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Pergamon
0031-9384(95)00096-8
Physiology & Behavior, Vol. 58, No. 3, pp. 573-585, 1995
Copyright ¢~ 1995 Elsevier Science Lid
Printed in the USA. All rights reserved
0031-9384/95 $9.50 + .I)0
Estrus- and Steroid-Induced Changes in Circadian
Rhythms in a Diurnal Rodent,
Octodon degus
SUSAN E. LABYAK* AND THERESA M. LEE? l
Departments of*Nursing and ~Psychology, 1103 E. Huron, Neuroscience Laboratory Building, University of
Michigan, Ann Arbor, MI 48104-1687, E-Mail: terrilee@ UMICH.edu
Received 2 August 1993
LABYAK, S. E. AND T. M. LEE.
Estrus and steroid-induced changes in circadian rhythms in a diurnal rodent, Octodon degus.
PHYSIOL BEHAV 58(3) 573-585, 1995.--Diurnal
Octodon degus
exhibited marked alterations in activity and temperature in
conjunction with the 3 wk estrous cycle when housed in LD 12:12 light cycle. On the day of estrus, mean daily activity increases
109%, mean core temperature rises .4°(2, activity onset is advanced 2 h, and amplitudes of both rhythms decline compared with
the 3 days prior to estrus. On the day following estrus, activity onset was delayed 4.9 h, and mean activity and core temperature
fell below that of the preestrus period. Ovariectomy significantly reduced mean temperature (.98°C) but did not significantly alter
mean activity, and eliminated cyclic effects of estrus. Estrogen replacement led to a nonsignificant elevation in mean activity and
core temperature with no change in the phase angle of entrainment. Progesterone replacement significantly reduced mean core
temperature and mean activity, while only the phase angle difference between temperature minimum and activity onset was
significantly altered. Intact degus maintained in constant darkness displayed only transient fluctuations in activity onset and
temperature minimum during and after estrus. Estrogen or progesterone treatment of ovariectomized, free-running degus altered
mean temperature and activity levels, but did not influence tau. Changes in phase angle of entrainment during estrus are not the
result of hormone effects on the circadian clock but likely reflect increased or decreased levels of activity.
Octodon degus Estrus Circadian Activity Temperature Ovariectomy Free-running rhythm
Phase angle of entrainment
OVARIAN hormones influence the timing and expression of cir-
cadian activity and core temperature rhythms in nocturnal female
rodents (1,13,15,31). During proestrus and estrus (days three and
four of a four-day estrous cycle) when estrogen levels are high
(4), hamsters demonstrate an increase in mean daily activity (25),
a phase advance in the time of activity onset (22,24), and a rise
in mean core temperature (24). In blind, ovariectomized female
hamsters estradiol shortened the free-running circadian activity
period (21,22), and this effect could be blocked by the adminis-
tration of progesterone (31). These findings suggest that estradiol
and progesterone interact to modulate the timing and expression
of circadian activity in the hamster.
Rats display similar estrous-related changes in circadian ac-
tivity (26) and core temperature (20,35) in their five-day estrous
cycle. On the day of estrus, total activity and activity duration
increase in Sherman and LEW/ZTM rats. Activity onset is phase
advanced and the circadian period is notably shortened in both
entrained and free-running animals (1,20,35). In other strains of
rats (e.g., Holtzman and Sprague-Dawley), increased running-
wheel activity and temperature elevations occur on the night of
proestrus (2,15,37), accompanied by a phase advance in the cir-
cadian activity and core temperature rhythms. In contrast to stud-
ies in which animals were housed with running-wheels, Kent et
al. (15) found that sedentary Sprague-Dawley rats, as well as
those housed in cages with locked wheels, displayed a significant
phase delay in the temperature rhythm on each day of the estrous
cycle. They concluded that estrous-related changes in core tem-
perature were largely a function of increased activity, and that
ovarian hormones had little influence on temperature in Sprague-
Dawley rats.
While an estrous effect on circadian activity and core tem-
perature rhythms has been documented in nocturnal rodents, few
investigators have studied the impact of estrus on circadian
rhythms in diurnal mammals. Since species-typical patterns are
evident in nocturnal species, estrous-related alterations in circa-
dian rhythms may be distinctly different in diurnal species. For
example, in one case study, the circadian activity of a premeno-
pausal 46 yr old woman was monitored for one year via wrist-
actograph. At the time of ovulation, the subject demonstrated a
significant decrease in mean activity coupled with a significant
phase delay in activity onset (7). The findings suggest that cir-
cadian responsiveness to changing ovarian hormones is distinctly
different in diurnal humans when compared to nocturnal rats or
hamsters. Wever (34) studied changes in the free-running core
temperature rhythm across the menstrual cycle in young women,
and compared them to mean temperatures in men of similar ages.
While preovulatory mean temperatures in young women were
similar to those of males, postovulatory mean temperatures were
To whom requests for reprints should be addressed.
573
574 LABYAK AND LEE
higher. Such postovulatory temperature changes have not been
reported for hamsters and rats, and may be the result of nocturnal/
diurnal differences or the difference in length of the luteal stage.
Diurnal female degus
(Octodon degus)
display altered circa-
dian activity and core temperature rhythms at approximately 20
day intervals in this laboratory. The purpose of these experiments
was to determine whether periodic circadian changes were the
result of estrus, and if so, to determine the potential role of es-
trogen and progesterone in these changes. The daily mean, phase,
and amplitude of the circadian activity and core temperature
rhythms were monitored. If data from diurnal degus are consis-
tent with those from nocturnal rodents then we would expect that
(i) female degus would demonstrate a phase advance in circadian
activity and core temperature rhythms during proestrus and es-
trus, coupled with an increase in mean daily activity and core
temperature; (ii) mean core temperature during proestrus/estrus
might rise in conjunction with increased mean activity; (iii) ovar-
iectomy would eliminate the cyclic alterations in circadian activ-
ity and core temperature associated with estrus; and (iv) estrus
or replacement hormones would influence 7- consistent with hor-
mone-induced changes in entrained phase.
METHODS
Animals and Housing
Fourteen young, adult female degus (6-16 mo.; average life-
span 5-7 yr) reared in a laboratory colony at the University of
Michigan were used in these experiments (N = 5-10 per exper-
iment). Parental stock was derived from colonies in five locations
in the U.S., and outbreeding was used to maintain genetic diver-
sity in the population. The degus were individually housed in
47.5 × 26.25 × 20 cm cages fitted with running-wheels (9 cm
wide and 34.5 cm in diameter). Food and water were available
ad lib, and room temperature was maintained at 22 ° ___ 2°C. Degus
were maintained in 12 h of light per day (LD 12:12; lights on at
0600 h EST) for 3 wk prior to beginning data collection.
Surgery
The degus were implanted intraperitoneally with Mini-mitter
transmitters (model VM-FH; Mini-mitter, Inc., Sun River, OR)
to measure core temperature and general activity with Dataquest
III hardware and software. Degus were anesthetized with Keta-
mine HCL (30 mg/kg of body weight) and Xylazene (2.5 mg/kg
of body weight). The animals required approximately three hours
to recover, and Yohimbine (2.5 mg/kg of body weight, IP) was
administered immediately following the surgery to shorten the
recovery period.
Data Collection and Analysis
The animals were given at least 1 wk to recover prior to ex-
perimentation. Running-wheel activity was recorded as the num-
ber of wheel revolutions and was stored at 10 min intervals by
Dataquest. General activity and core temperature were monitored
via Mini-mitter transmitters at 10 min intervals. The estrous cycle
was monitored daily by microscopic examination of washes from
vaginal lavage (11).
Spectral analysis and Chi square periodograms were used to
verify the 24 h period of the entrained activity and core temper-
ature rhythms (10). The Tan and Dataquest III computer software
programs (Mini-Mitter, Inc., Sunriver, OR) were used for data
display (actograms and frequency histograms) and analysis. Re-
peated measures ANOVA tests were used to compare activity
and core temperature across different stages of the estrous cycle
with posthoc paired comparisons used to test differences between
specific stages of the estrous cycle. Summary statistics are re-
ported as the mean + SEM, and differences considered signifi-
cant ifp < 0.05.
Four parameters for the activity and core temperature rhythms
were examined: the daily mean, the phase angle of entrainment of
activity onset (~Ao) and temperature minimum (k~TM) with the tim-
ing of lights-on, the phase angle of entrainment of the first morning
peak of activity ('~AP) and morning peak in temperature (k~-rp) with
the timing of lights-on, and the daily rhythm amplitude (from mean
to peak value). The time of activity onset and temperature mini-
mum were determined by examining frequency histograms (Fig.
1). Activity onset was defined as the time when an animal first
demonstrated a minimum of 40 counts of activity in a 10 min
interval, and was active for at least four consecutive 10 min inter-
vals. Temperature minimum was defined as the time of the lowest
daily temperature readings (for three to four consecutive 10 min
intervals). ~AO and ~TM were derived by calculating the difference
between the time of lights-on and the time of activity onset and
temperature minimum, respectively, kOAp and ~TP were derived by
calculating the difference between the time of lights-on and the
time of the morning peak in activity and core temperature, respec-
tively (Fig. 1). The phase angle relationship between temperature
minimum and activity onset (~TA), calculated by k~TM--~'AO, was
alSO compared across the estrous cycle. Rhythm amplitudes were
determined by calculating the difference between the rhythm peak
(maximum value) and the rhythm mean, where the 24 h mean was
computed from midnight each day.
EXPERIMENT 1
Twenty-one estrous cycles from ten degus (i.e., each animal
contributing two or three estrous cycles to the data base) were
used to examine the effect of estrus on the circadian activity and
core temperature rhythms. Data were analyzed in four periods:
(i) preestrns consisted of the three days preceding estrus; (ii)
estrns consisted of one to two days; (iii) metestrus consisted of
the day immediately following estrus; and (iv) postestrus con-
sisted of the two days following metestrus. The data were similar
across animals and were therefore pooled.
Results
The animals displayed stable entrainment under LD 12:12 and
demonstrated vaginal changes indicating estrous cycles of 20.5
-+ .5 days. Vaginal lavage could only be carried out for three to
five days per cycle; during the remainder of the cycle the vaginae
were not patent. While daily vaginal lavage distinguished nones-
trous from proestrous and estrous days, proestrus and estrus could
not always be distinguished because the animals were often in
transition between these stages at the time of lavage.
Estrous smears correlated with a marked rise in mean daily
activity and core temperature (Figs. 2 and 3). The animals dem-
onstrated a significant increase in mean running-wheel activity
(109% increase; p < 0.001) from preestrus to the day of estrus
(Fig. 3A). This was followed by a significant decline in mean
activity on the day of metestrus (67% decrease from estrus; p <
0.001), and recovery during the postestrous period to preestrous
baseline. Mean changes in activity across the estrous cycle were
not the result of changes in daily peak activity levels.
On the day of estrus, there was a 1.9 h advance in ~PAO (P <
0.007; Table 1), followed by a 4.9 h delay (p < 0.001; Table 1)
on the day of metestrus, and a return to baseline preestrous state
during the postestrous period. ~AP was advanced 1.5 h (p --
0.055; Table 1) on the day of estrus, and delayed 3.5 h during
metestrus, although this shift did not reach significance (Table
1). In conjunction with the advance in ~PAo and ~AP, the ampli-
ESTRUS-INDUCED CHANGES IN CIRCADIAN RHYTHMS 575
60o
z
500 ~,
400
° i
,,J 300
re
..a 200 "
-r
loo J
36.0
A
4,080 8.000 12.00 lS. Oe 20.00
Timl
(HOurs)
39.0 4
S
38.5
o.. 38.0
lgl
re
k.- 37.5
ilu
tfi 37.0
p-
ud
re
O 36.5
0
A
4o88e 8. ee8 12.8o 16.88 28.ee
Timl (Hourm)
FIG. 1. Frequency histograms of one day of (A) running-wheel activity
and (B) core temperature for a single female degu. The arrows indicate
phase reference points for activity onset (kOAo) and temperature minimum
(~TM)- The asterisks indicate the phase reference points for first activity
(~AP) and temperature (~TP) peaks after midnight. The bar in the center
of the graph indicates the light:dark schedule.
tude of the circadian activity rhythm declined 11.3% during es-
trus (p < 0.008; Fig. 4A), but did not differ from baseline during
metestrus when kOAo was significantly delayed. The decline in
amplitude during estrus was due to the increased duration of el-
evated activity.
Mean daily core temperature rose .4°C during estrus (p <
0.001 ; Fig. 3B), and then declined .66°C from estrus to metestrus
(p < 0.001). While there was no significant change in the daily
minimum temperature across the estrous cycle, temperature max-
imum declined significantly between preestrus or estrus and me-
testrus (.4 and .5°C; p < 0.03 and p < 0.001, respectively).
In contrast to the advance observed in the kOAo and
~AP on
the
day of estrus, there was a nonsignificant .59 h advance in ~vM
on the day of estrus. The changes in ~TP across the estrous cycle
approached significance (2-tailed posthoc tests of significance);
temperature peak advanced 1.2 h on the day of estrus (p = 0.065)
and delayed 3.5 h between estrus and metestrus (p -- 0.075; see
Table 1). The amplitude of the circadian temperature rhythm de-
clined 16.4% between preestrus and estrus (p < 0.02; Fig. 4B).
The decline in rhythm amplitude during estrus was due to an
increase in the duration of elevated temperatures.
Thus, on the day of estrus mean activity and core temperature
rose as the amplitude of those rhythms declined. In addition, the
onset of activity (k~Ao) and the first morning peak of activity (kOAp)
were phase advanced on the day of estrus. Metestrus was marked
by a reduction in mean activity and core temperature, and a phase
delay in LOgo. Recovery of the temperature amplitude during me-
testrus and postestrus was gradual, while the amplitude of the
activity rhythm returned to baseline during metestrus. The phase
angle difference between temperature minimum and activity on-
set (~TA) was significantly altered during estrus. ~w was reduced
by 1.6 h from preestrus to estrus (p < 0.03), but increased to 4
h during metestrus (p < 0.005) before returning to the preestrous
relationship (Table 1). The alteration in ~'vg was primarily due
to changes in ~AO, while kOVM remained essentially constant.
EXPERIMENT 2
Circadian activity and core temperature rhythms in the degu
are tightly coupled. Activity onset begins as temperature rises,
and activity/temperature elevations throughout the day are strik-
ingly similar. This strong association between circadian activity
and core temperature rhythms makes it difficult to evaluate the
effects of ovarian hormones on the temperature rhythm of intact
animals with running-wheels. Increases in mean activity correlate
well with temperature elevations. However, mean temperature
B
5.880 XS.e8 X5.88 2e.88 5.888 Xe.ee lb. ee ~.~u
T.i. ml
(Hours)
FIG. 2. Entrained animal's (A) activity and (B) core temperature actograms during preestrus,
estrus and postestrus changes. Each line represents one day of data. Asterisk denotes the day of
estrus. Bar at the bottom of the actograph indicates the light:dark schedule. The lowest 25% of
temperature data are not displayed to assist in viewing the daily rise in temperature.
576 LABYAK AND LEE
A
2;
O
2;
o
[..,
o
e~
-1
m
::11:
2;
i/J
275
220
165
110
55
PRE ESTRUS MET POST
B 38.70
38.48
~
38.26
38.04
0
r..)
,~
37.8:2
37.60
PRE ESTRU8 MET POST
FIG. 3. Variations in mean (4- SEM) (A) running-wheel activity and (B)
core temperature across the estrous cycle. Based on vaginal lavage, PRE
= 3 days preestrus, ESTRUS = 24 to 36 h around estrus, MET = 24 h
following estrus, and POST = 2 days following metestrus. All days begin
at midnight. Asterisks represent significant differences from preestrus
using posthoc paired comparisons (p < 0.001). Letter "a" represents
significant differences between estrus and metestrus with posthoc paired
comparisons (p < 0.001).
can rise without increasing activity, although this has not been
reported in other rodents unless they have fevers (15,24,28). To
further evaluate the influence of running-wheel activity on core
temperature, the mean total activity (gathered by the Mini-mitter/
Dataquest system) and core temperature of seven female degus
(6-8 mo old), with and without running-wheels available, were
examined across two or three estrous cycles. Effects of the es-
trous cycle on the circadian activity and core temperature
rhythms were evaluated across the four periods described in Ex-
periment 1: preestrus, estrus, metestrus, and postestrus. Repeated
measures ANOVA tests with posthoc paired comparisons were
used to examine differences between the running-wheel and non-
running-wheel treatments, and differences were considered sig-
nificant if p < 0.05.
Results
During the time that animals had access to running-wheels,
they demonstrated a significant rise in general activity during
estrus (p < 0.001; Fig. 5A), followed by a significant decline in
activity from estrus to metestrus (p < 0.001) which continued
into the postestrous period (p < 0.001). Mean core temperature
also rose between preestrus and estrus (p < 0.001; Fig. 5B), and
fell significantly between estrus and metestrus (p < 0.001) and
between estrus and postestrus (p < 0.001).
Although total activity (includes activity generated while in a
running wheel) was considerably diminished (54%, p < 0.001)
when running-wheels were removed, the animals without run-
ning-wheels continued to demonstrate a significant rise in activity
during estrus (p < 0.05; Fig. 5A). The percent change in mean
daily activity on the day of estrus was comparable with or without
a running-wheel (54% and 40.4%, respectively). Activity de-
clined between estrus and metestrus (28.9%; p < 0.05), and re-
mained lower during the postestrous period (p < 0.05). Although
mean core temperature declined .32°C following removal of the
wheel (p < 0.001; Fig 5B), the degus continued to display a
significant .4°C rise in core temperature during estrus (p < 0.02),
a significant .4°C decline between estrus and metestrus (p <
0.001), and a .55°C decline between estrus and postestrus (p <
0.01; Fig. 5B). Again, the percent change in core temperature on
the day of estrus was comparable with or without running-wheel
activity (1.14% and 1.19%, respectively). Thus, the heavy activ-
ity associated with the running-wheel was not responsible for
estrous-related elevations in mean activity and core temperature.
EXPERIMENT 3
The influence of estrogen and progesterone on circadian ac-
tivity and core temperature rhythms was evaluated by first elim-
inating and then restoring those hormones. Nine female degus
from Experiment 1 were anesthetized as previously described,
and bilateral ovariectomy was performed. After monitoring run-
ning-wheel activity and core temperature rhythms for approxi-
mately 2 too, each animal was anesthetized and implanted with
estradiol benzoate (EB). Crystalline EB (Sigma) was adminis-
tered in 20 mm silastic capsules (Dow-Corning; 1.98 mm ID,
3.15 mm OD), filled to an effective length of 15 mm with hor-
mone, and sealed at both ends with Medical Adhesive Silicone
Type A (Dow-Corning). Capsules were implanted subcutane-
ously in the interscapular region (18). Vaginal opening and
smears consistent with estrus were restored in eight of nine ani-
mals, and circadian activity and core temperature were examined
in those eight animals. One animal's implant evidently failed to
work since there was no vaginal opening following EB implan-
tation. Data from this animal were not included in the analysis.
After one week, estradiol capsules were removed.
Five animals were then given 1 wk to recover from the effects
of estradiol before being implanted with progesterone. As pre-
viously described for EB administration, crystalline progesterone
(Sigma) was administered in 20 mm silastic capsules filled to an
effective length of 15 mm with hormone, and implanted subcu-
taneously in the interscapular region. Capsules remained in place
for 1 wk.
The daily mean, phase angle of entrainment, and amplitude
of the circadian activity and core temperature rhythms were eval-
uated across four time periods: intact (non-estrus), postovariec-
tomy (OVX), preimplant of each hormone, and postimplant of
each hormone. Five to seven days representative of each period
were selected for analysis as previously described. These data
were also compared to estrous and metestrous data collected from
these same animals while intact (Experiment 1).
Results
After ovariectomy, the cyclic effects of estrus on activity and
core temperature were completely absent. Although animals
ESTRUS-INDUCED CHANGES IN CIRCADIAN RHYTHMS 577
TABLE
1
PHASE ANGLE OF ENTRAINMENT (Time in h)
Intact Animals (Experiment
I)
STAGE OF ESTRUS
(N = 10)
PRE-ESTRUS +0.4 ± 0.6' +2.3 _ 0.3 -1.9 ± 1.0 +0.2 _ 0.1 +2.0 _+ 0.5
ESTRUS +2.3 ± 0.3 b +2.9 _ 0.4 -0.4 ___ 1.0 ~ + 1.3 ± .4 4-0.4 4- 0.5 f
METESTRUS -2.6 ± I.@ +2.2 + 0.7 -3.9 _ 2.9 -2.2 ± 1.2 +4.3 ± 1.48
POST-ESTRUS -0.1 ± 0.7 d +2.2 _ 0.5 -3.9 _+ 1.3 -0.9 ± 0.8 +2.4 _+ 0.5 h
Ovariectomized Animals
(Experiment 3)
PRE-EB (N = 8) +1.3 ± 0.1 +2.5 ± 0.2 +0.3 ± 0.1 +0.1 _+ 0.1 +1.2 _ 0.1
PLUS EB +2.2 _ 0.8 +3.6 _ 0.4 -0.4 ± 0.7 +I.0 ± 0.4 +1.3 _ 0.6
PRE-PROGEST (N = 5) -2.1 __- 1.8 +0.6 _ 1.4 -3.7 ± 2.0 -3.0 ± 1.6 +2.7 ± 0.6
PLUS PROGEST -4.1 ~ 1.6 +1.0 ± 0.8 -5.3 ± 1.7 -1.7 ± 1.3 +5.2 ± 1.4 ~
Mean _ SEM; bp < 0.07 from Pre-estrus; ~p < 0.001 from Estrus, p < 0.003 from Pre-estrus; dp < 0.02 from Metestrus, p < 0.002 from Estrus;
~p = 0.055 from Pre-estrus; ~p < 0.03 from Pre-estrus; ~p < 0.005 from Estrus, p < 0.045 from Pre-estrus; "p < 0.003 from Estrus; 'p < 0.05 from
Pre-progest; kOAO = Phase angle activity onset to lights on (LO); 9AP = Phase angle morning activity peak to LO; 9ru = Phase angle temperature
minimum to LO; ~TP = Phase angle morning temperature peak to LO; kOTA = Phase angle between ~TM and ~ao; EB = Estradiol Benzoate; Progest
= Progesterone (crystalline); Pre-progest = week following EB removal.
demonstrated a 16.9% decline in activity compared with the no-
nestrous period for intact animals, the decline was not significant
(Fig. 6A). Also, no significant alterations were seen in kOao, kOap
or the amplitude of the circadian activity rhythm compared with
the nonestrous state. In contrast, mean core temperature declined
.98 ° _ .07°C following ovariectomy (Fig. 6B; p < 0.02). This
alteration in mean temperature was sustained throughout the re-
mainder of the study, despite the fact that animals continued to
have access to running-wheels. There were no significant
changes in ~tm, kOTP, or the amplitude of the temperature rhythm
following ovariectomy.
Treatment with estradiol caused a nonsignificant increase in
mean activity (35.7%; Figs. 6A and 7A). There were no signifi-
cant changes in ~AO
or ~I/Ap
following estradiol implant (see Table
1, Fig. 7A), and activity amplitude did not differ significantly
between intact, nonestrous or OVX states.
Estradiol did nor affect mean core temperature (Figs. 6B and
7B). As previously noted, there was a .98°C decline in core tem-
perature following ovariectomy, and this newly established mean
temperature was not influenced by the estrogen implant. Phase
angles of entrainment, ~vm, k~Tp, and
~'ITA were
not altered by EB
(Fig. 7, Table 1). The administration of estradiol was associated,
however, with a decline in the amplitude of the temperature
rhythm (10.6%; p < 0.01) from the OVX, preEB condition.
One week following removal of the estradiol, progesterone
was implanted. The degus demonstrated an immediate, decline
in mean activity (54%; p < 0.001, Fig. 6A) within the first 24 h
of progesterone implantation, which continued throughout the
remaining 4 days (p < 0.005; Fig. 6A). @go, k~Ap, and activity
amplitude were not significantly altered following progesterone
treatment (Fig. 7A, Table 1).
Mean core temperature also declined (.21°C; p < 0.005; Fig.
6B) following the administration of progesterone, k~TM, ~TP, and
temperature amplitude did not differ significantly from the pre-
progesterone state (Fig. 7B, Table 1). However, kVVA was in-
creased by progesterone treatment (p < 0.05, Table 1), as de-
scribed for intact animals during metestrus (Experiment 1).
Therefore, as predicted, degus did not display cyclic alter-
ations associated with estrus in the circadian activity and core
temperature rhythms after ovariectomy. Although estradiol re-
placement reinstated vaginal estrus in these animals, it did not
significantly increase either mean activity or core temperature.
The only significant effect observed in EB-implanted animals
was damping of the temperature rhythm amplitude. In con-
trast, progesterone replacement led to significant reductions in
both mean activity and temperature. Neither hormone treat-
ment significantly altered entrained phase angles of activity or
temperature. However, after EB and progesterone treatments
~AO advanced or delayed (p < .10) consistent with estrus or
metestrus (Table 1). The delay in ~AO after progesterone
resulted in a significant increase in
~kT/Tg
similar to that of
metestrns.
EXPERIMENT 4
Experiments 1-3 demonstrate that estrus has a pronounced
effect on the timing and expression of entrained circadian activity
and core temperature rhythms in the diurnal degu. The daily
mean and amplitude of both rhythms were altered during estrus,
and the phase of activity onset was advanced. While it is possible
that ovarian hormones act directly on the central circadian pace-
maker, an alternative possibility is that gonadal hormones pro-
duce transient increases or decreases on mean activity and tem-
perature with no direct impact on period length (7-). The short
reproductive cycles of rats and hamsters (four to five days; I l,
26) make it difficult to evaluate whether the effects of estrus on
~kO are the result of transient increases in mean activity, or if
they reflect changes in the ~-. The longer estrous cycle of degus
provides more time to determine whether hormonal changes alter
~-, or alter the duration of the active relative to the inactive phase
of the circadian rhythm.
To further evaluate the influence of cyclic changes in steroids
on the circadian pacemaker, seven intact female degus were
placed in constant darkness (DD), and their free-running loco-
motor activity and core temperature rhythms were evaluated
across the estrous cycle. Fourier and Chi square periodograms
were used to determine the period of the activity and core tem-
perature rhythms for 6-10 days before and after estrus. Using
578 LABYAK AND LEE
m
0
m
0
Z
.<
=
O
eL 1
0
el.
Z
0
100
1
ol
i
-100 i i
PRE ESTRUS
/-
MET POST
-1
I I I I
PRE ESTRUS MET POST
FIG. 4. Changes in rhythm amplitudes from preestrus (PRE) for circadian
(A) running-wheel activity and (B) core temperature rhythms. Group
designations as in Fig. 3 (* p < 0.05, ** p < 0.01 with posthoc paired
comparisons to preestrus).
the time of activity onset and temperature minimum as phase
reference points, daily changes in ~- were examined during prees-
trus, estrus, metestrus, and postestrus (see Experiment 1 for def-
initions). Postestrus included up to six days following metestrus
to determine the stable postestrous T. The average, overall phase
shift during estrus was calculated 4-6 days postestrus, and
equalled the difference between the "actual" phase shift and the
"predicted" phase shift based on preestrous 7- derived from 7-
10 days of data. Paired t-tests were used to examine actual vs.
predicted changes in time of activity onset and temperature min-
imum on the day of estrus and metestrus.
Results
Thirteen estrous cycles (each animal contributing 1-3 estrous
cycles to the data base) were used to examine the effect of estrus
on the circadian activity and core temperature rhythms in DD.
The average estrous period was 22.3 _ 1 days in DD, not sig-
nificantly different from that in LD 12:12 (p = 0.063). In DD
female degus displayed changes in activity and core temperature
across the estrous cycle similar to those of intact animals in LD
12:12, although mean activity was somewhat depressed (10%).
Mean activity and core temperature rose on the day of estrus
(80%, p < 0.02 and .41°C, p < 0.001, respectively; Figs. 8 and
9). Metestrus was associated with a decline in mean activity
(60%, p < 0.008) and core temperature (.56°C, p < 0.001) com-
pared with estrus (Figs. 8 and 9).
The average free-running ~- was 23.7 ___ 0.09 h for both the
activity and core temperature rhythms during the preestrous in-
terval, and did not differ significantly during the postestrous in-
terval (23.8 +__ 0.08 h). Compared to the average daily advance
in the free-running rhythm, degus demonstrated a nonsignificant
advance in activity onset on the day of estrus (1.70 + .66 h, p =
0.071), followed by a significant delay (2.97 _+ 1.05 h, p < 0.05)
on the day of metestrus. However, the average overall phase shift
in activity onset (determined 4-6 days after estrus) from the time
predicted by preestrous ~- was negligible (0.013 _ .137 h; Fig.
8A). No significant changes from those predicted by preestrous
7- were observed in the timing of daily temperature minimum or
in ~TA across the estrous cycle (Fig. 8B).
These data suggest that estrous-induced changes in ovarian
hormones produce increases or decreases in mean temperature
and activity, but only a transient effect on circadian phase of
entrained animals. Alternatively, estrous-induced phase advances
may be completely (and perfectly) reversed by metestrous delays.
A
450
-'~
360
z
O
270
;,.,,
180 i
Z
90
-
/
/
t;
r"
PRE
A
//
/
// '\
',
It
")i,- Q
\
• - a
, !
J r
ESTRUS MET POST
B
38.7
38.3
O
v
m
37.9
37.5
/' ,,
/ \
/ ,,
37.1
'
PRE ESTRUS MET POST
FIG. 5. Daily mean (_+ SEM) (A)
activity and (B) core temperature in female
degus with (O) and without (D) mnning wheels across the estrous cycle.
Group designations as in Fig.
3. Paired
comparison differences from
PRE
are designated by asterisks
(* p < 0.05, *** p < 0.001). Paired
comparison
differences between estrus and metestrus are designated by
"a."
ESTRUS-INDUCED CHANGES IN CIRCADIAN RHYTHMS 579
A
240
z
202
Z
o
i- 164
0
,..1 1~'6
~ 88
Z
X 50
PItE
esrkvs
a
I
[
~'t
OVX OVX+B2 O'VX4F
B
38.80 -
Q
nd
31,44
~ 38.~
IL,
• 1 37.72
8
37.35
37.00
L a
,1
PIlE ~ITkU$
MET
OVX OYX+B2 OVX+p
FIG. 6. Variation in mean daily (A) running-wheel activity and (B) core
temperature before and after ovariectomy, estradiol implant, and proges-
terone implant. PRE, ESTRUS and MET as in Fig. 3, OVX = ovariecto-
mized state, OVX+E2 = plus estradiol implant, OVX+P = plus proges-
terone implant. The same animals were used in each treatment; open bars
= intact, dark bars = OVX. * designates significant difference between
PRE and ESTRUS, "a" designates significant difference between
ESTRUS and MET, "b" designates significant difference between nones-
trus-intact and OVX, ** designates significant difference between OVX
(after EB removal) and OVX+P.
EXPERIMENT 5
This experiment evaluated the influence of estrogen and
progesterone on free-running circadian activity and core tem-
perature rhythms by first eliminating and then restoring the
hormones. Five female degus, previously ovariectomized in
Experiment 3, were housed in constant darkness (DD). After
monitoring running-wheel activity and core temperature for 2
wk, each animal was anesthetized and implanted, under dim
red light, with capsules containing estradiol benzoate (EB), as
described in Experiment 3. After one week capsules were re-
moved, and animals were given 10 days to recover before be-
ing implanted with progesterone capsules (remained in place
for 1 wk). The daily mean and amplitude of the circadian ac-
tivity and core temperature rhythms were evaluated across
four time periods: Preovariectomy (non-estrous days), post-
ovariectomy, preimplant and postimplant. Fourier and Chi-
square periodograms were used to determine activity and core
temperature ~- across these four periods.
Results
Following ovariectomy, female degus housed in DD demon-
strated a 27.8% decline in mean activity (p < 0.02, Fig. 9A) and
a .68°C decline in mean temperature (p < 0.008, Fig. 9B) com-
pared with free-running, intact, nonestrous animals. Estrous-re-
lated alterations in activity and core temperature were no longer
apparent (Fig. 10).
Compared with the OVX state, mean activity (55%; p <
0.008; Figs. 9A and 10A) and mean temperature (.28°C; p <
0.025; Figs. 9B and 10B) rose significantly after estradiol was
implanted. Rhythm amplitudes were not altered by EB treatment.
Progesterone implantation brought about a 20% decline in
mean activity from the week prior to implantation (p < 0.05;
Figs. 9A and 10A), however, there was no significant decline in
mean temperature (Figs. 9B and 10B). As with EB, rhythm am-
plitudes were unaffected by hormone treatment.
While estradiol replacement significantly increased mean ac-
tivity and core temperature and progesterone replacement signif-
A
(- EB "9
t'- PROG -1)
B
__~,,,,m,..+..,L+a~ .aU,.,,L+
L+..-Z"--~
Lm L L~L,d._l,., •
- .
~,,-.A~,
• i...
-- *.n ,,Till& - ~.d~l ..dk,,k
+
.d~ _ ~.~m _.t,.~
......
.L,dL..,di&
_.Jl, Jr,,It .JUL.IL .
~,,...JIL _ ,,. + .,I,_ .,dl mm~ _,w. --
....
LL~ ~It.A..IIL •
. . ~ •
....
~l._L_,~._~~
uk_
__ ,,.ilk
~LA ~
__
. ,ll.i'- ----d.m+--,dl~ ~ ~L
..
3,1
~4 __L .,Jndlu~&JA~.,-,,dk,~--...--
11
- LA ~JLll/L~
~LL
~ ...hLJ~, . _
:,: _ • iktld~u~ . ~ &,,k
.LJ
~ A,L . _i .~_
A_
me
~l'i. ..~
_& .ik_ iLL AL /I~L~
31 + & ~ *.IL jL J~J
3J ~ ~,L A.JL adL
_ ~L~ L~L L..]
3,1 k&Jk ,eL_ L. ,,
,.,~A&&i~. Jill .Ab .m.~
...... LLL_----,L-.L!I~,-,'
. _A ,,m,~..t.. _
L&lbi~
.al~ &~,~Lm.
_,,. A,s,. an, _.It - A.
. _ ~ ,lldL ,-S
~L • +LkLI JIk -a~
• .LI LL&Lll ~ AL_ _,~
|
mug,eL L _.i[ _
dk.
,~ • L J~A JLmLA
6.400 Ii.00 16.00 ~:0o00 6.000 10.O0 ,IG,,~lll ~0.00
T],I~ (~) TLN fla,.Jru)
IIIII
FIG. 7. Entrained, ovariectomized animal's (A) double-plotted activity and (B) core temperature actograms before, during and after exposure to estradiol benzoate
(EB) and progesterone (PROG). Dark arrows indicate day that Silastic capsule was implanted, open arrows indicate day of capsule removal. Animals were anesthetized
for initial EB implant, but anesthesia was not required for implant removal and insertion thereafter.
ESTRUS-INDUCED CHANGES IN CIRCADIAN RHYTHMS 581
A
~m
I nun nllm |1 I n imm Ilni il
m ill him mn I m Im
_m!
•
I mR II n I m mum In
5. Oee 2O.O0 IG*OO 2O*OO G, OOO 2O.OO 2ES, OO 20.00
Time (Houru)
JGL ilL_
. L --J
~
f nil
"-'-
-- _lil diaL J.L did- J iil JL .
L ~i
-
. L t IRaJB j JLimmm. _ rail
L • nimJk
j_..m.llm • ._ • .m, dm _, .~-.
_ - - m
.I---,--,,- _ _ _ ,i, "m,mL ."-"-'- .,t_JR,. i
-- .L-mJ .J... jm__ mA,- JJ. .L,
..... ~-- -. ~----
~dm..mm-
• ~m __ mm
•
~d__ ..iidnJk .L
L eL
• i
J n
k[ t
m~
~t_ -nm,~m ~ [_ ~&nhnd. ~"~ .m.
I ".nnJIL _'""" ~ .I m |lt,,m,m n~
"
G.eO0 2e.gO £G. eo 20,QO B.@O0 20.00 2G.9@ 20.00
T£mt (MOURN)
FIG. 8. Actogram of twelve days of double-plotted (A) wheel-running and (B) core
temperature data for a single female degu maintained in constant darkness (DD). As-
terisk denotes the time of estrus.
icantly decreased mean activity in OVX degus, the circadian ac-
tivity and core temperature 7-'s were not altered by either
hormone treatment (Fig. 10).
DISCUSSION
These experiments support previous findings that circadian
activity and core temperature rhythms are markedly altered dur-
ing estrus in rodents. However, these experiments do not support
the hypothesis that estrous-induced circadian changes in phase
are the result of alterations in the period of the circadian clock.
Rather, it seems more likely that alterations of entrained activity
phase are the result of hormonally controlled alterations in the
amount of locomotor behavior. Mean core temperature also is
influenced by ovarian hormones, at times independent of effects
those hormones have on activity.
On the day of estrus, entrained female degus demonstrated an
increase in mean daily activity and core temperature, a phase
advance in the circadian activity rhythm (kOAo, kOAp), a reduction
in the amplitude of both the circadian activity and core temper-
ature rhythms, and a reduction in the phase angle difference be-
tween temperature minimum and activity onset (kOTA). On the day
following estrus, k~Ao was phase delayed, the mean activity and
core temperature fell below that of the preestrous period, and
~T/TA
was greatly increased (Experiment 1). These estrous-related
changes in entrained circadian activity and core temperature in
female degus are similar to those reported for other rodents. For
example, female rats initiate activity earlier and display a signif-
icant increase in daily running-wheel activity and mean core tem-
perature during proestrus/estrus (1,15,20,26,35). Furthermore,
Kent et al. (15) noted that 50% of the female rats in their study
demonstrated a consistent phase advance (at least .5 h) of the
core temperature rhythm.
The rise in mean core temperature during proestrus in rats
was positively correlated with the increase in activity, suggesting
that the thermogenic response was induced by activity. Kent et
al. (15) found that female Sprague-Dawley rats did not display
significant elevations in mean core temperature during proestrus
when running-wheels were locked or removed. In contrast, re-
moving the running-wheels from degus did not alter estrous-re-
lated changes in core temperature (Experiment 2). While degus
deprived of wheels demonstrated a decline in mean daily core
temperature, it rose .4°C on the day of estrus, exactly the same
increase as when a wheel was present. Degus also demonstrated
a decline in mean daily activity after wheel removal, however
activity increased 40.4% on the day of estrus, similar to the in-
crease of 54% with a wheel. Thus, removal of the running-wheel
does not alter the degu's thermogenic response on the day of
estrus, and there is only a weak association between temperature
changes on the day of estrus and the absolute amount of activity.
As expected, ovariectomized degus did not demonstrate the
estrous-related cyclic alterations in circadian activity and core
temperature previously noted in intact animals. Furthermore, the
mean core temperature declined significantly following ovariec-
tomy and this decline persisted throughout the remainder of the
experiments (Experiments 3 and 5). Similar reductions in mean
core temperature following ovariectomy have been recorded in
female rats (35) and rabbits (16), as well as in hypophysectom-
ized mice (12). Furthermore, postmenopausal women do not dis-
play the postovulatory rise in mean core temperature seen in pre-
582 LABYAK AND LEE
A
270
o
m ~ 226
~ 112
-~ 94
<
50
.Ic
I
i i
PRE ESTRU$
a
MET
OVX
OVX+E2
OVX+P
B
"o
Da
N
:E
~a
1~
n~
Z
311.9
311.2
37.$
36.8
i , i , i i i
PRE F.~TRU$ MET OVX OVX+lt2 OVX+P
FIG. 9. Mean daily (A) running-wheel activity and (B) core temperature before
and after ovariectomy, estradiol implant, and progesterone implant in female de-
gus maintained in DD. Group and significance designations are as in Fig. 6, with
** now designating significant difference between OVX and OVX + hormone
treatment.
menopausal women, and their mean core temperatures are
permanently lowered (34). Results from these experiments indi-
cate that core temperature of degus, but not mean activity, is
sensitive to basal levels of ovarian hormones.
Ovariectomy of entrained females caused a nonsignificant de-
cline in activity (Experiment 3). In contrast, OVX, free-running
females (Experiment 5) displayed a significant 27.8% decline in
activity with the decline in temperature. The apparently greater
affect of OVX on activity of females in DD was likely the result
of an additive influence of constant darkness (10% decline in
Experiment 4 compared with Experiment 1) and ovariectomy
(16.9% decline in Experiment 3) on activity, neither of which
was significant alone. Core temperature of intact females housed
in DD (Experiment 4) was exactly the same when compared with
intact females in LD12:12 (Experiment 1), while OVX signifi-
cantly decreased temperature (Experiment 3). We conclude that
core temperature is influenced by basal ovarian hormones and
not lighting conditions, while locomotor activity can be signifi-
cantly altered only by decreasing both basal hormones and lights.
Estradiol replacement reinstated vaginal estrus in degus
housed in LD12:12 and DD, but mean core temperature and ac-
tivity were significantly elevated only in Experiment 5 (p < .10
for Experiment 3). However, in both experiments, EB implants
produced far less of a response than did natural estrus. The lack
of a significant phase advance in activity after EB implant might
have been the result of depressed sensitivity to estrogen follow-
ing long-term ovariectomy (19), compared with intact estrous
females. In contrast, progesterone caused significant declines in
mean temperature and activity for animals housed in LD12:12
and DD comparable to (or greater than) that of intact metestrus
animals, yet k~AO of entrained animals was still not significantly
delayed. However, ~'tg was significantly increased after proges-
terone, suggesting that the nonsignificant phase delay in ~AO of
entrained animals (p < 0.08) might have represented a small
hormonal influence on the circadian clock. Thus, the results of
Experiment 3 were inconclusive for determining whether steroid
hormone changes during estrus were responsible for changes in
~AO -
In these studies, metestrus or the administration of progester-
one to ovariectomized degus resulted in a significant decline in
mean activity and core temperature. This finding was surprising
since progesterone has a thermogenic effect in women (8,9,14).
Women experience a rise in core temperature of .5 °- I°F follow-
ing ovulation. Pregnanediol, a progesterone metabolite, is
thought to exert a thermogenic effect on thermoregulatory centers
in the hypothalamus (29). A similar thermogenic response to pro-
gesterone has been demonstrated in rats (13,23) and cattle (36).
Following ovariectomy, female rats continued to display a ther-
,J~
A
t- EB "-)
(- PROG
IL~ IlL ~ _~
B
_&Az . _~lll _,,It .A, ~ ,,L
L.dZ
.
_JdlL ..Jr ~I~LJt~, a~_ ~aaaZak ~ a~,.
dLdlm -a. aL ~ .Inlm,.._ ~ • ,I,4. Jb..RAdt dIR. ~ Am_
b ~lk*a*Ja~ ~Jk JL Am. A /IJl~tlllt ~Jl~_
• Jr, Aat,~s..,L'L~',_
_.ill
,t/,~,i ~ a.,._~
.
,tl,d~,l ~ .,ik._x.
_
AL Adt~dulkA Jm_
JJL ,t ,ilul. dl~ ,f, dllm _ ~ A ,LL~. ~ltlL ~ JIlL
LJlILJt dt~LttLtaLILIk.Lt AkLimt LLLjt~_Jk~li&~La~ AL~L--~LILIJk ~ _
,IL.dllk..,z,,.*..,z~--=,i.,,. ~_~t.. _ .I,. ak" ,,JL .dlt *A-'.- M,..,.~u,_ _. _
11 ~.i _d~t~ ---'~ ,i.--_ j~ _ z, * JL --.~IL ; _ AL •
/
~mLdlkdl-dlJL .Ik ~ . dK.
S.OOO te.oe
xS.IIII all.ee S.ooo tll,;oo t~-.li
TLlll (Hourll) Tlnml (Hl:lurll)
FIG. 10. Ovariectomized animal's (A) double-plotted activity and (B) core temperature actograms before, during and after exposure to estradiol benzoate (EB) and
progesterone (PROG) while housed in DD. Animals were anesthetized for initial EB implant, but anesthesia was not required for removal and insertion thereafter.
Arrows and designations as in Fig. 7.
584 LABYAK AND LEE
mogenic response to injections of progesterone, however, the
hormone was less effective when compared to the response of
intact animals (13). Why temperature responses to progesterone
should differ in degus from other species so far studied is unclear,
but the effect of progesterone or metestrus on temperature was
consistent across all five experiments.
Although data from Experiments 2 and 3 indicate that tem-
perature changes are not the result of changes in activity, the
converse may be true. Significant changes in core temperature
were accompanied by appropriate increases or decreases in mean
activity and, for entrained cycling females, a phase change in
activity onset. The one time this was not true, was maintenance
of near normal levels of activity after ovariectomy caused core
temperature to drop nearly I°C (Experiment 3). However, from
a given baseline core temperature, estrous-related changes in core
temperature were associated with comparable changes in activity.
To further determine whether the estrous-induced changes in
entrained circadian rhythms were the result of changes in period
length, degus were maintained in DD while undergoing estrus
(Experiment 4). Most animals in DD displayed transient fluctu-
ations in the timing of daily activity onset and temperature min-
imum during, and immediately after estrus, however, the circa-
dian period was not affected when examined 4-6 days after
estrus. Ovariectomy eliminated all estrous-related fluctuations in
activity and temperature, and subsequent treatment with estrogen
and progesterone did not induce significant alterations in activity
or core temperature ~-'s (Experiment 5). Therefore, the estrous
cycle and small hormone-induced changes in phase of entrained
rhythms do not appear to be caused by hormonal effects on the
circadian clock. Rather it is more likely that hormones act upon
brain regions directly controlling locomotor activity (for review,
5) and temperature. Hormonally influenced activity control cen-
ters include the striatum (6,27) and cerebellum (30). Changes in
phase of activity might then be likened to changes in levels of
arousal to become more or less active, without changes in the
clock.
The lack of changes in kOAO or 7- after ovariectomy or proges-
terone treatment caused significant declines in body temperature
were surprising. Both theoretical (3) and empirical data suggest
that changes in core temperature can cause alterations in kOAo and/
or 7- (e.g., 17,32,33). Thomas et al. (32) reported that even short
periods of lowered core temperature during torpor, that were in-
sufficient to significantly lower mean daily core temperature,
were sufficient to significantly alter 7- in Siberian hamsters. Per-
haps degus have a Q~o closer to 1.0 than some other mammals,
such that a larger change in core temperature than occurred in
these experiments is required before an effect of change can be
measured in ~PAO or 7-.
Data on estrous cycle and hormone-induced changes in cir-
cadian period for hamsters might also be interpreted as having
insignificant effects on the clock. The circadian activity period
of free-running, intact, estrous hamsters advanced by about 20
min (21,38), and the period shortened by 16 min in blind, ova-
riectomized, estrogen-treated hamsters (31). In contrast to ham-
sters, 7- was unchanged in degus following ovariectomy and treat-
ment with steroids, and the nonsignificant transient change in
phase on the day of estrus for degus in Experiment 4 was larger
than that of hamsters. Hamsters demonstrate changes in activity
level primarily at the onset of activity in response to endogenous
or exogenous hormones (31). Increases and decreases in activity
are not restricted to the onset of activity in degus, but are quite
pronounced during that time. Data from both species suggest that
ovarian hormones alter activity levels at the onset of the active
period, causing changes in circadian phase, but have no perma-
nent or major effect on 7-. This interpretation is supported by the
lack of any significant change in temperature phase under any
conditions for the degus.
In conclusion, we find cyclic changes in ovarian hormones
have a pronounced effect on the timing and expression of the
circadian activity and core temperature rhythms in the diurnal
degu. Compared with rats and hamsters, the activity/temperature
relationship has several unique characteristics: (i) access to run-
ning-wheels is not responsible for estrous-related circadian
changes in core temperature; (ii) ovariectomy reduces mean tem-
perature without a significant reduction of mean activity or
change in phase for entrained animals, and without a change in
7- for free-running animals; (iii) estrogen replacement in en-
trained, OVX degus brought about a nonsignificant increase in
core temperature and activity, while progesterone replacement
led to an immediate significant reduction in core temperature and
mean activity, with neither hormone significantly altering "PAt;
and (iv) in free-running conditions (DD), estrous-related fluctu-
ations in the timing of daily activity onset and temperature min-
imum are transient, and there were no significant changes in 7-
with hormone treatment of OVX females. It is likely in the degus
that hormonal affects on circadian activity and temperature do
not directly involve the central circadian neural structures.
Rather, hormone-induced changes in core temperature and, per-
haps, also in brain circuits controlling locomotor activity, alter
mean activity and activity phase in entrained, intact females dur-
ing the estrous cycle.
ACKNOWLEDGEMENTS
We wish to thank Dr. Bill Xanten at the Washington National Zoo,
John Gramieri at the Lincoln Park Zoo, Dave and Charlesetta Webster
at the Scoviile Children's Zoo, Dr. Milton Stetson at the University of
Delaware, and Dr. Robert Davis at State University of N.Y. at Platts-
burgh for their advice and assistance in establishing a breeding colony
of O.
degus.
We also wish to thank Dr. Philip Meyers and Dr. Barbara
Lundrigan at the University of Michigan's Museum of Zoology for pro-
viding space for housing the colony; and two anonymous reviewers for
their comments. This research was supported in part by NCNR grant
NRO6397; NIMH grant MH49089; Wyeth-Ayerst Laboratories Fund of
the American Nurses' Foundation; Rackham Graduate School, the Office
of the Vice President of Research, and Continuing Education for Women
at the University of Michigan; and Sigma Theta Tau (Rho Chapter).
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