- Email: [email protected]
Time required for temperature-induced changes in gonadal aromatase activity and related gonadal structure in turtle embryos
Differentiation (1992) 52: 13-18 Ontogeny, heoplssia and DifferentiationTherapy
0 Springer-Verlag 1992
Time required for temperature-induced changes in gonadal aromatase activity and related gonadal structure in turtle embryos Gidle Desvages and Claude Pieau lnstitut Jaques Monod, C.N.R.S. et UniversitC Paris 7,2. Place Jussieu, F-75251 Paris Cedex 05, France Accepted in revised form I1 August 1992
Abstract. In the turtle Emys orbicuiaris, sexual differentiation of gonads is temperature-dependent. Oestrogens have been shown to be involved in this phenomenon and temperature has been expected to act, directly or indirectly, on regulation of synthesis or activity of cytochrome P-450aromatase (P-450arom). We have studied the effects of temperature shifts and of exposure at female- or male-producing temperatures for different times on gonadal aromatase activity and gonadal structure. In a first series of experiments, eggs were incubated at 25" C (masculinizing temperature) up to stage 18 and then exposed for 1 to 8 days at 35" C, a highly feminizing temperature. The response was exponential : aromatase activity increased clearly only after 4 day exposure at 35" C , then it was considerably enhanced. After 1 and 2 days at 35" C, the structure of gonads was not moditied. With longer exposures at 35" C, gonads were progressively feminized : medullary epithelial cords disappeared, whereas an ovarian cortex was forming. In another type of experiment, eggs incubated at 30" C (feminizing temperature) until stage 19 were transferred at 25°C for 6 days. In embryos of these shifted eggs, gonadal aromatase activity was about ninefold lower than that in control embryos (maintained at 30" C). However, this activity did not fall to the level measured in embryos of the same stage incubated at 25" C from egg-laying and was about twofold higher than that measured at the time of transfer. Gonads exhibited a cortex anlage but the medulla was more voluminous than that of controls and epithelial cords were beginning to form within. Together these results show that changes in gonadal aromatase activity and in gonadal structure are correlated, and that temperature acts on regulation of P-450 arom synthesis. Amplification of this synthesis during the thermosensitive period at higher temperatures could reflect amplification of expression of the P-450 arom gene.
Correspondence to: G. Desvages
Key words: Cytochrome P-450aromatase - Temperature differentiation - Turtle embryos
- Gonadal
Introduction
As in many other species of reptiles, the sexual differentiation of gonads in the European pond turtle (Emys orbicularis) is influenced by the incubation temperature of eggs during a certain period (thermosensitive period) of embryonic development [ 15, 18, 191. Experiments combining the effects of oestradiol, oestrone and tamoxifen (an antioestrogen) with those of temperature implied the involvement of oestrogens in gonadal differentiation. During the early stages of the thermosensitive period, the amounts of oestrogens were found higher at 30" C (feminizing temperature) than at 25" C (masculinizing temperature), suggesting that the activity of cytochrome P-450 aromatase (P-450arom), the enzyme responsible for the aromatization of androstenedione into oestrone and of testosterone into oestradiol-17 p, is different at these temperatures [4].Gonadal aromatase activity was low at 25" C and 30" C at the beginning of the thermosensitive period, but was somewhat higher at 30" C than at 25OC. Afterwards, it increased at 30" C to form a marked peak when germ cells underwent meiotic prophase, whereas at 25"C, it remained very low up to hatching [2]. Moreover, it was shown that a 6 day shift of temperature from 25" C (masculinizing) to 35" C (highly feminizing) during the thermosensitive period strongly increased aromatase activity, whereas a similar shift was ineffective after this period. In the case of shifts of the same durations from 30" C to 35" C (both feminizing temperatures), a slight increase of aromatase activity was obtained during as well as after the thermosensitive period. Moreover, temperature did not have a direct important effect on aromatase activity measurements, since very close values were obtained when the assays were performed at 25" C , 30" C or 36" C . These results suggested that temperature does not act directly on activity
14
of P-450arom, but rather on the regulation of its synthesis [ 2 ] . In this case, the response to temperature shifts during the thermosensitive period would not be immediate. Changes in aromatase activity resulting from shifts from a masculinizing to a feminizing temperature and vice versa would require a few days. We report here the results of such temperature shifts in Emys orbicularis, and examine whether temperature-induced changes in aromatase activity are correlated with changes in the histological structure of the gonads.
Methods Collection and incubation of eggs. The eggs of Emys orbiculuris were obtained from gravid females by intracoelomic injection of 5 IU oxytocin [8]. They were incubated as previously described [15]at either 25" C (a 100% male-producing temperature), 30" C (a 100% female-producing temperature) or at 35" C (a highly feminizing temperature [ 171). The thermosensitive stages (or period) for sexual differentiation of gonads extend from stage 16 to stage 22 of the embryonic development [ 18). In a first series of experiments, 194 eggs from 22 clutches, all collected the same day, were first incubated at 25" C until stage 18, i.e. about the first third of the thermosensitive period [18]. Eighteen embryos were taken to measure gonadal aromatase activity at that stage. Then, in each clutch, some eggs were shifted to 35" C for 1, 2, 4, 6, or 8 days, whereas the other eggs were maintained at 25" C for the same periods. In a second type of experiment, 34 eggs from 6 clutches were first incubated at 30" C up to stage 19, i.e. about the middle of the thermosensitive period [18]. Two eggs of each clutch were used to measure the gonadal aromatase activity at that stage. Fourteen of the other eggs, 1-3 per clutch, were then shifted to 25" C for 6 days, whereas the remainder were maintained at 30" C. At the end of these incubations, embryos were decapitated and their developmental stage was determined, based on criteria described for the snapping turtle, Chelydra serpentina [18, 261. In each series, embryos incubated at the higher temperature, even in the case of short exposures, were more developed than those incubated at the lower one. Measurement of gonadal aromatase activity. All assays were carried out in duplicate. In the first series of experiments, aromatase activity was measured on pools of 14 to 18 gonads. In the second experiment, assays were performed on pools of respectively: 12 gonads of embryos incubated at 30" C until stage 19; 8 gonads of embryos maintained 6 more days at 30" C; and 14 gonads of embryos shifted to 25" C for 6 days. Gonads were removed and placed in cold medium (RPMI 1640, N-[2-Hydroxyethyl]piperazine-N'-[2-ethancsulfonic acid] H EPES buffer) until incubation. Pools of gonads were then incubated for 4.5 h at 36" C in 0.4ml RPMI containing 0.5pM [l P-3H] androstenedione (27.5Ci/mml, New England Nuclear) as substrate. At the end of the incubation, radioactivity was measured in thc medium after stcroid extraction by chloroform followed by dextran-charcoal adsorption. Aromatase activity was calculated from the ratc of [3H] water rcleased from the 1 /?-position [l]. It is expressed in femtomoles per gonad per hour. Histology. Pooled gonads were fixed in Bouin's solution within the tube used for incubation. They were examined under a dissecting microscope and four from each pool were studied histologically. After dehydratation, gonads were embedded in paraffin, serially sectioned at 7.5 pm and stained with hematoxylin and eosin.
Results
Shifts from 25" C to 35" C Eflkts on gonadal aromatase activity. To determine the time required to induce a definite increase of gonadal aromatase activity by increasing the incubation temperature, eggs of Emys were shifted from 25" C to 35" C at stage 18 and exposed at 35" C for varying times (1 to 8 days). Results are given in Fig. 1. The 1 and 2 day exposures at 35" C had no detectable effect on aromatase activity. After 4 days at 35" C, aromatase activity was about threefold that measured in control embryos (maintained at 25" C). The 6 and 8 day exposures at 35" C markedly enhanced the aromatase activity : about fortyfold and seventyfold respectively as compared with controls. These results show that more than 2 days of exposure at a female producing temperature are required to induce increase in aromatase activity. Moreover, the response to increasing times of exposure corresponds to an exponential function (Fig. 1). Elfects on gonadal structure. At stage 18, the testicular differentiation had started in embryos incubated at 25" C : the medullary part of gonads contained thin epithelial cords including germ cells; the germinal epithelium was still present but thinner than in the undifferentiated gonads 1151. During the following stages (19 to Aromatase activity moldgonadhour)
+
15
-
10
-
T
5 -
25OC I
I
I
I
I
0
1
2
4
6
8
18
19-
19
20-
20+
21
18
19
19+
20+
21
22-
*
Days Stages at 25°C Stages at 35'C
Fig. 1. Aromatase activity as a function of the duration of exposure at 35" C (1 to 8 days) in gonads of Emys orbicularis embryos shifted from 25 to 35" C at stage 18 (day 0) and in controls maintained at 25" C for the same times. The embryonic stages reached at the different times of incubation at 25°C and 35°C are indicated. Values shown represent mean +/- SD of assays carried out on two distinct pools of gonads. The response at 35" C fits well ( r = 0.99) with an exponential function: aromatase activity=0.0469 x 1 0 ' 0 . 3 I34 x d r y s )
Fig, 2A-H. Transverse sections through gonads of Emys orbicularis embryos obtained from eggs transferred from 25" C to 35" C at stage 18 and then incubated at 35" C for 2(B), 4(D), 6(F) and 8(H) days, and from eggs maintained at 25" C for the same times (A, C, E, C). Note that in controls epithelial cords are present in the medulla, and the germinal epithelium is becoming flat (A, C, E, C). In E and chiefly in C, some medullary cords are tubular. After 2 days at 35" C, the structure of the gonad is not very differ-
ent from that of control (compare B with A). After 4 and 6 days at 35" C, thin epithelial cords are still present, but the volume of the medulla is reduced and a cortex anlage is formed (compare D and F with C and E respectively). After 8 days at 35" C, medullary epithelial cords are no longer recognizable and the cortex anlage is more developed (compare H with G). rc. rete cord; Bur, 50 pm
16
21, Fig. 1) in control embryos maintained at 25" C, the medullary cords (future seminiferous cords) progressively enlarged and some of them acquired a lumen and became tubular (Fig. 2 G). The germinal epithelium continued to flatten (Fig. 2A, 2C, 2 E and 2G), however, in some individuals it remained slightly higher (not shown). In embryos exposed for 1 day and 2 days at 35" C , gonads did not display marked histological differences with those of controls (compare Fig. 2B with Fig. 2A). After 4 and 6 days at 35" C, reduced and disorganized epithelial cords were still present in the medulla, and an ovarian-like cortex had begun to form at the surface by multiplication of germ cells in the germinal epithelium (Fig. 2D and 2F). After 8 days of exposure at 35" C, gonads had acquired a more obvious ovarian structure : epithelial cords were no longer recognizable in the medulla except rete cords in the dorsal region and the cortex was thicker (Fig. 2H, compare with Fig. 2G). This study shows that changes in gonadal structure are correlated with changes in gonadal aromatase activity. Indeed, the first histological signs of gonadal feminization coincide with the beginning of the increase of aromatase activity (4 days after transfer from 25 to 35" C), and then the higher the aromatase activity, the clearer the ovarian structure.
Shifts from 30" C to 2.5" C Efsects on gonadal aromatase activity. To determine whether the aromatase activity in gonads decreases, remains stable or continues to increase in relation to a decreasing incubation temperature, eggs of Emys were shifted from 30" C to 25' C at stage 19 and exposed at 25" C for 6 days. Stage 19 was chosen for the shift because the gonadal aromatase activity becomes clearly higher at 30" C than at 25" C at that stage, although it is still low ([2] and Fig. 3).
Fig. 4A-C. Transverse sections through gonads of Emys orbiculuris embryos obtained from eggs: A, incubated at 30" C, stage 19; B, incubated at 30" C, slage 22- (6 days older than A); C, incubated at 30" C up to stage 19, and then at 25" C for 6 days (stage 21). In A and B, the medulla is reduced (chiefly in A) and does not
Aromatase activity (10-15 moldgonadhour)
10
5
0
-25°C 30°C
25°C
30°C
1
30°C
25°C
Stage 19
Stage 21
Stage 22-
Fig. 3. Effect of a shift of temperature from 30" C to 25" C at stage 19, followed by a 6 day exposure at 25" C on gonadal aromatasc activity in embryos of Emys orbiculuris. 'At the end of the experiment, embryos shifted to 25" C had reached stage 21, whereas control embryos maintained at 30° Chad reached stage 22-. Figure shows aromatase activity at stages 19 and 22-' in embryos incubated at 30" C and at stage 21 in embryos shifted to 25" C for 6 days. As a comparison, it also shows arornatase activity at stages 19 and 21 in embryos incubated at 25" C. Values represent mean +/- SD of assays carried out on two distinct pools of gonads
In embryos exposed for 6 days at 25' C, the aromatase activity in gonads was about ninefold lower than in control embryos left at 30" C (Fig. 3). However, it was about fivefold that measured in embryos of the same stage (stage 21) incubated at 25" C since egg-laying. In
exhibit any epithelial cords; many germ cells are in the anlage of the future cortex. In C, the cortex anlage is as developed as in B,but the medulla is more voluminous and medullary epithelial cords are beginning to form (arrows). Bur, 50 pm
17
addition, it was twofold that of embryos at stage 19 incubated at 30" C (Fig. 3). Therefore, a 6 day shift from a female- to a male-producing temperature did not result in an immediate decrease in aromatase activity. Rather, the aromatase activity continued to increase slightly, at least for the length of exposure. Effects on gonadal structure. At stage 19, the gonads of embryos incubated at 30" C were much thinner than the gonads of embryos at the same stage incubated at 25" C (Fig. 4A, compare with Fig. 3A). The volume of the medulla was significantly reduced due to a strong inhibition of the initial sex cords. Most germ cells were in the anlage of the cortex derived from the germinal epithelium, but some of them remained in the medulla (Fig. 4A). Control embryos maintained 6 days at 30" C reached a stage between 21 and 22. Their gonads were approximately twofold bigger than at stage 19 but presented the same structure : the medulla, devoid of epithelial cords, was composed of a mass of small cells and of some larger germ cells; in the differentiating cortex, germ cells were dividing but had not yet entered meiosis (Fig. 48). In embryos shifted for 6 days at 25" C, gonads exhibited a cortex similar to that existing in controls. However, the volume of the medulla was greater due to a more significant mass of cells from which some epithelial cords were beginning to form; these cords indicating a slight masculinization of the gonads (Fig. 4C, compare with Fig. 4B).
Discussion In embryos of the turtle Emys orbicularis, oestrogens have been shown to be involved insonadal differentiation and temperature has been expected to act, directly or indirectly, on the regulation of P-450 arom synthesis [4]. Results presented in this study show that changes in gonadal aromatase activity induced by shifts of temperature from a masculinizing to a feminizing temperature and vice versa require several days. Thus, in shifts from 25" C (masculinizing) to 35" C (highly feminizing) with different times of exposure at 35" C, the response is an exponential function: the increase in gonadal aromatase activity, not detectable at 1 and 2 days, becomes marked (threefold) at 4 days, and considerable (seventyfold) at 8 days. In shifts from 30" C (feminizing) to 25"C, the gonadal aromatase activity after a 6 day transfer does not fall immediately to a testicular level, but continues to increase slightly. These data agree with an effect of temperature on P-450 arom synthesis. The response observed in the first type of experiment reflects amplification of the enzyme production as the duration of exposure at feminizing temperature increases. The second type of experiment reflects the continuation of the enzyme production for a certain time after transfer from feminizing to masculinizing temperature. Several factors are known to modify the aromatase activity in other systems, such as human adipose cells [7, 211 and human and rat ovarian granulosa cells [lo, 20, 221. Some of these factors, including gonadotropins,
cyclic AMP and forskolin, increase aromatase activity [5, 7, 9, 10, 201 whereas other factors including prolactin and epidermal growth factor inhibit it [12, 21, 221. In most cases, changes in activity are correlated with changes in the levels of mRNA encoding P-450 arom [9, 12, 20-221. In Emys orbicularis, temperature-induced changes in synthesis and activity of P-450 arom could also result from changes in the levels of mRNA encoding the enzyme. In this view, regulation of expression of the P-450 arom gene would be temperature-sensitive. The expression of P-450 arom gene in human tissues has been shown to be regulated by tissue-specific promoters [13, 141. In Emys orbicularis as in other vertebrates, the sites of steroid synthesis in gonads are not the same in embryos as in adults. In embryos, steroids are synthesized in the epithelial cells (future Sertoli cells) of the testicular cords (or tubes) in testes, and in the cells derived from the medullary sex cords in ovaries [15]. Therefore, in Emys orbicularis, the promoter responsible for the expression of P-450 arom in gonads could be different in embryos and in adults, leading to different responses to factors regulating P-450 arom synthesis. The transcriptional initiation complex could be thermosensitive, or a thermosensitive factor could be implicated in the formation of this complex in embryos, but not in adults. However, temperature could act upstream on regulation of the expression of another gene. We had previously suggested that the expression of the gene encoding the anti-Mullerian hormone (AMH) could be thermoregulated [4]. Indeed, in mammals, AMH inhibits aromatase activity in differentiated fetal ovaries [24]. Likewise, partially purified chick AMH represses the aromatase activity in homospecific embryonic ovaries as well as in 16-day-old rat fetal ovaries. Also purified bovine AMH exhibits an anti-aromatase activity upon ovaries of Emys orbicularis embryos at stage 24, i.e. after the thermosensitive period [3]. In phenotypic males of Emys orbicularis, the regression of Miillerian ducts begins at stage 22, i.e. at the end of the thermosensitive period, and continues up to hatching [I 51. High amounts of AMH at these stages could explain the failure to increase aromatase activity by shifts of temperature from 25" C to 35" C [2]. At 25" C, lower amounts of the hormone are probably produced earlier, during the thermosensitive period and may downregulate P-450 arom synthesis. However, the expression of AMH gene itself does not appear to be temperature-sensitive. Indeed, when eggs are shifted from 25" C to 30" C during the thermosensitive period, as early as stages 16 to 19, Miillerian ducts degenerate in some individuals [ 181, showing that AMH can be produced at 30" C. In male chick embryos, raising the incubation temperature from 38" C to 40.5" C results in the retention of both Miillerian ducts [23]. This phenomenon does not appear as a consequence of a decrease of AMH secretion [25]. It could be due to a slight increase of aromatase activity and therefore of oestrogen production in the gonads. Indeed, oestrogens have been shown to prevent Mullerian duct regression [ll]. It was previously shown that in Emys orbicularis shifts from 30" C (feminizing) to 35" C (even more feminizing) increase aromatase activity, during as well as after the thermosensitive period 121. This response indi-
18
cates that high temperatures activate expression rather than prevent repression of P-450arom gene. Recently, the implication of P-450 arom enzyme on gonadal differentiation has been clearly demonstrated in chickens [6]. As already suggested [4],the regulation of expression of P-450arom gene probably plays a key role in sex differentiation of gonads in all vertebrates by regulating the levels of oestrogens in gonads and, therefore, allowing their male or female differentiation. Our study shows that in Emys orbicularis, changes in gonadal structure are well correlated with changes in gonadal aromatase activity. Testicular cords differentiate and the germinal epithelium regresses while aromatase activity remains low. Inversely, testicular cords regress and an ovarian cortex develops while aromatase activity increases. Thus, gonads of embryos incubated at 25" C up to stage 19 and then transferred at 30" C for 6 days, exhibit an ovarian-like cortex and anlagen of medullary epithelial cords indicating that they would have evolved towards ovotestes. Aromatase activity in these gonads is, as expected, higher than in typical testes at 25" C but lower than in typical ovaries at 30" C. Incubation of eggs within the transitional range of temperature between male- and female-producing temperatures (28-29" C)has been shown to result in phenotypic males, phenotypic females and intersexes [ 161. Therefore, the turtle Emys orbicularis offers a good model to define the different levels of aromatase activity and consequently of oestrogens - at which a testis, an ovotestis or an ovary does differentiate. Acknowledgements. We thank M. Girondot for his help with the figures and L. Guillon for her help with the manuscript. Animals were captured with permission of the French Environment Ministry.
References 1.Ackerman GE, Smith ME, Mendelson CR, Mac Donald PC. Simpson ER (1981) Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J Clin Endocrinol Metab 53:412417 2. Desvages G , Pieau C (1992)Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs. J Steroid Biochem Molec Biol41:851-853 3. Di Clemente N, Ghaffari S, Pepinsky RB, Pieau C, Josso N, Cate RL, Vigier B (1992) A quantitative and interspecific test for biological activity of anti-Mullerian hormone: the fetal ovary aromatase assay. Development 114:721-727 4.Dorizzi M, Mignot TM, Guichard A, Desvages G, Pieau C (1991) Involvement of oestrogens in sexual differentiation of gonads as a function of temperature in turtles. Differentiation 47:9-1 7 5.Dorrington JF, Moon YS,Armstrong DT (1975)Estradiol-17p biosynthesis in cultured granulosa cells from hypophysectomized immature rats: stimulation by follicle-stimulating hormone. Endocrinology 97: 1328-1 331 6.Elbrecht A, Smith RG (1992)Aromatase enzyme activity and sex determination in chickens. Science 255 :467-470 7. Evans CT, Corbin CJ, Saunders CT, Merril JC, Simpson ER, Mendelson CR (1987)Regulation of oestrogen biosynthesis by
human adipose stromal cells: effects of dibutyryl cyclic AMP, epidermal growth factor, and phorbol ester on the synthesis of aromatase cytochrome P-450.J Biol Chem 262:6914-6920 8.Ewert MA, Legler MA (1978) Hormonal induction of oviposition in turtles. Herpetologica 34:314-31 8 9. Fitzpatrick SL, Richards JS (1991)Regulation of cytochrome P-450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129~1452-1462 10. Hsueh AJW, Adashi EY, Jones PBC, Welsh TH (1984) Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5 :76-1 27 11. Hutson J, Ikawa H, Donahoe PK (1982)Oestrogen inhibition of Miillerian inhibiting substance in the chick embryo. J Pediatr Surg 17:953-959 12. Krasnow JS, Hickey GJ, Richards JS (1990)Regulation of aromatase mRNA and estradiol biosynthesis in rat ovarian granulosa and luted cells by prolactin. Mol Endocrinol4: 13-21 13.Mahendro MS, Means GD, Mendelson CR, Simpson ER (1991) Tissue-specific expression of human P-450 arom : the promoter responsible for expression in adipose tissue is different from that utilized in placenta. J Biol Chem 266:11276 11281 14. Means GD, Kilgore MW, Mahendroo MS, Mendelson CR, Simpson ER (1991)Tissue-specific promoters regulate aromatase cytochrome P-450 gene expression in human ovary and fetal tissues. Mol Endocrinol 5:2005-201 1 15. Pieau C (1974)Differenciation du sexe en fonction de la temperature chez les embryons d'Emys orbiculuris L. (ChClonien); effets des hormones sexuelles. Ann Embryol Morphog 7:365-394 16. Pieau C (1976)Donnees recentes sur la differenciation sexuelle en fonction de la temperature chez les embryons d'Emys orbiculurk L. (Chelonien). Bull SOCZoo1 Fr 101 (Suppl4):46-53 17. Pieau C (1978) Effets de temperatures d'incubation basses et elevees sur la diffkrenciation sexuelle chez des embryons d'Emys orbiculuris L. (Chelonien). CR Acad Sci Paris. krie D 286:121124 18.Pieau C,Dorizzi M (1981)Determination of temperature sensitive stages for sexual differentiation of the gonads in embryos of the turtle Emys orbicularis L. J Morphol 170:373-382 19.Raynaud A, Pieau C (1985) Embryonic development of the genital system. In: Gans C, Billett F (eds) Biology of the reptiha. Vol XV, Development B. J Wiley and Sons, New York, pp 149-300 20. Richards JS,Hickey GJ, Chen S, Shively JE, Hall PF, GaddyKurten D, Kurten R (1987) Hormonal regulation of estradiol biosynthesis, aromatase activity and aromatase mRNA in rat ovarian follicles and corpora lutea. Steroids 50: 393-409 21. Simpson ER, Merrill JC, Hollub AJ, Graham-Lorence S, Mendelson CR (1989) Regulation of estrogen biosynthesis by human adipose cells. Endocr Rev 10:136148 22. Steinkampf M,Mendelson CR, Simpson ER (1988)Effects of epidermal growth factor and insulin-like growth factor -I on the levels of mRNA encoding aromatase cytochrome P-450 in human ovarian granulosa cells. Mol Cell Endocrinol 59:9399 23.Stoll R (1948)Action de quelques agents physiques et chimiques sur les canaux de Miiller de I'embryon de Poulet. Arch Anat Microsc Morphol Exp 37:333-346 24. Vigier B, Forest MG, Eychenne B, Bezard J, Garrigou 0, Robel P, Josso N (1989)Anti-Miillerian hormone produces endocrine sex-reversal of fetal ovaries. Proc Natl Acad Sci USA 86:36843688 25. Weniger JP, Thiebold JJ (1981)On the mechanism of the retention of the Miillerian ducts in the chick embryo incubated at a temperature above normal. Anat Embryol 163:345-350 26. Yntema CL (1968)A series of stages in the embryonic development of Chelydra serpentina. J Morphol 125:219-252
0 Springer-Verlag 1992
Time required for temperature-induced changes in gonadal aromatase activity and related gonadal structure in turtle embryos Gidle Desvages and Claude Pieau lnstitut Jaques Monod, C.N.R.S. et UniversitC Paris 7,2. Place Jussieu, F-75251 Paris Cedex 05, France Accepted in revised form I1 August 1992
Abstract. In the turtle Emys orbicuiaris, sexual differentiation of gonads is temperature-dependent. Oestrogens have been shown to be involved in this phenomenon and temperature has been expected to act, directly or indirectly, on regulation of synthesis or activity of cytochrome P-450aromatase (P-450arom). We have studied the effects of temperature shifts and of exposure at female- or male-producing temperatures for different times on gonadal aromatase activity and gonadal structure. In a first series of experiments, eggs were incubated at 25" C (masculinizing temperature) up to stage 18 and then exposed for 1 to 8 days at 35" C, a highly feminizing temperature. The response was exponential : aromatase activity increased clearly only after 4 day exposure at 35" C , then it was considerably enhanced. After 1 and 2 days at 35" C, the structure of gonads was not moditied. With longer exposures at 35" C, gonads were progressively feminized : medullary epithelial cords disappeared, whereas an ovarian cortex was forming. In another type of experiment, eggs incubated at 30" C (feminizing temperature) until stage 19 were transferred at 25°C for 6 days. In embryos of these shifted eggs, gonadal aromatase activity was about ninefold lower than that in control embryos (maintained at 30" C). However, this activity did not fall to the level measured in embryos of the same stage incubated at 25" C from egg-laying and was about twofold higher than that measured at the time of transfer. Gonads exhibited a cortex anlage but the medulla was more voluminous than that of controls and epithelial cords were beginning to form within. Together these results show that changes in gonadal aromatase activity and in gonadal structure are correlated, and that temperature acts on regulation of P-450 arom synthesis. Amplification of this synthesis during the thermosensitive period at higher temperatures could reflect amplification of expression of the P-450 arom gene.
Correspondence to: G. Desvages
Key words: Cytochrome P-450aromatase - Temperature differentiation - Turtle embryos
- Gonadal
Introduction
As in many other species of reptiles, the sexual differentiation of gonads in the European pond turtle (Emys orbicularis) is influenced by the incubation temperature of eggs during a certain period (thermosensitive period) of embryonic development [ 15, 18, 191. Experiments combining the effects of oestradiol, oestrone and tamoxifen (an antioestrogen) with those of temperature implied the involvement of oestrogens in gonadal differentiation. During the early stages of the thermosensitive period, the amounts of oestrogens were found higher at 30" C (feminizing temperature) than at 25" C (masculinizing temperature), suggesting that the activity of cytochrome P-450 aromatase (P-450arom), the enzyme responsible for the aromatization of androstenedione into oestrone and of testosterone into oestradiol-17 p, is different at these temperatures [4].Gonadal aromatase activity was low at 25" C and 30" C at the beginning of the thermosensitive period, but was somewhat higher at 30" C than at 25OC. Afterwards, it increased at 30" C to form a marked peak when germ cells underwent meiotic prophase, whereas at 25"C, it remained very low up to hatching [2]. Moreover, it was shown that a 6 day shift of temperature from 25" C (masculinizing) to 35" C (highly feminizing) during the thermosensitive period strongly increased aromatase activity, whereas a similar shift was ineffective after this period. In the case of shifts of the same durations from 30" C to 35" C (both feminizing temperatures), a slight increase of aromatase activity was obtained during as well as after the thermosensitive period. Moreover, temperature did not have a direct important effect on aromatase activity measurements, since very close values were obtained when the assays were performed at 25" C , 30" C or 36" C . These results suggested that temperature does not act directly on activity
14
of P-450arom, but rather on the regulation of its synthesis [ 2 ] . In this case, the response to temperature shifts during the thermosensitive period would not be immediate. Changes in aromatase activity resulting from shifts from a masculinizing to a feminizing temperature and vice versa would require a few days. We report here the results of such temperature shifts in Emys orbicularis, and examine whether temperature-induced changes in aromatase activity are correlated with changes in the histological structure of the gonads.
Methods Collection and incubation of eggs. The eggs of Emys orbiculuris were obtained from gravid females by intracoelomic injection of 5 IU oxytocin [8]. They were incubated as previously described [15]at either 25" C (a 100% male-producing temperature), 30" C (a 100% female-producing temperature) or at 35" C (a highly feminizing temperature [ 171). The thermosensitive stages (or period) for sexual differentiation of gonads extend from stage 16 to stage 22 of the embryonic development [ 18). In a first series of experiments, 194 eggs from 22 clutches, all collected the same day, were first incubated at 25" C until stage 18, i.e. about the first third of the thermosensitive period [18]. Eighteen embryos were taken to measure gonadal aromatase activity at that stage. Then, in each clutch, some eggs were shifted to 35" C for 1, 2, 4, 6, or 8 days, whereas the other eggs were maintained at 25" C for the same periods. In a second type of experiment, 34 eggs from 6 clutches were first incubated at 30" C up to stage 19, i.e. about the middle of the thermosensitive period [18]. Two eggs of each clutch were used to measure the gonadal aromatase activity at that stage. Fourteen of the other eggs, 1-3 per clutch, were then shifted to 25" C for 6 days, whereas the remainder were maintained at 30" C. At the end of these incubations, embryos were decapitated and their developmental stage was determined, based on criteria described for the snapping turtle, Chelydra serpentina [18, 261. In each series, embryos incubated at the higher temperature, even in the case of short exposures, were more developed than those incubated at the lower one. Measurement of gonadal aromatase activity. All assays were carried out in duplicate. In the first series of experiments, aromatase activity was measured on pools of 14 to 18 gonads. In the second experiment, assays were performed on pools of respectively: 12 gonads of embryos incubated at 30" C until stage 19; 8 gonads of embryos maintained 6 more days at 30" C; and 14 gonads of embryos shifted to 25" C for 6 days. Gonads were removed and placed in cold medium (RPMI 1640, N-[2-Hydroxyethyl]piperazine-N'-[2-ethancsulfonic acid] H EPES buffer) until incubation. Pools of gonads were then incubated for 4.5 h at 36" C in 0.4ml RPMI containing 0.5pM [l P-3H] androstenedione (27.5Ci/mml, New England Nuclear) as substrate. At the end of the incubation, radioactivity was measured in thc medium after stcroid extraction by chloroform followed by dextran-charcoal adsorption. Aromatase activity was calculated from the ratc of [3H] water rcleased from the 1 /?-position [l]. It is expressed in femtomoles per gonad per hour. Histology. Pooled gonads were fixed in Bouin's solution within the tube used for incubation. They were examined under a dissecting microscope and four from each pool were studied histologically. After dehydratation, gonads were embedded in paraffin, serially sectioned at 7.5 pm and stained with hematoxylin and eosin.
Results
Shifts from 25" C to 35" C Eflkts on gonadal aromatase activity. To determine the time required to induce a definite increase of gonadal aromatase activity by increasing the incubation temperature, eggs of Emys were shifted from 25" C to 35" C at stage 18 and exposed at 35" C for varying times (1 to 8 days). Results are given in Fig. 1. The 1 and 2 day exposures at 35" C had no detectable effect on aromatase activity. After 4 days at 35" C, aromatase activity was about threefold that measured in control embryos (maintained at 25" C). The 6 and 8 day exposures at 35" C markedly enhanced the aromatase activity : about fortyfold and seventyfold respectively as compared with controls. These results show that more than 2 days of exposure at a female producing temperature are required to induce increase in aromatase activity. Moreover, the response to increasing times of exposure corresponds to an exponential function (Fig. 1). Elfects on gonadal structure. At stage 18, the testicular differentiation had started in embryos incubated at 25" C : the medullary part of gonads contained thin epithelial cords including germ cells; the germinal epithelium was still present but thinner than in the undifferentiated gonads 1151. During the following stages (19 to Aromatase activity moldgonadhour)
+
15
-
10
-
T
5 -
25OC I
I
I
I
I
0
1
2
4
6
8
18
19-
19
20-
20+
21
18
19
19+
20+
21
22-
*
Days Stages at 25°C Stages at 35'C
Fig. 1. Aromatase activity as a function of the duration of exposure at 35" C (1 to 8 days) in gonads of Emys orbicularis embryos shifted from 25 to 35" C at stage 18 (day 0) and in controls maintained at 25" C for the same times. The embryonic stages reached at the different times of incubation at 25°C and 35°C are indicated. Values shown represent mean +/- SD of assays carried out on two distinct pools of gonads. The response at 35" C fits well ( r = 0.99) with an exponential function: aromatase activity=0.0469 x 1 0 ' 0 . 3 I34 x d r y s )
Fig, 2A-H. Transverse sections through gonads of Emys orbicularis embryos obtained from eggs transferred from 25" C to 35" C at stage 18 and then incubated at 35" C for 2(B), 4(D), 6(F) and 8(H) days, and from eggs maintained at 25" C for the same times (A, C, E, C). Note that in controls epithelial cords are present in the medulla, and the germinal epithelium is becoming flat (A, C, E, C). In E and chiefly in C, some medullary cords are tubular. After 2 days at 35" C, the structure of the gonad is not very differ-
ent from that of control (compare B with A). After 4 and 6 days at 35" C, thin epithelial cords are still present, but the volume of the medulla is reduced and a cortex anlage is formed (compare D and F with C and E respectively). After 8 days at 35" C, medullary epithelial cords are no longer recognizable and the cortex anlage is more developed (compare H with G). rc. rete cord; Bur, 50 pm
16
21, Fig. 1) in control embryos maintained at 25" C, the medullary cords (future seminiferous cords) progressively enlarged and some of them acquired a lumen and became tubular (Fig. 2 G). The germinal epithelium continued to flatten (Fig. 2A, 2C, 2 E and 2G), however, in some individuals it remained slightly higher (not shown). In embryos exposed for 1 day and 2 days at 35" C , gonads did not display marked histological differences with those of controls (compare Fig. 2B with Fig. 2A). After 4 and 6 days at 35" C, reduced and disorganized epithelial cords were still present in the medulla, and an ovarian-like cortex had begun to form at the surface by multiplication of germ cells in the germinal epithelium (Fig. 2D and 2F). After 8 days of exposure at 35" C, gonads had acquired a more obvious ovarian structure : epithelial cords were no longer recognizable in the medulla except rete cords in the dorsal region and the cortex was thicker (Fig. 2H, compare with Fig. 2G). This study shows that changes in gonadal structure are correlated with changes in gonadal aromatase activity. Indeed, the first histological signs of gonadal feminization coincide with the beginning of the increase of aromatase activity (4 days after transfer from 25 to 35" C), and then the higher the aromatase activity, the clearer the ovarian structure.
Shifts from 30" C to 2.5" C Efsects on gonadal aromatase activity. To determine whether the aromatase activity in gonads decreases, remains stable or continues to increase in relation to a decreasing incubation temperature, eggs of Emys were shifted from 30" C to 25' C at stage 19 and exposed at 25" C for 6 days. Stage 19 was chosen for the shift because the gonadal aromatase activity becomes clearly higher at 30" C than at 25" C at that stage, although it is still low ([2] and Fig. 3).
Fig. 4A-C. Transverse sections through gonads of Emys orbiculuris embryos obtained from eggs: A, incubated at 30" C, stage 19; B, incubated at 30" C, slage 22- (6 days older than A); C, incubated at 30" C up to stage 19, and then at 25" C for 6 days (stage 21). In A and B, the medulla is reduced (chiefly in A) and does not
Aromatase activity (10-15 moldgonadhour)
10
5
0
-25°C 30°C
25°C
30°C
1
30°C
25°C
Stage 19
Stage 21
Stage 22-
Fig. 3. Effect of a shift of temperature from 30" C to 25" C at stage 19, followed by a 6 day exposure at 25" C on gonadal aromatasc activity in embryos of Emys orbiculuris. 'At the end of the experiment, embryos shifted to 25" C had reached stage 21, whereas control embryos maintained at 30° Chad reached stage 22-. Figure shows aromatase activity at stages 19 and 22-' in embryos incubated at 30" C and at stage 21 in embryos shifted to 25" C for 6 days. As a comparison, it also shows arornatase activity at stages 19 and 21 in embryos incubated at 25" C. Values represent mean +/- SD of assays carried out on two distinct pools of gonads
In embryos exposed for 6 days at 25' C, the aromatase activity in gonads was about ninefold lower than in control embryos left at 30" C (Fig. 3). However, it was about fivefold that measured in embryos of the same stage (stage 21) incubated at 25" C since egg-laying. In
exhibit any epithelial cords; many germ cells are in the anlage of the future cortex. In C, the cortex anlage is as developed as in B,but the medulla is more voluminous and medullary epithelial cords are beginning to form (arrows). Bur, 50 pm
17
addition, it was twofold that of embryos at stage 19 incubated at 30" C (Fig. 3). Therefore, a 6 day shift from a female- to a male-producing temperature did not result in an immediate decrease in aromatase activity. Rather, the aromatase activity continued to increase slightly, at least for the length of exposure. Effects on gonadal structure. At stage 19, the gonads of embryos incubated at 30" C were much thinner than the gonads of embryos at the same stage incubated at 25" C (Fig. 4A, compare with Fig. 3A). The volume of the medulla was significantly reduced due to a strong inhibition of the initial sex cords. Most germ cells were in the anlage of the cortex derived from the germinal epithelium, but some of them remained in the medulla (Fig. 4A). Control embryos maintained 6 days at 30" C reached a stage between 21 and 22. Their gonads were approximately twofold bigger than at stage 19 but presented the same structure : the medulla, devoid of epithelial cords, was composed of a mass of small cells and of some larger germ cells; in the differentiating cortex, germ cells were dividing but had not yet entered meiosis (Fig. 48). In embryos shifted for 6 days at 25" C, gonads exhibited a cortex similar to that existing in controls. However, the volume of the medulla was greater due to a more significant mass of cells from which some epithelial cords were beginning to form; these cords indicating a slight masculinization of the gonads (Fig. 4C, compare with Fig. 4B).
Discussion In embryos of the turtle Emys orbicularis, oestrogens have been shown to be involved insonadal differentiation and temperature has been expected to act, directly or indirectly, on the regulation of P-450 arom synthesis [4]. Results presented in this study show that changes in gonadal aromatase activity induced by shifts of temperature from a masculinizing to a feminizing temperature and vice versa require several days. Thus, in shifts from 25" C (masculinizing) to 35" C (highly feminizing) with different times of exposure at 35" C, the response is an exponential function: the increase in gonadal aromatase activity, not detectable at 1 and 2 days, becomes marked (threefold) at 4 days, and considerable (seventyfold) at 8 days. In shifts from 30" C (feminizing) to 25"C, the gonadal aromatase activity after a 6 day transfer does not fall immediately to a testicular level, but continues to increase slightly. These data agree with an effect of temperature on P-450 arom synthesis. The response observed in the first type of experiment reflects amplification of the enzyme production as the duration of exposure at feminizing temperature increases. The second type of experiment reflects the continuation of the enzyme production for a certain time after transfer from feminizing to masculinizing temperature. Several factors are known to modify the aromatase activity in other systems, such as human adipose cells [7, 211 and human and rat ovarian granulosa cells [lo, 20, 221. Some of these factors, including gonadotropins,
cyclic AMP and forskolin, increase aromatase activity [5, 7, 9, 10, 201 whereas other factors including prolactin and epidermal growth factor inhibit it [12, 21, 221. In most cases, changes in activity are correlated with changes in the levels of mRNA encoding P-450 arom [9, 12, 20-221. In Emys orbicularis, temperature-induced changes in synthesis and activity of P-450 arom could also result from changes in the levels of mRNA encoding the enzyme. In this view, regulation of expression of the P-450 arom gene would be temperature-sensitive. The expression of P-450 arom gene in human tissues has been shown to be regulated by tissue-specific promoters [13, 141. In Emys orbicularis as in other vertebrates, the sites of steroid synthesis in gonads are not the same in embryos as in adults. In embryos, steroids are synthesized in the epithelial cells (future Sertoli cells) of the testicular cords (or tubes) in testes, and in the cells derived from the medullary sex cords in ovaries [15]. Therefore, in Emys orbicularis, the promoter responsible for the expression of P-450 arom in gonads could be different in embryos and in adults, leading to different responses to factors regulating P-450 arom synthesis. The transcriptional initiation complex could be thermosensitive, or a thermosensitive factor could be implicated in the formation of this complex in embryos, but not in adults. However, temperature could act upstream on regulation of the expression of another gene. We had previously suggested that the expression of the gene encoding the anti-Mullerian hormone (AMH) could be thermoregulated [4]. Indeed, in mammals, AMH inhibits aromatase activity in differentiated fetal ovaries [24]. Likewise, partially purified chick AMH represses the aromatase activity in homospecific embryonic ovaries as well as in 16-day-old rat fetal ovaries. Also purified bovine AMH exhibits an anti-aromatase activity upon ovaries of Emys orbicularis embryos at stage 24, i.e. after the thermosensitive period [3]. In phenotypic males of Emys orbicularis, the regression of Miillerian ducts begins at stage 22, i.e. at the end of the thermosensitive period, and continues up to hatching [I 51. High amounts of AMH at these stages could explain the failure to increase aromatase activity by shifts of temperature from 25" C to 35" C [2]. At 25" C, lower amounts of the hormone are probably produced earlier, during the thermosensitive period and may downregulate P-450 arom synthesis. However, the expression of AMH gene itself does not appear to be temperature-sensitive. Indeed, when eggs are shifted from 25" C to 30" C during the thermosensitive period, as early as stages 16 to 19, Miillerian ducts degenerate in some individuals [ 181, showing that AMH can be produced at 30" C. In male chick embryos, raising the incubation temperature from 38" C to 40.5" C results in the retention of both Miillerian ducts [23]. This phenomenon does not appear as a consequence of a decrease of AMH secretion [25]. It could be due to a slight increase of aromatase activity and therefore of oestrogen production in the gonads. Indeed, oestrogens have been shown to prevent Mullerian duct regression [ll]. It was previously shown that in Emys orbicularis shifts from 30" C (feminizing) to 35" C (even more feminizing) increase aromatase activity, during as well as after the thermosensitive period 121. This response indi-
18
cates that high temperatures activate expression rather than prevent repression of P-450arom gene. Recently, the implication of P-450 arom enzyme on gonadal differentiation has been clearly demonstrated in chickens [6]. As already suggested [4],the regulation of expression of P-450arom gene probably plays a key role in sex differentiation of gonads in all vertebrates by regulating the levels of oestrogens in gonads and, therefore, allowing their male or female differentiation. Our study shows that in Emys orbicularis, changes in gonadal structure are well correlated with changes in gonadal aromatase activity. Testicular cords differentiate and the germinal epithelium regresses while aromatase activity remains low. Inversely, testicular cords regress and an ovarian cortex develops while aromatase activity increases. Thus, gonads of embryos incubated at 25" C up to stage 19 and then transferred at 30" C for 6 days, exhibit an ovarian-like cortex and anlagen of medullary epithelial cords indicating that they would have evolved towards ovotestes. Aromatase activity in these gonads is, as expected, higher than in typical testes at 25" C but lower than in typical ovaries at 30" C. Incubation of eggs within the transitional range of temperature between male- and female-producing temperatures (28-29" C)has been shown to result in phenotypic males, phenotypic females and intersexes [ 161. Therefore, the turtle Emys orbicularis offers a good model to define the different levels of aromatase activity and consequently of oestrogens - at which a testis, an ovotestis or an ovary does differentiate. Acknowledgements. We thank M. Girondot for his help with the figures and L. Guillon for her help with the manuscript. Animals were captured with permission of the French Environment Ministry.
References 1.Ackerman GE, Smith ME, Mendelson CR, Mac Donald PC. Simpson ER (1981) Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J Clin Endocrinol Metab 53:412417 2. Desvages G , Pieau C (1992)Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs. J Steroid Biochem Molec Biol41:851-853 3. Di Clemente N, Ghaffari S, Pepinsky RB, Pieau C, Josso N, Cate RL, Vigier B (1992) A quantitative and interspecific test for biological activity of anti-Mullerian hormone: the fetal ovary aromatase assay. Development 114:721-727 4.Dorizzi M, Mignot TM, Guichard A, Desvages G, Pieau C (1991) Involvement of oestrogens in sexual differentiation of gonads as a function of temperature in turtles. Differentiation 47:9-1 7 5.Dorrington JF, Moon YS,Armstrong DT (1975)Estradiol-17p biosynthesis in cultured granulosa cells from hypophysectomized immature rats: stimulation by follicle-stimulating hormone. Endocrinology 97: 1328-1 331 6.Elbrecht A, Smith RG (1992)Aromatase enzyme activity and sex determination in chickens. Science 255 :467-470 7. Evans CT, Corbin CJ, Saunders CT, Merril JC, Simpson ER, Mendelson CR (1987)Regulation of oestrogen biosynthesis by
human adipose stromal cells: effects of dibutyryl cyclic AMP, epidermal growth factor, and phorbol ester on the synthesis of aromatase cytochrome P-450.J Biol Chem 262:6914-6920 8.Ewert MA, Legler MA (1978) Hormonal induction of oviposition in turtles. Herpetologica 34:314-31 8 9. Fitzpatrick SL, Richards JS (1991)Regulation of cytochrome P-450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129~1452-1462 10. Hsueh AJW, Adashi EY, Jones PBC, Welsh TH (1984) Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5 :76-1 27 11. Hutson J, Ikawa H, Donahoe PK (1982)Oestrogen inhibition of Miillerian inhibiting substance in the chick embryo. J Pediatr Surg 17:953-959 12. Krasnow JS, Hickey GJ, Richards JS (1990)Regulation of aromatase mRNA and estradiol biosynthesis in rat ovarian granulosa and luted cells by prolactin. Mol Endocrinol4: 13-21 13.Mahendro MS, Means GD, Mendelson CR, Simpson ER (1991) Tissue-specific expression of human P-450 arom : the promoter responsible for expression in adipose tissue is different from that utilized in placenta. J Biol Chem 266:11276 11281 14. Means GD, Kilgore MW, Mahendroo MS, Mendelson CR, Simpson ER (1991)Tissue-specific promoters regulate aromatase cytochrome P-450 gene expression in human ovary and fetal tissues. Mol Endocrinol 5:2005-201 1 15. Pieau C (1974)Differenciation du sexe en fonction de la temperature chez les embryons d'Emys orbiculuris L. (ChClonien); effets des hormones sexuelles. Ann Embryol Morphog 7:365-394 16. Pieau C (1976)Donnees recentes sur la differenciation sexuelle en fonction de la temperature chez les embryons d'Emys orbiculurk L. (Chelonien). Bull SOCZoo1 Fr 101 (Suppl4):46-53 17. Pieau C (1978) Effets de temperatures d'incubation basses et elevees sur la diffkrenciation sexuelle chez des embryons d'Emys orbiculuris L. (Chelonien). CR Acad Sci Paris. krie D 286:121124 18.Pieau C,Dorizzi M (1981)Determination of temperature sensitive stages for sexual differentiation of the gonads in embryos of the turtle Emys orbicularis L. J Morphol 170:373-382 19.Raynaud A, Pieau C (1985) Embryonic development of the genital system. In: Gans C, Billett F (eds) Biology of the reptiha. Vol XV, Development B. J Wiley and Sons, New York, pp 149-300 20. Richards JS,Hickey GJ, Chen S, Shively JE, Hall PF, GaddyKurten D, Kurten R (1987) Hormonal regulation of estradiol biosynthesis, aromatase activity and aromatase mRNA in rat ovarian follicles and corpora lutea. Steroids 50: 393-409 21. Simpson ER, Merrill JC, Hollub AJ, Graham-Lorence S, Mendelson CR (1989) Regulation of estrogen biosynthesis by human adipose cells. Endocr Rev 10:136148 22. Steinkampf M,Mendelson CR, Simpson ER (1988)Effects of epidermal growth factor and insulin-like growth factor -I on the levels of mRNA encoding aromatase cytochrome P-450 in human ovarian granulosa cells. Mol Cell Endocrinol 59:9399 23.Stoll R (1948)Action de quelques agents physiques et chimiques sur les canaux de Miiller de I'embryon de Poulet. Arch Anat Microsc Morphol Exp 37:333-346 24. Vigier B, Forest MG, Eychenne B, Bezard J, Garrigou 0, Robel P, Josso N (1989)Anti-Miillerian hormone produces endocrine sex-reversal of fetal ovaries. Proc Natl Acad Sci USA 86:36843688 25. Weniger JP, Thiebold JJ (1981)On the mechanism of the retention of the Miillerian ducts in the chick embryo incubated at a temperature above normal. Anat Embryol 163:345-350 26. Yntema CL (1968)A series of stages in the embryonic development of Chelydra serpentina. J Morphol 125:219-252