Shimpei Tojo

Structures of the Asparagine-Linked Sugar Chains of Human Chorionic Gonadotropin

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.

The biochemical properties of urinary human chorionic gonadotropin from the patients with trophoblastic diseases

Human chorionic gonadotropin (hCG) was extracted and purified from urine of normal pregnant women and patients with hydatidiform mole and choriocarcinoma using the same methods. Both hCG-hydatidiform mole and hCG-choriocarcinoma as well as hCG-normal pregnancy were separated into α and β subunits by SDS disc electrophoresis upon treatment with 2-mercaptoethanol and showed the same immunoreactivities against anti-hCG, -αhCG, and -β hCG as hCG in each radioimmunoassay. In vivo bioassay, bioactivities of hCG-normal pregnancy and hCG-hydatidiform mole were approximately 7, 000 IU/mg (2nd IS), while that of hCG-chorio-carcinoma was only 400 IU/mg. Conversely, the receptor binding activities in vitro of hCG-chorio-carcinoma was about 3 times more effective than the other 2. Although the amino acid composition of these hCG preparations were practically identical, a great difference in the carbohydrate composition was observed. The significant difference was that while sialic acid was undetectable in hCG-choriocarcinoma approximately 8.5% of sialic acid was found in hCG-normal pregnancy and hCG-hydatidiform mole. A parallel finding was that iodinated hCG-choriocarcinoma was taken up in large quantities by the liver in comparison to the ovary which differed from that observed with hCG-normal pregnancy and hCG-hydatidiform mole in Parlow rats. The present findings support the thesis that neoplastic or malignant transformation of trophoblasts may result in an alteration of the glycosylation process, especially the sialylation, in the biosynthesis of hCG rather than the translation steps.

“Triggering” of Ovulation after Infusion of Synthetic Luteinizing Hormone Releasing Factor (LRF)

Abstract. Synthetic luteinizing hormone releasing factor (LRF) was infused intravenously for 6 hours into two women who had donor insemination. Supplemental LRF was injected subcutaneously after a 6 hours continuous infusion. Ovulation was triggered by LRF infusion and two successful pregnancies were achieved. It is suggested that ovulation might be triggered by a continuous intravenous infusion of synthetic LRF, if follicular development of the ovary is sufficient. It is concluded that “triggering” of ovulation by a continuous infusion of LRF might be a convenient means for controlling the timing of ovulation in case of donor insemination.

Production and Secretion of hCG and hCG Subunits by Trophoblastic Tissue

Advanced techniques have brought great success in purification and characterization of glycoprotein hormones. The human glycoprotein hormones, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and human chorionic gonadotropin (hCG) are all composed of a protein core derived from two non-identical alpha and beta subunits, with branched carbohydrate side chains radically attached to asparagine, serines, and/or threonine. In contrast to the essentially identical or nearly identical amino acid sequence among the α-subunits, the biological properties of native hormones are greatly contributed by the β-subunit (which also carries specific antigenic sites) and are structurally unique for each species. Antisera generated to the β-subunit of hCG discriminate comparatively well between hLH and hCG, while most of those derived from intact hormones do not (Amir, 1972).

Ovarian response to exogenously administered human gonadotropins during the postpartum period

Eighteen normal puerperal women received a combined administration of human menopausal gonadotropin (HMG) and human chorionic gonadotropin (HCG) and the ovarian response to these human gonadotropins was evaluated by the daily estimation of the 24 hour urinary excretion of total estrogens. Fourteen of the 18 subjects studied were responsive to the exogenously administered gonadotropins with a rise in the urinary estrogen excretion. Moreover, plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels during the postpartum period were low compared to normal cycle gonadotropin levels. Thus, it might be concluded that puerperal anovulation and amenorrhea during lactation might be due to hypophyseal gonadotropic dysfunction rather than to ovarian refractoriness.

Abortifacient effect and uterine cervix-dilating action of 16,16-dimethyl trans Δ2 PGE1 methyl ester (ONO 802) in the form of a vaginal suppository (a randomized, double-blind, controlled study in the second trimester of pregnancy)

A newly synthesized 16, 16-dimethyl trans delta 2 PGE1 methyl ester (ONO 802) was clinically applied in the form of a vaginal suppository for therapeutic abortion of second trimester pregnancies. Its effects were studied in a double-blind test comparing it with an inactive placebo suppository. The study was conducted at 12 Japanese university hospitals. The number of patients was 125 in total, i.e. 63 receiving ONO 802 vaginal suppository (containing 1.0 mg ONO 802) and 62 receiving inactive placebo suppository. ONO 802 was more effective than placebo with a success rate of 87% (complete (71%) and incomplete (16%) abortions). The onset of uterine contractions was observed in 154.3+/-18.1 min. and the onset of uterine bleeding in 323.6+/-41.0 min. The expulsion of the fetus and placenta was observed in 955.4+/-97.0 and 961.6+/-97.0 min., respectively. The cervix dilating effect of ONO 802 was observed in 63.5% of the patients at 3 hours after the start of administration. Nausea, vomiting, abdominal cramps, diarrhea and pyrexia were noticed. However, all these side effects were transient and mild, requiring no treatment. No abnormality was observed in the puerperal course, duration of uterine bleeding or onset of subsequent menstruation following the therapeutic abortion. Therefore, the present study demonstrated that ONO 802 vaginal suppository was an effective and valuable drug for therapeutic abortion of second trimester pregnancies.

Collagenolytic activity and steroid levels after administration of dehydroepiandrosterone sulfate.

Seventy‐five pregnant women in their 38th to 41st weeks of gestation were given a single intravenous injection of 200 mg of dehydroepiandrosterone sulfate (DHAS). The changes in estriol, 17β‐estradiol and progesterone levels in the serum, the uterine cervix and the myometrium of the placenta‐implanting site were then determined. Estriol levels remained unchanged both in serum and tissue, but the level of 17β‐estradiol increased sharply both in serum and tissue after four hours. The increases of 17β‐estradiol in the serum and the portio vaginalis of the same patient were well correlated (r = 0.79898), but the percentage of increase was much higher in the portio vaginalis than in the serum (p < 0.001). Serum progesterone levels did not change initially, but always decreased within four or eight hours in cases in which labor had started or delivery was accomplished within 24 hours (p < 0.01 at four hours). The total amount of collagenase was determined in ten subjects before and after the administration of DHAS. The total collagenase activity was elevated by an average of 152.3%. The peak of activity was accelerated from the fourth to the second day (p < 0.001) of the culture. A probable mechanism of DHAS action in accelerating cervical ripening is presented.

Response to Luteinizing Hormone Releasing Factor (LRF) in Normal Subjects and Anovulatory Patients

Recently the following decapeptide sequence for porcine luteinizing hormone releasing factor (LRF) was proposed by Schallys group: (Pyro) Glu‐His‐Trp‐Ser‐Gly‐Leu‐Arg‐Pro‐Gly‐NH2. The polypeptide corresponding to this structure has been synthetized and this synthetic LRF is now available for clinical study. Intravenous administration of synthetic LRF in dose of 200 μg stimulated a rise in serum LH and FSH in normal subjects. Most anovulatory patients showed approximately the same degree of response to LRF. No rise in serum LH or FSH occurred in an anovulatory patient with panhypopituitarism following injection of 200 μg of LRF. It may be concluded that synthetic LRF is convenient means for testing the integrity of the human pituitary for LH and FSH secretion.

A Study on the Effect of Dehydroepiandrosterone Sulfate on So-Called Cervical Ripening

Abstract. Dehydroepiandrosterone sulfate (DHAS) is now used for a dynamic test of placental function by many obstetricians. While practicing this test, the authors found that DHAS markedly promoted so‐called “cervical ripening”. To study this problem, DHAS of 50 or 100 mg in multiple doses were injected into 132 Japanese pregnant women in their 38th–42nd week of gestation. The change in Bishop score was carefully recorded. Bishop score in the injected groups of primiparae (100 mg) began to rise much sooner than the control groups (p<0.01 on seventh day and 14th day). However, such significant difference in the rise of Bishop score was not noted in the multiparae and primiparae with 50 mg. Although the rise of score is not significant, the duration (day) from injection to delivery was shorter in the injected group than the control group (t=2.1529, p<0.05 in primiparae with 50 mg, t=3.8829, p<0.01 with 100 mg, t=2.1029, p<0.05 in multiparae with 50 mg). In some of these cases, labor began or delivery was finished within 24 hrs. Among the factors of Bishop score, mainly the effacement, consistency and dilatation of the cervix were remarkably improved by DHAS injection (p<0.01 and <0.05). Side effects of any type were not seen in the mothers and foetuses. As a conclusion, DHAS injection is considered to produce favorable conditions for delivery in women with “unripe cervix” by softening the soft birth canal. Furthermore, it is suggested that DHAS might play an important role in triggering labor.

GONADOTROPHIN RESPONSE TO LUTEINIZING HORMONE RELEASING FACTOR (LRF) IN PUERPERAL WOMEN

Abstract. Synthetic luteinizing hormone releasing factor (LRF) was administered intravenously in a dose of 200 μg to twenty normal lactating puerperal women and serum follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) response to LRF was measured by double antibody radio‐immunoassay. Synthetic LRF failed to stimulate FSH secretion in all the eight volunteers in the 1st postpartum week. About half of the six subjects in the 3rd postpartum week responsed to LRF with a rise of serum FSH and LH. All the six lactating women in the 5th postpartum week were responsive to LRF and there was a concomitant rise in serum FSH and LH. These results suggest that gonadotrophic activity of the anterior pituitary is suppressed by an unknown mechanism during the first few weeks of puerperium and gonado‐trophin reserve function recovers completely around the 5th postpartum week. Thus, it might be assumed that puerperal anovulation or amenorrhoea is due to hypo‐thalamic‐pituitary dysfunction during the first few postpartum weeks and due to hypothalamic disorders after the 5th postpartum week.