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Detection of urinary gonadotropins in callitrichid monkeys with a sensitive immunoassay based upan a unique monoclonal antibody.

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American Journal of Primatology 31:181-188 (1993)
Detection of Urinary Gonadotropins in Callitrichid
Monkeys With a Sensitive lmmunoassay Based Upon a
Unique Monoclonal Antibody
TONI E. ZIEGLERl.', ROBERT L. MATTER12~3,AND FREDERICK H. WEGNER'
'Department of Psychology and 'Wisconsin Regional Primate Research Center, University of
Wisconsin, Madison, Wisconsin; 3United States Department of Agriculture, Agricultural
Research Service, 5-143 Animal Sciences Research Center, University of Missouri,
Columbia, Missouri
A radioimmunological method for measuring urinary luteinizing hormone
(LH) and chorionic gonadotropin (CG) excretion in the family Callitrichidae is described. The method uses a monoclonal antibody that will be
available in virtually unlimited quantity. Several polyclonal antisera that
have been useful for the detection of callitrichid gonadotropins are near
depletion. The monoclonal antibody-based RIA provided similar results
when compared with the mouse Leydig cell bioassay for LH and a previously validated polyclonal antibody-based RIA. When the monoclonal antibody is used for immunodetection of Saguinus oedipus LH by non-reducing SDS-PAGE, a single entity is recognized that corresponds with the
molecular weight range of bioactive LH and appears to be in the normal
range for LH in nonhuman primates. LH and CG were detected by the
monoclonal antibody-based RIA in urine from representatives of species
from all genera of callitrichids. Hormonal profiles of daily urine samples
revealed the detection of the preovulatory LH surge by both RIA methods
in the cotton-top tamarin (Saguinus oedipus) and pygmy marmoset (Cebuella pygmaea) and the increase in CG due to pregnancy in both species.
Serial dilutions of midcycle and pregnancy urine from Suguinus, Callithrix, Leontopithecus, and Cebuella exhibited parallelism when compared
with our in-house reference standard of rhesus monkey CG. Cultured
Saguinus oedipus pituitary cells showed an increased release of LH when
challenged by gonadotropin releasing hormone (GnRH) providing further
support that the monoclonal antibody-based RIA measures LH in this
species. 0 1993 Wiley-Liss, Inc.
Key words: marmosets, tamarins, urinary LH and CG, imnaunoassay
INTRODUCTION
The family Callitrichidae is comprised of the New World marmosets and tamarins. Several species in this family have been studied extensively due to their
immunologic uniqueness and due to the social influences on fertility that occur.
Received for publication September 3, 1992; revision accepted March 20, 1993.
Address reprint requests to Dr. Toni E. Ziegler, Dept. of Psychology, 1202 W. Johnson St., University of
Wisconsin, Madison, WI 53706.
0 1993 Wiley-Liss, Inc.
182 / Ziegler et al.
However, because of the endangered status of many of the species, their small size
(weights of different species range from 100 to 800 g), and excitable nature, invasive methods of obtaining data are undesirable.
The measurement of urinary hormones has been proven to be an effective
method for determining reproductive status in studies requiring long-term or repeated evaluation of reproductive hormones [Epple & Katz, 1984; French & Stribley, 1985; Ziegler et al., 1987a,b, 19901. Gonadotropic hormones are excreted into
the urine in callitrichids and have been monitored by both bio- and immunoassays
[Hodges et al., 1979; Ziegler et al., 1987a,b, 19901. Bioassays for gonadotropins
require the use of live rodent tissue and are expensive and time consuming. The
use of immunologic assays have been more efficient but without purified gonadotropins available for reagent development, the assays must rely on the use of
heterologous reagents and nonspecies-specific antisera.
With this in mind, we wanted to establish a method that was useful for measuring gonadotropins in the urine of all species of callitrichids for comparative
studies. Additionally, we needed an assay that would provide an unlimited quantity of antisera for an indefinite period of time, thereby eliminating the depletion
of antisera during long-term studies. Although the use of the monoclonal antibody
described here has been reported for many mammalian species, this is the first
report in New World primates.
MATERIALS AND METHODS
Urine Samples
Urine was collected from species representing the four genera of the family
Callitrichidae. The species used were Saguinus oedipus, Leontopithecus rosalia,
Leontopithecus chrysomelas, Callithrix jacchus, and Cebuella pygmaea. Urine from
five cotton-top tamarins (Saguinus oedipus) and four pygmy marmosets (Cebuella
pygmaea) was collected a t the University of Wisconsin Psychology Department
Marmoset and Tamarin Colony by the method described in Ziegler et al. [1987a,
19901. The common marmosets (Callithrix jacchus) were maintained a t the Wisconsin Regional Primate Research Center and urine from three female marmosets
was kindly donated by Dr. David H. Abbott. The Leontopithecus urine was collected from four females and generously donated by Dr. Jeffrey A. French from the
University of Nebraska at Omaha’s Callitrichid Colony. Tamarin and pygmy marmoset urine was collected daily for a t least 40 days during the postpartum period
to detect the postpartum LH peak and the subsequent increase in CG with pregnancy. The donated urine from the common marmosets and the Leontopithecus
species was collected a t the time of the LH peak associated with ovulation and
during early pregnancy. Samples from individual females were pooled together on
the day of the LH peak for each species (Callithrix, n = 3; Leontopithecus, n = 4)
and for pregnancy in each species (Callithrix, n = 3; Leontopithecus, n = 2) and
assayed in serial dilutions to determine parallelism.
Assay Development and Procedures
A monoclonal antibody raised against bovine LH was used for assay development (Clone #518B7; Monoclonal Antibodies, Inc., Mountain View, CA; readily
available and distributed by Dr. Janet Roser, Department of Animal Science, University of California, Davis, CA). This antibody has been extensively characterized
and is known to cross-react with LH from a wide variety of nonprimate mammalian species [Matteri et al., 19871. Human CG (hCG, CR-125) was iodinated as
previously described [Matteri et al., 19871 for use as the radioligand (specific activity = 65.28 pCi/kg). Best detection was determined to occur at a concentration
LWCG RIA for Callitrichid Monkeys / 183
of the antisera a t 0.9 ng IgG/tube and 40,000 cpm of the radioligand. A pool of
concentrated and partially purified pregnant rhesus urine was used as the inhouse standard reference preparation (HSP: 5-14-77)a t doses ranging from 0.2 to
4.6 pl/assay tube to produce the standard curve. The biopotency of the HSP = 10.3
IU of hCG, CR-l25/ml. The second antibody (goat anti-mouse gamma globulin, P4
titer; Antibodies, Inc., Davis, CA? was diluted 1:lOO in phosphosaline buffer with
5%polyethylene glycol (PEG).
Reference standard or sample plus assay buffer were added for a combined
volume of 300 p1. Immediately thereafter, the primary antibody was added in 100
~1 of assay buffer with 5%normal mouse serum (Arne1 Products, Inc., New York,
NY). After an overnight incubation a t room temperature, 100 p1 of radioligand in
assay buffer was introduced and the mixture was incubated for 24 hours at 4°C. On
the third day of the procedure, 1 ml of the secondary antibodyPEG solution was
introduced. After 1hour at 4"C, the tubes were centrifuged for 30 minutes (1,9OOg,
7°C). The supernatant was then aspirated and the tubes counted for gamma decay.
The assay was validated by contrast with analyses of identical samples with a
previously characterized RIA based on a polyclonal antiserum (H26, anti-ovine
LH-beta subunit; Ziegler et al., 1987a) and the in vitro mouse Leydig cell bioassay
for LH [Ziegler et al., 1987al.
Further proof of LH detection was achieved by both bioassay and immunoassay of LH with the monoclonal assay subsequent to separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in over ten urine samples collected on the day of the preovulatory LH peak. This method separates
proteins by their molecular weight and its use for the tamarin has been reported
in Matteri and Ziegler [19921.
Excretion of LH from pituitary cells also provided evidence of LH crossreactivity with the monoclonal antiserum. A pituitary gland from a cotton-top tamarin
female who was euthanized for medical reasons was processed for cell culture by
the methods of Matteri et al. [19901. Dispersed pituitary cells were either used as
controls (n = 2 wells with the addition of serum-free culture medium) or as stimulated cells (n = 2 wells with the addition of 1 nM GnRH) to induce LH release
from the cells. The incubated secretions of the pituitary cells in serum-free medium
before GnRH challenge were serially diluted for parallelism against the standard
curve for assay validation.
Statistical Analyses
Quality control data was provided by a pool of tamarin urine collected during
pregnancies. The pool was run in triplicate for eight assays and intra- and interassay coefficients of variation were computed as 8.17 and 14.14%,respectively. These
values were higher than those values reported from RIAs using polyclonal antisera
due to the polyethylene glycol required for the second antibody which causes a
higher background.
Parallelism was determined by computed linear regression lines analyzed by
t-test for determining the differences between slopes [Brownlee, 19601. For comparison, correlation analysis for the two RIAs were performed on 120 tamarin
urine samples.
RESULTS
The assay developed using the monoclonal antiserum compared well with previous methods of detecting gonadotropin activity. Although a callitrichid standard
was not available for assay comparison, comparison of the same rhesus monkey
reference standard by the presently developed RIA and the previously described
184 / Ziegler et al.
assay based on the H-26 antiserum indicated that there was a 3.5-fold improvement in level of detection. Comparisons between sample concentrations measured
by both RIA techniques indicated that a strong correlation occurred between the
two RIA results. For 120 samples from 3 cotton-top tamarin females, the line of
best fit was y = 0.738 + 4.873x, r2 = 0.85. Assay detection limits for the monoclonal-based RIA was > 0.4 pl/ml and for the H-26 based RIA was >5 pllml. The
comparison of all three LH assay techniques used in this study on identical daily
urine samples from a cotton-top tamarin female following parturition and showing
the first postpartum ovulatory cycle also indicates that the monoclonal antiserum
reliably detects the LH peak (Fig. 1). Panel A contrasts the LH profiles determined
by the two immunoassays. Panel B shows the coincident profile of bioactive LH and
the expected increase in concentration of estrone-conjugates following the preovulatory LH surge in callitrichid monkeys. Urinary estrogens typically rise a t the
time of the LH peak and remain elevated during the luteal phase of the cycle in
callitrichid species [Ziegler et al., 19891. All three assay techniques produced
equivalently valid results.
Figure 2 shows representative profiles of LH/CG using both gonadotropin RIAs
in the cotton-top tamarin and the pygmy marmoset. In the tamarin (panel A) both
the LH peak from a postpartum conceptive cycle and the resultant increase in CG
concentration are shown. Both assays reflected the rise of CG at the same time and
term pregnancies occurred for three of the five tamarins. Two tamarins did not
conceive from the first postpartum ovulation. For the pygmy marmoset (panel B),
the LH peak associated with ovulation and the resultant increase in CG concentration are shown by both assay techniques. Hormonal profiles from two of the
pygmy marmosets showed two postpartum ovulation cycles prior to a rise of CG
indicative of pregnancy and profiles from two pygmy marmosets showed an increase in CG following the first LH peak associated with ovulation.
Volumes of urine samples from midcycle and pregnant S. oedipus, L. rosalia,
L. chrysomelas, C .jacchus, and C . pygmaea were tested for parallelism as well as
medium from cultured S. oedipus pituitary cells. No differences in parallelism
were detected among any of the samples and the reference standard (P < 0.05).
The molecular weight separation of tamarin urinary LH by bioactivity and
immunoreactivity for one urine sample is shown in Figure 3. Both results detected
a molecular entity corresponding to LH a t 38.35 kD. The mean bio- and immunological molecular weight determined from four different midcycle LH peak samples
from four tamarins was 38.51 2 0.15 S.E.M.
Further support that tamarin LH is measured by the present RIA system is
provided by the response of cotton-top tamarin pituitary cells to an in vitro challenge with GnRH. The addition of GnRH caused an increase in LH concentration
(5.62 pUm1 5 0.30 S.D., n = 2) over the control level (3.70 pl/ml h 0.48, n = 2) in
the secreted medium. The concentration of intracellular LH was lower in the
GnRH stimulated wells (10.75 pl/ml 2 0.48, n = 2) than the controls (12.89 pl/ml
2.8, n = 2) indicative of LH depletion.
*
DISCUSSION
The development of an immunoassay for callitrichid urinary LH and CG based
on a monoclonal antibody provides a reliable and efficient tool for reproductive
studies and the antibody will be available virtually indefinitely. Long-term studies
of reproductive functioning can be completed without the depletion of antisera.
Therefore, a reliable and consistent LH assay will support studies of these animals
that will impact on conservation biology, as well as basic reproductive biology.
The new immunoassay we have developed can be used as a fully validated LH
LWCG RIA for Callitrichid Monkeys / 185
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a
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b
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B
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2
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E
Days Postpartum
Fig. 1. Daily urine samples from a female cotton-top tamarin demonstrating the postpartum ovulatory surge
of LH concentration. Panel A graphs the concentration of LH as measured by the monoclonal antibody RIA
(518B7,open circles) and the polyclonal antibody RIA (H26, closed circles) per day postpartum. Panel B graphs
the concentration of LH measured by the bioassay (open circles) and estrone conjugates (ElC, closed circles) as
hormone concentration per day postpartum.
186 / Ziegler et al.
A
-E
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a
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B
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150
4-
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3-
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In
2-
Days Postpartum
Fig. 2. Concentrations of urinary gonadotropin excretion from a cotton-top tamarin female showing the post
partum LH peak and the subsequent rise in CG concentrations (A), and a pygmy marmoset female showing the
postpartum LH peak and the subsequent rise in chorionic gonadotropin (CG) concentrations (B).The urinary
gonadotropinswere measured by the monoclonal RIA (518B7,open circles) and the polyclonal RIA (H26, closed
circles).
assay technique for callitrichid LH and CG. We have shown that the 518B7 monoclonal antibody recognizes LH and CG from callitrichid monkeys, which is consistent with its known cross-reactivity with LH from many other mammals [Matteri
et al., 1987; Bravo et al., 19903. Previous characterization of the antibody has
conclusively demonstrated high specificity for LH [Matteri et al., 19871. The 518B7
monoclonal antisera has also been used for the development of a sensitive enzyme-
LWCG RIA for Callitrichid Monkeys / 187
38.35 kD
V
15
10
5
n
0.0
0.2
0.4
0.8
0.8
1.o
Relative to Dye Front
Fig. 3. Gel electrophoresis by SDS-PAGE for a midcycle urine sample from a cotton-toptamarin. The gels were
assayed by the monoclonal RIA (closed circles) and the bioassay (open circles). Both methods demonstrated a
peak in activity in the gels that indicate a molecular weight of 38.35 kD.
linked immunosorbent assay (ELISA) for measuring LH in cattle, sheep, rat and
mouse LH [Spearow & Trost, 19871 but without an effective reduction in time or
expense. The specificity of the antibody for tamarin LH was confirmed by the
present immunodetection of a single molecule by SDS-PAGE which was also bioactive. Furthermore, excellent agreement exists among the new assay, the existing
polyclonal antibody-based RIA [Ziegler et al., 1987al and the LH bioassay when
identical urinary samples are analyzed (see Fig. 1).In vitro, the LH response to
GnRH is readily detected accompanied by the expected depletion of intracellular
LH and even though this was demonstrated in only one tamarin pituitary the
response was consistent to the measurement of LH. There were no instances of
nonparallelism between biological samples and the reference standard. Compared
to the RIA presently in use for callitrichid LH/CG based on the nearly depleted
H-26 antibody, a 3.5-fold improvement in detection limit has been achieved.
The development of an RIA or an ELISA using this antibody allows the detection and measurement of CG in Old World primates as well such as the rhesus
monkey (Macaca rnulutta) (Matteri, in preparation) but does not detect circulating
or urinary LH. The usefulness of the antibody for detecting gonadotropin excretion
in other New World primates has not been reported.
CONCLUSIONS
1. A reliable and efficient RIA for callitrichid LH has been developed. Due to
the monoclonal nature of the antibody, the assay system will be available indefinitely.
2. LH and CG are detected by the immunoassay.
3. The immunoassay can be used for the detection of LHKG in all four genera
of callitrichids.
ACKNOWLEDGMENTS
This research was supported by grants NIMH 35,215 to Professor C.T. Snowdon and NIH RR 00167 to the Wisconsin Regional Primate Research Center
188 I Ziegler et al.
(WRPRC).We thank Dr. W.E. Bridson for consultation, Dr. G.D. Hodgen for the
H26 antiserum, and Drs. D.H. Abbott and R.J. Hutz for reviewing earlier drafts of
this manuscript. We also thank T. Porter, R. Roush, and Dr. N. Schultz-Darkenfor
the collection of the marmoset and tamarin urine. This is WRPRC publication
32-037.
REFERENCES
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C.H.; Lasley, B.L. Endocrine responses in
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SCIENCE & ENGINEERING. New York,
J. Wiley, Inc., 1960.
Epple, G.; Katz, Y. Social influences on estrogen excretion and ovarian cyclicity in
saddle back tamarins (Suguinus fuscicollis). AMERICAN JOURNAL OF PRIMATOLOGY 6:215-227,1984.
French, J.A.; Stribley, J.A. The reproductive
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Hodges, J.K.; Czekala, N.M.; Lasley, B.L.
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1979.
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Matteri, R.L.; Dierschke, D.J.; Bridson,
W.E., Rhutasel, N.S.; Robinson, J.A.
Regulation of the biopotency of primate
luteinizing hormone by gonadotropin-releasing hormone in vitro and in vivo. BIOLOGY OF REPRODUCTION 43:10451049, 1990.
Matteri, R.L.; Ziegler, T.E. Detection of
nonhuman primate gonadotropins in polyacrylamide gels: an alternative to western
blotting. AMERICAN JOURNAL OF PRIMATOLOGY 26:155-166, 1992.
Spearow, J.L.; Trost, B.A. Development of a
sensitive enzyme-linked immunosorbent
assay for cattle, sheep, rat, and mouse
luteinizing hormone. BIOLOGY OF REPRODUCTION 37:595-605, 1987.
Ziegler, T.E.; Bridson, W.E.; Snowdon, C.T.;
Eman, S. Urinary gonadotropin and estrogen excretion during the postpartum estrus, conception, and pregnancy in the cotton-top tamarin (Suguinus oedipus
oedipus). AMERICAN JOURNAL OF PRIMATOLOGY 12:127-140,1987a.
Ziegler, T.E.; Savage, A.; Scheffler, G.;
Snowdon, C.T. The endocrinology of puberty and reproductive functioning in female cotton-top tamarins (Suguinus oedipus) under varying social conditions.
BIOLOGY OF REPRODUCTION 37:618627, 1987b.
Ziegler, T.E.; Sholl, S.A.; Scheffler, G.; Haggerty, M.A.; Lasley, B.L. Excretion of estrone, estradiol, progesterone in the urine
and feces of the female cotton-top tamarin
(Suguinus oedipus oedipus). AMERICAN
JOURNAL OF PRIMATOLOGY 17:185195,1989.
Ziegler, T.E.; Snowdon, C.T.; Bridson, W.E.
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urinary estrogens and gonadotropins in the
female pygmy marmoset (Cebuellu pyg~ w u ) AMERICAN
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JOURNAL OF PRIMATOLOGY 22~191-203.1990.
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