Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle.

In vitro produced, 2-cell bovine embryos were cultured in serum-free medium supplemented with various combinations of growth factors to test the hypothesis that these polypeptide factors are able to signal preimplantation development. The developmental arrest that occurs during the 8-cell stage with typical culture methods might be relieved by a growth factor-dependent mechanism that would stimulate expression of the embryonic genome, thereby mimicking events that occur in vivo in the oviduct during the fourth cell cycle (8- to 16-cell stage). Subsequently, other growth factors might promote compaction and blastulation, processes which normally occur in the uterus. The effects of growth factors on early embryos were evaluated using phase contrast microscopy to monitor progression to the 8-cell stage, completion and duration of the fourth cell cycle, and blastocyst formation. Platelet derived growth factor (PDGF) promoted development beyond the 16-cell stage in 39.1% of the 2-cell embryos examined in all experiments. The duration of the fourth cell cycle among these embryos was approximately 26 hours. During development after the 16-cell stage, PDGF reduced the proportion of embryos bastulating from 12.7% to 5.8%; in contrast, transforming growth factor alpha (TGF alpha), acting during the same developmental time period, increased the proportion of embryos blastulating from 8.6% to 40.6%. These results, using serum-free medium, indicated that PDGF signalled completion of the fourth cell cycle. TGF alpha, and perhaps basic fibroblast growth factor (bFGF), promoted blastulation of 16-cell embryos during subsequent culture.     

Effect of fibronectin on early embryo development in cows.

Two-cell bovine embryos produced in vitro were cultured in serum-free medium containing the soluble glycoprotein fibronectin (50 micrograms ml-1) to study the function of the extracellular matrix in early development. Some of the embryos (48/164, 29.3%), developed beyond the 16-cell stage compared with none of the 179 controls. Fibronectin at lower (5 micrograms ml-1) or higher (300 micrograms ml-1) concentrations did not promote embryo development (0/89 and 0/82, respectively). Indirect immunofluorescence demonstrated the presence of both fibronectin and its receptor on the surface of eight-cell embryo blastomeres, and biotinylated fibronectin demonstrated that exogenous fibronectin could cross the zona pellucida. These results, demonstrating the successful culture of bovine embryos in serum-free medium, support the hypothesis that the extracellular matrix, specifically fibronectin, plays a role in early development of bovine embryos.     

Transforming growth factor beta 1 and adrenocorticotropin differentially regulate the synthesis of adrenocortical cell heparan sulfate proteoglycans and their binding of basic fibroblast growth factor.

Adrenocortical differentiated functions are under the control of both endocrine hormones such as ACTH and local factors such as transforming growth factor beta (TGF beta) or basic fibroblast growth factor (bFGF). Besides their regulatory actions on the synthesis of corticosteroids, these two classes of factors also exert some important effects on the cellular environment. We have examined here the regulation by ACTH and TGF beta of adrenocortical cell proteoglycan synthesis and secretion. Under basal conditions, adrenocortical cells synthesized and secreted several species of sulfated proteoglycans, 80% of them being recovered in solution in the culture medium. When analyzed by ion exchange chromatography, the cell extracts and the media from cells metabolically labeled with 35S-sulfate were found to contain two and three species of radioactive sulfated proteoglycans, respectively. All species were proteoheparan-sulfates. Treatment of adrenocortical cells with TGF beta 1 or ACTH resulted in a significant increase of the incorporation of 35S into both secreted and cell-associated proteoglycans. ACTH stimulated more than three times the amount of secreted proteoglycans eluting from DEAE-Trisacryl as peak B, whereas TGF beta preferentially increased the amount of peak C. No important modification of the size of the synthesized proteoglycans was observed. The subpopulation of heparan sulfate proteoglycans capable to bind bFGF was also largely increased after ACTH or TGF beta treatment and paralleled the variation in overall proteoheparan sulfate synthesis. Thus those effects of TGF beta and ACTH on proteoglycan synthesis may participate in an increased ability of adrenocortical cells to bind and respond to bFGF.     

Modulation, by transforming growth factor beta (TGF beta), of the mitogenic effect of fibroblast growth factor (FGF) on rat oligodendrocytes in culture

The transforming growth factor beta (TGF beta) is a weak mitogen for rat oligodendrocytes grown in serum-free chemically defined medium. When these cells were treated by basic fibroblast growth factor (bFGF), which is the most potent known mitogen for cultured oligodendrocytes, together with TGF beta we observed that at low doses TGF beta potentiates the mitogenic effect of bFGF while at higher concentrations it partly inhibits this effect.     

Effect of the association of IGF-I, IGF-II, bFGF, TGF-β1, GM-CSF, and LIF on the development of bovine embryos produced in vitro

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-β1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-β1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P < 0.05) on Day 8 after in vitro fertilization and similar results to use of SOF + 10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-β1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.     

The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor.     

Nicotine regulates basic fibroblastic growth factor and transforming growth factor beta1 production in endothelial cells

Nicotine, a constituent of cigarette smoking, may induce atherosclerosis through the production of growth factors. The pattern of bFGF and TGF beta1 production and release by bovine aortic endothelial cells (EC) stimulated with nicotine (from 6 x 10(-4) to 6 x 10(-8) M) was studied. EC viability and count were assessed. The presence of bFGF and TGF beta1 in serum-free conditioned media was determined by the inhibition antibody-binding assay and Western blot analysis. Mitogenic activity of nicotine on EC was also determined. Polymerase chain reaction (PCR) was used to study the expression of bFGF and TGF beta1. The bFGF release after nicotine stimulation was greater than controls, whereas TGF beta1 release was lower. At a nicotine concentration of 6 x 10(-6) M we noted the greatest mitogenic activity. The addition of monoclonal antibody anti-bFGF decreased the tritiated thymidine uptake of EC exposed to nicotine but the addition of monoclonal antibody anti-TGF beta1 had no significant effect. bFGF mRNA expression was significantly higher in EC exposed to nicotine than in controls, whereas TGF beta1 mRNA expression was not modified. From these data we concluded that nicotine regulates bFGF production and release and TGF beta1 release and may have a key role in the development and progression of atherosclerosis.     

Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro

Experiments were carried out to investigate putative beneficial effects of adding epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in chemically defined media. Presumptive zygotes (18 h post-insemination) were randomly assigned to culture treatments. In experiment 1, treatments involved additions of recombinant human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were seen in numbers of 4-cell stage embryos between groups. A concentration of 5 ng/ml EGF but not 1 or 25 ng/ml during embryo culture improved percentages of 4-cell stage embryos reaching blastocysts compared to the control (P<0.05). Numbers of inner cell mass (ICM) cells and trophoblast cells of day 8 blastocysts were similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no differences in numbers of 4-cell stage embryos between groups. When compared to controls, IGF-I treatments at 10 and 50 ng/ml improved proportions of 4-cell stage embryos that reached blastocysts (P<0.05). In experiment 3, numbers of ICM cells of day 8 blastocysts were significantly higher after being cultured with 50 ng/ml of IGF-I compared to those of the controls (P<0.05). No additive effect of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) was seen when results were compared to those following supplementation of the media with either EGF or IGF-I alone. In conclusion, both EGF and IGF-I could independently enhance bovine preimplantational development in chemically defined media and IGF-I but not EGF may play a mitogenic role during early bovine development.     

Transforming growth factor beta-1 positively modulates the bioactivity of fibroblast growth factor on corneal endothelial cells

Transforming growth factor beta-1 (TGF beta-1), known as an inhibitor of vascular endothelial cell proliferation in vitro, stimulates bovine corneal endothelial cells (BCE) proliferation. It also positively modulates the response of BCE cells to fibroblast growth factor (FGF) and epidermal growth factor (EGF). This effect is concentration dependent within a physiological range of TGF beta-1, but it is blocked if cells are cultured on extracellular-matrix-coated dishes instead of plastic. TGF beta-1 does not modify the number or the affinity of bFGF receptors on BCE cell surface but increases the bFGF content of these cells. This suggests that TGF beta-1 might act through regulation of bFGF synthesis in BCE cells.     

A growth factor for cardiac myocytes is produced by cardiac nonmyocytes.

Cardiac nonmyocytes, primarily fibroblasts, surround cardiac myocytes in vivo. We examined whether nonmyocytes could modulate myocyte growth by production of one or more growth factors. Cardiac myocyte hypertrophic growth was stimulated in cultures with increasing numbers of cardiac nonmyocytes. This effect of nonmyocytes on myocyte size was reproduced by serum-free medium conditioned by the cardiac nonmyocytes. The majority of the nonmyocyte-derived myocyte growth-promoting activity bound to heparin-Sepharose and was eluted with 0.75 M NaCl. Several known polypeptide growth factors found recently in cardiac tissue, namely acidic fibroblast growth factor (aFGF), basic FGF (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF beta 1), also caused hypertrophy of cardiac myocytes in a dose-dependent manner. However, the nonmyocyte-derived growth factor (tentatively named NMDGF) could be distinguished from these other growth factors by different heparin-Sepharose binding profiles (TNF alpha, aFGF, bFGF, and TGF beta 1) by neutralizing growth factor-specific antisera (PDGF, TNF alpha, aFGF, bFGF, and TGF beta 1), by the failure of NMDGF to stimulate phosphatidylinositol hydrolysis (PDGF and TGF beta 1), and, finally, by the apparent molecular weight of NMDGF (45-50 kDa). This nonmyocyte-derived heparin-binding growth factor may represent a novel paracrine growth mechanism in myocardium.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis.

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGF beta), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGF beta and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGF beta to bFGF determines the magnitude of the Schwann cell response.     

Exogeneous substances affecting development of in vitro-derived bovine embryos before and after embryonic genome activation

Three experiments were conducted to evaluate how exogenous substances [fetal bovine serum (FBS), arachidonic acid (AA), glutathione (GSH), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF), fibroblast growth factor (FGF), insulin, transferrin and selenium (ITS)] affect preimplantation bovine embryo development. Cumulus-oocyte complexes (COC) were matured and fertilized in vitro, and their development was monitored up to 192 h post insemination in a two-step culture system. In Experiment 1, inseminated oocytes were cultured in modified bovine embryo culture medium (mBECM) supplemented with FBS or BSA for up to 60 h post insemination, and the resultant 8-cell embryos were then cultured singly in mBECM supplemented with AA+GSH+PDGF+TGF. More (P<0.005) blastocysts were derived from 8-cell embryos produced in media containing FBS than BSA. In Experiment 2, the 8-cell embryos produced in mBECM supplemented with FBS were cultured singly in mBECM as follows: 1) no supplementation; 2) AA and GSH or 3) AA, GSH, PDGF and TGF. Compared with no supplementation, a significant (P<0.05) increase in the proportion of 16-cell embryos and morulae was obtained after the addition of either AA+GSH or AA+GSH+PDGF+TGF. In Experiment 3, oocytes were cultured singly in mBECM as follows: 1) no supplementation; 2) AA+GSH+PDGF+TGF; 3) AA+GSH+PDGF+TGF and FGF; 4) AA+GSH+PDGF+TGF and ITS; 5) AA+GSH+PDGF+TGF, FGF and ITS or 6) FBS. Eight-cell embryos grown in each system were subsequently cultured singly in mBECM with AA+GSH+PDGF+TGF. More (P<0.05) 16-cell embryos were obtained in medium supplemented with either AA+GSH+PDGF+TGF and ITS or FBS than in unsupplemented medium. Fewer (P<0.05) oocytes developed to the 8-cell stage with the addition of AA+GSH+PDGF+TGF and FGF than without. In conclusion, embryo development to the blastocyst stage is regulated by exogenous AA, GSH, PDGF, FGF and ITS in a stage-specific manner.     

Growth-factor-related proteins that are inducers in early amphibian development may mediate similar steps in amniote (bird) embryogenesis

Xenopus and murine activin A homologues (XTC-MIF and WEHI-MIF) and Xenopus and bovine basic fibroblast growth factor (bFGFs) are potent inducers of mesodermal and endodermal pathways of development in amphibian blastular animal cap cells. Porcine transforming growth factor beta 2 (TGF beta 2) is a weaker inducer in the same assay but human platelet-derived growth factor (PDGF) is inactive. We have assayed these factors for evidence of homologous effects in bird development. Unlike amphibians, bird embryos never exhibit a clean segregation of a cell layer that has a uniform specification when uninduced, and can be cultured in isolation as an assay after exposure to soluble factors. We have therefore performed less direct experiments, of three types. We have briefly cultured early chick epiblast cells with and without factors and then assayed their capacity to attach and spread upon fibronectin, in comparison with young streak and substreak hypoblast cells. We have asked whether similar microculture with factors alters the ability of quail epiblast cells to disrupt morphogenesis, and to integrate into the structure, of host chick blastoderms into which they are seeded. Finally, whole early chick blastoderms have been preincubated with or without factors for a brief period before setting them up to develop in vitro under circumstances usually permitting successful formation of axial pattern. Strong effects of the activin-like factors, of bFGF and of TGF beta 2 were seen in all three procedures, while PDGF was essentially inactive. In epiblast cells, effective factors at picomolar concentrations induced stable spreading upon fibronectin, and a capacity to adhere and spread upon basal epiblast surface and prevent morphogenesis in host blastoderms. Preincubation of whole early blastoderms with these factors led to characteristic deviation from normal development over the subsequent 24 h. We therefore suggest that peptides from the particular families that are active as inducers in amphibian blastula ectoderm may mediate homologous or closely related steps in respecification throughout vertebrates.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGFβ), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGFβ and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGFβ to bFGF determines the magnitude of the Schwann cell response.     

Fibronectin-associated transforming growth factor

We have studied the ability of fibronectins to induce anchorage-independent growth of NRK-49F cells in serum-free medium. Cells were seeded in soft agar in the presence of various concentrations of plasma fibronectins, and colonies were counted after 10 days. It was found that, with some exceptions, human plasma fibronectins induced anchorage-independent growth at concentrations in 20-100 micrograms/ml range. The ability of exogenously supplied fibronectins to promote anchorage-independent growth of NRK cells is attributed to a transforming growth factor (TGF) activity associated with gelatin-agarose affinity purified plasma fibronectins. This TGF activity required epidermal growth factor (EGF) in our serum-free assay system. The TGF-like activity appears to either co-purify or to be associated with fibronectin at neutral pH during molecular sieve chromatography and during ultracentrifugation through sucrose density gradients. The TGF activity "dissociates" from fibronectin at extremes of pH, however, and can be separated from fibronectin by molecular sieve chromatography in 1 M acetic acid. Under these conditions, the TGF-like activity chromatographed as a single peak with an apparent molecular weight of approximately 30 kDa. The physical-chemical properties, chromatographic behavior, and biological activity of this TGF suggest that it is type-beta transforming growth factor/growth inhibitor (beta-TGF/GI). The TGF activity has been observed in fibronectin isolated from fresh human plasma as well as in fibronectins from several other species obtained from commercial suppliers. Our results would suggest that caution be applied in the interpretation of experiments in which gelatin affinity purified fibronectins are used at micrograms/ml concentrations.