Immunohistochemical localization of insulin-like growth factor 1 and 2 in the endocrine pancreas of rat, dog, and man, and their coexistence with classical islet hormones

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Immunohistochemical techniques were used to study the occurrence and distribution of insulin-like growth factor 1 (IGF-1) and IGF-2 in the pancreas of man, dog, and rat and their possible coexistence with insulin (INS), glucagon (GLUC), somatostatin
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  Placenta (1993), 14, 1-12 Immunohistochemical Localization of Insulin-like Growth Factors (IGFs) and IGF Binding Proteins--1, -2 and -3 in Human Placenta and Fetal Membranes D. J. HILL b, D. R. CLEMMONS a, S. C. RILEY, N. BASSETT J. R. G. CHALLIS MRC Group in Fetal and Neonatal Health and Development, Lawson Research Institute, St Joseph's Health Centre, Departments ~ f Physiologv, Medicine and Obstetrics and Gynaecology, University of Western Ontario, London, Ontario. a Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, 27599, USA b To whom correspondence should be addressed at: Lawson Research Institute, St Joseph's Health Centre, 268 Grosvenor Street, London, Ontario, Canada, N6A 4V2 Paper accepted 5.5.1992 SUMMARY Insulin-like growth factor (IGF) I and II are synthesized within the placenta and are believed to play an important role in the regulation of placental growth and endocrine function. IGF bioavailability is determined at a cellular level by several spedfic binding proteins (IGF BPs), which are widely but selectively distributed in all developing tissues. We have used immunohistochemistry to localize IGF I and II peptides, and IGF BP-1, -2, and -3 in human placentae, fetal membranes and umbilical cord at 6-8 weeks after therapeutic termination and at term after spontaneous delivery. Primary antisera were directed against human IGF I, human IGF BP-1, bovine IGF BP-2, and human IGF BP-3 respectively. Immunoreactive IGF BP-2 was found in association with the syncytiotrophoblast, intermediate trophoblasts of the etal villi and chorion, amnion and decidua; while weaker staining was seen in some but not all cytotrophoblasts. A similar but less intense staining pattern was observed or IGF BP- 1 and IGF peptides in placenta and amnio-chorion. Strong immuno-staining for IGF BP-1 was seen in deddual cells. No immuno- reactive IGF BP-3 was found in placenta or membranes. A co-distribution of lGF BP-2, BP-1 and IGF peptides in placenta suggests a role for these IGF BPs in determining the localization of the IGFs for actions on target tissues. 0143-4004/93/010001 + 12 $08.00/0 9 1993 Bailli~re TindaU Ltd  2 Placenta (1993), VoL 14 INTRODUCTION The insulin-like growth factors (IGFs) I and II have the potential for diverse biological actions within placenta. IGF I has a mitogenic action on placental stromal fibroblasts (Fant, Munro and Moses, 1986) and insulin-like metabolic effects such as an increase in amino acid transport by human placenta cells (Bhaumick and Bala, 1984). Both !GF I and II stimulated 3fl-hydroxysteroid activity within isolated human cytotrophoblasts (Nesder, 1989, 1990), supporting a role for IGFs in the maintenance of placental progesterone production but inhibited aromatase activity (Nestler, 1987, 1990). Exposure to IGF I was also found to increase placental lactogen release from human placental explants (Bhaumick, Dawson and Bala, 1987). The tissue source of IGF I and II that might exert placental effects is not well defined. Although both peptides are present in the human fetal circulation they are also synthesized by the placenta itself(Fant, Munro and Moses, 1986; Han et al, 1988; Brice et al, 1989). IGFs are found in association with specific binding proteins, of which there are at least six species (Shimasaki et al, 1991) that have been detected in blood and extracellular fluids, tissue extracts and cell-conditioned culture medium (Ooi and Herington, 1988). Human IGF BP-1 is a 28-30 kDa protein first purified from amniotic fluid with similar affinity for IGF I and II (Povoa et al, 1984). It is identical to proteins previously termed placental protein 12, or pregnancy-associated endometrial al-globulin (Bell and Keyte, 1988; Brewer et al, 1988; Julkunen et al, 1988). IGF BP-2 is a 34 kDa peptide with greater affinity for IGF II than I which, like IGF BP-1, is present in normal, adult serum (Binkert et al, 1989; Hossenlopp et al, 1990). Human IGF BP-3 exists in postnatal serum as a 150 kDa IGF:BP complex, comprising a glycosylated IGF-binding subunit with a greater affinity for IGF II than I (IGF BP-3) and an acid-labile subunit that will only bind to IGF BP-3 in the presence oflGF I or II at neutral pH (Baxter and Martin, 1989). Some 150 kDa complex is found in term human blood but it is absent in second trimester (D'Ercole et al, 1980). However, IGF BP-3 is abundant in fetal serum at this time (Lassarre et al, 1991) suggesting that the acid- labile subunit is lacking during early development. Human IGF BP-4 to -6 have recently been cloned from a placental library and their amino acid sequences deduced (Shimasaki et al, 1990, 1991). However, the physiological role of these proteins is at present unknown. While the various IGF BPs enhance the biological stability of IGF I and II (Ooi and Herington, 1988) recent observations suggest that they have key roles in determining IGF bioavailability. Exogenous IGF BP- 1 or -3 were able to modulate the mitogenicity oflGF I in isolated cell cultures, in either an augmentive or an inhibitory manner (Elgin et al, 1987; De Mellow and Baxter, 1988; Adashi et al, 1991). Similarly, Rutanen, Pekonen and Makinen (1988) demonstrated that IGF BP-1 inhibited the binding of IGF I to human endometrial tissue and blocked IGF I bioactivity, while Rit-vos et al (1988) found that IGF BP- 1 decreased the mitogenic potency oflGF I on choriocarcinoma cells. The differential ability oflGF-BPs to regulate IGF bioactivity is likely to be related to their association with cell membranes, leading to modifications of IGF:receptor interactions (Clemmons et al, 1987). For instance, IGF BP-3 has a different affinity for IGF I in the soluble versus the membrane-bound state (McCusker et al, 1990). Analysis of the anatomical distribution of IGF BPs in placenta and fetal membranes is therefore important in defining the possible target sites and magnitude of IGF action in vivo. In this study, we have used immunohistochemical techniques to determine the presence and distribution of IGF BPs-1, -2 and -3, together with IGF peptides, in human placenta, fetal membranes and umbilical cord after therapeutic termin- ation in first trimester and after spontaneous term delivery.  Hill et ah IGF Binding Proteins in the Human Placenta METHODS AND MATERIALS Sources of material Human placentae, fetal membranes with adherent decidua, and umbilical cord were obtained following 12 full-term spontaneous deliveries (40-42 weeks gestation) at St Joseph's Health Centre, London, Ontario dUring the routine collection of tissues for pathological examination. Tissue was also obtained from five patients undergoing therapeu- tic abortion between 6 and 8 weeks of gestation (performed under the approval of the Abortion Committee, Victoria Hospital, London, Ontario). Additional tissues from five patients who underwent therapeutic abortion at 10-12 weeks gestation were kindly provided by Dr C. G. Goodyer, Children's Hospital, Montreal, Quebec. Placentae, membranes and cord were fixed for 24 h in 10 per cent (v/v) neutral-buffered formalin, dehydrated in ascending ethanol series (70, 90 and 100 per cent) and xylene, and embedded in paraffin. The immunodetection ofIGF I or II in tissue sections was found to be improved by fixation in 2 per cent (w/v) paraformaldehyde and 2 per cent (v/v) glutaraldehyde in 0.01 M phosphate- buffered saline (PBS; pH 7.4) at 4~ for 18-24 h. Following two rinses in PBS, each of 24 h, tissues were dehydrated and embedded as described above. This technique was used for tissues from five term deliveries. Tissue sections (5/~m) were prepared using a rotary microtome and mounted on glass slides, before baking at 45~ for 48 h and storage at room temperature for immunohistochemistry. Immunohistochemistry Immunohistochemistry was performed using the avidin-biotin-peroxidase method of Hsu, Raine and Fanger (1981). Briefly, tissue sections were de-paraffinized in xylene, rehydrated in a descending ethanol series (100, 90 and 70 per cent) into PBS and incubated in 1 per cent (v/v) hydrogen peroxide in PBS for 15 min to eliminate endogenous peroxidase activity. Non-specific binding was blocked by incubation in 2 per cent (v/v) normal sheep serum in PBS. Tissue sections were incubated with a 1:1000 dilution of each of the primary antisera (except those recognizing a 1 anti-chymotrypsin at 1:800 and lysozyme at 1:400) in 0.01 M PBS containing 1 per cent (v/v) normal sheep serum and 0.01 per cent (w/v) sodium azide (pH 7.4) for 48 h at 4~ in a humidified chamber. Following washing in PBS, 100/~1 of affnity-pufified biotinylated goat anti-rabbit IgG (1:20 dilution in PBS; Sigma Chemical Co., St Louis, MO) was applied to each section and the incubation continued for 40 min at room temperature. Following a further wash with PBS the sections were covered with 100#1 of Extravidin peroxidase (1:20 dilution in PBS; Sigma) and incubated for a further 40 rain at room temperature. The antibody-bound peroxidase was visualized by the addition of 0.075 per cent (w/v) 3, 3' diaminobenzidine (Aldrich Chemicals Inc., Milwaukee, WI) in Tris-HC1 buffer (0.05 M, pH 7.6) containing 0.02 per cent (v/v) of 30 per cent hydrogen peroxide for between 2 and 5 min. Sections were lightly counterstained with Carazzi's haematoxylin, dehydrated in an ascending ethanol series and xylene, and mounted under glass coverslips with Permount (Fisher Scientific, Toronto, Ontario). Tissues were examined by light microscopy. For comparison, some tissue sections were also examined by light microscopy following immunohistochemistry using the peroxidase anti-peroxidase technique as de- scribed previously (Sternberger et al, 1970); or were subjected to immunofluorescent staining. Immunofluorescent staining was performed as described above except that follow- ing exposure to the primary antiserum slides were washed in PBS and exposed to 1:100 dilution of goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC, Sigma)  4 Placenta H993), Vol. 14 for 1 h. Slides were then washed in three changes of PBS and mounted in a non-quenching mountant. Antibodies The following primary antisera were used. 1) A rabbit anti-human IGF BP- 1 raised against a 31 kDa IGF BP-1 purified to homogeneity from amniotic fluid as described by Busby, Klapper and Clemmons (1988a). This antibody is identical to that described and character- ized by Busby, Snyder and Clemmons (1988b). 2) A rabbit anti-bovine IGF BP-2 raised against a 34 kDa IGF BP-2 purified from serum-free culture medium conditioned by the Madin-Darby bovine kidney (MDBK) cell line as reported by Bourner et al (1991). This antiserum recognized a single 34 kDa protein following Western bl0t analysis of human serum. 3) A rabbit anti-human IGF BP-3 raised against a de-glycosylated form of human recombinant IGF BP-3 produced and purified as described by McCusker et al (1991). 4) A rabbit anti-human IGF I antiserum raised against natural, human IGF I and kindly provided by Dr D. Morrell, Department of Growth and Development, Institute of Child Health, London, England. 5) A rabbit anti-bovine antiserum recognizing bovine muzzle cytokeratins (Dako Corp., Santa Barbara, CA) which was used to identify epithelial cell types in placenta and fetal membranes. 6) Rabbit anti-human a 1 anti-chymotrypsin and rabbit anti-human lysozyme (Dako) which were used to identify placental Hofoauer calls. Antisera against bIGF BP-2 and hlGF BP-3 were prepared by injecting rabbits intradermally with the pure peptides in complete Freund's adjuvant (Sigma). Rabbits were re-injected subcutaneously 4 weeks later with IGF BP in 50% incomplete Freund's adjuvant. Immunospecificity Several criteria were used to assess the specificity of staining for IGF BPs and IGF peptides. Immunostaining was abolished when a) non-immune rabbit serum was substituted for the primary antiserum; b) the biotinylated secondary antiserum was omitted; and c) the primary antiserum was pre-incubated overnight at 4~ with an excess of homologous ligand. In order to assess the cross-reactivity between each IGF BP antiserum and heterologous BP ligands two methods were used. First, increasing amounts of purified hIGF BP-1, bIGF BP-2 or hIGF BP-3 were subjected to Western blot analysis following 12.5 per cent sodium dodecyl sulphate polyacrylamide gel electrophoresis and transfer to nitrocellulose. Filters were incubated with 1:1000 final dilution of each antiserum in Tris-HC1 buffer (pH 7.2) for 90 min before washing and the visualization of BP:antibody complex using an alkaline phosphatase-conjugated antibody detection system (Sigma). The antiserum against hIGF BP-1 showed approximately 2 per cent cross-reaction with bIGF BP-2 and no cross- reactivity with hlGF BP-3; that against blGF BP-2 showed a 1.5 per cent cross-reaction with hIGF BP-1 and less than 0.1 per cent cross-reaction with hIGF BP-3; while the antiserum against hIGF BP-3 demonstrated an approximate 2 and 5 per cent cross-reactivity with hIGF BP-1 and bIGF BP-2 respectively. Specificity of staining for IGF BPs were also examined on sections of placenta and fetal membranes, and is described under Results. Immunostaining for IGF BPs was not abolished when the relevant primary antiserum was pre-incubated with 130 nM recombinant hIGF I or II (Bachem Inc., Torrance, CA). In order to assess the cross-reactivity of the antiserum against hIGF I with IGF II, increasing amounts oflGF I or II were subjected to Western blot analysis as described above. The antiserum showed approximately a 10 per cent cross-reactivity with IGF II by this technique, although in solution assay the cross-reactivity was only 4 per cent. This suggests that both IGF I and IGF II are likely to be detected in tissue sections by the anti-hlGF I  Hill et al. IGF Binding Proteins in the Human Placenta 5 antiserum. Specificity studies on tissue sections are described under results. When the IGF I antiserum was used within a radioimmunoassay to detect IGF I in fractions eluting from a Sephadex G-200 chromatographic separation of human fetal fibroblast-conditioned culture medium, at neutral pH, immunoreactivity was detected with a Mr of 30-40 kDa, consistent with the presence of IGF:BP complexes, as demonstrated previously (Hill et al, 1987). This suggests that the antiserum recognizes IGFs when associated with IGF BPs. Immunostain- ing for IGF peptides on placental sections was not blocked following pre-incubation of the anti-hIGF I antiserum with 30 nM hIGF BP-1, bIGF BP-2, or hIGF BP-3. RESULTS Positive staining was consistently observed in placenta and fetal membranes from mid- and late first trimester until term with specific antisera recognizing IGF BP-1, BP-2 and IGF peptides utilizing the avidin-biotin-peroxidase method. Specificity of staining was deter- mined for sections from term placenta 9 Pre-incubation of anti-blGF BP-2 antiserum with 40 nM blGF BP-2 completely abolished the staining pattern [Figure 1 a) versus Figure 1 (b)]. Staining was not blocked following pre-incubation of the anti-serum with 40-200 nM hlGF BP-1 [Figure 1 (a) versus Figure. 1 (c)] or hlGF BP-3 (not shown). Similarly, pre-incubation 9 9 9 ~ of anti-hlGF BP-1 antiserum with 40 nM hlGF BP-1 abolished positive staining while pre- incubation with up to 200 nM blGF BP-2 or hlGF BP-3 was ineffective. Positive staining achieved with anti-hlGF I antiserum was blocked following pre-incubation with either 100 nM hlGF I or 500 nM hlGF II, confirming the inability of this antiserum to discriminate adequately between IGF I and II in this tissue type. When these specificity studies were repeated with sections of fetal membranes taken at term Or first trimester similar findings were obtained. Positive staining of placenta and membranes was also obtained with antisera recognizing IGF BP-1, BP-2 and IGF peptides using the peroxide anti-peroxidase or immunofluorescent techniques. These did not offer better definition of immunopositivity than did the avidin-biotin-peroxidase method. Placenta Immunocytochemical localization of IGF BP-2 in term placenta showed a major presence in the syncytiotrophoblast with strong cytoplasmic staining but no staining of the nuclei [Figures 1 (a) and 2(a)]. Occasional large cytotrophoblasts with prominent nucleus which were seen adjacent to the syncytium showed a mixed pattern of staining, some being weakly immuno- positive while others were immunonegative. Weaker staining for IGF BP-2 was seen in the fetal placental stroma and associated with the extracellular matrix. The cytoplasm of fetal stromal cells was largely immunonegative, but positive staining was found in the cytoplasm of Hofbauer cells and endothelial cells of the fetal capillaries [Figures 2(a) and Co)]. Hofbauer cells were identified by their immunopositive staining with antisera against a 1 anti- chymotrypsin or lysozyme. A population of rounded cells with prominent nuclei was seen in the placental villi which stained strongly for IGF BP-2 [Figure 2(b)]. These cells were also immunopositive for cytokeratins when staining was performed on adjacent sections, sugges- ting that they were epithelial and probably intermediate trophoblasts. Weak immunopositive staining was observed with anti-hIGF BP-1 antiserum in term placenta with a distribution similar to that of IGF BP-2 [Figure l(d)]. Only weak staining, which could not be convincingly distinguished from non-specific staining, was observed when using the anti-
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