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S J Kimber

Publications and source records attributed to S J Kimber.

At least 19 recordsLinked to original sources
PubMed2005

Characterization of the uterine phenotype during the peri-implantation period for LIF-null, MF1 strain mice.

Leukemia inhibitory factor plays a major role in the uterus and in its absence embryos fail to implant. Our knowledge of the targets for LIF and the consequences of its absence is still very incomplete. In this study, we have examined the ultrastructure of the potential implantation site in LIF-null MF1 female mice compared to that of wild type animals. We also compared expression of proteins associated with implantation in luminal epithelium and stroma. Luminal epithelial cells (LE) of null animals failed to develop apical pinopods, had increased glycocalyx, and retained a columnar shape during the peri-implantation period. Stromal cells of LIF-null animals showed no evidence of decidual giant cell formation even by day 6 of pregnancy. A number of proteins normally expressed in decidualizing stroma did not increase in abundance in the LIF-null animals including desmin, tenascin, Cox-2, bone morphogenetic protein (BMP)-2 and -7, and Hoxa-10. In wild type animals, the IL-6 family member Oncostatin M (OSM) was found to be transiently expressed in the luminal epithelium on late day 4 and then in the stroma at the attachment site on days 5-6 of pregnancy, with a similar but not identical pattern to that of Cox-2. In the LIF-null animals, no OSM protein was detected in either LE or stroma adjacent to the embryo, indicating that expression requires uterine LIF in addition to a blastocyst signal. Fucosylated epitopes: the H-type-1 antigen and those recognized by lectins from Ulex europaeus-1 and Tetragonolobus purpureus were enhanced on apical LE on day 4 of pregnancy. H-type-1 antigen remained higher on day 5, and was not reduced even by day 6 in contrast to wild type uterus. These data point to a profound disturbance of normal luminal epithelial and stromal differentiation during early pregnancy in LIF-nulls. On this background, we also obtained less than a Mendelian ratio of null offspring suggesting developmental failure.

Animals↗
PubMed2004

Trophoblast differentiation in vitro: establishment and characterisation of a serum-free culture model for murine secondary trophoblast giant cells.

Differentiation of trophoblast giant cells is an early event during the process of murine embryo implantation. However, differentiation of secondary trophoblast giant cells in the rodent is still only partially understood, probably because of the lack of suitable in vitro models and cell markers. In order to advance our understanding of trophoblast differentiation, suitable in vitro models and markers are required to study their development. The objectives of this study were to establish and characterise a serum-free in vitro model for murine secondary trophoblast cells. Secondary trophoblast giant cells growing in vitro and paraffin sections of day 8.5 postcoitum mouse embryos were processed for immunostaining to establish the expression of potential markers using antibodies to blood group antigens, E-cadherin, alpha(7) integrins and activator protein-gamma, as well as placental lactogen-II. Within 3 days in serum-free culture, ectoplacental cone-derived secondary trophoblast cells underwent simultaneous induction of both morphological and functional differentiation. Secondary trophoblasts grew in vitro as a monolayer of cells with giant nuclei and expressed B and Le-b/Le-y blood group antigens, alpha(7) integrins and placental lactogen-II, as well as activator protein-gamma. Transcripts for activator protein-gamma and placental lactogen-II were detected in cultures by RT-PCR and for placental lactogen-II by in situ hybridisation. At later time-points apoptosis increased. A fibronectin substrate significantly increased secondary trophoblast cell numbers and surface area of outgrowth. The increase in cells with giant nuclei coincided with induction of placental lactogen-II expression. A relationship was found between the nuclear area of secondary trophoblast cells and expression of placental lactogen-II.

Animals↗
PubMed2004

Role of leukemia inhibitor factor (LIF) in decidualisation of murine uterine stromal cells in vitro.

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

Alkaline Phosphatase↗
PubMed2002

Expression of cell adhesion molecules during human preimplantation embryo development.

Formation of a fully differentiated, implantation competent blastocyst requires the expression of a complex repertoire of molecules. However, the events that drive morphogenesis are poorly elucidated in the human embryo. In this work, we describe the amplification of representative cDNAs from morphologically and developmentally normal, individual human embryos at all stages from pronucleate to blastocyst. These cDNAs were probed to reveal the temporal expression pattern of cell adhesion molecules thought to play a key role in murine preimplantation embryo development. We demonstrated constitutive expression of beta actin, beta 1 and alpha 6 integrins, ZO-1 and E-cadherin, as shown previously in mouse embryos. No expression of beta 3, alpha 2, alpha 3 or alpha 7 integrins nor of L or P selectin was detected at any stage of preimplantation development. beta 5 integrin showed a regulated pattern of expression and was not expressed in blastocysts, while desmocollin-2 could only be detected at the blastocyst stage. Expression and localization of beta 1, beta 5 and alpha 6 integrins and ZO-1 and E-cadherin proteins was confirmed in blastocyst stage embryos by immunocytochemistry. We have identified differences in the expression of integrin molecules between mouse and human embryos, and propose a role for alpha v beta 5 and alpha 6 beta 1 integrin dimers in the human embryo at implantation.

Blastocyst↗
PubMed2001

Glycosylation changes during differentiation of the murine uterine epithelium.

In mouse uterine luminal epithelium (LE) several terminal carbohydrate structures are regulated by ovarian steroids and show stage-specific expression during early pregnancy. We have demonstrated that expression of H-type-1 antigen (Fucalpha1-2Galbeta1-3GlcNAcbeta1-) is regulated by oestrogenic stimulation of alpha1-2fucosyltransferase (fut1) mRNA levels in LE. H-type-1 expression is high after ovulation but becomes negligible after implantation. In contrast, NeuNAcalpha2-3Galbeta1-, and specifically sialyl Le-x (NeuAcalpha2,3Galbeta1,4[Fucalpha1-3]GlcNAcbeta1-), is stimulated by progesterone. It is not expressed on LE after ovulation but is expressed maximally on apical LE at the time of and after implantation. However, mRNA levels for 4 out of 5 known Gal/GalNAc alpha2-3sialyltransferases appear not to change in LE during early pregnancy, suggesting an alternative level of control.

Animals↗
PubMed2000

Blastocyst implantation: the adhesion cascade.

This review covers the sequence of cell adhesion events occurring during implantation of the mammalian embryo, concentrating on data from mouse and human. The analogy is explored between initial attachment of trophoblast to the uterine lining epithelium and that of neutrophils to the endothelial lining of blood vessels at sites of inflammation. The possible role of various carbohydrate ligands in initial attachment of the blastocyst is reviewed. The evidence for subsequent stabilization of cell adhesion via integrins or the trophinin-tastin complex is discussed.

Animals↗
PubMed2000

Molecular interactions at the maternal-embryonic interface during the early phase of implantation.

In mammals the embryo must implant in the uterus and develop a placenta to gain nutrition and facilitate gas exchange. In this article, the earliest events in this process are reviewed. The embryo can implant only when it has reached the blastocyst stage. The blastocyst is composed of an inner clump of cells, the inner cell mass, that gives rise to the fetus and an outer layer of trophectoderm (TE), the precursor of the placenta. Both blastocyst and uterus must differentiate in parallel to reach the appropriate state of maturity (activated blastocyst and receptive uterus) at which implantation can occur. Interaction between TE and the luminal epithelium (LE) lining the uterus initiates implantation, and both soluble signals and association between molecules on apposed surfaces appear to be involved. A number of cell surface molecules have been implicated in the initial attachment between TE and LE. These include HSPG, Le-y and the H-type-1 antigen, HB-EGF, trophinin-tastin-bystin complex, integrins, and extracellular matrix molecules such as osteopontin and laminin. Others, such as mucins, may need to be removed or modified to allow adhesion to proceed. Evidence for the role of these components is discussed.

Animals↗
PubMed2000

Desmosomes are reduced in the mouse uterine luminal epithelium during the preimplantation period of pregnancy: a mechanism for facilitation of implantation.

Dynamic regulation of intercellular junctions is an essential aspect of many developmental, reproductive, and physiological processes. We have shown that expression of the desmosomal protein desmoplakin decreases in the luminal uterine epithelium during the preimplantation period of pregnancy in mice. By the time of implantation (between Days 4.5 and 5 of pregnancy), desmoplakin protein can barely be detected by SDS-PAGE and Western blotting, and by immunocytochemistry, it is restricted to well-spaced, punctate dots at the apicolateral junction. Using confocal XZ series and electron microscope quantitation, both the density and distribution of desmosomes along the lateral cell surfaces of luminal epithelial cells were observed to change during early pregnancy. On Day 1 of pregnancy, desmosomes were found at high density in the apicolateral junctional complex, being present here in 79% of ultrathin sections examined, whereas on Day 5, the density was much reduced (present in only 18% of ultrathin sections examined). Desmosomes were found along the lateral surfaces, at or below the level of the nucleus, in 15% of ultrathin sections examined on Day 1 of pregnancy but in only 1% on Day 5. Desmoplakin mRNA declined during the first 4-5 days of pregnancy, along with the protein, suggesting that these changes are controlled at the level of mRNA. This study shows that desmosomes are regulated during early pregnancy, and we propose that a reduction in desmosome adhesion facilitates penetration of the luminal epithelium by trophoblast cells at implantation.

Animals↗
PubMed1999

Hormonal control of H-type alpha(1-2)fucosyltransferase messenger ribonucleic acid in the mouse uterus.

The H epitope, an alpha(1-2)fucosylated carbohydrate structure, has been implicated in initial attachment of the murine blastocyst to luminal uterine epithelial cells in vitro. In this study, the expression of the H-type alpha(1-2)fucosyltransferase (FUT1) gene was examined in endometrium of mice. Northern blotting of luminal epithelial RNA identified a single 6.2-kilobase transcript. In situ hybridization studies showed a signal for FUT1 mRNA on Days 1-3 of pregnancy in glands and luminal epithelium. The signal diminished by Day 4 and could not be detected on Day 5 of pregnancy. The in situ signal in endometrial epithelia was highest at estrus and metestrus and was absent at diestrus. Estrogen treatment after ovariectomy gave strong FUT1 mRNA expression in epithelia, but with progesterone, progesterone + estrogen, or vehicle, no message could be detected. A semiquantitative reverse transcription-polymerase chain reaction (PCR) analysis of FUT1 mRNA from luminal epithelium generated large amounts of PCR product on Day 1 of pregnancy; this diminished on Days 2, 3, and 4, and the product was barely detectable on Day 5. A kinetic analysis of FUT1 activity on Day 1 of pregnancy suggested a single enzyme with a Michaelis-Menten constant (Km) of 0.29 mM towards phenyl-beta-D-galactoside and of 1.75 mM towards Galbeta(1-3)GalNAc. These results suggest that expression of the H epitope is regulated at the level of FUT1 transcription and that transcription is stimulated by estrogen in the endometrial epithelium.

Animals↗
PubMed1999

Demonstration of oestrogenic control of H-type-1 carbohydrate antigen in the murine endometrial epithelium by use of ICI 182,780.

The carbohydrate H-type-1 antigen has been implicated in attachment of the murine blastocyst to the endometrial epithelium. Monoclonal antibody 667/9E9 was used to investigate the steroidal dependency of expression of this antigen in the murine endometrial epithelium using intact or ovariectomized mice treated with oestrogen or the pure anti-oestrogen, ICI 182,780. The effects of this anti-oestrogen were also investigated in the endometrial epithelium from intact rats. In both ovariectomized, oestrogen-supplemented and intact mice after treatment with ICI 182,780, staining with monoclonal antibody 667/9E9 was abolished in the luminal epithelium on both the apical and lateral cell surfaces, whereas basal staining remained. Glandular staining in mice was not affected in the same manner. In intact rats, where H-type-1 antigen expression has been reported to be predominantly controlled by progesterone, the anti-oestrogen had little effect, in accordance with previous reports.

Animals↗
PubMed1998

Morphological evidence for a morphogenetic field in gastropod mollusc eggs.

Eggs of the marine gastropod Crepidula fornicata examined by confocal imaging of FITC-lectin binding to the surface, and cryoscopic-SEM both reveal a surface architecture of linear structures organized around the animal-vegetal axis, which is spatially related to the anterior-posterior (a-p) axis of the subsequent embryo. A series of structures is also orientated with reference to specific micromere quartets formed during spiral cleavage. Thus, the surface architecture may provide a visible marker for a morphogenetic field which generates the a-p axis and organizes the cleavage pattern. Moreover, this architecture is co-extensive with that found on the vegetal, polar lobe-bearing region of eggs, as described by others, and which varies between gastropod taxa with varied types of body form. Confocal imaging reveals a distinct localization of F-actin to the architecture of the lobe region. However, the integrity of this F-actin is not responsible for the maintenance of the surface architecture. The significance of these findings to our understanding of the generation of diversity within the Gastropoda and general ontogenic mechanisms is discussed.

Actins↗
PubMed1996

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

Animals↗
PubMed1996

Decidual sialylation shows species-specific differences in the pregnant mouse and rat.

Biotinylated lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA), which bind to alpha 2,6-linked and alpha 2,3-linked sialyl residues, respectively, were used as probes to study glycan terminal modifications associated with decidualization in the uterine stroma of pregnant rats and mice. Binding of lectins from Erythrina cristagalli (ECA), Phaseolus vulgaris (leukoagglutinin, L-PHA), Triticum vulgaris (WGA) and Bandeiraea simplicifolia (BSA-1B4) was also examined. Tissues from rats between day 5 and day 8 of gestation and mice between day 5 and day 7 of gestation were fixed in Bouin's solution and embedded in wax prior to lectin histochemistry. On day 7 in rats and day 6 in mice, there was a marked reduction in the binding of SNA in the subluminal decidua surrounding the implantation site. In rats, MAA binding to enlarged decidual cells around the implantation chamber was increased markedly, but there was no change in mice. In both species there was de novo binding of ECA in the SNA-negative area, suggesting that the loss of alpha 2,6-linked sialyl residues unmasks terminal N-acetyl lactosamine. These findings are consistent with previous evidence of a close structural and functional similarity between the artificially induced deciduoma and true decidua of rats and show identical changes to the glycosylation patterns previously found in differentiating rat deciduoma. In both species, therefore, decidua exhibits regionally specific terminal glycosylation. However, the species-specific expression of alpha 2,3-linked sialyl residues suggests distinct patterns of steroidally modulated sialyl transferase expression.

Animals↗
PubMed1995

Cell adhesion molecules on the oocyte and preimplantation human embryo.

The presence of cell adhesion molecules on human oocytes, early embryos, and pre-hatched blastocysts was examined by indirect immunofluorescence and compared to the distribution found on first trimester villous placenta with the same antibodies. Six integrin subunits (alpha 3, alpha V, beta 1, beta 3, beta 4, beta 5) were observed consistently throughout preimplantation development. Evidence was also obtained for the presence of integrin subunits alpha 2, alpha 4, alpha L, beta 2, and beta 7 on a small number of oocytes. A more restricted developmental analysis of E-cadherin, ICAM-1, NCAM, and VCAM-1 demonstrated that these cell adhesion molecules are also present on oocytes and early embryos. L-selectin was detected on oocytes but was not found on 8-cell embryos. The oocyte and early blastomeres have complex surfaces in which the integrin and CAM families are represented.

Blastocyst↗
PubMed1995

CD44 is expressed throughout pre-implantation human embryo development.

The cell surface glycoprotein CD44 has been demonstrated in a variety of cell types in embryonic and adult tissues. We have established that CD44 is present on human oocytes, cumulus cells, early embryos and pre-hatched blastocysts by indirect immunofluorescence. We have also shown that CD44 is present on 8-11 week placental stroma cells, but not on the trophoblast. These findings demonstrate that CD44 is present throughout preimplantation development, and that down-regulation occurs on the embryonic surface after implantation.

Blastocyst↗
PubMed1995

Expression of carbohydrate antigens in the rat uterus during early pregnancy and after ovariectomy and steroid replacement.

Monoclonal antibodies were used to examine the expression of a number of carbohydrate antigens in the rat endometrial epithelium from day 1 to day 8 of pregnancy and in ovariectomized rats supplemented with ovarian steroids. Carbohydrate antigens based on the Gal beta 1-GlcNAc backbone structure were expressed and some of these (Le(y), Le(x), B antigen) were present at all stages of pregnancy and independent of ovarian steroids. The H-type-2 antigen was stimulated by progesterone and expressed in the sensitized and receptive uterus, but was not detected after implantation or, in ovariectomized rats, in the refractory phase. The H-type-1 antigen, which is stimulated by oestrogen in the mouse, appeared to be stimulated by progesterone in rats. It was expressed throughout the period of pregnancy but maximal expression was found on days 4-5. The histo-blood group A antigen appeared in ovariectomized rats only after treatment with progesterone followed by three daily injections of progesterone and oestrogen, and in the corresponding postimplantation period of pregnant rats. Its appearance corresponded to the loss of detectable H-type-2 antigen. This study shows that the rat endometrial epithelium expresses some carbohydrate antigens not expressed in mice (A and B antigen) or under completely different steroidal regulation (H-type-1). Moreover, the T antigen was expressed on the endometrial epithelium adjacent to decidua on days 7 and 8 of pregnancy, but not in rats given ovarian steroids to mimic the sensitized, receptive or refractory phase. Differences in expression between glandular and luminal epithelial indicated differences in steroidal regulation, as found in mice.

Animals↗
PubMed1994

A new in vitro model of murine mesoderm migration: the role of fibronectin and laminin.

Examination of the factors involved in primary mesodermal migration in the mouse has been complicated by the lack of a suitable in vitro model. We have developed a new culture system using primitive streak stage embryos denuded of primitive endoderm, which allows easy observation and manipulation of the outgrowing cells. The cells migrating away from these explants were shown by immunocytochemistry to express vimentin and an epitope of the I antigen recognised by the antibody C6, both of which are present on the newly emerged mesoderm and not on the embryonic ectoderm in sections of embryos in utero. Conversely, cytokeratin, stage-specific embryonic antigen 1 (SSEA-1), E-cadherin and desmoplakin are expressed by the embryonic ectoderm but lost during mesoderm formation in vivo. They are absent or expressed very weakly by the migrated cells in vitro. In addition, only explants of the ectoplacental cone (EPC) and visceral endoderm alone, expressed a carbohydrate epitope (recognised by monoclonal antibody BOO6), characteristic of the EPC and primitive endoderm in utero, but absent from mesoderm. Thus we conclude that the cells which outgrow in this system are indeed mesodermal in phenotype. We have confirmed the work of others in demonstrating the presence of fibronectin (FN) and laminin (LN) in the migratory path of the mesoderm, at the ectoderm-visceral endoderm interface. We also report that the beta 1 integrin subunit of the FN and LN receptor is expressed by mesodermal cells at this interface. Using our in vitro model we have examined the role of the extracellular matrix (ECM) in mesodermal migration. Mesodermal cells migrate further and faster on substrates coated with FN or LN, and this increased migration is abolished by appropriate blocking antibodies. We conclude that the ECM, in particular FN and LN, plays an important role in the migration of primary mesodermal cells during gastrulation in the mouse embryo.

Animals↗