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PubMed1999

Low doses of ultraviolet A radiation stimulate adhesion of human dermal fibroblasts by integrins in a protein kinase C-dependent pathway.

In this work, we have studied the modulation of fibroblast-extracellular matrix interactions by physiological doses of ultraviolet A (UV-A) radiation using an adhesion assay on collagen. We have shown that low doses of UV-A (20 kJ/m2) stimulate fibroblast adhesion while higher doses (100 and 200 kJ/m2) inhibit it. By measurement of the thiobarbituric acid reactive substances (TBARS) and use of the chain-breaking antioxidant vitamin E, no role of lipid peroxidation can be detected in these effects. By incubating fibroblasts with a specific protein kinase C (PKC) inhibitor, GF109203X, we have demonstrated that the stimulation of the adhesion by low doses of UV-A involves, at least in part, a PKC-dependent mechanism. In addition, using function-blocking antibodies of alpha 1, alpha 2 or alpha 5 integrin chains involved in extracellular matrix anchorage, we have shown that they decrease the stimulation of adhesion following low doses of UV-A radiation, demonstrating the involvement of these three integrin chains in this UV-A effect. In parallel, 20 kJ/M2 of UV-A are found to rapidly stimulate membrane expression of alpha 1, alpha 2 and alpha 5 integrin chains. This work, which underlines the involvement of integrins in UV-A effects, contributes to the evaluation of the mechanisms by which cell-matrix interactions modulate cell behaviour.

Adolescent↗
PubMedDate not supplied

Mesothelium expression of integrins in vivo and in vitro.

OBJECTIVE: To characterize the expression of alpha subunits of integrin adhesion molecules in peritoneal tissue in vivo and in vitro. METHOD: Peritoneum from the anterior abdominal wall (n = 22) and the serosa of the posterior uterus (n = 11) was obtained from women of reproductive age without endometriosis who were undergoing surgery for benign conditions. Immunohistochemical studies were performed on serial sections of peritoneum from the anterior abdominal wall, the uterine serosa, mesothelial monolayer cultures, and peritoneum explants from the abdominal wall using monoclonal antibodies to alpha subunits of integrin adhesion molecules. Electron microscopy was performed to localize these adhesion molecules in the mesothelium. RESULTS: The mesothelial expression of alpha integrin subunits was identical in the anterior peritoneum and uterine serosa. In vivo the mesothelium strongly expressed alpha 2 and alpha 3 and variably expressed alpha 6. In the monolayer cultures there was moderate/strong staining for alpha 2, alpha 3, and alpha 5; there was minimal expression of alpha v. In the explants there was moderate/strong expression of alpha 2, alpha 3, alpha 5, and alpha v; alpha 6 was variably expressed. The ultrastructure of the mesothelium was unique in the anterior peritoneum, uterine serosa, and the monolayer cultures. The integrin subunits were distributed throughout the cytoplasm, were expressed in the plasma membrane, and were present on the surface (i.e., towards the peritoneal cavity) of the mesothelium. CONCLUSION: Integrins are expressed by the mesothelium of the peritoneum. The mesothelium expression of integrins in vivo differs from that of the mesothelium integrin expression in monolayer culture and explant culture.

Antigens, CD↗
PubMed2004

Rolling adhesion through an extended conformation of integrin alphaLbeta2 and relation to alpha I and beta I-like domain interaction.

In vivo, beta(2) integrins and particularly alpha(L)beta(2) (LFA-1) robustly support firm adhesion of leukocytes, but can also cooperate with other molecules in supporting rolling adhesion. Strikingly, a small molecule alpha/beta I-like allosteric antagonist, XVA143, inhibits LFA-1-dependent firm adhesion, while at the same time it enhances adhesion in shear flow and rolling both in vitro and in vivo. XVA143 appears to induce the extended conformation of integrins as shown by increased activation epitope exposure. Fab to the beta(2) I-like domain converts firm adhesion to rolling adhesion, but does not enhance adhesion. Residue alpha(L)-Glu-310 in the linker following the I domain is critical for communication to the beta(2) I-like domain, rolling, integrin extension, and activation by Mn(2+) of firm adhesion. The results demonstrate the importance of integrin extension in rolling, and suggest that rolling and firm adhesion are mediated by extended conformations of alpha(L)beta(2) that differ in the affinity of the alpha(L) I domain for ICAM-1.

Animals↗
PubMed2006

Importance of force linkage in mechanochemistry of adhesion receptors.

The alpha subunit-inserted (I) domain of integrin alphaLbeta2 [lymphocyte function-associated antigen-1 (LFA-1)] binds to intercellular adhesion molecule-1 (ICAM-1). The C- and N-termini of the alpha I domain are near one another on the "lower" face, opposite the metal ion-dependent adhesion site (MIDAS) on the "upper face". In conversion to the open alpha I domain conformation, a 7 A downward, axial displacement of C-terminal helix alpha7 is allosterically linked to rearrangement of the MIDAS into its high-affinity conformation. Here, we test the hypothesis that when an applied force is appropriately linked to conformational change, the conformational change can stabilize adhesive interactions that resist the applied force. Integrin alpha I domains were anchored to the cell surface through their C- or N-termini using type I or II transmembrane domains, respectively. C-terminal but not N-terminal anchorage robustly supported cell rolling on ICAM-1 substrates in shear flow. In contrast, when the alphaL I domain was mutationally stabilized in the open conformation with a disulfide bond, it mediated comparable levels of firm adhesion with type I and type II membrane anchors. To exclude other effects as the source of differential adhesion, these results were replicated using alpha I domains conjugated through the N- or C-terminus to polystyrene microspheres. Our results demonstrate a mechanical feedback system for regulating the strength of an adhesive bond. A review of crystal structures of integrin alpha and beta subunit I domains and selectins in high- and low-affinity conformations demonstrates a common mechanochemical design in which biologically applied tensile force stabilizes the more extended, high-affinity conformation.

Cell Adhesion↗
PubMed1997

Importance of the tubulointerstitium in human glomerulonephritis. II. Distribution of integrin chains beta 1, alpha 1 to 6 and alpha V.

Accumulation of extracellular matrix is important in the progression of glomerulonephritis. Since adherent cell types utilize integrins to bind and organize extracellular matrix proteins, we have assessed expression of the beta 1 integrins in sequential sections from 85 human renal biopsies and 4 normal kidneys by immunohistochemical staining. Our results demonstrate strong correlations between expression of the alpha 5 chain within the interstitium, the alpha V chain on proximal and distal tubular epithelium and the presence of chronic histological damage. Moreover, staining for interstitial alpha 5 and proximal and distal tubular alpha V were also strongly associated with expression of certain adhesion molecules (ICAM-1, VCAM-1, E-selectin and L-selectin) and the presence of macrophages within the interstitium, which have been linked, in an earlier study, with the degree of chronic histological damage and disease progression. However, in contrast to our earlier study of adhesion molecules, there were also associations between expression of integrin chains within the glomerulus and tubulointerstitium. For example, there were strong positive associations between staining for alpha 5 on glomerular endothelium and its expression on extraglomerular vascular endothelium and between both mesangial alpha 1 and podocyte alpha 3 and tubular staining for the common beta 1 subunit. While the functional significance of these associations is obscure, they suggest some kind of communication between cells in different sites in the kidney. There were also positive associations between staining for different integrins within the glomerulus, notably mesangial cell staining for alpha 2, glomerular endothelial cells staining for alpha 5 and glomerular epithelial cell alpha 3. These results suggest that there is a coordinated upregulation of integrin expression both within the tubulointerstitium and the glomerulus and that at least some of these integrins (interstitial alpha 5 and distal tubular alpha V) are associated with the expression of other adhesion molecules, macrophage infiltration and the presence of markers of disease progression (interstitial fibrosis and tubular atrophy).

Antigens, CD↗
PubMed2000

Modifications of the fiber in adenovirus vectors increase tropism for malignant glioma models.

Recombinant adenovirus (Ad) vectors provide a means of local, therapeutic gene delivery to a wide range of neoplasms. Ad-mediated gene therapy trials in malignant glioma models have been limited by the need for high viral titers and multiple dosages. In an attempt to improve Ad vector gene transfer, we studied human (U87, D54) and rodent (GL261, C6) malignant glioma cell lines transfected with various doses of unmodified Ad vectors (AdZ), Ad vectors that contain an alteration of the fiber-coat protein and that direct virus binding to heparan sulfate receptors (AdZ.F(pK7)), and Ad vectors with modifications of the fiber-coat protein that direct virus binding to alpha1, integrin cellular receptors (AdZ.F(RGD)). AdZ.F(pK7) increased the frequency of cells expressing the reporter gene, beta-galactosidase, and improved transduction by 2- to 20-fold compared with AdZ in U87, D54, and GL261 cells. In U87, D54, GL261, and C6 tumors, AdZ.F(pK7) increased gene transfer by 10- to 100-fold compared with AdZ. AdZ.F(RGD) increased gene expression in C6 xenografts compared with AdZ, but had reduced transduction compared with the C6 xenografts of AdZ in all other glioma tumors. These findings suggest that the increased tropisms resulting from alterations of the Ad vector fiber-coat protein as in AdZ.F(pK7) and AdZ.F(RGD) offer a feasible approach to improving in vitro and in vivo transduction efficiencies in certain malignant glioma cell lines.

Adenoviridae↗
PubMed2006

Wound healing is defective in mice lacking tetraspanin CD151.

The tetraspanin CD151 forms complexes in epithelial cell membranes with laminin-binding integrins alpha6beta4, alpha3beta1, and alpha6beta1, and modifies integrin-mediated cell migration in vitro. We demonstrate in this study that CD151 expression is upregulated in a distinct temporal and spatial pattern during wound healing, particularly in the migrating epidermal tongue at the wound edge, suggesting a role for CD151 in keratinocyte migration. We show that healing is significantly impaired in CD151-null mice, with wounds gaping wider at 7 days post-injury. The rate of re-epithelialization of the CD151-null wounds is adversely affected, with significantly less wound area being covered by migrating epidermal cells. Our studies reveal that although laminin levels are similar in wild-type and CD151-null wounds, the organization of the laminin in the basement membrane is impaired. Furthermore, upregulation of alpha6 and beta4 integrin expression is adversely affected in CD151-null mice wounds. In contrast, we find no significant effect of CD151 gene knockout on alpha3 and beta1 integrin expression in wound repair. We suggest that mice lacking the CD151 gene are defective in wound healing, primarily owing to impairment of the re-epithelialization process. This may be due to defective basement membrane formation and epithelial cell adhesion and migration.

Animals↗
PubMed2001

Expression of beta 1 integrins in human dental pulp in vivo: a comparative immunohistochemical study on healthy and chronic marginal periodontitis samples.

AIM: The objective of this study was to determine the tissue distribution of beta 1 integrin chains in sound human dental pulps and to compare the findings with connective tissue compartments of other organs and to pulp tissue in teeth extracted due to periodontal disease. METHODOLOGY: Freshly frozen pulp tissue samples from teeth extracted for orthodontic reasons were examined and compared to samples from teeth extracted due to chronic (marginal) periodontitis. beta 1 integrin chains were determined using an indirect-immunoperoxidase technique. Seven monoclonal antibodies recognizing alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and beta 1 chains of Very Late Activation Antigen (VLA) integrins were used for this purpose. RESULTS: VLA-1, VLA-2, VLA-3 and VLA-5 were expressed by vascular endothelium and vascular smooth muscle in varying intensities in both groups. VLA-6 reactivity was observed in the basal surfaces of arterial, venous and capillary endothelia. Our results indicate that there was no significant difference in the expression of VLA integrins in sound pulp tissue when compared to the samples from chronic (marginal) periodontitis and the connective tissue compartments of other viscera. CONCLUSION: The present findings suggest that human dental pulp tissue is not different from other connective tissue compartments in the body with respect to VLA integrin expression, and chronic marginal periodontitis does not affect pulp tissue to a histopathologically detectable extent.

Antibodies, Monoclonal↗
PubMed1997

Localization of integrin beta 1, alpha 1, alpha 5 and alpha 9 subunits in the rat testis.

Very late activation (VLA, beta 1; alpha 1; alpha 5, alpha 9) integrins were studied by immunoblotting and immunohistochemistry in the testes of sexually mature rats. All integrin subunits were present in membrane fractions of homogenized testes. Immunohistochemistry revealed that the anti beta 1 antibody recognized peritubular cells and the basement membrane of blood vessels. Immunoreactivity was also demonstrated in the lamina propria, basement membrane, and the basal cytoplasm of Sertoli cells. In elongating spermatids, beta 1 integrin was localized to the acrosome. The alpha 1 subunit was expressed in peritubular cells and in the lamina propria. In the adluminal compartment, round spermatids were stained diffusely for the alpha 1 subunit. Immunoreactivity for alpha 1 integrin was found additionally in the acrosomes of elongating spermatids shortly before their release into the seminiferous tubule lumen. The alpha 5 subunit was expressed in the acrosomes of elongating spermatids as well as in their distal cytoplasm during stages III-VI; the cytoplasmic lobes of elongate spermatids and/or residual bodies also appeared to be immunostained in seminiferous tubules at stages VII-VIII. The alpha 9 subunit was immunolocalized only in the basement membrane and in peritubular cells. These data suggest that integrins are involved in spermatogenesis, in particular in the process of spermatid maturation.

Animals↗
PubMed1997

Expression of integrins during axolotl limb regeneration.

Limb regeneration in urodeles is achieved through the dedifferentiation of tissues at the amputation plane and through the production of the blastema. This tissue breakdown is possible by extensive alterations in molecules of the extracellular matrix. In this respect we describe the regulation of several integrins during such events. It was found that alpha 1 and beta 1 integrins were down-regulated as blastema formation proceeded. In contrast, the expression of alpha 3, alpha 6 and alpha v integrins were upregulated in the blastema. These data are consistent with the roles of integrins in developmental phenomena and are discussed in light of the mechanisms of dedifferentiation.

Ambystoma↗
PubMedDate not supplied

Fibroblast subpopulations in intra-oral wound healing.

The objective of this study was to characterize fibroblasts at sequential time points during intra-oral wound healing in the rat. Experimental wounds were made at several time points in the mucoperiosteum of the palate of 35-day-old Wistar rats. Fibroblasts were cultured from the biopsies under standard conditions for the same number of passages. The expression of the integrin subunits alpha 1, alpha 6, and beta 1; and the intermediate filaments alpha-smooth muscle actin and vimentin were analyzed by flow cytometry. Western blot analysis was performed at 0, 8, and 60 days postwounding to confirm the expression of both intermediate filaments. The phenotypic profiles of fibroblasts cultured from subsequent stages in the wound healing process differed considerably. We conclude that distinct fibroblast phenotypes can be isolated from different stages in wound healing. These phenotypes remained stable during in vitro culturing. In addition, cryosections of the wound areas were made at identical time points and were immunohistochemically stained for the same antigens. The immunohistochemical staining correlated well to the flow-cytometric data. These results suggest the occurrence of multiple subpopulations of fibroblasts with a specialized function during wound healing. We hypothesize that undesirable consequences of wound healing might be prevented through the modulation of specific fibroblast subpopulations.

Animals↗
PubMed1997

Expression of beta-1 integrins in ganglionic and aganglionic segments of patients with Hirschsprung's disease.

An immunocytochemical study was performed to assess the role of beta-1 integrins in the pathogenesis of Hirschsprung's disease. Fresh tissue samples from both aganglionic and ganglionic segments of five patients who were undergoing surgery for Hirschsprung's disease were obtained. Samples were rapidly frozen in liquid nitrogen. Sections were cut and stained using anti alpha-1, 2, 3, 4, 5, 6 and beta-1 monoclonals according to the indirect immunoperoxidase method. The evaluation did not reveal any significant change of pattern in the distribution of beta-1 integrins in the non-neural elements of both aganglionic and ganglionic segments of colon, such as the epithelium, muscularis mucosa, muscularis externa, connective tissues and blood vessels. Nerve fibres in both aganglionic and ganglionic segments strongly expressed the alpha-6 chain of very late activation antigen which led to their increase in the aganglionic segment. In addition to revealing the increase, alpha-6 monoclonals also had in situ positive control due to their presence in non-neural elements. Hence, immunostaining of the suction biopsies with anti alpha-6 monoclonals may be employed as a new and simple method in the diagnosis of Hirschsprung's disease. On the other hand, beta-1 integrins do not seem to play a role in the defective migration of ganglion cells occurring in Hirschsprung's disease.

Antigens, CD↗
PubMed1996

The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins.

Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin.

Adhesins, Bacterial↗
PubMed1996

The recognition sites of the integrins alpha1beta1 and alpha2beta1 within collagen IV are protected against gelatinase A attack in the native protein.

The susceptibility of three different solubilized forms of type IV collagen to gelatinase A cleavage and the concomitant effects on cell and integrin binding have been assessed. Dithiothreitol-solubilized Engelbreth-Holm Swarm (EHS) type IV collagen with disrupted intramolecular disulfide bonds in the CB3[IV] region was cleaved N-terminally to the CB3[IV] region into the two characteristic 100-300-nm fragments at 30 degrees C and was totally degraded at 37 degrees C. This was reflected in the partial or total loss of the alpha1beta1 and alpha2beta1 integrin binding sites within this region. The ability of gelatinase A to cleave EHS type IV collagen preparations with intact interchain disulfide bonds in CB3[IV] only occurred at higher temperatures. Furthermore, no effect on binding of cells or isolated integrins to the gelatinase-treated collagen could be detected after treatment at 37 degrees C. Dimeric collagen IV of human placenta with intact disulfide bonds in the CB3[IV] region was not degraded at all by gelatinase A at 37 degrees C.

Amino Acid Sequence↗
PubMed2001

Selective binding of collagen subtypes by integrin alpha 1I, alpha 2I, and alpha 10I domains.

Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.

Antigens, CD↗
PubMed2004

The fibril-associated collagen IX provides a novel mechanism for cell adhesion to cartilaginous matrix.

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.

Amino Acid Sequence↗
PubMed2000

An oncogenic form of the Flt-1 kinase has a tubulogenic potential in a sinusoidal endothelial cell line.

We have previously reported a constitutively activated form of the Flt-1 kinase (BCR-FLTm) molecularly engineered based on the structural backbone of the activated tyrosine kinase BCR-ABL. Here we show that it can induce not only growth stimulation but also tubulogenic differentiation of non-tubulogenic NP31 (non parenchymal) sinusoidal endothelial cells of rat liver in basement membrane matrix. Tubules formed in vitro were accompanied by fenestration structures and allowed circulation when transplanted into syngeneic animals. This biological response was not observed in other activated forms of kinases constructed in a similar fashion, which include Trk (BCR-TRK), KDR (BCR-KDR), and the parental BCR-ABL. Interestingly, formation of fine tubules was accomplished with lower but not higher expression levels of BCR-FLTm. Compared to NP cells in primary culture NP31 is deficient in expression of alpha1 integrin subunit, which was restored by expression of BCR-FLTm that had tubulogenic ability. Matrix-induced tyrosine phosphorylation of an adaptor protein Shc with recruitment of Grb-2 was observed even when tubulogenesis was nearly completed at G1 stage of the cell cycle in 2-3 weeks. Activation of matrix metalloproteinase 2 (MMP-2) and expression of urokinase type plasminogen activator (uPA) was observed with cellular invasion into matrix at the depth of 200-300 microm. Inhibitors for MAP kinase activator MEK1 and for serine proteases showed deleterious effects on the tubulogenesis. We suppose that matrix ligand-induced integrin signals cooperate with a low level of Flt-1 kinase activity to promote tubulogenic behaviors of endothelial cells in this system.

3T3 Cells↗
PubMed2005

Measures of maternal-fetal interaction in day-30 bovine pregnancies derived from nuclear transfer.

Embryonic mortality and abnormal placental morphology have been reported by most researchers studying nuclear transfer (NT), and it now is accepted that placental anomalies and poor development of cloned embryos are related. As early as day 50 of gestation, cloned bovine concepti exhibit poor structural organization of the developing placentomes. These experiments were designed to identify alterations in maternal-fetal interactions during establishment of the placentas of NT-derived embryos at day 30 of gestation. Bovine NT embryos were produced using cultured fibroblast cells from a single Hereford donor cow, and control embryos were derived from in vitro fertilization (IVF). Following in vivo culture in ligated sheep oviducts, day-8 blastocysts were transferred to synchronized recipient heifers. Tissues recovered from viable day-30 pregnancies were analyzed by real-time RT-PCR, immunohistochemistry, and quantitative histological techniques. Immunoperoxidase staining of caruncular tissue from NT- and IVF-derived pregnancies revealed no significant differences in expression of the extracellular matrix proteins, collagen type IV and laminin, or the receptor subunits, integrins alpha1 and alpha3, suggesting that altered expression of these proteins at day 30 of gestation is not a primary cause of abnormal placentome structure in cloned concepti. Percentage of binucleate cells (BNC) within the trophoblast also was similar in NT- and IVF-derived pregnancies; however, expression of the BNC-specific placental lactogen (PL) transcript was elevated in NT-derived concepti (p < 0.05). These results indicate that regulation of PL transcription was altered in cloned day-30 placental tissues, suggesting the presence of irregular fetal-maternal signaling patterns that might undermine continued development of NT-derived concepti.

Animals↗