Gisèle Desvages

Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs.

In embryos of the European pond turtle, sexual differentiation of gonads is temperature-dependent. Production of oestrogens appears to play a key role in this phenomenon. Gonadal aromatase activity was measured in embryos incubated at 25 degrees C (masculinizing temperature) and at 30 degrees C (feminizing temperature). At the beginning of the thermosensitive period, the aromatase activity was low at both temperatures but was somewhat higher at 30 than at 25 degrees C. Afterwards, it remained low in differentiating testes at 25 degrees C, whereas it increased in differentiating ovaries at 30 degrees C to form a marked peak when germ cells underwent meiotic prophase. Eggs were shifted either from 25 to 30 degrees C (highly feminizing) or from 30 to 35 degrees C for 6 days at different stages of embryonic development. The 25-35 degrees C shifts performed during the thermosensitive period strongly increased the aromatase activity but were ineffective after this period. The 30-35 degrees C shifts increased the aromatase activity at all stages. Altogether, results indicate that, in differentiating gonads of turtle embryos, temperature acts on the regulation of synthesis (and therefore activity) of cytochrome P-450 aromatase (P-450-aro). The expression of the P-450-aro gene itself could be temperature-dependent. However, temperature could also act upon the expression of another gene involved in P-450-aro regulation.

Sexual differentiation of gonads as a function of temperature in the turtle Emys orbicularis: Endocrine function, intersexuality and growth

Emys orbicularis is a freshwater turtle with temperature-dependent sex determination. Estrogens play a major role in gonadal differentiation; when they are produced at high levels during the thermosensitive period (TSP), ovaries differentiate; when their synthesis is very low, testes differentiate. Estrogens are synthesized from androgens through the activity of aromatase. We examine here two aspects of gonadal differentiation, intersexuality and growth, in E. orbicularis. For gonadal intersexuality, we studied the relationship between gonadal aromatase activity and gonadal structure at 28.5 degrees C (pivotal temperature), from the beginning of TSP to hatching, and compared results to those obtained at 30 degrees C (producing 100% females) and 25 degrees C (producing 100% males). At 28.5 degrees C, both males and females are obtained. However, histological differentiation of gonads is delayed compared to that at 25 degrees C and 30 degrees C, and an ovarian-like cortex of various thicknesses often develops at the surface of the male gonads; thus, several individuals display ovotestes at hatching. Despite important individual variations, the aromatase activity in ovaries differentiating at 28.5 degrees C increases during development as in ovaries differentiating at 30 degrees C. In most cases, however, activity is slightly lower than at 30 degrees C, and at the end of embryonic life, it becomes similar to that at 30 degrees C. In testes or ovotestes differentiating at 28.5 degrees C, aromatase activity remains low but is generally slightly higher than in testes at 25 degrees C; however, at the end of embryonic development, it becomes similar to that at 25 degrees C. Oocytes in the cortex of ovotestes begin to degenerate around hatching and continue to degenerate after hatching. Therefore, ovotestes evolve as testes. However, some oocytes may persist at the surface of testes up to the adult age. To estimate gonadal growth, the protein content was measured at different embryonic stages at 25 degrees C and at 30 degrees C. Testis growth is fast during TSP, somewhat slower after TSP, and decreases around hatching. Ovary growth is much slower than testis growth during TSP and then accelerates up to the end of embryonic development. This differential growth is well correlated with gonadal aromatase activity--much higher at 30 degrees C than at 25 degrees C--and can be explained by the fact that during TSP, testicular cords develop at 25 degrees C whereas they are inhibited at 30 degrees C; the ovarian cortex begins to form during this period but grows chiefly after TSP. Both inhibition of testicular cord development and stimulation of cortex development are under the control of endogenous estrogens. In the case of ovotestes, slight increases in estrogen synthesis, compared to that in typical testes, are sufficient to induce the transient formation of an ovarian-like cortex although they do not inhibit the development of testicular cords.

Steroid metabolism in gonads of turtle embryos as a function of the incubation temperature of eggs

In embryos of many reptiles, the sexual differentiation of gonads is temperature-dependent. In the turtle Emys orbicularis, all individuals become phenotypic males at 25 degrees C, whereas 100% phenotypic females are obtained at 30 degrees C. Steroid metabolism in embryonic gonads was studied at both temperatures, during and after the thermosensitive period for sexual differentiation. Pools of gonads were incubated for various times, with 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), progesterone, dehydroepiandrosterone or 4-androstene-3,17- dione as substrates. The analysis of metabolites combined two successive chromatographies (HPLC and TLC) and autoradiography. Conversion of pregnenolone to progesterone and of dehydroepiandrosterone to 4-androstene-3,17-dione was more important in testes at 25 degrees C than in ovaries at 30 degrees C. In ovaries, a large amount of 5-pregnene- 3 beta,20 beta-diol was formed from pregnenolone, and 5-androstene-3 beta,17 beta-diol was produced from dehydroepiandrosterone. In both testes and ovaries, 5 alpha-pregnane and 5 alpha-androstane derivatives were the main metabolites obtained from progesterone and 4-androstene-3,17-dione, respectively. Progesterone was also converted to 20 beta-hydroxy-4-pregnen-3-one. Dehydroepiandrosterone and 4-androstene-3,17-dione were also metabolized into 11 beta-hydroxy-4-androstene-3,17-dione (only in testes), testosterone, 11 beta,17 beta-dihydroxy-4-androstene-3-one, 17 beta-hydroxy-4-androstene-3,11-dione (low amounts in testes, traces in ovaries), 17 alpha-hydroxy-4-androstene-3-one, estrone and estradiol-17 beta (traces).