HORMONES AND BEHAVIOR 12, 232-242 (1979)

Diurnal Variation of Heat Intake in Ovariectomized,

Steroid-Treated Rats

H.J. CARLISLE,' C. W. WILKINSON,~ M. L. LAUDENSLAGER,

AND L. D. KEITH

Department of Psychology, University of California, Santa Barbara, California 93106

Ovariectomized rats trained to work for radiant heat reward in a cold environ-
ment were implanted with subcutaneous Silastic capsules containing either es-
tradiol, progesterone, both estradiol and progesterone, or no hormone. The hor-
mone treatments produced an average plasma estradiol concentration of 41 pglml
and progesterone concentration of 20-50 rig/ml. All groups obtained more heat
behaviorally when tested during the light phase of the LD cycle than when tested
in the dark. Body temperatures and metabolic rates were higher during the night
than during the day. There were no differences between groups in behavioral heat
intake or body temperature. All hormone-treated groups showed a greater reduc-
tion in core temperature than the control group when an exogenous source of heat
was not available, but there was no substantial effect of the hormone treatments
on metabolic rate except for a 6-7% increase in metabolism of the estrogen group.
The increased cooling rate of all hormone-treated groups may indicate a
nonspecific steroid-induced increase in heat loss in the cold. The diurnal variation
in heat intake establishes the LD cycle as a significant variable in thermoregula-
tory behavior of the rat. Thus, behavioral heat intake is high during the day when
metabolism and body temperature are low, and low at night when metabolism and
body temperature are high in this nocturnal species.

Variations in body temperature have long been associated with cyclic

changes in ovarian activity (Rubenstein, 1937). The thermogenic activity
of progesterone seems to be well established because body temperature is
high both during the luteal phase of the menstrual cycle (Davis and Fugo,
1948) and following the injection of progesterone in the human (Buxton
and Atkinson, 1948; Israel and Schneller, 1950) and rat (Freeman,
Crissman, Louw, Butcher, and Inskeep, 1970; Marrone, Gentry, and
Wade, 1976). Although numerous studies have verified the thermogenic
effect of progesterone (Kappas and Palmer, 1%3), the correlation be-

’ To whom correspondence should be addressed at: Department of Psychology, Univer-

sity of California, Santa Barbara, Calif. 93106.
’ Present address: Department of Physiology, University of California, San Francisco,
Calif. 94143.

232
0018-506X/79/030232-11$01.00/O
Copyright @ 1979 by Academic Press, Inc.
All rights of reproduction in any form reserved.
 STEROIDS AND THERMOREGULATION 233

tween progesterone secretion and an increase in body temperature is far

from perfect since 20% of the human cycles in which progesterone rose
following ovulation failed to show the predicted increase in body tempera-
ture (Moghissi, 1976). Body temperature is low during the follicular phase
of the human menstrual cycle (Davis and Fugo, 1948; Fischer, 1954) and
at proestrus in the rat (Brobeck, Wheatland, and Strominger, 1947; Mc-
Lean and Coleman, 1971; Yochim and Spencer, 1976), which supports the
idea that estrogen is associated with reduced body temperature. How-
ever, Marrone et al. (1976) have reported an increase in body temperature
of rats both at proestrus and following the injection of estradiol benzoate.
It may be that the thermogenic properties of the ovarian steroids are not
clear because much of the available information is based on the measure-
ment of body temperature alone rather than on the measurement of
underlying behavioral and autonomic mechanisms of heat dissipation and
heat conservation. Temperature-dependent behavior is an important as-
pect of energy balance that has received little attention in ovarian steroid
research. Cunningham and Cabanac (1971) reported a preference for
warmer temperatures as well as an increase in body temperature during
the luteal phase in humans, supporting the notion that progesterone has
both behavioral and temperature-elevating effects.
Changes in activity and in food intake during the estrous cycle have
been studied in detail (Wade, 1972, 1976), but not the behavioral ther-
moregulatory effects of the ovarian steroids. Kinder (1927) reported that
female rats maintained in cool environments decreased nest-building at
estrus. This could be interpreted as a behavioral thermoregulatory re-
sponse (either a decreased need to conserve heat or an increased need to
dissipate heat) or as a secondary consequence of the increased activity
associated with estrus.
A previous study in this laboratory found that estrogen-treated ovariec-
tomized rats showed a significant increase in behavioral heat intake com-
pared to nontreated controls (Wilkinson, Carlisle, and Reynolds, 1976).
Curiously, this increase in heat intake was not reflected as an increase in
body temperature which was lower both pre- and post-test in the
estrogen-treated animals. This observation could mean that estrogen
treatment is associated with either an increased rate of heat loss or a
reduced metabolic rate. Estrogen administered via slow-release Silastic
capsules in the Wilkinson et al. study produced plasma estradiol concen-
trations of 120 pgiml, which are perhaps two to three times higher than
observed proestrus concentrations of 40-50 pg/ml (Goodman, 1978; Hen-
derson, Baker, and Fink, 1977; Smith, Freeman, and Neill, 1975).
The purpose of the present study was to measure behavioral heat intake
of ovariectomized rats with moderate concentrations of estradiol and
progesterone. The animals were tested in both the light and dark phases of
the LD cycle to determine if the effects of steroid treatments are depen-
234 CARLISLE ET AL.

dent on circadian factors. Metabolic rate was also measured at three

ambient temperatures in the light and dark for similar comparisons.
Method
Animals and hormone treatments. Twenty-four female Sprague-
Dawley rats were housed in individual cages in colony rooms maintained
at 23 ? 2°C and 50% relative humidity. The 1ight:dark cycle was 12:12,
with lights on in one colony room at 0800 hr and off in a second room at
this same time. Bilateral ovariectomies were performed under sodium
pentobarbital anesthesia (Nembutal, 40 mg/kg, ip) when the animals were
approximately 60 days of age with body masses of 200 g; 1 week was
allowed for recuperation.
Previous work in this laboratory noted that Silastic (Dow Corning)
capsules containing estradiol (E) produced sustained high hormone con-
centrations for several months, whereas 20-mm-long capsules of proges-
terone (P) produced low concentrations which decreased rapidly within
several weeks after implantation. Our goal was to produce plasma con-
centrations of E approximating 40-50 pg/ml, and P concentrations ap-
proximating endogenous levels which are 25-30 r&ml at diestrus and
45-60 @ml during the proestrus surge (Goodman, 1978; Smith et al.,
1975).
Silastic capsules containing estradiol (17@estradiol, Sigma Chemical
Co.) were constructed from lo-mm lengths of No. 602-285 tubing (1.57
mm i.d. x 3.18 mm o.d.) with 4-mm wood applicator sticks inserted into
each end so that a 2-mm length of surface was available for steroid
diffusion. The capsules were packed with an average of 2.6 mg crystalline
E, and the ends were sealed with Silastic adhesive. Progesterone (Sigma
Chemical Co.) capsules were constructed from 12.5mm lengths of No.
602-265 tubing (1.57 mm i.d. x 2.41 mm o.d.) sealed with Silastic adhesive
but without wood sticks at each end. An average of 23.8 mg crystalline P
was packed into each capsule, and eight capsules were implanted per P
subject so that the total hormone administered was approximately 190 mg
per animal. A single E capsule was given per E subject, while control
animals received either one or eight empty capsules. Combined EP ani-
mals received one E and eight P capsules. All capsules were implanted
subcutaneously at the nape of the neck.
Apparatus. The animals were shaved and trained to press a lever for
radiant heat in a -8 5 2°C environment in an apparatus which consisted of
a 24-cm-diameter, hardware-cloth cage with Plexiglas-rod flooring and a
Plexiglas lever. Depression of the lever activated two 250-W red-bulb
infrared lamps mounted outside the cage and focused on the area in front
of the lever. The lamps were activated as long as the lever was depressed.
The power dissipated by the lamps was 300 W, which produced a radiant
flux density, measured with an Eppley thermopile, of 180 mW/cm2 in the
 STEROIDS AND THERMOREGULATION 235

center of the cage. Constant dim illumination (1.6 lx) was provided by a
7-W red-bulb incandescent lamp mounted outside the cage. The cage and
heat lamps were placed in a 17-ft3 freezer, with programming and control
equipment located in a room adjacent to the test room. The number of
responses and total seconds of heat received were recorded on elec-
tromechanical counters; a cumulative recorder provided a visual record of
the incidence and duration of responding. Rectal temperatures were ob-
tained pre- and post-test with a Model 46 Telethermometer (Yellow
Springs Instrument Co.) and a No. 402 probe inserted 6 cm.
Metabolic rate was determined from the rate of oxygen consumption in
an open-flow system. The shaved animal was placed in a sealed 8-liter
Plexiglas chamber in a temperature-controlled room. Dry air was drawn
through the animal chamber at the rate of 200 ml/min, redried, and passed
to a Beckman OM- 1I polarographic oxygen analyzer. The percentage
oxygen depletion was determined, and oxygen consumption was ex-
pressed as milliliters of 0%per gram per hour (STPD). A metabolism test
lasted 6 hr, with 2 hr at each ambient temperature of 25, 15, and SC, in
this order. Oxygen consumption measurements were taken at the end of
each 2-hr period. Pre- and post-test rectal temperatures were taken as
described previously. Food was available except during a test, and light-
ing was the same as the maintenance L/D condition.
Procedure. The animals were tested in groups of eight; half were
maintained with the light phase of the cycle occurring between 0800 and
2000 hr and for the other half the dark phase occurred during this time. All
tests were conducted during the middle 4-6 hr of the LD cycle. The
ovariectomized animals were first trained to work for radiant heat reward
in the cold and then given two additional tests of 2-hr duration to assure
stable responding before data were collected. Four tests of 2-hr duration
were then given with 2-3 days intervening between each test. The rats
were ranked on the basis of the average seconds of heat obtained per
minute in these tests, and four groups of three subjects each were formed
for both the light and dark conditions. Animals were assigned to either the
estradiol (E), progesterone (P), estradiol and progesterone (EP), or control
(C) groups such that there were no differences between groups in prehor-
mone rates of obtaining radiant heat. One week after hormone implanta-
tion, a practice test was given followed by four data tests as before. The
light/dark conditions were then reversed and 2 weeks allowed for adapta-
tion. A single practice test was again given followed by four data tests, as
before.
Radioimmunoassay of plasma progesterone and estradiol. Blood sam-
ples for progesterone determinations were collected from all animals at 7,
14, and 52 days after capsule implantation. A small incision was made at
the tip of the tail and approximately 300 yl of blood was taken in
heparinized microhematocrit tubes. Blood samples (3-3.5 ml) for es-
236 CARLISLEETAL.

tradiol determinations were collected from all animals at 52 days post-

implant by cardiac puncture under ether anesthesia using EDTA-treated
Vacutainers (Becton-Dickinson Co.). Following centrifugation, plasma
was stored at -10°C until assayed by radioimmunoassay.
Preliminary purification of plasma progesterone and estradiol by celite
microchromatography was modified from the clinical procedure of Abra-
ham, Hopper, Tulchinsky, Swerdloff, and Ode11(1971). Ether-extracted
progesterone was transferred to the microcolumn via 1 ml 2% (ethyl
acetate:solvent) solution. Progesterone was eluted with 3 ml 5% solution.
Cross-reacting steroids 17-hydroxyprogesterone, 11-deoxyprogesterone,
and corticosterone were not eluted (~1%) from the column. Micro-
columns for estradiol purification used a more polar stationary phase
(ethylene glycokwater, 8: 1) to decrease solvent effects. Ether-extracted
estradiol was transferred to the microcolumn via 1 ml 10% solution. A
3-ml 10% solution eluted progesterone, androstenedione, 17-
hydroxyprogesterone, testosterone, and estrone. A 3-ml 20% solution
eluted estradiol (56 +- 3%). Estriol and corticosterone were not eluted
(< 1%) from the microcolumn.
Antisera for radioimmunoassay of progesterone (S-49 No. 6), estradiol
(S-52 No. 5), and corticosterone (S-150 No. 4) were supplied by Dr. G.
Abraham, Harbor General Hosptial, Torrance, California. Radioim-
munoassay of plasma progesterone was as previously described (Abra-
ham et al., 1971). Radioimmunoassay of plasma estradiol utilized high
specific activity [3H]estradiol [N-(2,4,6,7,16,17-3Hlestradiol, SA = 145
Ci/mmol, NET-517, New England Nuclear] to maximize the sensitivity of
the procedure. If the chromatographic prepurification was omitted (be-
low) then steroid-stripped plasma (100 ~1) was substituted for gelatin and
the tracer was preincubated 1 hr at 22°C with the antibody.
Since extremely low E concentrations were observed in pilot experi-
ments, efforts were made to achieve adequate specificity without chro-
matography thereby eliminating the inherent procedural losses and vari-
ability associated with purification procedures: In ovariectomized female
rats, corticosterone, the principal adrenal steroid, is the only steroid
capable of significant displacement of estradiol tracer. Extracted non-
chromatographed determinations agreed with chromatographed determi-
nations when corticosterone interference was subtracted (corticosterone
concentration determined by radioimmunoassay, Keith, Winslow, and
Reynolds, 1978).
RESULTS
The mean heat intake of the four groups in the light and in the dark is
given in Table I. Analysis of variance revealed a significant light/dark
effect (F (1, 16) = 56, P < 0.001) but no effect of the hormone treatments.
Thus the rat, a nocturnal animal, obtains more heat behaviorally during
 STEROIDS AND THERMOREGULATION 237

TABLE 1
Mean (aSEM) Behavioral Heat Intake and Pre- and Post-Test Rectal Temperature in the
Light and in the Dark

Rectal temperature (“C)

Heat intake (secimin) Light Dark

Group Light Dark Pretest Post-test Pretest Post-test

E 17.2 (k2.32) 13.0 (k1.75) 37.3 (20.13) 38.0 (20.12) 38.0 (kO.17) 38.6 (20.10)
P 16.2 (22.58) 13.1 (21.34) 37.2 (kO.21) 38.0 (20.12) 38.1 (50.14) 38.4 (k-0.15)
EP 19.6 (50.79) 11.8 (21.07) 37.3 (kO.21) 38.0 (20.09) 38.2 (kO.09) 38.4 (20.06)
C 17.1 (k1.24) 13.5 (k1.51) 37.1 (20.10) 38.2 (kO.13) 37.7 (20.16) 38.3 (50.09)

the light phase of the LD cycle than during the dark. The difference in
behavioral heat intake between light and dark conditions was accounted
for by a change in the average duration of a response and not a change in
the frequency of responding. The average seconds of heat obtained per
response was 13.2 in the light and 8.8 in the dark, while the frequency of
responses was 1.7 per minute in the light and 1.5 per minute in the dark.
There were no differences between hormone groups in the frequency of
responding or in the duration of a response.
Pre- and post-test rectal temperatures are given in Table 1. There was
no overall difference between groups in the analysis of variance. Temper-
atures were higher in the dark than in the light (F (1, 16) = 53, P < O.OOl),
and higher post- compared to pretest (F (1, 16) = 140, P < 0.001).
Average metabolic rates as a function of ambient temperature and
light/dark testing conditions are given in Table 2. Analyses of variance

TABLE 2
Mean (?SEM) Rates of Oxygen Consumption as a Function of Ambient Temperature and
Pre- and Post-Test Rectal Temperature in the Light and in the Dark

O,(mI g-’ hr-I) Rectal temperature (“C)

Group 5°C 15°C 25°C Pretest Post-test

Light
E 2.66 (20.12) 1.97 (20.06) 1.19 (20.07) 37.7 (kO.40) 35.5 (20.36)
P 2.45 (20.05) 1.82 (20.07) 1.13 (a0.04) 37.5 (20.21) 35.1 (kO.11)
EP 2.53 (50.14) 1.92 (-r-0.11) 1.16 (kO.05) 38.2 (20.36) 35.1 (kO.49)
C 2.49 (20.08) 1.84 (20.07) 1.16 (50.06) 37.2 (20.37) 36.0 (20.42)

Dark
E 2.71 (20.16) 2.12 (kO.11) 1.35 (k0.07) 37.7 (kO.27) 34.5 (20.46)
P 2.51 (20.09) 1.89 (eO.09) 1.24 (-+0.04) 37.9 (20.24) 35.0 (,0.45)
EP 2.53 (?O. 17) 2.02 (r0.16) 1.30 (20.10) 37.9 (kO.21) 34.4 (20.29)
C 2.60 (20.12) 1.93 (20.12) 1.22 (kO.07) 37.5 (20.35) 35.7 (20.45)
238 CARLISLE ET AL.

showed significant effects of both the light/dark condition (F (1,20) = 4.6,

P < 0.05) and ambient temperature (F (2, 40) = 117, P < O.Ol), but no
effect of hormone treatment could be demonstrated. Oxygen consumption
was thus higher during the dark tests than during the light tests, and it
increased as ambient temperature decreased under both light and dark
conditions. Although there was no overall effect of the hormone treatment
in the analysis of variance, it should be noted that the estrogen-treated
group had consistently higher metabolic rates than the control group in
both the light (5.9%) and the dark (7.6%) at all ambient temperatures.
Pre- and post-test rectal temperatures for the metabolism tests are given
in Table 2. All hormone-treated groups had higher pretest but lower
post-test temperatures compared to the control group in both the light and
the dark. These differences are apparent when the cooling rate, or the
difference between pre- and post-test temperature, is considered. Thus,
for the E, P, EP, and C groups, the mean rates of cooling were 2.42,2.47,
3.08, and 1.22”C during the 6-hr tests in the light, and these rates increased
to 3.17, 2.85, 3.57, and 1.73”C in the dark, respectively. Analyses of
variance of the cooling rates were significant for both the light (F (3,20) =
3.1, P < 0.05) and the dark (F (3,20) = 3.9, P < 0.05). When cooling rates
are averaged irrespective of the light/dark condition, the various groups
can be ordered as follows: EP > E > P >> C.
As noted in numerous studies (Wade, 1976), estrogens retard an in-
crease in body mass. The body mass of the animals given estradiol alone
average 285 g at the time of hormone implantation and 286 g 7 weeks later.
The average increase in mass of other animals was 23 g, and there were no
significant differences between the EP, P, and C groups.

TABLE 3
Assay Parameters

Estradiol

progesterone Chromatographed Nonchromatographed

Minimum detectable steroid

Standard curve” 10 Pg 2 PB 2 Pg
Plasma* 0.25 rig/ml 8 &ml 4 @ml

Coefficient of variationC
Intraassay 15% 20% 11%
Interassay 20% - -

o Minimum detectable mass is defined as the smallest mass of steroid capable of sig-
nificant displacement of tracer at the 95% confidence level.
* Masses interpolated from the standard curves are corrected for nonspecific binding,
solvent effects, milliliter fractions, and procedural losses.
c Coefficient of variation is defined as [(standard deviation/mean) x lOO%].
 STEROIDS AND THERMOREGLJLATION 239

Radioimmunoassay parameters are listed in Table 3. Pooled (37 ? 5, 34

+- 3 pg/ml) and individual samples showed good agreement between
chromatographed and nonchromatographed determinations. Addition-
ally, the nonchromatographed determinations, as can be inferred from
Table 3, had both higher precision and lower sample volume require-
ments. Overall, the assay parameters appear comparable to other proce-
dures (Abraham er al., 1971). Estradiol concentrations measured by the
nonchromatographic procedure for plasma samples from the E and EP
groups were 41.2 + 9.1 (SD) pg/ml. Chromatography on four of these
samples yielded E concentrations that averaged only 4 pg/ml less than
nonchromatographed samples; the range of differences was between -8.8
and 3.5 pg/ml. The E concentration in plasma samples from animals
receiving P or no hormone averaged 8.2 t 6.4 pg/ml measured by the
nonchromatographic procedure. Progesterone concentrations sampled at
7, 14, and 52 days postimplant were 49.5 ? 13.9, 36.1 + 10.3, and 21.5 _t
6.4 rig/ml for animals receiving P or EP, and 4.1 + 3.4, 4.6 + 3.7, and 3.5
-+ 2.2 rig/ml for animals receiving E or no hormone, respectively. Proges-
terone concentrations thus averaged between 20 and 50 rig/ml during the
course of this study for P and EP groups, while estradiol was approxi-
mately 41 pg/ml for E and EP groups.
DISCUSSION
Behavioral heat intake is high during the day when metabolic rate and
body temperature are low, and low at night when metabolic rate and body
temperature are high. These observations show that behavioral heat in-
take is out of phase with respect to the well-known circadian increases in
metabolism, body temperature, activity, and food intake noted during the
night in rats (Aschoff, 1970). The diurnal rhythm of metabolism exists
independently of the variation in food intake and activity (Aschoff and
Pohl, 1970). In spite of the inverse relation between metabolism and
behavioral heat intake, body temperature was lower both pre- and post-
test during the day compared to the night. Thus metabolism and be-
havioral heat intake do not vary such that body temperature is maintained
at a constant level. Rather, since metabolism is high at night in a nocturnal
animal, less behavioral heat intake is required to maintain a higher tem-
perature. Conversely, more exogenous heat is required during the day
when metabolism is low to maintain a lower body temperature than at
night. A complementary outcome has been noted by Schmidt, Graf, and
Rautenberg (1978), who found that day-active pigeons obtained more heat
during the night than during the day.
Hormone treatments made very little difference in behavioral heat
intake in the present study. This is surprising since a previous study
(Wilkinson, Carlisle, and Reynolds, 1976)found that estrogen-treated rats
showed an increase in heat intake with reduced body temperatures rela-
240 CARLISLEETAL.

tive to control animals. There was no indication of reduced body tempera-

tures in E-treated rats in the present study. The main difference between
the E and C groups was the greater cooling rate of the E group during cold
exposure without a source of exogenous heat. All hormone-treated groups
cooled more rapidly than the C group, which raises the possibility of a
nonspecific steroid-induced increase in heat loss. This interpretation is
consistent with the greater cooling rate of animals given both E and P as
opposed to either E or P; Ethinyl estradiol-treated rats also cool faster
than control rats (Fregly, Black, Kelleher, and MacArthur, 1977). An
increased behavioral heat intake would be expected if steroid treatment is
associated with an increased cooling rate in the cold, but this was not
observed in the present study.
The simplest explanation for the discrepancy between the results of the
present study and those of Wilkinson et al. is that estradiol concentrations
of 120 pg/ml observed in the latter study are associated with increased
behavioral heat intake and reduced body temperatures while concen-
trations of 40 pglml in the present study are not. We recently verified
(unpublished observations) that this interpretation is correct. Further, the
Wilkinson et al. study predicted an increase in heat loss or a decrease in
heat production in E-treated animals. A decrease in heat production can
be ruled out since there was no evidence of low metabolism in the
E-treated group; rather, metabolic rates were 6-7% higher in E than in C
groups. Recent work has noted an increase in both heat production and
dry heat loss in E-treated rats relative to untreated controls
(Laudenslager, Wilkinson, Carlisle, and Hammel, 1979). The increase in
heat production in estrogen-treated animals could be compensatory to the
increased rate of heat loss. An increase in the rate of heat loss would be
consistent with the decrease in temperature of E compared to C groups in
the present study, the faster cooling rates of ethinyl estradiol-treated rats
(Fregly et al., 1977), and the increased behavioral heat intake with re-
duced body temperature in the Wilkinson study.
Progesterone treatments produced plasma P concentrations which de-
creased from approximately 50 @ml at 1 week postimplant to 20 r&ml at
2 months postimplant. There were no substantial differences between the
P and C groups in behavioral heat intake or metabolism. Body tempera-
tures were not significantly higher in the P than the C groups. If P is a
thermogenic hormone, either an increased rate of heat production or a
diminished rate of heat loss would be expected in P compared to C
animals. Neither alternative is supported by the present data.
Several possibilities can be suggested for the lack of effect of the
ovarian steroids on behavioral heat intake, and the lack of a differential
effect of estrogen and progesterone on body temperature. First, the
plasma concentrations of estradiol and progesterone were moderate;
higher concentrations may be required for thermogenic effects. gecondly ,
 STEROIDS AND THERMOREGULATION 241

the testing procedure interposed a delay of about a month between

ovariectomy and hormone treatment; delayed hormone treatment affects
the magnitude of the lordosis response (Damassa and Davidson, 1973),
and could also influence temperature regulation. Finally, the administra-
tion of steroids in Silastic capsules results in steady-state hormone con-
centrations; fluctuating concentrations may be important for the ther-
mogenic properties of estradiol and progesterone. As noted above, the
first possibility is correct for estradiol, but the latter two alternatives
cannot be evaluated at this time.
ACKNOWLEDGMENTS
This research was supported by NIH Research Grant HD-10473. Statistical analyses were
aided by computer programs developed at the Health Science Computing Facility, UCLA,
supported by NIH Special Research Grant RR-3. We wish to thank Pat Webb and Minne
Ness for expert assistance.

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