Detection of urinary gonadotropins in callitrichid monkeys with a sensitive immunoassay based upan a unique monoclonal antibody.код для вставкиСкачать
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 A -E -. J -E -aa 9 U : a h b co B - E . 2 . I E 0 a 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 -. a f a I- m 2 In B 5- -E I Q 150 4- L a f K 3- I- m 2 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 Bravo, P.W.; Fowler, M.E.; Stabenfeldt, C.H.; Lasley, B.L. Endocrine responses in the llama to copulation. THERIOGENOLOGY 33:891-899, 1990. Brownlee, K.A. Pp. 291-298 in STATISTICALTHEORYANDMETHODOLOGYIN 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 cycle of the golden lion tamarin (Leontopithecus rosuliu rosuliu):Patterns of urinary oestrogen excretion. JOURNAL OF REPRODUCTION AND FERTILITY 75:537546,1985. Hodges, J.K.; Czekala, N.M.; Lasley, B.L. Estrogen and luteinizing hormone secretion in diverse primate species from simplified urinary analysis. JOURNAL OF MEDICAL PRIMATOLOGY 3:349-364, 1979. Matteri, R.L.; Roser, J.F.; Baldwin, D.M.; Lipovetsky, V.; Papkoff, H. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. DOMESTIC ANIMAL ENDOCRINOLOGY 4:157-165,1987. 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. Reproductive performance and excretion of urinary estrogens and gonadotropins in the female pygmy marmoset (Cebuellu pyg~ w u ) AMERICAN . JOURNAL OF PRIMATOLOGY 22~191-203.1990.