the same stages is depicted in panels F,G,H. Aromatase is differentially localized into round structures, possibly surrounding germ cells, at the FPT (C,D), and this difference is seen even during the early, bipotential stages of development (C,F). FPT = female-producing temperature (31° C). MPT = male-producing temperature (26° C). Redrawn from [67, 80].

Figure 5

Steriod signaling component expression is altered by temperature shift in the red-eared slider turtle (Trachemys scripta). The left column (Panels A–L) depict gene expression following temperature shifts from MPT → FPT, for AR (A,B,C), aromatase (D,E,F), ERα (G,H,I), ERβ (J,K,L). The right column (Panels M-X) depict gene expression following temperature shift from FPT → MPT, for AR (M,N,O), aromatase (P,Q,R), ERα (S,T,U), ERβ (V,W,X). Temperature shifts occurred at Stage 17, and expression changes were assayed at Stage 19. For each box, horizontal arrows indicate continued incubation at the original temperature, while diagonal arrows indicate a shift to the opposite temperature. Abbreviations as in Figure 4. Redrawn from [67, 85].

Figure 6

Ovarian differentiation in response to a female-producing temperature versus exogenous estrogen at a male-producing temperature in the red-eared slider turtle (Trachemys scripta). Feminizing temperature and estrogen are not equivalent signals for estrogen signaling components during ovarian differentiation (Stage 23). Aromatase (A,D), ERα (B,E), and ERβ (C,F) are depicted in ovaries created at constant incubation at a female-producing temperature versus ovaries created as a result of estradiol (E₂) treatment of eggs at Stage 17. Dotted lines indicate the basement membrane separating the medullary and cortical compartments. Arrows in (D,E,F) highlight the shift in localization towards the cortical region. Abbreviations as in Figure 4. Redrawn from [67, 85].