Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society

Vol. 46, No. 6 Printed in U.S.A.

Comments Content of Chorionic Gonadotropin in Human Fetal Tissues* ILPO T. HUHTANIEMI,t| CAROL C. K0RENBR0T4 AND ROBERT B. JAFFE§ Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143 ABSTRACT. As part of a study on the physiological role of hCG in the human fetus, the hCG concentrations in homogenates of various fetal tissues were measured using a hCG /? subunit RIA. The mean concentrations (picograms of hCG per mg wet tissue ± SEM; n > 10, unless otherwise indicated) found in human fetuses of 12-20 weeks were: ovary, 46.9 ± 4.3; testis, 8.2 ± 1.7; kidney, 20.3 ± 2.8; thymus, 11.5 ± 1.2; adrenal, 2.6 ± 0.4; lung, 3.4 ± 0.7; liver, 1.8 ± 0.2; spleen, 1.4 ± 0.4 (n = 5); muscle, 2.4 ± 0.8 (n = 6); and meconium, 356 ± 104. That the immunoreactive material measured behaved like hCG was determined by RIA of the supernatants. Parallelism was demonstrated between dilution curves for the tissue homogenates and the hCG standard for all tissues except meconium. A rat Leydig cell in vitro bioassay was used to demonstrate that there was hCG biological activity in the supernatants in ovarian,

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thymic, and renal tissues. The mean ratios of biological to immunological activities were 5.3 in kidney (n = 4), 1.6 in thymus (n = 3), and 1.3 in ovary (n = 2). Blood content of the tissues was determined from measurements of hemoglobin levels and it was found that for the ovary, testis, kidney, and thymus, hCG concentrations were higher than could be explained by the presence of circulating hCG in the tissues. These results, together with our previous results of the binding and effects of hCG in the human fetal testis, support the fact that the fetal testis is a target organ for hCG in the stimulation of steroidogenesis. The presence of high levels of hCG in the ovary, thymus, kidney, and meconium poses questions for further study of the possible physiological role of hCG. (J Clin Endocrinol Metab 46: 994, 1978)

extent to which concentrations of hCG in testicular tissue compare with those of other fetal tissues, we quantitated this hormone in various human fetal tissues.

CTIVE production of hCG throughout L gestation is a characteristic feature of human pregnancy. Relatively little, however, is known about the physiological role of this placental hormone. Although the majority of hCG is secreted from the placenta to the mother, significant concentrations of this hormone also are present in the fetal circulation (1, 2). Recently, we demonstrated that the human fetal testis (16-20 weeks) can bind hCG and respond with maximal testosterone production to physiologic levels of hCG in vitro (3). To determine whether the human fetal testis concentrates hCG in vivo and the

Materials and Methods

Received June 20, 1977. * These studies were supported in part by NIH Grant HD08478 and a grant from The Rockefeller Foundation. f Recipient of NIH International Research Fellowship F05-TW-2243. Postdoctoral Fellow in Reproductive Endocrinology. Present address: Department of Clinical Chemistry, University of Oulu, SF-90220 Oulu 22, Finland. | Postdoctoral Fellow in Reproductive Endocrinology. § To whom requests for reprints should be addressed.

Human fetuses (12-20 weeks gestation) were obtained from prostaglandin-induced abortions. Ovary, testis, thymus, adrenal, kidney, and lung tissues were removed as rapidly as possible, usually within 30-120 min after delivery. Meconium was obtained by removing the intestine and emptying its contents with a blunt instrument pressed and drawn along its length. The dissected tissues were placed on ice, weighed, and homogenized in a glass homogenizer in 10 vol (1 g/10 ml) 0.1 N NaCl acidified with HC1 (pH 2.3). Homogenates were then incubated in a Dubnoff metabolic incubator for 16 h at 4 C with continuous shaking. The samples were centrifuged at 3000 X g for 10 min. Duplicate 200-jul aliquots of each supernatant were removed and analyzed for hCG with an hCG ft subunit RIA (courtesy of

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COMMENTS National Pituitary Agency, NIAMDD; antiserum /?hCG-SB6, hCG for iodination batch CR 119, 10,000 IU/mg). The reference standard was the Second International Reference Preparation of hCG obtained from the WHO. Extracts of kidney, thymus, and ovary also were analyzed with an in vitro Leydig cell LH-hCG bioassay (4). In these cases, the homogenization and subsequent incubation took place in 50 mM Tris-HCl buffer (pH 7.0). Serial dilutions of meconium, ovary, testis, kidney, and thymus extracts were analyzed in the hCG /? subunit RIA. The inhibition curves generated were tested for parallelism with the hCG standard by the method of Rodbard (5). Immunological similarity was considered to be present when the lines were parallel (6). Blood content of fetal tissues was measured according to Hohorst et al. (7). The tissues were homogenized in hemolysate reagent (Helena Laboratories, Beaumont, TX), and the spectrophotometric measurements were carried out with a Carey 118 spectrophotometer.

Results As seen in Fig. 1, the slopes of RIA dilution curves for saline extracts of fetal ovarian, testicular, renal, and thymic tissues were indistinguishable from that of the diluted hCG standard, according to the statistical criteria

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described by Rodbard (5). The dilution curve for meconium appeared biphasic. The recovery of hCG from the tissue homogenates was assessed by adding known amounts of highly purified hCG (CR 119) to the homogenates. Nearly 100% of this exogenous hormone was measured by RIA in each tissue tested. To determine whether the hCG immunoactivity measured in the tissue extracts was associated with biological activity, extracts of kidneys (n = 4), thymuses (n = 3), and ovaries (n = 2) were analyzed individually with the rat Leydig cell LH-hCG in vitro bioassay. In these cases, tissue extraction was performed at pH 7, as low pH was found to decrease the biological LH-hCG activity of the homogenate, perhaps due to the dissociation of the a and /? subunits of glycoprotein hormones (8). The low pH did not have any effect on immunological activity measured with the hCG /? subunit RIA. The ratios of biological to immunological hCG activity varied in the kidneys from 3.74-6.89 (mean, 5.3), in the thymuses from 1.45-1.64 (mean, 1.6), and in the two ovaries the ratios were 1.15 and 1.51 (mean, 1.3). The concentrations (picograms of hCG per mg wet tissue ± SEM) detected in different

95 90 80

2 60 thymus

a 403020kidney 10 5

0.1

hCG standard

1

10

100

1000

pi (tissue extract) or ng (hCG standard)

FIG. 1. RIA dilution curves for the hCG reference preparation (Second International Reference Preparation, WHO) and extracts of human fetal meconium, ovary, testis, kidney, and thymus.

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COMMENTS

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However, other functions of hCG in pregnancy, particularly in the fetus, remain largely unexplored. As hCG reaches peak circulating levels at a time of gestation (10-12 weeks) just before circulating male fetal testosterone lev40 els reach a peak (12-14 weeks) (2, 9), the role of hCG in stimulating fetal testicular testosterone has been postulated (10,11). Our recent studies demonstrating that physiologic concentrations of hCG can stimulate fetal testicular testosterone production in vitro and that human fetal testes can specifically bind hCG support this thesis (3). It also has been postulated that hCG may stimulate dehydroU epiandrosterone sulfate production by the feJC tal adrenal gland, and our recent superfusion _L 10 studies have demonstrated that this can occur J_ in the fetal zone of the fetal adrenal gland (12, 13). In the current study, an hCG /? subunit RIA IJ ftl rti Ll was used to measure immunoreactive hCG in various tissues from fetuses at 12-20 weeks FIG. 2. Concentration of hCG in human fetal tissues (pi- gestational age. To facilitate the dissociation cograms per mg wet tissue) measured with an hCG fi of hCG from its possible receptors, a low pH subunit RIA (mean ± SEM). The number of individual was used in the homogenization and extracsamples is indicated in the lower portion of each bar. The tion of hCG from tissue samples (14). Paralgestational ages of the fetuses varied between 12-20 lelism of dilution curves of the immunoreacweeks. tive hCG /? subunit from a variety of these fetal tissues are shown in Fig. 2. The highest tissues indicated that the material measured concentration per wet weight was found in was not heterogeneous by this RIA, except meconium, where it was 356 ± 104 (SEM) perhaps in meconium. Thus, although there is pg/mg. partial dissociation of the a and /3 subunit at As it is possible that the hCG concentration low pH during the extraction, there is evimeasured could result from the hormone pres- dently a reassociation of the subunits at the ent in the blood in the tissue, the blood con- neutral pH used in the RIA (15). tent of fetal tissues was measured. The blood The biological potency of the supernatants content of fetal parenchymal tissues (includ- of fetal kidney, thymus, and ovary homogeing kidney, lung, thymus, adrenal, ovary, and nates was demonstrated in a rat Leydig cell testis) varied between 3-7%. The hCG concen- LH in vitro bioassay. In each tissue analyzed, tration in the serum of 12-20-week-old fetuses the biological LH-hCG activity exceeded that is about 50 ng/ml (2), which would yield tissue measured in the RIA. The ratios of biological concentrations of 2.0-4.5 pg/mg. Thus, it is to immunological activities were high in kidlikely that the hCG detected in the fetal lung, ney (6.4), while lower and essentially the same liver, spleen, muscle, and adrenal was a reflec- for ovary and thymus (1.3, 1.6, respectively). tion of the blood in these tissues. However, The reasons for the differences are not apparthis cannot explain the hCG concentrations of ent and await further study. the ovary, kidney, thymus, and testis. The presence of concentrations of hCG in the fetal testis above that in the circulation is Discussion not surprising and is probably associated with hCG has a role in the stimulation of mater- testosterone production involved in genital denal ovarian steroidogenesis in early gestation. velopment. The ability of the human fetal 50

I

ft

1?

11

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COMMENTS testis to bind hCG and produce testosterone in response to physiologic concentrations of hCG in vitro (3) corroborates the suggestion that the human fetal testis, at least during the first half of gestation, is a target organ for hCG. The significance of high hCG concentrations in the kidney, ovary, thymus, and meconium awaits further investigation. As hCG is excreted via the kidneys in the adult, high concentrations of hCG in amniotic fluid (2) suggest that fetal kidneys also may excrete hCG. Fetal ovarian steroidogenesis has not been found to respond to hCG stimulation, although the capacity for androgen formation by the ovary is demonstrable by 12 weeks of gestation (16). The presence of hCG in the thymus raises the question of the possible connection of hCG with the development of the immune response (17, 18). However, whether the ovary and thymus are target organs of hCG or whether hCG is synthesized by these tissues remains unexplored. hCG is, like a-fetoprotein and carcinoembryonic antigen, one of the "tumor marker proteins" (19, 20). The latter two proteins are synthesized not only by tumor cells, but by a variety of normal embryonic cells as well. Thus, it is possible that fetal tissues other than the trophoblast may also possess the capacity of synthesizing hCG during fetal development. hCG may well have a broader physiological role in fetal development than has yet been acknowledged. The presence of significant amounts of hCG in a variety of fetal tissues raises possibilities for new roles of hCG in pregnancy. Acknowledgment We would like to thank Ms. Donna J. Piper for the hCG 0 subunit RIA measurements.

References 1. BRUNER, J. A., Distribution of chorionic gonadotropin in mother and fetus at various stages of pregnancy, J Clin Endocrinol Metab 11: 360, 1951.

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2. CLEMENTS, J. A., F. I. REYES, J. S. D. WINTER, and C.

FAIMAN, Studies on human sexual development. III. Fetal pituitary and serum, and amniotic fluid concentrations of LH, CG, and FSH, J Clin Endocrinol Metab 42: 9, 1976. 3. HUHTANIEMI, I. T., C. C. KORENBROT, and R. B. JAFFE, I.

hCG binding and stimulation of testosterone biosynthesis in the human fetal testis, J Clin Endocrinol Metab 44: 963, 1977. 4. MOYLE, W. R., and J. RAMACHANDRAN, Effect of LH on

steroidogenesis and cyclic AMP accumulation in rat Leydig cell preparations and mouse tumor Leydig cells, Endocrinology 93: 127, 1973. 5. RODBARD, D., Statistical aspects of radioimmunoassays, In Odell, W. D., and W. H. Daughaday (eds.), Principles of Competitive Protein-Binding Assays, Philadelphia, J. B. Lippincott Co., 1971, p. 204. 6. Ross, G. T., Factors governing choice of reference preparation for competitive binding assay, In Odell, W. D., and W. H. Daughaday (eds.), Philadelphia, J. B. Lippincott Co., 1971, p. 325. 7. HOHORST, H. J., F. H. KREUTZ, and T H . BUCHER, Uber

Metabolitgehalte und Metabolit-Konzentrationen in der Leber der Ratte, Biochem Z 332: 18, 1959. 8. PAPKOFF, H., and T. S. A. SAMY, Isolation and partial characterization of the polypeptide chains of ovine interstitial cell stimulating hormone, Biochim Biophys Ada 147: 175, 1967. 9. WINTER, J. S. D., I. A. HUGHES, F. I. REYES, and C. FAIMAN,

Pituitary-gonadal relations in infancy. II. Patterns of serum gonadal steroid concentrations in man from birth to two years of age, J Clin Endocrinol Metab 42: 679, 1976. 10. AHLUWAUA, B., J. WILLIAMS, and P. VERMA, In vitro testos-

terone biosynthesis in the human fetal testis. II. Stimulation by cyclic AMP and human chorionic gonadotropin (hCG), Endocrinology 95: 1411, 1974. 11. ABRAMOVICH, D. R., R. G. BAKER, and P. NEAL, Effect of

human chorionic gonadotropin on testosterone secretion by the foetal human testis in organ culture, J Endocrinol 60: 179, 1974. 12. JAFFE, R. B., M. SERON-FERRE, I. HUHTANIEMI, and C.

KORENBROT, Regulation of the primate fetal adrenal gland and testis in vitro and in vivo, J Steroid Biochem 8: 479, 1977. 13. SERON-FERRE, M., C. C. LAWRENCE, and R. B. JAFFE, Role

of hCG in the regulation of the fetal zone of the human fetal adrenal gland, Gynecol Invest 8: 64, 1977 (Abstract 87). 14. CATT, K. J., and M. L. DUFAU, Interactions of LH and hCG

with testicular gonadotropin receptors, In O'Malley, B., and A. Means (eds.), Receptors for Reproductive Hormones, New York, Plenum Press, 1973, p. 379. 15. GARNIER, J., R. SALESSE, and C. PERNOLLET, Reversible

folding of human chorionic gonadotropin at acid pH or upon recombination of the a and /? subunits, FEBS Lett 45: 166, 1974. 16. PAYNE, A. H., and R. B. JAFFE, Androgen formation from pregnenolone sulfate by the human fetal ovary, J Clin Endocrinol Metab 39: 300, 1974. 17. CONTRACTOR, S. I., and H. DAVIES, Effect of human chorionic somatomammotrophin and human chorionic gonadotrophin on phytohaemagglutinin-induced lymphocyte transformation, Nature [New Biol\ 243: 2§4, 1973. 18. CARTER, J., The effect of progesterone, oestradiol and hCG on cell-mediated immunity of pregnant mice, J Reprod Fertil 46: 211, 1976. 19. BRAUNSTEIN, G. D., J. L. VAITUKAITIS, P. P. CARBONE, and

G. T. Ross, Ectopic production of human chorionic gonadotrophin by neoplasms, Ann Intern Med 78: 39, 1973. 20. GOLDSTEIN, D. P., Chorionic gonadotropin, Cancer 38: 453, 1976.

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Content of chorionic gonadotropin in human fetal tissues.

Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society Vol. 46, No. 6 Printed in U.S.A. Comments Content of Chor...
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