Radioimmunoassay of Rhesus Monkey Chorionic Gonadotropin*

Vol. 26, No. 1, January 1975 Prinred in U.SA.

FERTILITY AND STERILITY Copyright<> 1975 The American Fertility Society

RADIOIMMUNOASSAY OF RHESUS MONKEY CHORIONIC GONADOTROPIN* WILLIAM HOBSON, PH.D.,t CHARLES FAIMAN, M.D.,t WILLIAM J. DOUGHERTY, PH.D.,t FRANCISCO I. REYES, M.D.,t AND JEREMY S. D. WINTER, M.DJ International Center of Environmental Safety, Albany Medical College, Hollo~n Air Force Base, New Mexico 88330, and Departme'}-ts of Phy~iology, ~ed~trics, and Obstetncs and Gynecology, Universlty of Mamtoba, Wmmpeg, Canada

Bioassay of urine and serum from pregnant rhesus monkeys (Macaca mulatta) has shown that monkey chorionic gonadotropin (mCG) is secreted only during the first trimester of pregnancy. 14 However, humans5 and chimpanzees6 •7 secrete this ·hormone throughout pregnancy. Although radioimmunoassay techniques have largely supplanted the bioassay of chorionic gonadotropin in humans and in chimpanzees, no radioimmunoassay techniques for mCG have yet been published. The greater sensitivity of radioimmunoassay for mCG in rhesus monkeys should permit earlier diagnosis of pregnancy and enable a closer study of mCG levels during late pregnancy. Experience in this and other laboratories indicates that consistently accurate pregnancy diagnoses cannot be made from bioassays of urine or blood samples collected before day 20, although pregnancy diagnosis by bioassay of mCG in rhesus monkeys is occasionally possible by day 17 of pregnancy (day 14 sample plus 3 days for assay). An accurate means of earlier pregnancy diagnosis would greatly benefit teratologic studies and animal husbandry techniques utilizing primates. This communication describes the development of radioimmunoassay for mCG,

its subsequent adaptation for pregnancy diagnosis, and data on serum levels of mCG during pregnancy in rhesus monkeys. MATERIALS AND METHODS

Animals. Mature female rhesus monkeys were individually caged and allowed free access to water and food. Menstrual cycle records, based on daily vaginal swabbing, were maintained. Females were mated for a 48-hour period betweec. day 7 and 13 of the menstrual cycle (day 1 = first day of menstruation). The specific day for mating was determined by the length of the previous menstrual cycle, according to the method of van Wagenen. 8 Samples. All blood samples were collected from the brachial vein during manual restraint. Blood samples (5 ml) for definition of the mCG peak were collected on days 10, 12, 14, 16, 19, 22, 25, 31, 35, 38, 41 (after mating), and randomly throughout the remainder of pregnancy. Blood samples (2 ml) for pregnancy diagnosis were collected on day 17 and 19 after mating. All blood samples were allowed to clot, they were centrifuged, and the serum was separated. Samples for pregnancy diagnosis were assayed immediately; samples for the Received March 19, 1974. mCG study were frozen (- 200 C) for later *Supported in part by Institut fiir Oekologische Chemie d. Gessellschaft fiir Strahlen-und Um- analysis. Monkey chorionic gonadotropin radioweltforschung mbH, Munich, Germany. immunoassay. The antiserum (GDN #15) t Albany Medical College. against ovine luteinizing hormone (LH) :!:University of Manitoba. 93

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and the procedures used are those described by Niswender et al, 9 with the following modifications: 1.5% normal rabbit serum (NRS)-barbital buffer (0.07M, pH 8.6)-was used as the diluent; the second antibody was at a final concentration of 6%. Ovine LH (LER-1056C2) was iodinated with either 125I or 131 I according to the method of Greenwood et al. 10 A partially purified e:· tract of mCG was used as assay standar1 Results were converted to units of tl: Second International Standard of huma chorionic gonadotropin (hCG); the mO standard equaled 41 IU/ml. 11 Assays were evaluated by the quality control system of Rodbard et aP 2 For pregnancy diagnosis, the reagents described by Niswender et al 9 were used but the concentrations and incubation times were changed as follows: NRS concentration was increased to 4%; the assays were conducted entirely at room temperature; unknown and labeled hormone were added at the same time and incubated for 12 hours; and the second antibody was added and incubated for four hours. Control samples from pregnant and nonpregnant animals were included in each assay. Diagnosis of pregnancy was based on comparison of unknown samples with pregnant and nonpregnant control values. Radioimmunoassay results were compared with diagnoses obtained by bioassay using a mouse uterine weight method. 1

VOLUME PREGNANCY SERUM (ml)

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1.0 OVINE LH ln9)

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mCG (miU)

FIG. 1. Comparison of standard curves obtained using ovine LH (NIH-LH-S16 standard) or a partially purified preparation of mCG with dilutions of serum from a pregnant monkey. MCG units are expressed in terms of the Second International Standard of hCG.

It was previously demonstrated9 that this antiserum does not cross-react with thyroid-stimulating hormone (TSH) or follicle-stimulating hormone (FSH) or rhesus pituitary origin. However, both rhesus LH and mCG depress the binding oflabeled hormone. Results are expressed as mCG on the assumption that LH levels are low throughout pregnancy. Similar assumptions are indeed made for most hCG radioimmunoassay systems. The sensitivity of the assay, expressed as the smallest amount significantly different from zero, was approximately 2 miU. The coefficient of variation between duplicate determinations at low, medium, and high levels ranged from 7% to 10%.

Serum levels. Serum levels of mCG began to rise rapidly at about day 16 of pregnancy, reached a peak at day 25, and declined thereafter, reaching base line levels by day 35 (Fig. 2). Levels of mCG RESULTS became detectable in a few monkeys as Monkey chorionic gonadotropin radio- early as day 12; however, mCG was not immunoassay. Initial efforts to develop detectable in all pregnant monkeys until a radioimmunoassay for mCG using ovine day 16. Considerable variation was noted LH (NIH-LH-S16) for standards resulted in the maximum levels of mCG reached in significant nonparallelism between during early pregnancy in these monkeys dilutions of serum from pregnant rhesus (800 to 3,660 miU/ml). Analysis of monkeys and standards (Fig. 1). How- samples taken randomly after day 40 of ever, when mCG was used as a standard, pregnancy failed to measure mCG condilutions of pregnancy serum and stand- centrations which were distinguishable ards gave parallel inhibition curves. from menstrual cycle LH values.

BOO

By day 17 of pregnancy, mCG levels were elevated so that there was no difficulty in distinguishing them from ovulatory LH peak levels. Thus, routine diagnosis of pregnancy is now done in this colony on samples collected on day 17.

400

DISCUSSION

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0

20

40

60

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120

DAYS POST MATING

FIG. 2. Serum levels ofmCG during pregnancy in rhesus monkeys. Vertical bars represent standard errors of the mean. Dotted lines connect values which were not distinguishable from basal levels ofLH.

Pregnancy diagnosis. The shortened version of the mCG radioimmunoassay proved to be a simple and reliable method for diagnosis of pregnancy. Comparison of the radioimmunoassay method with the mouse uterine weight method on duplicate day 19 samples revealed that the radioimmunoassay could detect pregnancies that were missed by the bioassay (Table 1). Although a few pregnancies were diagnosed as early as day 12 after mating, the radioimmunoassay did not correctly diagnose all pregnancies until day 16. A false-positive diagnosis of pregnancy could be obtained before day 16 when a sample was collected from a nonpregnant monkey during the ovulatory surge of LH at midcycle. Since the ovulatory LH peak is transitory, 13 an assay on a second sample taken 48 hours later eliminated false-positive diagnoses. TABLE 1. Comparison of Radioimmunoassay and Mouse Uterine Weight Methods of Pregnancy Diagnosis on Serum Samples Collected 19 Days After Mating RadioMouse uterine immunoD_I_·agn_os_is_"_ _ _ _ _ _ _ _ _ _ w_ei:_gh_t_assay

Pregnant Not pregnant Incorrectly diagnosed pregnant Incorrectly diagnosed not pregnant

29 72 0 7

36 65 0 0

•Positive pregnancy diagnoses were confirmed by palpation or subsequent birth or abortion.

The radioimmunoassay for mCG presented in this paper is sensitive, accurate, and reproducible. The unique antiserum (GDN#15) has also been used for radioimmunoassay of LH in several species and has been shown not to cross-react with rhesus FSH or TSH. 9 Thus, in the monkey, it is specific for CG or LH. Monkey chorionic gonadotropin has been shown to have antigenic determinants in common with hCG. Not only can mCG biologic activity be neutralized by prior treatment with anti-hCG antisera, 14- 16 but mCG can compete in some radioimmunoassay systems for hCG. 14·17 Nevertheless, hCG radioimmunoassay characteristics, such as inadequate sensitivity17 or only partial cross-reactivity15·16 may preclude their practical use for the measurement of mCG in serum. In addition, many antisera directed against hLH or hCG18-20 fail to recognize rhesus LH or CG at all. Results obtained from this radioimmunoassay for serum mCG levels during early pregnancy are in agreement with those obtained from bioassay. 1·3 Both methods indicate that mCG begins to increase by day 12 to 16 of pregnancy, reaches a peak at about day 25, and declines to basal levels by day 35. Mean peak serum concentrations of mCG measured by radioimmunoassay in this study (1367 ± 262 miU/ml) are lower than, but within the range of, those measured by bioassay (1000 to 8000 miU/ml) 1·3 In general, studies utilizing bioassay methods have reported nondetectable levels of mCG in serum and urine beyond day 35 of pregnancy1·4; however, in

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one study 21 low levels of mCG, measured by bioassay of pooled urine extracts from a single monkey, were observed during late pregnancy. However, it is possible that the method was detecting basal LH levels. The results of the present experiment support this contention since LH-mCG concentrations measured by radioimmunoassay were not different from LH levels found in samples taken from nonpregnant monkeys during the follicular or luteal phases of their menstrual cycles. 22 Consequently, it appears that rhesus monkeys, unlike other higher primate species, do not secrete significant amounts of chorionic gonadotropin during the last two trimesters of pregnancy. A reliable method for the diagnosis of pregnancy in rhesus monkeys shortly after implantation greatly increases the value of this species for use in teratologic studies. It is now possible to expose pregnant females to possible teratagens earlier, and at a precise time of fetal organogenesis. Thus, risks of exposure to these compounds during early pregnancy can be more accurately assessed, and waste of experimental animals can be avoided. This method also affords definite advantages for husbandry and breeding in primate colonies. In addition to the benefits of earlier and more accurate pregnancy detection, it reduces both sampling amounts and assay costs. Moreover, preliminary results indicate that a similar mLH assay may be valuable for increasing fertility by estimating the optimum time for mating based on detection of the preovulatory LH peak.

nancy indicated that mCG began increasing as early as day 12 after mating, reached a peak by day 25, and declined to nondetectable levels around day 35. The assay procedure was adapted for use as a rapid method for pregnancy diagnosis; the results were available as early as 12 hours after collection of serum samples. The method is capable of detecting a few pregnancies by day 12 and all pregnancies by day 17. Routine use of this method provided accurate pregnancy diagnosis four days earlier than was possible with the mouse uterine weight bioassay method previously used in this laboratory. Acknowledgments. We are grateful for: the antiserum from Dr. Gordon Niswender; the mCG standard from Dr. W. D. Peckham; the ovine LH for iodination from Dr. Leo Reichert; the NIH-LHS16 from NIAMDD; the technical assistance of G. Salazar; and, the editorial assistance of J. Lowry. REFERENCES 1. Tullner WW, Hertz R: Chorionic gonadotropin

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3.

4.

5.

SUMMARY

A radioimmunoassay for rhesus monkey chorionic gonadotropin (mCG) employing an antiovine LH antiserum, 125! or 131 !-ovine LH tracer, and mCG for standards was developed. Radioimmunoassay of serum levels of mCG during preg-

January 1975

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levels in the rhesus monkeys during early pregnancy. Endocrinology 78:204, 1966 Hodgen GD, Dufau ML, Catt KJ, et al: Estrogens progesterone and chorionic gonadotropin in pregnant rhesus monkeys. Endocrinology 91: 896, 1972 Arslan M, Meyer RK, WolfRC: Chorionic gonadotropin in the blood and urine of pregnant rhesus monkeys (Macaca mulatta). Proc Soc Exp Bioi Med 125:349, 1967 van Wagenen G, Simpson ME: Gonadotropin hormone excretion of the pregnant monkey (Macaca mulatta). Proc Soc Exp Bioi Med 90: 346, 1955 Borth R Jr: Chorionic gonadotropin. In Endocrinology of Pregnancy. Edited by F Fuchs, A Klopper. New York, Harper and Row, 1971, p 16 gonadotropin prolactin, estrogen and progesterone levels throughout the menstrual cycle and pregnancy in the chimpanzee. Endocrinology (In press) Nixon WE, Hodgen GD, Neimann WH, et al: Urinary chorionic gonadotropin in middle and late pregnancy in the chimpanzee. Endocrinology 90:1105, 1972 van Wagenen G: Mating in relation to pregnancy in the monkey. Yale J Bioi Med 17: 745, 1945

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9. Niswender GD, Monroe SE, Peckham WD, et al: Radioimmunoassay for rhesus monkey luteinizing hormone (LH) with anti-ovine LH serum and ovine LH- 131 1. Endocrinology 88: 1327, 1971 10. Greenwood FC, Hunter WM, Glover JS: The preparation of 1311-labelled human growth hormone of high specific radioactivity. Biochem J 89:114, 1963 11. Peckham WD: Personal communication 12. Rodbard D, Rayford PL, Cooper JA, et al: Statistical quality control of radioimmunoassays. J Clin Endocrinol 28:1412, 1968 13. Weick RF, Dierschke KJ, Karsch JF, et al: Periovulatory time courses of circulating gonadotropic and ovarian hormones in the rhesus monkey. Endocrinology 93:1140, 1973 14. Tullner WW, Rayford PL, Ross GT: Evidence for similar antigenic determinants in gonadotropins from urine of man and monkey (Macaca mulatta). Endocrinology 84:908, 1969 15. Nixon WE, Tullner WW, Rayford PL, et al: Similarity of antigenic determinants in pituitary and chorionic gonadotropin from primates.

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Endocrinology 88:702, 1971 16. Bagshawe KD, Orr AH, Godden J: Crossreaction in radioimmunoassay between human chorionic gonadotropin and plasma from various species. J Endocrinol 42:513, 1968 17. Hobson B, Wide L: A comparison between chorionic gonadotropin extracted from human, rhesus monkey and marmoset placentae. J Endocrinol 55:363, 1972 18. Neill JD, Peckham WD, Knobil E: Apparent absence of immunological cross-reactivity between human and simian gonadotropic hormones as determined by radioimmunoassay. Nature 213:1014, 1967 19. Faiman C, Ryan RJ, Greslin JG, et al: Species specificity of FSH and LH as determined by radioimmunoassay. Proc Soc Exp Bioi Med 125:1232, 1967 20. Faiman C: Unpublished data 21. Hobson BM: New observations on the excretion of chorionic gonadotropin during pregnancy in the rhesus monkey (Macaca mulatta). Folia Primato 18:463, 1972 22. Hobson W, Faiman C: Unpublished data