- Email: [email protected]
Histomorphological effects of epidermal growth factor on skin and oral mucosa in neonatal mice
Arch.7 oral Biol Vol. 25. pp Pergamon
37 to 43
Press Ltd 1980. Prmted
in Great
Britain
HISTOMORPHOLOGICAL EFFECTS OF EPIDERMAL GROWTH FACTOR ON SKIN AND ORAL MUCOSA IN NEONATAL MICE N. E. STEIDLERand P. C. READE Department of Dental Medicine and Surgery, University of Melbourne, 711 Elizabeth Street, Melbourne 3C00 Victoria, Australia Summary-Epidermal growth factor (EGF), isolated from the submandibular salivary glands of adult male mice, was injected subcutaneously into newborn mice, at dosages of 0.5, 1, 2, 3, and 6 pg/g body wt per day for up to 14 days. Control, weight-matched littermates were injected with 0.15 M saline solution. EGF increased the thickness of the epithelium and keratin of both skin and oral mucosa but respiratory epithelium was unchanged. EGF stimulated the incorporation of DNA precursors and mitosis in epidermal basal cells, but not in the dermis. The lack of promotion of DNA synthesis in the dermis in uivo contradicted the previously reported mitogenic effect of EGF on fibroblasts in cell culture. Thus EGF can modulate some epithelial tissues in oiuo, but is selective in its effect, depending probably on the influence of the underlying connective tissue.
aorn mice were left with their mothers during the experiments and there were no statistically significant differences in the weights of the animals selected for the experiments.
INTRODUCTION Epidermal growth factor (EGF) is a 53-amino acid polypeptide, mol wt 6045 daltons (Savage, Ingami and Cohen, 1972) which was first isolated from the submandibular salivary glands of mice by Cohen (1962). Gregory (1975) demonstrated that there is a great similarity in the amino acid sequence of urogastrone, human EGF as isolated by Cohen and Carpenter (1975) and mouse EGF, indicating that urogastrone is probably EGF. Cohen (1962) showed that injection of EGF into neonatal mice produces premature eruption of the incisors and early eyelid separation. The in vitro effects of EGF have received a great deal of attention; it stimulates both nucleic acid and protein synthesis in a wide variety of cell types, including fibroblasts (Hollenberg & Cuatrecasas, 1973), epithelial cells (Cohen, 1965) and human vascular endothelial cells (Gospodarowicz et al., 1978). EGF is mitogenic to epithelial cells and fibroblasts (Lembach, 1976). Increased keratinization of skin has been reported in neonatal mice given the polypeptide parenterally (Cohen and Elliott, 1963). Mouse EGF increases epithelial proliferation in embryonic chicken skin in vitro showing that EGF is not species-specific, and affects skin directly, rather than by a systemically mediated process (Cohen, 1965). The physiological role of EGF is not yet fully understood, most research having been on cell culture systems. The effects of EGF in viuo have been little studied and nothing is known of its effects on oral mucosa. MATERIALS AND
Purification of EGF EGF was isolated as described by Savage and Cohen (1972). The salivary glands were homogenized, and an acetic acid extract prepared. Following centrifugation at lOO,OOOg, resuspension in HCl and recentrifugation at 100,000 g, the EGF was separated by Biogel-P-10 gel chromatography (Bio-rad Laboratories, California, U.S.A.). The EGF fraction was neutralized, and ultrafiltered under pressure through a Diaflo UM2 membrane (Amicon Corporation, Mass., U.S.A.). Diethylaminoethyl-cellulose ion-exchange gel chromatography (Whatman, Kent, England) was used for the final purification; the pure protein was then desalted, lyophilised and stored as a dry powder at -20°C until used. Administration
METHODS
Animals Swiss White mice, fed and watered ad libitum, used. Adult male mice, 10&120 days old, were for EGF extraction, because the submandibular vary glands of mature male mice contain the EGF (Savage and Cohen, 1972; Roberts, 1974).
and assessment
Purified EGF dissolved in 0.15 M NaCl was injected subcutaneously into the dorsal neck skin of 150 new-born mice at dosages of 0.5, 1, 2, 3, and 6 pgg/g body weight/day. Littermates were injected with an equivalent volume of 0.15 M NaCl. Daily injections were commenced within 8 h of birth (Day 0), and continued at the same time each day. Each day, the body weights were recorded and assessments made of incisor eruption, eyelid separation and of macroscopic external changes. At eight days, animals were given intraperitoneal injections of colchicine (0.2 mg/lOOg body weight), killed by decapitation under halothane anaesthesia 45 min later; specimens were obtained of the heads, palatal and lingual mucosae, caudal dorsal skin remote from the site of injection, ventral skin, and a variety of other organs. In a separate experiment, the neonatal mice were injected with C3H]-thymidine (1 &i/g body weight) and killed 60 min later. The tissue specimens
were used salimost New31
N. E. Steidler and P. C. Reade
38
Table 1. Effect of EGF on times of mandibular
and eyelid separation
Mandibular incisor eruption (age in days)
Dosage of EGF @g/g/day) 0 0.5 1 2 3 6
Eyelid separation (age in days) 12-13 10 9 8 778 7
I@-11 9-10 8-9 7-8 667 667
were fixed in form01 acetate, embedded in paraffin wax by routine methods and sections stained with haematoxylin and eosin. For radioautography, mounted sections were dipped in Nuclear Track Emulsion NTB-2 (Eastman, Kodak, Rochester, New York, U.S.A.) and exposed at room temperature in a lead-lined box with a dessicant for 14 days. The sections were then developed in D-19 developer (Eastman-Kodak, Rochester, New York U.S.A.) and stained with haematoxyhn and eosin. Nuclei were counted as labelled if covered by 5 grains or more (Baserga and Malamud, 1969). All measurements and mitotic figure counts were done by the same observer, who did not know which experimental group the section belonged to. Assessments of epithelial and keratin thicknesses were made with a graduated ocular micrometer grid, which had been previously calibrated against a known length. A minimum of 50 measurements per section were taken, of at least 3 widely spaced sections of tissue. Mitotic indices were obtained by counting the number of colchicine-arrested mitotic figures in at least 1000 basal epithehal cells per section of 3 sections per animal, and expressed as a percentage. Tritiated thymidine labelling indices were derived from counts of the number of cells, in either the basal layer of the epithehum or the fibroblasts of the dermis, which were labelled in at least 1000 cells per section of 3 sections per animal. The proportion of labelled cells was expressed as a percentage. Statistical analysis was by Student’s test. RESULTS
Growth
incisor eruption
eflects
The most striking early and continuing effect of EGF administration in the experimental animals was a marked reduction in total body weight gain (Fig. 1) which was dose-related, in that the greater inhibition
occurred with the higher dosages. The skin was wrinkled and scaling, and hair growth seemed to be impaired. No deaths occurred during the experimental period. Incisor
eruption
and eyelid
separation
Incisor eruption and eyelid separation occurred prematurely; both, especially eyelid separation, were dose-related (Table 1). Histologically, both teeth and eyelids appeared normal. Skin
Keratinization of both ventral and dorsal skin increased in EGF-treated animals (Figs. 2 and 3; Table 2) and the cellular layer of the epidermis was thicker in animals given 2 pg--6pg EGF/g body weight/day. Even though the administration of EGF appeared to have a retarding effect on hair growth, no gross histological changes was seen in the hair follicles. The total skin thickness was markedly decreased despite the increased thickness of keratinized and nucleated epidermal layers. The thickness of the skin, as measured from the external limit of the stratum granulosum to the external margin of the paniculus carnosus, was decreased by approximately one-third in animals given 2 pg EGF/g body weight per day for 8 days (Table 2). This difference in thickness was more marked with higher dosages of EGF and due to a decrease in the thickness of the dermis, as well as a decrease in the adipose tissue content of the skin. To study how EGF affects the dermis, some experimental and control animals were injected with C3H]-thymidine (1 nCi/g body weight) and the skin examined by radioautography. EGF increased the labelling index of basal epithelial cells (EGF:9.9 k 2.4; Control: 3.8 &- 1.6, p < 0.001) compared with controls, but
Table 2. Effect of EGF on dorsal skin EGF (pg/EGF/g body wt/day) (n = 60) Control 3 pg 6pg 2pg (n = 20) (n = 20) (n = 20) (n = 20)
Measurement Thickness of skin Thickness of nucleated cellular layer Keratin thickness Mitotic index (%)
* p < 0.001. ** p < 0.01.
(compared
750 + 50 19 * 11 41 + 12 3.3 f 1.2
580 k 45*
550 f 50*
42 f 12* 42 + 14* 84 &- 22* 89 k 26* 4.7 f 1.8** -
with control group).
460 + 120* 43 * 12* 85 + 25*
EGF and neonatal murine oral mucosa and skin
39
Table 3. Effect of EGF on oral mucosal epithelium EGF (pg/EGF/g Measurement
Type Palatal
Lingual
Thickness of nucleated cellular layer Keratin thickness Mitotic index (%) Thickness of nucleated cellular layer Keratin thickness Mitotic index (%)
body wt/day) (n = 60) 3 pg 6pg (n = 20) (n = 20)
Control (n = 20)
2% (n = 20)
30 + 6 9+2 1.3 + 0.4
38 + I* 11 + 2** 2.2 f 0.6*
42 + 6* 12 f 3*
48 _t 8* 12 _t 3***
31 * I 16 f 3 1.2 f 0.4
40 f 8* 18 k 3**** 4,0 + 2.-l*
59 * 10* 18 f 4*****
59 * 9* 20 * 4*
* p < 0.001. ** p < 0.005. *** p < 0.02. **** p < 0.05. ****i 0.05 < p < 0.10.
(compared
with control
group).
did not significantly alter the labelling mdex of the fibrous connective tissue cells (EGF:2.8 f 1.5; Control:3.4 f 1.8; p > 0.1). Mitotic indices of basal epithelial cells, as determined by the colchicine method, were increased in EGF-treated animals (EGF: 4.7 f 1.8; Control: 3.3 f 1.2; p < 0.05) (Table 2). Oral mucosa The effect of EGF on the epithelium of the oral mucosa was not as marked as that seen in skin, but was qualitatively similar. Dorsal lingual mucosa from EGF-treated animals was more highly keratinized than control tissue, and the epithehum was thicker, especially with higher dosages of EGF (Figs. 4 and 5). The keratin peaks on the lingual papillae were higher but because of the difficulty in determining the level of section of each papilla, the keratin thickness was recorded only in the regions between the papillae, where the keratin was of more even thickness. A significant increase in keratin thickness did not, however, occur consistently in all experimental animals, although the nucleated cellular component of the epithelium was significantly thicker @ < 0.001) in all mice given 2 pg-6 pg EGF/g body weight/day. There were similar changes in the palatal mucosa (Figs. 6 and 7). With EGF, the cellular nucleated component of the epithelium was thicker. This was apparently a result of cellular proliferation, because the number of cell layers was increased and the mitotic index was greater (EGF:2.2 f 0.6; Control: 1.3 + 0.4; p < 0.001) (Table 3). The keratin layer was thicker in EGF-treated animals, and in these areas, the stratum granulosum was more prominent. There was a tendency for the thickness of the keratin and nucleated cellular layers to be greater with higher doses of EGF. The palatal epithelium in both control and EGFtreated animals was orthokeratinized; there was no evidence of a change to parakeratinization associated with the increase in epithelial proliferation. Respiratory
epithelium
The mucosa lining the upper respiratory tract and maxillary sinuses showed no difference in morphology or mitotic index between control and experimental animals.
DISCUSSION
The changes in the dorsal and ventral skin were similar to those reported by Cohen and Elliott (1963). The apparent retardation by EGF of mesenchymal growth, as measured by the decreased rate of total body growth, and decreased thickness of the dermis in EGF-treated animals might be due to a specific mesenchymal retarding effect or might be part of a systemic effect of EGF on metabolism or nutrition. Heimberg et al. (1965) found that in animals given l-2 pg EGF/g body weight per day for 10 days, the liver became fatty, with a net accumulation of triglycerides. Both epithelial cells (Cohen, 1965) and fibroblasts (Lembach, 1976) respond to EGF in vitro by increased DNA synthesis and mitosis. Canalis and Raisz (1978) showed that EGF appears to inhibit collagen synthesis in bone in vitro, as assessed by incorporation of [3H]-proline into collagenase-digestible protein. Our findings suggest collagen synthesis may be similarly affected in vivo. The inter-dependence of epithehum and mesenthyme is of potential importance in the regulation of the response to EGF. However Cohen (1965) found that in vitro, EGF-induced changes in epithelium occur independently of the presence of connective tissue. On the other hand, EGF does not induce proliferation of bovine cornea1 or lens epithehal cells in cell culture, despite the presence of specific plasma membrane receptors for that substance (Gospodarowicz et al., 1977) whereas topically applied EGF induces cornea1 epithelial proliferation in viva in damaged corneas (Frati et al., 1972). Similarly, Tyler and Pratt (1979), showed that the stimulation of DNA synthesis induced by EGF in the presumptive fusional zone of the epithelium of the palatal shelves in the rat embryo is dependent on the presence of the underlying mesenchyme. Thus the response of epithelium to EGF can be modified by connective tissue, and it is possible that the response of connective tissue cells is similarly controlled. We show here that, in the neonatal mouse, EGF does not alter the incorporation of DNA precursors into cells of the dermis, in contrast to the mitogenic and DNA synthesis-pro-
N. E. Steidler and P. C. Reade
40
moting effects of EGF on fibroblasts in t&o, but is mitogenic to basal epidermal cells. In an in uitro study, Paul et al. (1978) showed that cells possessing receptors for EGF on their surfaces may not be capable of reacting to it, probably because of an inability to progress through the sequence of events that follow polypeptide-receptor binding. A similar mechanism might explain the lack of effect of EGF on the morphology or mitotic index of respiratory epithelium. A more probable explanation of the lack of effect in our study is that there may be a modifying effect mediated by the connective tissue. which controls the response of the respiratory epithehum to EGF. In general, the changes m oral mucosa were similar to those in skin, with an increase in keratinization, epithelial thickness, mitotic index and prominence of the stratum granulosum. Mucosa which is rapidly proliferating tends to produce parakeratin rather than orthokeratin, whereas more slowly proliferating epithelium tends to be orthokeratinized. In our study, the association of more mitosis with orthokeratinization may be related to the short duration of the EGF administration. and implies that EGF specifically affects both processes, and therefore maintains orthokeratinization even when the mitotic rate increases, Webster and Rubright (1978) found that removal of the submandibular salivary glands from adult mice does not affect the keratinization or mitotic rates in oral mucosa. Our findings seem at first to be out of accord with this. However, our animals were neonatal and the responsiveness to EGF of the oral mucosa and skin may change with age; injections of EGF do not produce discernible changes in dorsal skin in adult mice (Cohen and Elliott, 1963), and Bertsch and Marks (1974) found that DNA synthesis in explants of chick epidermis in vitro is not increased by EGF, whereas explants of chick embryonic epidermis respond by increased DNA synthesis, On the other hand, a possible explanation is that removal of the submandibular salivary gland complex does not necessarily eliminate EGF production. Serum concentrations of EGF in male mice, determined by radioimmunoassay, remain unchanged for at least 3 days after sialadenectomy (Byyny et al., 1974) indicating that there is an accessory site for EGF production, which may supplement that of the submandibular glands; alternatively endogenous plasma EGF produced before salivary gland removal may remain active for a time after sialadenectomy. The Jong interval of 55 days between sialadenectomy and examination in Webster and Rubright’s (1978) study makes it more probable that there is a non-salivary gland site of EGF production.
ques and Application. Chap. 5, p, 134. Harper & Rowe, New York. Bertsch S. and Marks F. 1974. Etfect of foetal calf serum and epidermal growth factor on DNA synthesis in explants of chick embryo epidermis. Nature 251, 517 -519.
Byyny R. L., Orth D. N.. Cohen S. and Doyne E. S. 1974. Epidermal growth factor: effects of androgens and adrenergic agents. Endocrinology 95. 776782. Canalis E. and Raisz L. G. 1979. EtTect of epidermal growth factor in oitro. Endocrinology 104, 862-869. Cohen S. 1962. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J. biol. Chem. 237, 155551562. Cohen S. 1965. The stimulation of epidermal proliferation by a specific protein (EGF). Deul Biol. 12, 394407. Cohen S. and Carpenter G. 1975. Human epidermal growth factor: isolation and chemical and biological orouerties. Proc. natn. Acud. Sci. U.S.A. 72. 1317-1321. Cohen S. and Elliott G. A. 1963. The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J. invest. Derm. 40, I-5. Frati L., Daniele S.. De Logu A. and Covelli 1. 1972. Selective binding of the epidermal growth factor and its specific effects on the epithelial cells of the cornea. Expl Eye Res. 14, 135-141. Gospodarowicz D., Brown K. D., Birdwell C. R. and Zetter B. R. 1978. Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor. epidermal growth factor and thrombin. J. cell Eiol. 77, 77478X. Gospodarowicz D., Mescher A. L., Brown K. D. and Birdwell C. R. 1977. The role of fibroblast growth factor and epidermal growth factor in the proliferative response of the cornea1 and lens epithelium. Expl Eye Res. 25, 631-649. Gregory H. 1975. Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 257. 3255327. Heimberg M., Weinstein I., Le Quire V. S. and Cohen S. 1965. The induction of fattv liver in neonatal animals by a purified protein (EGF) from mouse submaxillary gland. Ltfe Sci. 4, 162551633. Hollenberg M. D. and Cuatrecasas P. 1973. Epidermal growth factor: receptors in human fibroblasts and modulation of action by cholera toxin. Proc. natn. Acad. Sci. U.S.A. 70, 29642968.
Lembach K. J. 1976. Induction of human tibroblast proliferation bv_ epidermal growth factor (EGF): enhance_ ment by an EGF-binding arginine esterase and by ascorbate. Proc. natn. Acad. Sci. U.S.A. 13, 1833187. Paul D.. Brown K. D.. Rupniak H. T. and Ristow H. J. 1978. Cell cycle regulation by growth factors and nutrients in normal and transformed cells. In vitro 14, 76-84. Roberts M. L. 1974. Testosterone-induced accumulation of epidermal growth factor in the submandibular salivary glands of mice, assessed by radioimmunoassay. Biochem. Pharmac.
Acknowledgemrnts~This study was supported by a grant from the National Health and Medical Research Council of Australia. We thank Mr. D. Rowler for the excellence of his technical assistance in preparing the histological specimens. We are grateful to Mr. T. Dobrostanski for photographic assistance and to Mrs. E. Gates for typing the manuscript. REFERENCES
Baserga
R. and
Experimentul
Malamud Pathology
D. 1969. Modern Methods ift ctnd Autoradiography Terhni-
23, 3305~3308.
Savage C. R. and Cohen S. 1972. Epidermal growth factor and a new derivative. J. biol. Chem. 247, 7609761 I. Savage C. R., Ingami T. and Cohen S. 1972. The primary structure of epidermal growth factor. J. biol. Chem. 247, 7612-7621. Tyler M. S. and Pratt R. M. 1979. Effect of epidermal growth factor on the isolated palatal epithelium in vitro. J. dent. Rex 58, 385 (abstract). Webster’ A. K. and Rubright W. C. 1978. Histologic measurements of the effects of submandibular gland removal on oral and skin epithelia of mice. Archs oral Biol. 23, 597-599.
EGF and neonatal murine oral mucosa and skin
Plate 1 overleaf.
41
42
N. E. Steidler and P. C. Reade
Plate 1 Fig. 1. A control mouse (above) and an experimental mouse to which was administered 2pg EGF/g body wt/day for 8 days (below), showing retardation of hair growth and stunting due to EGF. x 1.5 approx. Figs 2 and 3. Sections of normal murine dorsal skin (Fig. 2) and dorsal skin from a mouse given 2 pg EGF/g body wt/day for 8 days (Fig. 3) demonstrating decrease in skin thickness as measured from the paniculus carnosus (P), in&ased keratin and nucleated cellular epithelial thickness due to the effects of EGF administration. x 45 Figs 4 and 5. Sections of dorsal lingual mucosa from a control mouse aged 8 days (Fig. 4) and a mouse given 2 pg EGF/g body wt/day for 8 days (Fig. 5). The EGF-treated animal has thicker lingual epithelium and keratin, and a more prominent granular cell layer. x 450 Figs, 6 and 7. Sections showing palatal mucosa from a control mouse aged 8 days (Fig. 6) and a mouse of the same age who had received 2 pg EGF/g body wt/day since birth (Fig. 7). The palatal epithelium from the EGF-treated animal has thicker keratin and a thicker nucleated cellular layer than control tissue. x 450
EGF and neonatal murine oral mucosa and skin
Plate 1.
43
37 to 43
Press Ltd 1980. Prmted
in Great
Britain
HISTOMORPHOLOGICAL EFFECTS OF EPIDERMAL GROWTH FACTOR ON SKIN AND ORAL MUCOSA IN NEONATAL MICE N. E. STEIDLERand P. C. READE Department of Dental Medicine and Surgery, University of Melbourne, 711 Elizabeth Street, Melbourne 3C00 Victoria, Australia Summary-Epidermal growth factor (EGF), isolated from the submandibular salivary glands of adult male mice, was injected subcutaneously into newborn mice, at dosages of 0.5, 1, 2, 3, and 6 pg/g body wt per day for up to 14 days. Control, weight-matched littermates were injected with 0.15 M saline solution. EGF increased the thickness of the epithelium and keratin of both skin and oral mucosa but respiratory epithelium was unchanged. EGF stimulated the incorporation of DNA precursors and mitosis in epidermal basal cells, but not in the dermis. The lack of promotion of DNA synthesis in the dermis in uivo contradicted the previously reported mitogenic effect of EGF on fibroblasts in cell culture. Thus EGF can modulate some epithelial tissues in oiuo, but is selective in its effect, depending probably on the influence of the underlying connective tissue.
aorn mice were left with their mothers during the experiments and there were no statistically significant differences in the weights of the animals selected for the experiments.
INTRODUCTION Epidermal growth factor (EGF) is a 53-amino acid polypeptide, mol wt 6045 daltons (Savage, Ingami and Cohen, 1972) which was first isolated from the submandibular salivary glands of mice by Cohen (1962). Gregory (1975) demonstrated that there is a great similarity in the amino acid sequence of urogastrone, human EGF as isolated by Cohen and Carpenter (1975) and mouse EGF, indicating that urogastrone is probably EGF. Cohen (1962) showed that injection of EGF into neonatal mice produces premature eruption of the incisors and early eyelid separation. The in vitro effects of EGF have received a great deal of attention; it stimulates both nucleic acid and protein synthesis in a wide variety of cell types, including fibroblasts (Hollenberg & Cuatrecasas, 1973), epithelial cells (Cohen, 1965) and human vascular endothelial cells (Gospodarowicz et al., 1978). EGF is mitogenic to epithelial cells and fibroblasts (Lembach, 1976). Increased keratinization of skin has been reported in neonatal mice given the polypeptide parenterally (Cohen and Elliott, 1963). Mouse EGF increases epithelial proliferation in embryonic chicken skin in vitro showing that EGF is not species-specific, and affects skin directly, rather than by a systemically mediated process (Cohen, 1965). The physiological role of EGF is not yet fully understood, most research having been on cell culture systems. The effects of EGF in viuo have been little studied and nothing is known of its effects on oral mucosa. MATERIALS AND
Purification of EGF EGF was isolated as described by Savage and Cohen (1972). The salivary glands were homogenized, and an acetic acid extract prepared. Following centrifugation at lOO,OOOg, resuspension in HCl and recentrifugation at 100,000 g, the EGF was separated by Biogel-P-10 gel chromatography (Bio-rad Laboratories, California, U.S.A.). The EGF fraction was neutralized, and ultrafiltered under pressure through a Diaflo UM2 membrane (Amicon Corporation, Mass., U.S.A.). Diethylaminoethyl-cellulose ion-exchange gel chromatography (Whatman, Kent, England) was used for the final purification; the pure protein was then desalted, lyophilised and stored as a dry powder at -20°C until used. Administration
METHODS
Animals Swiss White mice, fed and watered ad libitum, used. Adult male mice, 10&120 days old, were for EGF extraction, because the submandibular vary glands of mature male mice contain the EGF (Savage and Cohen, 1972; Roberts, 1974).
and assessment
Purified EGF dissolved in 0.15 M NaCl was injected subcutaneously into the dorsal neck skin of 150 new-born mice at dosages of 0.5, 1, 2, 3, and 6 pgg/g body weight/day. Littermates were injected with an equivalent volume of 0.15 M NaCl. Daily injections were commenced within 8 h of birth (Day 0), and continued at the same time each day. Each day, the body weights were recorded and assessments made of incisor eruption, eyelid separation and of macroscopic external changes. At eight days, animals were given intraperitoneal injections of colchicine (0.2 mg/lOOg body weight), killed by decapitation under halothane anaesthesia 45 min later; specimens were obtained of the heads, palatal and lingual mucosae, caudal dorsal skin remote from the site of injection, ventral skin, and a variety of other organs. In a separate experiment, the neonatal mice were injected with C3H]-thymidine (1 &i/g body weight) and killed 60 min later. The tissue specimens
were used salimost New31
N. E. Steidler and P. C. Reade
38
Table 1. Effect of EGF on times of mandibular
and eyelid separation
Mandibular incisor eruption (age in days)
Dosage of EGF @g/g/day) 0 0.5 1 2 3 6
Eyelid separation (age in days) 12-13 10 9 8 778 7
I@-11 9-10 8-9 7-8 667 667
were fixed in form01 acetate, embedded in paraffin wax by routine methods and sections stained with haematoxylin and eosin. For radioautography, mounted sections were dipped in Nuclear Track Emulsion NTB-2 (Eastman, Kodak, Rochester, New York, U.S.A.) and exposed at room temperature in a lead-lined box with a dessicant for 14 days. The sections were then developed in D-19 developer (Eastman-Kodak, Rochester, New York U.S.A.) and stained with haematoxyhn and eosin. Nuclei were counted as labelled if covered by 5 grains or more (Baserga and Malamud, 1969). All measurements and mitotic figure counts were done by the same observer, who did not know which experimental group the section belonged to. Assessments of epithelial and keratin thicknesses were made with a graduated ocular micrometer grid, which had been previously calibrated against a known length. A minimum of 50 measurements per section were taken, of at least 3 widely spaced sections of tissue. Mitotic indices were obtained by counting the number of colchicine-arrested mitotic figures in at least 1000 basal epithehal cells per section of 3 sections per animal, and expressed as a percentage. Tritiated thymidine labelling indices were derived from counts of the number of cells, in either the basal layer of the epithehum or the fibroblasts of the dermis, which were labelled in at least 1000 cells per section of 3 sections per animal. The proportion of labelled cells was expressed as a percentage. Statistical analysis was by Student’s test. RESULTS
Growth
incisor eruption
eflects
The most striking early and continuing effect of EGF administration in the experimental animals was a marked reduction in total body weight gain (Fig. 1) which was dose-related, in that the greater inhibition
occurred with the higher dosages. The skin was wrinkled and scaling, and hair growth seemed to be impaired. No deaths occurred during the experimental period. Incisor
eruption
and eyelid
separation
Incisor eruption and eyelid separation occurred prematurely; both, especially eyelid separation, were dose-related (Table 1). Histologically, both teeth and eyelids appeared normal. Skin
Keratinization of both ventral and dorsal skin increased in EGF-treated animals (Figs. 2 and 3; Table 2) and the cellular layer of the epidermis was thicker in animals given 2 pg--6pg EGF/g body weight/day. Even though the administration of EGF appeared to have a retarding effect on hair growth, no gross histological changes was seen in the hair follicles. The total skin thickness was markedly decreased despite the increased thickness of keratinized and nucleated epidermal layers. The thickness of the skin, as measured from the external limit of the stratum granulosum to the external margin of the paniculus carnosus, was decreased by approximately one-third in animals given 2 pg EGF/g body weight per day for 8 days (Table 2). This difference in thickness was more marked with higher dosages of EGF and due to a decrease in the thickness of the dermis, as well as a decrease in the adipose tissue content of the skin. To study how EGF affects the dermis, some experimental and control animals were injected with C3H]-thymidine (1 nCi/g body weight) and the skin examined by radioautography. EGF increased the labelling index of basal epithelial cells (EGF:9.9 k 2.4; Control: 3.8 &- 1.6, p < 0.001) compared with controls, but
Table 2. Effect of EGF on dorsal skin EGF (pg/EGF/g body wt/day) (n = 60) Control 3 pg 6pg 2pg (n = 20) (n = 20) (n = 20) (n = 20)
Measurement Thickness of skin Thickness of nucleated cellular layer Keratin thickness Mitotic index (%)
* p < 0.001. ** p < 0.01.
(compared
750 + 50 19 * 11 41 + 12 3.3 f 1.2
580 k 45*
550 f 50*
42 f 12* 42 + 14* 84 &- 22* 89 k 26* 4.7 f 1.8** -
with control group).
460 + 120* 43 * 12* 85 + 25*
EGF and neonatal murine oral mucosa and skin
39
Table 3. Effect of EGF on oral mucosal epithelium EGF (pg/EGF/g Measurement
Type Palatal
Lingual
Thickness of nucleated cellular layer Keratin thickness Mitotic index (%) Thickness of nucleated cellular layer Keratin thickness Mitotic index (%)
body wt/day) (n = 60) 3 pg 6pg (n = 20) (n = 20)
Control (n = 20)
2% (n = 20)
30 + 6 9+2 1.3 + 0.4
38 + I* 11 + 2** 2.2 f 0.6*
42 + 6* 12 f 3*
48 _t 8* 12 _t 3***
31 * I 16 f 3 1.2 f 0.4
40 f 8* 18 k 3**** 4,0 + 2.-l*
59 * 10* 18 f 4*****
59 * 9* 20 * 4*
* p < 0.001. ** p < 0.005. *** p < 0.02. **** p < 0.05. ****i 0.05 < p < 0.10.
(compared
with control
group).
did not significantly alter the labelling mdex of the fibrous connective tissue cells (EGF:2.8 f 1.5; Control:3.4 f 1.8; p > 0.1). Mitotic indices of basal epithelial cells, as determined by the colchicine method, were increased in EGF-treated animals (EGF: 4.7 f 1.8; Control: 3.3 f 1.2; p < 0.05) (Table 2). Oral mucosa The effect of EGF on the epithelium of the oral mucosa was not as marked as that seen in skin, but was qualitatively similar. Dorsal lingual mucosa from EGF-treated animals was more highly keratinized than control tissue, and the epithehum was thicker, especially with higher dosages of EGF (Figs. 4 and 5). The keratin peaks on the lingual papillae were higher but because of the difficulty in determining the level of section of each papilla, the keratin thickness was recorded only in the regions between the papillae, where the keratin was of more even thickness. A significant increase in keratin thickness did not, however, occur consistently in all experimental animals, although the nucleated cellular component of the epithelium was significantly thicker @ < 0.001) in all mice given 2 pg-6 pg EGF/g body weight/day. There were similar changes in the palatal mucosa (Figs. 6 and 7). With EGF, the cellular nucleated component of the epithelium was thicker. This was apparently a result of cellular proliferation, because the number of cell layers was increased and the mitotic index was greater (EGF:2.2 f 0.6; Control: 1.3 + 0.4; p < 0.001) (Table 3). The keratin layer was thicker in EGF-treated animals, and in these areas, the stratum granulosum was more prominent. There was a tendency for the thickness of the keratin and nucleated cellular layers to be greater with higher doses of EGF. The palatal epithelium in both control and EGFtreated animals was orthokeratinized; there was no evidence of a change to parakeratinization associated with the increase in epithelial proliferation. Respiratory
epithelium
The mucosa lining the upper respiratory tract and maxillary sinuses showed no difference in morphology or mitotic index between control and experimental animals.
DISCUSSION
The changes in the dorsal and ventral skin were similar to those reported by Cohen and Elliott (1963). The apparent retardation by EGF of mesenchymal growth, as measured by the decreased rate of total body growth, and decreased thickness of the dermis in EGF-treated animals might be due to a specific mesenchymal retarding effect or might be part of a systemic effect of EGF on metabolism or nutrition. Heimberg et al. (1965) found that in animals given l-2 pg EGF/g body weight per day for 10 days, the liver became fatty, with a net accumulation of triglycerides. Both epithelial cells (Cohen, 1965) and fibroblasts (Lembach, 1976) respond to EGF in vitro by increased DNA synthesis and mitosis. Canalis and Raisz (1978) showed that EGF appears to inhibit collagen synthesis in bone in vitro, as assessed by incorporation of [3H]-proline into collagenase-digestible protein. Our findings suggest collagen synthesis may be similarly affected in vivo. The inter-dependence of epithehum and mesenthyme is of potential importance in the regulation of the response to EGF. However Cohen (1965) found that in vitro, EGF-induced changes in epithelium occur independently of the presence of connective tissue. On the other hand, EGF does not induce proliferation of bovine cornea1 or lens epithehal cells in cell culture, despite the presence of specific plasma membrane receptors for that substance (Gospodarowicz et al., 1977) whereas topically applied EGF induces cornea1 epithelial proliferation in viva in damaged corneas (Frati et al., 1972). Similarly, Tyler and Pratt (1979), showed that the stimulation of DNA synthesis induced by EGF in the presumptive fusional zone of the epithelium of the palatal shelves in the rat embryo is dependent on the presence of the underlying mesenchyme. Thus the response of epithelium to EGF can be modified by connective tissue, and it is possible that the response of connective tissue cells is similarly controlled. We show here that, in the neonatal mouse, EGF does not alter the incorporation of DNA precursors into cells of the dermis, in contrast to the mitogenic and DNA synthesis-pro-
N. E. Steidler and P. C. Reade
40
moting effects of EGF on fibroblasts in t&o, but is mitogenic to basal epidermal cells. In an in uitro study, Paul et al. (1978) showed that cells possessing receptors for EGF on their surfaces may not be capable of reacting to it, probably because of an inability to progress through the sequence of events that follow polypeptide-receptor binding. A similar mechanism might explain the lack of effect of EGF on the morphology or mitotic index of respiratory epithelium. A more probable explanation of the lack of effect in our study is that there may be a modifying effect mediated by the connective tissue. which controls the response of the respiratory epithehum to EGF. In general, the changes m oral mucosa were similar to those in skin, with an increase in keratinization, epithelial thickness, mitotic index and prominence of the stratum granulosum. Mucosa which is rapidly proliferating tends to produce parakeratin rather than orthokeratin, whereas more slowly proliferating epithelium tends to be orthokeratinized. In our study, the association of more mitosis with orthokeratinization may be related to the short duration of the EGF administration. and implies that EGF specifically affects both processes, and therefore maintains orthokeratinization even when the mitotic rate increases, Webster and Rubright (1978) found that removal of the submandibular salivary glands from adult mice does not affect the keratinization or mitotic rates in oral mucosa. Our findings seem at first to be out of accord with this. However, our animals were neonatal and the responsiveness to EGF of the oral mucosa and skin may change with age; injections of EGF do not produce discernible changes in dorsal skin in adult mice (Cohen and Elliott, 1963), and Bertsch and Marks (1974) found that DNA synthesis in explants of chick epidermis in vitro is not increased by EGF, whereas explants of chick embryonic epidermis respond by increased DNA synthesis, On the other hand, a possible explanation is that removal of the submandibular salivary gland complex does not necessarily eliminate EGF production. Serum concentrations of EGF in male mice, determined by radioimmunoassay, remain unchanged for at least 3 days after sialadenectomy (Byyny et al., 1974) indicating that there is an accessory site for EGF production, which may supplement that of the submandibular glands; alternatively endogenous plasma EGF produced before salivary gland removal may remain active for a time after sialadenectomy. The Jong interval of 55 days between sialadenectomy and examination in Webster and Rubright’s (1978) study makes it more probable that there is a non-salivary gland site of EGF production.
ques and Application. Chap. 5, p, 134. Harper & Rowe, New York. Bertsch S. and Marks F. 1974. Etfect of foetal calf serum and epidermal growth factor on DNA synthesis in explants of chick embryo epidermis. Nature 251, 517 -519.
Byyny R. L., Orth D. N.. Cohen S. and Doyne E. S. 1974. Epidermal growth factor: effects of androgens and adrenergic agents. Endocrinology 95. 776782. Canalis E. and Raisz L. G. 1979. EtTect of epidermal growth factor in oitro. Endocrinology 104, 862-869. Cohen S. 1962. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J. biol. Chem. 237, 155551562. Cohen S. 1965. The stimulation of epidermal proliferation by a specific protein (EGF). Deul Biol. 12, 394407. Cohen S. and Carpenter G. 1975. Human epidermal growth factor: isolation and chemical and biological orouerties. Proc. natn. Acud. Sci. U.S.A. 72. 1317-1321. Cohen S. and Elliott G. A. 1963. The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J. invest. Derm. 40, I-5. Frati L., Daniele S.. De Logu A. and Covelli 1. 1972. Selective binding of the epidermal growth factor and its specific effects on the epithelial cells of the cornea. Expl Eye Res. 14, 135-141. Gospodarowicz D., Brown K. D., Birdwell C. R. and Zetter B. R. 1978. Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor. epidermal growth factor and thrombin. J. cell Eiol. 77, 77478X. Gospodarowicz D., Mescher A. L., Brown K. D. and Birdwell C. R. 1977. The role of fibroblast growth factor and epidermal growth factor in the proliferative response of the cornea1 and lens epithelium. Expl Eye Res. 25, 631-649. Gregory H. 1975. Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 257. 3255327. Heimberg M., Weinstein I., Le Quire V. S. and Cohen S. 1965. The induction of fattv liver in neonatal animals by a purified protein (EGF) from mouse submaxillary gland. Ltfe Sci. 4, 162551633. Hollenberg M. D. and Cuatrecasas P. 1973. Epidermal growth factor: receptors in human fibroblasts and modulation of action by cholera toxin. Proc. natn. Acad. Sci. U.S.A. 70, 29642968.
Lembach K. J. 1976. Induction of human tibroblast proliferation bv_ epidermal growth factor (EGF): enhance_ ment by an EGF-binding arginine esterase and by ascorbate. Proc. natn. Acad. Sci. U.S.A. 13, 1833187. Paul D.. Brown K. D.. Rupniak H. T. and Ristow H. J. 1978. Cell cycle regulation by growth factors and nutrients in normal and transformed cells. In vitro 14, 76-84. Roberts M. L. 1974. Testosterone-induced accumulation of epidermal growth factor in the submandibular salivary glands of mice, assessed by radioimmunoassay. Biochem. Pharmac.
Acknowledgemrnts~This study was supported by a grant from the National Health and Medical Research Council of Australia. We thank Mr. D. Rowler for the excellence of his technical assistance in preparing the histological specimens. We are grateful to Mr. T. Dobrostanski for photographic assistance and to Mrs. E. Gates for typing the manuscript. REFERENCES
Baserga
R. and
Experimentul
Malamud Pathology
D. 1969. Modern Methods ift ctnd Autoradiography Terhni-
23, 3305~3308.
Savage C. R. and Cohen S. 1972. Epidermal growth factor and a new derivative. J. biol. Chem. 247, 7609761 I. Savage C. R., Ingami T. and Cohen S. 1972. The primary structure of epidermal growth factor. J. biol. Chem. 247, 7612-7621. Tyler M. S. and Pratt R. M. 1979. Effect of epidermal growth factor on the isolated palatal epithelium in vitro. J. dent. Rex 58, 385 (abstract). Webster’ A. K. and Rubright W. C. 1978. Histologic measurements of the effects of submandibular gland removal on oral and skin epithelia of mice. Archs oral Biol. 23, 597-599.
EGF and neonatal murine oral mucosa and skin
Plate 1 overleaf.
41
42
N. E. Steidler and P. C. Reade
Plate 1 Fig. 1. A control mouse (above) and an experimental mouse to which was administered 2pg EGF/g body wt/day for 8 days (below), showing retardation of hair growth and stunting due to EGF. x 1.5 approx. Figs 2 and 3. Sections of normal murine dorsal skin (Fig. 2) and dorsal skin from a mouse given 2 pg EGF/g body wt/day for 8 days (Fig. 3) demonstrating decrease in skin thickness as measured from the paniculus carnosus (P), in&ased keratin and nucleated cellular epithelial thickness due to the effects of EGF administration. x 45 Figs 4 and 5. Sections of dorsal lingual mucosa from a control mouse aged 8 days (Fig. 4) and a mouse given 2 pg EGF/g body wt/day for 8 days (Fig. 5). The EGF-treated animal has thicker lingual epithelium and keratin, and a more prominent granular cell layer. x 450 Figs, 6 and 7. Sections showing palatal mucosa from a control mouse aged 8 days (Fig. 6) and a mouse of the same age who had received 2 pg EGF/g body wt/day since birth (Fig. 7). The palatal epithelium from the EGF-treated animal has thicker keratin and a thicker nucleated cellular layer than control tissue. x 450
EGF and neonatal murine oral mucosa and skin
Plate 1.
43