백서태반의 Chorionic Gonadotropic Hormone의 소재에 관한 면역조직화학적 연구

Abstract

[한글]

[영문]

That the syncytium is a source of placental steroids has not seriously been questioned since Wislocki(1955) demonstrated the histochemical localization in the syncytiotrophoblast of sudanophilic droplets, which he associated with estrogen and

progestrone.

The origin of chorionic gonadotropin, on the other hand, has engendered a current controversy. Over a quarter of a century ago Gey et al.,(1938) extracted from placental tissue cultures a gonadotropin that they assumed to originate from the cytotrophoblast. Several years later Wislocki(1943)localized PAS-positive material, thought to represent the glycoprotein, in the Kanghans cells. Clinical support for the origin of Chorionic Gonadotropin from the cytotrophoblast was provided by the temporal concidence of maximal cytotrophoblastic proliferation with the highest titers of human chorionic gonadotropin in the urine and serum.

Quite recently, however, Thiede and Choate localized chorionic gonadotropin by immunofluorescent technics in the syncytium, and to a much lesser extent in the amnion, but failed to detect specific fluorescence in the cytotrophoblast. In combined ultrastructural and immunofluorescent studies, Pierce and Midgley, working with human chriocarinoma, likewise detected the human chorionic gonsdotropin in the syncytium but not in the cyotrophoblast. In a similar study of both transplanted human choriocarcinoma and hydatidiform mole, Wynn and Davies(1965), having demonstrated by electronmicroscope that the benign and malignant trophoblastic growths maintain the fine structural features of their normal counterparts, indicated that only the syncytium contained the subcellular organelles required for

synthesis of proteins, with particularly abundant endoplasmic reticulum and well developed Golgi complexes, whereas the cytotrophoblast was ultrastructurally simple.

The purpose of this study is to characterize the patterns of tissue chorionic gonadotropin localization in the placenta at different stage of pregnancy.

Materials and Methods

A total of 114 albino rats, each around 200gms, were used for the experiments. Of them, 35 rats were used for preliminary experiments to determine estrus cycle, to achieve successful matching and to determine the duration of normal pregnancy.

Estrus cycle was determined by the vaginal smear methods of Long and Evans(1922), and matching was achieved by housing a male and female rat together in the pre-estrus cycle and rechecking the vaginal smear for the presence of spermatozoa.

In this way it was found that the average estrus cycle was 104 hours and the duration of normal prognancy was 21 days.

The placentas were obtained by removing of the entire uterus of 3 to 5 rats at 24 hours intervals through pregnancy. A total of 496 placentas were observed in the 66 pregnant rats.

From each removed uterus the numbers of conceptuses, the size of pregnant segments, and size of placentas were determined. Then, the specimens were divided into 2 parts. One part was embedded in paraffin after fixation in 4% neutral formalin, and the other was used for frozen section by cryostat.

Antibody:

Rabbit anti-human chorionic gonadotropin (HCG) of high specificity and potency was used in the immunofluorescent studies. This antiserum was obtained from the Organon Pharmacutical Corporation.

This anti-HCG was proved to react with rat chorionic gonadotropin by a preliminary test against sera and urnie of pregnant rats.

Preparation of conjugate:

The rabbit anti-human chorionic gonadotropin gamma-globulin was prepared by DEAE chromatograpy. The purified anti-sera solution was conjugated with fluorescen isothiocyanate (FITC) (Coons and Kaplan 1950). The conjugate was purified by the column of sephadex G-25 with 0.01 M. PH 7.4 phosphate buffer solution (Sober, 1958).

Fluorescent staining Procedures:

Immediately prior to staining, the blocks of qick-frozen tissue were sectioned at 5 to 6 microns with the cryostat at -25C. The sections were thawed and air-dried at room temperature for 10min. the tissue sections were overlaid with FITC conjugated rabbit anti-HCG and placed in petri dished contain moistened filter paper for 45 min. at room temperature. The slides were rinsed in 0.5M. phosphate buffered saline, PH 7.2, for 10min. and placed in a 50% solution of glycerol and phosphate-buffered saline.

The preparations were examined for fluorescence with a A.O. Spencer microscope using an Osram HBO 200 mercuryarc lamp with appropriate ultraviloet filters.

The blocking test was carried out in each set of sections by pretreatment with unconjugated anti-sera followed by fluorescent staining.

Results and Summary

Intense apple-green fluorescence was observed in the cytoplasm of trophoblast giant cells of the junctional zone from the 12th to 19th day of pregnancy as well as the syncytiotrophoblastic cells of human placenta. The trophospongial cells and glycogen cells of the junctional zone, and decidual cells showed no distinct green fluorescence, but granular green fluorescence was observed in the cytoplasm of the trophoblast cell of the labyrinth on the 16th to 18th days of pregnancy.

The epithelium of the amnion investing the fetal surface of the placenta and the umbilical cord, which is covered with a single layer of amnion cells, showed no fluorescence. No specific fluorescence was hound in blood vessels of the chorionic plate, the connective tissue of the labyrinth or blood vessels of the umbilical cord, or glandular epithelium, blood vessels, or stroma of the decidua. The nuclei and areas of fibrinoid degeneration, all exhibited no fluorescence. Blocking tests in each set gave a consistently negative reaction.

Methyl-green pyronin positive substance (RNA) appeared in relatively large amounts from the 8th day of pregnancy in the cells of yolk sac, labyrinth, junctional zone, and decidua.

The trophospongial cells and trophoblast giant cells revealed intense positive staining (RNA) from the 12th to 14th day of pregnancy, and the amount decreased somewhat in the late stage of pregnancy.

In summary, albino rat chorionic gonadotropin was localized mostly in the cytoplasm of the trophoblast giant cells in the junctional zone and a smaller amount in the trophoblast cells of the labyrinth by means of the immunofluorescent techinc.