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Estimation of immunoreactive testicular androgen metabolites in the urine of saddle-back tamarins.

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American Journal of Primatology 2387-98 (1991)
Estimation of lmmunoreactive Testicular Androgen
Metabolites in the Urine of Saddle-Back Tamarins
G. EPPLE, I. KUDERLING, A.M. BELCHER, A SCHAFER, AND A. LERCHL
German Primate Center, Gottingen, Federal Republic of Germany (G.E.,I.K., A.M.B., A S . ,
A.L.); Monell Chemical Senses Center, Philadelphia, Pennsylvania (G.E.,A.M.B.)
Hydrolysis, extraction, and radioimmunoassay techniques for the estimation of excreted testosterone metabolites in the urine of saddle-back tamarins have been validated and are described. The steroids measured with
the testosterone antiserum used are mostly present as glucuronides and
sulfates. Immunoreactivity in high-performance liquid chromatography
(HPLC) fractions of urinary extracts and in a standard mixture of cortisol,
testosterone, and dihydrotestosterone (DHT) were compared. The fractions
with the same retention data as testosterone accounted for the major part
of the immunoreactivity . Several other immunoreactive compounds of unknown identity were present in low concentrations. These results suggest
that testosterone conjugates are the major steroid metabolites measured
with this method in Saguinus fuscicollis. Urinary testosterone levels of
castrated males were much lower than those of intact males. Testosterone
treatment of castrated males resulted in a temporary superphysiological
increase in the levels of urinary testosterone and in an individually variable increase in the levels of the minor immunoreactive compounds. These
results suggest that estimation of testosterone metabolite levels in urine is
a valid method for the assessment of testicular activity in Saguinus fuscicollis.
Key words: eallitrichids, urinary testosterone, testosterone, radioimmunoassay
INTRODUCTION
A number of studies have shown that the social environment strongly influences several reproductive endocrine parameters in callitrichid monkeys. Many of
these studies deal with social suppression of ovarian cyclicity in females [Abbott,
1986; Abbott et al., 1987; Epple & Katz, 1984; French et al., 1983, 1989; Tardif,
1984; Ziegler et al., 1987133,but work by Abbott [19861has suggested a relationship
between the social environment and endocrine responses in males as well.
These studies document the value of marmosets and tamarins as subjects for
research in socioendocrinology. Unfortunately, their use in manipulative experiments is somewhat restricted by their small size and the fact that some species are
Received for publication March 20, 1990; revision accepted September 6, 1990.
Address reprint requests to Dr. Gisela Epple, Monell Chemical Senses Center, 3500 Market Street,
Philadelphia, PA 19104-3308.
0 1991 Wiley-Liss, Inc.
88 / Epple et al.
very susceptible to handling stress. For these reasons, the possibility of obtaining
large blood samples at frequent intervals is quite limited. Investigations that
require frequent measurements of hormone levels have successfully employed the
quantification of gonadotropins and gonadal steroid metabolites in urine. To date,
the use of these methods in research on callitrichids has been limited to females
[Epple & Katz, 1984; French et al., 1983,1989; Heger & Neubert, 1987; Hodges &
Eastman, 1984; Hodges et al., 1981; Ziegler et al., 1987a,bl. Studies on humans
[Gupta & Butler, 1969; Gupta et al., 1975; Ismail, 19761, mice [Tyler et al., 19781,
and tree shrews [Collins et al., 19891, however, have shown that the quantification
of testosterone conjugates in urine can be used to document endocrine events in
males.
This paper describes the quantification of testosterone conjugates and some
other urinary metabolites in the tamarin Saguinus fuscicollis and documents that
these metabolites can be used as rough indicators of testicular activity. The catabolism and excretion of circulating testosterone in callitrichids is poorly understood, and the major urinary testosterone metabolites of Saguinus are unknown. In
humans, testosterone is mainly excreted in the form of 17-ketosteroids and, to a
lesser degree, in the form of testosterone conjugates [Ismail, 19761. However, the
quantification of urinary testosterone conjugates is considered a more precise indicator of testicular activity than the measurement of 17-ketosteroids, since the
latter also include metabolites of adrenal androgens [Ismail, 19761. Because of
these findings, we elected t o evaluate the estimation of immunoreactive testosterone in urine from tamarin males as a measure of testicular activity.
MATERIALS AND METHODS
Subjects and Their Maintenance
Urinary androgen excretion was monitored in six adult, laboratory-born
males. Three of the males were 5 years old, one 8 years old, one 9 years old, and one
12 years old. Four castrated males were also studied under a number of conditions.
Two of the castrates were wild-born, had been castrated 15 years prior to this
study, and were approximately 20 years old. The other two castrates were laboratory-born, ll-year-old males. They had been castrated 3 years prior to this study.
In addition, 15 laboratory-born males between the ages of 2 and 12 years served as
donors for a large pool of urine employed in assay quality control. Some of these
males also donated urine used in the procedural validations and in the fractionation studies.
All males used in this study lived as members of permanent male-female pairs,
some with offspring. All groups lived in modular cages, consisting of several stainless steel compartments (50 x 80 x 130 cm). Depending on group size, cages
consisted of three (for pairs) to six (for small families) compartments. All cages
were equipped with natural branches and a nest box. Sliding partitions made it
possible to divide the compartments, so that urine donors could be separated from
their group mates without removing the donor from the home cage.
Up to four different groups of tamarins were housed in one room, but visual
contact among groups was prevented by screens. The animal rooms were maintained on a 12 hr light-12 hr dark schedule at a temperature of about 26°C and a
relative humidity of 50%. The animals received a mixed diet of fruits, vegetables,
cereals, meat, and a commercial callitrichid chow.
Estimation of Urinary Androgens
Radioimmunoassay (RIA) was used to estimate the levels of urinary androgen
metabolites in male tamarins. The procedure involved enzymatic hydrolysis of
Androgen Metabolites in Tamarin Urine / 89
urinary glucuronides and sulfates and extraction of the hydrolysates, followed by
RIA of extracts. Hormone levels were related to the creatinine content of the urine
sample to control for variation in urine concentration
Collection of urine samples. For the purpose of urine collection, males were
separated from their group mates in one of the compartments of their home cage.
Stiff, corrugated plastic matting, featuring cup-like indentations at regular intervals, was placed on the cage floor for urine collection. The indentations effected
some separation of urine from feces and other debris. Only uncontaminated urine
was collected. Urine was aspirated with a Pasteur pipette at least every 2 hr except
during the night. All samples were kept on ice or in the refrigerator until collection
was completed. The duration of urine collection varied according to the experiment
(see below). Urine samples were centrifuged at 4°C and 4,5009 for 15 min, divided
into several aliquots, and stored at -20°C until analysis. In addition to individual
samples, aliquots of a large volume of pooled urine from intact males were stored
to provide samples for quality control of each assay.
Hydrolysis. A semipurified glucuronidase from Helixpomatia (Sigma G1512)
with both glucuronidase and sulfatase activity was used. Urine samples (30-100
p1) were incubated in sodium acetate buffer (0.5 M, pH 4.6) with 2000 IU of
glucuronidase at 37°C for 18 hr.
Extraction. Each hydrolysate was extracted by vortexing 1min with 10 ml of
fresh t-butyl methylether. The extracts were snap frozen in a mixture of acetone
and dry ice. The ether phase was decanted, dried under nitrogen, and dissolved in
2 ml of Tris buffer (0.05 M tris [hydroxymethyll-amino methane, 0.1 M NaCl, 0.1%
NaN,, 0.1% gelatine, pH 8.0). Except for the fractionation studies described below,
no fractionation steps were performed, since our initial goal was to measure all
immunoreactive androgens.
RIA. Triplicate aliquots of all samples were prepared for RIA. For each aliquot, 400 pl of urinary extract was incubated with tritiated testosterone (100 pl)
and 200 pl of testosterone antiserum. Tritiated testosterone [1,2,6,7-3H(N)1(New
England Nuclear NET-370) was diluted in Tris buffer so that 100 (1.1 contained 0.05
pCi (approximately 35,000 cpm). The antiserum, provided by E. Nieschlag, was
also diluted in Tris buffer and used at a dilution of 1:8,000 so that it bound about
22% of the radioactive hormone in the absence of a sample. Cross reactivity of the
antiserum is listed in Table I.
All aliquots were incubated for 18 hr at 4"C, folllowed by separation of free
from bound steroids. For this purpose, 200 p1 of a suspension of dextran-coated
charcoal (charcoa1:dextran 1 O : l ) in Tris buffer (kept at 4°C) was added to each
sample. The samples were incubated for 15 min at 4°C following the addition of
charcoal to the last sample, and then centrifuged for 20 min at 4,500g and 4°C.
From each aliquot 700 p1 of the supernatant was pipetted into a scintillation vial
containing 3 ml of scintillation cocktail (Zinsser Analytic Quickszint). All tubes
were counted in a scintillation counter for 5 min.
A standard curve was constructed using three aliquots each of testosterone
standards of 25, 50, 100, 250, 500, 1,000, and 2,000 pg. A number of blanks containing only sodium acetate and hydrolysis enzyme and several aliquots of the
quality control pool of urine from intact males were included in triplicate with each
batch of unknown samples. These samples were processed through hydrolysis,
extraction, and RIA. Assay sensitivity was monitored, based on counts obtained for
the blanks. Only assays that gave blank values below the lowest concentration of
standard used (25 pg) were accepted.
Creatinine assay. Urinary creatinine was determined, using a modification
of the method of Heineghrd and Tiderstrom [1973]. A color reagent was prepared,
90 / Epple et al.
TABLE I. Cross Reactivity of the
Testosterone Antiserum [Nieschlag &
Loriaux, 19721
Cross reactivity
(%I
Steroid
Testosterone
Dihydrotestosterone
Epitestosterone
Androstenediol
Androstenedione
Dehydroepiandrosterone
Estradiol
Estriol
Estrone
Progesterone
17-OH-progesterone
Deoxycorticosterone
100
100
0.1
1.6
0.3
0.002
0.003
0.001
0.001
0.05
0.003
<0.001
using a filtered 50% aqueous picric acid solution, containing 0.02 M sodium borate
and 0.06 M sodium dodecyl sulfate (SDS). Just before use, this solution was diluted
6:l with 1 N NaOH. Of this reagent, 900 pl was added to 10 p1 urine in 140 pl
distilled water, and the absorption was measured at 485 nm after 15 min incubation at room temperature. Sixty percent acetic acid (30 p1) was then added and the
absorption remeasured after 5 min. The change in absorption was calculated and
the creatinine concentration determined by subtraction of values for blanks and
interpolation on a standard curve. All samples, blanks, and standards were run in
duplicate in each assay.
Procedural Validations
The effectiveness of 2,000 IU glucuronidase to hydrolyze the androgen conjugates in different amounts of urine was determined by hydrolysis, extraction, and
RIA of aliquots of pooled quality control urine from intact males. Three aliquots
each of urine volumes between 20 p1 and 100 pl in increments of 10 p1 were
hydrolyzed with 2,000 IU glucuronidase, extracted and assayed. The samples
yielded an average of 74.5
2.0 pg testosterone/pl urine, indicating that the
enzyme activity gives consistent results within the range of volumes used.
The efficiency of combined hydrolysis, extraction, and RIA was determined by
the recovery of testosterone glucuronide (Sigma) dissolved in Tris buffer. Twenty
aliquots, prepared so that each 400 pl RIA sample contained 500 pg of testosterone
glucuronide, equivalent to 310 pg testosterone, were assayed in triplicate. The
recovery of testosterone was 73.0% 2 2.1%.
The linearity of measurement with dilution was ascertained by hydrolysis,
extraction, and RIA of serial dilutions from three pools of quality-control urine
from intact males. Three aliquots each of 5 pl of urine and three aliquots each of
volumes between 10 pl and 100 p1 in increments of 10 pl were hydrolyzed and
extracted. From each extract, three aliquots were assayed. They were prepared so
that the assay tubes contained 1, 2, 4, 8, 12, 16, and 20 pl urine.
Figure 1 compares the standard curve and the binding inhibition values resulting from the serial dilutions. Table I1 shows the testosterone level per microliter urine for each dilution. It appears that below 4 pl, linear measurements are
not obtained. The mean testosterone level of urine in volumes between 4 pl and 20
*
Androgen Metabolites in Tamarin Urine / 91
xiOJ,
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54-
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321I
I
25
pg testosterone
50
250
100
I
pI urine assayed
1
2
I
500
I
I
1000
I
4
I
10
I
2000
1
1
1
1
20
Fig. 1. The solid line depicts binding values for testosterone standards in counts per minute (cpm) corrected for
nonspecific binding (nsb). Single points represent values obtained from serial dilutions of quality control urine.
TABLE 11. Testosterone (pg/pl of urine) in
Serial Dilutions
Urine (p1) in RIA
1
2
4
6
8
10
12
14
16
18
20
Testosterone (pg/pl)
98.0
100.7
76.2
72.3
71.8
80.5
79.8
69.4
62.4
79.5
78.6
p1 was 75.2 2 2.3 pg/Fl. This value agrees very well with the mean value obtained
from 30 aliquots of the same pool when intraassay variability was determined
(73.5 2 2.2 pglpl). The amount of testosterone per assay tube was correlated significantly (r = 0.98) with the volume of urine assayed, indicating that the estimate
of hormone levels is not affected by urinary volume when volumes above 4 p1 are
used.
Intraassay variability was determined by hydrolysis, extraction, and RIA of 39
aliquots of the quality-control pool of urine from intact males, each aliquot measured in triplicate. The intraassay variability, expressed as the coefficient of variation (%CV) was 11.3%. Interassay variability, based on 15 separate assays of
samples from the quality control pool, was 12.6%.
The possibility that the androgen levels in urine would change, perhaps by
bacterial action, during the period of time the samples were exposed to the environmental conditions of the animal rooms was investigated. A urine sample each
92 I Epple et al.
was collected from four adult males within 15 min of separation for collection. The
samples were pooled and then divided in half. The first half was centrifuged and
frozen immediately, the second half was kept on clean plastic sheeting in the
empty collection compartment of a cage for 2 hr, then centrifuged and frozen.
Urinary testosterone in four aliquots from the fresh pool measured 421.7 t 27.3
ng/mg creatinine; urinary testosterone in four aliquots from the 2-hr-old pool measured 450.7 2 85.8 ng/mg creatinine. Testosterone levels in fresh and aged urine
did not differ significantly from each other (t test). These results indicate that the
time that elapsed before urine collection in these procedures was not associated
with any obvious changes in the immunoreactive androgen levels of the urine.
To confirm the assumption that most of the steroids measured in the assay
were present in the conjugated form, aliquots of two urine pools, one from seven
intact adults and one from four castrated adults, were assayed with and without
hydrolysis. The final volume in each assay tube was calculated to yield values
within the range of the standard curve. Assay tubes contained 6 pl of hydrolyzed,
extracted urine from intact males; 60 pl of hydrolyzed, extracted urine from castrates; 120 p1 of nonhydrolyzed, extracted urine from intact males; and 400 pl of
nonhydrolyzed, extracted urine from castrates. Mean testosterone levels in hydrolyzed urine were 100.5 ? 4.3 pgipl for intact males and 2.3 ? 0.2 pglpl for castrated
males. Mean levels for nonhydrolyzed urine were 1.8 0.2 pg/pl for intact males
and 0.06 t 0.01 pg/pl for castrated males. These results show that in intact as well
as in castrated males, free androgens make up an insignificant portion of the
metabolites measured with the present method.
*
Fractionation Studies
Since the antibody cross reacts with other steroids, we cannot assume that all
the immunoreactivity is due to testosterone alone. To ascertain whether testosterone is indeed the major androgen metabolite that we measure, fractionation of the
urine was carried out by high-pressure liquid chromatography (HPLC), and RIA
was performed on each fraction. The immunoreactivity of each fraction was plotted
vs. the fraction number and compared with a chromatogram of a mixture of known
standards.
For this purpose, a pool of urine from intact males (6.9 ml) was hydrolyzed and
extracted. The ether extract was evaporated to dryness and taken up in 690 p1
HPLC-grade methanol. A 50 pl aliquot, equivalent to 500 pl urine, was fractionated using a Chrom Spher C18 column, 200 mm length, 3 mm ID, 9 mm OD, with
particle size 5 pm. The elution solvent was acetonitrile/water (40:60, v/v) at a flow
rate of 0.3 ml/min.
The fractionated eluate was collected in 30 sec fractions for 30 min. The absorbance was monitored at 254 nm for about 16 min and then changed to 280 nm
to detect dihydrotestosterone (DHT) or other nonpolar steroids that show no absorbance at 254 nm. All fractions were evaporated to dryness using a freeze dryer,
and each fraction was redissolved in 2 ml of Tris buffer (pH 8.0). These solutions
were stored in 0.5 ml aliquots at -20°C until analysis by RIA.
Fractionation of a 50 pl standard mixture of cortisol (33 ng), testosterone (33
ng), and DHT (333 ng) was performed under identical conditions before each urine
fractionation to compare retention times of immunoreactive components with
known compounds. The same fractionation procedures were used to determine
immunoreactive components of urine from castrated males that had been treated
with testosterone. The two adult males who were castrated 15 years prior to this
study were used as urine donors. The urine was collected 1day after each male was
injected with testosterone propionate as described below to measure the androgen
Androgen Metabolites in Tamarin Urine I 93
metabolites a t their highest levels. Volume of urine fractionated for one male was
250 p,l; for the other male the volume was 214 p,l.
Urinary Androgen Levels in Intact and Castrated Males
To ascertain that the immunoreactive urinary androgens measured with the
present methodology are mainly of testicular origin, urinary androgen levels of
intact males were compared with those of castrates before and after testosterone
treatment. Six intact adult males and four castrated adult males were used as
urine donors. The first morning urine was collected from each intact male on 5
consecutive days. Each male was separated from his group for 1 hr of urine collection immediately after emerging from his nest box in the morning.
Urine from each of the four castrated males was collected before and during
testosterone treatment. After 9 consecutive days of urine collection from untreated
castrates, each male received one subcutaneous injection of testosterone propionate suspended in sterile saline, equivalent to 10 mg testosterone. A second injection of the same dosage was given 13 days after the first one. Urine was collected
daily from each male until 21 days after the second testosterone treatment. During
this period, each castrate spent the night in the urine collection compartment of his
home cage and was reunited with his female partner after morning urine had been
collected.
RESULTS
Fractionation Studies
From the fractionation studies, it appears that testosterone is indeed the major, but not the sole, immunoreactive component in urine from intact males. Figure
2A displays the results of RIA of fractionated urine from the pool of intact male
donors. The elution of cortisol, testosterone, and DHT, as determined by HPLC
fractionation under identical conditions, is indicated on the chromatograms.
The retention time for testosterone, as determined by HPLC, corroborates that
derived from the RIA data. Together with the fact that the antibody is highly
specific for testosterone and DHT, this strongly suggests that testosterone is indeed the major urinary immunoreactive androgen. The other two immunoreactive
peaks elute a t retention times near but not identical to those of cortisol and DHT.
These data suggest that steroids of polarity similar to that of cortisol and DHT, but
not identical to these hormones, are also present to some degree.
Fractionation and RIA of urine from testosterone-treated, castrated males produce similar results, although hormone levels are much higher than in intact
males. Testosterone appears to be the major urinary androgen metabolite detected
here as well (Fig. 2B,C). Testosterone seems to be responsible for the only peak
seen in the urine of one of the two castrates (Fig. 2B). In the urine from the second
castrate, several additional peaks are present. One of them (fraction 13) may be
identical to the minor peak seen in the corresponding fraction from intact male
urine. The other peaks do not correspond to any peaks found in intact male urine
(Fig. 2 0 .
Testosterone Levels in Intact and Castrated Males
Intact adult males show much higher urinary testosterone levels than castrated males. Figure 3 depicts daily testosterone levels in the six intact males. As
Figure 3 shows, there was pronounced individual variability in testosterone levels.
In addition, four of the six intact males also exhibited high day to day variability
in urinary testosterone levels.
Testosterone injection resulted in a dramatic increase in the urinary testosterone levels of each castrated male. Figure 4 shows the mean daily testosterone
94 / Epple et al.
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Androgen Metabolites in Tamarin Urine I 95
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DAYS
Fig. 3. Testosterone levels (ng/mg creatinine) in urine samples from six intact males (AN, BR, NE, RO, TE,
HA) collected on 5 consecutive days. 'The sample for this day was lost.
levels of the four castrates before and during treatment. On the second day following the first injection of 10 mg testosterone, urinary testosterone levels were
over three times higher than the highest levels measured in intact males. Urinary
testosterone levels declined rapidly during the days following injection but remained a t levels above those of intact males for the first 4 days. Following this
period, testosterone levels declined further and by 10 days had fallen below the
lowest level seen among the six intact adults.
DISCUSSION
The results of our combined HPLC/RIA fractionation studies with urine from
intact males and from testosterone-treated castrates strongly suggest that in
Saguinus fuscicollis some of the circulating testosterone is excreted in the form of
testosterone conjugates. The enzyme employed in the hydrolysis has glucuronidase
as well as sulfatase activity. The relative contributions of testosterone glucuronides and sulfates to the total immunoreactivity is unknown. Very little free
testosterone is present in the urine.
RIA of all HPLC fractions of intact male urine indicates that testosterone is
indeed the major immunoreactive component, but two other components that react
with the antibody are present. These elute near, but do not coelute with, cortisol
and DHT, as determined by HPLC fractionation and RIA of a mixture of cortisol,
testosterone, and DHT standards. The retention data for cortisol were determined
from the HPLC data alone, because it showed no immunoreactivity with the antibody.
Data for fractionated urine from castrated males, 1 day after treatment with
testosterone, show that testosterone is also the major immunoreactive peak, al-
Fig. 2. A: Immunoreactivity (pgifraction)in HPLC fractions of intact male urine and of a standard steroid mix.
The fractions of maximum intensity for elution of standards are indicated by C, cortisol;T, testosterone; DHT,
dihydrotestosterone. B: Immunoreactivity (pg/fraction)in HPLC fractions of urine from a testosterone treated
castrated male and of a standard steroid mix. The fractions of maximum intensity for elution of standards are
indicated as in A. C: Immunoreactivity (pg/fraction) in HPLC fractions of urine from a second testosterone
treated castrated male and of a standard steroid mix. The fractions of maximum intensity for elution of standards are indicated as in A.
96 / Epple et al.
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5
10
15
20
25
days
30
35
40
45
Fig. 4. Mean (?SEMI testosterone levels (ngimg creatinine) in daily urine samples from four castrated males
before and after treatment with testosterone. The arrows indicate the days of testosterone treatment. For some
samples with low testosterone levels, additional assays were performed with larger volumes of urine to obtain
in-range values. Therefore, sufficient urine was not always available for more than one measurement.
though at highly elevated levels. In urine from one of the two castrated males,
several other immunoreactive components are also present, and some of these
appear different from those found in urine from intact males. It is possible that
these components are metabolites of testosterone that are present in normal males
as well, but, due to the much lower androgen levels overall, the values for intact
males are well below threshold. These components did not appear in the analysis
of urine from the second castrated male. They may reflect individual variability.
It is possible, of course, that urinary testosterone conjugates represent only a
minor portion of all excreted testosterone metabolites, i.e., that only a small fraction of this steroid is excreted in the conjugated form. In humans for example,
testosterone conjugates form a small percentage of all excreted testosterone metabolites, whereas the major urinary testosterone metabolites are 17-ketosteroids
[Ismail, 19761. Additional studies on saddle-back tamarins are needed to elucidate
testosterone catabolism in this species, to identify its major excretory products, and
to determine whether this hormone is mainly excreted via urine as is the case in
Homo [Ismail, 19761.
The levels of immunoreactive androgen metabolites in tamarin urine roughly
reflect testicular activity. This is indicated by the finding that urinary hormone
levels were much higher in intact adult males than in castrates. Hormone levels
increased strongly after injection of testosterone and declined to levels lower than
those of intact males within 10 days following injection of the hormone. These
results suggest that quantification of the immunoreactive urinary testosterone
metabolites is a useful noninvasive tool for the assessment of testicular activity in
those studies in which long-term monitoring of hormone levels is necessary. However, future studies must determine how accurately changes in the levels of these
urinary androgen metabolites reflect changes in circulating plasma testosterone
levels.
In the five intact males, interindividual as well as day-to-day variability in
Androgen Metabolites in Tamarin Urine I 97
urinary testosterone levels was quite pronounced. At present, we cannot identify
the cause of this variability. However, several factors can be excluded. Since all
urine samples were collected at the same time of day, diurnal rhythms of testosterone secretion, which have been documented in several primate species, including Callithrix jacchus [Kholkute, 19841, are unlikely to have influenced the
results of the present study. Moreover, testosterone levels do not appear to be
related to the age of the subjects. The average testosterone level of the oldest male,
12-year-old AN (157.5 5 54.7 nglmg creatinine), was in the same range as that of
5-year-old male RO (161.7 25.6 ng/mg creatinine). Differences in social status
can also be excluded as the cause of individual variability. All males were highranking adults who had sired offspring previously.
It is quite possible that urinary androgen levels reflect a high variability in
circulating testosterone levels. However, we have not monitored plasma testosterone levels over extended periods of time to detect such variations. Variability in
urinary testosterone levels has also been seen in healthy human males, where
urinary testosterone excretion per 24 hr peaked at intervals of 5-6 days [Ismail,
19761.
Because of the pronounced variability in urinary androgen levels of saddleback tamarins, the quantification of these metabolites is not useful for evaluating
the effects of short-term experimental manipulations on testosterone levels, based
on a small number of urine samples. However, when animals are monitored over
extended periods of time, relatively stable, individually characteristic urinary testosterone levels can be documented (Epple et al., unpublished data). Therefore,
monitoring of urinary testosterone levels can be of value in long-term studies in
which each individual serves as its own control. Such studies include the assessment of maturational changes, as was done in tree shrews [Collins et al., 19891,
assessment of long-term effects of social status on male testosterone levels, and
similar projects.
A long-term study by Lerchl et al. (in preparation) provides an example of the
utility of urinary testosterone determinations for the detection of diurnal changes
in testosterone levels in Saguinus fuscicollis. Urine was collected from five males
for the duration of 2 hr periods, which covered the 12 hours between lights on and
lights off in the animal rooms. No urine was collected during the night. There were
pronounced individual differences in testosterone levels among the five males.
However, in the group of males, testosterone levels were highest in urine collected
during the first 2 hr of daylight, which included the first morning void. The findings suggest that testosterone is excreted mainly during the night and early in the
morning. It remains to be seen, of course, whether this reflects increased nocturnal
levels of circulating testosterone. Circulating testosterone levels are higher during
the nightly rest period in another diurnal callitrichid, the common marmoset
[Kholkute, 19841.
*
CONCLUSIONS
1. The method validated in these studies provides a tool for the estimation of
urinary testosterone levels in Saguinus fuscicollis and can be used for a rough
assessment of testicular activity.
2. Estimation of testosterone metabolite levels in urine is a noninvasive technique that permits frequent and long-term assessment of gonadal hormone activity.
98 / Epple et al.
ACKNOWLEDGMENTS
T h i s study w a s supported by the German Primate Center and by NIH grant
R 0 1 HD23918. We thank Dr. E. Nieschlag for supplying the testosterone antiserum.
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