GENERAL AND COMPARATIVE

ENDOCRINOLOGY

www.elsevier.com/locate/ygcen

General and Comparative Endocrinology 134 (2003) 131–138

Sex and season influence gonadal steroid biosynthetic pathways,end-product production and steroid conjugation in blotched

blue-tongued lizards (Tiliqua nigrolutea)

Ashley Edwards,a,* Susan M. Jones,a and Noel W. Daviesb

a School of Zoology, University of Tasmania, GPO Box 252-05, Hobart, Tasmania 7001, Australiab Central Science Laboratory, University of Tasmania, GPO Box 252-74, Hobart, Tasmania 7001, Australia

Accepted 12 June 2003

Abstract

We examined differences in gonadal steroid production and biosynthetic pathway activity with changing reproductive condition

and between sexes in the scincid lizard, Tiliqua nigrolutea. We observed clear seasonal and sexual variation in the production of

androgens and steroid conjugates, but detected no 17b-estradiol or 5a-dihydrotestosterone produced by the gonads. An alternative

steroid, more polar than estradiol, was detected: an investigation of this steroid is reported separately [Gen. Comp. Endocrinol.

129 (2002) 114]. There were seasonal and sex-related differences in steroid biosynthetic pathway activity. The D5 pathway me-

tabolite, dehydroepiandrosterone, was detected only in males, and only from incubations using regressed testicular tissue. There

was also a seasonal difference between the sexes in rates of progesterone accumulation, although the absence of corresponding

elevated plasma concentrations suggests that the role of progesterone switches from a directly acting hormone to a precursor for

others during the reproductive cycle in females. These results suggest that within the traditional view that vertebrate biosynthetic

pathway activity and end-products are phylogenetically conserved, there is likely to be considerably species- and/or genus-specific

variation.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Conjugation; End-product; Ovary; Reptile; Steroid biosynthesis; Testis; Tiliqua nigrolutea

1. Introduction

There is considerable conservation of the major

end-products of reproductive steroid biosynthesis

within vertebrate classes. Among the reptiles, birds,and mammals, testosterone (T) and 17b-estradiol (E2)are usually the major end-products of gonadal ste-

roidogenesis in males and females, respectively (Kime,

1987). In several classes of lower vertebrates, however,

T and E2 are instead converted by the gonads to

forms that are more biologically active. Such modifi-

cations can occur at the number five (anuran am-

phibian: Kime, 1987), six and seven (agnathans: Kime

* Corresponding author. Fax: +61-3-6226-2745.

E-mail address: Ashley.Edwards@utas.edu.au (A. Edwards).

0016-6480/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0016-6480(03)00243-0

and Hews, 1980), 11 (fish: Huang et al., 1985; Leitz

and Reinboth, 1987, urodele amphibians: Lupo Di

Prisco et al., 1971, 1972), or 15 (agnathans: Kime and

Callard, 1982; Kime and Rafter, 1981) positions on

the steroid nucleus.When measuring plasma steroid concentrations, re-

searchers usually target the most common reproductive

steroids, T, E2, and progesterone (P4), with little con-

sideration for species-specific variations. However,

these patterns of preferred steroid end-product pro-

duction show some species-specific deviations within

several vertebrate classes, including the reptiles. For

example, in the lizard Tiliqua rugosa the major testic-ular androgen is not T but epitestosterone (epiT)

(Bourne et al., 1985), while in females of the snake

Thamnophis sirtalis parietalis a significant proportion

(30–60%) of the total plasma estrogen is 6a- and

132 A. Edwards et al. / General and Comparative Endocrinology 134 (2003) 131–138

6b-hydroxylated estradiol (6a- and 6b-OH-E2) ratherthan E2 (Whittier and Hess, 1992). Such deviations

may provide important information about the evolu-

tion of the activity of steroidogenic pathways within

and between vertebrate groups.

Traditionally, contributions of the two main bio-

synthetic pathways (D4 and D5) to T and E2 pro-

duction are also regarded as conserved within

vertebrate classes. While both pathways may operate,fish and amphibians use predominantly the D4 path-

way from pregnenolone (P5) via 17a-hydroxyproges-terone and androstenedione (AD), while the reptiles,

birds, and mammals use the D5 route from P5, via

17a-hydroxypregnenolone and dehydroepiandrosterone

(DHA) (Kime, 1987; Norris, 1997) preferentially.

However, a number of studies challenge this historical

premise: some reports have suggested the possibility ofchanges in pathway preference within a species. In

vertebrates, reproductive condition can affect the rel-

ative contributions and overall activity of steroid

biosynthetic pathways (reptile: Bourne and Licht,

1985; Lofts, 1972; fish: Borg et al., 1992; lungfish: Joss

et al., 1996; elasmobranch: Callard and Leathem,

1965; Kime and Hews, 1982; amphibian: Canosa and

Ceballos, 2002; mammal: Bedrak et al., 1983). Ste-roidogenic activity can also depend on sex (fish: Kime

and Groves, 1986) and temperature, particularly in

ectothermic species (fish: Kime, 1979; Kime and Hy-

der, 1983; Lofts, 1987; Manning and Kime, 1985;

reptile: Huf et al., 1989; Xavier, 1982).

There is relatively little information available on

steroid biosynthesis in reptiles. The question of pathway

preference for reproductive steroid production by rep-tilian gonads has been partially addressed in only a few

species, and usually only in animals of one sex (Bourne

and Licht, 1985; Chan and Callard, 1974; Callard, 1967;

Hews and Kime, 1978; Lofts, 1972). Variation in path-

way preference with changing reproductive condition

has not been addressed. Cautious extrapolation from

the available literature suggests that the D4 pathway is

more active, but some authors have reported the de-tection of small amounts of D5 pathway intermediates

(Bourne and Licht, 1985; Bourne, 1981; Chan and

Callard, 1974; Huf et al., 1989; Lupo Di Prisco et al.,

1968).

This study examined variation in the activity of the

steroid biosynthetic pathways and end-product for-

mation with sex and reproductive condition in a rep-

tile, the viviparous skink, Tiliqua nigrolutea. Thisspecies was chosen because information on the annual

reproductive cycle, patterns of change of plasma ste-

roid concentrations (Edwards and Jones, 2001a,b) and

androgen excretion (Atkins et al., 2002) are already

established, and because the closely related species, T.

rugosa, was the focus of a series of studies of steroid

metabolism several decades ago (e.g., Bourne and

Seamark, 1973, 1978; Huf et al., 1989; Vinson et al.,1975).

2. Materials and methods

2.1. Incubations

Animals were maintained as previously described(Edwards and Jones, 2001a,b). Lizards were killed by

ketamine injection (0.4ml im, (1ml kg�1)) and simulta-

neous inhalation of Halothane gas, in accordance with

ANZCCART recommendations for reptile euthanasia

(ANZCCART, 1993). Gonadal tissue was collected

from adult male (N ¼ 4) and female (N ¼ 4) blue-ton-

gued lizards at times of year corresponding with repro-

ductive activity/gonadal hypertrophy (spring) andreproductive quiescence/gonadal regression (autumn).

Tissues from each animal were treated separately. Right

and left gonads were combined and minced finely with

scissors. The tissue from each animal was then divided

evenly between 10 incubation flasks, each containing

15 ll [3H]pregnenolone ([3H]P5) (sp. act. 777GBq

mmol�1) as substrate. Pregnenolone was specifically

selected as the incubation substrate as it lies immediatelyabove the point of branching between the D4 and D5steroid biosynthetic pathways. All incubations of male

tissue used 200mg tissue per flask. Incubations of female

tissue varied from 60mg per flask (regressed ovaries) to

300mg tissue per flask (hypertrophied vitellogenic ova-

ries). Yolk was expressed from vitellogenic follicles, and

follicles were rinsed with incubation medium prior to

incubation. Both thecal and granulosa (and corporalutea (CLs) when present) tissues were included in the

incubations as we wished to assess the total biosynthetic

ability of the ovaries, rather than that of component

tissues. Leibovitz culture medium (Hepes-buffered: pH

¼ 7.6) (5 ml) was added to each flask: these were held

on ice until all flasks were prepared. Samples were in-

cubated at 35 �C in an air environment in a gently

rocking water bath. The incubation temperature reflectsthe previously published preferred body temperature of

this species (34.8 �C, Rawlinson, 1974). Incubations

were terminated at 10, 30, 60, 120, and 180min (N ¼ 2

flasks at each time) by rapid freezing and media were

stored at )20 �C. Free (non-conjugated) steroids were

later extracted from thawed media in 2� 10ml dichlo-

romethane (DCM). These DCM washes were combined,

and stored at )20 �C until further analysis as describedbelow. A 100 ll aliquot of the original medium from

each incubation was assayed for radioactivity in 3ml

scintillation cocktail following the extraction of free

steroids. This allowed the determination of the total

tritiated steroid in each incubation and the proportion

of tritiated substrate that became conjugated during the

incubation.

A. Edwards et al. / General and Comparative Endocrinology 134 (2003) 131–138 133

2.2. Thin layer chromatography

Steroids synthesised from [3H]P5 were first identified

tentatively using sequential thin layer chromatography

(TLC). The DCM washes containing free steroids ex-

tracted from the incubation medium were evaporated to

dryness, redissolved in 2� 200 l DCM washes and

spotted onto pre-coated Merck (0.2mm silica gel)

20� 20 cm plastic 60 F254 plates using a capillary tube.Initially, steroids were co-eluted with authentic steroids

(Sigma) in solvent system I (chloroform (CHCl3):meth-

anol (MeOH)) (95:5 (v/v)). Plates were air-dried and

standards were visualised by standard techniques, using

UV light (254 nm) (Petrino et al., 1993) or sublimed

iodine crystals (Lupo Di Prisco et al., 1968).

Fractions from the TLC plate corresponding to the

standards (and the spaces between them) were scrapedoff the plate separately and eluted through columns

packed with cotton wool and a layer of acid-washed

Celite in two column volumes of MeOH (approximately

20ml). Eluates were evaporated to dryness and redis-

solved in 5ml MeOH. To identify peaks of radioactivity

in each eluate, a 100 ll aliquot was assayed for radio-

activity in 3ml scintillation cocktail (Ecolite(+), ICN) in

a Beckman LS 5801 counter. Each fraction containing aradioactive peak was evaporated to dryness, spotted

onto a second TLC plate in 200 ll DCM and the pro-

cedure repeated using solvent system II (DCM: diethyl

ether (DEE) (5:2 (v/v)). Individual radioactive peaks

that had co-eluted twice with authentic standard were

stored in MeOH at )20 �C for further analysis. Any

radioactive peaks isolated from regions of the TLC plate

that did not co-elute with an authentic standard werealso rechromatographed in the second solvent system;

peaks were kept separate for further analysis. A char-

acteristic elution coefficient (Rf (distance travelled from

the point of origin by the authentic steroid/distance

travelled by the solvent front)) was calculated for each

steroid in each TLC solvent system.

Table 1

The Rfs (elution coefficient, see text) in thin layer chromatography (TLC)

romethane:diethyl ether (5:2 (v/v))), and high performance liquid chromatogra

of gonadal tissue from male and female T. nigrolutea

Steroid Rf, TLC system I Rf, TLC system II H

Pregnenolone 0.41 0.57 1

Progesterone 0.53 0.73

5a-Dihydrotestosterone 0.40 0.60

Dehydroepiandrosterone 0.41 0.43

Testosterone 0.34 0.57

Androstenedione 0.47 0.73

17b-Estradiol 0.36 0.29

6a-Hydroxyestradiol 0.05 0.07

Estriol 0.03 0.10

6b-Hydroxyestradiol 0.07 0.10

SE, standard error; N, sample size; and ND, not detected as an incubati

2.3. High performance liquid chromatography with radio-

metric detection

Steroids isolated as peaks in the two TLC systems

were identified using high performance liquid chroma-

tography (HPLC) with on-line radiometric detection.

The HPLC system used was a Waters Alliance 2690

liquid chromatograph attached to a Packard 500TR

Series radiometric detector. Radiometric detection ofHPLC runs allowed comparison of elution times be-

tween authentic standards and the isolated steroid

products of incubation with [3H]P5. A Waters 996

Photodiode Array Detector (resolution 1.2 nm) recorded

the UV spectra over the 190–300 nm range every second.

The column was a Waters Nova-Pak C18 reversed-

phase column (dimensions: 3.9� 150mm, 4 lm particle

size). The mobile phase was initially (4min) isocratic(MeOH:water (70:30)) and then ramped to 85% MeOH

over the next 8min at a flow rate of 0.8mlmin�1. Run

time was 12min per sample, and at the conclusion of

each run the system was equilibrated back to MeOH:

water (70:30) for 6min at the same flow rate. Authentic

standards were run through the system to determine the

elution time for 10 steroids with different polarities.

Samples in MeOH from TLC analysis were concen-trated to 1.2ml and filtered through 2 lm filters into

HPLC vials from which 15 ll was injected for HPLC

analysis. Those steroids for which radioactive peaks co-

eluted with authentic standards in both TLC solvent

systems were regarded as presumptively identified if the

HPLC elution time also corresponded to that of the

authentic standard (SE< 0.05, see Table 1). The unme-

tabolised [3H]P5 recovered from each incubation servedas a positive control. Yields of metabolites detected after

incubation of tissues with [3H]P5 as substrate are ex-

pressed as a percentage of the total radioactivity added

as [3H]P5 (i.e., radioactive counts isolated in a discrete

fraction/(total radioactive counts extracted as free ste-

roids + total radioactive counts remaining in incubation

solvent systems I (chloroform:methanol (95:5 (v/v))) and II (dichlo-

phy (HPLC) elution times of steroids isolated from in vitro incubations

PLC elution time (min) Repeatability of elution times (�1 SE (N))

0.660 �0.02 (18)

8.619 �0.01 (17)

6.400 ND

5.305 �0.01 (21)

4.716 �0.02 (21)

3.948 �0.03 (4)

3.571 ND

2.100 ND

1.913 ND

1.820 ND

on product.

134 A. Edwards et al. / General and Comparative Endocrinology 134 (2003) 131–138

medium as conjugated metabolites)). These values weredetermined by measuring radioactive counts present in

100 ll aliquots at each stage of the procedure.

3. Results

The amount of [3H]P5 present in incubation media

declined over the duration of the incubations as it wasconverted to other pathway intermediates. Corre-

spondingly, relative proportions of downstream metab-

olites varied during the incubations as they were first

synthesised, and then further metabolised. The steroids

identified as products of gonadal steroid incubations

and their TLC Rf and HPLC elution time are sum-

marised in Table 1. The metabolism of [3H]P5 by the

hypertrophied and regressed gonadal tissue of male and

Fig. 1. Mean proportion (%) of [3H]P5 metabolised in vitro over time

by hypertrophied and regressed the gonadal tissue of male (N ¼ 4) and

female (N ¼ 4) T. nigrolutea. Values are means, errors not calculated

due to small sample sizes.

Fig. 2. Mean proportion (%) of original substrate conjugated in vitro

over time by hypertrophied and regressed the gonadal tissue of male

(N ¼ 4) and female (N ¼ 4) T. nigrolutea. Values are means, errors not

calculated due to small sample sizes.

female lizards is illustrated in Fig. 1. [3H]Pregnenolonewas more rapidly and more completely metabolised in

the incubations of hypertrophied than regressed tissue

and most completely by hypertrophied testis, in which

only 6.1% (range 4.0–8.9%) of the original [3H]P5 re-

mained after 180min.

In both sexes, conjugated steroids were detected in

greater absolute amounts after 180min of in vitro in-

cubation by hypertrophied than regressed gonadal tissue(Fig. 2). Additionally, the pattern of production differed

with reproductive condition in both sexes. Hypertro-

phied tissue continued to produce conjugated steroids

throughout the 180min incubations, while production

of steroid conjugates by regressed ovarian tissue was

greatest at 120min (mean 45.7%, range 37.7–65.4%) and

declined slightly by 180min. Incubations of regressed

testicular tissue rapidly conjugated approximately 30.0%(range 20.5–36.1%) of the original substrate (by 10min),

but subsequently showed little further increase in con-

jugate formation.

Seasonal differences in in vitro conversion of labelled

substrate to P4 by gonadal tissue from both sexes are

presented in Fig. 3. Incubations with regressed ovarian

tissue collected from post-parturient females displayed

the greatest and most rapid increase, which comprised28.6% (range 13.5–39.8%) of the total tritiated steroids

present by 180min. Conversely, incubations with hy-

pertrophied, late vitellogenic-stage ovarian tissue rap-

idly (by 10min) converted 12.6% (range 8.0–17.9%) of

the total 3[H]P5 to P4, but showed no increase in the

proportion of tritiated steroid present as P4 over time.

By 10min, the proportion of tritiated steroid present as

P4 in incubations of both active and quiescent ovariantissue was greater than in incubations of testicular tissue

from either group and remained so until 180min of in-

cubation. At no time during any incubation of male

Fig. 3. Mean proportion (%) of original substrate present as P4 over

time in in vitro incubations with the hypertrophied and regressed go-

nadal tissue of male (N ¼ 4) and female (N ¼ 4) T. nigrolutea. Values

are means, errors not calculated due to small sample sizes.

A. Edwards et al. / General and Comparative Endocrinology 134 (2003) 131–138 135

tissue was tritiated P4 detected as more than 4.0% of thetotal tritiated free steroids metabolised from [3H]P5.

The patterns of AD production in vitro by hyper-

trophied and regressed gonadal tissue of both sexes are

shown in Fig. 4. There were no clear seasonal differences

in the proportion of radioactivity present as AD after

incubation of gonadal tissue of either sex. The propor-

tion of total radioactivity present as AD fluctuated over

time, but was never greater than 8.0% in any incubation.More AD was detected in incubations of testicular than

ovarian tissue in both seasons, and the proportion of

AD was greater in incubations with regressed testis.

The in vitro production of T from tritiated precursors

over time by hypertrophied and regressed gonadal

tissue of male and female lizards is illustrated in Fig. 5.

Testosterone production was greater by testis than

ovary throughout the incubations for both gonadalstates. Testosterone continued to accumulate through

time in the incubation of regressed testicular tissue,

rapidly until 120min of incubation and then more

slowly, until, after 180min, 46.0% (range 33.9–59.8%)

of the radioactivity originally added as [3H]P5 was

present as T. The proportion of T present in incubations

of hypertrophied testicular tissue increased rapidly until

60min (mean 47.0%, range 41.7–52.6%), then declinedmarkedly from 60 to 180min. Testosterone was also

synthesised by ovarian tissue, to a greater extent by

hypertrophied than regressed ovaries, reaching a maxi-

mum of 14.6% (range 12.0–16.7%) of the total radio-

activity present following 180min of incubation with

late-vitellogenic stage, hypertrophied, ovarian tissue.

Dehydroepiandrosterone was detected only in small

amounts in incubations of regressed testicular tissue:DHA 4.4% (range 3.5–5.3%) of tritiated free steroids in

the incubation by 180min. No DHA was detected in

Fig. 4. Mean proportion (%) of original substrate present as AD over

time in in vitro incubations with the hypertrophied and regressed go-

nadal tissue of male (N ¼ 4) and female (N ¼ 4) T. nigrolutea. Values

are means, errors not calculated due to small sample sizes.

incubations of hypertrophied testis, or from any incu-bations using ovarian tissue. Furthermore, E2 and

5a-dihydrotestosterone (5a-DHT) were not detected as

metabolites of incubation of gonadal tissue with [3H]P5.

One additional and presently unidentified polar steroid

was synthesised in relatively large proportions by go-

nads of both males and females (Edwards et al., 2002).

4. Discussion

Tiliqua nigrolutea utilises the conserved pathways

observed in most higher vertebrates (Kime, 1987; Nor-

ris, 1997), probably reflecting the early evolution of an

efficient system of chemical communication (Bentley,

1998); intermediates of both the D4 (AD, P4) and D5(DHA) steroid biosynthetic pathways were detected asproducts of gonadal biosynthesis in T. nigrolutea. The

more rapid and more complete metabolism of [3H]P5

to downstream metabolites by hypertrophied than

regressed gonadal tissue of both male and female

T. nigrolutea almost certainly reflects the greater mass of

tissue present and, thus, the increased biosynthetic

ability of the gonads during the spring reproductive

period. This, in turn, reflects the increased concentra-tions of steroids to which other tissues are exposed

during the period corresponding to final gamete matu-

ration, ovulation, and mating. These observations cor-

relate well with reported changes in plasma steroid

concentrations throughout the annual cycle in males

(Edwards and Jones, 2001a) and females (Edwards and

Jones, 2001b) of this species, in both of which plasma

T and estrogen (E) concentrations are significantlyelevated in spring. Few generalisations from the pub-

lished literature are possible, as previous results are

Fig. 5. Mean proportion (%) of original substrate present as T over

time in in vitro incubations of with the hypertrophied and regressed

gonadal tissue of male (N ¼ 4) and female (N ¼ 4) T. nigrolutea.

Values are means, errors not calculated due to small sample sizes.

136 A. Edwards et al. / General and Comparative Endocrinology 134 (2003) 131–138

mixed. Often only males are examined, or reproductivecondition is not considered. Reports range from exclu-

sive use of the D4 pathway (Callard, 1967; Hews and

Kime, 1978), to contributions from both D4 and D5pathways in varying proportions (Bourne and Seamark,

1978; Chan and Callard, 1974; Huf et al., 1989; Lupo Di

Prisco et al., 1968).

The shape of the percentage-yield curve for the pro-

duction of T from [3H]P5 by regressed testicular tissue,and the absence of any other androgens as incubation

end-products all confirm that T is the major product of

[3H]P5 metabolism in male gonads in the post-repro-

ductive period. This supports previous results which

found no evidence for an alternative to T in T. nigro-

lutea, despite the gonadal synthesis of epiT by the clo-

sely related T. rugosa (Bourne et al., 1985). However, in

incubations with hypertrophied testis from T. nigrolutea

(spring reproductive period), T is rapidly metabolised

further, although not, as expected, to E2. 17b-Estradiolwas not detected as a product of gonadal steroid bio-

synthesis (in either sex) in this species. Instead, a dif-

ferent, very polar, steroid was synthesised by both

testicular (hypertrophied) and ovarian (hypertrophied

and regressed) tissue. Further analyses, reported sepa-

rately strongly suggest that it is an estrogen, an alter-native to E2, and a major end-product of gonadal

steroid biosynthesis in this species (Edwards et al.,

2002). Testosterone appears to be a precursor for this

polar steroid.

The detection of estrogens other than E2 in reptiles is

not without precedent. In the lizard Dipsosaurus dorsalis

and the turtle Pseudemys scripta elegans both E2 and

estrone (E1) were detected after in vitro incubation ofovarian tissue using experimental conditions similar to

those employed in this study (Chan and Callard, 1974).

Additionally, both E2 and E1 have been identified from

extracts of ovarian tissue from the lizard Lacerta sicula

(Lupo Di Prisco et al., 1968), and Whittier and Hess

(1992) reported 6-hydroxylated E2 in the plasma of the

snake T. s. parietalis. Unfortunately, most previous

studies of reptilian steroid biosynthesis examined malesonly and did not attempt to identify estrogens as incu-

bation products (Bourne and Licht, 1985; Bourne and

Seamark, 1978; Callard, 1967; Lofts and Choy, 1972;

Tam et al., 1969).

Conversely, the proportion of labelled steroid present

as T during in vitro incubations of ovarian tissue in-

creased over time despite accounting for only a small

proportion of the total tritiated steroid present (Fig. 5).However, given that circulating T concentrations vary

meaningfully during the reproductive cycle in female T.

nigrolutea (Edwards and Jones, 2001b), T is likely to be

a physiologically important metabolite of steroid bio-

synthesis by the ovaries, in addition to being a precursor

for estrogen production, as has been reported in other

reptiles (Guillette et al., 1997; Owens, 1997; Staub and

De Beer, 1997). A physiological role for T in ovulationin female T. nigrolutea has been postulated (Edwards

and Jones, 2001b). Additionally, 5a-dihydrotestosterone(5a-DHT) was not detected as a product of the gonads

in either sex, but appears to be synthesised peripherally

by several different tissues (Edwards, Jones, and Davies,

unpublished results).

Steroid conjugates also constituted a significant pro-

portion of the end-products of steroid production in thisstudy and those proportions varied seasonally. Total

gonadal steroid conjugate formation was greater by

hypertrophied than regressed gonads. Male T. nigrolutea

conjugate a large proportion (80%) of the steroids ex-

creted in feces (Atkins et al., 2002) and steroids are be-

lieved to act as semiochemicals in other terrestrial

vertebrates (Drickamer, 1989; Madison, 1976). Cloacal

scent trailing behaviour is reported in other reptiles(Cooper and Vitt, 1984; Ford, 1986), including the re-

lated skink, T. rugosa (Bull et al., 1993). Seasonal vari-

ation in conjugated steroid metabolite production by the

gonads may reflect their potential to act as semio-

chemicals during the mating period in T. nigrolutea.

There was a clear difference between the sexes in the

extent of the production and metabolism of P4, as well

as seasonal variation in production in females. Proges-terone appears to be physiologically important in female

T. nigrolutea during spring: there is an increase in

plasma P4 concentrations at this time (Edwards and

Jones, 2001b). Interestingly, however, incubations of

regressed ovary, despite reduced tissue mass, produced

markedly more P4 than incubations with hypertrophied

ovary. The absence of a corresponding increase in

plasma P4 (Edwards and Jones, 2001b) strongly suggeststhat P4 is rapidly converted to downstream metabolites

at this time (autumn). In male lizards, P4 seems to act

only as an intermediate in testicular steroid biosynthesis.

Very little (<3.0%) of the total radioactivity was present

as P4 during in vitro incubations of testicular tissue in

any sample from either season. This presumably reflects

the position of P4 as a precursor for T or E production,

and correlates with the lack of a pronounced annualpattern of changes in plasma P4 concentrations de-

scribed for males, in which plasma P4 concentrations are

low throughout the annual reproductive cycle (Edwards

and Jones, 2001a). Additionally, these patterns of P4

production and metabolism demonstrate clearly marked

activity of the D4 steroid biosynthetic pathway in the

hypertrophied and regressed gonads of both sexes of this

lizard.The detection of AD, synthesised by the gonadal

tissue of males and females, confirms the activity of the

D4 steroid biosynthetic pathway in T. nigrolutea of both

sexes. Increased relative amounts of AD present in in-

cubations with regressed compared with hypertrophied

testis probably reflect a shift in the position of the

equilibrium between T and AD (Kime, 1987), to favour

A. Edwards et al. / General and Comparative Endocrinology 134 (2003) 131–138 137

AD at a time of year when plasma T is low (Edwardsand Jones, 2001a). Dehydroepiandrosterone was de-

tected from testicular incubations, and not from any

incubations with ovarian tissue, suggesting a sex-related

difference in the activity of the D4 and D5 steroid bio-

synthetic pathways. There is also seasonal variation in

the relative contributions of these two pathways in male

lizards: DHA was detected from incubations using re-

gressed testis only. When present, DHA comprised aconsiderably smaller proportion of total radioactivity

than AD throughout the incubations. It is most likely

that in T. nigrolutea the D4 pathway predominates in the

biosynthesis of gonadal steroids and that the D5 path-

way may make only a small contribution to total steroid

production by the gonads. This is consistent with the

small number of studies on other reptile species that

have used [3H]P5 or [3H]cholesterol as a substrate for invitro incubation (Callard, 1967; Chan and Callard,

1974; Hews and Kime, 1978; Huf et al., 1989). However,

these observations may, instead, be a reflection of in-

creased D5 pathway activity in spring, with larger pro-

portions of DHA being produced and metabolised too

rapidly to be detected by the sampling regime used here.

This study using the lizard T. nigrolutea, reports the

detection of intermediates of both the D4 and D5 steroidbiosynthetic pathways. D5 Pathway activity was only

detected in males, suggesting a variation in pathway

preference between the sexes. This study also reports clear

variations in steroid biosynthetic pathway preference

between hypertrophied and regressed gonadal tissue from

males and females. Contrary to some established litera-

ture, steroid biosynthesis appears to be a dynamic and

plastic process, with scope for variation according tophysiological conditions. It is likely that as more in-depth

studies of this type are completed, an alternative will

emerge to the traditional view of steroid biosynthetic

patterns as being phylogenetically conserved; it is likely

that there are greater, albeit subtle, variations in the

patterns, and products of steroid biosynthesis, both

within and between species, than previously suspected.

Acknowledgments

This researchwas funded by aUniversity of Tasmania,

School of Zoology postgraduate scholarship to A.E., and

a University of Tasmania Small ARC grant to S.M.J. We

thank Jane Girling and Brett Gartrell and two anony-

mous reviewers for helpful comments on the manuscript.This work was conducted under University of Tasmania

Animal Ethics Approval Numbers 95046 and 98015.

References

Anzccart, 1993. Euthanasia of animals used for scientific purposes. In:

Reilly, J.S. (Ed.), ANZCCART, Australia.

Atkins, A., Jones, S.M., Edwards, A., 2002. Fecal steroids may not be

useful for monitoring reproductive status in male blue-tongued

lizards (Tiliqua nigrolutea: scincidae). J. Herpetol. 36, 106–109.

Bedrak, E., Rosenstrauch, A., Kafka, M., Friedlander, M., 1983.

Testicular steroidogenesis in the camel (Camelus dromedarius)

during the mating and the nonmating season. Gen. Comp.

Endocrinol. 52, 255–264.

Bentley, P.J., 1998. Comparative Vertebrate Endocrinology, third ed.

Cambridge University Press, New York, USA.

Borg, B., Mayer, I., Lambert, J., Granneman, J., Schulz, R., 1992.

Metabolism of androstenedione and 11-ketotestosterone in the

kidney of the three-spined stickleback, Gasterosteus aculeatus. Gen.

Comp. Endocrinol. 86, 248–256.

Bourne, A.R., 1981. Blood metabolites of injected [14C]progesterone in

the lizard Tiliqua rugosa. Comp. Biochem. Physiol. B 70, 661–664.

Bourne, A.R., Licht, P., 1985. Steroid synthesis in turtle testes. Comp.

Biochem. Physiol. B 81, 793–796.

Bourne, A.R., Seamark, R.F., 1973. The synthesis of corticosterone by

the adrenal tissue of the lizard Tiliqua rugosa. Comp. Biochem.

Physiol. B 45, 275–277.

Bourne, A.R., Seamark, R.F., 1978. Seasonal variation in steroid

biosynthesis by the testis of the lizard Tiliqua nigrolutea. Comp.

Biochem. Physiol. B 59, 363–367.

Bourne, A.R., Taylor, J.L., Watson, T.G., 1985. Identification of

epitestosterone in the plasma and testis of the lizard Tiliqua

(Trachydosaurus) rugosa. Gen. Comp. Endocrinol. 58, 394–401.

Bull, C.M., Bedford, G.S., Schulz, B.A., 1993. How do sleepy lizards

find each other? Herpetologica 49, 294–300.

Callard, I.P., 1967. Testicular steroid synthesis in the snake, Natrix

sipedon pictiventris. J. Endocrinol. 37, 105–106.

Callard, I.P., Leathem, J.H., 1965. in vitro steroid synthesis by

the ovaries of elasmobranchs and snakes. Arch. Anat. Microsc. 54,

35–48.

Canosa, L.F., Ceballos, N.R., 2002. Seasonal changes in testicular

steroidogenesis in the toad Bufo arenarum H. Gen. Comp.

Endocrinol. 125, 426–434.

Chan, S.W.C., Callard, I.P., 1974. Reptilian ovarian steroidogenesis

and the influence of mammalian gonadotropins (follicle-stimulat-

ing hormone and luteinizing hormone) in vitro. J. Endocrinol. 62,

267–275.

Cooper, W.E., Vitt, L.J., 1984. Conspecific odor detection by the male

broad-headed skink, Eumeces laticeps: effects of sex and site of

odor source and of male reproductive condition. J. Exp. Zool. 230,

199–209.

Drickamer, L.C., 1989. Pheromones: behavioural and biochemical

aspects. In: Balthazart, J. (Ed.), Advances in Comparative and

Environmental Physiology. Molecular and Cellular Basis of Social

Behavior in Vertebrates, vol. 3. Springer, Berlin and Heidelberg,

pp. 269–348.

Edwards, A., Jones, S.M., 2001a. Changes in plasma testosterone,

estrogen and progesterone concentrations throughout the annual

reproductive cycle in male viviparous blue-tongued skinks, Tiliqua

nigrolutea, (Scincidae), in Tasmania. J. Herpetol. 35, 293–299.

Edwards, A., Jones, S.M., 2001b. Changes in plasma progesterone,

estrogen and testosterone concentrations throughout the repro-

ductive cycle in female viviparous blue-tongued skinks, Tiliqua

nigrolutea, (Scincidae), in Tasmania. Gen. Comp. Endocrinol. 122,

260–269.

Edwards, A., Jones, S.M., Davies, N.W., 2002. A possible alternative

to 17b-estradiol in a viviparous lizard, Tiliqua nigrolutea. Gen.

Comp. Endocrinol. 129, 114–121.

Ford, N.B., 1986. The role of pheromone trails in the sociobiology of

snakes. In: Duvall, D., Muller-Schwarz, D., Silverstein, R.M.

(Eds.), Chemical Signals in Vertebrates IV. Encology, Evolution

and Comparative Biology. Plenum Press, New York, pp. 261–278.

Guillette Jr., L.J, Woodward, A.R., Crain, D.A., Masson, G.R.,

Palmer, B.D., Cox, S.M., You-Xiang, Q., Orlando, E.F., 1997.

138 A. Edwards et al. / General and Comparative Endocrinology 134 (2003) 131–138

The reproductive cycle of the female American alligator (Alligator

mississippiensis). Gen. Comp. Endocrinol. 108, 87–101.

Hews, E.A., Kime, D.E., 1978. Testicular steroid biosynthesis by the

green lizard Lacerta viridis. Gen. Comp. Endocrinol. 35, 432–435.

Huf, P.A., Bourne, A.R., Watson, T.G., 1989. The in vitro biosyn-

thesis of epitestosterone and testosterone from C19 steroid precur-

sors in the testis of the lizard Tiliqua rugosa. Gen. Comp.

Endocrinol. 75, 280–286.

Huang, F.-L., Liu, T.-C., Lo, T.-B., 1985. Biosynthesis of 11-

ketotestosterone by carp testis in vitro. In: Lofts, B., Holmes,

W.N. (Eds.), Current Trends in Comparative Endocrinology.

Hong Kong University Press, Hong Kong, pp. 233–234.

Joss, J.M.P., Edwards, A., Kime, D.E., 1996. in vitro biosynthesis of

androgens in the Australian lungfish, Neoceratodus forsteri. Gen.

Comp. Endocrinol. 101, 256–263.

Kime, D.E., 1979. The effect of temperature on the testicular

steroidogenic enzymes of the rainbow trout, Salmo gairdneri.

Gen. Comp. Endocrinol. 39, 290–296.

Kime, D.E., 1987. The steroids. In: Chester-Jones, I., Ingleton, P.M.,

Philips, J.G. (Eds.), Fundamentals of Comparative Vertebrate

Endocrinology. Plenum Press, New York, pp. 3–56.

Kime, D.E., Callard, G.V., 1982. Formation of 15a-hydroxylatedandrogens by the testis and other tissues of the sea lamprey,

Petromyzon marinus, in vitro. Gen. Comp. Endocrinol. 4, 267–270.

Kime, D.E., Hews, E.A., 1980. Steroid biosynthesis by the ovary of the

hagfish Myxine glutinosa. Gen. Comp. Endocrinol. 42, 71–75.

Kime, D.E., Hews, E.A., 1982. The effect of temperature on steroid

biosynthesis by testes of the dogfish, Scyliorhinus caniculus. Comp.

Biochem. Physiol. B 71, 675–679.

Kime, D.E., Hyder, M., 1983. The effect of temperature and

gonadotropin on testicular steroidogenesis in Sarotherodon (Tila-

pia) mossambicus in vitro. Gen. Comp. Endocrinol. 50, 105–115.

Kime, D.E., Groves, D.J., 1986. Steroidogenesis by gonads of a

viviparous teleost, the sailfin molly (Poecilia latipinna), in vitro and

in vivo. Gen. Comp. Endocrinol. 63, 125–133.

Kime, D.E., Rafter, J.J., 1981. Biosynthesis of 15-hydroxylated

steroids by gonads of the river lamprey, Lampetra fluviatilis, in

vitro. Gen. Comp. Endocrinol. 44, 69–76.

Leitz, T., Reinboth, R., 1987. The biosynthesis of 11-ketotestosterone

by the testis of the Siamese fighting fish Betta splendens Regan

(Anabantoidei, Belontiidae). Gen. Comp. Endocrinol. 66, 145–157.

Lofts, B., 1972. The sertoli cell. Gen. Comp. Endocrinol. Supp. 3, 636–

648.

Lofts, B., 1987. Testicular function. In: Norris, D.O., Jones, R.E.

(Eds.), Hormones and Reproduction in Fishes, Amphibians and

Reptiles. Plenum Press, New York, pp. 283–325.

Lofts, B., Choy, L.Y.L., 1972. Steroid synthesis by the seminiferous

tubules of the snake Naja naja. Gen. Comp. Endocrinol. 17,

588–591.

Lupo Di Prisco, C., Delrio, G., Chieffi, G., 1968. Sex hormones in the

ovaries of the lizard Lacerta sicula. Gen. Comp. Endocrinol. 10,

292–295.

Lupo Di Prisco, C., Delrio, G., Chieffi, G., Cardellini, L.B., Magni,

A.P., 1971. Identification and biosynthesis of steroid hormones in

the ovary and fat bodies of female Triturus cristatus carnifex.

Comp. Biochem. Physiol. B 40, 53–60.

Lupo Di Prisco, C., Baslie, C., Delrio, G., Chieffi, G., 1972. In vitro

metabolism of cholesterol-4-14C and testosterone-4-14C in testes

and fat bodies of Triturus cristatus carnifex. Comp. Biochem.

Physiol. B 41, 245–249.

Madison, D.M., 1976. Chemical communication in amphibians and

reptiles. In: M€uuller-Schwarze, D., Mozell, M.M. (Eds.), Chemical

Signals in Vertebrates. Plenum Press, New York and London, pp.

135–168.

Manning, N.J., Kime, D.E., 1985. The effect of temperature

on testicular steroid production in the rainbow trout, Salmo

gairdneri, in vivo and in vitro. Gen. Comp. Endocrinol. 57, 377–

382.

Owens, D.W., 1997. Hormones in the life history of sea turtles. In:

Lutz, P.L., Musick, J.A. (Eds.), The Biology of Sea Turtles.

CRC Press, Boca Raton, New York, London, Tokyo, pp. 314–

315.

Norris, D.O., 1997. Vertebrate Endocrinology, third ed. Academic

Press, New York.

Petrino, T.R., Lin, Y.-W.P., Netherton, J.C., Powell, D.H., Wallace,

R.A., 1993. Steroidogenesis in Fundulus heteroclitus V.: Purifica-

tion, characterization, metabolism of 17a, 20b-dihydroy-4-preg-nen-3-one by intact follicles and its role in oocyte maturation. Gen.

Comp. Endocrinol. 91, 1–15.

Rawlinson, P.A., 1974. Biogeography and ecology of the reptiles of

Tasmania and the Bass Strait area. In: Williams, W.D. (Ed.),

Biogeography and Ecology in Tasmania. W. Junk, The Hague, pp.

230–269.

Staub, N.L., De Beer, M., 1997. The role of androgens in female

vertebrates. Gen. Comp. Endocrinol. 108, 1–24.

Tam, W.H., Phillips, J.G., Lofts, B., 1969. Seasonal changes in the in

vitro production of testicular androgens by the cobra (Naja naja

Linn.). Gen. Comp. Endocrinol. 13, 117–125.

Vinson, G.P., Braysher, M., Whitehouse, B.J., 1975. in vitro steroi-

dogenesis by the nonzoned adrenocortical tissue of the skink,

Tiliqua rugosa. Gen. Comp. Endocrinol. 26, 541–549.

Whittier, J.M., Hess, D.L., 1992. The occurrence of 6-substituted

estradiol 17b in the plasma of female garter snakes (Thamnophis

sirtalis parietalis). Adv. Comp. Endocrinol. 1, 77–82.

Xavier, F., 1982. Progesterone in the viviparous lizard Lacerta

vivipara: ovarian biosynthesis, plasma levels and binding to

transcortin-type protein during the sexual cycle. Herpetologica

38, 62–70.