BIOLOGY OF REPRODUCTION 54, 91-99 (1996)
Metabolism of Reproductive Steroids during the Ovarian Cycle in Two Species
of Callitrichids, Saguinus oedipus and Callithrixjacchus,
and Estimation of the Ovulatory Period from Fecal Steroids'
Toni E. Ziegler, 2'3 '4 Guenther Scheffler,3 Daniel J. Wittwer,3 Nancy Schultz-Darken 3
Charles T. Snowdon, 4 and David H. Abbott 3 5'
Wisconsin RegionalPrimate Research Center, 3 Department of Psychology4
and Departmentof Obstetricsand Gynecology,5 University of Wisconsin, Madison, Wisconsin 53715
ABSTRACT
Gonadal steroids were measured in daily fecal samples providing comparative data on steroid metabolism in two genera of New
World primates. Circulating bioactive LH and progesterone concentrations and fecal progesterone, pregnanediol, estradiol, and estrone
concentrations were measured by collecting blood and daily fecal samples from four captive common marmoset females and four cottontop tamarin females for 30 days. High recoveries (> 80%) of labeled steroids that were added directly to the feces before extraction were
recovered from feces of both species. Because of the presence of complex steroid conjugates, only one fifth the amount of estradiol was
measured without solvolysis as compared to the amount measured with solvolysis. In tamarins, steroids were metabolized rapidly, with
all postovulatory increases occurring within two days after the circulating LH peak (an increase of 2 SD higher than mean follicular levels).
In marmosets, steroid excretion was slower; increased steroid levels occurred 2-4 days after the LH peak except in the case of estrone,
which did not consistently increase after the LH peak. Circulating estrone and estradiol both contributed to the high excretion of estradiol
inthe feces from both species. The timing inthe delay inexcretion of fecal steroids was used to accurately determine the ovulatory period
to within a 2-day window. This degree of accuracy is possible when the duration of the delay to the LH peak is known for agiven species.
0.07 SEM), indicating
Additionally, steroid concentrations were highly correlated between frozen and lyophilized fecal samples (0.81
that fluid removal from the feces did not effectively alter steroid profiles.
INTRODUCTION
ovulatory peaks. Circulating estrone, however, rose after
ovulation, with sustained elevations throughout the luteal
phase of the cycle [3, 81. Therefore, circulating estradiol appears to reflect follicular function, but circulating estrone
may reflect luteal function. In cotton-top tamarins, profiles
of urinary estrogen concentrations indicate that both estrone and estradiol increase after the serum and urinary LH
peak [8]. However, urinary estrone concentrations were
found to be approximately 100 times higher than urinary
estradiol concentrations [8] due to the metabolism of nearly
50% of circulating estradiol into estrone before excretion
into urine and feces [4]. Therefore, circulating estradiol is
contributing to the already abundant excretion of estrone.
In feces, radiolabeling studies have indicated that estradiol
is converted to estrone before excretion in the tamarin [4].
However, Heistermann et al. [2] measured estradiol in feces,
and the levels appeared high throughout the ovarian cycle.
The relationship between concentration of excreted fecal
estradiol and estrone is still unknown.
The common marmoset and the cotton-top tamarin are
both biomedically important nonhuman primate species
[9, 10], representing two different genera within the family
Callitrichidae. To date, few studies have compared steroid
metabolism of reproductive hormones in these two species,
which show a pronounced social regulation of fertility. Determining the metabolism of reproductive hormones into
urine and feces enables noninvasive monitoring of reproduction without disturbing the social and behavioral influ-
Estrogen metabolism and excretion during the ovulatory
cycle of New World primates are uniquely different from
these processes in Old World primates, apes, and humans.
In all New World species examined-the cotton-top tamarin, Saguinus oedipus [1, 2], the common marmoset, Callithrixjacchus[2, 3], the Goeldi's monkey, Callimicogoeldi
[4, 5], the saddle-back tamarin, Saguinusfuscicollis[2], the
golden lion tamarin, Leontopithecus rosalia[6], the muriqui
monkey, Brachyteles arachnoides[7], and the white-faced
saki, Pitheciapithecia[81]-urinary or fecal estrogen profiles
do not reveal the pattern found in Old World monkeys and
apes of a follicular surge prior to ovulation. Instead, estrogens increase similarly to progesterone metabolites with a
sustained elevation throughout the presumed luteal phase
of ovulatory cycles. Most information on steroid metabolism
in New World primates comes from work on the common
marmoset and the cotton-top tamarin. Comparisons of circulating hormones to urinary hormones in common marmosets [3] and cotton-top tamarins [8] indicated that for both
species, circulating estradiol concentrations did show preAccepted August 23, 1995.
Received March 13, 1995.
'This research was supported by grants NIMH 35.215 to C.T.S. and T.E.Z. and NIH RR
00167 to the Wisconsin Regional Primate Research Center. This is WRPRC publication #35012.
2
Correspondence: Dr. Toni E. Ziegler, Wisconsin Regional Primate Research Center,
1223 Capitol Court, University of Wisconsin, Madison, WI 53715. FAX: (608) 263-4031; email: [email protected]
91
92
ZIEGLER ET AL.
ences on fertility. Hodges and Eastman [11] have compared
the relative levels of estrone and estradiol in urine from
common marmosets and cotton-top tamarins during the follicular and luteal phases of ovulatory cycles. In common
marmosets, the predominant estrogen appears to be estradiol 17f, while cotton-top tamarins excrete primarily estrone. However, a large portion of estradiol was unmeasurable in the cotton-top tamarin, presumably because of the
presence of complex conjugates. These results suggest that
measurement of estrogens in the urine and feces of marmosets and tamarins may require more comprehensive
methods such as solvolysis to liberate the estrogens from
conjugates. Ziegler et al. [4] found that conjugated steroids
accounted for up to 85% of total estrogens in both the urine
and feces of the cotton-top tamarin. Hydrolysis alone does
not liberate all of the estradiol from the conjugated form.
The following study was designed to determine the metabolism and excretion of ovarian steroids into the feces of
cotton-top tamarins and common marmosets by measuring
both circulating and fecal steroids during the ovulatory cycle.
This information was used to determine the relative delay of
fecal steroid excretion following ovulation in order to estimate
the periovulatory period from fecal sampling alone. Fecal
analysis has the potential for monitoring reproductive steroids
and determining reproductive function in free-ranging marmosets and tamarins if the steroids that best reflect ovarian
changes can be identified. Additionally, a series of samples
were analyzed as dried feces to determine the importance of
variability of fecal fluid content on steroid levels.
logue (Estrumate; Mobay Corp., Shawnee, KS; i.m. at 0.75
jtg/female) to end the luteal phase. Prostaglandin F2, causes
luteolysis, which inhibits progesterone secretion [14].
Sample Collection
Tamarin feces were collected daily during the 30 days as
the first fecal void of the day between 0800 and 0900 h and
were frozen immediately until steroid analyses. All tamarin
feces were collected by holding a bucket underneath the
female until she defecated. For blood sampling, tamarin females were captured, and 0.5-1 ml blood was withdrawn
without anesthesia three times weekly for 30 days. The resultant serum was stored at - 20°C.
To collect feces from marmoset females each morning,
the female was either captured and held at the time of blood
sampling or was captured and placed into a small cage with
fresh paper on the cage floor until she defecated. Marmosets
would defecate readily when held. For blood sampling, females were captured, placed in a restraint tube, and bled
unanesthetized at twice-weekly bleedings for 30 days until
the periovulatory period, at which time blood sampling occurred every day for 5 days followed by sampling three
times weekly for the remaining days. The onset of the periovulatory period was predicted by monitoring plasma progesterone levels, which decreased to levels < 10 ng/ml during the follicular phase. Blood was collected between 0800
and 0900 h; 0.1-0.3 ml was collected per sample from the
femoral vein into a heparinized syringe. Blood samples
were centrifuged at 500 X g for 10 min, and the plasma
fraction was frozen until hormonal analyses.
MATERIALS AND METHODS
Sequential Hydrolysis and Solvolysis
Animals
Blood and fecal samples were collected from four female
cotton-top tamarins and four female common marmosets.
The cotton-top tamarins were housed at the University of
Wisconsin Psychology Department's Marmoset and Tamarin Colony in cages measuring 1.5 X 0.85 X 2.3 m. All
females were adult (2.5-10 yr of age) multiparous and all
had been cycling prior to the study. Two females were
paired with males and were hysterectomized with ovaries
left intact; one female was paired with a vasectomized male,
and one was living alone. Details of colony husbandry have
been reported previously [12]. The common marmosets
were maintained at the Wisconsin Regional Primate Research Center as either the female in male-female pairs or
as the dominant female in heterosexual adult groups. Cages
measured either 0.89 X 0.85 x 0.85 m or 0.75
0.70 x
0.69 m, or three cages (each measuring 0.88
0.85 X 0.86
m) were joined together. Details of colony management
have been reported elsewhere [13]. For all marmoset females (1.5-6 yr of age), sustained pregnancy was prevented
by injection of cloprostenol sodium, a prostaglandin F2,, ana-
To determine the level of conjugation of fecal steroids,
both tamarin and marmoset samples were subjected in triplicate to sequential enzyme hydrolysis and acid solvolysis.
These procedures allow for the separation of steroids according to those that occur in the free form and are soluble
in solvents, those that are water-soluble due to conjugation
to simple glucuronides and sulfates (liberated by enzyme
hydrolysis), and those that are conjugated to double sulfates
and glucuronides (liberated by solvolysis). The steroids extracted serially by diethyl ether from the aqueous portion
of the samples were subsequently combined to provide the
total estimate of each steroid. Additionally, 20 000 cpm of
tritiated estrone-glucuronide (Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London,
UK) was added in triplicate to blank water tubes, tamarin
fecal pools, and marmoset fecal pools; these were incubated for 30 min prior to the initial extraction to determine
the efficiency of the hydrolysis technique.
Fecal steroids in 0.1 g of feces were separated from the
fecal solids through use of the citrate buffer complex (citrate
buffer: 0.05 M citrate in 0.15 M NaCl [pH 5], 0.1% sodium
BLOOD AND FECAL HORMONES IN FEMALE CALLITRICHIDS
azide, 0.1% gelatin) including 0.1% Brij and 20% methanol.
The techniques involved solubilizing the feces in 20 ml of
citrate buffer complex. From this, 1) an aliquot of 500 ll
was extracted with 5 ml of diethyl ether to separate the free
steroids. The ether was dried, and the sample was reconstituted in 500 pl of ethanol and stored until chromatography. To the remaining aqueous phase, 2) 25 pil -glucuronidase (H2, containing sulfatase activity; Sigma Chemical
Company, St. Louis, MO) was added and hydrolysis was
performed overnight at 37°C in a water bath. The next day,
the liberated steroids from the hydrolyzed sample were extracted with diethyl ether. The ether portion was dried and
resuspended in ethanol. 3) The aqueous phase was subjected to acid solvolysis by a modification of the technique
reported by Eastman et al. [3]. To the sample, 100 Il of saturated NaCl, 50 pl of 2.5 M H2SO4, and 4 ml of ethyl acetate
were added, and the sample was vortexed for 1 min and
incubated overnight at 40°C in a water bath. The following
day, 4 ml of ethyl acetate was added; the samples were
vortexed for 5 min and centrifuged for 2 min at 1000 X g,
and the solvent phase was pipetted off. The ethyl acetate
was neutralized by washing with 2.5 ml distilled H2 0O,separated, and then dried. All fractions of free steroid were
resuspended in column solvents, and the steroids were separated through column chromatography and assayed by the
procedures reported below.
Assays
Blood samples were analyzed for both LH and progesterone. Bioactive LH was measured in both tamarin serum
and marmoset plasma through use of the mouse interstitial
cell bioassay [1, 8]. For the marmoset plasma samples, the
cell reaction was stopped with 1 ml absolute ethanol. The
ethanol was decanted and dried in a water bath at 50°C and
rehydrated with 1 ml of gel PBS (pH 7.0), and 50-pL1 amounts
were used for the testosterone RIA. Intra- and interassay
coefficients of variation (CV) were 3.4% and 13.7%, respectively, for tamarin LH (n = 4); for marmoset LH, the intraand interassay CV were 5.87% and 9.7%, respectively (n =
9). Serum progesterone in the tamarin was analyzed by RIA
using the technique reported by Ziegler et al. [8] with intraand interassay CV of 2.9% and 3.5%, respectively (n = 3).
Plasma progesterone for the marmoset was analyzed by
ELISA as reported by Saltzman et al. [131, with intra- and
interassay CV of 2.8% and 12.1%, respectively (n = 126).
Tamarin and marmoset feces were extracted and assayed
under identical conditions. The extraction procedure was a
modification of the procedure reported by Ziegler et al. [4].
The method was revised until all tritiated steroids (New
England Nuclear, Boston, MA) gave a recovery of over 80%
by increasing the volume of the buffer and adding methanol
(Mallinckrodt, Paris, KY) and a surfactant (Brij 35; Sigma).
The steroids were extracted from 0.1 g feces at room tem-
93
perature into a citrate buffer complex (see above). Tritiated
steroids (added as 40 000-80 000 cpm of estrone, estradiol,
progesterone, pregnanediol) were added directly to the
feces before extraction to determine recoveries. High
counts were used since only 2.5% of the sample was analyzed because of high steroid levels. Fecal pools were run
in duplicate for each steroid to provide an external recovery. Samples were vortexed for 5 min and centrifuged for
20 min at 2000 X g. Five hundred microliters of the supernatant was used for external recoveries of the initial extraction procedure, and 500 pl was used for each sample. Solvolysis by the procedure described above was performed
on each sample, and the sample was stored in 500 pll ethanol. Celite chromatography was used to separate the steroids by polarity as previously described [4] with the following modifications. Samples were applied in 1 ml iso-octane/
ethyl acetate (96:4) and rinsed with 0.5 ml of the same
application solvent. Progesterone eluted slightly early, so
the 0.5-ml sample rinse was combined with the 3.5-ml isooctane for the progesterone fraction. The estrone fraction
was eluted with 4.5 ml of 15% ethyl acetate in iso-octane
and contained both estrone and pregnanediol. All fractions
were dried and reconstituted in 500 pl1 ethanol and stored
refrigerated until assayed.
The RIAs for estrone, estradiol, and progesterone have
been reported previously [1, 8]. Validations for tamarin and
marmoset fecal pools were as follows. Serial dilutions of the
tamarin fecal pool (n = 6) gave parallelism to the standards
for estrone, estradiol, and progesterone with no differences
in slopes (p > 0.05), and accuracy ranged between 96.2%
and 105.8% for the three steroids. Serial dilutions of the marmoset fecal pool gave parallelism to the standards for estrone, estradiol, and progesterone, with no differences in
slopes (p > 0.05), with an accuracy between 102.5% and
108.7%.
An RIA was adapted for pregnanediol measurement by
the use of pregnanediol standards in the range of 10012 800 pg (Sigma). The assay uses labeled 20a-hydroxyprogesterone and antiserum to 20a-hydroxyprogesterone but
cross-reacts equally to the two steroids. The assay would
therefore measure both steroids, since they elute in the
same fraction after celite chromatography, but is referred to
as a pregnanediol assay. Both steroids are metabolites of
progesterone. Tritiated 20a-hydroxyprogesterone ([1,2-3H]
20a-hydroxy-4-pregnen-3-one; New England Nuclear) was
used at 15 000 cpm/100 H. The antibody, a monoclonal
anti-20-hydroxyprogesterone, was provided by Dr. Robert
Chatterton (Northwestern University, Evanston, IL) and used
at a 1:450 dilution/100 p1. Cross reactivities for other steroids
were 164% for pregnanediol glucuronide, 41% for 20a-hydroxy-4-pregnen-3-one 3-oxime, 10% for 203-hydroxy-5,pregnane-3-one, 4% for 5a-pregnane-3,2003-diol, 2% for
progesterone, 0.2% for androsterone, and less than 0.1% for
estrone, estradiol-170, cortisol, and other steroids. Serial di-
ZIEGLER ET AL.
94
TABLE 1. Mean percentage ± SE recovery of steroids from tamarin and marmoset
feces through use of sequential hydrolysis and solvolysis.a
Species
Steroid
Cotton-top
tamarin
Progesterone
Pregnanediol
Estrone
Estradiol
Progesterone
Pregnanediol
Estrone
Estradiol
Common
marmoset
Free
Hydrolysis
Solvolysis
76 + 1.2
95 ± 0.08
90 + 1.1
12 +±1.1
64 + 1.1
95 ±+0.78
88 ± 1.5
16 ± 0.51
12 + 1.6
2 + 0.24
9 + 1.2
3 ± 0.27
19 + 0.87
4 ± 1.3
10 + 2.1
1 +0.41
12 + 0.42
3 + 0.31
1 ± 0.21
84 ± 1.4
17 ± 1.2
1 ± 0.55
2 + 0.96
83 + 0.77
steroids were extracted with diethyl ether from an aqueous phase to determine the percentage of steroids nonconjugated; the remaining aqueous phase was
hydrolyzed and then extracted to determine the simple conjugates, and the remaining aqueous phase underwent solvolysis and extraction to liberate the di- or
triconjugates.
a Free
lutions of the tamarin fecal pool (n = 6) were parallel to
the standards with no differences in slopes (t = 0.54, p >
0.05), and accuracy was 106.3%. Serial dilutions of the marmoset fecal pool (n = 6) were parallel to the standards (t
= 1.54, p > 0.05), and accuracy was 110.9%.
Estimation of Ovulatory Period and Day of the Rise in
Steroid Hormone Concentrations
The day of ovulation was estimated as the day of the
circulating or urinary LH peak. The day of the LH peak was
selected as the day of highest concentration of LH with a
subsequent rise of circulating progesterone into the normal
range for the luteal phase of the ovarian cycle. The decline
in progesterone was used to indicate the onset of the follicular phase, since no external signs of bleeding occur in
these species. Once the day of the circulating or urinary LH
peak was determined, the rises in concentrations of circulating progesterone and fecal steroids were presented as
days from the LH peak. All fecal steroids showed an increase after the LH peak and remained elevated during the
luteal phase (high circulating progesterone), as was seen
previously with urinary steroids in tamarins [81. Since the
tamarins were bled only three times per week, LH concentrations from daily urine samples helped provide evidence
for designating the day of the LH peak. For two of the tamarin females, the day of the circulating LH peak was known
to have occurred on the day of the blood sample, since the
serum LH concentration was 5-13 times higher than on the
remaining days sampled, and urinary LH concentration on
the same day was 4-5 times higher than on the remaining
days. For the remaining two females, a urinary LH peak
occurred at a day on which there were no blood sample for
comparison. However, circulating LH levels from blood
samples collected the day before and after the urinary LH
peak did not increase more than 2 times in relation to the
remaining days sampled. The first day of a significant rise
of steroid concentration greater than 2 SD from the mean
follicular levels (the mean concentration of the 5 previous
days) was selected as the critical day following the LH peak
for determining the first steroid rise.
Comparison of Wet Weight Vs. Dry Weight Feces
For 21 serially collected fecal samples from one female
tamarin, a portion (0.1 g) of wet feces was lyophilized to
dryness (Vacu-Freeze; Vertis VF Sentry, Gardiner, NY) prior
to analysis for comparison with the same samples (0.1 g)
used for direct analysis from frozen feces. The percentage
dry weight was determined for the lyophilized samples.
StatisticalAnalyses
Means and SEM were calculated for recoveries. CVs were
calculated for internal and external variation of fecal pools
by the method of Robard [15]. Determination of the onset
of early steroid elevation was made by computing the 95%
confidence interval of the mean follicular levels for each
steroid and the time at which steroid concentration rose
above that level. Correlations between frozen and lyophilized samples were determined by computing r 2.
RESULTS
Technique Evaluation
Recoveries of added tritiated steroids by the extraction
technique in which a large volume of citrate buffer is combined with a small percentage of a surfactant and methanol
were between 84% and 94% for all steroids for both species.
Recoveries for the entirety of the extraction and chromatography steps were between 60% and 70% for all four steroids
for both species. Intra- and interassay CVs for the tamarin
pool were, respectively, 7% and 18% for estrone, 5% and
16% for estradiol, 4% and 14% for progesterone, and 4%
and 16% for pregnanediol. Intra- and interassay CVs for the
marmoset pool were, respectively, 9% and 10% for estrone,
10% and 18% for estradiol, 6% and 21% for progesterone,
and 5% and 12% for pregnanediol.
Table 1 reports the mean percentage of each fecal steroid
as it was found in the feces, i.e., free, conjugated to simple
glucuronides and sulfates, or conjugated to double or com3
TABLE 2. Mean percentage ± SE of liberated H estrone-glucuronide added to
marmoset and tamarin feces.a
Sample
Free
Hydrolysis
Solvolysis
Total recovery
Blankb
TFPC
CMPd
7.26 ± 0.21
78.00 ± 1.7
56.53 + 1.8
71.00 ± 1.9
4.49 + 0.23
21.74 ± 1.6
2.53 + 0.23
1.37 ± 0.29
2.43 + 0.05
81.89 + 1.5
88.60 ± 1.9
84.57 ± 3.2
aAfter a 30-min incubation, steroids were extracted with diethyl ether from an aqueous phase to determine the percentage of steroids nonconjugated; the remaining
aqueous phase was hydrolyzed and then extracted with ether to determine the
simple conjugates, and the remaining aqueous phase underwent solvolysis to liberate the di- or triconjugates and extracted.
bWater blank, no feces.
CCotton-top tamarin fecal pool.
dCommon marmoset fecal pool.
BLOOD AND FECAL HORMONES IN FEMALE CALLITRICHIDS
95
-90
+
-80
30000- 70
I
50
20000
U
40
00
-30
a.
E
10000-
,
20
10
...
-
i
...
5
...
...
ii
15
10
20
E
m
eEP
I$
-v
25
Days
FIG. 1. Comparison of estradiol concentrations during ovarian cycling in serial
fecal samples from a female cotton-top tamarin whose samples had been analyzed
as frozen feces or as lyophilized feces. The percentage dry weight of the lyophilized
samples is indicated by the starred points.
plex glucuronides and sulfates. The tamarin and the marmoset were similar in that the majority of progesterone,
pregnanediol, and estrone were found in the feces in the
free fraction. In both species, however, most estradiol was
measurable only after solvolysis. Without solvolysis, estimates of estradiol would be only 20% of the total measured
estradiol in each species. Table 2 indicates the mean recovery of tritiated estrone-glucuronide from blanks and fecal
pools. As expected, the highest recoveries for tritiated estrone-glucuronide were after hydrolysis and extraction for
the blank, but labeled estrone-glucuronide added directly
to tamarin and marmoset feces was found mainly in the free
fraction prior to hydrolysis and extraction. For the tamarin
and marmoset, a liberation of steroids simply conjugated to
glucuronides and sulfides may occur directly in the feces.
Patterns of concentrations from frozen and lyophilized
feces were similar for all steroids analyzed. For the 21 samples, correlation of steroid concentrations from frozen samples vs. lyophilized samples prior to assay were r 2 = 0.93
for pregnanediol, r 2 = 0.74 for progesterone, r2 = 0.92
for estradiol, and r2 = 0.65 for estrone. Profiles of estradiol
concentrations from wet analysis feces vs. lyophilized feces
are shown in Figure 1. The profiles for the two methods are
similar. The percentage dry weight of the lyophilized fecal
samples, also shown, indicates that the fluid content did not
change much for the 21 samples. Adjusting the lyophilized
samples for percentage dry weight did not alter the estradiol
profile even though the concentrations were much higher.
FecalExcretion in Cotton-Top Tamarins and Common
Marmosets
Ovulation occurred for each female during the sampling
period. Both species showed the typical New World pattern
of excreted estrogens in that all estrogen metabolites in-
I+
I
so
0
0
U
c
bo
a
0
0
To
so
U
0t
I
U
100000
0
A
c
W
75000
+
50000
c
.o
25000
m
hi
-a
LI,
cV
0
Days from Serum LH peak
FIG. 2. Serum bioactive LH and progesterone levels (top) ina representative cotton-top tamarin female during the ovarian cycle as compared to fecal progesterone
and pregnanediol (middle) and fecal estradiol and estrone (bottom). All graphs are
expressed as day from the serum LH peak.
creased concurrently with the progesterone metabolites and
showed sustained elevations during the luteal phase. None
of the excreted steroids for either species rose prior to the
circulating LH peak. Figures 2 and 3 illustrate the patterns
of circulating LH and progesterone compared with those for
the excreted steroids for an individual cotton-top tamarin
and an individual common marmoset. Excreted progester-
96
ZIEGLER ET AL.
Common Marmoset
150
{
10o
0
+
C.
50
o
=
.o
0
c-
0
TABLE 3. Steroid rise in days from the circulating LH peak.
Species
Cir. P
Fecal P
Fecal Pd
Fecal El
Fecal E2
Cotton-top tamarins
TIN
0
1
1
1
ASH
0
2
2
2
QUB
1
2
2
2
BAB
0
0
0
1
Mean ± SE 0.25 ±0.25 1.25 ±0.45 1.25 ±0.45 1.5± 0.3
1.5
Common marmosets
2
4
6
009
3
098
2
3
2
5
3
7
202
2
3
030
1
3
5
4
Mean + SE 2.0 ±0.4 2.75 +0.25
3.5± 0.65 5.5 + 0.65 3.75
1
2
2
1
± 0.3
4
4
4
3
0.25
P.
higher than those of progesterone. Total progesterone excretion was much higher in the tamarins than in the marmosets. Delays in the excretion of progesterone and preg15000
nanediol were similar for the tamarins (see Table 3), but for
the marmosets, the increase in concentration of pregnanediol following the LH peak varied between individuals. The
cq
10000
.°
5000 progesterone increase occurred 3 days after the LH peak in
0
27
three of the four marmosets and 2 days after the LH peak
o
to
in the other female. Additionally, the profiles of marmoset
5000
P
A2
pregnanediol were less clear in illustrating sustained elevations of the steroid over the luteal phase level. In the
a.
tamarin, both progesterone and pregnanediol were equally
consistent and displayed similar profiles for all females.
Concentrations of excreted estradiol were considerably
higher than those of estrone for both species. In tamarin
females, estradiol levels were generally 10 times the levels
of estrone and provided a clearer pattern of ovarian cycling.
In marmosets, estradiol levels were 3 to 6 times higher than
Co
estrone levels. Estradiol patterns were more consistent than
estrone patterns between female marmosets, showing a
o
well-defined increase following ovulation. Estrone concen'0
C.)
trations in one female showed no consistent increase after
ovulation, while estradiol levels rose and remained elevated
a)
throughout the luteal phase.
UDifferences in the metabolism of steroids between the
-1D
-IU
-3
U
D
I
13
/U
two species became apparent on examination of the initial
onset of steroid increase after the LH peak as displayed in
Days from Plasma LH peak
Table 3. Within 2 days of the circulating LH peak, all fecal
FIG. 3. Plasma bioactive LH and progesterone levels (top) in a representative comsteroids had increased in cotton-top tamarins; in contrast,
mon marmoset female during the ovarian cycle as compared to fecal progesterone
for the marmoset the increase in steroids was delayed by 2
and pregnanediol (middle) and fecal estradiol and estrone (bottom). All graphs are
expressed as day from the plasma LH peak. Note: Day -11 indicates the day the
to 7 days, with fecal estrone showing the greatest variability
female was given a prostaglandin F2,,analogue to end the conceptive cycle.
in increase from the LH peak. The delay in the steroid increase was consistent between females of each species and
therefore may provide an estimate of the ovulatory period.
one followed the pattern of circulating progesterone in all
For the tamarins in this study, the time of ovulation could
the females of both species, with a lag in fecal excretion.
be estimated to within 2 to 3 days prior to the onset of the
Levels of pregnanediol were much higher than those of profecal steroid increase while for the marmoset, the time of
gesterone in the feces for both species. In cotton-top tamovulation could be estimated to occur 2 to 3 days before
arins, pregnanediol levels were approximately 50 times
the progesterone increase and 3 to 4 days before the estrahigher than progesterone levels. For the common marmodiol increase.
sets, pregnanediol levels were approximately 30 times
20000
+
to
C.)
c
BLOOD AND FECAL HORMONES IN FEMALE CALLITRICHIDS
DISCUSSION
Even considering the delay in fecal excretion of steroids,
fecal estrogens do not show an increase in concentration
during the follicular phase of the ovulatory cycle for the
marmoset and the tamarin. This pattern is consistent with
findings from other studies on New World primates
[2, 5, 16]. Previously reported results for cotton-top tamarins
and common marmosets as well as the present findings suggest an explanation. A large percentage (47%) of radiolabeled estradiol injected into cotton-top tamarins was converted to estrone in the urine and feces [4]. Radiolabeled
estrone was excreted into the feces as 11% free and 89%
conjugated. The free portion appears to be estrone and the
larger conjugated portion to be di- or triconjugates. Circulating estrone concentrations were high and remained elevated throughout the luteal phase in both marmosets and
tamarins [8, 17]. This may indicate a luteal origin of circulating estrone in contrast to estradiol, which was elevated
and which peaked during the follicular phase or on the day
of the LH peak. Estradiol appeared in the urine, however,
after the circulating and urinary LH peak [8], but in low levels compared to estrone. The present study reveals that estradiol is found in the feces in much higher levels than estrone for both the marmoset and tamarin. Thus, circulating
estrone may also be metabolized into estradiol, since fecal
estradiol levels are so much higher than fecal estrone levels
in the tamarin and since much of the fecal estradiol occurs
as di- or triconjugates derived from estrone.
Both secreted estrone and estradiol are known to interconvert as a consequence of peripheral metabolism [18]. Additionally, interconversion of estradiol and estrone occurs in
the intestines due to 173-dehydrogenase activity from microbacterial flora [19]. These results indicate that profiles of estradiol and estrone due to secretion from the ovary during
different ovarian phases will not be observed after metabolism
and excretion into the urine and feces. Interconversion of estrone and estradiol along with higher levels of estrone may
explain the high luteal phase levels of both estrogens along
with low levels during the follicular phase. High luteal secretion of estrone may account for the urine and fecal estrogen
profile seen in all New World primates.
The present techniques provided estimates of the major
ovarian steroids by measuring both conjugated and free
steroids. Since steroid recoveries from the fecal solids were
high, and since both free and conjugated steroids were estimated, the profiles of steroid excretion should represent
the majority of each steroid examined. This study not only
provided an estimate of the ovulatory period but also indicated which of the steroids measured offered the most
reliable pattern of excretion over the ovulatory cycle. For
tamarins, fecal estradiol, progesterone, and pregnanediol
showed obvious and consistent changes in concentration
over the ovarian cycle, but for estrone, the pattern was less
97
clear. In marmosets, even though pregnanediol was seen in
higher concentrations than progesterone, progesterone
showed a more consistent onset of steroid increase and a
clearer profile. This contrasts with findings by Heistermann
et al. [2], who observed less variation between individual
females using fecal pregnanediol concentrations. Estradiol
exhibited a consistent, clear profile for marmosets in this
study as well as in that of Heistermann et al. [2].
Underestimation of fecal estradiol in both species would
have occurred without the solvolysis procedure to liberate
the steroid from complex estradiol conjugates. Over 80% of
estradiol was measurable only after solvolysis. This compares well with our previous study of metabolism of radiolabeled estradiol in the cotton-top tamarin, in which up to
85% of the fecal steroid was conjugated [4]. These complex
conjugated estrogens, such as estradiol-3,17 disulfate,
which is found in squirrel monkey urine (Saimiri scuirus)
[201, cannot be measured directly by standard RIA and ELISA
techniques. Only after cleavage of the steroid from the conjugate are antibody binding sites available for competitive
binding assays. When Heistermann et al. [2] measured free
estradiol, they did not find clear profiles of ovarian cycling
in the cotton-top tamarin. Our results, however, indicate
that total estradiol profiles after solvolysis, which liberates
both mono-, di-, and triconjugates, provided clearer and
less variable profiles than did estrone. Procedural differences between the two laboratories may account for some
differences in the profiles, especially since samples at the
Heistermann laboratory were lyophilized. However, since
the proportion of steroid conjugates can change from one
phase of the ovarian cycle to another [11], our method of
measuring both free and conjugated estrogens eliminates
any pattern changes that might occur as a consequence of
measuring free steroids only. In addition, the resulting estrogen profiles appeared to be as reliable as progesterone
metabolites for assessing luteal function in both species.
Added radiolabeled estrone-glucuronide indicated that
some hydrolysis of conjugates occurred directly in the feces
for these two species. Hydrolytic activity from intestinal [3glucuronidase has been reported in primates [21] and is dependent on dietary fiber. Hydrolytic activity in the intestines
explains the relatively low percentage of steroids obtained
after hydrolysis for these two species in the present study.
Enzymatic activity in the feces may account for the higher
percentage of free steroids in many primate species.
Steroids were excreted into the feces 1 to 2 days sooner in
the cotton-top tamarins than in the common marmosets. All
steroids measured showed an increase within 2 days following the LH peak, and fecal progesterone and pregnanediol
increases occurred only 1 day after circulating progesterone
concentrations increased in all but one female. In contrast,
common marmoset circulating concentrations of progesterone generally were found to increase 1-2 days following the
circulating LH peak ([22]; current data), but fecal steroid con-
98
ZIEGLER ET AL.
centrations rose several days later. The diets of the tamarins
and marmosets were similar in this study and therefore were
unlikely to contribute to the differences found in excretion of
steroids. Differences in steroid metabolism between marmosets and tamarins, however, do exist: only 5% of excreted
progesterone is found in the urine while the remaining 95%
is excreted into the feces in cotton-top tamarins [41, whereas
progesterone metabolites are measurable in much higher
amounts in urine from common marmosets [3]. These results
indicate that estimation of the periovulatory period will be
more precise if the relationship of fecal steroids to the LH surge
are known for the species of interest. Information from one
species, therefore, may not be applicable to another species,
even within the same family.
Between-female variation with respect to the onset of
steroid increase after the LH peak was low in both species.
However, variability between species was high. For the cotton-top tamarin and common marmoset, one can estimate
a 2- to 3-day window during which ovulation should have
occurred. Thus such noninvasive measures of ovulation can
be compared with behavioral events. For captive common
marmosets and cotton-top tamarins whose diets differed little, steroid excretion into the feces was consistent enough
to determine the delay in fecal steroid excretion from the
time of ovulation. Whether wild marmosets and tamarins
with more dietary variation show more variation in excretion time of steroids needs to be determined.
Conflicting results have been reported concerning the
need to eliminate the effect of fluid variability on steroid
concentration in the feces. Wasser et al. [23] found that lyophilizing samples to correct for fluid variability improved
fecal-to-serum correlation of steroid concentration. Shideler
et al. [24] reported that diarrhea in cynomolgus monkeys
did not reduce the levels of fecal steroids, while Bamberg
et al. [25] found that diarrhea in gorillas did influence estrogen levels. In our study, removing the water content in individual fecal samples did not change the profiles of steroid
concentration from the pattern seen in samples analyzed as
wet feces. The water content did not alter much between
the samples, and the variations that did occur in water content did not alter the interpretation of the profile. None of
the monkeys in the current study had noticeable soft or
diarrhea-type stools that might have diluted the steroids. In
captive primates that are healthy and have consistently solid
stools, variability in fluid content may not alter the steroid
levels. This may not be true for free-ranging primates whose
diet and parasitic condition may change over the course of
a study. For field collection of feces, the condition of the
sample should be noted at the time of collection.
The ability to monitor fecal steroids in free-ranging tamarins and marmosets will provide useful comparative data
for captive studies in several areas. Comparative data on
concentrations of fecal neutral sterols and acidic steroids in
laboratory and free-ranging cotton-top tamarins may pro-
vide information on dietary effects on the spontaneous colon cancer that occurs in this species [26]. Elucidating the
mechanisms of fertility suppression requires an understanding of how these mechanisms work in nature where the
proximity between group members is not limited. Also, environmental regulators of fertility and their mechanisms of
action can be studied in these species, in which fecundity
is very high in captivity but often reduced in the wild.
Our current techniques have been useful in elucidating
the major steroids to be analyzed and those that appear to
provide the most consistent patterns of steroid excretion.
Large-volume extractions and chromatography separations
are costly and time-consuming. However, using the present
study as a basis of comparison, we can now improve the
methods to provide for a more efficient system. To be practicable for field collection in developing countries, it will be
necessary to develop efficient methods for preserving feces
for transport to the lab and inexpensive, precise methods
for laboratory analyses of steroids from feces.
In summary, estradiol is excreted in high levels in the
feces of both marmosets and tamarins, and this may be due
to high levels of circulating estrone. Solvolysis is required
to measure fecal estradiol in cotton-top tamarins and common marmosets. For comparisons between the species,
progesterone and estradiol provided for more consistent increases in concentration following ovulation. If steroid excretion rate is similar in wild cotton-top tamarins and common marmosets, then the day of ovulation can be estimated
as occurring 1 to 2 days before the progesterone and estradiol increase in cotton-top tamarins, and 2 to 3 days before
the increase of progesterone in common marmosets and 3
to 4 days before the increase in estradiol.
ACKNOWLEDGMENTS
The authors would like to thank the members of the University of Wisconsin Psychology
Department Marmoset and Tamarin Colony for assistance in sample collection and care of
the monkeys, particularly P. Cofta, L.Converse, T. Dreyfus, and R. Rousch. For the assistance
with the marmoset samples and care, we thank the Wisconsin Regional Primate Research
Center (WRPRC) animal care staff and W. Saltzman. We thank F. Wegner of WRPRC Assay
Services for technical support with the LH assays, C. Kapke for assistance in fecal analysis.
and Robert Chatterton for the donation of the 20a-hydroxyprogesterone antibody. A.A Carlson provided criticism of the manuscript.
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