Immunohistochemical staining techniques used on an 11-week-old fetus showed that fetal antigen 2 (FA2) was present intracellularly in endochondral and perichondral osteoblasts, and the immunoreaction was extended into the adjacent bone matrix. Osteoclasts and chondroblasts were found to be FA2 negative. A granular perinuclear intracytoplasmic FA2 immunoreaction was found in cultured osteoblasts and osteogenic osteosarcoma cells, and immunoelectron-microscopical examination revealed a granular immunoreaction product in the rough endoplasmic reticulum. These findings indicate that FA2 is synthesized by osteoblasts and osteogenic osteosarcoma cells. A reaction of immunological identity was found between FA2 purified from second trimester amniotic fluid and serum-free supernatants of cultured osteogenic osteosarcoma cells. This shows that an antigen recognized by the anti FA2 antibody is secreted by these malignant cells. Thus, FA2 may represent a marker for altered bone metabolism, and have a potential in the classification of osteogenic osteosarcoma/chondrosarcoma.

Foetal antigen 2 (FA-2) is a connective-tissue-associated antigen isolated from second trimester human amniotic fluid. FA-2 has an alpha-electrophoretic mobility and is a single-chain molecule with a molecular weight of 26 kDa as determined by polyacrylamide gel electrophoresis (PAGE). Using indirect immunofluorescence and the immunoperoxidase technique, FA-2 was found to be in the lamina densa/sublamina densa region of the basement membrane zone (BMZ) in adult as well as in foetal skin. FA-2 was found throughout the dermis in foetal skin, whereas in adult skin it was found to be associated with the BMZ and around the blood vessels, hair follicles and eccrine glands. Intracellular FA-2 antigen was demonstrated in proliferating fibroblasts by the indirect immunoperoxidase technique and immunoelectron microscopy of the fibroblasts revealed staining of the antigen in the cisternae of the rough endoplasmatic reticulum at the trans-side of the Golgi complex as well as in vesicles close to the plasma membranes. FA-2, a hitherto undescribed antigen associated with human BMZ, is probably being synthesized by proliferating fibroblasts.

Monospecific antisera against two fetal antigens (FA-1 and FA-2), alphafetoprotein (AFP) and two endometrial proteins (PP12 and PP14) were used to examine the distribution of these proteins and antigens in human trophoblast and gestational endometrium in first and third trimesters of pregnancy, normal human ovary and fetal tissues by indirect immunoperoxidase histochemical localisation techniques. Fetal liver stained exclusively for FA-1 and AFP which was used as a reference protein. Staining for FA-2 was seen in fetal connective tissue, in particular the basement membrane. FA-1 and FA-2 did not stain positively in decidua, trophoblast or ovarian tissue. Gestational endometrium stained positively for PP14 exclusively in the glandular epithelium, whilst staining for PP12 was seen only in the stromal cells. Trophoblast, both early and late, and ovarian tissue did not stain positively for any of the four substances tested.

Circulating human chorionic gonadotrophin (hCG), Schwangerschaftsprotein 1 (SP-1) and pregnancy-associated plasma protein A (PAPP-A) were examined in 10 women with ectopic gestation in relation to distribution and intensity of staining for these molecules using immunohistochemical techniques applied to matched trophoblastic and decidualized endometrial tissues. All 10 women revealed strong staining for hCG and SP-1 in the syncytiotrophoblast, which was apparently unrelated to maternal levels of hCG and SP-1, levels being less than the 10th centile of the normal range in 9/10 and 7/10 women, respectively. By contrast maternal PAPP-A levels seemed to correlate with the intensity and the distribution of staining for PAPP-A. Circulating PAPP-A was undetectable (less than 5 mIU/l) in 4 patients, and below the 10th centile in the remaining 6. Using immunohistochemical techniques hCG, SP-1 and PAPP-A could not be demonstrated in any of the decidualized endometrial tissues studied.

The influence of antibody absorption procedures and proteolytic pre-treatment of formaldehyde-fixed placental tissue on the localization of pregnancy-associated plasma protein A by immunoperoxidase technique was examined.

The immunoperoxidase technique was used to study the localization of pregnancy-associated plasma protein A (PAPP-A) in formaldehyde-fixed paraffin-embedded tissue from normal human placentae at the gestational age of 8, 15 and 40 weeks. Sections of formaldehyde-fixed tissue treated with a proteolytic enzyme and incubated in antiserum against PAPP-A either raised in goats or rabbits showed that PAPP-A was distributed in the cytoplasm of the syncytiotrophoblast. The protein was not found in the cytotrophoblast. Sections without pretreatment with trypsin and incubation in goat anti-PAPP-A showed no staining reaction, whereas incubation in rabbit anti-PAPP-A revealed a staining of the syncytiotrophoblast surface. The results indicate that PAPP-A is probably synthesized in the syncytiotrophoblast.

During early pregnancy, placentation occurs in a relatively hypoxic environment which is essential for appropriate embryonic development. Intervillous blood flow increases at around 10–12 weeks of gestation and results in exposure of the trophoblast to increased oxygen tension (Po2). Prior to this time, low oxygen appears to prevent trophoblast differentiation towards an invasive phenotype. In other mammalian systems, oxygen tension effects are mediated by hypoxia inducible factor-1 (HIF-1). We found that the ontogeny of HIF-1α subunit expression during the first trimester of gestation parallels that of transforming growth factor-β3(TGFβ3), an inhibitor of early trophoblast differentiation. Expression of both molecules is high in early pregnancy and falls at around 10 weeks of gestation when placental Po2levels are believed to increase. Antisense-induced inhibition of HIF-1α inhibited the expression of TGFβ3, and stimulated extravillous trophoblast (EVT) outgrowth and invasion. Of clinical significance we found that TGFβ3expression was increased in pre-eclamptic placentae when compared to age-matched controls. Significantly, inhibition of TGFβ3by antisense oligonucleotides or antibodies restored the invasive capability to the trophoblast cells in pre-eclamptic explants. We speculate that if oxygen tension fails to increase, or trophoblasts do not detect this increase, HIF-1α and TGFβ3expression remain high, resulting in shallow trophoblast invasion and predisposing the pregnancy to pre-eclampsia.Effective fetal–maternal interactions during early placentation are critical for a successful pregnancy. Optimal placental perfusion requires the controlled invasion of trophoblast cells deep into the decidua to the spiral arteries. Trophoblast stem cells, also referred to as cytotrophoblast cells, reside in chorionic villi of two types, floating and anchoring villi. Floating villi, which represent the vast majority of chorionic villi, are bathed in maternal blood and primarily perform gas and nutrient exchange for the developing embryo. During early placentation, cytotrophoblast cells in the floating villi proliferate and differentiate by fusing to form the multinucleate syncytiotrophoblast layer. Cytotrophoblast cells in anchoring villi either fuse to form the syncytiotrophoblast layer, or break through the syncytium at selected sites and form multilayered columns of non-polarized extravillous trophoblast cells, which physically connect the embryo to the uterine wall (Figure 1). The extravillous trophoblast cells invade into the uterine wall as far as the first third of the myometrium and its associated spiral arteries, where they disrupt the endothelium and the smooth muscle layer and replace the vascular wall. This results in the conversion of the narrow calibre arteries into distended uteroplacental arteries, thereby increasing blood flow to the placenta and allowing an adequate supply of oxygen and nutrients to the growing fetus. The invasive activity of the extravillous trophoblast cells is at a maximum during the first trimester of gestation, peaking at around 10–12 weeks and declining thereafter. Insufficient invasion contributes to the development of pre-eclampsia, which often results in fetal intrauterine growth restriction, maternal hypertension and proteinuria. In contrast, unrestricted invasion is associated with premalignant conditions, such as invasive mole, and with malignant choriocarcinoma.Invading trophoblast cells undergo striking and rapid changes in cellular functions that are temporally and spatially regulated along the invasive pathway (Figure 1) (Cross, Werb and Fisher, 1994. The formation of the anchoring villi is accompanied by changes in synthesis and degradation of extracellular matrix proteins and their receptors, and changes in the spatial distribution of extracellular matrix proteins, as well as changes in the expression of adhesion molecules (Damsky, Fitzgerald and Fisher, 1992; Bischof et al., 1993). For example, during invasion the expression of laminin is lost and the expression of fibronectin is acquired. Extravillous trophoblast cells lose the expression of E-cadherins, downregulate the expression of α6β4integrin (a laminin receptor) and acquire the expression of α5β1integrin (a fibronectin receptor). Subsequently extravillous trophoblast cells differentiate to acquire an invasive phenotype, which is indicated by the expression of markers such as gelatinase B/MMP9 and α1integrin(a collagen/laminin receptor) as well as markers typical of a vascular adhesion phenotype. Thus, specific changes in extracellular matrix proteins and their receptors are associated with the acquisition of an invasive phenotype by the extravillous trophoblast cells.

Summary. Four antibody preparations against pregnancy‐associated plasma protein (PAPP‐A) were compared in order to find an explanation for the contradictory results published on tissue localization, clinical usefulness and biological function of PAPP‐A. One of the preparations studied was a rabbit anti‐PAPP‐A antiserum which has been offered for general scientific use (Bischof et al. 1979). Only the IgG fraction of anti‐PAPP‐A antisera which appeared to be monospecific and had been further absorbed with fetal connective tissue gave specific uniform staining of the cytoplasm of the syncytiotrophoblast exclusively. Circulating PAPP‐A could not be detected by RIA employing this IgG preparation in the non‐pregnant state, or before 18 days after conception. Circulating PAPP‐A could be detected in all seven pregnant women studied within 4 weeks after conception. Identical results were obtained with a commercially available IgG fraction against PAPP‐A.

The immunoperoxidase technique was used to study the localization of pregnancy-associated plasma protein A (PAPP-A) in formaldehyde-fixed paraffin-embedded tissue from normal human placentae at the gestational age of 8, 15 and 40 weeks. Sections of formaldehyde-fixed tissue treated with a proteolytic enzyme and incubated in antiserum against PAPP-A either raised in goats or rabbits showed that PAPP-A was distributed in the cytoplasm of the syncytiotrophoblast. The protein was not found in the cytotrophoblast. Sections without pretreatment with trypsin and incubation in goat anti-PAPP-A showed no staining reaction, whereas incubation in rabbit anti-PAPP-A revealed a staining of the syncytiotrophoblast surface. The results indicate that PAPP-A is probably synthesized in the syncytiotrophoblast.

The localization of human placental lactogen (hPL), pregnancy-specific beta-1 glycoprotein (Schwangerschaftsprotein 1, SP1) and pregnancy-associated protein A (PAPP-A) was examined in intrauterine and tubal ectopic gestation (n=5) by the immunoperoxidase technique. The distribution of hPL and SP1 was identical in placental tissues obtained from intra- and extrauterine pregnancies, being uniformly seen throughout the syncytiotrophoblast. hPL and SP1 were not demonstrated in uterine decidual tissue from ectopic pregnancies. During early (week 8) intrauterine pregnancy, PAPP-A was not restricted to the mature syncytiotrophoblast, being observed also in some trophoblast-like cells adjacent to islands of syncytiotrophoblast. In contrast, in ectopic gestation, PAPP-A was observed in these cells at six weeks' gestation only. We were unable to detect PAPP-A in trophoblastic tissue of chorionic villi and uterine decidual tissue from ectopic gestation.

The synthesis of murine alpha-foetoprotein (m-AFP) was induced in 43 out of 47 adult mice of both sexes by sc administration of 100 micrograms oestradiol-17 beta every second day. The m-AFP serum levels of the oestrogen treated male and female mice were 7 and 17%, respectively, of the levels in mice during late pregnancy. Immunohistochemical examination of liver tissue revealed the intracellular presence of m-AFP in less than 0.5% of the hepatocytes scattered throughout the liver of the oestrogen treated mice.

Intravenous injection of small amounts of monospecific rabbit IgG against pregnancy-associated murine protein I (PAMP-I) induced abortion in mice where there was a histocompatibility difference between mother and fetuses. No abortion could be induced in inbred mice by a similar treatment. The maternal serum level was found to be higher in inbred than in outbred mice. The abortive dose of antibodies did not influence the serum levels of PAMP-I. Histological examination of uterine, placental and liver tissue showed only morphological changes in the placental tissue of mice which aborted due to the treatment with anti-PAMP-I antibodies.

The concentration of human platelet lactogen (hPL), pregnancy specific beta-1 glycoprotein (SP-1) and pregnancy-associated plasma protein A (PAPP-A) were analysed in consecutive serum samples from a patient who gave birth to a child with Cornelia de Lange syndrome. HPL and SP-1 were present in normal concentrations from week 20 to week 35 of gestation whereas PAPP-A could not be detected in any of the samples examined. Immunohistochemical examination of two placentae from Cornelia de Lange syndrome revealed normal localization of hPL and SP-1 but the absence of PAPP-A from the syncytiotrophoblast. The significance of association between Cornelia de Lange syndrome and compromised synthesis of PAPP-A is discussed.

The role of the endothelium and subendothelial connective tissue in the permeability of a healing intima was studied by vital staining with Evans blue and transmission electron microscopy after severe mechanical lesion of the rabbit aorta. Reendothelialization, decreasing permeability, and organization of neointimal connective tissue were concomitant events in the healing processes. The determinant factor in decreasing permeability was reendothelialization with the formation of endothelial flaps and junctions. Changes in the subendothelial connective tissue seemed also to be factors that influenced the permeability.

ABSTRACT: Two pregnancy‐associated murine proteins, PAMP‐1 and PAMP‐2, have been found to be immunologically cross‐reacting analogues to the human proteins pregnancy zone protein (PZP) and pregnancy‐specific beta1‐glycoprotien (SP‐1), respectively. In this study, the physiochemical and biological properties of the proteins are compared and the effect on pregnancy of administration of monospecific antibodies against the proteins is reported.

This report describes the intracellular and extracellular localization of fibronectin at the ultrastructural level in primary cultures of aortic smooth muscle cells. Fibronectin was present in all the cisternae of the rough endoplasmic reticulum except the perinuclear cisterna, in large vesicles associated with the trans side of the Golgi complex, and in single large vesicles in the cytoplasm often associated with microtubules. The extracellular microfibrils were heavily stained. In sections parallel to the plane of growth bundles of extracellular microfibrils in continuity with arrays of intracellular microfilaments were observed (fibronexus). The basement membrane around the aortic smooth muscle cells was discontinuous and diffusely stained. The results indicate that fibronectin is localized in the cytoplasmic membranous apparatus of protein synthesis, processing, and secretion. The lack of reaction product in the flat cisternae of the Golgi complex let suggest either that fibronection may not be present in significant amounts within the flattened cisternae or that the method is insufficient in detecting the glycoprotein in this subcompartment off the Golgi complex.

This report describes a transmission electron microscopic study on hybrids between subhexaploid mouse melanoma cells and subtetraploid mouse fibroblasts. The melanoma cell line was heterogeneous in terms of pigment production, but all cells contained melanosomes, although in different stages of development. Characteristic features of the fibroblasts included the cytoskeleton, endocytic vesicles, and occurrence of dilated cisternae of granular endoplasmic reticulum. Despite the gene dosage in favour of the melanoma parent cell, the hybrids were devoid of melanosomes, and their phenotype was typically fibroblastic in character.