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 ),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. 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