[Is scanning electron microscopy a pertinent tool for the analysis of periprostethic breast capsules?]. / Le microscope électronique à balayage environnemental est-il un outil pertinent pour l'analyse des capsules périprothétiques mammaires ?

PURPOSE: Scanning electron microscopy (SEM) is a powerful analytical tool that allows the study of interactions between commonly used biomaterials and the human body. In conventional SEM (HiVac), hydrated biological samples cannot be analyzed in their natural state and must be dried and metallized. The primary goal of this study is to present recent developments in SEM, notably Environmental SEM (ESEM). The secondary objective is to define the potential utility of these new technologies in the study of periprosthetic breast capsules. MATERIALS AND METHODS: Our pilot study group prospectively included 10 patients with breast cancer undergoing 2-stage expander to implant reconstruction. Periprosthetic breast capsule specimens were sampled during expander removal. Each sample was analyzed using both HiVac and ESEM modalities. Energy dispersive X-ray (EDX) studies were also conducted in order to assess the chemical composition of the capsular tissue samples. Under each observation mode, comparisons of samples' three-dimensional surface relief, cellular composition and biofilm presence were made. For each image, a score from 1-3 on a Likert scale was attributed by three independent experts in electron microscopy. RESULTS: HiVac mode was found to be superior to ESEM for the assessment of the three main study parameters (surface relief, cellularity, biofilm). The quality of the EDX analysis was equivalent under both SEM modalities. CONCLUSION: HiVac mode was shown to be more appropriate than ESEM for the global analysis of periprosthetic breast capsules. EDX analysis permits the identification of atypical chemical elements in tissue samples.

Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts.

This study was undertaken to characterize uterine immune factors involved in the establishment of pregnancy in gilts. Thirty crossbred Yorkshire-Landrace gilts of similar age and weight were observed twice a day for oestrous behaviour with intact boars. On the day of first standing oestrus (Day 0) and 12h later, 15 gilts were inseminated with pooled semen from Duroc boars of proven fertility. Pregnant gilts were slaughtered either on Days 10, 15 or 25 of gestation (n=5 per day). The other 15 gilts were not inseminated and were slaughtered on either Days 0, 10 or 15 of the oestrous cycle (n=5 per day). Immediately after slaughter, endometrial tissue samples from the mesometrial side were removed for gene expression using RNase protection assay and in situ hybridization methodologies. The other uterine horn was flushed with 20 ml of PBS to collect the uterine fluid. In pregnant gilts, endometrial interleukin (IL)-6 mRNA expression was higher on Day 15 than on Days 10 and 25 (P<0.01 and P<0.1, respectively). On Day 15, IL-6 expression was also significantly higher (P<0.01) in pregnant gilts than in cyclic gilts. In both pregnant and cyclic gilts, transforming growth factor (TGF)-beta2 in uterine fluid was significantly higher (P<0.0001) on Day 15 than on Day 10. At the gene expression level, TGF-beta2 also increased between Days 10 and 15 in both cyclic and pregnant gilts but differences were not significant. On Day 15, concentrations of interferon-gamma and prostaglandin E(2) (PGE(2)) in uterine fluid were markedly higher (P<0.001) in pregnant gilts than in cyclic gilts, whereas the total amount of TGF-beta2 in uterine fluid and its endometrial expression were approximately 70% higher although this increase was not significant. Finally, tumour-necrosis factor-alpha and granulocyte-macrophage/colony-stimulating factor mRNA expressions were undetectable in all endometrial samples. In conclusion, production and/or expression of uterine TGF-beta2, IL-6 and PGE(2) increased during the embryonic attachment period and are coincidental with embryonic interferon-gamma production.

P34H sperm protein is preferentially expressed by the human corpus epididymidis.

During epididymal transit, mammalian spermatozoa acquire new surface proteins that are necessary for gamete interaction. We have previously described a 34-kDa human epididymal sperm protein, P34H, that has been shown to be involved in sperm-zona pellucida interaction. In the present study, we report the cloning and characterization of the full-length complementary DNA encoding human P34H. The predicted amino acid sequence revealed 65% identity with P26h, the hamster counterpart of the P34H. The deduced P34H amino acid sequence revealed a 71% similarity with a pig lung tetrameric carbonyl reductase, a member of the short chain dehydrogenase/ reductase family proteins. Northern blot analysis revealed that P34H messenger RNA (mRNA) was highly expressed in the human epididymis, principally in the corpus region. A single 912-bp P34H transcript was detected. In situ hybridization experiments showed that the P34H mRNA was predominantly expressed in the proximal and distal sections of the corpus epididymidis. The staining was restricted to the principal cells of the epididymal epithelium. The localization of P34H mRNA was in agreement with the appearance of P34H protein along the male reproductive tract. Western blot analysis revealed that recombinant P34H expressed by a yeast expression system, is antigenically related to the native P34H sperm protein. Based on its pattern of expression and its function in one of the key steps leading to fertilization, P34H can be considered as a marker of epididymal sperm maturation in humans.

CD8 membrane expression is down-regulated by transforming growth factor (TGF)-beta 1, TGF-beta 2, and prostaglandin E2.

PROBLEM: CD8 T-cells are present at a lower frequency in human decidua than in peripheral blood. Because transforming growth factor (TGF)-beta 2 down-regulates CD4 membrane expression, its contribution, as well as the contribution of TGF-beta 1 and prostaglandin (PG) E2, to the modulation CD8 expression was studied using human peripheral blood lymphocytes (PBLs). METHOD OF STUDY: PBLs were cultured with TGF-beta 1, TGF-beta 2, PGE 2, PGI 2, or day-12 rabbit blastocoelic fluid (BF) that was or was not depleted of TGF-beta 2 and/or PGE 2. Quantum Simply Cellular Microbeads were then used to evaluate CD8 membrane expression levels. RESULTS: This study is the first demonstration that treatment of PBLs with TGF-beta 1, TGF-beta 2, and PGE 2 leads to a dose-dependent decrease in CD8 expression. A significant inhibition was observed at 2.5 mg/mL for TGF-beta 2, 5 ng/mL for TGF-beta 1, and 10 ng/mL for PGE 2. In contrast, PGI 2 had no effect. Treatment of PBLs with BF day-12 decreased CD8 expression. This effect, however, was not observed when BF was depleted of TGF-beta 2 and/or PGE 2. CONCLUSIONS: Our results suggest that TGF-beta s and PGE 2 are important modulators of CD8 membrane expression in human lymphocytes. Because TGF-beta 1, TGF-beta 2, and PGE 2 are produced by the conceptus and by uterine cells and because the effect is observed after only 3 days of treatment, the present data suggest that these substances can locally modulate the phenotype of lymphocytes at the fetomaternal interface. Such modulation may explain, at least partly, the changes observed in the population of decidual lymphocytes during pregnancy.

Characterization of endoglin on mouse uterine stromal cells.

During the oestrous cycle and early pregnancy, the uterus undergoes a variety of morphological and physiological modifications involving uterine cell proliferation and differentiation as well as extensive tissue remodelling. Transforming growth factor beta (TGF-beta) has powerful effects on these events and thus is thought to have a critical role in uterine physiology. Endoglin is a transmembrane glycoprotein that binds TGF-beta 1 and -beta 3 and interacts with TGF-beta signalling receptors to modulate many effects of this growth factor in different types of cell. Studies in mice revealed the highest concentrations of endoglin in the reproductive tract, notably on stromal cells of cyclic and pregnant uteri. The objective of the present study was to investigate the role of endoglin expressed on uterine stromal cells in binding TGF-beta and in the cellular responses induced by this growth factor. Highly purified populations of uterine stromal cells were isolated by cell affinity to the monoclonal antibody MJ7/18, which is specific to mouse endoglin. Affinity labelling of these cells with 125I-labelled TGF-beta followed by immunoprecipitation with endoglin-specific polyclonal 1256:4b antiserum indicated that endoglin expressed at the surface of uterine stromal cells binds TGF-beta 1 and interacts with TGF-beta signalling receptors. Treatment of uterine stromal cells with different concentrations of TGF-beta 1 induced a biphasic proliferative response and addition of MJ7/18 as well as neutralizing TGF-beta antibodies showed endoglin to be a modulator of TGF-beta-induced stromal cell proliferation. Given the importance of TGF-beta in the regulation of uterine physiology, these results indicate a role for endoglin during uterine tissue remodelling and decidualization.

Hormonal control of the biosynthesis of hamster oviductin.

In several mammalian species, the epithelial secretory cells of the oviduct synthesize and secrete specific glycoproteins that become associated with the zona pellucida of the ovulated oocyte. These glycoproteins are collectively designated as oviductins. A monoclonal antibody directed against hamster oviductin was used to study the ontogeny of this glycoprotein. Indirect immunofluorescence experiments performed on sections of hamster oviduct revealed that the glycoprotein begins to be secreted in 10-day-old females and that all of the oviductal secretory cells showed fluorescent staining by day 14. The intensity of the immunofluorescence reaction reached a maximum in the 28-day-old females. The oviducts of the 7-day-old hamster incorporated [35S]methionine in vitro into several proteins; however, the production and secretion of detectable amounts of radiolabeled oviductin only began at 14 days of age and reached a maximum at day 28 of age. It appears that the ontogeny of oviductin parallels the hormone dependent changes leading to sexual maturation and that its maximum secretion is already established at the time of the first ovulatory cycle. These results are substantiated by the fact that the production of oviductin is induced in estradiol-treated, but not progesterone or non-treated prepubertal animals, as determined by indirect immunofluorescence experiments.

Localization of endoglin, a transforming growth factor-beta binding protein, and of CD44 and integrins in placenta during the first trimester of pregnancy.

Endoglin is an integral membrane glycoprotein that binds transforming growth factor-beta 1 (TGF beta 1) with high affinity and is predominantly expressed on human endothelial cells. Characterization of this homodimeric protein from human term placenta has shown that it is particularly abundant on the syncytiotrophoblast. Immunofluorescence staining of sections of first trimester placenta now reveals that endoglin is found at even higher levels on the syncytiotrophoblast of samples ranging from 6 to 12 wk of gestation. Very low levels are observed on the undifferentiated cytotrophoblast cells that can be identified by their expression of the alpha 6 beta 4 integrin, a receptor for laminin. Within the villi, blood vessels and stromal cells are negative for endoglin but positive for alpha 1 beta 1 integrin, a receptor for collagens and laminin. Stromal cells also express CD44, a hyaluronic acid receptor. Of particular interest is the up-regulation of endoglin expression in the transition from polarized undifferentiated to non-polarized intermediate cytotrophoblasts (CTB) as the cells align in columns to invade the uterus. This occurs in parallel with the acquisition of alpha 5 beta 1 integrin (fibronectin receptor) and precedes the loss of alpha 6 beta 4 integrin. CD44 and alpha 1 beta 1 integrin are noticeably absent from the CTB within the columns but are expressed at very high levels throughout the placental bed. Endoglin is undetectable within the decidua; thus, intermediate CTB that have invaded the placental bed express alpha 5 beta 1 integrin and cytokeratins but not endoglin.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization and in situ localization of murine endoglin reveal that it is a transforming growth factor-beta binding protein of endothelial and stromal cells.

Endoglin is an integral membrane glycoprotein predominantly expressed on human endothelial cells and recently shown to bind transforming growth factor-beta 1 (TGF beta 1) with high affinity. We now report the cloning and sequencing of a full-length murine endoglin complementary DNA of 2902 base pairs which hybridizes specifically with a single messenger RNA (mRNA) species. The polypeptide of 653 amino acids has an overall identity of 72% with human and porcine endoglin. The transmembrane and cytoplasmic domains of all three proteins differ by two to four amino acids and are 70% identical to the corresponding regions of the TGF beta binding protein, betaglycan. Relative levels of murine endoglin mRNA were estimated by polymerase chain reaction and found to be high in ovary and uterus, intermediate in heart and muscle, and low in placenta and spleen. In situ hybridization and immunofluorescence confirmed that murine endoglin, like its human counterpart, is present in blood vessels and capillaries in all tissues examined. In addition, the stromal cells in the connective tissue of intestine, stomach, heart, muscle, uterus, ovary, and testis were strongly and specifically reactive with complementary RNA probes and with a polyclonal antibody to endoglin; epithelial cell layers were distinctly unreactive. This distribution is similar to that of extracellular TGF beta 1, particularly in heart and uterus, and suggests that endoglin on stromal fibroblast-like cells might be regulating access of TGF beta 1 to the signaling receptor complex. NCTC-2071 fibroblasts in culture were shown to express high levels of endoglin mRNA by polymerase chain reaction. After chemical cross-linking with [125I]TGF beta 1 and immunoprecipitation with the polyclonal antihuman endoglin serum, a radiolabeled band of mol wt 180,000 corresponding to dimeric endoglin was observed under nonreducing conditions, whereas a single band of mol wt 90,000 was seen under reducing conditions. Thus murine fibroblast endoglin is capable of binding TGF beta 1. Future studies should establish the specialized role of endoglin in the TGF beta receptor complex of endothelial and stromal cells.

CD44 in human placenta: localization and binding to hyaluronic acid.

CD44, a receptor for hyaluronic acid (HA), has been identified in the stroma of stem and terminal chorionic villi of human term placenta. The CD44 glycoprotein antigen, isolated from placenta by affinity to monoclonal antibody (mAb) 50B4, consisted mainly of species of M(r) 85,000 and 200,000. Radiolabelled CD44 bound specifically to HA attached to plastic, predominantly via the M(r) 85,000 species; this binding was inhibited by soluble HA and hyaluronidase. The binding of CD44 to HA was also inhibited by mAb 50B4 and IM7.8.1, which recognize epitopes of cluster I and II respectively, but was not blocked by a polyclonal antibody to peptide 18-30 of the B loop (residues 12-101). These results suggest that the portion of the B loop of CD44 implicated in the binding to HA is between amino acids 31-101 and that epitopes located outside the B loop, such as that recognized by mAb IM7.8.1 (between residues 132-215), contribute to this interaction. The presence of a functional CD44 molecule in the human term placenta suggest a role for this molecule in situ in the stabilization and orientation of HA network important in the maintenance of the structural integrity of the placenta.

Assignment of the human endoglin gene (END) to 9q34-->qter.

Endoglin is a homodimeric membrane glycoprotein primarily associated with human vascular endothelium. It is also found on bone marrow proerythroblasts, activated monocytes and on lymphoblasts in childhood leukemia. Endoglin has recently been described as a component of the transforming growth factor-beta (TGF-beta) receptor system as it can bind TGF-beta 1 with high affinity. We now report on the localization of the human endoglin gene (END) to human chromosome 9, by Southern blot analysis of BglII fragments of DNA from human-hamster somatic cell hybrids. This chromosomal localization was confirmed by fluorescent in situ hybridization coupled with Distamicin A (DA)/4',6-diamidino-2-phenylindole (DAPI) banding on human chromosomes. The regional localization was assigned to 9q34-->qter by GTG-banding (G-banding by trypsin using Giemsa stain), indicating a telomeric position with respect to the Philadelphia breakpoint.

The zona pellucida binds the mature form of an oviductal glycoprotein (oviductin).

The use of a monoclonal antibody (MAb) specific for the oviductal zona pellucida (ZP) of the hamster has demonstrated that a new antigen (oviductin) is acquired by the ZP during transit of the oocyte in the oviduct. The epitope that is recognized by the MAb bears a terminal N-acetyl-D-galactosamine residue. We conducted a study in order to determine whether this immunoreactivity of the oviductal ZP results from the addition of the terminal sugar residue to a preformed ZP protein or from the transfer of the mature glycoprotein produced by oviductal secretory cells. We measured the incorporation of [35S]methionine into proteins using four different incubation systems: cumulus oophorus (CO) alone, CO in the presence of oviductal fluid, CO co-incubated with empty oviducts, and CO within intact oviducts. At the end of the incubation period, the ZP, vitelli, dispersed cumulus without oocyte, oviducts, and culture medium were isolated and analyzed for their protein content by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiography, and immunodetection. The cumulus cells synthesized several proteins, independently of the oviductal environment; however, none of these proteins corresponded to oviductin. The ZP and the vitelli of cumulus oophorus that were incubated either alone or in the presence of oviductal fluid did not contain radioactive oviductin. When the oviduct (empty or intact) was present in the incubation system, radiolabeled oviductin was synthesized and secreted into the incubation medium. The ZP picked up a detectable amount of radioactive antigen only in the system in which intact oviducts were incubated.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of distinct epitopes of endoglin, an RGD-containing glycoprotein of endothelial cells, leukemic cells, and syncytiotrophoblasts.

Endoglin is a glycoprotein expressed predominantly on human endothelial cells. It was first identified with mAb 44G4, produced against the pre-B acute lymphoblastic HOON cell line. We now report that four mAbs independently produced against human umbilical vein endothelial cells (HUVECs), chronic myelogenous leukemia in blast crisis, or U-937 pro-monocytic cells stimulated with phorbol myristate acetate also react with endoglin. High levels of reactivity of all mAbs were observed with HUVEC, while intermediate levels were seen with HOON and U-937 cells. By sequential immunoprecipitation from HUVEC and U-937 cell extracts, it was established that RMAC8, HEC-19, 8E11, and 1G2 mAbs react with the same protein as 44G4. Three distinct epitopes recognized by 44G4, RMAC8, and 1G2 mAbs were identified by competitive radioimmunoassay and flow cytometry. The HEC-19 epitope is spatially related to the 44G4 epitope, whereas the 8E11 epitope is most closely related to the 1G2 epitope. Western blot analysis showed that all antibodies react with the endoglin dimer (Mr = 170,000) purified from placenta. Immunostaining of sections of full-term placenta revealed reactivity not only with fetal vessels but also with the syncytiotrophoblast, the fetal cell layer which interfaces with maternal blood. When HUVEC monolayers were treated with the different mAbs to endoglin, prior to incubation with U-937 cells, a 5- to 10-fold stimulation of adhesion was observed. A fibronectin hexapeptide containing RGD, but not the corresponding RGE peptide, was capable of inhibiting the increased adhesion, when tested with mAb 44G4 and RMAC8. However, the same peptides had no effect on the binding of any of the five anti-endoglin mAbs to cells. Since 44G4 and RMAC8 recognize two distinct epitopes of endoglin, and since all five mAbs stimulated adhesion, the results suggest that a signal has been triggered through endoglin on HUVECs. Endoglin might be implicated either directly, by binding to a specific integrin-like ligand, or indirectly, by regulating the level of adhesion between certain integrins and their receptors.

Demonstration by lectin-gold cytochemistry of transfer of glycoconjugates of oviductal origin to the zona pellucida of oocytes after ovulation in hamsters.

We have previously localized an antigen of oviductal origin to the zona pellucida of superovulated hamster oocytes (Kan et al.: Journal of Histochemistry and Cytochemistry 36:1441-1447, 1988) and described the intracellular distribution of this antigen in the oviductal epithelium (Kan et al.: Biology of Reproduction 40:585-598, 1989). These results led to our hypothesis that the oviduct is a bona fide site of origin of certain components of the zona pellucida. In this report, using the high resolution lectin-gold approach with Helix pomatia lectin (HPL)-colloidal gold complex, we present cytochemical evidence to show that glycoconjugates containing terminal N-acetyl-D-galactosamine residues are absent from the zona pellucida of ovarian oocytes but are synthesized and secreted by the nonciliated secretory cells of the oviduct and later become associated with well-defined structural elements of the zona matrix of oocytes during passage through the oviduct. The nature of the HPL-binding glycoconjugates was determined by biochemical analyses. Electrophoretic and immunological experiments demonstrated that the glycoconjugates correspond to the high molecular weight polydispersed glycoprotein that we have previously described. We have designated this glycoprotein "hamster oviductin 1" (Hm OV-1). Our results further substantiate the belief that the oviduct is a source of origin of zona pellucida constituents.

Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation.

The zona pellucida (ZP), a glycoprotein layer that encloses the mammalian oocyte, is formed during follicular development in the ovary, persists at the time of fertilization within the oviduct, and then surrounds the embryo until implantation in the uterus. Although the structure and chemical properties of the ZP have been extensively studied, the precise site of origin of the ZP remains a matter of controversy. Moreover, the mechanism of synthesis and secretion of the ZP constituents is not fully elucidated. We have recently developed monoclonal antibodies (MAbs) against oviductal ZP of the golden hamster. We have used one of these MAbs (an immunoglobulin G) and the protein A-gold technique to study the localization of the corresponding antigenic sites, and we report here their distribution in the oviduct and within the cumulus oophorus complex of the superovulated hamster. In the oviductal epithelium, immunolabeling was observed in non-ciliated secretory cells in structures involved in protein secretion. In the cumulus masses collected from the oviduct, the sites of immunoreactivity were localized exclusively in the ZP encompassing the oocyte. Gold particles were evenly distributed throughout the entire thickness of the ZP. Treatment of the cumulus masses with hyaluronidase prior to preparation of isolated oocytes for immunocytochemistry did not affect this uniformity. The ZP of the preovulatory oocytes in ovarian follicles was not labeled. Our study provides immunocytochemical evidence for the secretion of an oviductal antigen that becomes intimately associated with the ZP of the oocytes during their passage through the oviduct.

Immunoelectron microscopic localization of an oviductal antigen in hamster zona pellucida by use of a monoclonal antibody.

The zona pellucida is an extracellular matrix of glycoproteins which surrounds the mammalian oocyte and preimplantation embryo. We have recently developed monoclonal antibodies against oviductal zona pellucida of the golden hamster. We applied the post-embedding immunocytochemical method using a monoclonal antibody (IgGl,k) to determine the precise location of antigenic sites in the cumulus oophorus complex of the superovulated hamster. By applying the high-resolution protein A-gold technique, we demonstrated that the sites of immunoreactivity were exclusively in the zona pellucida encompassing the oocyte. Other structures within the oocyte and neighboring cumulus cells were not labeled by gold particles. Moreover, gold particles were evenly distributed throughout the entire thickness of the zona pellucida, indicating that this extracellular layer is at least in part made up of an antigen recognized by the monoclonal antibody that is uniformly distributed in the zona matrix.

Characterization of an oviductal glycoprotein associated with the ovulated hamster oocyte.

Hamster oviducts in culture incorporate [35S]-methionine into secretory proteins. One of these proteins is immunoprecipitated by a monoclonal antibody specific to an antigen found in oviductal oocytes but not in ovarian oocytes. This antigen, called oviductin, is progressively added to the oocyte during its transit through the oviduct. Oviductin migrates as a diffuse band with a molecular mass between 160 and 250 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The electrophoretic behavior of this protein suggests the presence of polysaccharide side chains. Chemical deglycosylation causes a decrease in molecular mass and removes the antigenic determinant originally present on the glycoprotein. By using the radiation inactivation method, the molecular mass of the core protein has been found to be approximately 44 kDa. These results indicate that the oviduct is an actual site of synthesis of the oviductin. This glycoprotein contains a high proportion of sugar residues, which account for antigenic determinant recognized by the monoclonal antibody.

Monoclonal antibodies specific for an oviductal component associated with the hamster zona pellucida.

Five monoclonal antibodies (MAbs) were produced against oviductal zona pellucida (ZP) of the hamster. They were purified from ascitic fluid by high pressure liquid chromatography (HPLC) on hydroxylapatite (HPHT) and anion exchange columns. All five MAbs reacted selectively with oviductal ZP and oviductal secretions, no binding was observed to intra-ovarian ZP. A study of the tissue specificity, as evaluated by indirect immunofluorescence, revealed binding of all of these Abs only to the oviduct and, to a lesser extent, to the uterus. A cytosolic fraction from hamster oviduct was subjected to SDS-PAGE under reducing conditions and electro-transfer to a nitrocellulose membrane; immuno-enzymatic staining showed a reaction with a polydispersed oviductal component of high molecular weight (approx. 200,000). The native antigen has a molecular weight higher than 400,000 as determined by molecular sieve chromatography. These results suggest that an oviductal antigen is added to the hamster ZP during its transit through the oviduct. This antigen, called oviductin, is a heavily glycosylated protein of high molecular weight.