close

Вход

Забыли?

вход по аккаунту

?

Characterization and comparison of testicular LHhCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops).

код для вставкиСкачать
American Journal of Primatology 2:285-290 (1982)
Characterization and Comparison of Testicular
LHlhCG Receptors of Rhesus Monkeys (Macaca
rnulafta) and Green Monkeys ( Cercopithecus aethiops)
P. ZAIDI A N L ~E.J. WICKINGS
Max Planck Clinical Research Unit for Reproductive Medicine, University Women's Hospital,
Munster, Federal Republic of Germany
M. ARSLAN
Department of Biological Sciences, Quaid-i-Aeam Uniuersity, Islamabad Pakistan
E. NIESCHLAG
Mar Planck Clinical Research Unit for Reproductive Medicine, University Women's Hospital,
Miinster, Federal Republic of Germany
Testicular luteinizing hormone (LHlhCG)receptors were characterized in seven
green monkeys and compared with those of four rhesus monkeys. Testicular
tissue showed high binding affinity for lZ5I-hCG,(O.9-2.5X1O9 M-', and
0.7-1.64X109 M-', respectively, for green and rhesus monkeys) and low binding
capacity (0.343-0.682 fmollmg and 0.198-0.355 fmolimg testicular
homogenate, respectively). There was no difference in binding affinity between
the two groups. Testicular LHlhCG receptors in both species bound human LH
(hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular
tissue showed similar high binding affinity (6.4X109 M - I ) and low binding
capacity (1.04 fmollmg tissue homogenate) for Iz5II-hCG.
Rat LHlhCG receptors
bound hLH, hCG, and oLH to a similar degree.
Key words: testicular LHlhCG receptors, green monkey, rhesus monkey, testosterone
INTRODUCTION
The broad similarity in testicular functions between humans and nonhuman primates
makes it increasingly desirable to use primate models rather than small laboratory
rodents in reproduction research. To date, the primate model best characterized in terms
of testicular morphology and function is the rhesus monkey [Davies et al, 1979; Dym,
1980;Nieschlag & Wickings, 19801. However, as it is becoming increasingly difficult to
obtain Asian macaques, attention is being focused on alternative primate species. In
view of this we have selected the African green monkey (Cercopithecus aethiops) as
another Old World monkey species and have characterized the testicular luteinizing
hormone (LHlhCG) receptors and other testicular parameters in a group of seven
immature animals. The hCG binding characteristics and specificity of the testicular
LHlhCG receptors were compared with those in the rhesus monkey and both species
were then compared with the adult male rat.
Received October 15, 1981; accepted November 2, 1981
Address reprint requests t o E. Nieschlag, Max Planck Clinical Research Unit for Reproductive Medicine,
University Women's Hospital, Steinfurter Str. 107,4400 Munster, Federal Republic of Germany.
0275-256518210203-025$02.000 1982 Alan R. Liss, Inc.
286
Zaidietal
MATERIALS AND METHODS
Subjects
The 7 green monkeys (C. uethiops) used in this study were sacrificed as kidney donors
for viral cultures, and testes were removed on that occasion. The animals had been obtained from Kenya and had been quarantined in individual cages in a temperature- and
humidity-controlled environment for 3 months. All animals were immature as judged
from body weights of 2.0-3.0 kg (adult animals typically weighing 4.0-7.0 kg; Napier &
Napier [1967]),but they were a t different stages of pubertal development as indicated by
testicular weight and coloration of scrotal skin (rangingfrom pale pink to dark blue).The
animals were sacrificed using an overdose of ketamine (Ketanest, Parke-Davis,
Freiburg) between 7:OO and 8:OO a.m.
Four laboratory-maintained rhesus monkeys (Mucuca rnuluttu) were used. Three
animals were adult (more than 5 yr, body weight 9.2-14.2 kg) and the fourth was 3.5-4.0
yr, as determined from dentition [Haigh & Scott, 19651 and body weight (4.4 kg). The
animals were bilaterally castrated under ketamine anaesthesia (8-12 m g k g body
weight).
Testes were removed from six adult Wistar rats (60days, 200-250 g) immediately after
decapitation.
Methods
Blood samples were obtained where possible for radioimmunological determination of
testosterone [Nieschlag & Loriaux, 19721. Testicular testosterone concentrations were
measured in small pieces of monkey tissue as previously described for rabbits
[Nieschlag et al, 19751 and humans [Nieschlag et al, 19791. The remaining tissue was used for receptor analysis by radioligand receptor assay.
Radioligand Receptor Assay
The assay was essentially performed as previously described by Geisthovel et al(19811
for rat testicular tissue. Testes were decapsulated immediately after removal, minced,
and homogenized in ice-cold buffer using a glass tissue grinder (12 strokes at 100 rpm).
The testicular homogenate was filtered through a nylon gauze and centrifuged at 2100 g
for 10 min at 4 "C. The supernatant was discarded and the pellet was resuspended in icecold buffer (0.05 M TrisiHCl, pH 7.2, containing 0.25 M sucrose, 0.01 M MgCI,, 0.1%
BSA) and the volume was adjusted to an appropriate dilution for the binding assay (50
mg tissue homogenate (wet weight)/ZOOpl buffer).
Highly purified hCG (biologicalpotency 11.2 I.U.ipg, Serono, Frieburg) was iodinated
using a modified lactoperoxidase method and purified by vacuum ultrafiltration. The
specific activity, determined by self-displacement in the radioligand receptor assay and
also by calculating the total radioactivity associated with the protein fraction, was in
the range of 16.5-18.1 fiCi/pghCG. The biological potency of a similarly prepared batch
of tracer was evaluated in an in-vitro LH bioassay system [Wickings et al, 19791, and
found to be identical with that of the unlabeled preparation [Geisthovel et al, 19811. The
tracer was freshly prepared for each animal.
Duplicate aliquots of 200 pl containing 15 mg (rat),50 mg (green monkey), and 100 mg
(rhesus monkey) tissue homogenate, respectively, were incubated with 25,000 cpm
IZ5II-hCG
(10p1 buffer) in the presence of increasing amounts of unlabelled highly purified
hCG (0-10 ngilO pl buffer, biological potency 11.2 1.U.lfig)in displacement studies. Nonspecific binding was calculated using an excess of unlabeled hCG (2500 I.U.
F'regnesidtube; Serono, Freiburg). The maximum percentage binding was corrected for
specific activity.
After 60 min, the incubation at 34°C in a shaking waterbath (37 oscillationslmin) was
stopped by the addition of 2 ml ice-cold buffer. Bound and free hCG were separated by
Testicular LHlhCG Receptors in Two Monkey Species
287
immediate centrifugation at 2100 g for 10 min at 4°C. The pellet was washed twice and
the radioactivity of the pellet was then counted. Binding affinity and capacity were calculated by Scatchard plot analysis of the displacement curves [Scatchard, 19491.
The nonspecific binding was 0.5-2.0%. The intra-assay coefficient of variation for the
maximum hCG binding was 2.1-3.5% (n=9) and the interassay coefficient of variation
was 2.0-4.5% (n=9), as determined from a deep frozen pool of rat testicular tissue
prepared as described [Geisthovel et al, 19811.
Receptor Specificity
Specificity of the LHlhCG receptors was analyzed in the receptor assay by addition of
increasing amounts of hCG (biologicalpotency 11.2 I.U./pg; Serono, Freiburg), human
LH (hLH, LER 960), and ovine LH (oLH, LER 1056 C 2 ) .
RESULTS
In preliminary experiments to standardize the radioligand receptor assay for primate
tissue, the maximum specific binding of LzSI-hCG
was found to be directly proportional
to the amount of the testicular homogenate per tube for both rhesus and green monkey
tissue (Fig. 1).The amount of tissue used in the binding studies was extrapolated from
these curves, i.e., 50 mg per tube for green monkey and 100 mg per tube for rhesus
monkey tissue. Since the total amount of the tissue homogenate available for these
studies, particularly from the immature monkeys, was very limited, these amounts
represent the minimum tissue required to perform displacement studies.
The binding affinities of the LHihCG receptors were similar in green and rhesus
monkeys. The binding capacity for 1z51-hCG
was significantly lower for rhesus as compared with green monkeys (P<0.005) (Table I). The binding of "51-hCG to both green
and rhesus monkey testicular tissue was significantly inhibited by unlabeled hCG
(P<0.0005) and hLH (P<O.O005) but not by oLH. The binding characteristics in
monkeys were similar to those measured in a pooled homogenate from adult rats (binding affinity 6.4X1OY M-', binding capacity 1.04 fmollmg). By comparison, binding of
1251-hCG
to rat testicular tissue was significantly displaced by hCG, hLH, and oLH
(P< 0.005, Fig. 2).
50
100
150
200
250
tissue weight (mg)
Fig. 1. Correlation between maximum specific hCG binding ('Po total radioactivity boundper tube) and the
amount of testicular tissue homogenate per tube from green and rhesus monkeys.
288
Zaidi et a1
TABLE I. Testicular Parameters From Seven Green and Four Rhesus Monkevs
Body weight
Green monkey
2.0
2.0
2.7
2.5
3.0
2.6
3.0
Rhesus monkey
4.4
9.2
10.2
14.2
,\"
-
Agelcolour of Testicular
scrotal skin weight (g)
Testosterone
Testicular Serum
(nglg)
(ngiml)
Binding
affinity Binding capacity
X 10'"
(fmollmg)
light blue
light blue
dark blue
dark blue
dark blue
dark blue
dark blue
1.76
1.74
3.56
4.46
4.86
5.45
12.38
18.5
38.4
46.8
10.8
11.2
60.4
104.1
2.82
3.64
1.16
1.09
5.24
4.62
0.249
0.145
0.140
0.190
0.110
0.250
0.090
0.528
0.682
0.546
0.493
0.616
0.343
0.352
3.5-4.0
adult
adult
adult
13.9
42.8
46.0
65.8
70.6
128.0
71.3
104.0
13.50
17.20
8.90
0.072
0.081
0.125
0.164
0.198
0.253
0.355
0.220
Rat
-
Green monkey
R hesus monkey
100.
The testicular testosterone concentrations showed a wide scatter between 10.8-104
nglg wet weight in green monkey and 71-128 nglg wet weight in rhesus monkey tissue.
There was a correlation between testicular weight and testicular testosterone concentrations in green monkeys (r=0.78; P s 0.05),but not in rhesus monkeys (r=0.68; P> 0.2).
Serum testosterone concentrations in the immature green monkeys were significantly
lower than values measured in the adult rhesus monkeys (P< 0.001).
DISCUSSION
The binding affinities of LHihCG receptors in the two primate species investigated
here were very similar, and there was no apparent change in affinity in the green
monkeys at different developmental stages. Hence it could be postulated that hCGbinding affinities in adult green monkeys would also be similar to those in the adult
Testicular LHlhCG Receptors in Two Monkey Species
289
rhesus monkeys. The binding capacities were, however, significantly lower in rhesus
monkey than green monkey tissue. This may have been due to age differences between
the two populations investigated or it could be an actual species difference. It has been
reported in rats that the affinity constant of hCG receptors does not change with different stages of development but that there is a change in the total number of binding
sites with age [Morris & Sexena, 1980; Pahnke et al, 19751.
The specificity of the receptors as tested here is very similar in green and rhesus
monkeys and is in good agreement with that already presented for rhesus monkeys and
humans [Davies et al, 19791. Both rhesus monkey and human LH/hCG receptors bound
human gonadotropins selectively, whereas they did not cross react with nonprimate hormones. By contrast, rat testicular receptors have been found to be less discriminating
than those of primates; they bind not only homologous gonadotropins, but also those
from rabbits, ovine, porcine, and human gonadotropins [Huhtaniemi & Catt, 19811.Both
Old World monkey species, M. mulatta and C. aethiops show good agreement with
humans.
The testicular and serum levels of testosterone measured in green monkeys were in
good agreement with those from immature rhesus monkeys [Arslan, et al, 19781. The
testosterone concentrations in testis tissue showed great variability and were not
related to hCG binding or serum testosterone levels. In the group of green monkeys, a
correlation between testicular testosterone and testicular weight could be established,
as would be expected. This correlation, however, could not be observed in the rhesus
monkeys, which is probably due the small number of animals.
CONCLUSIONS
1. Testicular tissue from green and rhesus monkeys binds hCG with similar affinity.
2. hCG-binding capacity is lower in the adult rhesus monkey than in the immature green
monkey, probably due to age differences.
3. The specificity of receptor binding is similar in green and rhesus monkeys, and is in
agreement with that reported for humans.
ACKNOWLEDGMENTS
We are indebted to Dr. H. Muller, Landesuntersuchungsamt Munster, for providing
the green monkey tissue. P. Zaidi is the recipient of a scholarship from the Deutscher
Akademischer Austauschdienst (DAAD). The study was supported by the Deutsche
Forschungsgemeinschaft
(Ni 13019). Ms. Ch. Seiler’s and Ms. A. Jung’s secretarial
_ _
assistance is gratefully acknowledged.
REFERENCES
Arslan, M.; Zaidi, A.A.;Qazi, M.H. Effect of gonadotropins and LH-RH on functional differentiation of immature monkey testis. INT E R N A T I O N A L J O U R N A L O F ANDROLOGY (SUPPI) 2:319-328, 1978.
Davies, T.F.; Walsch, C.P.; Hodgen, D.G.;
Dufau, M.L.; Catt, K.J. Characterization of
primate luteinizing hormone receptor in testis
homogenate and Leydig cell. JOURNAL OF
CLINICAL ENDOCRINOLOGY &
METABOLISM 48:680-685,1979.
Dym, M. Morphology of the monkey testis:
Comparison with human and with lower mammalian species, pp. 116-128 in NONHUMAN PRIMATE MODELS FOR STUDY
OF HUMAN REPRODUCTION. T.C. Anand
Kumar, ed. Basel, Karger. 1980.
Geisthovel F.; Brabant, G.; Wickings, E.J.;
Nieschlag, E. Changes in testicular hCGbinding and Leydig cell function in rats
throughout life. HORMONE RESEARCH 14:
47-55, 1981.
Haigh, V.M.; Scott, A. Some radiological
and other factors for assessing age in the
rhesus monkey using animals of known age.
LABORATORY ANIMAL CARE 15:57-73,
1965.
Hsu, A.F.; Stratico, D.; Hosaka, M.; Troen,
P. Studies of the human testis. Properties of
human chorionic gonadotrophin receptors in
adult testis and relation to intratesticular
testosterone concentration. JOURNAL OF
CLINICAL ENDOCRINOLOGY &
METABOLISM 47~529-536,1978.
290
Zaidietal
Huhtaniemi, I.T.; Catt, K.J. Differential
binding affinities of rat testis luteinizing hormone (LH) receptors for human chorionic
gonadotropin, human LH and ovine LH.ENDOCRINOLOGY 108:1931-1938, 1981.
Morris, P.L.; Sexena, B.B. Dose and age dependent effects of Prolactin (Prl) on luteinizing hormone and prolactin binding sites in
r a t leydig cell homogenate. ENDOCINOLOGY 107:1639-1645,1980.
Napier, J.R.; Napier, P.H. A HANDBOOK
OF PRIMATES. p. 100, London, Academic
Press, 1967.
Nieschlag, E.; Loriaux, D.L. Radioimmunoassay for plasma testosterone. ZEITSCHRIFT KLINISCHE CHEMIE UND
KLINISCHE BIOCHEMIE 101164-168,
1972.
Nieschlag, E.; Wickings, E.J. Does the
rhesus monkey provide a suitable model for
human testicular functions? pp. 103-119 in
ANIMAL MODELS IN HUMAN REPRODUCTION. M. Serio, ed. New York, Raven
Press, 1980.
Nieschlag, E.; Tekook, W.; Usadel, K.H.;
Kley, H.K.;Krdskemper, H.L. Testicular testosterone concentration and in-uitro response
to hCG in normal and in testosterone immunized rabbits. STEROIDS 25:379-385,
1975.
Nieschlag, E.; Wickings, E.J.; Mauss, J. Endocrine testicular functions in-viuo and invitro in infertile men. ACTA ENDOCRINOLOGICA 90:544-551,1979.
Pahnke, V.G.; Leidenberger, F.A.; Kunzig,
H.J. Correlation between hCG (LH)-binding
capacity, Leydig cell number and secretory activity of r a t testis throughout pubescence.
ACTA ENDOCRINOLOGICA 79~610-618,
1975.
Scatchard, G. The attraction of proteins for
small molecules and ions. ANNALS OF THE
NEW YORK ACADEMY OF SCIENCES
51:660-672, 1949.
Wickings, E.J.; Qazi, M.H.; Nieschlag, E.
The determination of biologically active LH in
the serum of male rhesus monkeys (Macaca
mulattal. JOURNAL OF REPRODUCTION
AND FERTILITY 57:497-504.1979.
Документ
Категория
Без категории
Просмотров
1
Размер файла
367 Кб
Теги
greek, monkey, rhesus, lhhcg, testicular, characterization, receptors, mulatta, macaca, aethiopsis, comparison, cercopithecus
1/--страниц
Пожаловаться на содержимое документа