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In vitro bioactive luteinizing hormone assay shows cyclical seasonal hormonal changes and response to luteinizing-hormone releasing hormone in the squirrel monkey (Saimiri boliviensis boliviensis).

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American Journal of Primatology 14:167-175 (1988)
In Vitro Bioactive Luteinizing Hormone Assay Shows
Cyclical, Seasonal Hormonal Changes and Response to
Luteinizing-Hormone Releasing Hormone in the Squirrel
Monkey (Saimiri boliviensis boliviensis)
Departments of 'Obstetrics an'd Gynecology and 'Comparative Medicine, University of
South Alabama College of Medicine, Mobile, Alabama
Studies on the reproductive mechanisms of the squirrel monkey have been
hampered by inadequate measurements of luteinizing hormone (LH). The
mouse interstitial cell bioassay, which measures testosterone production as
the endpoint, was validated for use in the squirrel monkey by parallel
responses of serum to LH standards and by in vivo responses to a n LHreleasing hormone (LHRH) analogue. The LH surge profile, as determined
by daily blood sampling, was found to be of 1-2 days duration and comparable in amplitude to those of other primates. A 9.7day ovulatory cycle
length was also calculated and was similar to previous estimates based on
other hormonal and behavioral methods. A 150-fold decrease in basal LH
was found in the nonbreeding season, as was a decreased LHRH response.
This assay makes possible future studies on hypothalamic-ovarian mechanisms in this species.
Key words: bioactive luteinizing hormone, estrous cycle, seasonal changes, releasing
hormone challenge
Investigations into the reproductive cycle of the squirrel monkey have included
attempts to improve the limited yield of offspring from a breeding colony [Lorenz et
al, 1973; Kaplan, 19771, development of in vitro fertilization techniques [Dukelow,
1970; Dukelow, 19831, and studies of hormonally stimulated behavior [Wilson, 1977;
Coe et al, 1981; Willams et al, 19861. Although the current market demand for use
of this species in clinical studies [Langston et al, 1984; Pindak et al, 19851 has
underscored the need to understand further and to optimize its reproductive mechanisms, relatively little is known. Until recently, gonadal steroids were the only
hormones measurable in reproductive research. Measurements of gonadotropin levels have been limited to a preliminary report of a rise in luteinizing-hormone-like
Received October 25, 1986; revision accepted September 20, 1987.
Address reprint requests to Richard R. Yeoman, PhD, Department of Obstetrics-Gynecology, University
of South Alabama, Room 336, Clinical Science Building, Mobile, AL 36688.
0 1988 Alan R. Liss, Inc.
168 / Yeoman et al.
activity occurring at the time of the estradiol rise [Ghosh et al, 19821. This report
used a radioimmunoassay with an antibody capable of measuring human luteinizing
hormone (LH) but not previously validated in the squirrel monkey. The antibody for
this assay is no longer available, and similar assays have failed t o show sensitivity
to squirrel monkey LH in our hands. Irregularities with radioimmunoassay measurements also have been found in rhesus monkeys [Neill et al, 19771, suggesting
that an in vitro bioassay is better suited for gonadotrophin measurements in certain
primate species.
The present study used the testosterone production of a mouse Leydig cell
preparation to combine the sensitivity of a radioimmunoassay endpoint with the
measurement of the biological activity of LH [Wickings et al, 1979; Van Damme et
al, 19741. The bioassay was validated in the squirrel monkey by comparing dose
responses to serum and standard LH preparations. Bioactive LH patterns were then
quantitated in the breeding and nonbreeding seasons, and also after stimulation
with an LH-releasing hormone analogue.
Subjects were reproductively proven female squirrel monkeys (Saimiri boliviensis boliviensis) that were wild-caught and housed in laboratory breeding groups for
several years. Breeding groups ranged from seven to nine females with one to two
males per pen. Details of housing have been described previously [Diamond et
al, 1984al. Animals were housed indoors but had limited access to the outdoors
during the summer months. The building temperature was regulated at 22°C with
a 15-h:9-h 1ight:dark schedule. Animals received a high-protein monkey chow and
vitamin supplements daily. Animals were captured and manually restrained in the
same sequence at each blood sampling time. One milliliter of blood was collected
from the femoral vein within 3 minutes of capture. Serum was separated and stored
frozen until assayed.
The in vitro bioassay of LH is based upon the stimulation of testosterone
biosynthesis by LH in dispersed mouse interstitial cells [Van Damme et al, 1974;
Wickings et al, 19791. Four- to six-week-old male mice of the C3H inbred strain
(Charles River, Wilmington, MA) were sacrificed with COa, and the testes were
quickly removed on ice. Cells were physically dispersed in Medium 199 (Gibco,
Grand Island, NY) containing 0.12 mM methyl isobutyl xanthene and 0.2% bovine
serum albumin (Sigma, St. Louis). Cells were then gently agitated in a Dubnoff
incubator for 1hour at 34°C. After centrifugation (150g x 10 min), resuspension of
the pellet, and filtration (300 pm mesh), the solution was diluted to provide 2 x lo5
cells per 200 pl assay volume, as measured with hemocytometer. The NIH monkey
pituitary standard, LER 1909-2,was used in a range of 0.5-10 ngltube. All serum
samples were assayed in duplicate at 1.25, 2.5, and 5 pI per tube. The testosterone
produced after a 4-hour incubation while agitated at 34°C was assayed as previously
described [Wiebe et al, 19841, using antibody #250 from Dr. G.D. Niswender (Colorado State University, Fort Collins). The potency estimates for serum samples
relative to the LH standard were calculated by parallel line analysis [Brownlee,
19651 adapted for computer by Dr. W.E. Bridson (Wisconsin Regional Primate Research Center, Madison). Sensitivity was 0.5 ng LER 1909-2/tube,and coefficients of
variation were 9.6% within assays and 19.1% between assays. Series of multiple
samples from individual animals were always assayed in a single assay to preserve
the hormonal trends maximally. Parallelism was tested among LER 1909-2, the
First International Standard for Bioassay and serial dilutions of squirrel monkey
serum. Studies of cyclic LH patterns during the breeding season involved daily blood
draws between 0900 and 1100 h for 19-to 20-day periods from late January to early
BIOLH in the Squirrel Monkey / 169
March. Twenty-two animals were sampled in this aspect of the study. Samples for
determination of nonbreeding LH levels were collected in a different group on an
October morning in conjunction with the evaluation of pituitary responsiveness.
Pituitary responsiveness was evaluated with an LH analogue (LHRH-A)(WY/
40972, Wyeth, Philadelphia) that had been previously characterized [Corbin, 19821.
Challenges in comparable groups during nonbreeding (n = 4) and breeding (n = 6)
seasons involved administration of 0.1 pg LHRH-A in 0.2 ml saline IV preceded by
baseline blood sampling and followed by further sampling at 20-minute intervals for
100 minutes. A second LHRH-A injection was given 60 minutes after the first
injection in the nonbreeding season to evaluate a priming effect in the pituitary.
Progesterone was also measured in breeding animals challenged to aid in cycle stage
determination. The radioimmunoassay involved extraction and binding with antibody kindly provided by Dr. V. Mahesh (Medical College of Georgia, Augusta).
Details have been reported previously [Diamond et al, 1984al.
Results from the mouse interstitial cell in vitro bioassay for LH showed that a
serial dilution of squirrel monkey serum was linear and parallel to the monkey
standard, LER 1909-2 (Fig. 1).Dilutions that contained as little as 1.25 pl serum
were able to stimulate testosterone production above baseline. The First International Standard for Bioassay also stimulated testosterone production in a parallel
manner at a potency of 35.6 pIU/ng LER 1909-2.
Sera from cycling monkeys were analyzed for LH, and increases of greater than
2 SD of preceding day values were detected in normal cycles prior to pregnancy and
after abortion. Incidental observations of matings corroborated the onset and duration of the annual breeding season. Animals suspected of being pregnant, as judged
by sustained elevated bioactive levels, were not included in these analyses. The first
day of LH rise was marked and consistently greater than the following day and thus
was termed the peak. The data were normalized to the peak of the LH release and
compared from 4 days before peak through 3 days after. The mean peak LH serum
levels for the 17 LH surges encountered was 2,865.6 425.7 ng/ml (mean k SEMI,
as compared to 487.4 & 31.9 ng/ml measured on the preceding day (Fig. 2). Thus, the
levels rose dramatically to a peak, yet fell slowly so that values were still above
baseline 24 hours later. In ten animals, two surges were detected in each animal
during the 20-day blood draw, and a cycle period of 9.7 & 0.4 days was determined.
Stimulation of an LH rise with LHRH-A in the nonbreeding season produced a
highly significant (P < .Ol) elevation in serum LH at 20 minutes postinjection (Fig.
3). The response to a second injection at 60 minutes while the levels were still
elevated was negligible, which is consistent with the effects of a long-acting agonist.
This stimulation trial was done in October, before the breeding season, as suggested
by the low basal levels of LH. When an equivalent LHRH-A injection was repeated
during the breeding season in a comparable group (n = 6), a much greater response
was observed, and the baseline levels were more than two orders of magnitude
greater (Table I). The estrous cycle phase during breeding season testing was random, but an analysis of daily LH and progesterone levels in serum collected before
the LHRH-A challenge showed that four of the six animals were tested 8 to 10 days
after their last LH surge and had 219.5 & 15.5 ng progesterone per ml serum. The
other two animals had an LH surge 2 days previously with 173.5 i-7.5 ng progesterone per ml. On the day of the challenge, preinjection LH levels were comparable in
all animals tested (1,790.2 & 132.6 ng/ml); however, the early luteal animals responded with a larger increase in LH (1,100 100 ngiml) than did the late luteal
170 I Yeoman et al.
LER 1909-2
800 -
2 400c3
200 -
5 10 20
ng or pl or pIU
50 100 200
Fig. 1. Dose-response curves from the monkey pituitary extract LER 1909-2(NIH),female monkey serum,
and 1st International Standard for Bioassay (MCR 70145).
Days from LH surge
Fig. 2. LH surge profile from 17 cycles normalized to the day of LH surge (mean f SEM).
BIOLH in the Squirrel Monkey / 171
0.1 ug
50 -
40 -
20 -
Minutes After First LHRH-A
Fig. 3. LH responses from four individual animals administered 0.1 pg LHRH-A IV at time 0 and 60
minutes later during the nonbreeding season.
TABLE I. Comparison of Bioactive LH Levels (nglml)at 0 and 20 Minutes After 0.1 pg
LHRH-A Administration Intravenously in the Nonbreeding Season to One Group and in
the Breeding Season to a Comparable Group
t = O
(n = 4)
+ OBa
1,790.2 + 132.6
= 20
Net increase
51.8 f 8.2
2,572.5 f 259.9
(n = 6)
animals (598.5 91.4 ng/ml). All breeding season animals released much more LH
when stimulated than did animals during the nonbreeding season.
In the study reported here, the in vitro LH bioassay with mouse interstitial cells
was validated for use in the squirrel monkey by obtaining consistent parallel
responses with recognized pituitary and urinary LH standards. The specificityof the
assay was further established by stimulating the pituitary with LHRH-A and observing an appropriate rise in serum LH. Previous studies have shown no interfer-
172 I Yeoman et al.
ence from serum levels of follicle-stimulatinghormone in the interstitial cell response
[Van Damme et al, 19741. A particular advantage of this assay for use with squirrel
monkeys, whose body weight is only 500-800 g, is the small sample volume (20 p1)
needed to prepare the dilution curve.
Application of this assay to serum samples collected during the breeding season
showed short-duration LH surges occurring frequently in cyclic patterns. These daily
samples, although limited in precision by the collection frequency, revealed that LH
was markedly elevated for only 1 day and decreased to less than one-half the peak
level on the 2nd day. This pattern contrasts with the 2- to 3-day marked elevation
detected with radioimmunoassay in this species [Ghosh et al, 19821. This difference
may represent a more dramatic cyclic pattern in biological LH activity than in
immunological activity. A change in the bio- to immuno-activity ratio during the
ovarian cycle has been reported previously in rhesus monkeys [Neill et al, 19771.
Another difference in these studies was housing conditions, since our samples were
taken from animals in breeding pens, whereas the previous study used females
housed without males. The sample-collecting technique may be another source of
difference, since our study collected 1 ml of blood per day with manual restraint,
and the previous study collected 2 ml blood per day from Nebutal-anesthetized
animals. In another New World species, the marmoset, marked LH elevations of 1day duration have been reported [Harlow et al, 19831, similar to elevations observed
in certain Old World species, rhesus [Weick et al, 19731 and stumptail macaques
[Wilks, 19771.
The magnitude of the LH surge, as sampled with daily morning blood draws,
was remarkable in that the change from the previous day was comparable percentagewise to other species and similar in concentration to human ovulatory LH levels
measured in the same assay [Abuzeid et al, 19861. Other peptide hormones in the
squirrel monkey such as growth hormone, thyroid-stimulating hormone, and adrenal corticotrophic hormone also have been found to be in concentrations comparable
to other primates [Kaack et al, 1980; Brown et al, 1970; Coe et al, 19781. The
conventional range of LH levels in squirrel monkeys is notable when considering
the extremely high levels of estrogen and progesterone that have been measured in
this species [Diamond et al, 1984a; Chrousos et al, 19821 and may relate t o a
defective receptor mechanism [Diamond et al, 1984133.
Observations of ovulatory cycle length also could be determined in some animals
from the LH patterns, and our calculation of 9.7 days was comparable to previous
estimates based on ovarian steroids, estrous behavior, and vaginal cytology [Diamond et al, 1984a; Wolf et al, 1977; Wilson, 1977; Castellanos & McCombs, 19681. A
comparison of cycle lengths reveals that the squirrel monkey has the shortest
ovulatory cycle of all monkeys reported as a result of its brief follicular phase [Pohl
& Hotchkiss, 19831.
The annual occurrence of a nonbreeding, noncyclic state in squirrel monkeys
housed indoors is reflected in LH measurements. LH levels were 150-foldless at this
time of the year than during the breeding season. Seasonal reproductive mechanisms likely involve dynamic interactions within the entire hypothalamic-pituitaryovarian axis, with the response of each component facilitated by prior stimulation.
Thus, it is difficult to identify the cause of reduced hormonal activity during this
nonbreeding season. In our colony of squirrel monkeys, serum levels of estrogen and
progesterone are markedly reduced in the nonbreeding season [Diamond et al,
1984al. Other studies have shown that ovarian sensitivity to an ovulation-inducing
regimen of exogenous gonadotropins decreases during the summer nonbreeding
season [Harrison & Dukelow, 19731. This decreased sensitivity was explained as
possibly due to a decreased endogenous gonadotropin background. The expected
BIOLH in the Squirrel Monkey / 173
decrease in LH during the nonbreeding season was confirmed in the present study.
The decrease in serum LH levels observed with the change from breeding to nonbreeding status is comparable to but much greater than the twofold decrease observed in nonbreeding rhesus monkeys [Walker et al, 19841. Although marmosets
are New World monkeys, as are squirrel monkeys, they are not seasonal breeders
[Hearn & Lunn, 19751. However, within a breeding group of marmosets, only the
dominant male and female breed. These dominant animals have measurable levels
of LH, while subordinate animals have undetectable levels [Abbott et al, 19811.
Follicle-stimulating hormone is another pituitary gonadotropin with major importance in ovarian stimulation, yet to date it has not been measured in New World
monkeys because of lack of an appropriate assay. Folliclestimulating hormone has
been found to decrease significantly in rhesus monkeys during the nonbreeding
season [Walker et al, 19841, suggesting that this hormone also has an involvement
in seasonal reproductive mechanisms.
The seasonal change in serum LH of the squirrel monkey may involve a diminished pituitary responsiveness to releasing hormones in the nonbreeding season.
The present study has shown that equivalent amounts of LHRH-A induce much less
LH release in the nonbreeding season than in the breeding season. This decrease in
pituitary responsiveness may be due to markedly reduced background levels of
estrogen, since estrogen has been shown to enhance gonadotroph responses t o luteinizing hormone-releasing hormone [Higuchi & Kawakami, 19821. A more important
mechanism responsible for decreased serum LH in the nonbreeding season may be
a decrease in the frequency of luteinizing hormone-releasing hormone pulses from
the hypothalamus. Seasonally breeding sheep exhibit a decrease in the number of
luteinizing hormone pulses during the nonbreeding season [Scaramuzzi & Baird,
19771, and LH pulses are highly associated with pulses of releasing hormone [Clarke
& Cummins, 19821. Thus, the dramatic contrast in baseline LH levels observed
between breeding and nonbreeding periods may suggest a hypothalamic-pituitary
mechanism of seasonal reproductive change in this species. A similar conclusion has
been reached concerning seasonality in other mammals [Clarke, 19811 and is nicely
demonstrated in ovariectomized sheep, which have a seasonal increase in LH while
receiving a constant level of estradiol treatment [Legan et al, 19771.
1.The mouse interstitial cell in vitro bioassay for LH, which measures testosterone production as the endpoint, was validated for squirrel monkeys by parallel
responses of serum and LH standards. Appropriate serum LH increases in animals
in response to stimulation with an LHRH analogue were also observed.
2. The LH surge profile determined by daily blood sampling was found to be of
1-2 days duration and comparable in amplitude t o those of other primates.
3. A 9.7-day ovulatory cycle length was also calculated and was similar to
previous estimates using other hormonal and behavioral methods.
4.A 100-folddecrease in basal LH was found in the nonbreeding season.
5. This assay makes possible future studies on hypothalamic-ovarian mechanisms in this species.
These studies were supported by NIH grant RR01254. The authors wish to
thank Dr. William E. Bridson of the Wisconsin Regional Primate Research Center,
University of Wisconsin, and Dr. David R. Mann of Morehouse School of Medicine
174 I Yeoman et al.
for technical guidance on this assay. We also wish to thank the Keproductive
Endocrinology Laboratory staff, the Primate Research Laboratory staff, and Mr.
Robert Ricker for their excellent technical assistance. The proficient secretarial
assistance of Mrs. Mary Lou Russell and Mrs. Melissa Gillis is very much appreciated. We are indebted to Dr. G. Bialy, NIH, and Dr. P.L. Storring, WHOMBSB, for
standards; to Dr. G. Niswender, Colorado State University, for testosterone antibody;
to Dr. V. Mahesh, Medical College of Georgia, Augusta, for progesterone antibody;
and to Dr. A . Corbin, Wyeth Laboratories, for the LHRH analogue. All experiments
presented in this manuscript were performed in accordance with standards established by the Animal Welfare Act and by the document entitled “Guide for the
Humane Care and Use of Laboratory Animals.”
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