Bombesin and other growth factors activate cell proliferation in chick embryo otic vesicles in culture

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 87-96 ◽  
Author(s):  
J.J. Represa ◽  
C. Miner ◽  
E. Barbosa ◽  
F. Giraldez
Keyword(s):  
Cell Proliferation ◽  
Growth Factors ◽  
Chick Embryo ◽  
Calf Serum ◽  
Cell Number ◽  
Otic Vesicle ◽  
Thymidine Uptake ◽  
Normal Pattern ◽  
Proliferative Growth ◽  
Otic Vesicles

The ability of the mitogenic peptide bombesin and other growth factors to trigger and support early development of the inner ear was studied on chick embryo otocysts in culture. The normal pattern of development was preserved in cultured otic vesicles in the presence of 20% fetal calf serum in the medium. Differentiation proceeded from stage 18 to 22 during the first 24 h and further to stage 24 in 48 h. Estimates of cell number and mitotic rates revealed a distinct period of proliferative growth which was maximum at the 24 h period of incubation. This was coincident with a high rate of DNA synthesis as measured by the acid-precipitable incorporation of [3H]thymidine. Development could be arrested by deprivation of serum during 24h. It could then be reactivated by readmission of serum to proceed with the normal pattern of morphological differentiation and cell proliferation. Bombesin (100 nM) was able to reactivate development in growth-arrested vesicles. Its effect was dose-dependent, saturable and potentiated by insulin (5 micrograms ml-1) which was ineffective if used alone. When associated with insulin, bombesin carried differentiation to stage 21 and stimulated mitotic activity above the level of serum as judged from estimates of cell number and [3H]thymidine uptake. EGF and PDGF were also effective in reinitiating development although their potency was smaller than bombesin. The reactivation by serum or bombesin was blocked by amiloride. The results show that (1) the otic vesicle can provide a useful model for studying the mechanisms that control proliferative growth and differentiation during normal development and (2) bombesin and other growth factors are able to activate growth in embryonic developing tissues.

scholarly journals STUDIES IN THE BIOLOGY OF METALS

1928 ◽  
Vol 48 (5) ◽  
pp. 659-665 ◽  
Author(s):  
Frederick S. Hammett ◽  
Vilma L. Wallace
Keyword(s):  
Cell Proliferation ◽  
Chick Embryo ◽  
Body Length ◽  
Rapid Growth ◽  
Cell Number ◽  
Point Of View ◽  
Lead Ion ◽  
Head Region ◽  
Differential Development

Our study of the effect of the lead ion on the development of the chick embryo has brought out the following facts: 1. Gross growth is retarded. 2. Somite growth is retarded to a degree greater than that exhibited by body length and width. 3. The head and optic anlagen are regions of particular sensitivity. Their differential development is markedly inhibited. From the purely biological point of view these results are in line with the findings of Child (10) and his school as to the sensitivity of the head end of rapidly growing organisms to harmful influences, and with those of Stockard (11) as to the peculiar sensitivity of the optic anlagen. It is almost too well known to need repetition that the head region and the somites of embryos are specific areas of intense growth by increase in cell number. Therefore, turning from the general to the particular, the differential retardation of these regions which is caused by lead, is evidence justifying the conclusion that it is areas of rapid growth by cell proliferation which are selectively inhibited by this metallic ion.

Retinoic acid modulation of the early development of the inner ear is associated with the control of c-fos expression

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1081-1090 ◽  
Author(s):  
J. Represa ◽  
A. Sanchez ◽  
C. Miner ◽  
J. Lewis ◽  
F. Giraldez
Keyword(s):  
Cell Proliferation ◽  
Retinoic Acid ◽  
Inner Ear ◽  
Early Development ◽  
Otic Vesicle ◽  
Low Concentrations ◽  
Full Effect ◽  
20 Nm ◽  
Otic Vesicles

The effects of retinoic acid (RA) on the early development of the inner ear were studied in vitro using isolated chick embryo vesicles. Low concentrations of RA (1–50 nM) inhibited vesicular growth in stage 18 otic vesicles that were made quiescent and then reactivated by either serum or bombesin. Growth inhibition was concentration-dependent and was paralleled by a reduction in the rate of DNA synthesis as measured by [3H]thymidine incorporation. Half-inhibition occurred between 1 and 10 nM RA, and the full effect at 20 nM. Retinoic acid, in the presence of serum, induced the precocious differentiation of (1) secretory epithelium, the tegmentum vasculosum and endolymphatic sac and (2) early sensory and supporting epithelia. These structures were positioned in their corresponding normal presumptive areas. The overall direction of growth was reversed by RA and the ratio of the internal to the external vesicular surface area increased with RA concentration. The expression of the nuclear proto-oncogene c-fos in the developing otic vesicle was transient and stage-dependent. High levels of c-fos mRNA were positively correlated with cell proliferation. Incubation of growth-arrested otic vesicles with bombesin plus insulin at concentrations that induced cell proliferation produced a strong induction of c-fos. This mitogen-induced expression was suppressed by 25 nM RA. The results suggest (1) a role for retinoic acid in controlling the early development of the inner ear and (2) that this control is effected through the regulation of the proto-oncogene c-fos.

Polarization and density of Na-pumps in the inner ear of the chick embryo during early stages of development

Development ◽  
1987 ◽  
Vol 100 (2) ◽  
pp. 271-278
Author(s):  
F. Giraldez ◽  
J.J. Represa ◽  
L. Borondo ◽  
E. Barbosa
Keyword(s):  
Binding Sites ◽  
Fluid Transport ◽  
Developmental Stages ◽  
Otic Vesicle ◽  
Transient Stage ◽  
Highly Sensitive ◽  
Proliferative Growth ◽  
K Concentration ◽  
Otic Vesicles ◽  
Vectorial Transport

The otic vesicle consists of a pseudostratified epithelium with some features of transporting epithelia. The present work questions whether Na-pumps are polarized in this epithelium and what is the relation between the location or density of pumps and development. This was done by measuring the binding of [3H]ouabain to isolated otic vesicles in developmental stages 16 to 22. The results show the presence of specific ouabain-binding sites located in the inward-facing membrane of the otic vesicle epithelium. Binding was saturable at increasing concentrations of ouabain and was highly sensitive to the external K+ concentration with half-maximal inhibition below 0.5 mM, indicating that the binding sites and Na-pump sites are identical. A transient stage-dependent increase in the density of Na-pumps during the period that precedes growth was observed. Evidence is given against this being directly related to an increased net fluid transport rate despite the fact that pump sites were always polarized throughout these stages. The conclusions are that (1) the otic vesicle epithelium is a polarized structure with the possibility of vectorial transport of solutes and water and (2) the increased number of pumps may operate as a regulatory mechanism during normal proliferative growth.

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells

2002 ◽  
Vol 80 (8) ◽  
pp. 790-795 ◽  
Author(s):  
Shirley C Paski ◽  
Zhaoming Xu
Keyword(s):  
Cell Proliferation ◽  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Number ◽  
3T3 Cells ◽  
Intracellular Pool ◽  
Igf I ◽  
Epidermal Growth ◽  
Number Counts

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I) - stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I) - stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.Key words: PDGF, EGF, IGF-I, labile intracellular pool of zinc, cell proliferation, DNA synthesis, 3T3 cells.

scholarly journals Culture of prostate epithelial cells of the rhesus monkey on extracellular matrix substrate: influence of steroids and insulin-like growth factors

1999 ◽  
Vol 162 (3) ◽  
pp. 443-450 ◽  
Author(s):  
TS Udayakumar ◽  
DA Jeyaraj ◽  
M Rajalakshmi ◽  
RS Sharma
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Growth Factors ◽  
Epithelial Cells ◽  
Rhesus Monkey ◽  
Secretory Activity ◽  
Cell Number ◽  
Cell Physiology ◽  
Prostate Epithelial Cells ◽  
Igf I

Rhesus monkey prostate epithelial cells from the cranial lobe were isolated and cultured in flasks coated either with collagen IV or laminin. The effects of stromal cell medium, androgens and growth factors on cell number, thymidine incorporation and secretory activity were assessed. The results indicate that dihydrotestosterone (DHT) and androstenedione have stimulatory influences on cell proliferation and secretion in coated flasks. DHT was more effective in increasing cell number but the induction of secretory activity was similar with both steroids. The combination of IGF-I and -II resulted in inducing better cell proliferation and secretory activity than the individual IGFs but, of the two IGFs, IGF-I was more effective than IGF-II. DHT with IGFs was more potent in inducing proliferation, differentiation and secretion than androstenedione. Even in the absence of steroids or growth factors, colony formation and confluence occurred in coated flasks but cell differentiation and secretion only to a limited extent. In conclusion, we were able to establish an in vitro primary culture of prostate epithelial cells from rhesus monkey using extracellular matrix proteins, steroids and growth factors as additional supplements. This culture system may be useful to study prostate cell physiology and to identify drugs that can inhibit cell proliferation.

Lung fibroblasts from animals breathing 100% oxygen produce growth factors for alveolar type II cells

1990 ◽  
Vol 259 (4) ◽  
pp. L247-L254 ◽  
Author(s):  
M. M. Everett ◽  
R. J. King ◽  
M. B. Jones ◽  
H. M. Martin
Keyword(s):  
Growth Factors ◽  
Conditioned Medium ◽  
Calf Serum ◽  
Cell Number ◽  
Mitotic Division ◽  
Lung Fibroblasts ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Cell Densities

Type II cells were isolated from rats with a purity of 80–95% with less than 4% macrophages. These cells, after plating for approximately 16 h, were cultured with 50% RPMI 1640 and 50% (vol/vol) conditioned medium obtained from confluent hamster lung fibroblasts, together with 0.1% fetal calf serum (FCS). Conditioned media were obtained from either fibroblasts derived from normal hamsters breathing room air [normoxic-conditioned medium (NCM)] or from hamsters exposed for 4 days to 100% O2 [hyperoxic-conditioned medium (HCM)]. Controls consisted of 100% minimal essential medium (MEM) containing 0.1% FCS. Over a 96-h culture period, NCM stabilized cell populations but was unable to induce proliferation. In contrast, at low cell densities, HCM could cause a two- to threefold increase in type II cell number within 24–48 h after introduction. This effect could not be demonstrated at high cell densities. When tested with FCS concentrations ranging from 0 to 10%, maximum effects were obtained using 0.1–0.2% FCS. We conclude that lung fibroblasts from oxidant-injured hamsters produce growth factors that can stimulate at least one mitotic division in cultured type II cells, which are plated at low density. These factors are absent, or present in much lower concentration, in lung fibroblasts from normal animals.

Culture of Arterial Endothelial Cells

1975 ◽  
Vol 34 (03) ◽  
pp. 825-839 ◽  
Author(s):  
Francois M Booyse ◽  
Bonnie J Sedlak ◽  
Max E Rafelson
Keyword(s):  
Endothelial Cells ◽  
Calf Serum ◽  
Intercellular Junctions ◽  
Cell Number ◽  
Population Doubling ◽  
Homogeneous Population ◽  
Bovine Endothelial Cells ◽  
Pinocytotic Vesicles ◽  
Arterial Endothelial Cells ◽  
Doubling Times

SummaryArterial endothelial cells were obtained from bovine aortae by mild treatment with collagenase and medium perfusion. These cells were cultured in RPMI-1640 medium containing 15 mM Hepes buffer and 35% fetal calf serum at pH 7.35. Essentially ah (90–95%) the effluent cells were viable and 80% of these cells attached to the substratum within 1 hour. Small patches of attached cells coalesced to form confluent monolayers in 3–5 days. Confluent monolayers of endothelial cells consisted of a homogeneous population of tightly packed, polygonal cells. Selected cultures were serially subcultured (trypsin-EDTA) for 12–14 months (30–35 passages) without any apparent change in morphology or loss of growth characteristics. Primary and three-month old (15 passages) cultures had population doubling times of 32–34 hours and 29–31 hours, respectively. These cells (primary and subcultures) did not require a minimum cell number to become established in culture. Bovine endothelial cells (primary, first, fifth and thirteenth passages) were characterized ultrastructurally by the presence of Weibel-Palade bodies, pinocytotic vesicles and microfilaments and immunologically by the presence of thrombosthenin-like contractile proteins and Factor VIII antigen. The intercellular junctions of post-confluent cultures stained specifically with silver nitrate. From these data, we concluded that identifiable endothelial cells could be obtained from bovine aortae and cultured and maintained for prolonged periods of time.

scholarly journals EFFECTS OF CYCLIC AMP AND SEPHADEX FRACTIONS OF CHICK EMBRYO EXTRACT ON CLONED RETINAL PIGMENTED EPITHELIUM IN TISSUE CULTURE

1974 ◽  
Vol 61 (2) ◽  
pp. 369-382 ◽  
Author(s):  
D. A. Newsome ◽  
R. T. Fletcher ◽  
W. G. Robison ◽  
K. R. Kenyon ◽  
G. J. Chader
Keyword(s):  
Molecular Weight ◽  
Chick Embryo ◽  
Adenosine Monophosphate ◽  
Cell Number ◽  
Retinal Pigmented Epithelium ◽  
Culture Dish ◽  
Adhesive Properties ◽  
Embryo Extract ◽  
Chick Embryo Extract ◽  
Pigmented Epithelium

The effects of dibutyryl cyclic 3',5'-adenosine monophosphate (BcAMP) and Sephadex G-25 fractions of chick embryo extract on the growth rate, morphology, and pigmentation of normal chick retinal pigmented epithelium (PE) were investigated. Seven cloned PE cell lines were each grown in modified Ham's F-12 medium alone (F-12), or in F-12 supplemented with either high molecular weight (H) or low molecular weight (L) fractions of chick embryo extract. Cells grown in F-12 alone or in L medium formed compact epithelial sheets, whereas cells grown in H had a fibrocytic appearance and formed poorly organized monolayers. In H plus BcAMP, cell morphology was more epithelioid than in H alone, and generally the monolayers appeared more differentiated. Under each of these three culture conditions, 2 x 10-4 M BCAMP retarded the increase in cell number and decreased the final number of cells per culture dish, but had little effect on plating efficiency. BcAMP also increased the rate of cell adhesion to a plastic substratum. Pigmentation was marked in cultures grown in F-12 or in L alone, but the addition of BcAMP dramatically reduced visible pigmentation. This effect was reversed when BcAMP was removed from the culture medium. Thus BcAMP modifies cell and colonial morphology, rate of cell accumulation, adhesive properties, and pigmentation of normal PE cells.

scholarly journals Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1813-1819 ◽  
Author(s):  
Eri Shiraishi ◽  
Norifumi Yoshinaga ◽  
Takeshi Miura ◽  
Hayato Yokoi ◽  
Yuko Wakamatsu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sex Differentiation ◽  
Somatic Cells ◽  
Oryzias Latipes ◽  
Cell Number ◽  
Gonadal Differentiation ◽  
Loss Of Function ◽  
Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

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