Immunohistochemical localization of new placental proteins (PP20, PP25, PP26) in normal pregnancy and gestational trophoblastic disease

Trophoblast Research 8:355-365, 1994

I M M U N O H I S T O C H E M I C A L LOCALIZATION OF N E W PLACENTAL PROTEINS (PP20, PP25, PP26) IN NORMAL PREGNANCY AND GESTATIONAL TROPHOBLASTIC DISEASE Hitoshi Funayama 1, Seiko Iidzuka 1, Yoshichika SuzukiL Keiichi Isaka}, Shizuko YamabeL Masaomi TakayamaL and Hans Bohn 2 1Department of Obstetrics and Gynecology Tokyo Medical College Tokyo 160, Japan 2Behringwerke AG Marburg, Germany

INTRODUCTION

Twenty six proteins have been immunochemically isolated and physically characterized from soluble and solubilized fractions from term human placenta by Bohn and his coworkers (Bohn, 1973, 1975, 1982; Bohn and Klaus, 1976, 1979, 1980, 1985, 1991; Bohn and Winckler 1977a, 1977b, 1980, 1987; Bohn et al., 1980; Bohn et al., 1983). These proteins are called placental proteins (PP1-PP26). Many of these placental proteins have identified production sites and biological functions. Some of these placental proteins are enzymes (PP5, PP7, PP9, and PP24), binding proteins (PP2, PP12, PP22), immunomodulating proteins (PP14, PP21), coagulation inhibitor (PP4), plasminogen activator inhibitors (PP10) (Bohn et al., 1991), and calcium binding proteins (PP19) (Takayama et al., 1991). Placental proteins 20, 25, and 26 (PP20, PP25, PP26) are immunochemically isolated from the soluble fraction of term placenta and partially characterized by Bohn and Winckler (1986, 1991). These three proteins are glycoproteins with molecular weight determined by SDS-PAGE as 2.7, >20, and >15 KDa, respectively. The estimated amounts of the proteins are 0.5, <10, and <10 mg per term placenta. The origin and biological role of the proteins have not been revealed. The current study investigates the localization of these three proteins with immunohistochemical techniques. MATERIALS AND METHODS

Six early placentae were obtained after legal abortion and six term placentae were obtained after natural delivery. In two ectopic pregnancies, decidualized endometria were obtained by diagnostic endometrial scraping. Three proliferative and three secretory phase endometria were obtained after hysterectomy from uterine fibroid patients. Three total hydatidiform mole tissues were obtained after evacuation. One uterine invasive lesion of a patient of invasive mole was obtained

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after hysterectomy. Two lung metastatic lesions of choriocarcinoma were obtained after lobectomy. Every specimen was obtained after the patient's consent. Each specimen was immediately fixed in 20% formalin solution for three days, rinsed in water, and embedded in paraffin. These blocks were sliced 8 micrometer thick and mounted on lysine coated slide glasses. After deparaffinization with xylene, each section was pretreated with 0.3% hydrogen peroxide in phosphate-buffered saline for 20 minutes to inhibit the activity of internal peroxidase. Anti-PP-20, 25, and 26 rabbit sera (lots No. 461ZA, 740ZA, 189ZB, respectively: Behringwerke, Marburg, Germany) were used for the first antibodies at dilutions of 1:100-200. The reaction time was 30-60 minutes. Horseradish peroxidase conjugated anti-rabbit swine IgG (Dako, Copenhagen, Denmark) reacted as the second antibody in 1:40-100 dilution for 15-30 minutes. DAB (3,3'-diaminobenzidine tetrahydrochloride and 0.03% hydrogen peroxide reacted in 0.05 M Tris-HCl buffer for about ten minutes. Anti-hCG, anti-HPL, and anti-SP1 rabbit polyclonal antibodies (Dako) were used for positive control staining. Staining for hPL was used for identifying of extravillus trophoblastic cells. In order to rule out nonspecific staining, nonimmunized normal rabbit serum (Dako) was used instead of the first antibody for negative control with all staining procedure. The staining intensity of test section was graded arbitrarily as +, clear contrast like positive control; +, dispersed or weak staining; -, negative staining. RESULTS Normal Placenta Each protein was localized in cytoplasm of syncytiotrophoblasts (ST) of early and term placenta. Cytoplasm of cytotrophoblasts (CT was occasionally stained for PP25 and PP26 (Figures 1 and 2). Positive staining for PP25 and PP26 was recognized in extravillus trophoblasts (ET) of the basal plate and chorionic membrane (Figure 3). The staining was more noticeable in early placenta. PP20 and 25 staining were occasionally demonstrated in decidual cells. Staining for PP26 was observed in some decidual cells. Amnion, villus stroma, and maternal blood cells were negative for PP20, 25, and 26. Nucleated fetal erythrocytes in the vessels of early placental villi were occasionally positive for PP26 but were negative for the other two proteins (Figure 4 and Table1). Endometrium Proliferative and secretory phase endometria of normal nonpregnant women were not stained for these proteins. PP25 and PP26 were weakly positive on glandular cells in the sponge layer of decidualized endometrium from ectopic pregnant patients (Figure 5). Gestational Trophoblast Disease Placental proteins (PP25 and PP26) were localized in trophoblastic cells of the outer margin of molar vesicles of total hydatidiform mole. PP20 was not observed in molar vesicles. PP25 and 26 were localized in cytoplasm of syncytio and intermediate trophoblastic cells of invasive mole and metastatic lesions of choriocarcinoma (Figure 6). The staining for PP26 was clearer than that for PP25. PP20 was not observed in invasive moles or choriocarcinoma (Table 2).

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Figure 1A-C. Localization of PP20, PP25, and PP26 in early placental villi. A: PP20, B: PP25, C: PP26. X200. Hematoxylin counterstaining.

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Figure 2A-C. Localization of PP20, PP25, PP26 in term placental villi. A: PP20, B: PP25, C: PP26. X200. Hematoxylin counterstaining.

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Figure 3A-B. Localization of PP25 and PP26 in basal plate of normal placenta. Cytoplasm of ET were positive for PP25 (A) and PP26 (B). X200. Hematoxylin counterstaining.

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Figure 4. Nucleated fetal blood cells in capillaries of early placental villi. Hematoxylin counterstaining.

X200.

Table 1

Localization of PP20, 25, and 26 in H u m a n Placental Tissue

Villi Sync~tiotrophoblast ~tto]~lasm

PP20

PP25

PP26

+

+

+

+

+

M en'lDr"~'~e

Cytotrophoblast C~ol~lasm M~VnDl'ane

Stroma Hofbauer Cell Blood Vessel Fetal Blood Cell Umbilical Cord Blood Vessel Walton's jelly Membrane Amnion Chorion Trophoblast Stroma Basal Plate Trophoblast Decidual Cell Stroma Maternal Blood Cell White Blood Cell Red Blood Cell +: Positive staining +: Occasionally or weak positive staining -: Negative staining

+

+

+

+

+

+

+ q-

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Figure 5A-B. Staining of PP25 (A) and PP26 (B) on glandular cells in sponge layer of endometrium obtained from ectopic pregnant patients. X200. Hematoxylin counterstaining.

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Figure 6A-B. Localization of PP26 in metastatic legion of choriocarcinoma (A). X100. Infiltrating trophoblastic cells were also positive (B). X200. Hematoxylin counterstaining.

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Table 2

Localization of PP20, 25, and 26 in Gestational Trophoblastic Disease PP20

PP25

PP26

+

+

+

+

-+

+

+

+

Invasive Mole ST CT ST Choriocarcinoma ST CT IT ST: syncytiotrophoblast cell CT: cytotrophoblast cell IT: intermediate trophoblastic cell +: positive staining occasionally or weak positive staining -: negative staining

DISCUSSION PP20 was reported to be localized in cytoplasm of syncytiotrophoblast of villi and extravillus trophoblast in the basal plate and chorionic membrane in human and cynomolgus monkey placentae by an ABC immunohistochemical technique with 90% ethanol acetone mixture fixation (lnaba et al., 1987). They observed staining in early placental villi but not term placenta. In the current study, PP20 staining of early placental villi was more intense, and term placental villi stained very weakly. PP20might play some reproductive role in early pregnancy rather than during late pregnancy. No positive staining was observed on decidual cells for PP20 in formalin fixed paraffin embedded specimen. However, PP20 was positive on decidual cells after ethanol acetone fixation (Inaba et al., 1987). It might be necessary to utilize frozen sections for immunochemical staining and to develop a more sensitive assay system in order to conclude whether PP20 is localized in decidua. On the other hand, PP20 was demonstrated in the tissue extracts of adult spleen by the gel diffusion method (Bohn and Winckler, 1985). While PP20 was not pregnancy specific protein however, it might be a pregnancy-associated protein. PP25 and PP26 were observed in the cytoplasm of syncytiotrophoblast cells of normal placenta. Extravillus trophoblast cells in the basal plate and chorionic membrane were also positive for the proteins. The production sites of the proteins were proposed to be syncytiotrophoblast cells and extravillus trophoblast cells. Also, positive staining for PP25 and PP26 was observed in cytoplasm of cytotrophoblast cells. It was possible that cytotrophoblast cells produce PP25 and PP26 before differentiating to syncytiotrophoblasts.

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It has already been described that there was no occurrence of PP25 and PP26 in extracts of various tissues from human fetus and adults by the gel diffusion test (Bohn and Winckler, 1991). However, positive staining for PP25 and PP26 was observed in glandular cell of decidualized endometrium of ectopic pregnancy, and decidual cell for PP26 in the current study. Thus PP25 and PP26 could not be placenta specific. The findings indicated that endometrium might produce PP25 a n d / o r PP26 under the influence of large amounts of progesterone, although it was not demonstrated in early and mid secretory phase endometria in this study. It is widely accepted that hCG and SP1 are good chemical and histochemical markers of trophoblastic disease. We can usually demonstrate trophoblastic cells in invading sites in myometrium and in metastasis by immunohistochemical staining for hCG or SP1. However, it is often difficult to discern intermediate type trophoblastic cells in such legions with hCG or SP1 staining, because these two proteins are mainly demonstrated in syncytiotrophoblast cells. PP25 and PP26, especially PP26, stain not only in syncytiotrophoblast cells but also in intermediate trophoblastic cells so that it can be easier to determine invading trophoblastic cells like PP19 (Takayama et al., 1989). Therefore, PP26 may be helpful in evaluating tumor extent and the effect of preceding chemotherapy. PP20 was not observed in trophoblastic cells of gestational trophoblastic disease in the current study. However, more investigation (e.g., frozen section techniques) must be done before we conclude that trophoblastic disease is negative for PP20. SUMMARY Placental Proteins 20, 25, and 26 (PP20, PP25, PP26) were isolated from a soluble fraction of term placenta and characterized by Bohn (1985, 1991). To clarify the localization of these proteins, normal placenta, ectopic pregnancy, endometrium, and trophoblastic disease were studied by indirect immunohistochemical method. Each protein was localized in cytoplasm of syncytiotrophoblast (ST) of early and term placenta. Positive staining for PP25 and PP26 was recognized in the extravillus trophoblast (ET) of basal plate and membrane. Proliferative and secretory phase endometria were not stained for these proteins. However, PP25 and PP26 were localized on decidualized endometrium from ectopic pregnant patients and PP26 showed positive staining on decidua. Although PP20, 25, and 26 were localized on ST of placenta, there was a possibility of extra placental production. PP26 was localized in syncytio and intermediate trophoblastic cells of gestational trophoblastic disease. PP26 is proposed to be a useful new histochemical marker of trophoblastic disease. REFERENCES Bohn, H. (1973) Isolierung des Plazenta-Proteins PP2 and seine identifizierung als ferritin. Arch. Gyneak. 215, 263-275. Bohn,

H. (1975) Isolierung und c h a r a k t a r i s i e r u n g eines plazentaproteins (PP6). Arch. Gyneak. 218, 131-142.

hochmolekularen

Bohn, H. and Klaus, W. (1976) Isolierung und charaktarisierung des plazentaproteins PP1. Arch. Gyneak. 221, 165-175.

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Bohn, H. and Winckler, W. (1977a) Isolierung und charaktarisierung eines neuen gewebsproteins aus menschlichen plazenten. Arch. Gyneak. 22, 5-13. Bohn, H. and Winckler, W. (1977b) Isolierung und charaktarisierung des plazentaproteins PP5. Arch. Gyneak. 223, 179-186. Bohn, H. and Klaus, W. (1979) Isolierung und charaktarisierung eines neuen plazentaspezfischen proteins (PP10). Arch. Gyneak. 227, 125-134. Bohn, H. and Winckler, W. (1980) Isolierung und charaktarisierung eines neuen plazenta-gewebsproteins (PPll). Arch Gyneak. 229, 293-301. Bohn, H. and Klaus, W. (1980) Isolierung und charaktarisierung eines neuen plazentaspezfischen proteins (PP12). Arch. Gyneak. 229, 279-291. Bohn, H., Johansen, R., and Klaus, W. (1980) Neues plazentaprotein (PP15) mit immunosupressiver wirkung. Arch. Gyneak. 230, 167-172. Bohn, H. (1982) New soluble placental proteins. Ric. Clin. Lab. 12, 221-230. Bohn, H., Klaus, W., and Winckler, W. (1983) Purification and characterization of two new soluble placental proteins (PP13 and PP17). Oncodev. Biol. Med. 4, 343-350. Bohn, H. and Winckler, W. (1985) Isolation and characterization of four new placental tissue proteins (PP18, PP19, PP20, PP21). Arch. Gynecol. 240, 13-19. Bohn, H. and Winckler, W. (1987) Isolation and characterization of a flavin-containing placental protein (PP3). Arch. Gyneak. 240, 201-206. Bohn, H. and Winckler, W. (1991) Isolation and characterization of five new soluble placental tissue proteins (PP22, PP23, PP24, PP25, PP26). Arch Gynecol. 248, 111-115. Bohn, H., Winckler, and Grundmann, U. (1991) Immunochemically detected placental proteins and their biological functions. Arch Gynecol. Obstet. 249, 107-118. Inaba, N., Sato, N., Fukazawa, I., Ota, Y., Shirotake, S., Takamizawa, H., Nozawa, S., and Bohn, H. (1987) The immunocytochemical localization of new soluble placental tissue proteins (PP14, 16, 17, 19, 20 and 21) in human and cynomolgus monkey placenta. Arch. Gynecol. 240, 13-19. Takayama, M., Isaka, K., Suzuki, Y., Funayama, S., Suzuki, Y., Akiya, K., and Bohn, H. (1989) Comparative study of placental protein 19, human chorionic g o n a d o t r o p h i n and pregnancy-specific b e t a l - g l y c o p r o t e i n as immunohistochemical markers for extravillous trophoblast in pregnancy and trophoblastic disease. Histochemistry 93, 167-173. Takayama, M., Kitani-Yasuda, T., Isaka, K., Suzuki, Ya., Suzuki, Yo., Sakuma, S., and Bohn, H. (1991) Placental-tissue protein 19: Localization, dynamics, and molecular biological characteristics. In: Placenta: Basic Research for Clinical Application, (ed.), H. Soma, BasahKarger, pp. 126-146.