Reciprocal interactions between epithelium, mesenchyme, and epidermal growth factor (EGF) in the regulation of mandibular mitotic activity in the embryonic chick

Mandibular epithelia and mesenchyme from chick embryos of Hamburger and Hamilton (H.H.) stage 18-25 were cultured intact, in isolation, or in recombinations in the presence or absence of 5-40 ng/ml epidermal growth factor (EGF). 3H-thymidine labelling demonstrated that mesenchyme influenced epithelial mitotic activity and vice versa. EGF can substitute for the epithelial effect. The stimulation of mesenchymal proliferation by H.H. 18 and 22 epithelia correlated with high levels of epithelial proliferation. Epithelial proliferation was low at H.H. 25 and unaffected by mesenchyme or by EGF. Epithelial stimulation of mesenchymal proliferation began earlier (H.H. 18) than did mesenchymal stimulation of epithelial proliferation (H.H. 22); i.e., within the ages tested, the epithelium initiated these reciprocal mitogenic interactions. That epithelial dependence on mesenchyme coincided with epithelial bone-evoking properties, suggested a) that mesenchyme promotes or maintains epithelial bone-promoting activity and b) that the critical differentiative influence of epithelium on mesenchyme is a mitogenic one. The temporal correlation between a sharp decline in mesenchymal proliferation and termination of the osteogenic epithelial-mesenchymal interaction at H.H. 25 further supports a connection between epithelial maintenance of mesenchymal proliferation and epithelial evocation of osteogenesis.     

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro

Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.     

Genistein represses the induction of prostatic buds by testosterone]

Genistein, a phytoestrogen and a kind of endocrine disrupters, inhibits tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor. It is also effective both in the suppression of the prostatic cell proliferation and the prostate carcinogenesis. We have recently demonstrated that several growth factors, like EGF, transforming growth factor-alpha (TGF-alpha), or keratinocyte growth factor (KGF), can induce prostatic bud formation in the absence of androgen. The present study was performed to investigate whether genistein can suppress testosterone-induced prostatic bud formation. Urogenital sinuses of 16.5-day male rat fetuses were cultured organotypically for 5 days in a serum-free medium containing 10 or 100 ng/ml genistein and 50 ng/ml testosterone. The number and total volume of prostatic buds were analyzed by laser scanning microscopy and computerized. We found that genistein inhibits significantly testosterone-induced prostatic bud formation. In the presence of genistein, cell proliferation of the sinus epithelium was suppressed and the number of prostatic buds and total volume of the buds were reduced as compared with those in the sinuses cultured with testosterone alone. Genistein did not appear to cause necrosis of the sinus. These results support our hypothesis that growth factors like EGF secreted from the sinus mesenchyme activated by testosterone are involved in the induction and stimulation of growth of the prostatic buds.     

Control of proliferation of bovine vascular endothelial cells.

The effects of Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) on the proliferation of bovine vascular endothelial cells has been examined. FGF induces the initiation of DNA synthesis and cell proliferation in cloned endothelial cells of fetal and adult origin at concentrations as low as 1 ng/ml and is saturating at 50 ng/ml. EGF had no effect over the same range of concentrations. The mitogenic effect of FGF is blocked by a crude extract of cartilage. Platelet extract is also mitogenic for vascular endothelial cells although to a lesser extent than the purified FGF. In contrast to vascular endothelial cells, both EGF and FGF are mitogenic for vascular smooth muscle cells although EGF is less mitogenic than FGF at 100 ng/ml. The mitogenic effect of EGF and FGF on vascular smooth muscle is not blocked by the addition of a crude extract of cartilage, thus demonstrating the specificity of the chalone like effect of cartilage crude extract for endothelial cells.     

Lack of either chondrocyte hypertrophy or osteogenesis in Meckel's cartilage of the embryonic chick exposed to epithelia and to thyroxine in vitro

Osteogenesis was not initiated when Meckel's cartilages from embryonic chicks of Hamburger and Hamilton (H. H.) stages 38 and 39 were recombined with mandibular epithelia obtained from embryos of H. H. stage 22 (a stage when an epithelial-mesenchymal interaction elicits osteogenesis from mandibular mesenchyme) and grafted to the chorioallantoic membranes of host embryos for 7 to 21 days. Failure of osteogenesis was not because the cartilage inhibited or blocked the osteogenesis-initiating capabilities of mandibular epithelium for mandibular epithelia could still elicit osteogenesis when removed from Meckel's cartilages and recombined with mandibular mesenchyme. Chondrocyte hypertrophy is associated with osteogenesis in other cartilages, including Meckel's cartilage from rodent embryos. However, Meckel's cartilages from chick embryos of H. H. stages 34, 38, and 39 failed to hypertrophy when cultured in the presence of 7.5 nM thyroxine (3,3',5-triiodo-L-thyroxine), although H. H. stage 28 tibial chondrocytes cocultured with Meckel's cartilage did hypertrophy. Therefore, avian Meckelian chondrocytes fail to hypertrophy or to produce osteoprogenitor cells in response to stimuli known to evoke these events in other skeletal cells.     

Stage-related chondrogenic potential of avian mandibular ectomesenchymal cells

We have examined the in vitro stage-related chondrogenic potential of avian mandibular ectomesenchymal cells using micromass cultures. Our results indicate that mandibular ectomesenchymal cells as early as stage 16, soon after the formation of the mandibular arches and well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture. There is an increase in the total area of the cultures occupied by cartilage when cells from increasing stages of development are used. The nodular pattern of chondrogenesis in these cultures indicates that mandibular ectomesenchymal cells are a heterogenous population from the time of mandibular arch formation. In addition, we studied the temporal expression of the genes for extracellular matrix proteins during in vitro chondrogenesis and correlated the morphological changes with the pattern of gene expression. Low levels of type II collagen mRNA are present in the cultures prior to detection of any stainable cartilage matrix and increase 5 fold just before the onset of chondrogenesis in vitro. On the other hand mRNA for cartilage proteoglycan core protein was not detected until the second day of culture when stainable cartilage matrix was present and progressively increased thereafter. Messenger RNA for type I collagen was present at the time of initiation of cultures and continuously increased during the culture period. Our experiments also indicated that embryonic epithelia can inhibit the in vitro chondrogenesis of mandibular ectomesenchymal cells and that the inhibitory effect of embryonic epithelia is independent of its age and site of origin.     

Epidermal growth factor inhibits morphogenesis of the embryonic quail uropygial gland cultured in vitro

Formation of the uropygial papilla and glandular lumena was inhibited when the uropygial rudiment of a day 8 1/3 quail embryo was cultured for 2 days in a chemically defined medium in the presence of 50 ng/mL of epidermal growth factor (EGF). The epithelium of EGF-treated explants remained at the placode stage, or underwent minor invagination into the mesenchyme and became stratified like that of a 12- or 13-day-old embryo. EGF promoted cellular proliferation in the uropygial epithelium and the epidermis adjacent to the gland and it shortened the lag phase of proliferation and markedly stimulated epithelial DNA synthesis, detected immunocytochemically by labeling explants with 5-bromodeoxyuridine (BrdU). The maximal labeling index in EGF-treated uropygial epithelium was 55% higher than in the control. Electron microscopic observation revealed that the basal lamina had become irregular in the EGF-treated explants and that epithelial cytoplasmic processes penetrated through the basal lamina toward the mesenchyme. These same phenomena are observed in vivo when the glandular buds are formed during day 12–13. Some precocious changes occurred in the uropygial epithelium when the rudiment was cultured in the presence of EGF.     

Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization

The present study examined the effect of epidermal growth factor (EGF) during in vitro maturation (IVM) and embryo culture on blastocyst development in the pig. In experiment 1, cumulus oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, hormonal supplements, and with or without EGF (0–40 ng/ml) for 20–22 hr. They then were cultured for an additional 20–22 hr without hormones. After maturation, cumulus-free oocytes were co-incubated with frozen-thawed spermatozoa for 5–6 hr. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 hr. In experiment 2, oocytes were matured in medium containing 10 ng/ml EGF, inseminated, and putative embryos were cultured in the presence of 0–40 ng/ml EGF. In experiment 3, oocytes were cultured in the presence of 0, 10 and 40 ng/ml EGF to examine the kinetics of meiotic maturation. In experiment 4, 2- to 4-cell and 8-cell to morula stage embryos derived from oocytes matured with 10 ng/ml EGF were transferred to the oviduct and uterus, respectively, of each of three recipient gilts (3 and 4 days post-estrus, respectively). The presence or absence of EGF during IVM did not affect cumulus expansion, nuclear maturation, fertilization parameters, or cleavage rate. However, compared to no addition (21%), presence of 1 (33%) and 10 ng/ml EGF (42%) during IVM increased (P < 0.01) the rate of blastocyst development in a concentration-dependent manner. Compared to 10 ng/ml EGF, higher concentrations (20 and 40 ng/ml) reduced (P < 0.01) blastocyst development in a concentration-dependent manner (35% and 24%, respectively). No difference was observed between no addition and 40 ng/ml EGF (22%). Compared to no addition and 10 ng/ml EGF, a significantly (P < 0.001) higher proportion (25% vs. 55%) of oocytes reached metaphase II stage 33 hr after IVM with 40 ng/ml EGF. However, no difference was observed at 44 hr. Transfer of embryos to six recipient gilts resulted in three pregnancies and birth of 18 piglets. The results show that EGF at certain concentrations in IVM medium can influence the developmental competence of oocytes. However, addition of EGF during the culture of pig embryos derived from oocytes matured in the presence of EGF is without effect. Birth of piglets provides evidence that embryos derived from oocytes matured in a medium containing EGF are viable. Mol. Reprod. Dev. 51:395–401, 1998. © 1998 Wiley-Liss, Inc.     

Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondrogenesis

Bone morphogenetic protein 4 (BMP4) induces, whereas epidermal growth factor (EGF) inhibits chondrogenesis. We hypothesize that BMP4 and EGF mediated intracellular signals are both coupled in the regulation of Meckel's cartilage development. Two chondrogenic experimental model systems were employed to test the hypothesis: (1) an ex vivo, serum-free, organ culture system for mouse embryonic mandibular processes, and (2) a micromass culture system for chicken embryonic mandibular processes. Chondrogenesis was assayed by alcian blue staining and expression of Sox9 and type II collagen. Exogenous EGF inhibited and BMP4 induced ectopic cartilage in a dose-dependent manner. When BMP4- and EGF-soaked beads were implanted in juxtaposition within embryonic day 10 mouse mandibular processes, the incidence and amount of ectopic cartilage, and Sox9 and type II collagen expression induced by BMP4, were significantly reduced as the concentration of EGF was increased. Similarly, in chicken serum-free micromass cultures, expression of a constitutively active BMP receptor type IB by replication competent avian retrovirus system promoted the rate and extent of chondrogenesis; however, exogenous EGF attenuated this effect. In micromass cultures, BMP signaling resulted in nuclear translocation and accumulation of the signaling molecule Smad1, whereas the addition of EGF inhibited this event. Our results suggest that BMP4 and EGF function antagonistically, yet are coupled in the regulation of initial chondrogenesis. Smad1 serves as a point of convergence for the integration of two different growth factor signaling pathways during chondrogenesis.     

Platelet-derived growth factor A modulates limb chondrogenesis both in vivo and in vitro

Cartilage formation in the chick limb follows rapid proliferation, condensation and differentiation of limb mesenchyme. The control of these early events is poorly understood. Platelet-derived growth factor receptor alpha (PDGFR-alpha) is present throughout the mesenchyme of early chick limb buds, while its ligand, PDGF-A, is expressed in the surrounding epithelium. PDGFR-alpha is down-regulated in areas that will not give rise to cartilage and is then lost from cartilage forming areas after they begin to differentiate. PDGF-A increases chondrogenesis in micromass cultures of stage-20-24 limb buds, but not stage 25, where it inhibits chondrogenesis. Ectopic PDGF-A in the chick wing can lead to either a localized increase in cartilage formation, or an inhibition. Inhibition of PDGF signalling in the chick limb results in the loss of cartilage. These data demonstrate that PDGF-A functions to promote chondrogenesis at early stages of limb development and suggest that it inhibits chondrogenesis at later stages.     

Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes

We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences, probably inhibitory in nature, must regulate chondrogenesis in mesectodermal derivatives. (ABSTRACT TRUNCATED AT 250 WORDS)     

Dexamethasone and retinyl acetate similarly inhibit and stimulate EGF- or insulin-induced proliferation of prostatic epithelium

Prostatic epithelium proliferates in a defined medium consisting of basal medium RPMI1640 containing transferring (1 microgram/ml), EGF (10 ng/ml), and insulin (3.7 micrograms/ml or 0.1 IU/ml). Although neither dexamethasone nor retinyl acetate affected the proliferation of prostatic epithelium in RPMI1640 containing transferrin alone, they modify the mitogenic effect of EGF and insulin. Dexamethasone at 10(-10) M or retinyl acetate at about 3 X 10(-9) M inhibits proliferation stimulated by EGF. Higher concentrations of dexamethasone (10(-8) - 10(-6) M) or retinyl acetate (3 X 10(-8) - 10(-7) M) enhance the mitogenic activity of EGF. Dexamethasone had a similar effect in the presence of insulin. However, retinyl acetate stimulated, but did not significantly inhibit, proliferation in the presence of insulin. These results suggest that both dexamethasone and retinyl acetate, and possibly other glucocorticoids and retinoids, may regulate the proliferation of prostate epithelium by a dose-dependent modification of the activity of insulin and EGF.     

Effects of epidermal growth factor on maturation, fertilization and development of bovine follicular oocytes

When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 0 to 50 ng/ml EGF, the rate of metaphase II oocytes of 30 ng/ml EGF (97%) was significantly (P < 0.05) higher than that of the control (77%), 10 (85%), and 50 ng/ml EGF (82%). After in vitro fertilization, the rate of monospermic oocytes of 30 ng/ml (75%) and 50 ng/ml EGF (77%) was significantly (P < 0.05) higher than that of the control (56 %). When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 30 ng/ml EGF and/or 10% FCS and fertilized with frozen-thawed spermatozoa, the rate of monospermic oocytes was significantly (P < 0.05) higher in EGF + FCS (82%) than in EGF (61%) and FCS (67%). The rate of oocytes with 2 pronuclei was significantly (P < 0.05) higher in EGF + FCS (54%) than in EGF (27%). When in vitro-fertilized bovine embryos were cultured for 8 d with granulosa cells in TCM 199 containing 0, 10, 30 and 50 ng/ml EGF, the rate of embryos developing to the blastocyst stage was not significantly different among the control (22%), 10 ng/ml (20%), 30 ng/ml (18%), and 50 ng/ml (20%) EGF groups. These results indicate that EGF has a beneficial effect on in vitro maturation and fertilization of oocytes and that EGF plus FCS also have a beneficial effect on normal fertilization of oocytes. However, EGF had no beneficial effect on in vitro development of embryos when they were co-cultured with granulosa cells in medium with FCS.     

Comparative effects of cytosine arabinoside and a prostaglandin E2 antagonist, AH6809, on chondrogenesis in serum-free cultures of chick limb mesenchyme

The present study was designed to compare effects of an established inhibitor of cell proliferation and growth, cytosine arabinoside (Ara C), with that of a prostaglandin E2 (PGE2) antagonist, AH6809, on chondrogenesis in cultured mesenchyme derived from stage 25 chick limb buds. Continuous treatment of cell cultures with 10(-4) M AH6809 prevented completely the twofold increases in DNA content of control cultures which occurred between Day 1 and Day 5 of culture and also produced 90% inhibition of chondrogenesis occurring in control cultures during this same period. Treatment of cells with Ara C (0.1-0.5 microgram/ml) produced equivalent inhibition of DNA content during the same time period; however, chondrogenesis, as evaluated on Day 5 of cell culture, remained at approximately 90% of control cultures. These results indicate that the inhibitory effect of PGE2 receptor blockade on cell growth in these cultures cannot account for the potent inhibitory effects observed on differentiation of cartilage and provide further evidence in support of the notion that PGE2 plays an important initiating role in the process of chondrocyte differentiation within limb mesenchyme.     

Development of the mandibular skeleton in the embryonic chick as evaluated using the DNA-inhibiting agent 5-fluoro-2'-deoxyuridine

Mandibular development was examined in embryonic chicks following administration of 5-fluoro-2'-deoxyuridine (FUDR, 0.001-1.0 microgram/egg), an inhibitor of both DNA synthesis and of cell division. FUDR was injected in ovo at one of three developmental stages corresponding to 1) the migration of mandible-destined, midbrain-level neural crest cells (Hamburger and Hamilton [H.H.] stage 10); 2) midway through the epithelial-mesenchymal interaction required to initiate mandibular osteogenesis (H.H. stage 22), which is also after the epithelial-neural crest cell interaction required for the initiation of chondrogenesis in Meckel's cartilage; and 3) when prechondroblasts of Meckel's cartilage are beginning to differentiate (H.H. stage 25). Micromelia was induced following the administration of FUDR at either H.H. stages 22 or 25 but not when FUDR was given at H.H. stage 10. Although the micromelic mandibles were shorter than normal, Meckel's cartilage and the mandibular membrane bones both differentiated and grew along the full proximodistal length of the shortened mandibles. In contrast to the situation previously described by Ferguson for alligator embryos exposed to FUDR, the migration of neural crest cells in the embryonic chick was not inhibited by FUDR. In contrast to the situation previously described for rat embryos exposed to FUDR, differentiation of Meckel's cartilage was not inhibited in embryonic chicks exposed to FUDR. Differentiation of the membrane bones was also normal following either in ovo administration of FUDR or when mandibular processes were maintained in FUDR in vitro. Therefore, FUDR does not produce micromelia in the embryonic chick by interfering with the epithelial-mesenchymal/neural crest cell interactions, which are prerequisites or differentiation of cartilage or bone, nor by inhibiting the differentiation of chondrogenic or osteogenic mesenchymal cells after completion of these tissue interactions. Neither did the growth-inhibiting action of FUDR result from an inhibition of growth of Meckel's cartilage during the several days following initial chondrogenic differentiation. Rather, subsequent growth of the entire mandibular process was delayed. This mechanism of action differs from that in the alligator embryo, in which FUDR inhibits mandibular growth by removing mandible-destined, migrating neural crest cells, and in the rat, in which FUDR inhibits the differentiation of Meckel's cartilage but catch-up growth restores growth of the mandible to normal.     

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.     

Effects of platelet-contained growth factors (PDGF, EGF, IGF-I, and TGF-β) on DNA synthesis in porcine aortic smooth muscle cells in culture

Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-β (TGF-β) are potent mitogens present in human platelets. Since they are likely to be released simultaneously at the site of vessel injury, their combined effects on vascular smooth muscle cells are more relevant physiologically than their individual actions. Therefore, we added various concentrations of growth factors to quiescent porcine aortic smooth muscle cells cultured in lowserum (0.5%) medium and measured the amount of [³H]thymidine incorporated into DNA. Effect of TGF-β alone was concentration-dependent: stimulatory (1.5-fold increase over the basal) at 0.025 ng/ml and inhibitory at 0.1 ng/ml. Effects of the other three growth factors on DNA synthesis were only stimulatory; their maximally effective concentrations were 20 ng/ml for PDGF (eightfold over the basal), 40 ng/ml for EGF (sixfold increase), and 20 ng/ml for IGF-I (fourfold increase). When PDGF, EGF, and IGF-I were added at submaximally effective concentrations, their effects were additive. TGF-β at 1 ng/ml inhibited at least 50% of the effects of 20 ng/ml EGF and of 10 ng/ml IGF-I, whereas inhibition of the effect of 10 ng/ml PDGF required 10 ng/ml of TGF-β. The concentration of TGF-β needed to inhibit 50% of the combined effect of EGF, IGF-1, and PDGF was 5 ng/ml. These results show complex interrelationships between the growth factors contained in the α-granules of human platelets in their effects on porcine aortic smooth muscle cells.     

Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes

This study was conducted to examine the effect of epidermal growth factor (EGF) and 17beta-estradiol (E2) on nuclear and cytoplasmic (male pronuclear formation and early embryo development) maturation of porcine oocytes. Oocytes were aspirated from antral follicles and cultured in modified TCM-199 medium supplemented with 0.57 mM cysteine, 10 IU/ml eCG, 10 IU/ml hCG, with or without EGF and/or E2. In vitro fertilisation of matured oocytes was performed in a modified Tris-buffered medium (mTBM) with frozen-thawed ejaculated spermatozoa. Oocytes were transferred to NCSU-23 supplemented with 0.4% bovine serum albumin at 6 h after in vitro fertilisation. Significantly higher (p < 0.05) rates of nuclear maturation, pronuclear formation and cleavage (91.7%, 65.2% and 37.3%, respectively) were observed when oocytes were cultured in the medium containing both EGF (10 ng/ml) and E2 (1 microg/ml) than in the medium supplemented with either EGF or E2 or without both. Intracellular glutathione concentration in the oocytes cultured in the medium containing both E2 and EGF was also significantly higher (12.1 pmol per oocyte) than that of oocytes cultured in the medium with E2 or EGF alone or without both. These findings suggested that EGF and E2 have a synergestic effect on both nuclear and cytoplasmic maturation of porcine oocytes.