Download Offspring Sex Is Not Related to Maternal Allocation of Yolk Steroids

220
Offspring Sex Is Not Related to Maternal Allocation of Yolk Steroids
in the Lizard Bassiana duperreyi (Scincidae)
Rajkumar Radder1
Sinan Ali2
Richard Shine1,*
1
School of Biological Sciences A08, University of Sydney,
Sydney, New South Wales 2006, Australia; 2Department of
Biological Sciences, Macquarie University, Sydney, New
South Wales 2109, Australia
Accepted 10/23/2006; Electronically Published 1/3/2007
ABSTRACT
The eggs of birds and reptiles contain detectable levels of several
steroid hormones, and experimental application of such steroids can reverse genetically determined sex of the offspring.
However, any causal influence of maternally derived yolk steroids on sex determination in birds and reptiles remains controversial. We measured yolk hormones (dihydrotestosterone,
testosterone, and 17 b-estradiol) in newly laid eggs of the montane scincid lizard Bassiana duperreyi. This species is well suited
to such an analysis because (1) offspring sex is influenced by
incubation temperatures and egg size as well as by sex chromosomes, suggesting that yolk hormones might somehow be
involved in the complex pathways of sex determination, and
(2) experimental application of either estradiol or fadrozole to
such eggs strongly influences offspring sex. We obtained yolk
by biopsy, before incubating the eggs at a temperature that
produces a 50 : 50 sex ratio. Yolk steroid levels varied over a
threefold range between eggs from different clutches, but there
were no significant differences in yolk steroids, or in relative
composition of steroids, between eggs destined to become male
versus female. Further, yolk steroid concentrations were not
significantly related to egg size. Thus, yolk steroid hormones
do not appear to play a critical role in sex determination for
B. duperreyi.
Introduction
In most animal species, sex is one of the primary axes of morphological and behavioral variation within a population. Ac* Corresponding author; e-mail: [email protected].
Physiological and Biochemical Zoology 80(2):220–227. 2007. 䉷 2007 by The
University of Chicago. All rights reserved. 1522-2152/2007/8002-6402$15.00
cordingly, the factors that determine an individual’s sex have
been debated for more than 3,000 years (Mittwoch 2000). Although genotypic sex determination (GSD) is the best-understood system, environmental conditions during development
are known to bias offspring sex ratio in many insect, fish,
amphibian, and reptile species (reviewed by Korpelainen
[1990]). More surprisingly, recent data suggest that environmental sex determination (ESD) of this kind is compatible with
GSD because numerous epigenetic factors can influence offspring sex even in species with GSD. For example, offspring
sex ratio and/or sexual phenotype are influenced by food availability to reproducing females (in Seychelles warblers and zebra
finches: Komdeur et al. 1997; Kilner 1998), the proximity of
same- versus opposite-sex embryos in utero (in house mice
and Mongolian gerbils: Clark et al. 1994; Vandenbergh and
Huggett 1994), and the timing of insemination with respect to
ovulation (in numerous mammals: James 1996; see Lovern and
Wade 2003b for a review of such effects).
What factors interact with sex-specific genetic features to
influence the sex of offspring? Maternal allocation of steroid
hormones to eggs is a plausible factor because (1) eggs of birds
and reptiles often contain significant amounts of maternally
derived steroid hormones and (2) experimental application of
excess hormone to eggshells of birds and reptiles can switch
genetically determined sex (Wade et al. 1997; Freedberg et al.
2006). Reports of sex differences in yolk hormone levels suggest
some kind of functional link between maternally derived yolk
hormones and offspring sex: either hormones determine sex
or mothers allocate different amounts of steroid hormones to
eggs destined to carry male versus female embryos (see, e.g.,
Gil et al. 1999; Petrie et al. 2001; Lovern and Wade 2002, 2003a,
2003b; Correa et al. 2005; Pike and Petrie 2005). Such a sexspecific allocation seems feasible on physiological grounds because of widespread transfer of nutrients, gases, water, and
steroid hormones from pregnant mothers to their developing
embryos (Janzen et al. 1998, 2002; Rutstein et al. 2005). All
steroid hormones are lipophilic and therefore highly yolk soluble, and they readily cross biological membranes. Thus, steroids can easily pass from mother into the yolk during vitellogenesis and females could potentially control allocation to
each egg (Rutstein et al. 2005).
Previous research on sex-specific allocation of yolk steroids
and their potential role in sex determination has been conducted primarily on birds; much less is known about the role
of maternally contributed steroid hormones in the other major
lineage of oviparous vertebrates (i.e., reptiles). Within the rep-
Yolk Hormones in a Lizard 221
tiles, interest has focused on species of turtles, crocodilians, and
lizards that exhibit a specific form of ESD whereby offspring
sex is determined by the incubation temperatures experienced
by eggs (temperature-dependent sex determination [TSD]).
This work on reptiles has shown that yolk hormones are deposited during vitellogenesis and has suggested that relative
levels of hormones in the yolk can influence the developing
gonads (Elf 2004). As in birds, yolk is a source of both estradiol
and testosterone in TSD turtles, alligators, and leopard geckos,
and initial levels of both steroids are correlated with the sex of
the hatchlings (Elf 2004). In contrast, little is known about the
levels of yolk steroids, or the possibility of sex-specific allocation
by mothers, in GSD species of reptiles. A recent study on the
lizard Anolis carolinensis has suggested that mothers allocate
yolk steroids relative to the destined sex of the egg (Lovern and
Wade 2003b). Interestingly, however, recent studies dispute the
validity of earlier reports of sex differences in yolk steroid concentrations between male and female eggs in birds (see, e.g.,
Eising et al. 2003; Pilz et al. 2005; Gil et al. 2006). These studies
cast significant doubt on the general conclusion that reproducing females strategically modify yolk steroid levels relative
to offspring sex and suggest instead that the observed correlations might reflect embryonically produced rather than maternally derived steroids. Clearly, there is a need for further
observations on a wide variety of animals from different lineages and with different modes of sex determination.
The recent discovery of both GSD and TSD within a single
reptile population (of the montane lizard Bassiana duperreyi
[Gray 1838]; Shine et al. 2002) provides a unique opportunity
to explore the possibility of differential maternal allocation of
steroids into egg yolk and their possible sex-determining effects
and potential interaction with genetic factors. Briefly, when eggs
of B. duperreyi are incubated under relatively warm conditions
(optimal incubation regimes), hatchling sex is determined by
genetics: XX individuals develop as females and XY individuals
as males (Shine et al. 2002). However, this system breaks down
when eggs are incubated at the lower temperatures characteristic of high-elevation nests in the wild. Such an incubation
regime produces sex ratios skewed toward males (i.e., some XX
individuals develop as phenotypic males). Intriguingly, sex in
B. duperreyi is also affected by a third factor; regardless of
incubation temperature, larger eggs are more likely to produce
female offspring (Shine et al. 2002). Finally, our laboratory
experiments have shown that we can sex-reverse B. duperreyi
by applying either the aromatase inhibitor fadrozole (to produce males) or estradiol (to produce females) to eggshells partway through incubation (R. Radder and R. Shine, unpublished
data). Thus, we can use this unique study system to address
the following questions: (1) Do eggs destined to become males
versus females differ in their steroid profiles, as might be expected either if B. duperreyi mothers allocate yolk steroids differentially into genetically determined sons versus daughters or
if steroids play a causal role in sex determination? (2) Is the
amount or relative composition of sex steroids related to egg
size, as might be expected if the effect of egg size on offspring
sex is mediated by steroid allocation? and (3) Do yolk steroid
levels vary primarily among eggs within a clutch or among
clutches produced by different females (i.e., what is the magnitude of inter- vs. intraclutch variation)? Our study provides
the first data on yolk steroid levels in eggs of any scincid lizard
species.
Material and Methods
Study Species and Collection
Bassiana duperreyi are medium-sized (to 80 mm snout-vent
length [SVL]) oviparous scincid lizards that are widely distributed through southeastern Australia. Extensive research on this
species has focused on populations in the Brindabella Range
40 km west of Canberra (1,240 m a.s.l.; 148⬚50⬘E, 35⬚21⬘S) in
the Australian Capital Territory (see, e.g., Shine et al. 1995,
1997, 2002; Shine and Harlow 1996; Flatt et al. 2001; Shine
2002, 2004). These cool, high-elevation sites are close to the
upper elevational limits for oviparous reproduction by Australian lizards. Most female B. duperreyi nest communally during
early summer (usually in the last week of November and the
first two weeks of December), and individual clutches cannot
be distinguished with confidence in the field. Each female produces a single clutch (3–11 eggs) during the annual breeding
season. We collected gravid B. duperreyi from the Brindabella
Range on November 24 and 29, 2004, and brought the animals
to the University of Sydney. After they were weighed and measured, the females were housed in individual cages (each 22
cm # 13 cm # 7 cm) and allowed to oviposit. Each cage contained moist vermiculite, shelter items, and a water dish. The
lizards were fed live crickets three times a week. The room was
kept at an air temperature of 20⬚C and a 12L : 12D photoperiod.
Heating was provided by means of an underfloor heating element to maintain a thermal gradient of 20⬚–35⬚C within each
cage for 8 h/d and falling to ambient room temperature overnight (see Shine and Harlow 1996 for details). The lizards had
ample opportunity for behavioral thermoregulation. All cages
were visually inspected twice every day.
Egg Incubation
As soon as eggs were found they were removed from the cages.
All eggs were weighed and yolks sampled (for hormone assay)
using a sterile syringe with a 24-gauge needle. Approximately
10% (25–30 mg) of yolk was removed from each egg (determined by reweighing eggs after yolk removal). The yolk was
transferred into a 2-mL plastic Eppendorf tube and homogenized with 500 mL of distilled water with vigorous shaking and
stored at ⫺80⬚C until radioimmunoassay. After yolk removal,
the eggs were placed individually in 64-mL jars containing
moist (⫺200 kPa) vermiculite. The jars were kept in a cycling-
222 R. Radder, S. Ali, and R. Shine
temperature incubator with a sinusoidal diurnal thermal cycle
of 20⬚ Ⳳ 7.5⬚C. This thermal regimes mimics conditions that
eggs usually experience in the field and that produces sex ratios
close to 50 : 50 (Shine 2002; Shine et al. 2002). Incubation
under these conditions takes approximately 10–11 wk (Shine
et al. 2002). Incubators were checked twice daily for hatchlings.
Any hatchlings found were removed and sex was determined
by squeezing the tailbase to manually evert hemipenes (Harlow
1996; Shine et al. 2002). This sexing method was confirmed by
gonadal histology of 12 randomly selected samples at the age
of 8–10 wk. Our hemipene sexing method was 100% congruent
with gonadal histology.
samples, we removed endogenous steroids by treatment of yolk
with 1 mL of charcoal solution (10 mg/mL). We then added
known amounts of exogenous steroids (0, 100, and 250 pg/mL
of DHT, T, and E2; from Sigma) to each sample. After equilibration for 24 h, steroids were extracted and subjected to
radioimmunoassay. The measured steroid levels were similar
to expected values indicating negligible interference arising
from antibody/steroid interactions with egg yolk substances
(Table 1).
Data Analyses
Radioimmunoassay for Yolk Steroids
We used competitive-binding steroid radioimmunoassays (RIA)
to measure levels of the three principal sex steroids: dihydrotestosterone (DHT), testosterone (T), and 17 b-estradiol (E2). We
followed the RIA procedure of Wingfield and Farner (1975) as
modified by Schwabl (1993) for yolk steroid measurements. The
yolk samples were made up to 1 mL using distilled water. To
determine extraction recoveries, approximately 2,000 cpm of tritiated DHT, T, and E2 (PerkinElmer, Victoria, Australia) was
added to each sample to serve as tracer. After the samples were
vortexed and allowed to equilibrate overnight at 4⬚C, the hormones were extracted using petroleum ether and diethyl ether
(30 : 70 vol : vol). The hormones were reconstituted with 90%
ethanol and allowed to stand overnight (⫺20⬚C) for sedimentation of neutral lipids, which were removed by centrifugation.
The supernatant was dried and resuspended in (1 mL) 10% ethyl
acetate in isooctane. The yolk extract was transferred onto a
diatomaceous earth microcolumn consisting of a celite : ethylene
glycol : propolyne glycol upper phase and celite : water lower
phase. The samples were directly applied to columns, and hormone separation was completed by eluting each fraction with a
unique ethyl acetate : isooctane ratio (10% for DHT, 20% for T,
and 40% for E2). Elutes were collected and dried at 45⬚C under
a stream of nitrogen gas. The dried elutes were suspended in
(550 mL) phosphate buffer and an aliquot used to determine
hormone concentrations by competitive-binding radioimmunoassay using specific antibodies for each hormone of interest
(antibodies for T and DHT from Research Diagnostic, Flanders,
NJ; antibodies for E2 from Immuno Diagnostics, St. Peters,
Australia).
Yolk samples were assayed in duplicate, and hormone concentrations were compared to a standard curve from 1 to 250
pg/mL for T and E2 and 2 to 500 pg/mL for DHT. Steroid
extraction recoveries averaged 54.16% for DHT, 56.88% for T,
and 51.09% for E2. The intra-assay variation, calculated as the
coefficient of variation for standards, was 6.56% for DHT,
10.2% for T, and 4.9% for E2.
Specificity and accuracy of the hormone assay were examined
following the procedures of Schwabl (1993). From additional
We calculated the sex ratio as the number of males divided by
the total number of hatchlings (i.e., no. males/[no. males ⫹
no. females]; Wilson and Hardy 2002). We investigated possible
sex differences in yolk steroid levels by two methods: initially
using unpaired t-tests and later by Wilcoxon matched-pair
signed-rank tests to compare males and females within each
clutch. The latter method was adopted to avoid confounding
effects that might arise because of among-clutch variation in
yolk steroid levels (Elf 2004). For this purpose, we calculated
clutch mean values for male and female eggs separately, and
these mean values were used for data analyses. In some species,
the sex of the developing embryo may be determined by ratios
of T : E2 or DHT : E2 rather than by absolute amounts of any
single hormone (Bogart 1987). Therefore, we calculated T : E2
and DHT : E2 ratios and analyzed them as described above.
Relationships between maternal traits (SVL and body mass),
egg size, and yolk steroid levels, and among different steroid
levels within the egg, were elucidated by Pearson correlation
coefficient analyses. Since we detected no significant relationships between maternal traits and steroid levels, among-clutch
variation in yolk steroid levels was analyzed by one-way
ANOVA. Significance level was accepted at P ! 0.05. All data
analyses were performed using Statview 5.0 software (SAS Institute, Cary, NC).
Table 1: Amounts of the steroids dihydrotestosterone (DHT),
testosterone (T), and 17 b-estradiol (E2) in yolk from eggs of
the lizard Bassiana duperreyi
Hormone
Added to
Yolk (pg)
0
100
250
Hormone Measured in Yolk after
Addition (pg)
DHT
ND
108 Ⳳ 3.5
268 Ⳳ 2.8
T
ND
122 Ⳳ 5.0
260 Ⳳ 4.8
E2
ND
118 Ⳳ 2.6
276 Ⳳ 5.3
Note. Amounts are expressed as mean Ⳳ SE and were measured by radioimmunoassay after removing endogenous steroids by treatment with charcoal
solution (10 mg/mL) and addition of known amount of exogenous steroids.
n p 3 for each measurement. ND p not detectable.
Yolk Hormones in a Lizard 223
Results
Sex Differences in Yolk Steroid Levels
A total of 55 eggs from 12 clutches were used to obtain the
data, yielding a sex ratio of 0.62 (n p 21 female and 34 male
offspring), not significantly different from 50 : 50 (x 2 p 1.57,
df p 1, P p 0.21). At the time of oviposition, eggs destined to
become male or female did not differ significantly in concentrations of any of the steroids that we assayed (Table 2; Fig. 1)
or in the ratio measures that we calculated (Table 2).
The same pattern (or lack thereof) was evident from analyses
based on mean values for male versus female eggs per clutch.
Compared with their sisters, male offspring emerged from eggs
with similar postlaying levels of yolk DHT (Table 2; Fig. 2a),
T (Table 2; Fig. 2b), E2 (Table 2; Fig. 2c), and ratios such as
T : E2 and DHT : E2 (Table 2).
Within- and Among-Clutch Variations in Yolk Steroid Levels
Clutches laid by different females varied significantly in terms of
yolk T levels within eggs (Table 3) but not in DHT or E2 levels
(Table 3). Similarly, we did not detect significant interclutch variation in ratios such as T : E2 (Table 3) and DHT : E2 (Table 3).
Neither maternal body length nor maternal body mass was significantly correlated with mean yolk steroid levels (maternal body
length: r p 0.28, P p 0.39 for DHT, r p 0.43, P p 0.17 for T,
and r p 0.42, P p 0.18 for E2; maternal body mass: r p 0.14,
P p 0.67 for DHT, r p ⫺0.04, P p 0.90 for T, and r p 0.17,
P p 0.60 for E2).
In a comparison among clutches, a strong correlation was
evident between mean levels of T and E2 (r p 0.80, P p
Table 2: Statistical analyses of sex differences in levels of the
yolk steroids dihydrotestosterone (DHT), testosterone (T),
and 17 b-estradiol (E2) in eggs of the lizard Bassiana
duperreyi
Yolk Steroid
DHT
T
E2
DHT : E2 ratio
T : E2 ratio
Unpaired t-Test
(df p 53)
Wilcoxon SignedRank Test (n p 12)
t Value
Z Value
⫺.51
⫺.75
⫺.69
⫺.32
.02
P
.61
.46
.49
.75
.98
⫺.24
⫺.47
⫺.78
⫺1.95
⫺1.48
P
.81
.63
.43
.06
.14
Note. The left columns of the table provide the results of unpaired t-tests
comparing male and female offspring based on a total of 21 daughterproducing and 34 son-producing eggs. The right columns of the table provide
the results from nonparametric (Wilcoxon signed-rank) tests on data on mean
values for male and female offspring per clutch from 12 clutches. Regardless
of which statistical test was used, there was no significant difference in yolk
steroid levels between eggs producing male offspring and those producing
female offspring.
Figure 1. Sex steroid concentrations (mean Ⳳ SE ) in yolks of newly
laid eggs of the montane lizard Bassiana duperreyi compared to the
sex of the hatchling eventually emerging from that egg. The levels of
sex steroids in freshly oviposited eggs did not differ significantly between eggs destined to produce males and females (unpaired t-test,
t53 p ⫺0.51, P p 0.61 for dihydrotestosterone [DHT]; t53 p ⫺0.75,
P p 0.46 for testosterone [T]; and t53 p ⫺0.69, P p 0.49 for 17 bestradiol [E2]; n p 34 male eggs and 21 female eggs).
0.0009) as well as between DHT and E2 (r p 0.63, P p 0.026).
These correlations mean that overall, some clutches displayed
higher concentrations of all three steroids than did other
clutches. However, egg mass was not significantly related to
yolk steroid levels (r p ⫺0.23 , P p 0.09 for DHT, r p ⫺0.21,
P p 0.13 for T, and r p ⫺0.17, P p 0.13 for E2, n p 55 eggs)
or to ratio measures such as DHT : E2 (r p ⫺0.21, P p 0.13,
n p 55 eggs) or T : E2 (r p ⫺0.20, P p 0.13, n p 55 eggs).
Discussion
Eggs of Bassiana duperreyi contain measurable quantities of
yolk steroids (T, DHT, and E2) shortly after oviposition, with
levels (in picograms per milligram) within the range reported
previously for other reptile species (Elf 2003, 2004; Lovern and
Wade 2003b). Eggs from some clutches contained much higher
steroid levels than eggs from other clutches, but we found no
clear links between steroid levels and offspring sex in comparisons either within or among clutches. Thus, females of this
species apparently do not allocate steroids differentially to male
and female offspring, and maternal allocation of yolk steroid
hormones does not influence offspring sex in B. duperreyi.
Although early research produced conflicting results as to
whether maternal steroids are transferred to embryos via egg
yolk (Altmann and Hutton 1938; Hertelendy and Common
1965), more recent work using radioactively labeled steroids
and steroid radioimmunoassays makes it clear that maternally
derived steroids are common yolk constituents not only in birds
(Arcos 1972; Elf and Fivizzani 2002) but also in fishes, turtles,
alligators, and lizards (see, e.g., Conley et al. 1997; McCormick
224 R. Radder, S. Ali, and R. Shine
1998; Jennings et al. 2001; Bowden et al. 2002; Lovern and
Wade 2003a, 2003b; Elf 2004). However, the functional role of
maternally derived steroids and their effects (if any) on sex
determination remain controversial. Some empirical demonstrations of sexually dimorphic sex steroid concentrations (e.g.,
Petrie et al. 2001) rely on sampling eggs after embryonic gonads
have differentiated and thus may be actively involved in producing or sequestering steroids (Pilz et al. 2005). In support
of this interpretation, later studies demonstrated no sex difference in yolk steroid concentrations of avian eggs at the time
of laying (e.g., chicken eggs: Müller et al. 2002; Eising et al.
2003; quail eggs: Pilz et al. 2005; swallow eggs: Gil et al. 2006).
By day 10 of incubation, male embryos of these species produce
more T whereas females produce more E2 (Pilz et al. 2005).
The sexes also differ in steroid sequestration, with female embryos metabolizing androgens from the yolk more rapidly than
males, whereas males sequester/metabolize E2 from the yolk
more rapidly (Pilz et al. 2005). In recent work on a GSD reptile,
however, Lovern and Wade (2003b) reported sex differences in
yolk steroid levels in recently laid eggs of the anoline lizard
Anolis carolinensis.
We collected yolk samples from B. duperreyi eggs on day 0,
immediately after oviposition. As in the majority of reptiles,
fertilized eggs of B. duperreyi contain embryos at developmental
stage 30 at oviposition (Shine 1983; R. Radder, personal observation), and there is no trace of the gonadal-adrenalmesonephric kidney complex (GAM) in B. duperreyi at this
stage. The GAM complex is thought to be the source of steroid
biosynthesis in developing embryos during later stages of development (Elf 2004). Also, during the early stages of development, the reptilian GAM is quiescent and does not produce
any significant quantity of steroid hormones (Lovern and Wade
2003a; Elf 2004). Thus, the yolk steroids we quantified in B.
duperreyi eggs are of maternal origin. Yolk steroids were indeed
transferred from mother to embryo (as documented previously
in oviparous vertebrates), but interestingly, similar levels of yolk
Table 3: Among-clutch variation in levels of the yolk
steroids dihydrotestosterone, testosterone, and
17 b-estradiol in eggs of the lizard Bassiana duperreyi,
based on data from 12 clutches
One-Way ANOVAa
Figure 2. Yolk steroid hormone levels (mean Ⳳ SE ) of newly laid eggs
in relation to hatchling sex and clutch identity in the lizard Bassiana
duperreyi for (a) dihydrotestosterone (DHT), (b) testosterone (T), and
(c) estradiol (E2). Overall, there was no sexual difference in yolk steroids
between brothers and sisters within each clutch (Wilcoxon signed-rank
test, Z p ⫺0.24, P p 0.81 for DHT; Z p ⫺0.47, P p 0.63 for T;
Z p 0.78, P p 0.43 for E2 levels). Numbers above the bars in a represent sample sizes for each sex in each clutch and remain the same
for b and c.
Yolk Steroid
Dihydrotestosterone (DHT)
Testosterone (T)
17 b-estradiol (E2)
DHT : E2 ratio
T : E2 ratio
a
df p 11, 43.
* Significant differences (P ! 0.05).
F Value
1.63
2.83
1.33
.89
.95
P
.12
.007*
.24
.55
.50
Yolk Hormones in a Lizard 225
steroids were allocated by mothers into eggs destined to become
female and male.
If maternally derived yolk hormones do not determine offspring sex and are not allocated in a sex-specific way, what is
the biological role of such hormones? We can only speculate
that sex steroids in the yolk may influence other aspects of
offspring development, perhaps in ways that either minimize
or enhance sibling competition (Schwabl et al. 1997; Lipar and
Ketterson 2000; Eising et al. 2001; Navara et al. 2006). Alternatively, steroids may be present in the yolk simply as the byproduct of hormonal regulation of maternal physiology because
of passive uptake during yolk formation (see, e.g., Bowden et
al. 2002; Janzen et al. 2002). This second (nonadaptive) explanation fits well with our observation that B. duperreyi clutches
with higher levels of androgens (T and DHT) also had higher
levels of E2, perhaps reflecting simple variation among reproducing female lizards in overall hormone titers. We note that
some bird species differentially allocate yolk steroids to male
and female eggs, but sex steroids do not influence sex determination (Müller et al. 2002; Rutstein et al. 2005), so the functional roles (if any) of sex steroids in yolk remain unclear.
Our results provide a strong contrast to the only previous
study of yolk hormones in a reptile with sex chromosomes.
Lovern and Wade (2003b) demonstrated sex-specific allocation
of yolk steroids in a lizard, the green anole A. carolinensis.
However, their study species differs from ours (and indeed,
from most other reptiles) in producing only a single egg at a
time. The left and right ovaries are used in alternation, and
ovulation occurs frequently (at an interval of 7–14 d; Smith et
al. 1973; Andrews 1985). This unusual reproductive mode
would allow reproducing female anoles to allocate different yolk
hormones to different eggs. In contrast, female B. duperreyi
ovulate multiple eggs from each ovary simultaneously and produce only a single clutch of eggs during the breeding season
(Shine 2002). Unlike those of birds and A. carolinensis, all follicles for a given clutch in B. duperreyi (and most other reptiles)
are yolked simultaneously and hence are exposed to the same
maternal hormonal environment (Bowden et al. 2002). This
mode of reproduction may constrain sex-specific maternal allocation of steroids because it would require a reproducing
female to somehow produce a different quantity of androgens
as well as estrogens for different eggs, without compromising
her own reproductive physiology. Successful reproduction is a
fine-tuned balance between various sex steroids and especially
between androgens and estrogens (Whittier and Tokarz 1992).
Because sex steroid production is under the control of hypothalamic and pituitary hormones, mechanisms for selective local enhancement of specific hormone levels for differential sex
steroid allocation are difficult to visualize. Thus, the lack of
sex-specific yolk steroid allocation in B. duperreyi may reflect
physiological constraints.
Recent studies have attempted to link interspecific variation
in the T : E2 ratio with modes of sex determination (see, e.g.,
Lovern and Wade 2003a). Birds and lizards with GSD have
T : E2 ratios 11, and experimental manipulations of yolk steroid
levels in these species suggest that T may be more effective than
E2 in affecting early development (Schwabl 1996; Lipar and
Ketterson 2000; Lovern and Wade 2003a). In contrast, T : E2
ratios !1 have been reported in alligators and turtles with TSD,
and experimental manipulations of yolk steroids suggest that
E2 plays an important role in sex determination (Wibbels et al.
1994; Bowden et al. 2000). Is the T : E2 ratio indicative of functional differences in steroid roles in different clades or simply
a reflection of phylogenetic history (Lovern and Wade 2003a)?
Interestingly, B. duperreyi (which exhibits both GSD and TSD)
has a T : E2 ratio of 1.37 Ⳳ 0.18 (mean Ⳳ SE; i.e., 11, as in GSD
birds and lizards), and our exogenous application of E2 elicited
a massive shift of sex ratios. These apparently unusual features
of B. duperreyi may be related to its unusually complex multifactorial mode of sex determination.
Significant among-clutch variation in B. duperreyi for yolk
T levels mirrors the results of earlier studies on alligators, several
turtle species, and the leopard gecko (Elf 2004). Variation in
yolk steroids among clutches might be the consequence of varying levels of circulating hormones present in different mothers
during yolking of the follicles in B. duperreyi, as suggested for
other reptiles (Elf 2004). These differences among individual
females in yolk hormone levels also might engender corresponding variation in growth rates of offspring independent of
egg mass as in the case of other reptiles (Elf 2004) and birds
(Navara et al. 2006). Several factors (such as maternal age, body
size, diet, photoperiod, mating season, clutch interval, social
status, etc.) may contribute to individual variation in levels of
yolk hormones (Müller et al. 2002; Elf 2004; Rutstein et al.
2005). However, in this study we did not find any correlation
between yolk androgen or estrogen levels in B. duperreyi and
factors such as maternal body size, clutch size, or average egg
mass. Similar observations were reported for two species of
turtles, Chrysemys picta and Chelydra serpentina (Elf 2004).
Further, more detailed studies are warranted to shed light on
these aspects.
Overall, our study suggests that yolk steroids of maternal
origin are not critical for sex determination in B. duperreyi.
Androgen and estrogen concentrations were not correlated with
either offspring sex or egg size. However, it remains possible
that other yolk steroids (e.g., progesterone, corticosterone, etc.;
Correa et al. 2005; Pike and Petrie 2005) play some role in sex
determination, or that yolk androgens or estrogens are involved
but in a more complex way, in combination with other factors
(Pilz et al. 2005). Regardless, the multiple sex-determining systems within B. duperreyi make it an ideal model system with
which to further investigate the mechanisms by which reproducing females can influence the sex of their offspring.
226 R. Radder, S. Ali, and R. Shine
Acknowledgments
We thank Jai Thomas and Melanie Elphick for assistance in
the field and for the collection and maintenance of gravid lizards in the laboratory. The study was supported by research
grants to R.R. and R.S. from the Australian Research Council.
The study was approved by the Animal Ethics Committee of
the University of Sydney (AEC approval L04/7-2004/3/3885).
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