Differential expression of extracellular matrix components in the Fallopian tubes throughout the menstrual cycle

ABSTRACT: BACKGROUND: One of the unique characteristics of the female genital tract is the extensive tissue remodeling observed throughout the menstrual cycle. Multiple components of the extracellular matrix take part in this tissue rebuilding; however, the individual components involved have not been identified. METHODS: In the present study, the expression of extracellular matrix proteins and selected matrix metalloproteinase (MMP) activities in Fallopian tubes (FT) throughout the menstrual cycle were examined by PCR array, immunocytochemistry, zymography and bioinformatics. RESULTS: Of the eighty-four genes analyzed, eighty-three were expressed in the FT during at least one stage of the menstrual cycle. We observed a significant increase (>/=2-fold) in ADAMTS1, ADAMTS13, COL7A1, MMP3, MMP9, PECAM1, and THBS3 in the periovulatory phase compared to the follicular phase. Meanwhile, we observed a significant decrease (>/= 2-fold) in COL7A1, ICAM1, ITGA8, MMP16, MMP9, CLEC3B, SELE and TIMP2 in the lutheal phase compared to the periovulatory phase. Immunocytochemistry showed that MMP-3 and MMP-9 were localized in the endosalpinx during all phases of the menstrual cycle. Gelatin zymograms detected non-cycle-dependent protease activity. CONCLUSIONS: Several extracellular matrix components were regulated throughout the menstrual cycle in a cyclic pattern, suggesting a possible steroid regulation and a role in tissue remodeling and FT functions.     

Differential temporal and spatial progerin expression during closure of the ductus arteriosus in neonates

Closure of the ductus arteriosus (DA) at birth is essential for the transition from fetal to postnatal life. Before birth the DA bypasses the uninflated lungs by shunting blood from the pulmonary trunk into the systemic circulation. The molecular mechanism underlying DA closure and degeneration has not been fully elucidated, but is associated with apoptosis and cytolytic necrosis in the inner media and intima. We detected features of histology during DA degeneration that are comparable to Hutchinson Gilford Progeria syndrome and ageing. Immunohistochemistry on human fetal and neonatal DA, and aorta showed that lamin A/C was expressed in all layers of the vessel wall. As a novel finding we report that progerin, a splicing variant of lamin A/C was expressed almost selectively in the normal closing neonatal DA, from which we hypothesized that progerin is involved in DA closure. Progerin was detected in 16.2%±7.2 cells of the DA. Progerin-expressing cells were predominantly located in intima and inner media where cytolytic necrosis accompanied by apoptosis will develop. Concomitantly we found loss of α-smooth muscle actin as well as reduced lamin A/C expression compared to the fetal and non-closing DA. In cells of the adjacent aorta, that remains patent, progerin expression was only sporadically detected in 2.5%±1.5 of the cells. Data were substantiated by the detection of mRNA of progerin in the neonatal DA but not in the aorta, by PCR and sequencing analysis. The fetal DA and the non-closing persistent DA did not present with progerin expressing cells. Our analysis revealed that the spatiotemporal expression of lamin A/C and progerin in the neonatal DA was mutually exclusive. We suggest that activation of LMNA alternative splicing is involved in vascular remodeling in the circulatory system during normal neonatal DA closure.     

Cell-type-specific expression of transforming growth factor-alpha in the mouse uterus during the peri-implantation period.

Immunohistochemistry as well as in situ and Northern blot hybridization were employed to determine temporal and cell-type-specific expression of transforming growth factor-alpha (TGF-alpha) in the mouse uterus during the peri-implantation period. The co-localization of TGF-alpha (by immunohistochemistry) with its mRNA (by in situ hybridization) in the luminal and glandular epithelia on Days 1-4 of pregnancy (Day 1 = vaginal plug) and also in many stromal cells on Days 3 and 4 indicates that these cells are the primary sites of TGF-alpha synthesis during the preimplantation period. The higher levels of TGF-alpha mRNA in total uterine RNA on Day 4, as shown by Northern blotting, is consistent with the recruitment of stromal cells expressing this gene. During the post-implantation period (Days 5-8), the co-localization of the mRNA and protein in the decidua at the implantation sites suggests that the decidualizing stromal cells synthesize TGF-alpha. Although in situ hybridization showed the presence of mRNA in embryos on Days 5-8, immunostaining was noted in the embryo only on Days 5 and 6. These results suggest that uterine and embryonic expression of TGF-alpha during the peri-implantation period could be involved in embryonic development, preparation of the uterus for implantation, and decidualization.     

Differential spatial distribution and temporal regulation of tissue inhibitor of metalloproteinase mRNA expression during rat central nervous system development

The elucidation of the cellular and molecular mechanisms governing the maturation of the central nervous system (CNS) is rapidly emerging. Cell-cell and cell-matrix interactions play critical roles in all phases of developmental tissue remodeling. Throughout development, an intricate balance between extracellular matrix synthesis and degradation is preserved by the opposing actions of matrix metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Although recent evidence suggests that TIMPs exert diverse cell biological functions distinct from their MMP-inhibitory activities, few studies have investigated MMP or TIMP expression during CNS development. The present report analyzes the mRNA expression of the four known TIMPs throughout the course of embryonic and postnatal rat CNS development. The results clearly demonstrate the unique spatial distribution and temporal regulation of TIMP expression and suggest a distinct role for each TIMP during CNS development.     

Differential expression of insulin-like growth factors in the uterine epithelium and extracellular matrix during early pregnancy

We have studied the simultaneous expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) in the uterine epithelium and extracellular matrix during the time of trophoblast attachment and implantation. These studies reveal that IGF-I and IGF-II display different spatial and temporal patterns of expression during early pregnancy, and suggest a role for them in the process of attachment and implantation. Specifically, IGF-I is strongly expressed in the basal lamina which is the site of trophoblast invasion into the maternal stroma, and also in the apical epithelium, the site of initial trophoblast attachment. IGF-II is expressed to a lesser extent in the basal lamina, lateral plasma membranes and apical epithelium on day 3 but is only prominent apically at the time of implantation, suggesting a role in attachment.     

Constitutive expression of uterine receptors for insulin-like growth factor-I during the peri-implantation period in the pig.

Insulin-like growth factor-I (IGF-I), synthesized by the uterine endometrium of cyclic and early pregnant gilts, accumulates in the uterine luminal fluid, where it comes in contact with the developing conceptus and the rapidly growing uterus. The uterus and the conceptus thus represent potential target sites for the biological effects of IGF-I, provided high-affinity Type I receptors are present. This study was undertaken to evaluate the expression of functional IGF-I receptors in the endometrium and myometrium of pregnant (Day 10, 12, and 15) gilts and in the endometrium of cyclic (Day 15) and pseudopregnant (Day 15) gilts and to correlate levels of these receptors with temporally regulated uterine production of IGF-I. Specific binding of 125I-IGF-I to endometrial membranes pretreated with MgCl2 (4 M) at 4 degrees C for 16 h, was saturable and membrane concentration-dependent. Competition of 125I-IGF-I binding to endometrial membranes was highest with unlabeled IGF-I greater than IGF-II much greater than insulin, whereas porcine relaxin was noncompetitive. Affinity cross-linking of endometrial membranes with 125I-IGF-I followed by SDS-PAGE and autoradiography revealed two labeled bands of Mr greater than 200,000 and Mr 135,000, with the major band being the Mr 135,000 species. Scatchard analysis of 125I-IGF-I binding to endometrial membranes from Day 12 pregnant gilts revealed a single class of binding sites with a dissociation constant (Kd) = 4.08 +/- 0.09 nM. Membranes prepared from endometrium of Day 10, 12, and 15 pregnant gilts exhibited comparable 125I-IGF-I binding (p greater than 0.05) that was higher (p less than 0.001) than that for the corresponding myometrial membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential extracellular matrix gene expression by fibroblasts during their proliferative life span in vitro and at senescence.

Increasing evidence supports the idea that the finite proliferative life span of normal fibroblasts is a differentiation-like phenomenon. If this were correct, an ordered sequence of differential gene expression should be associated with the in vitro progression of cells from low passage to high passage (senescence). To define the pattern of expression of fibroblast differentiation-associated genes during this in vitro progression, we have determined the temporal pattern of expression of extracellular matrix (ECM) genes in Syrian hamster dermal fibroblasts as a function of passage level and percentage of proliferative life span in vitro. Steady-state mRNA levels were determined by Northern and dot blot analyses of total cellular RNA hybridized with cDNA probes specific for fibronectin, procollagen alpha 1III, and procollagen alpha 1I. Cells were analyzed at 24 hr postconfluence to minimize the presence of actively proliferating cells, and because maximal levels of fibronectin, alpha 1III, and alpha 1I mRNAs were observed 24 hr postconfluence. Unique, multiphasic patterns of expression of each of these ECM components were observed as the cells progressed from low passage to high passage. As the cells reached midhigh passage, fibronectin mRNA levels increased. This midpassage increase in fibronectin was followed by an increase in the level of alpha 1III mRNA as the cells reached the end of their in vitro proliferative life span, and then alpha 1I when the cells entered the postmitotic senescent phase, at which time the level of fibronectin mRNA also declined. A similar overlapping cascade pattern of up-regulation of these genes is seen during development and wound repair. This suggests that as cultured fibroblasts reach the end of their proliferative life span, they reinitiate a gene expression program used in tissue development and repair.     

Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats

It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13–20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-(I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.     

Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases

As a member of the phylum Cnidaria, the body wall of hydra is organized as an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM). Previous studies have established the general molecular structure of hydra ECM and indicate that it is organized as two subepithelial zones that contain basement membrane components such as laminin and a central fibrous zone that contains interstitial matrix components such as a unique type I fibrillar collagen. Because of its simple structure and high regenerative capacity, hydra has been used as a developmental model to study cell-ECM interaction during epithelial morphogenesis. The current study extends previous studies by focusing on the relationship of ECM biogenesis to epithelial morphogenesis in hydra, as monitored during head regeneration or after simple incision of the epithelium. Histological studies indicated that decapitation or incision of the body column resulted in an immediate retraction of the ECM at the wound site followed by a re-fusion of the bilayer within 1 hour. After changes in the morphology of epithelial cells at the regenerating pole, initiation of de novo biogenesis of an ECM began within hours while full reformation of the mature matrix required approximately 2 days. These processes were monitored using probes to three matrix or matrix-associated components: basement membrane-associated hydra laminin beta1 chain (HLM-beta1), interstitial matrix-associated hydra fibrillar collagen (Hcol-I) and hydra matrix metalloproteinase (HMMP). While upregulation of mRNA for both HLM-beta1 and Hcol-I occurred by 3 hours, expression of the former was restricted to the endoderm and expression of the latter was restricted to the ectoderm. Upregulation of HMMP mRNA was also associated with the endoderm and its expression paralleled that for HLM-beta1. As monitored by immunofluorescence, HLM-beta1 protein first appeared in each of the two subepithelial zones (basal lamina) at about 7 hours, while Hcol-I protein was first observed in the central fibrous zone (interstitial matrix) between 15 and 24 hours. The same temporal and spatial expression pattern for these matrix and matrix-associated components was observed during incision of the body column, thus indicating that these processes are a common feature of the epithelium in hydra. The correlation of loss of the ECM, cell shape changes and subsequent de novo biogenesis of matrix and matrix-associated components were all functionally coupled by antisense experiments in which translation of HLM-beta1 and HMMP was blocked and head regeneration was reversibly inhibited. In addition, inhibition of translation of HLM-beta1 caused an inhibition in the appearance of Hcol-I into the ECM, thus suggesting that binding of HLM-beta1 to the basal plasma membrane of ectodermal cells signaled the subsequent discharge of Hcol-I from this cell layer into the newly forming matrix. Given the early divergence of hydra, these studies point to the fundamental importance of cell-ECM interactions during epithelial morphogenesis.     

Temporal and spatial expression of genes for cartilage extracellular matrix proteins during avian mandibular arch development

We have examined the temporal expression of genes for extracellular matrix proteins (type I collagen, type II collagen, and the cartilage specific proteoglycan core protein) during the development of the avian mandibular arch. We detected low levels of type II collagen mRNA in the mandibular arch as early as stage 15. Type II collagen mRNA remained low but increased slightly as development progressed from stage 15 to stage 25. More dramatic increases occurred after stage 25 coincident with overt chondrogenesis. In contrast, mRNA for the core protein of cartilage specific proteoglycan was not detected prior to the onset of chondrogenesis, appeared at stage 25, and increased thereafter. Type I collagen mRNA was also present as early as stage 15 and dramatically increased after stage 28/29, coincident with initiation of osteogenesis. Using in situ hybridization, we found that type II collagen mRNA became detectable in the center of the mandible around stage 24/25 coincident with the initiation of chondrogenesis. At later stages (26-32) type II collagen mRNA was localized in the cartilaginous rudiment. The pattern of hybridization observed with the proteoglycan core protein probe at later stages of development was essentially identical to that observed with the type II collagen probe. In contrast, the probe for the alpha 1 (I) collagen mRNA was localized over the perichondrium, over differentiated bone, and in areas within the mandibular arch where bone formation had been initiated.     

Production of leukotrienes and prostaglandins in the rat uterus during peri-implantation period

We have measured by radioimmunoassay the production of leukotrienes (LTC4 and LTB4) and prostaglandins (PGE2 and PGF2 alpha) in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC4 or LTB4 remained unaltered on days 1-3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE2 and PGF2 alpha showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: vasodilators and agents that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation per se.     

Epidermal growth factor binding in rat uterus during the peri-implantation period

The profile of epidermal growth factor (EGF) binding to uterine membranes of rats on Day 1 through Day 7 of pregnancy was studied. The binding was lowest on Day 1 and increased gradually through the pre- and postimplantation periods. Binding affinity of the Day 7 uterine membranes was considerably higher than that of the Day 1. Apparent affinity constants (Ka) of Day 1 and Day 7 membranes were 0.29 X 10(-8) M and 1.03 X 10(-8) M respectively. To our knowledge, this is the first report of the modulation of EGF binding to uterine membranes by progesterone-estrogen interaction during early pregnancy.     

Spatial anisotropies and temporal fluctuations in extracellular matrix network texture during early embryogenesis

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks--(a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time-dependent rearrangements of a primordial biomaterial. We conclude that the ECM microenvironment changes markedly in time and space during the most important period of amniote morphogenesis--as determined by fluctuating textural properties.     

Temporal and spatial expression of integrins and their extracellular matrix ligands at the maternal-fetal interface in the rhesus monkey during pregnancy

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (alpha1, alpha5, alpha6, beta1, and beta4) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin alpha1, alpha5, and beta1 subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin alpha1, alpha5, beta1, and beta4 subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.     

Organization of extracellular matrix components during differentiation of adipocytes in long-term culture

Summary Scanning electron microscopy (SEM) observation showed that fully differentiated spherical adipocytes were embraced by a network of collagens and fibroblastic preadipocytes. The properties of both the collagen networks and the preadipocytes allow the adipocytes to be interconnected, forming a fat-cell cluster, which can anchor to the bottom of a culture dish. In this network structure, collagen fibrils and fibrillar bundles were closely arranged and stratified. We found that immunostained collagens appeared to form extracellular network structures, which can be observed by SEM. The extracellular network of fibronectin was the first to develop among the extracellular matrix (ECM) components, though it became degraded with the progress of adipocyte differentiation. The type I collagen network was the last to develop and remained well organized through the late stage of adipocyte differentiation. The extracellular networks of type III, V, and VI collagen developed by the mid-stage and remained in the late stage of adipocyte differentiation. The network structures of type IV collagen and laminin became degraded during the differentiation process and localized at the surface of spherical cells. In addition to these basement membrane components, types III, V, and VI collagens also showed pericellular spherical staining patterns. These results demonstrated that the constitution and distribution of the ECM are altered during adipocyte differentiation, suggesting that the organization of each ECM component into a suitable structure is a requirement for the differentiation and maintenance of unilocular adipocytes.     

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.     

Restricted spatial and temporal expression of G-protein alpha subunits during Drosophila embryogenesis.

Of the known signal transduction mechanisms, the most evolutionarily ancient is mediated by a family of heterotrimeric guanine nucleotide binding proteins or G proteins. In simple organisms, this form of sensory transduction is used exclusively to convey signals of developmental consequence. In metazoan organisms, however, the developmental role of G-protein-coupled sensory transduction has been more difficult to elucidate because of the wide variety of signals (peptides, small molecules, odorants, hormones, etc.) that use this form of sensory transduction. We have begun to examine the role of G-protein-coupled signaling during development by investigating the expression during Drosophila embryogenesis of a limited set of G proteins. Since these proteins are a common component of all G-protein-coupled signaling systems, their developmental pattern of expression should indicate when and where programmed changes in gene activity are initiated by, or involve the participation of, G-protein-coupled signaling events. We have focused on the spatial and temporal expression pattern of three different Drosophila G-protein alpha subunits by northern blot analysis, in situ hybridization and immunocytochemistry using antibodies directed to peptides specifically found in each alpha subunit. From the spatial and temporal restriction of the expression of each protein, our results suggest that different forms of G-protein-coupled sensory transduction may mediate developmental interactions during both early and late stages of embryogenesis and may participate in a variety of specific developmental processes such as the establishment of embryonic position, the ontogeny of the nervous system and organogenesis.