JOURNAL OF EXPERIMENTAL ZOOLOGY 293:58–66 (2002)
Experimental Manipulation of Steroid Concentrations
in Circulation and in Egg Yolks of Turtles
FREDRIC J. JANZEN,1* MATTHEW E.WILSON,2 JOHN K. TUCKER,3
4
AND STEPHEN P. FORD
1
Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011
2
Division of Animal and Veterinary Sciences,WestVirginia University, Morgantown,
WestVirginia 26506
3
Illinois Natural History Survey, Brighton, Illinois 62012
4
Department of Animal Science, University of Wyoming, Laramie,Wyoming 82071
Steroid hormones in egg yolks are increasingly recognized as an important compoABSTRACT
nent of maternal and o¡spring ¢tness in oviparous vertebrates.Yet, except for in birds, the mechanism
by which females allocate these resources is poorly understood.We manipulated systemic levels of hormones in reproductively mature female red-eared slider turtles (Trachemys scripta elegans) with silastic
implants to test the hypothesis that hormones are allocated to developing follicles as a quantitative
function of circulating levels in the females. Turtles exhibited similar amounts (o1 ng/ml) of circulating steroids (dihydrotestosterone, estradiol-17b, or testosterone) in early September immediately prior
to experimental manipulation. After treatment with silastic implants, circulating levels of steroids
increased markedly. By the following April after hibernation, circulating levels of dihydrotestosterone
had returned to preimplantation levels, but circulating levels of estradiol-17b and testosterone in estradiol-17b- and testosterone-treated turtles, respectively, remained substantially elevated through
April. Focusing on testosterone, we detected nearly six-fold higher concentrations in yolk from mature
follicles from testosterone-treated turtles than in yolk from mature follicles from control turtles. Our
results provide support for the hypothesis that concentrations of steroids in egg yolks of turtles re£ect
circulating concentrations of steroids during follicular development rather than the hypothesis that
females selectively allocate speci¢c amounts of steroid hormones to each egg separately. Our ¢ndings
also highlight an unambiguous physiological mechanism by which nongenetic maternal e¡ects in oviparous species can directly in£uence the nutritional milieu experienced by developing embryos. J. Exp.
Zool. 293:58^66, 2002.
r 2002 Wiley-Liss, Inc.
Maternal allocation of resources to eggs or developing o¡spring is a crucial component of maternal
and o¡spring ¢tness (Bernardo,’96; Mousseau and
Fox, ’98). In this regard, di¡erential allocation of
steroid hormones may be particularly important.
For example, studies with birds have shown that:
(1) hormone concentrations vary among eggs within
clutches and among clutches within populations
(e.g., Schwabl, ’93; Schwabl et al., ’97; Lipar et al.,
’99; Reed and Vleck, 2001); (2) this variation is due
to circulating levels of the hormones in the females
(e.g., Adkins-Regan et al.,’95; Schwabl,’96a; Wilson
and McNabb, ’97); and (3) di¡erential allocation of
hormones to eggs in£uences hatchling phenotypes
(e.g., Schwabl,’93,’96b; Adkins-Regan et al.,’95; Wilson and McNabb, ’97; Lipar and Ketterson, 2000)
and may enhance parental ¢tness (Gil et al.,’99). Similar ¢ndings overall have been reported for ¢sh as
well (e.g., McCormick,’98).
r 2002 WILEY-LISS, INC.
Substantial concentrations of steroid hormones
also occur in the egg yolk of alligators (Conley
et al., ’97) and turtles (Janzen et al., ’98; Bowden
et al., 2000) and may vary considerably among
clutches laid by di¡erent females and among species. Of particular note, variation in allocation of
steroid hormones to egg yolks may be linked to different sex-determining mechanisms in various turtle taxa and to important phenotypic variation in
the o¡spring, including developmental rate and gonadal sex determination (Janzen et al.,’98; Bowden
et al., 2000). All these results documenting steroid
Grant sponsor: National Science Foundation; Grant number: DEB9629529.
*Correspondence to. F. J. Janzen, Department of Zoology and Genetics, Iowa State University, IA 50011-3223. E-mail: fjanzen@iastate.edu.
Received 1 June 2001; Accepted 14 January 2002
Published online in Wiley InterScience (www.interscience.wiley.
com). DOI: 10.1002/jez.10092.
CIRCULATING AND YOLK STEROID LEVELS IN TURTLES
hormones in egg yolk and attendant phenotypic
consequences for a variety of oviparous vertebrates
thus have manifold implications regarding the potential impacts of phytoestrogens or steroid-like
pollutants on embryonic development (e.g., Willingham and Crews,’99).
But what is the mechanism behind maternal allocation of steroid hormones to egg yolks in turtles?
One obvious hypothesis is that hormones are allocated to developing follicles as a quantitative function of circulating levels in the females (e.g.,
Adkins-Regan et al.,’95). In species such as turtles,
where whole clutches consist of many follicles developed and ovulated simultaneously (e.g., Callard
et al., ’78), steroid hormone concentrations in egg
yolks are relatively similar within clutches (Janzen
et al., ’98; Bowden et al., 2000). This result is consistent with, but not de¢nitive proof for, the ‘‘circulating hormones’’ hypothesis. A contrasting hypothesis is that females selectively allocate speci¢c
amounts of steroid hormones to each egg separately, as could be true in birds where eggs are developed and ovulated sequentially (e.g., Schwabl
et al.,’97; Reed and Vleck, 2001).
Our objective was to test the ‘‘circulating hormones’’ hypothesis by manipulating systemic levels
of hormones in reproductively mature female turtles. We focused on red-eared slider turtles (Trachemys scripta elegans) for several reasons: (1) they
have served widely as a model species, particularly
in studies of comparative physiology (summarized
in Ernst et al., ’94; Willingham and Crews, 2000);
(2) we have previously documented substantial
among-clutch variation in yolk testosterone levels
in this species (Janzen et al., ’98); and (3) we have
shown that this variation may be causally linked
to important phenotypic variation in the o¡spring
(Janzen et al.,’98). In the present study, we ¢rst assessed the e⁄cacy of our method by measuring circulating hormone levels just prior to and at regular
intervals for more than six months after experimental implantation of mature females with either
dihydrotestosterone, estradiol-17b, or testosterone.
Because of results from prior research (Janzen
et al.,’98), we subsequently focused on the relationship between testosterone treatment and concentrations of this steroid in fully developed follicular
eggs.
MATERIALS AND METHODS
Experimental design
Thirty-¢ve gravid turtles were hand-captured at
¢eld sites in Calhoun and Jersey Counties, Illinois
59
while making nesting forays on 7^9 June 1996
(Tucker, ’97). Turtles were subsequently induced to
oviposit by injection of oxytocin (10 IU/kg) to assess
steroid concentrations in egg yolks (Janzen et al.,
’98). The spent females were transported to Iowa
State University on 9 June and were held in one of
three 300 -gallon Rubbermaid stock tanks containing 30 gallons of dechlorinated tap water at
271C with a 12 hr light:12 hr dark cycle and fullspectrum light. Turtles were fed a mix of Purina
Trout Chow and wild vegetation (e.g., fresh clover
and goldenrod). Six adult males were trapped from
the Mississippi River near the field sites on 1^8 July
and were housed along with the females beginning
on 15 July to promote subsequent reproductive activity. Individual turtles were identified by drilling
up to four holes in different combinations of marginal scutes.
A subset of 32 female turtles was divided into
four experimental groups (n=8 in each group) to investigate the relationship between systemic steroid
concentrations and subsequent steroid concentrations in egg yolks. Each turtle was injected with a
subcutaneous 1 ml silastic implant (polymer+
catalyst+physiological saline) containing either no
steroid (i.e., control group), 2.5 mg of dihydrotestosterone, 2.5 mg of estradiol 17-b, or 2.5 mg of testosterone on 4 September and received a second
implant of the same treatment on 2 October. We
sampled blood (1 ml) from the brachial sinus of each
turtle immediately prior to each implantation and
in late afternoon every two weeks from 4 September
until 31 October. Blood was collected in heparinized syringes and placed on ice until plasma was
harvested.
Turtles were moved to an environmental chamber for hibernation on 31 October. The initial temperature was 211C, which was gradually lowered to
61C over the course of four weeks. The turtles were
then held at 61C with 8 hr light:16 hr dark in one of
two 300 -gallon stock tanks containing 30 cm dechlorinated tap water. Turtles were brought out of
hibernation gradually, with the temperature of the
environmental chamber slowly increased beginning on 1 March 1997 until it reached 211C on 1
April when turtles were transported to a greenhouse facility. The light and temperature treatments during hibernation are similar to those
experienced by the turtles in the habitat from which
they were collected (Tucker, personal observation).
Blood was sampled from the brachial sinus of each
turtle on 1 April, as described above. Turtles were
retained under natural temperature and lighting
conditions (and feeding was resumed) with the
60
F.J. JANZEN ET AL.
Fig. 1. Autoradiograph of a clutch of shelled eggs in a reproductively mature female Trachemys scripta that
died accidentally just prior to the nesting season (see MATERIALS AND METHODS for details).
CIRCULATING AND YOLK STEROID LEVELS IN TURTLES
intent to have them shell and oviposit eggs in late
May and early June (the normal nesting period)
for experimental analysis. Unfortunately, failure
of the exhaust system in the greenhouse on 14^19
April caused room temperatures to rise as high as
441C, which caused substantial mortality. This tragedy is all the more unfortunate in that one female
had already shelled a clutch of eggs (Fig. 1) and the
females (n=5 controls and n=4 testosterone-implanted turtles) that we autopsied within a few
hours after death for analysis of yolk steroids had
fully developed follicles. We did not autopsy turtles
from the other two treatments, so we cannot provide information on follicle development in these
cases.
Assay validations
Dihydrotestosterone
The assay for determining dihydrotestosterone
in turtle plasma was based on the method described
by Werawatgoompa et al. (’82). The method utilizes
potassium permanganate to oxidize the 4,5 double
bond of testosterone to 4,5 dihydroxy-testosterone,
eliminating its a⁄nity for the antibody and allowing the direct radioimmunoassay of dihydrotestosterone. The antibody used has a crossreactivity
of 100% for testosterone, 70% for dihydrotestosterone, and o0.1% for androsterone, dehydroepiandrosterone,
androstenedione,
estradiol-17b,
estrone, and ethiocholanolone. Duplicate samples
of plasma (50 ml) were added to 950 ml of nonpyrogenic water and vortexed for 10 sec. Free dihydrotestosterone was then extracted with 3 ml of
anhydrous diethyl ether. The ether fractions were
decanted from the snap frozen plasma/water phase
and dried under ¢ltered air. The samples were oxidized by adding 1 ml of KMnO4 (32 mM) for 20 min
and then re-extracting with 3 ml anhydrous diethyl
ether. The second ether fraction was then decanted
from the snap frozen KMnO4 reaction and dried under ¢ltered air. Samples were dissolved in benzene:methanol (85%:15%, v:v) and puri¢ed on LH-20
columns conditioned in the same solvent. The puri¢ed fractions were then dried under ¢ltered air.
Fractions were evaluated by adding 100 ml of assay bu¡er (50 mM ethylenediaminetetraacetic acid,
10 mM Tris, 1 mM NaN3, 1% Knox gelatin (w/v, pH
7.3), 100 ml of antibody (diluted 1/1,000) and 100 ml of
assay tracer [50 -dihydro[1,2,4,6,7- 3H]-testosterone
(Amersham, Piscataway, NJ)]. The sensitivity of
the assay, de¢ned as the dihydrotestosterone standard which yielded 95% of the counts in the bu¡er
control tubes, was approximately 50 pg. Precision
61
and accuracy of the dihydrotestosterone assay were
evaluated by the addition of 0.1 ng/ml or 1.0 ng/ml of
dihydrotestosterone to plasma containing 0.04 ng/
ml dihydrotestosterone, resulting in 0.11 ng/ml or
0.95 ng/ml dihydrotestosterone, respectively. Extraction e⁄ciency, as determined by adding tracer
to plasma samples prior to extraction and counting
the fraction collected from the LH-20 column, averaged 75.5% 7 2.8%, and assay values were back corrected for this. All samples were evaluated in a
single assay and the intra-assay CV was 2.3%.
Estradiol-17b
The antibody used has a crossreactivity of 100%
for estradiol-17b, 5% for estradiol 3 -methyl ether,
4% for epiestriol, 3% for estrone, 0.3% for equilenin, and o0.1% for 2 -methoxyestradiol, equilin,
diethylstilbesterol, and testosterone. Duplicate
samples of plasma (50 ml) were added to 950 ml of
nonpyrogenic water and vortexed for 10 sec. Free
estradiol-17b was then extracted with 3 ml of anhydrous diethyl ether. The ether fractions were decanted from the snap frozen plasma/water phase
and dried under ¢ltered air. Samples were dissolved in benzene:methanol (85%:15%, v:v) and
puri¢ed on LH-20 columns conditioned in the same
solvent. The puri¢ed fractions were then dried under ¢ltered air.
Fractions were evaluated by adding 100 ml of
assay bu¡er (same as above), 100 ml of antibody
(diluted 1/30,000), and 100 ml of assay tracer [2,4,6,
7- 3H]-estradiol-17b (Amersham)]. The sensitivity of
the assay, de¢ned as the estradiol-17b standard
which yielded 95% of the counts in the bu¡er control tubes, was approximately 2 pg. Precision and
accuracy of the estradiol-17b assay were evaluated
by the addition of 25 pg/ml or 100 pg/ml of estradiol-17b to plasma containing 4.6 pg/ml estradiol17b, resulting in 21.9 pg/ml or 108.2 pg/ml estradiol17b, respectively. Extraction e⁄ciency, as determined by adding tracer to plasma samples prior to
extraction and counting the fraction collected from
the LH-20 column, averaged 68.5% 7 0.9%, and assay values were back corrected for this.Values for a
pool sample run in all assays resulted in intra- and
inter-assay CVof 5.5% and 18.3%.
Testosterone
Plasma testosterone was quanti¢ed using the
Coat-A-Count Total Testosterone Assay Kit (Diagnostic Products Corporation, Los Angeles, CA).
Precision and accuracy of the testosterone assay
62
F.J. JANZEN ET AL.
were evaluated by the addition of 0.2 ng/ml, 1ng/ml,
or 8 ng/ml of testosterone to plasma containing
nondetectable levels of testosterone, resulting in
0.24 ng/ml, 0.84 ng/ml, or 8.44 ng/ml testosterone,
respectively. Extraction e⁄ciency, as determined
by adding tracer to plasma samples prior to
extraction and counting the extract, averaged
90.2% 7 0.9%, and assay values were back corrected for this. Values for a pool sample run in all
assays resulted in intra- and inter-assay CVof 4.4%
and 16.0%.
RESULTS
Turtles exhibited similar amounts of circulating
steroids in early September prior to initiation of
the experiment (Figs. 2^4). All three focal hormones averaged o1 ng/ml in the blood at this time.
Subsequently, after treatment with silastic implants and prior to hibernation, circulating levels
of steroids increased markedly (P50.05) for each
experimental group (four-fold for dihydrotestosterone, eight-fold for estradiol-17b, and 100 -fold for
testosterone) when compared to preimplantation
values or to values for the control group (Figs. 2^4).
By the following April after hibernation, circulating levels of dihydrotestosterone in dihydrotestosterone-treated turtles had returned to near pre-
implantation levels (1.07 ng/ml 7 0.35 ng/ml vs.
0.85 ng/ml 7 0.16 ng/ml, respectively). In contrast,
circulating levels of estradiol-17b and testosterone
in estradiol-17b- and testosterone-treated turtles,
respectively, remained substantially elevated
through April (2.23 ng/ml 7 0.89 ng/ml and 15.63
ng/ml 7 8.51 ng/ml, respectively) compared to preimplantation levels (0.23 ng/ml 7 0.07 ng/ml and
0.87 ng/ml 7 0.76 ng/ml, respectively) and to circulating levels in April in turtles not receiving
implants of these steroids (0.31 ng/ml 7 0.13
ng/ml and 1.61 ng/ml 7 0.93 ng/ml, respectively)
(Figs. 2^4).
Several additional, interesting patterns emerged
from the steroid treatments. First, dihydrotestosterone and estradiol-17b levels were slightly
elevated in testosterone-treated turtles, especially
shortly after the testosterone was implanted
(Figs. 2 and 3, respectively), but these values
were not signi¢cantly di¡erent from those for control turtles for any assay date (P40.05 for all comparisons). Similarly, levels of testosterone
were somewhat elevated in dihydrotestosteronetreated turtles (not evident in Fig. 4 because of
the scale). These values were only signi¢cantly different from those for control turtles for the 16 and
30 October assay dates (F=4.65, P=0.05 and F=9.07,
P=0.01, respectively). For those two assay dates,
Fig. 2. Temporal changes in dihydrotestosterone in systemic circulation of reproductively mature female
Trachemys scripta as a function of experimental treatment (dihydrotestosterone-, estradiol-17b-, testosterone-,
or control-implanted). n=8 in each treatment group.
CIRCULATING AND YOLK STEROID LEVELS IN TURTLES
63
Fig. 3. Temporal changes in estradiol-17b in systemic circulation of reproductively mature female Trachemys
scripta as a function of experimental treatment (dihydrotestosterone-, estradiol-17b-, testosterone-, or controlimplanted). n=8 in each treatment group.
circulating levels of testosterone in dihydrotestosterone-treated turtles were about twice as high
(2 ng/ml) as those in control turtles (1 ng/ml).
To the contrary, control turtles and other experimental turtles generally exhibited consistent, near
baseline levels of the nontreatment steroids
(Figs. 2^4).
Testosterone concentrations were substantially
greater in yolk from mature follicles (12^22 mm diameter) from testosterone-treated turtles (six folli-
Fig. 4. Temporal changes in testosterone in systemic circulation of reproductively mature female Trachemys
scripta as a function of experimental treatment (dihydrotestosterone-, estradiol-17b-, testosterone-, or controlimplanted). n=8 in each treatment group.
64
F.J. JANZEN ET AL.
cles from each of four females) than in yolk from
mature follicles (13^20 mm diameter) from control
turtles (six follicles from each of five females). That
is, levels of testosterone in follicular yolk from testosterone-treated turtles were nearly six-fold higher than from control turtles (215 pg/mg 7 28 pg/mg
vs. 37 pg/mg 7 8 pg/mg, respectively, P50.05).While
substantive and highly significant, this difference
is an order of magnitude lower than the corresponding comparison of circulating testosterone concentrations between these same females shortly after
the experimental treatments during the previous
fall (Fig. 4). Still, more than six months elapsed between the last steroid implant placement and the
‘‘final’’ maturation of the follicles.
DISCUSSION
This experiment produced two main results: 1)
Circulating concentrations of steroid hormones increased markedly after turtles received experimental implants of particular steroids; and 2)
Testosterone concentrations in egg yolks were
greatly elevated in turtles that received testosterone-containing implants more than six months
prior to ‘‘complete’’ follicular maturation. Thus,
these results provide support for the hypothesis
that concentrations of steroids in egg yolks of turtles re£ect circulating concentrations of steroids
during follicular development rather than the hypothesis that females selectively allocate speci¢c
amounts of steroid hormones to each egg separately. Taken together, these ¢ndings demonstrate
the potential for using subcutaneous implants impregnated with steroids as an experimental method
for manipulating yolk steroid concentrations in
turtles.They also highlight an unambiguous physiological mechanism by which nongenetic maternal
e¡ects in oviparous species can directly in£uence
the nutritional milieu experienced by developing
embryos.
Of particular interest is the observation that circulating concentrations of both estradiol-17b and
testosterone remained elevated nearly seven
months after the last implantation of these steroids.The elevated levels of testosterone in testosterone-implanted turtles undoubtedly contributed to
the marked increase of this steroid in the follicular
yolk of these females. Further, the yolk testosterone
levels in testosterone-implanted females were 2^4
times higher than levels observed in freshly laid
eggs of Trachemys scripta elegans females collected
from the same area in a previous study (Janzen
et al., ’98). In addition to or instead of embryonic
physiological activity, it is conceivable that tem-
perature-dependent differences in yolk sac aromatase activity or expression (e.g., Jeyasuria and
Place, ’97), along with maternally derived differences in yolk testosterone, may function to initiate
differential gonadal activation (e.g., ovary or testes)
through alterations in the ratio of estrogen to testosterone in blood reaching the embryo. Such maternal transfer of a sex steroid to the egg yolk
would constitute a potentially significant source
of maternal influence over embryonic development
and adult phenotype in oviparous species with or
without temperature-dependent sex determination
(e.g., Bowden et al., 2000, 2001). Our model system
and technique would allow more critical experimental evaluation of this hypothesis.
Two of our steroid manipulations produced indirect e¡ects on nontarget steroids, but only one
of these e¡ects was statistically signi¢cant. Testosterone-treated turtles had slightly elevated circulating levels of dihydrotestosterone and estradiol17b, although these e¡ects were apparently due to
background noise. To the contrary, dihydrotestosterone-treated turtles had moderate but signi¢cantly elevated circulating levels of testosterone
during two of the assay dates. This result suggests
that, in response to unusually high concentrations
introduced by the experimental implants, dihydrotestosterone may have impacted a testosterone negative feedback loop in the turtles. In all other
cases, only circulating levels of the target steroids
were elevated by the experimental implants.
In Chrysemys picta, a freshwater turtle closely related to T. scripta, elevation of both estradiol-17b
and testosterone in the blood of adult females is observed in the spring at the time of maximal follicular growth, and again in the fall in association with
ovarian recrudescence (Callard et al.,’78). Interestingly, testosterone concentrations are markedly
higher than those of estradiol-17b during both the
spring and fall elevations of circulating steroids.
Research is needed to determine if T. scripta exhibits a similar pattern in nature.
We detected di¡erent circulating concentrations
of the three steroids used in this study even though
identical amounts of each kind were implanted in
the treated turtles. We suspect that dihydrotestosterone and estradiol-17b, because they are very potent steroids, were reduced and conjugated for
excretion rapidly compared to testosterone, which
often naturally circulates at several orders of magnitude higher concentrations (e.g., Callard et al.,’78).
Regardless, our results clearly show that levels of
circulating steroids, focusing on testosterone, are
re£ected in concentrations of these same steroids
CIRCULATING AND YOLK STEROID LEVELS IN TURTLES
in the yolk of mature follicles of turtles.Thus, the endocrinological state of the female during follicular
development, to the extent that it is a¡ected by genetic or environmental (e.g., stress) factors, can be
transmitted nongenetically to the eggs that she lays.
But, is such hormonal variation in the egg yolks
biologically relevant? Prior work with birds and
¢sh has clearly demonstrated that concentrations
of various hormones in the egg have profound
implications for the resulting o¡spring (e.g.,
Schwabl, ’93, ’96b; Adkins-Regan et al., ’95; Wilson
and McNabb,’97; McCormick,’98; Lipar and Ketterson, 2000) and may even enhance parental ¢tness
(Gil et al.,’99). Similar ¢ndings have been documented in turtles as well (Janzen et al., ’98; Bowden
et al., 2000, 2001). Indeed, in at least some species
of turtles, concentrations of testosterone and estradiol-17b in the egg yolk are related to rate of embryonic development (Janzen et al., ’98) and to
sexual di¡erentiation (Bowden et al., 2000). Such
endocrinological maternal e¡ects may in fact provide a common underlying explanation, in addition
to traditional intonations of genetic e¡ects,
for well-documented, among-clutch variation in
turtles for a wide variety of o¡spring traits (e.g.,
Brooks et al.,’91; Packard and Packard,’93; Janzen
et al.,’95).
Our ¢ndings have implications in a more practical, applied context as well. Because concentrations of hormones in egg yolks have a substantive
impact on o¡spring phenotypes in a diversity of vertebrates, it follows that exogenous substances that
mimic such hormones could also have a corresponding in£uence on these organisms. Of special interest, given the results of this study, females could
conceivably transmit so-called ‘‘environmental estrogens’’such as PCBs or DDT (and its metabolites)
to egg yolks during follicular development and
subsequently modify the phenotypes of the resulting o¡spring. Indeed, environmental estrogens
and other contaminants have been detected in
turtle and alligator eggs (e.g., Bishop et al., ’91;
Heinz et al.,’91), and exogenous application of those
xenobiotics can result in signi¢cant sex reversal in
turtles with temperature-dependent sex determination (e.g., Bergeron et al., ’94; Sheehan et al., ’99;
Willingham and Crews, ’99, 2000; but see Podreka
et al., ’98). And, there is increasing evidence
that nongenetic environmental e¡ects can be transmitted across generations (e.g., Francis et al.,
’99)! Clearly, bioaccumulation of xenobiotic
hormone mimics, in conjunction with endogenous
hormone levels, in female vertebrates deserves
further, in-depth experimental scrutiny because
65
there are implications for humans as well (e.g., Sonawane,’95).
ACKNOWLEDGMENTS
We thank Carole Hertz for technical expertise in
conducting the steroid radioimmunoassays, Nirvana Filoramo and Moynell Tucker for assistance in
collecting turtles, and Rachel Bowden and two
anonymous reviewers for constructive comments
on the manuscript. Turtles were obtained under Illinois Department of Conservation Scienti¢c Permit A93.0202 and extensions to JKT, and the
research was approved by Iowa State University’s
Animal Care Committee (Permit 6 - 6 -3258 -1-J). Financial support was provided in part by NSF DEB9629529 and an Iowa State University Research
Grant to FJJ and by the Illinois Natural History
Survey and the Long Term Resource Monitoring
Program for work done by JKT. Journal Paper No.
J-19841 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project Nos.
3369 and 3505, and supported by the Hatch Act
and State of Iowa Funds.
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