temporal difference between testis and ovary...

600 S. GANESH ET AL.JOURNAL OF EXPERIMENTAL ZOOLOGY 283:600–607 (1999)

© 1999 WILEY-LISS, INC.

Temporal Difference Between Testis and OvaryDeterminations With Possible Involvement ofTestosterone and Aromatase in Gonadal Differentiationin TSD Lacking Lizard, Calotes versicolor

SUBRAMANIAM GANESH, BIBHA CHOUDHARY, AND RAJIVA RAMAN*Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University,Varanasi 221005, India

ABSTRACT In the garden lizard, Calotes versicolor, which lacks identifiable sex chromosomes,incubation temperature also does not have a deterministic effect on the gender. However, theembryos reared at high temperature (33–35°C) have a shorter duration of incubation as well asgonadal differentiation. In contrast, exogenous application of the male hormone testosterone toembryos at ambient temperature (28°C) results in almost all individuals with only testis. Thusthe testosterone treatment reverts genic females to males and accelerates the differentiation oftestis, a feature similar to the high-temperature treatment. Treatment of eggs with estradiol showsno difference from that seen in the untreated eggs. The present series of experiments was done toestablish the “window” of testosterone sensitivity and to understand the interaction between sexhormones and high temperature on gonadal differentiation. The period between day 5 and 15 ofembryonic development was the window period of testosterone sensitivity for sex reversal. Thisperiod coincided with the formation of the genital ridge and its differentiation into cortex andmedulla. Treatment of the 33°C-reared embryos with testosterone resulted in hatchlings of boththe sexes, in contrast to only males at the ambient temperature. In contrast, at the same tempera-ture (33°C), all the dihydrotestosterone (nonaromatisable testosterone)–treated embryos hatchedinto males. However, those given estradiol showed no sex bias regardless of the day of applicationand the concentration of drug. Eggs were also treated with aromatase inhibitor, CGS 16949 A, atambient temperature and at 33°C. All the 33°C eggs to which the drug was given on day 25hatched into males. These results suggest that though high temperature has no direct effect onsex determination in this species, it may have a stimulatory effect on aromatase activity, leadingto the conversion of the exogenously applied testosterone into estradiol and permitting ovariandifferentiation in the genic females. It also follows from the present report that the pathway oftestis formation in Calotes versicolor is triggered much earlier, and irreversibly, than that for theovary. J. Exp. Zool. 283:600�607, 1999. © 1999 Wiley-Liss, Inc.

Recent studies on the effects of exogenously ap-plied sex hormones, their agonists, and their an-tagonists on reptilian embryos have demonstratedthat in a number of species hormones influencethe differentiation of developing gonads (reviewedby Crews et al., ’94; Pieau, ’96). As a general rule,estrogen induces ovary in all the embryos, turn-ing them into female. Though seen predominantlyin the TSD (temperature-dependent sex determi-nation) species, this feature has also been ob-served in a few of those endowed with the sexchromosomes (CSD) (Bull, ’83). In the TSD spe-cies, even testosterone treatment at the male-pro-ducing temperature results in the female (Crews,’94; Pieau, ’96). However, administration of thenonaromatisable androgen dihydrotestosterone

(DHT) to Chelydra serpentina at an intermediatetemperature induces development of testis in allthe individuals, resulting in all male hatchlings(Wibbels et al., ’92). It has therefore been inferredthat the feminizing effect of testosterone in thisspecies is due to its aromatisation into estrogenby the enzyme aromatase. This suggestion is for-tified by the observation that coadministration of

Grant sponsors: Department of Science and Technology; theIndian Council of Medical Research; the University Grants Com-mission.

S. Ganesh’s present address: Lab for Neurogenetics, Brain ScienceInstitute (RIKEN), 2-1 Hirosawa, Wako-Shi, Saitama, 351-0198, Japan.

*Correspondence to: Rajiva Raman, Lab for Neurogenetics, BrainScience Institute (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. E-mail: [email protected]

Received 5 May 1998; Accepted 28 September 1998.

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GONADAL DIFFERENTIATION IN C. VERSICOLOR 601

testosterone and an aromatase inhibitor resultsin the development of a male even at the female-producing temperature (Crews, ’94; Jeyasuria etal., ’94). These observations lead to the sugges-tion that in TSD reptiles (1) estradiol is essentialfor the determination of female sex, (2) aromataseis the key enzyme in gonadal differentiation, and(3) its activity is regulated by the incubation tem-perature of eggs (Crews et al., ’94; Pieau, ’96).

In the Indian garden lizard, Calotes versicolor,which lacks the identifiable sex chromosomes, theincubation temperature also does not have a de-terministic effect on the gonadal phenotype (Ga-nesh and Raman, ’95). Most of the naturally bornhatchlings have an indifferent gonad until about7 to 10 days after birth. Nevertheless, when rearedat a higher temperature, the incubation period ismarkedly shortened and the gonads in the major-ity of hatchlings are fully differentiated in boththe sexes within a day or two of birth. In con-trast, the application of testosterone to embryosin the first half of development at ambient tem-perature induces testis development in almost allthe embryos, though the duration of incubationis not shortened. Estrogen treatment to the em-bryos, however, has no effect either on the gonadaldifferentiation or on the duration of incubation(Ganesh and Raman, ’95). The present series ofexperiments has been done to define the windowperiod of testosterone sensitivity and to under-stand the interactive effects of temperature andsex hormones on gonadal differentiation by ap-plying hormones to the eggs reared at high tem-perature.

MATERIALS AND METHODSThe procedure of collection and maintenance of

eggs was essentially the same as reported previ-ously for this species (Ganesh and Raman, ’95).The eggs were recovered from gravid femalescaught during the breeding season (July–August).For all the experiments, eggs from different fe-males were mixed together and reallotted into dif-ferent groups for various experiments. The eggswere maintained in moist sand at a constant in-cubation temperature (28°C—ambient; 33°C,35°C—high) in BOD incubators. All the hormones,testosterone (17β-hydroxy 3-oxo-4-androstene;Sigma, St. Louis, MO), dihydrotestosterone (DHT:5α-androstan-17β-ol-3-one; Sigma), β-estradiol(Sigma), and the aromatase inhibitor CGS 16949A (Ciba-Geigy, Summit, NS), were dissolved in90% alcohol at the required concentration, and 5µl of the solutions was applied topically to the eggs

at different stages of embryonic development. Un-treated eggs at 33°C and those treated with test-osterone at the ambient temperature (28°C)served as two different controls.

RESULTSWindow of testosterone sensitivity in embryo

In the previously reported experiments on theeffect of testosterone on gonadal differentiation inC. versicolor, the hormone was applied on days 5,10, and 15 of embryonic development, and themajority of hatchlings were born as male (Ganeshand Raman, ’95). To check up to which stage ofdevelopment was testosterone effective in sex re-versal, the treatment of the hormone was ex-tended to the 20-, 25-, and 30-day embryos (atambient temperature; 10 embryos each). The sexof the individual was established by examininghistological sections of the gonads of 8- to 10-day-old hatchlings; only about 50% of the hatchlingswere male on day 20 and 25, and a few of themhad clear ovary. Most of the eggs treated on day30 did not survive till hatching. This was in con-trast to the 5- to 15-day embryos, in which morethan 80% hatchlings were males and there wereno females. Thus the testosterone-sensitive periodof C. versicolor, the “window,” appears to be be-tween day 5 and 15 of embryonic development(Fig. 1). Histological sections of the mesonephros-gonadal complex (MGC) of the “window” werestudied in 5-, 10-, 15-, and 20-day embryos. Al-though MGC was seen from day 5 itself, the geni-tal ridge was first seen in the day 10 embryo as athickening on the ventromedian surface of themesonephros, which further enlarged and differ-entiated into cortex and medulla in the day 15samples. In the day 20 MGC, sex cords (primor-dial seminiferous tubule of testis) were formed inthe medulla, but the cortical region did not dif-ferentiate any further at this stage (Fig. 2). Thusthe testosterone-sensitive period of the embryo co-incided with the formation and further differen-tiation of the genital ridge. Also, the formation ofsex cords in the medulla indicated a step towardtestis formation much in advance of cortical de-velopment.

The effect of testosterone,dihydrotestosterone, and aromataseinhibitor on gonadal differentiation

at high temperatureThe untreated embryos at ambient temperature

as well as at 33°C showed different degrees of go-

602 S. GANESH ET AL.

nadal differentiation; many had either testis orovary, but a few had an indifferent gonad, retain-ing both testicular as well as ovarian anlage(Ganesh and Raman, ’95). However, in the tes-tosterone-treated (100 µg/egg; applied on day 10of incubation) ambient temperature group of eggs,the majority of hatchlings had only testis (11 outof 14; Table 1). Both these results were in agree-ment with those previously reported for this spe-cies (Ganesh and Raman, ’95). But unlike theprevious group of experiments (Ganesh andRaman, ’95), in which more than 80% of the 35°Ceggs had survived, administration of testosterone(100 µg/egg) to the 35°C eggs led to very high mor-tality (>90%; data not given). Hence the presentset of experiments was done on the eggs rearedat 33°C, and the one-time application of testoster-one was given in two concentrations (50 µg/eggand 100 µg/egg) at three different stages of em-bryonic development (on day 10, 15, and 20 of em-bryonic development; see Table 1). Althougheven in these groups the survival rate was onlyabout 50%, it was better than that with the 35°Cgroup. The surviving eggs hatched on day 41 ± 1irrespective of the concentration of the hormone

and the stage of treatment. The results summa-rized in Table 1 show two significant departuresfrom the controls (testosterone at ambient tempera-ture; 33°C untreated); individuals of both the sexeswere produced (sex ratio slightly skewed toward fe-male) and the ovary in certain females had largefollicles surrounded by numerous supporting cells(Fig. 3b). Such advanced ovarian differentiation wasnever seen in the 7- to 10-day-old hatchlings. In-stead, they were comparable to the ovary of about2-month-old hatchlings (see Fig. 3c). When the eggswere treated with the nonaromatisable DHT (20µg/egg and 30 µg/egg; on day 15), all the survivinghatchlings born to the 20 µg DHT /33°C) were male.All the 30 µg eggs died at different stages of devel-opment (see Table 1).

The difference between the results of testoster-one and DHT hinted that the aromatisation of tes-tosterone to estrogen could have a role in thetestosterone-induced feminization. Therefore, an-other set of experiments was done with the Cyto-chrome P450 aromatase inhibitor, CGS 16949 A,which was applied (5 µg/egg) to two groups of eggsincubated at 28°C and 33°C, either on day 15 orday 25 of incubation. All the hatchlings of the 28°C

Fig. 1. Idiogram displaying the window period of testoster-one sensitivity in embryos. The hatched area displays the win-

dow. The idiogram is based on cumulative results of the presentinvestigation as well as those of Ganesh and Raman (’95).


Fig. 2. Transverse sections of 10-, 15-, and 20-day em-bryos through their MGC. Note the extension of the genitalridge in the day 10 embryo (a), the differentiation of cortex

and medulla in the genital ridge of the day 15 embryo (b),and the formation of sex cords in 20-day-old embryos. gc, germcells; c, cortex; m, medulla; sc, sex cords. Magnification ×900.


eggs, regardless of the day of application, wereunaffected by the drug. The same was true of theday 15, 33°C samples. In contrast, all the hatch-lings from the day 25, 33°C eggs turned out to bemales (Table 2).

Effect of estradiol at ambient and hightemperature in gonadal differentiation

The above results indicated that the testoster-one-induced feminization at high temperature

could be due to precocious availability of estra-diol. Therefore, fresh experiments were done withestradiol at ambient and high temperature. Sincein the previously reported series of experimentsthe 5-, 10-, and 15-µg estradiol, administered to5, 10, and 15 days ambient temperature–rearedembryos, did not show any effect on sex determi-nation (Ganesh and Raman, ’95), we applied thehormone (16 µg/egg) on day 30 at ambient tem-perature. The results were identical to those pre-

TABLE 1. Gonads in the hatchlings of the eggs treated with androgens reared at ambient and 33°C temperature

Incubation No. of hatchlings Gonadal differentiation Mean incubationGroup temperature Treated Survived Testis Ovary Indifferent period (days)

No treatment 33°C 20 18 9 7 2 41 ± 1Testosterone treatment

On day 10100 µg/egg 28 ± 2°C1 15 14 11 0 3 52 ± 150 µg/egg 33°C 15 6 2 1 3 41 ± 1

100 µg/egg 33°C 15 10 1 5 4 41 ± 1On day 15

50 µg/egg 33°C 15 6 1 3 2 41 ± 1100 µg/egg 33°C 15 6 1 1 4 41 ± 1

On day 2050 µg/egg 33°C 15 11 3 4 4 41 ± 1

100 µg/egg 33°C 15 4 3 0 1 41 ± 1Dihydrotestosterone

treatmentOn day 15

20 µg/egg 33°C 50 18 18 0 0 41 ± 130 µg/egg 33°C 30 0 — — — —

128°C = ambient temperature.

Fig. 3. Histological sections of ovary from 10-day-oldhatchlings after (a) high temperature (33°C; control) and (b)high temperature (33°C) + testosterone (100 µg/egg) treat-

ments, and (c) of around a 2-month-old normally grownhatchling. Note the large-sized ova in (b), which are compa-rable to those in (c). of, ovarian follicle. Bar = 50 µm.


TABLE 2. Gonads in the hatchlings of the eggs treated with CGS 16949 A (aromatase inhibitor)reared at ambient and 33°C temperature


On day 155 µg/egg 28°C 10 8 3 1 3 52 ± 15 µg/egg 33°C 10 9 4 3 2 41 ± 1

On day 255 µg/egg 28°C 10 10 5 1 4 52 ± 15 µg/egg 33°C 10 9 9 — — 41 ± 1

TABLE 3. Gonads in the hatchlings of the eggs treated with estradiol reared at ambient and 33°C temperature


On day 1510 µg/egg 28°C 10 7 3 0 3 52 ± 1

On day 2010 µg/egg 28°C 20 12 4 7 1 52 ± 1

On day 2510 µg/egg 28°C 10 8 4 3 1 52 ± 15 µg/egg 33°C 10 8 3 1 3 41 ± 1

10 µg/egg 33°C 10 10 5 5 — 41 ± 1

viously obtained except that in the ovaries thethickness of the germ cell layers of the cortex wasmuch greater than in the controls. However, when25 µg estradiol was given to the 20-day embryoat 33°C, only 2 out of the 30 embryos survived.Nearly the same was true with the embryos given16 µg of the drug on day 20 at 33°C (7 survivedout of 25). Gonadal sections from these 7 revealedthe presence of both male and female individu-als. Whereas in the females the ovary was hyper-trophied, in the 7-day-old males the testis stillretained a rudimentary layer of cortical epithe-lium. In another series of experiments, 10 µg and5 µg/egg estradiol was given on day 25 to the 33°C-reared eggs. As seen in Table 3, there was no strik-ing difference between the control (ambient) andthe experimentals. Thus estradiol in any combi-nation did not have any effect on the differentia-tion of testis, but it could have an effect on thematuration of ovary.

DISCUSSIONThe present study on the effect of testosterone

on the day 20, 25, and 30 eggs at ambient tem-perature reveals that, unlike the 5-, 10-, and 15-day embryos, the sex-reverting effect of testosteroneis reduced or absent. Thus the period between days5 to 15 appears to be the “window” for androgensensitivity and for determination of testis in Calotes.The histological examination of the MGC shows that

the window coincides with the evagination of thegenital ridge followed by its regionalization into themedulla (primordium of testis) and cortex (primor-dial ovary) and finally the formation of sex cords inthe medulla with not much differentiative activityin the cortex. It appears that the pathway of testisdifferentiation is initiated in the indifferent gonadduring the testosterone-sensitive window.

The most intriguing result in the present set ofexperiments is the differentiation of ovary in thewindow period–testosterone–treated eggs whenthey were incubated at high temperature. Evenmore, in a number of females, the ovary was en-dowed with enlarged follicles, as large as those ofapproximately 2-month-old naturally growing fe-males. Though in reptilian embryos the feminiz-ing effect of testosterone is not unusual (Pieau,’96), in C. versicolor it is, because in this speciesthe same hormone induces only testis at ambienttemperature, and the untreated eggs at high tem-perature show no preference for ovarian differen-tiation (Ganesh and Raman, ’95; see controlgroups in Table 1 of this study). In the presentset of experiments, where a substantial amountof mortality was encountered, on the whole morefemales were obtained. However, with the pres-ence of a fair proportion of males it is unlikelythat the relative abundance of females is becauseof sex reversal of genic males to females. At thesame time, the apparent decline in the frequency


of males, when viewed along with the high rateof mortality, could be due to preferential loss ofthe male embryo. Contrary to the testosterone ex-periments, treatments with its nonaromatisablederivative, DHT, and the aromatase inhibitor,CGS 16949 A (applied on day 25 at 33°C), pro-duced only males despite high temperature. Boththese experiments provide evidence that differen-tiation of ovary into the treated eggs was due toaromatisation of the exogenously given testoster-one, resulting into estradiol. It is also conceivablethat on day 25 both testosterone and the enzymearomatase were available only in the high-tem-perature eggs and not in those reared at ambienttemperature. It is to be noted that in C. versi-color histochemical analysis failed to detect sexsteroid synthesis till hatching (Gaintonde andGouder, ’84). That is, in C. versicolor, at high tem-perature the synthesis or activity of the enzymecytochrome P-450 aromatase is induced earlierthan at the lower, ambient temperature. The sameis true for a number of other reptiles. This wouldnot only lead to ovary formation but also advanceits differentiation and accelerate the subsequentsteps in ovarian maturation, leading to more ma-ture eggs in these females than in those natu-rally developed. However, since a fair proportionof hatchlings were males, either the precocioussynthesis or activity of aromatase occurred onlyin the “genic” female or else the male embryoswere refractory to the enzyme at this stage of de-velopment.

Temperature-induced aromatase activity hasbeen shown to mediate gonadal differentiation in anumber of TSD reptiles (Desvages and Pieau, ’92;Desvages et al., ’93). Even in the chromosomallysex determined (CSD) amphibian, Pleurodeles waltl(ZZ/ZW sex chromosomes), the aromatase levelshowed a temperature-specific change (Chardard etal., ’95). In these, the incubation temperature doesnot increase the rate of aromatase activity per se;instead it may induce fresh synthesis of the enzyme.C. versicolor appears to be among the first non-TSD/CSD reptiles to show aromatase-mediated testoster-one feminization under the influence of tempera-ture. Even though under indirect and artificial(temperature) conditions, the evidence is goodenough to conclude that aromatase has a crucialrole in the determination of ovary in C. versicolor.

The existence of the “window” period of tes-tosterone receptivity and the results with DHT,aromatase inhibitor, and estradiol collectively pro-vide some insight into the time and stage of de-velopment when the pathways toward testis and

ovary are fixed in C. versicolor. It is important tonote that, for both the male hormones, testoster-one (at ambient temperature) and DHT (even athigh temperature), the window period of their ef-fectiveness in inducing testis was between day 10to 15, the first quarter of embryonic development.On the other hand, since the aromatase inhibitorbecame effective much later (day 25) and only atelevated temperature (33°C), it can be safely as-sumed that the effective aromatase activity at theambient temperature would occur even later thanday 25. The same conclusion may be derived fromall the estradiol experiments; estradiol, unlike tes-tosterone, does not revert the indifferent gonadtoward ovary regardless of the stage (window orafterwards) or temperature at which it is applied.That is, in C. versicolor, estradiol does not seemto modulate gonadal differentiation; insteadaromatase may be the more critical molecule inovary differentiation. These results imply that inthis species the initial cue for sex determinationis genic rather than hormonal, and that male isthe heterogametic sex. These results also showthat during development the pathway toward tes-tis differentiation is fixed much earlier than thatof ovary; and the default female embryos remainin a flexible state by the time the pathway to tes-tis differentiation is irreversibly fixed in genicmales. Therefore, whereas the “female” embryoscan be reverted, the male embryos cannot.

These experiments add to our knowledge of sexdetermination and differentiation in the lizard, C.versicolor, which lacks both CSD and TSD, in thefollowing two ways: (1) the deterministic cues fortestis and ovary are temporally distinct, testis pre-ceding ovary by about 10 days, and (2) the en-zyme aromatase plays an important role in thedifferentiation of ovary. Whereas the first featurelooks very similar to that seen in mammals, theother is common to the TSD reptiles. Since thereare indications of the male-specific distribution ofthe SRY/ZFY family of genes in C. versicolor(Ganesh et al., ’97), could it be that this species isone among those on the crossroads of the CSD/GSD and TSD? Our present efforts are directedtoward cloning and characterizing the sex-relatedgenes of Calotes versicolor and deriving their se-quence of expression in gonadal differentiation.

ACKNOWLEDGMENTSWe thank M/s CIBA-Geigy for the gift of aroma-

tase inhibitor and Dr. Amitabh Krishna for thegift of testosterone and estradiol. The researchwas funded by a grant from the Department of


Science and Technology to R.R. S.G. and B.C. aresupported by research fellowships, respectively,from the Indian Council of Medical Research andthe University Grants Commission.

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