Influence of epithelial-mesenchymal interaction on the viability of facial mesenchyme. II: Synthesis of basement-membrane components during tissue recombination

The presence of basement-membrane components during tissue separation procedures was determined employing monoclonal antibodies to laminin and type IV collagen. In addition, the reconstitution of basement-membrane components and the formation of the basement-membrane were examined in isolated epithelium and mesenchyme and in tissue recombination. Epithelium and mesenchyme of maxillary processes of chick embryos were separated by a variety of protocols, including those employed in a prior study (Saber et al: Anat. Rec. 225:56-66, 1989). Results indicated that the protocol previously employed did not remove basement-membrane components after enzymatic tissue separation. A revised protocol in which the basement-membrane components (i.e., laminin and type IV collagen) were removed from isolated tissues prior to recombination revealed that a developmental compartment and a gradient of cell viability, comparable in size and dimensions to that observed in the study of Saber et al. (ibid.) was present in the mesenchyme of recombined explants. Type IV collagen and laminin, therefore, do not appear to be required initially during tissue recombination in order for subsequent growth-sustaining effects to be expressed. Additional studies revealed, however, that synthesis of basement-membrane components occurred not only in isolated tissues but was altered markedly by tissue recombination. Culture of isolated tissues demonstrated induction of laminin synthesis in separated epithelium by 24 hours and induction of collagen synthesis in isolated mesenchyme by 24 hours. Recombination of epithelium and mesenchyme, however, resulted in rapid induction of laminin synthesis within 1 hour. Recombination of epithelium and mesenchyme after 24 hours resulted in the presence of laminin not only in epithelium but in mesenchyme as well. Both tissues were required for basement-membrane formation which appeared to be fully reconstituted by 24 hours in culture. These observations indicate that recombination in culture alters the pattern of synthetic activity of these basement-membrane components. These can be characterized as "early" (temporal) and "late" spatial) responses by the recombined tissues.     

Influence of the substrate's hydrophilicity on the in vitro Schwann cells viability

A series of polymeric biomaterials including poly (methyl acrylate) (PMA), chitosan (CHT), poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate and hydroxyethyl acrylate monomeric units were tested in vitro as culture substrates and compared for their impact on the proliferation and expansion of Schwann cells (SCs). Immunocytochemical staining assay and scanning electron microscopy techniques were applied to perform a quantitative analysis to determine the correct maintenance of the cultured glial cells on the different biomaterials. The results strongly suggest that cell attachment and proliferation is influenced by the substrate's surface chemistry, and that hydrophobic biomaterials based on PMA, PEA, and the copolymers PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and proliferation of SCs in vitro.     

Differentiation of the mammalian retinal pigment epithelium in vitro: Influence of presumptive retinal neuroepithelium and head mesenchyme

The ancestor cells of the pigment epithelium of the mammalian eye are derived from the neuroepithelial cells of the neural plate. They are neurally determined in the process of neurulation but finally decide to follow the pigment cell lineage, whereas the adjacent tissue develops into the neuroretina and the optic stalk. This decision is most probably made in the developmental stage of eye cup formation. The pigment epithelium becomes restricted to the outer leaf of the eye cup and does not encroach on the adjacent neuroepithelial tissues of the internal leaf and the eye stalk. It is therefore supposed to be channelled by a locally confined determinant factor that has not yet been identified. In the present study, development of the mammalian eye and the neural versus pigment cell decision were investigated in mouse embryos. Three approaches were used to discover the source of the putative determinant involved in the process of neuroepithelial decision. First, eye primordia were cultured from stage 11 embryos (0 somites, early neural plate stage, embryonic day 7 1/2–8) to stage 16 embryos (34 somites, neural tube stage, ed 10); this is prior to pigment cell induction. The eye primordia were first cultured in head segments and their natural position. In these experiments, 50% of the ocular neuroepithelia developed along the nerve cell and glial cell lineage. However, the other 50% of the cultured specimens partly developed into pigment epithelia. In these specimens the determinant factors had obviously remained functionally intact in vitro. In the second type of experiment, the eye primordia were also cultured within the head segments, but with the prospective neuroretina selectively removed. This experiment should show whether the inner layer of the eye cup (the prospective neuroretina) is involved in the neuroepithelial lineage decision. In these experiments 90% of the cultured eye primordia failed to develop pigmented cells. The prospective neuroretina was therefore considered as a candidate for the production of an inductive factor. Finally, eye primordia from stage 14–15 embryos (13–29 somites, ed 9–9 1/2) were either transplanted into heterotopic tissues, such as mesenchymal organs, neuroepithelium or heterochronic muscle, or grown as controls in their natural position and tissue environment. In these conditions both transplanted eye primordia and controls bore pigmented epithelium. Hence, the lineage decision, whether to form neural or pigment cell, remained undisturbed in all epitopes tested. On the basis of these experiments, it seemed unlikely that the development of pigment cells was initiated by a mesenchyme-derived factor exclusively produced near the eye vesicle.     

Influence of microbial colonization on sperm-mucus interaction in vivo and in vitro*

Two-hundred-and-thirty-three asymptomatic couples with a meanduration of infertility of 5 years were submitted to postcoitaltesting (PT) and to sperm penetration meter test (SPMT) andsimultaneous microbial screening. Cervical swabs and semen specimenswere collected for culture of Mycophma homink, Ureaphma urealyticum,Chlamydia tmhomufis, Neisseria gonorrhoeae, other potentiallypathogenic and commensal aerobic and anaerobic bacteria, herpessimplex virus, vaginal swabs for Trichomonas vaginalis and yeasts.Results of microbial screening were analysed with regard tosperm penetration ability into wives' cervical mucus in vivoand in vitro, but no marked influence was revealed for mostmicmrganisms. Samples of only one of the 233 couples provedto be completely sterile. The findings suggest that in asymptomaticpatients microbial colonization is of minor importance for sperm-mucusinteraction.     

Ultrastructural characterization of the mesenchyme of the facial processes during development of the rat intermaxillary segment

This study quantifies ultrastructural changes within the mesenchyme of the maxillary and medial/lateral nasal processes before and after formation of the intermaxillary segment. At both day 11 and day 13 of development, 4 fetal rat heads were fixed with 3% glutaraldehyde and processed for electron microscopy. Differences between cells within the facial processes were discerned at day 11 of gestation. Significantly more rough endoplasmic reticulum was present in the cells of the maxillary processes than in the lateral nasal processes (p < .05), and a greater number of cell projections was found in the maxillary processes than in the medial nasal processes (p < .05). At day 13, no significant differences were found in the facial processes. Of particular note, a consistent increase in the number of cell projections within all the facial processes was found on day 13 when compared with day 11 (p < .05). This change may be related to intercellular signaling between the mesenchymal cells at the time of onset of major histogenic changes.     

过氧化氢对生育男性精子凋亡及核DNA完整性的影响

目的研究体外过氧化氢对生育男性精子凋亡及精子DNA完整性的影响。方法58例正常生育男性均来自吉林大学第二临床医院泌尿男科,精子Percoll优选后制作精子悬液,用伊红Y染色分析精子活率,用瑞-姬染色分析精子凋亡率,用精子核吖啶橙荧光染色检测精子DNA完整性。结果外加过氧化氢浓度为0.02mmol/l时,精子活率显著低于对照组（P〈0.05）。当浓度为0.20mmol/l时,精子活率显著低于对照组（P〈0.05）,精子凋亡率显著高于对照组（P〈0.05）。外加过氧化氢浓度为0.02mmol/l、0.2mmol/l时,精子DNA完整性均显著低于对照组（P〈0.05）,且精子DNA完整性随实验浓度增加而下降。结论体外过氧化氢对精子活率、凋亡率与DNA完整性有影响,高浓度的过氧化氢可促使精子凋亡,损伤精子DNA完整性,导致男性不育。     

Influence of cations on the viability of Halobacterium mediter-ranei cells

The effects of monovalent and divalent cations on the cell viability of an extremely halophilic bacteria H. mediterranei were examined. High magnesium concentrations (≧72 mM) were required for stabilization of H. mediterranei cells, although calcium ions were more potent than magnesium in preserving viability. On the other hand, H. mediterranei cells are also able to survive in the presence of monovalent cations, such as ammonium and potassium, at least 14 days. The implications for maintaining stability and neutrality of several cell envelope components by monovalent and divalent cations are discussed.     

Retinoic acid decreases the viability of mouse blastocysts in vitro

BACKGROUND: This study was designed to examine the cytotoxic effect of retinoic acid on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryo development in vitro. METHODS AND RESULTS: Mouse blastocysts were exposed for 24 h to doses of 0, 0.1 micromol/l and 10 micromol/l all-trans retinoic acid and observed for their capacity to implant and develop during the early postimplantation period in vitro. When retinoic acid-pretreated blastocysts were allowed to implant in vitro, significantly fewer embryos were able to reach a later stage of embryo development. Compared with the findings for the control blastocysts, exposure to retinoic acid resulted in a significant reduction in the average number of total cells in blastocysts and the trophectoderm/inner cell mass lineage. The effect was associated with a significant increase in the frequency of cells identified as being engaged in apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and Annexin V techniques. CONCLUSIONS: This is the first evidence that retinoic acid induces cell death (apoptosis) and inhibits cell proliferation in mouse blastocysts. This results in the retardation of early postimplantation blastocyst development and subsequent blastocyst death.     

The viability of mononuclear phagocytes in vitro is diminished by the interaction of cells with serum proteins bound to the culture substratum

The long-term viability of bone marrow-derived mononuclear phagocytes in vitro was inversely correlated with the capacity of the cells to attach to the culture substratum. Mononuclear phagocytes suspended in medium containing 10% fetal bovine serum and subcultured on substrata coated with a 0.1% solution of poly(2-hydroxyethyl methacrylate) formed a population of non-adherent or loosely attached cells that remained viable for a 7 day incubation period. In contrast, cells subcultured under otherwise identical conditions on substrata optimal for cell attachment exhibited a 40-fold decline in cell number during the same period of time. The survival of mononuclear phagocytes subcultured under conditions which promoted cell attachment was increased by reducing the concentration of serum in the medium. Thus, cells subcultured 7 days in the complete absence of serum exhibited only a two-fold decline in cell number. However, mononuclear phagocytes subcultured in the absence of serum exhibited a ten-fold decline in cell number when cultured on substrata coated with serum proteins. This decline was reversed by the addition of the mononuclear phagocyte-specific growth factor, colony stimulating factor-1 (CSF-1) to the medium. These results indicate that serum proteins bound to the culture substratum exert a significant influence on the viability of adherent mononuclear phagocytes in vitro and on the requirement of cells for CSF-1 in order to survive.     

Periostin is expressed within the developing teeth at the sites of epithelial-mesenchymal interaction.

Periostin was originally isolated as an osteoblast-specific factor that functions as a cell adhesion molecule for preosteoblasts and is thought to be involved in osteoblast recruitment, attachment, and spreading. The protein was renamed "periostin" because of its expression in the periosteum and periodontal ligament, indicating a potential role in bone and maintenance of tooth structure. Periostin has structural similarity to insect fasciclin-I and can be induced by TGF-beta and Bmp2. Because tooth and periodontium development is a well-described genetic model for organogenesis governed by a reciprocal set of epithelial-mesenchymal interactions, thought to be controlled by various TGF-beta superfamily members, we investigated whether periostin is present during tooth morphogenesis. Both periostin mRNA and protein expression were analyzed throughout normal tooth development (embryonic day [E] 9.5-newborn) and within both Bmp4- and Msx2-null embryos. Periostin mRNA is initially present within the E9.5 first branchial arch epithelium and then shifts to underlying ectomesenchyme. Both mRNA and protein are asymmetrically localized to the lingual/palatal and buccal side during the early epithelial-mesenchymal interactions. Periostin is also present in dental papilla cells and within the trans-differentiating odontoblasts during the bell and hard tissue formation stages of tooth development. We suggest that periostin plays multiple roles as a primary responder molecule during tooth development and may be linked to deposition and organization of other extracellular matrix adhesion molecules during maintenance of the adult tooth, particularly at the sites of hard-soft tissue interface.     

BMP signaling regulates the fate of chondro‐osteoprogenitor cells in facial mesenchyme in a stage‐specific manner

Background : Lineage tracing has shown that most of the facial skeleton is derived from cranial neural crest cells. However, the local signals that influence postmigratory, neural crest‐derived mesenchyme also play a major role in patterning the skeleton. Here, we study the role of BMP signaling in regulating the fate of chondro‐osteoprogenitor cells in the face. Results : A single Noggin‐soaked bead inserted into stage 15 chicken embryos induced an ectopic cartilage resembling the interorbital septum within the palate and other midline structures. In contrast, the same treatment in stage 20 embryos caused a loss of bones. The molecular basis for the stage‐specific response to Noggin lay in the simultaneous up‐regulation of SOX9 and downregulation of RUNX2 in the maxillary mesenchyme, increased cell adhesiveness as shown by N‐cadherin induction around the beads and increased RA pathway gene expression. None of these changes were observed in stage 20 embryos. Conclusions : These experiments demonstrate how slight changes in expression of growth factors such as BMPs could lead to gain or loss of cartilage in the upper jaw during vertebrate evolution. In addition, BMPs have at least two roles: one in patterning the skull and another in regulating the skeletogenic fates of neural crest‐derived mesenchyme. Developmental Dynamics 245:947–962, 2016. © 2016 Wiley Periodicals, Inc.     

Effect of epithermal neutrons on viability of glioblastoma tumor cells in vitro

We studied in vitro effect of epithermal neutrons in various doses on viability of glioblastoma U87 tumor cells. Increasing the dose from 1.9 to 4.1 Sv promoted cell death. Cytofluorimetric analysis revealed no activation of apoptosis in the irradiated cells, which attested to necrotic death of the tumor cells exposed to epithermal neutron radiation.     

不同强度永磁磁场对成纤维细胞的生物学活性影响研究

目的以磁源空间磁场定量计算为依据,研究正常培养条件下与缺氧培养条件下不同强度永磁磁场对人皮肤成纤维细胞的影响。方法选用人皮肤成纤维细胞,以5×104/mL密度每孔100μL种于96孔板。磁源N面中心实测磁感应强度分别为3.4、5.6、13.8、27.8、52.6、107.0、178.9、292.2 mT的8组磁源,前6组(钐钴合金)尺寸分别为Φ8.0 mm×2.0 mm,后两组(钕铁硼)尺寸分别为Φ8.0 mm×1.5 mm、Φ8.0 mm×2.5 mm,均轴向充磁,即为A、B、C、D、E、F、G、H组。将8组磁场分别作用于正常培养条件下与缺氧培养条件下的人皮肤成纤维细胞,并设对照组(加磁组与非加磁组),3 d后用四甲基偶氮唑盐(MTT)法分别检测细胞活力,做统计学分析。结果两种培养条件下8组不同强度的加磁组人皮肤成纤维细胞的MTT数值与非加磁组的对照组相比较均有升高,正常培养中磁场强度为B、C、D、E、F、G、H组与对照组相比较,差异有统计学意义(P<0.05),且随着磁场强度的增加活性值越高;缺氧培养中磁场强度为G、H组与对照组相比较,差异有统计学意义(P<0.05)。结论实验中选取的永磁磁场对于人皮肤成纤维细胞有增殖和降低损伤的作用。     

In vitro production of monoclonal antibodies to cultured embryonic chick limb mesenchyme

A simple, rapid protocol for the in vitro production of monoclonal antibodies (MAbs) that recognize native antigens in cultured chick limb mesenchyme during chondrogenic differentiation is described. Murine lymphocytes were stimulated by direct exposure to methanol-fixed micromass cultures of limb mesenchyme derived from the distal tip of stage 25 chick limb buds. Initial immunohistochemical characterization of two antibodies (DIDI and DIIA5) produced by this method showed preferential localization of reactivity with antigens in developing cartilage nodules during chondrogenesis in cultured chick limb mesenchyme. This study demonstrates the utility of in vitroimmunization of lymphocytes for the production of MAbs to native antigens expressed by differentiating embryonic limb cells in culture. Immunohistochemical data provided by DIDI and DIIA5 suggest that antigens bearing these epitopes may be important in early morphogenetic events during limb skeletal development.     

Effects of BMP-7 on mouse tooth mesenchyme and chick mandibular mesenchyme.

BMP-7 is a member of the BMP family of signaling molecules that are thought to play key roles in mediating inductive events during embryogenesis. In the present study the possible roles of BMP-7 in mediating inductive events during the initiation phase of odontogenesis and mandibular morphogenesis were investigated. To do so, we have examined the effects of agarose beads soaked in recombinant BMP-7 on E11 mouse molar-forming mesenchyme and stage 23 chick mandibular mesenchyme, and analyzed the patterns of expression of Bmp-7 in developing mouse and chick first branchial arches. Beads releasing BMP-7 induced a translucent zone, cellular proliferation, and expression of Msx-1, Msx-2, and Bmp-4 in molar-forming mesenchyme after 24 hr. The effects of BMP-7 on molar-forming mesenchyme are similar to the effects of BMP-4 and are consistent with their overlapping patterns of expression in the thickened epithelium of the early developing tooth buds, which is suggestive of cooperative and/or redundant roles of BMPs in mediating the inductive interactions during the early stages of odontogenesis. Our studies in the developing chick mandible showed that Bmp-7 is expressed in the mandibular epithelium. In the absence of mandibular epithelium, BMP-7 beads maintained cell proliferation and Msx expression in the medial mandibular mesenchyme and were able to induce cell proliferation, cell death, and Msx expression in the lateral chick mandibular mesenchyme. The effects of BMP-7 on the expression of Msx genes in lateral chick mandibular mesenchyme, although different from the effects of lateral mandibular epithelium, are similar to the effects of epithelium from the medial region where multiple Bmps are expressed. We also showed that laterally placed BMP-7 beads induced ectopic expression of Msx genes and changes in the development of posterior skeletal elements in the maxillary and mandibular arches. However, despite its proliferative effects on mandibular mesenchyme, BMP-7 did not support the directional outgrowth of the mandible. These observations suggest that epithelial-mesenchymal interactions in the medial region of the mandibular arch regulating directional outgrowth of the mandibular mesenchyme are mediated by cooperative interactions between BMPs and other growth factors. Our observations also indicated that EGF, another growth factor implicated in mediating epithelial-mesenchymal interactions in the initiation phase of odontogenesis and morphogenesis of the developing mandible, induces an extensive translucent zone and cellular proliferation in the E11 mouse molar-forming mesenchyme and stage 23 chick mandibular mesenchyme. However, in contrast to BMPs, EGF did not induce Msx-1, Msx-2, and Bmp-4, but modulated the effects of BMPs on the expression of Msx-1 and Msx-2 in these mesenchymes. Our combined data suggest that BMP-7 is a component of the signaling network mediating epithelial-mesenchymal interactions during the initiation phase of odontogenesis and morphogenesis of the mandibular arch.     

Epidermal growth factor can replace thymic mesenchyme in induction of embryonic thymus morphogenesis in vitro

The thymus is surrounded by a thin layer of mesenchyme and the epithelial-mesenchymal interaction is known to be essential for the thymus development. To clarify the roles of mesenchyme in the thymus lobule formation that occurs around embryonic days 14–15 in vivo, we set up a three-dimensional organ culture system. The epithelium of embryonic day 13 thymic primordium was separated from the mesenchyme and cultured in Matrigel (reconstituted basement membrane). Addition of the mesenchyme to a chamber separated by a membrane filter induced the lobule formation of the thymic epithelium in vitro. We found that epidermal growth factor (EGF) can replace the mesenchyme for lobulation of the embryonic thymus in vitro. Among other growth factors tested, only transforming growth factor (TGF)-α was as effective as EGF, in agreement with the fact that EGF and TGF-α bind to the same receptor. These results suggest that EGF or its family members may be involved in morphogenesis and differentiation of the thymus gland epithelium, although we cannot exclude the possibility that other unknown factors are required in vivo.     

Vimentin and epithelial-mesenchymal transition in human breast cancer--observations in vitro and in vivo

Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within these tumours is used for prognosis and selection of therapies, there is a continuing need for additional markers to be identified. A considerable amount of current literature, based predominantly on cell culture systems, suggests that a major mechanism responsible for the progression of breast cancer is due to tumour cells losing their epithelial features and gaining mesenchymal properties. These events are proposed to be very similar to the epithelial-mesenchymal transition (EMT) process that has been well characterised in embryonic development. For the developmental and putative cancer EMT, the cell intermediate filament status changes from a keratin-rich network which connects to adherens junctions and hemidesmosomes, to a vimentin-rich network connecting to focal adhesions. This review summarises observations of vimentin expression in breast cancer model systems, and discusses the potential role of EMT in human breast cancer progression, and the prognostic usefulness of vimentin expression.