Thyroid-stimulating activity of human chorionic gonadotropin in sera of normal pregnant


Masateru Horimoto, Norio Yoshikawa, Yorihiko Horikoshi, Isamu Sawaragi and Mitsuo Inada

Masayoshi Yoshimura, Mitsushige Nishikawa, Susumu Second Department


of Internal Medicine' and Department of Obstetrics and Gynecology2, Kansai Medical University, Moriguchi, Osaka, Japan

Abstract. To ascertain the thyrotropic activity of human gonadotropin in sera of normal pregnant


women, we examined the adenylate cyclase activation in the cultured FRTL-5 cells by extracted hCG from 7 normal pregnant women. hCG was extracted from the sera using anti-hCG-\g=b\ subunit monoclonal antibodycoated microwells, eluted with 2 mol/l guanidine-HCl, and reconstituted with hypotonic Hanks' solution. FRTL-5 cells were precultured in 5H medium, incubated for 2 h with the serum extracts, and the cAMP released into the medium was measured. hCG levels in serum extracts ranged from 1100 to 6800 IU/l; values corresponded to 1.4-19.8% compared with those in the original serum samples. Addition of the extracts to FRTL-5 cells resulted in significant increases in the cAMP accumulation, ranging from 9.8 to 59.0 nmol/l. cAMP levels were also increased in a dose-dependent manner by adding purified hCG as well as crude hCG and hTSH to FRTL-5 cells. These findings suggest that the thyroid gland of normal pregnant women may actually be stimulated by hCG itself.

Human chorionic

gonadotropin has been claimed thyroid-stimulating agent in patients with hydatidiform mole (1,2) or choriocarcinoma (3-6). Moreover, some investigators reported that in normal pregnancy TSH suppression temporarily




correlated with hCG elevation (7,8), and that free or free T3 levels were significantly elevated at the time when hCG levels were maximal (8-11). It was also reported that the thyroid gland of preg¬ nant women was truely stimulated, as evidenced by increasing thyroid volume and serum thyroglobu-

lin levels

(12,13). It seems well established that the stimulation thyroid during normal pregnancy is most likely not due to TSH since supersensitive serum TSH levels are lower or at least not increased during pregnancy as compared with post partum (13,14). Using the supernatant of polyethylene glycol precipitation of serum, we have recently re¬ ported that there exists thyroid-stimulating activity in sera of normal pregnant women (15). The ac¬ tivities showed a significant correlation with the serum hCG levels (15). Therefore, it was conceiv¬ able from these results that hCG has a thyrotropic activity in normal pregnancy. In the present study, we extracted hCG from sera of normal pregnant women using anti-hCG-ß subunit monoclonal antibody, and examined the thyrotropic activity therein to ascertain whether hCG in normal pregnant serum can really stimu¬ late the thyroid. In addition, we also examined the cAMP accumulation induced by purified hCG, and compared it with that induced by crude hCG or hCG-ß subunit as well as by hTSH.

Subjects and Methods





obtained from 7 normal pregnant

(5 in the first, 1 in the second, and 1 in the third trimester; age range 25-32 years). None of the women women


goitre or signs of hyperthyroidism.



of hCG

In advance of the extraction of hCG, serum was pretreated by applying 5 ml to an anti-human TSH-ß subunitcoated plastic tube (RIA-gnost human TSH kit, Hoechst, Tokyo, Japan) to remove TSH. The tubes were agitated overnight at room temperature. Extraction of hCG from the sera of normal pregnant women was performed by employing the anti-hCG-ß subunit monoclonal antibodycoated microwell used in the Delfia hCG kit (Pharmacia

Co, Tokyo, Japan). Each sample, 250 (il per well,


into 18 antibody-coated microwells. The microwells were then incubated with agitation for an hour at room temperature. After decanting the serum, wells were washed twice with 500 (il of 150 mmol/1 NaCl. The hCG bound to the antibody was eluted by adding 250 (il of 2 mol/1 guanidine-HCl solution (pH 3.2). The eluates were collected so that in 5 of the samples, the 18 aliquot-por¬ tions were grouped together, whereas the remaining sam¬ ples were divided into groups of 3, 6, and 9 wells. They were immediately dialysed against 10 mmol/1 phosphate buffer containing 150 mmol/1 NaCl (pH 7.4) for 4 h at 4°C, and again against distilled water overnight at 4°C (16). After the dialysates had been lyophilized, they were reconstituted with 800 (il of Hanks' solution without NaCl containing 1.5% (w/v) bovine serum albumin, 20 mmol/1 HEPES, and 0.5 mmol/1 3-isobutyl-l-methylxanthine (modified Hanks' medium) (17).


cAMP assay The cloned rat thyroid cell line FRTL-5, established from

Effects of hCG and TSH on cAMP accumulation hTSH, ranging from 10~'° to 5xl0"9 mol/1,

in¬ creased cAMP accumulation in the culture medium from 13.0 to 98.7 nmol/1, and reached a plateau at 10"8 mol/1 of hTSH. When crude or purified hCG was added, cAMP release also increased in a dosedependent manner. The cAMP response to com¬ mercial hCG was about twice the response to pu¬ rified hCG at the same concentration (Fig. 1). Ad¬ dition of hCG-ß subunit, however, showed no in¬ creases in the cAMP levels (Fig. 1). In a comparison of the effects of hTSH and purified hCG on cAMP accumulation on a molar basis, the potency of pu¬ rified hCG was less than one per cent of hTSH (Fig. 2). Addition of increasing concentrations of crude hCG (5000-100 000 IU/1) with 5 mU/1 of hTSH showed an additive effect on cAMP accumulation. In contrast, as shown in Fig. 3, the TSH-induced cAMP accumulation was inhibited by addition of more than 50 000 IU/1 of crude hCG with the suf¬ ficient dose of hTSH (500 mU/1).

Extraction of hCG from serum The hCG levels in the sera of normal pregnant women ranged from 31 000 to 110 000 IU/1. After the incubation with anti-hCG-ß subunit monoclo-

by Ambesi-Impiombato et al. (18), grown previously described (19). The cells (5 x 105 cells/well) were pre-incubated for 7 days in 5H medium, and further incubated in duplicate for 2 h at 37°C with varying concentrations of hTSH, hCG or serum extracts cells of





in 300 |il of modified Hanks' medium. After the incuba¬ tion, the cAMP in the incubation medium was measured by using a commercial RIA kit (Yamasa Shoyu Chemical



Thyroid-stimulating activity of human chorionic gonadotropin in sera of normal pregnant women.

To ascertain the thyrotropic activity of human chorionic gonadotropin in sera of normal pregnant women, we examined the adenylate cyclase activation i...
408KB Sizes 0 Downloads 0 Views