Correlation among Thermosensitive Period, Estradiol Response, and Gonad Differentiation in the Sea...

Correlation among Thermosensitive Period, EstradiolResponse, and Gonad Differentiation in the Sea TurtleLepidochelys olivacea

Horacio Merchant-Larios,* Salvador Ruiz-Ramirez,† Norma Moreno-Mendoza,*and Alejandro Marmolejo-Valencia**Department of Cell Biology, Instituto de Investigaciones Biomedicas, UNAM, Apartado Postal 70228, Mexico, D.F.,Mexico 04510; and †Centro de Ecologıa Costera, S. P. Melaque, Universidad de Guadalajara, Guadalajara, Mexico

Accepted May 28, 1997

Reptile embryos with temperature sex determinationhave a thermosensitive period (TSP). The finding thatexogenous estradiol (E2) overcomes the effect of male-promoting temperature led to the idea that temperaturemay regulate estrogen concentration in the gonad duringTSP. Since interspecific variations in TSP and in theeffect of exogenous E2 exist, we undertook a study in theolive ridley Lepidochelys olivacea. Four parameters werecorrelated: the TSP (time dimension), the thermosensi-tive stages (rate of development), gonad development(histological aspect), and the estradiol response. Twokinds of experiments were performed: (1) Eggs wereshifted once, at different stages of development, from amale-promoting temperature to a female-temperature (orvice versa) for the remainder of development. (2) Eggs atmale-promoting temperature were treated once with 6 or12 mg of estradiol (E2) at various times of incubation.Sex ratio was established around hatching in eachexperimental series. We found that the temporal dimen-sion of the TSP was around 7 days (Days 20–27 ofincubation) at a male-promoting or a female-promotingtemperature. The rate of development of the wholeembryo and gonadal growth was faster at female-promoting temperature than at male-promoting tempera-ture. Formation of the genital ridge began at stage 21–22and histological differentiation of the gonads occurredaround stage 26–27. Although these stages coincidedwith the TSP, at male-promoting temperature the thermo-

sensitive stages occurred earlier (from stages 20–21 tostages 23–24) than at female-promoting temperature(from stages 23–24 to stages 26–27). Thus, at malepromoting-temperature, sex was determined in embryoswith incipient or undifferentiated gonads. In contrast, E2treatment continued to feminize the gonads of embryosat a male-promoting temperature beyond the TSP up tostage 25–26, but the E2-induced ovaries were signifi-cantly smaller than temperature-induced ovaries. It issuggested that the doses of E2 used were higher than theconcentration of endogenous E2 required for normal sexdetermination. The lack of correlation between sexdetermination and gonad differentiation suggests thatirreversible molecular processes underlying sex determi-nation occur earlier at male- than at female-promotingtemperature. Results suggest that the male sex may bethe default state and that the female condition must beimposed upon it. r 1997 Academic Press

Sexual differentiation of the gonads is dependent onincubation temperature in five sea turtle species: Eret-mochelys imbricata (Dalrymple et al., 1985), Lepidochelysolivacea (Morreale et al., 1982) Caretta caretta (YntemaandMrosovsky, 1980, 1982), Chelonia mydas (Miller andLimpus, 1980, Morreale et al., 1982), and Dermochelyscoriacea (Rimblot et al., 1985). Pivotal temperatures(temperature during incubation at constant tempera-ture which gives 50% individuals of each sexual

General and Comparative Endocrinology 107, 373–385 (1997)Article No. GC976946

373

0016-6480/97 $25.00Copyright r 1997 by Academic PressAll rights of reproduction in any form reserved.

phenotype; Mrosovsky and Pieau, 1991) are known forthe last three species and have been placed around 29°.Temperatures around 27° or 32° are male- or female-promoting, respectively. In C. caretta, it was found thatthe thermosensitive period (TSP) was at stages 21–26(Yntema and Mrosovsky, 1982; Harry and Williams,1991).In L. olivacea, a descriptive study of the morphology,

ultrastructure and some histochemical aspects on theestablishment of the genital ridge and gonads incu-bated at male- or female-promoting temperatures wasmade but did not attempt to establish the TSP (Mer-chant-Larios et al., 1989). In D. coriacea, it has beenfound that higher aromatase activity at female- than atmale-promoting temperature may correspond to TSP(Desvages et al., 1993).Administration of exogenous estradiol to embryos

incubated at masculinizing temperatures overcame theeffect of temperature and resulted in all-femalehatchlings in several reptiles with temperature sexdetermination (TSD) (Bull et al., 1988; Crews et al., 1991;Dorizzi et al., 1991; Wibbels et al., 1991, 1993; Pieau etal., 1994). Moreover, treatment of eggs with aromataseinhibitors incubated at feminizing temperature dis-rupted ovary differentiation or induced testis differen-tiation at temperatures that otherwise yielded bothsexes (Lance and Bogart, 1992; Rhen and Lang, 1994;Crews and Bergeron, 1994; Dorizzi et al., 1994, 1996;Wibbels and Crews, 1994). The sensitive period forestrogen effects on gonadal development apparentlycoincided with the TSP which, in turn, coincided withthe establishment of an undifferentiated gonad and theonset of its histological sex differentiation (Gutzke andChymiy, 1988; Raynaud and Pieau, 1985; Dorizzi et al.,1991; Wibbels et al., 1991, 1993). However, interspecificvariations have been found and estrogen effects on seaturtles are not available.The TSP implies at least three related but different

aspects: the temporal dimension, the stage of develop-ment, and the histological differentiation of the gonad.Since the rate of embryonic development is dramati-cally affected by temperature (Yntema andMrosovsky,1980; Miller, 1985), and temperature shifts affect differ-ent stages of development depending on the back-ground temperature (Yntema, 1979; Pieau and Dorizzi,1981), the TSP (temporal dimension) and the thermo-sensitive stages may be different. Consequently, if the

developmental rate of the gonad follows the develop-mental rate of the embryo, the TSPmay affect differentstages of its histological differentiation at male- orfemale-promoting temperatures. Therefore, a compre-hensive study correlating the temporal dimension, thestage of development, and the histological differentia-tion of the gonad in different species will be the basis tounderstand the physiological basis of temperature-dependent sex determination.In the current work, we undertook a study correlat-

ing the TSP, the thermosensitive stages, and the histo-logical differentiation of the gonad in the sea turtle L.olivacea. The operational criterion for the TSP wasbased on the response of the embryos incubated atconstant temperature to one shift from male-promot-ing temperature to female-promoting temperature orvice versa. Moreover, the effect of exogenous estradioland feminizing temperature were compared. Our find-ings lay the ground work for our studies concernedwith the steroidogenic pattern in developing embryosof L. olivacea.

MATERIALS AND METHODS

Animals. L. olivacea eggs were obtained from ‘‘LaEscobilla’’ beach (96° 278169W, 15° 408369N), Oaxaca,Mexico, on the night they were laid and transported toMexico City within 12 hr by ground transportation.This study used eggs from 12 clutches obtained be-tween 1991 and 1995. After arrival, eggs were placed incovered plastic trays filledwithmoistened vermiculite.Containers of eggs from each clutch were placed inincubators at either 276 0.5° (male-promoting tempera-ture) or 32 6 0.1° (female-promoting temperature) fordifferent experiments.Experiments on critical periods. Nine experiments

using a total of 10 clutches (100–110 eggs/clutch) wereperformed. Containers with 10 eggs each were trans-ferred at different days of incubation from male-promoting temperature to female-promoting tempera-ture or the converse, from female-promotingtemperature to male-promoting temperature, and keptthere until hatching. In each experimental series, around80 eggs were used and four to eight exchanges weremade at different times. Since most exchanges wererepeated one or more times in different experiments,

374 Merchant-Larios et al.

Copyright r 1997 by Academic PressAll rights of reproduction in any form reserved.

eggs from several clutches were used for each ex-change.Controls: at least 20 eggs were left without transfer

in every experimental series to certify the stability ofincubation conditions for promoting 100% male orfemale hatchlings. One experimental series was dis-carded (95 eggs) where three females were found in theincubator set at male-promoting temperature. One ortwo embryos from each temperature were harvested atevery exchange, their gonadswere processed for histol-ogy, and the stage of development was establishedaccording to Miller’s criteria (1985).Estradiol (E2) treatment. Eggs from five clutches

were used for these experiments. Groups of 10–30 eggsincubated at 27 6 0.5° were treated once with 6 or 12µg of E2 diluted in 3 µl of oil and replaced into themale-promoting temperature. Simultaneously, 5 eggswere treated only with oil. Vehicles with or without E2were administered through the eggshell by droppingthe solution in a plastic ring sealed with paraffin andplacing it on the most vascularized region near theembryo. All groups were kept at male-promotingtemperature until hatching.Histology. Sex was determined by the microscopic

structure of the gonad. Samples were fixed withKarnovsky’s solution and embedded in Epon, as previ-ously described (Merchant-Larios et al., 1989). To com-pare the feminizing effects of E2 and temperature, amorphometric study was undertaken: cross sectionsfrom the medial region of the ovaries of hatchlingswere analyzed. Images obtained with a camera lucidawere outlined on a paper and the area of the cortex andmedulla were measured with a planimeter. Gonadsfrom 26 E2-treated embryos and 26 embryos incubated

FIG. 1. Four pictures showing the different developmental rate ofembryos of L. olivacea atmale (left side)- or female-promoting (right side)temperatures. (A) 20 days: stages 21 and 24. (B) 27 days: stages 24 and25–26. (C) 32 days: stages 24–25 and 27–28. (D) 40 days: stages 26–27 and29–30. Two stages for a single sample mean that mixed characteristicswere found according to the morphological criteria for marine turtlesproposed by Miller (1985), in which L. olivacea was omitted. Bar, 2 cm.

TABLE 1

Correspondence between Days of Incubation and DevelopmentalStages at Male- and Female-Promoting Temperatures in L. olivacea

No. days Stage at 27 6 0.5° Stage at 32 6 0.1°

15 18–19 21–2220 20–21 23–2424 22–23 24–2527 23–24 25–2632 24–25 27–2835 25–26 28–2940 26–27 29–3045 27–28 31

Temperature, Estradiol, and Gonad Differentiation 375


at female-promoting temperature were analyzed.Means and standard deviations were calculated foreach experimental condition. Student’s t test wasapplied using ,0.05.

RESULTS

Embryos incubated at 32° developed faster thanembryos incubated at 27° (Table 1 and Fig. 1). After 20days of incubation, the embryos incubated at highertemperature (female-promoting) reached stages 23–24,whereas at lower temperature (male-promoting) theyreached stages 20–21, around two or three stagesbehind (Fig. 1A). After 27 days, the embryos incubatedat female-promoting temperature were at stages 25–26,whereas those at male-promoting temperature hadreached stages 23–24 (Fig. 1B). After 32 days at female-promoting temperature the embryos reached stages27–28, whereas at male-promoting temperatures theembryos were at stages 24–25 (Fig. 1C). Embryosincubated at female- or male-promoting temperaturesfor 40 days were at stages 29–30 or 26–27, respectively.Embryos developing at feminizing temperature hatchedaround Day 45, while those developed at masculiniz-ing temperature were still at stages 27–28. The latterhatched after around 63–65 days of incubation.In correlation with the faster development of whole

embryos at female-promoting temperature than atmale-promoting temperature, the gonads grew fasterat female-promoting temperature (Fig. 2). The histologi-cal aspect of the genital ridges and gonads at differentstageswas studied in cross sections taken from samplesat 20, 27, 32, and 40 days from different regions.Figures 2 and 3 show the histology of gonads fromembryos at incubation. The histological changes forthe establishment of an undifferentiated gonad weresimilar at both male- and female-promoting tempera-tures: at stages 21–23, proliferating cells became con-densed as clusters near the coelomic epithelium of the

genital ridge (Fig. 2A). Then, around stages 24–26,medullary sex cords were formed and remained con-tinuous with the surface epithelium in some regions(Figs. 2B–2D, 3A, and 3B). Histological sex differentia-tion of the gonads was evident at or after stages 26–27.Gonads from embryos incubated at male-promotingtemperature showed a thin surface epithelium andprominent medullary cords at stage 27 (Fig. 3C). Ingonads at female-promoting temperature, the surfaceepithelium became thick and contained most of thegerm cells, although at stage 27 the medullary cordswere still evident (Fig. 3B). Medullary cords appearedfragmented in female gonads at stage 29 (Fig. 3D).

Temperature Exchanges and Sex DeterminationPeriods

Table 2 summarizes the results of response to oneshift of temperature at different developmental stages.All embryos initially incubated at male-promotingtemperature up to stages 17–19 were feminized whentransferred to 32°C. Between stages 20 and 23 adecreasing number of embryos incubated at male-promoting temperature responded to the female-promoting temperature: 73.6, 57.5, and 47.3%, respec-tively, responded at stages 20, 21, and 22, whereas only7.2% were feminized when transferred at stage 23.From stage 24 onward all hatchlings from embryosinitially incubated at 27° and then shifted to 32°developed as males.In contrast, the response of embryos initially incu-

bated at female-promoting temperature covered moreadvanced stages than embryos incubated at male-promoting temperature: all hatchlings from embryosinitially incubated at female-promoting temperatureand transferred to male-promoting temperature atstage 23, or earlier, developed intomales.When shiftedat stages 24, 25, or 26, the percentage of embryos whichresponded to the male-promoting temperature was94.5, 62.3, and 3.1%, respectively. Finally, embryos

FIG. 2. Histology of the gonads of embryos incubated at male (Aand C)- or female (B and D)-promoting temperature.A–B and C–D show crosssections of gonads at 20 and 27 days of incubation, respectively. This period of around 7 days corresponds to the TSP. Although at thefemale-promoting temperature the gonads are larger (stages 24 and 26) than at the male-promoting temperature (stages 21 and 24), they appearmorphologically undifferentiated. Prominent medullary cords (MC) and several germ cells (arrows) in the coelomic epithelium are observed ingonads from embryos incubated at both temperatures. Bar, 100 µm.



incubated at 32° up to stage 27 developed as femalesafter shift to male-promoting temperature.The different response to one shift of temperature

depending on temperature of incubation (male- orfemale-promoting temperatures) led us to investigatethe correlation between stages of development and

days of incubation. As shown in Fig. 4, although thethermosensitive stages appeared different, the thermo-sensitive periods (temporal dimension) were similar.During the first 20 days of incubation, all embryosresponded to the shifted temperature regardless of thestage of development reached during this period.Thereafter, during the next 7 days, a decreasing num-

FIG. 3. Aand B are gonads fixed at 32 days at male- or female-promoting temperatures, respectively. They were from embryos at stages 25 (A)and 27 (B) corresponding to the SDP. At female-promoting temperature (B) the gonad has initiated medullary cord fragmentation (stars); a thickcortex with abundant germ cells (arrow) indicates the start of ovarian differentiation. At male-promoting temperature (A), the gonad still has anundifferentiated aspect with medullary cords (MC) and several germ cells (arrows) in the coelomic epithelium. Pictures C and D show gonadsfixed after 40 days of incubation at stages 27 and 29 at male- or female-promoting temperature, respectively. The testis (C) shows a thin cortexand conspicuous medullary cords (MC) surrounded by stromal tissue (S). Picture D shows an ovary with a thick cortex (CX) and remnants ofmedullary cords (stars). Bar, 100 µm.

TABLE 2

Critical Periods (C.P.) for Sex Determination of L. olivacea

StageaNo.eggsb

No.hatchlings

No.males

No.females

%Responsec C.P.

Eggs shifted from 27 6 0.5° to 32 6 0.1°17 20 (2) 11 0 11 100 UP18 20 (2) 17 0 17 100 UP19 20 (2) 11 0 11 100 UP20 30 (3) 19 5 14 73.6 TSP21 50 (5) 33 14 19 57.5 TSP22 80 (4) 76 40 36 47.3 TSP23 100 (5) 97 90 7 7.2 TSP24 40 (4) 34 34 0 0 SDP25 10 (1) 8 8 0 0 SDP26 20 (2) 17 17 0 0 SDP27 10 (1) 9 9 0 0 SDP

StageNo.

EggsbNo.

HatchlingsNo.Males

No.Females

%Responsea C.P.

Eggs shifted from 32 6 0.1° to 27 6 0.5°17 20 (2) 19 19 0 100 UP18 20 (2) 11 11 0 100 UP19 20 (2) 11 11 0 100 UP20 40 (4) 18 18 0 100 UP21 20 (2) 9 9 0 100 UP22 30 (3) 18 18 0 100 UP23 20 (2) 18 18 0 100 UP24 100 (5) 74 70 4 94.5 TSP25 100 (5) 85 53 32 62.3 TSP26 40 (4) 32 1 31 3.1 TSP27 30 (3) 22 0 22 0 SDP

a According with Miller’s criteria (1985). UP, undeterminedperiod. TSP, temperature-sensitive period. SDP, sex-determinedperiod.

b Number of clutches.c Gonadal sex was determined by histology a few days after

hatching.

FIG. 4. Correlation among sex determination, days of incubation,and stage of development at female- or male-promoting tempera-tures. During the first 20 days of incubation, all embryos respondedto the shifted temperature and it was named the undeterminedperiod (UP). Thereafter, at around 7 days, a decreasing number ofembryos responded to the shifted temperature; this is the thermosen-sitive period (TSP). Afterward, none of the embryos responded tothe shifted temperature and this was considered the sex-determinedperiod (SDP). While at the female-promoting temperature(32 6 0.1°C) the TSP was from stages 23–24 to stages 26–27, at themale-promoting temperature (27 6 0.5°C) the TSP was from stages20–21 to stages 23–24. Moreover, the SDP was initiated at stages27–28 or 24–25 at female- or male-promoting temperature, respec-tively.



ber of embryos responded to the shifted temperatureat different stages of development: embryos at male-promoting temperature during these 7 days were atstages 20–23, whereas embryos at female-promotingtemperaturewere at stages 24–26. Finally, after approxi-mately 27 days of incubation, all embryos from bothmale- and female-promoting temperatures were unre-sponsive to the shifted temperature and maintainedthe sex expected with the initial temperature.Assuming that the percentage of embryos respon-

sive to the shifted temperature corresponded to em-bryos where sex was not determined by the initialtemperature, the embryonic development was dividedinto three periods: undetermined (UP), thermosensi-tive (TSP), and sex-determined (SDP) periods (Table 2and Fig. 4). During TSP, the embryos incubated atmale- or female-promoting temperatures were at stages20–23 or 24–26, respectively. At these stages the gonadswere histologically undifferentiated (Fig. 2). The onsetof SDP was around stage 24 for embryos incubated atmale-promoting temperature and around stage 27 forembryos incubated at female-promoting temperature.Although at the female-promoting temperature thehistological differentiation of the female gonad wasdetectable, at male-promoting temperature, the go-nads were still undifferentiated (Figs. 3A and 3B).

Effect of Estradiol (E2) at Different DevelopmentalStages

Embryos incubated at male-promoting temperatureup to stages 24–25 were feminized by E2 treatment.Although some embryos may still respond to exog-enous E2 at stage 26, they did not respond after thisstage (Table 3). The histological study of E2-treatedgonads revealed a reduction or complete disappear-ance of the medullary cords and a thick cortex, whichsuggest ovarian differentiation (Fig. 5). The morpho-metric analysis showed, however, that ovaries fromE2-treated hatchlings were significantly smaller thanovaries from hatchlings incubated at female-promot-ing temperature (Fig. 6).

DISCUSSION

There are some discrepancies between the resultsobtained in the present work and those published

previously (Merchant-Larios et al., 1989; Merchant-Larios and Villalpando, 1990). There is no correspon-dence between the developmental stages since inprevious studies we used the developmental stagesproposed by Cratz (1982) for L. olivacea, which aredifferent from Miller’s stages (Miller, 1985) used in thepresent study. Our conclusion that the female gonaddifferentiates earlier than themale gonadwas based onthe presence of germ cells on the surface epithelium inmale gonads up to hatching, whereas medullary cordsare fragmented in female gonads before hatching.However, in the current study, which uses far moresamples from embryos incubated under more stableconditions, it was found that histological differentia-tion occurs at stages 26–27 at both male- and female-promoting temperatures. The interesting finding inour recent study was that sex determination at male-promoting temperature occurs at stage 24, before thehistological differentiation of the gonad.It is known that in reptiles with TSD, temperature

changes affect sex determination only during a certainperiod of incubation. Mrosovsky and Pieau (1991)defined the thermosensitive period (or thermosensi-tive stages) as ‘‘that interval when variation of tempera-ture (any kind of temperature variation) has beenshown to have some significant effect (any kind ofeffect) on sex ratio.’’ Apart from the interspecificvariations that may exist, the sensitive periods alsovary with the experimental procedures. Nevertheless,there is agreement that reptiles with TSD have tempera-ture-sensitive periods placed around the middle one-

TABLE 3

Effect of Estradiol (6 µg/egg) Applied to Embryos at DifferentStages of Development in L. olivacea Incubated at 27 6 0.5°

Stage No. eggsa No. males No. females No. ovotestis

20 20 (2) 1 14 221 10 (1) 0 4 022 10 (1) 0 8 223 20 (2) 0 17 023–24 10 (1) 0 10 024 30 (2) 0 20 225 10 (1) 0 9 025–26 10 (1) 3 5 026 20 (2) 14 0 027 10 (1) 6 0 028 10 (1) 5 0 0

a Number of clutches.



third to one-half of embryonic development (Yntema,1979; Bull and Vogt, 1981; Pieau and Dorizzi, 1981;Yntema and Mrosovsky, 1982; Ferguson and Joanen,1983; Bull, 1987; Webb et al., 1987; Wibbels et al., 1991).Therefore, clearly, there are two periods not sensitiveto temperature: at the beginning and at the end ofembryonic development.During the first 20 days of incubation, all L. olivacea

embryos responded to one shift of temperature (male-to female-promoting temperatures or vice versa). Thus,during the first days of incubation, sex was not yetdetermined and all embryos responded to one shift oftemperature when they were left at the new tempera-ture until hatching. Double shifts of temperature dur-ing this period when all embryos were undetermineddid not modify the sex promoted by the backgroundtemperature. Thereafter, sex began to be determined insome embryos and gradually fewer embryos of theexperimental group responded to the shifted tempera-ture. Finally, during the last days of incubation, allembryos were determined. That is, they were insensi-tive to the shifted temperature.The present results suggest that the physiological

basis for the lack of sensitivity to temperature shiftsduring the first and the last days of embryonic develop-ment are different. In double shift experiments, ‘‘insen-sitivity’’ during the first days of development refers tothe period when the embryos were sexually undeter-mined. In contrast, at late developmental stages, ‘‘insen-sitivity’’ to temperature is due to the fact that theembryos were sexually determined.

We divided the development of L. olivacea embryosinto three periods: UP, TSP, and SDP. It must beemphasized that these periods refer to groups and notto individual embryos. Thus, the gradual sex determi-nation of a series of embryos during TSP suggests thatthe embryos became determined at different stages,probably due to genotypic variations. If sex determina-tion is defined as ‘‘the establishment of an irreversiblemolecular process which leads to male or femaledifferentiation,’’ it can be postulated that TSP refers tothe time required to set this process in a series ofdeveloping embryos of a species. Developmental‘‘time,’’ however, has two implications: the temporaldimension and the stage of development. Since thelatter are expressed in terms of morphology, underly-ing molecular events may follow different rates.Most studies on thermosensitive periods are ex-

pressed in terms of thermosensitive stages of develop-ment (Pieau an Dorizzi, 1981; Yntema and Mrosovsky,1982; Bull and Vogt, 1981; Wibbels et al., 1991; Harryand Williams, 1991). In the present study, we foundthat the thermosensitive stages in L. olivacea, arebetween stages 21–22 and stages 26–27. These stagesare similar to the thermosensitive stages reported inCaretta caretta, another sea turtle (Yntema and Mros-ovsky, 1982; Harry and Williams, 1991). Our presentresults, however, suggest that in L. olivacea, the thermo-sensitive stages are different at male- and female-promoting temperatures. Embryos incubated at male-

FIG. 5. Small ovary from a hatchling treated in ovo with 12 µg ofestradiol on Day 20 of incubation at male-promoting temperature. Athick cortex with several germ cells (GC) and remanents of medul-lary cords (MC) can be seen. Bar, 100 µm.

FIG. 6. Morphometric study of gonads from hatchlings incubatedat male-promoting temperature 16 or 12 µg of estradiol (E2) andfrom hatchlings incubated at female-promoting temperature.



promoting temperature start sex determination at stages21–22, whereas embryos incubated at female-promot-ing temperature begin to be determined around stage24. Moreover, embryos initially incubated at male-promoting temperature did not respond to female-promoting temperature around stage 24, whereas em-bryos initially incubated at female-promotingtemperature did not respond later, around stage 27.Results suggest that sexual fate at the male-

promoting temperature became irreversible in all em-bryos two or three stages earlier than at the female-promoting temperature. Although less dramatic (oneor two stages earlier), similar findings have beenreported in two freshwater turtles, Emys orbicularis andGraptemys ouchitensis (Pieau and Dorizzi, 1981; Bulland Vogt, 1981). However, in Eublepharis maculariusand Trachemys scripta it was reported that the sexdetermination at female-promoting temperature be-comes irreversible prior to that at male-promotingtemperature (Bull, 1987; Wibbels et al., 1991). Althoughdifferences in the chronology of male and female sexdetermination may represent interspecific variation,they could also be related to the sex-determiningpotencies of the specific temperatures used in eachstudy (Wibbels et al., 1991).According with the current experimental proce-

dures, in L. olivacea, TSPwas placed aroundDays 20–27 ofincubation.During this period (temporal dimension), indi-vidual embryosmay reach the threshold of an irreversiblemolecular process necessary for sex determination. Ex-periments of Lang and Andrews (1994) in A. mississip-piensis involving shifts of 33° to 31° and vice versafound that both the male-promoting temperature andthe female-promoting temperature were equally effec-tive over the same length of time (around 20 days).Interestingly, in A. mississippiensis, sex is determined

one to two stages earlier at female-promoting tempera-ture than at male-promoting temperature. Although intheAmerican alligator (A.mississippiensis) low tempera-ture is female-promoting, whereas in the olive ridley(L. olivacea) it is male-promoting, in both species, sexdetermination occurs earlier in low-growing embryosat lower temperature than in fast-developing embryosat higher temperature. An interaction between geno-type and environmental temperature may explain thedifferent outcomes: asynchronous development of

whole embryos at male- or female-promoting tempera-tures may trigger signals for either male or femaledetermination depending on the species genotype(Mittwoch, 1992).Of particular interest, present results showed that

some embryos of L. olivacea start sex determinationaround Day 20 and all became determined around Day27 of incubation. Differences in the rate of embryonicdevelopment at male- or female-promoting tempera-tures were also reflected in differences between sexdetermination and the histological differentiation ofthe gonads at both temperatures. Whereas at female-promoting temperature, sex determination occurredwhen ovaries had already begun their histologicaldifferentiation (around stage 26), at male-promotingtemperature, sex determination occurred early, whenthe gonads were still undifferentiated (around stage24). This at first appears counterintuitive, as the growthand metabolic rate is clearly greater at the warmertemperature. A possible explanation is that the malesex is the default state and that the female conditionmust be imposed upon it. The present results showedthat stages 24–25 mark the end or the onset of TSD atmale- or female-promoting temperature. Therefore, upto this stage an irreversible male-determining mecha-nism may be set in 100% of embryos at male-promotingtemperature, whereas in embryos incubated at female-promoting temperature, the female-determiningmech-anism may still be switched off.The lack of direct correlation between sex determina-

tion and histological differentiation of the gonad wasalso reported in A. mississippiensis. In this species, thethermosensitive period (shifts from 34° to 30° and viceversa, Ferguson and Joanen, 1983) occurs during go-nadal ridge stages and ends well in advance of thesexual differentiation of the gonads (Deeming andFerguson, 1988; Joss, 1989).Experiments on several reptiles with TSD have

shown that estrogens can overcome the effect ofmale-promoting temperatures and produce femalehatchlings. Furthermore, the use of compounds thatdisturb steroidogenesis, including aromatase inhibi-tors, has led to the idea that estrogen and temperatureact via a common physiological pathway (see reviews:Raynaud and Pieau, 1985; Crews et al., 1994; Dorizzi etal., 1996). Current results demonstrate that in L. oliva-



cea, as in other species, exogenous estradiol also‘‘feminized’’ the gonads of hatchlings incubated atmale-promoting temperature. Thus, exogenous estra-diol inhibited the development of medullary testicularcords, whereas the gonadal cortex was stimulated.Experiments in Emys orbicularis have shown that aro-matase activity is important for maintenance of differ-entiated ovaries after TSP (Dorizzi et al., 1996).Although in various species the sensitive stages to

exogenous estradiol correspond to the thermosensitiveperiod (Gutzke and Chymiy, 1988; Wibbels et al.,1991b; Dorizzi et al., 1991), we found that, in L. olivacea,the period of estradiol sensitivity was longer than thethermosensitive period from male- to female-promot-ing temperature. This may be explained by a doseeffect of the exogenous estradiol: at female-promotingtemperature, estradiol synthesis may be regulated at acritically lower concentration required for normal dif-ferentiation of the gonad than the concentrationsemployed in the present experiments. In fact, it wasfound that in the estradiol-treated embryos, the ‘‘femi-nized’’ gonads were considerably smaller than ovariesinduced by temperature. Abnormally small ovariesfrom estradiol-treated embryos, however, seemed torecover after hatching. It was found that in hatchlingsfrom estradiol-treated eggs, morphologically normalovaries developed sixmonths after hatching (Merchant-Larios et al., unpublished observations).It has been postulated that estrogen synthesis and,

therefore, aromatase activity may play a key role in sexdetermination of thermosensitive species (Pieau et al.,1994) but the actual mechanism remains to be eluci-dated. Species variations and different experimentalprocedures have complicated the finding of a commonmechanism if it exists at all. In Dermochelys coriacea,another sea turtle, aromatase activity was found to behigher at 30.5° (female-promoting temperature) than at27° (male-promoting temperature). When eggs wereshifted from 27 to 35° at different embryonic stages andexposed to 35° for 6 days, an increase in aromataseactivity was seen only between stages 23 and 27(Desvages et al., 1993). We found that in L. olivacea,embryos incubated at female-promoting temperatureare determined between stages 23–24 and 26–27. It willbe interesting to study if in this species gonadalaromatase activity is induced at female-promotingtemperature.

Collectively, interactions between temperature andgenetic factors may exist for sex determination in allTSD species. If enhanced aromatase activity is theinitial signal for ovary differentiation, at least twopossibilities can be postulated: (i) the enzyme aro-matase directly responds to temperature, and theestrogen produced exerts a positive feedback controlon the aromatase gene (Pieau, 1996) and (ii) a thermo-sensitive process (gene?) located upstream of thearomatase gene controls the female developmentalpathway, including the enhanced expression of thearomatase gene.The present findings that at male-promoting low

temperature sex determination occurs before that atfemale-promoting high temperature suggest that en-dogenous estrogens produced at female-promotinghigh temperature may prevent the expression of amale-determining gene in L. olivacea. Lack or lowconcentration of estrogen may allow accumulation of amale-determining gene product. On the other hand, inA. mississippiensis, exogenous E2 feminizes embryos atmale-promoting high temperature (Lang and An-drews, 1994), suggesting that, in this species, endog-enous estrogens are produced at female-promotinglow temperature. Thus, if feminizing estrogen is pro-duced at different temperatures (and/or developingrates), genetic predisposition may underlie differentmechanisms controlling aromatase activity.Moreover, since sex determination and morphologi-

cal differentiation of the gonad appear as dissociatedevents, female-promoting high temperature may en-hance estrogen production, which may prevent testisdifferentiation and lead the undifferentiated gonad toform an ovary.

ACKNOWLEDGMENTS

The Secretarıa de Pesca and Instituto Mexicano de Pesca gavepermission to collect eggs in La Escobilla, Oaxaca, a Mexican faunalpreserve.We thankMr. Jose G. Baltazar for technical assistance in thelaboratory and Biol. J. Vasconcelos, Director of the Centro Mexicanode la Tortuga, Mazunte, Oax., and Biol. Cuahutemoc Villanueva forassistance in the field work. Mrs. Isabel Perez-Montfort helped withthe English. This study was partially supported by the MexicanCONACYT (4037-N) and PAPIID-UNAM:IN209694.



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