Submandibular Glands Of Male Rats Research Articles

Expression of glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in the rat submandibular gland is influenced by pre- and post-natal high-fat diet exposure.

Background: Incretins, i.e., glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) promote insulin secretion to reduce postprandial blood sugar. Previous studies found incretins in the salivary glands. However, the role of GLP-1 and GIP in the submandibular gland (SMG) is unclear. This study investigates the effects of a high-fat diet (HFD) on the expression of GLP-1 and GIP throughout the development of rat SMG. Methods: Pregnant 11-week-old Wistar rats were divided into two groups: those fed on a standard diet (n = 5) and those fed on a HFD (n = 5). From day 7 of pregnancy and throughout the lactation period, all the rats were fed on either a chow diet or HFD. The newborns were divided into four subgroups (n = 6): standard diet males (SM), HFD males (HM), standard diet females (SF), and HFD females (HF). The SMGs of 3- and 10-week-old rats from each subgroup were collected under general anesthesia. Moreover, body weight, food intake, and fasting blood sugar were measured. The mRNA expression of GLP-1 and GIP was quantified, and the localization was observed using immunohistochemistry (p < 0.05). Results: GLP-1 mRNA expression was statistically significantly more upregulated in HM than in HF at 3 weeks. Moreover, GLP-1 mRNA expression was significantly higher in HM than in both SM and HF at 10weeks. Although a decreasing trend was observed in GIP mRNA expression in both 3- and 10-week-old rats fed on a HFD, a significant difference between HM and SM only occurred at 3weeks. Furthermore, the GIP mRNA expression of HM was lower than that of HF at 10weeks. Immunohistochemical staining revealed GLP-1 and GIP expression mainly in the SMG duct system. Moreover, vacuolated cytoplasm in the duct was observed in rats fed on a HFD. Conclusion: Exposure to HFD during pre- and post-natal periods increased GLP-1 mRNA expression in the SMGs of male rats. However, GIP expression decreased following the HFD in male newborns. Furthermore, a decreasing trend of GIP mRNA expression was observed in male newborns after HFD feeding. Sex influenced incretin hormones secretion and obesity-related conditions. HFD during pre- and post-natal periods reprograms the epigenome, contributing to subsequent disease development.

Experimental apical periodontitis alters salivary biochemical composition and induces local redox state disturbances in the salivary glands of male rats.

The objective was to evaluate the effects of experimental apical periodontitis on the inflammatory, functional, biochemical, and redox parameters of the parotid and submandibular glands in rats. Twenty 12-week-old male Wistar rats were randomly divided into two groups (n = 10): a control group and apical periodontitis group. After 28days, the saliva was collected for salivary flow rate and biochemistry composition. Both glands were sampled for quantification of the tumor necrosis factor-alpha (TNF-α) and biochemical analyses of redox state. TNF-α concentrations were higher in both salivary glands adjacent to the periapical lesions in animals with apical periodontitis and also compared to the control group. The apical periodontitis group increased the salivary amylase, chloride, potassium, calcium, and phosphate. The total oxidant capacity increased in the parotid gland adjacent to the periapical lesions in the same rat and compared to the control group. Conversely, the total antioxidant capacity of the parotid glands on both sides in the apical periodontitis group was lower than that in the control group. Furthermore, glutathione peroxidase activity increased in the submandibular gland adjacent to the apical periodontitis group compared to the control group. Experimental apical periodontitis alters salivary biochemical composition, in addition to increasing inflammatory marker and inducing local disturbances in the redox state in the parotid and submandibular glands of male rats. Apical periodontitis could exacerbate the decline in oral health by triggering dysfunction in the salivary glands.

Chronic Stress Induces Neurotrophin-3 in Rat Submandibular Gland

PurposePlasma neurotrophin-3 (NT-3) levels are associated with several neural disorders. We previously reported that neurotrophins were released from salivary glands following acute immobilization stress. While the salivary glands were the source of plasma neurotrophins in that situation, the association between the expression of neurotrophins and the salivary gland under chronic stress conditions is not well understood. In the present study, we investigated whether NT-3 levels in the salivary gland and plasma were influenced by chronic stress.Materials and MethodsExpressions of NT-3 mRNA and protein were characterized, using real-time polymerase chain reactions, enzyme-linked immunosorbent assay, and immunohistochemistry, in the submandibular glands of male rats exposed to chronic stress (12 h daily for 22 days).ResultsPlasma NT-3 levels were significantly increased by chronic stress (p<0.05), and remained elevated in bilaterally sialoadenectomized rats under the same condition. Since chronic stress increases plasma NT-3 levels in the sialoadenectomized rat model, plasma NT-3 levels were not exclusively dependent on salivary glands.ConclusionWhile the salivary gland was identified in our previous study as the source of plasma neurotrophins during acute stress, the exposure to long-term stress likely affects a variety of organs capable of releasing NT-3 into the bloodstream. In addition, the elevation of plasma NT-3 levels may play important roles in homeostasis under stress conditions.

Salivary glands as the source of plasma brain-derived neurotrophic factor in stressed rats engaged in biting behavior

Brain-derived neurotrophic factor (BDNF) is crucial for the survival and differentiation of the central and peripheral nervous systems. Recently, BDNF has been reported to exert broader biological activity on non-neural cells. A previous study examined the effect of immobilization stress on BDNF and its receptor tyrosine receptor kinase B in male rat submandibular glands. In the present study, we found that the rat submandibular gland is the major source of plasma BDNF during acute immobilization stress. Biting modulates the mRNA and protein levels of BDNF in the rat hippocampus, so we also investigated whether the plasma BDNF concentration is influenced by biting. Two hours of acute immobilization stress significantly increased the amount of BDNF mRNA within the rat submandibular glands. Moreover, allowing biting behavior for the second half of the 2-h stress exposure significantly increased the amount of salivary gland BDNF mRNA relative to stress alone. Similar results were found with plasma BDNF concentrations under the same conditions. We confirmed that biting during stress attenuates the increases in plasma adrenocorticotropic hormone and corticosterone concentrations, but this was not dependent on the submandibular glands. Increased BDNF, mRNA and protein expressions were observed in salivary duct cells as a result of immobilization stress and biting behavior, as demonstrated by real-time polymerase chain reaction, immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay. Taken together, the findings indicate that the submandibular glands evidently contribute to the increase in plasma BDNF upon biting.

Immobilization Stress Induces BDNF in Rat Submandibular Glands

Brain-derived neurotrophic factor (BDNF) promotes survival and differentiation of the cells of the central and peripheral nervous systems. BDNF has been identified in non-neural tissue, including the heart, lung, platelets, lymphocytes, and lacrimal glands. Immobilization stress modifies BDNF mRNA expression in some organs. The present study examines the effect of immobilization stress on BDNF, and its receptor TrkB, in male rat submandibular glands. Increased BDNF mRNA and protein expression were observed in duct cells as a result of immobilization stress, as demonstrated by real-time PCR, Western blot, immunohistochemistry, and analysis by microdissection. TrkB mRNA was not detected in salivary gland tissue, or oral or esophageal mucosa, by RT-PCR. Rat submandibular gland was thus identified as an organ which expresses BDNF. Furthermore, the results of this study suggest that increased salivary BDNF expression occurs following immobilization stress.

Enhanced immunocytochemical expression of antioxidant enzymes in rat submandibular gland after normobaric oxygenation

In order to clarify the role of antioxidant enzymes in the male rat submandibular gland against short-term normobaric oxygenation, we performed immunocytochemical staining of manganese-containing superoxide dismutase (Mn-SOD), copper- and zinc-containing SOD (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase, and glutathione S-transferases (GST alpha, GST mu, and GST pi) between days 1 and 7 after normobaric oxygenation. Ultrastructural alterations and immunoreactivities for malondialdehyde (MDA), a lipid peroxidation-related molecule, of the acinar and ductal cells after the oxygenation were also investigated. Immunoreactivity for MDA was exhibited in the acinar cells throughout the experiment. On the other hand, immunoreactivity for the SODs, CAT, and GSTs was not altered, when compared to that of controls, but was significantly elevated in the granular, striated, and excretory ductal cells. Since an increase of lipid peroxidation as indicated by enhanced immunoreactivity for MDA was detected in the acinar and intercalated ductal cells, the results indicate that the enhanced antioxidant enzymes in the granular, striated, and excretory ductal cells play a crucial role in the self-defense system of the male rat submandibular gland against normobaric oxygenation.

Morphological Aspects of the Postnatal Development of Submandibular Glands in Male Rats: Involvement of Apoptosis

We studied the involvement of the apoptotic mechanism(s) in cell differentiation in the developing male rat submandibular gland using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-labeling) assay in combination with light and electron microscopy. Whereas the proacinar cells were completely transformed into acinar cells within 2 weeks after birth, starting on postnatal Day 21, the terminal tubule cells formed vacuoles that disappeared by postnatal Day 35. During this period, positive TUNEL reactivity was seen in the terminal tubule cells, and electron microscopic analysis showed that certain morphological features of apoptosis, including fragmentation of nuclei and the presence of apoptotic bodies in the cytoplasm, were present in and restricted to the terminal tubule cells. These results indicate that, in addition to an autophagocytosis-mediated mechanism, apoptosis may also be involved in reducing the number of terminal tubule cells during postnatal development in the submandibular gland.

Immunoregulatory effects of glandular kallikrein from the salivary submandibular gland of rats.

A protein of 40 kD molecular weight was isolated from the salivary submandibular glands of male rats. The protein catalyzed the hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester. This esterase activity was inhibitable with the protease inhibitor aprotinin. The sequence of the first 25 amino acids of this protein was identical to that of rat glandular kallikrein (rGK). When added to cultures of murine lymph node cells suboptimally stimulated with the T cell mitogen concanavalin A, rGK markedly stimulated the proliferative activity of these cells. When injected into mice, rGK suppressed the contact sensitivity response to picryl chloride, a form of delayed-type hypersensitivity. Similar in vitro and in vivo effects were induced with GK from porcine pancreas (pGK). Moreover, the aforementioned in vitro and in vivo effects were abolished by aprotinin either added to the tissue culture medium or injected into the animals immediately before rGK or pGK. This demonstrates that the enzymatic activity of rGK and pGK is important for the induction of immunoregulatory effects. These results suggest that rGK is a systemic immunoregulatory enzyme with immunosuppressive potential. GK is the first example for systemic immunoregulation by an enzyme, the secretion of which is under neuroendocrine control.

Evidence for the Systemic Delivery of a Transgene Product from Salivary Glands

The aim of this study was to assess the feasibility of using gene transfer to salivary glands to direct the systemic delivery of therapeutic proteins in vivo. We used a replication-deficient recombinant adenovirus vector (Ad alpha 1AT) that encodes human alpha 1-antitrypsin (h alpha 1-AT), which we used as a marker protein. Ad alpha 1AT (5 x 10(9) pfu) was administered by retrograde ductal instillation to the submandibular glands of male rats. The amount of h alpha 1-AT found in the salivary glands, saliva, serum, and other tissues was analyzed by a sensitive enzyme-linked immunosorbent assay (ELISA). Maximal levels of the marker protein were detected at 24-48 hr post-virus administration for glands (274 ng/mg protein), saliva (approximately 313 ng/ml), and serum (approximately 5 ng/ml). Serum levels remained elevated for 96 hr, whereas the measured half-life for the marker protein was approximately 2 hr. Generally little to no h alpha 1-AT was detectable in most other organs. However, we were able to measure low levels of marker protein in tissues immediately surrounding infected glands. In all animals studied, levels of h alpha 1-AT were higher in the glandular venous effluent than in arterial blood. Similar results were found with parotid glands. The aggregate data demonstrate that salivary glands may be a target for the nonsurgical, systemic delivery of transgene-encoded therapeutic proteins for diseases that require relatively low circulating protein levels.

Immunoreactive insulin in rat salivary glands and its dependence on age and serum insulin levels.

The major salivary glands of adult rodents contain immunoreactive insulin (IRI). To determine if the concentration of IRI in salivary glands is modulated by the level of serum insulin, insulin immunoreactivity in the parotid and submandibular glands of male rats at different ages (sucklings, pubescent, mature, and elderly) was assayed and compared with corresponding serum insulin concentrations. Salivary glands from suckling rats contained 94 ng/g (wet weight) insulin, which is 1.6 times higher than the level in pubescent rats, and about 10 times higher than levels in mature and elderly rats. No direct relationship between salivary gland content and serum IRI levels was indicated by the data. In an attempt to increase insulin levels in serum, insulin-secreting pancreatic islet adenomas were induced in young male rats by injecting streptozotocin (an islet tumor-inducing drug) with nicotinamide (which reduces the drug's beta-cell cytotoxicity). The mean insulin content of salivary glands from drug-treated rats that had not yet expressed tumors was no higher than controls. After the development of visible tumors of pancreatic islet tissue, however, salivary gland IRI was markedly elevated, reaching 40 times control levels, whereas serum insulin, and the immunoreactive insulin content of two insulin-sensitive tissues (vis. hepatic, adipose), were elevated only 2-fold. Examination of histologic sections of the parotid and submandibular glands from drug-treated animals revealed no evidence for the formation of salivary tumors. The data indicate that salivary gland insulin content (i) is age-related, being highest in neonates and declining thereafter, (ii) is generally identical in parotid and submandibular glands at a given age, and (iii) is not modulated solely by the animal's serum insulin concentration. These results are discussed in regard to the possible sources of insulin detected in the major salivary glands.

Evidence of Gelatinase Secretion by the Submandibular Gland in Prepubescent Rats

The gelatin cleaving activities in secretions of cultured fragments of male rat submandibular glands were studied using zymography. Gelatinolytic activities of 88-, 64-, and 57-kDa proteins detected in the tissues from 22-28-day old animals were undetectable in 31-70-day old rats. The traces of gelatinolytic activity associated with 28-kDa protein were detectable from 22-day old rats in serum-free media, and this activity of the enzyme markedly increased with aging from 38-days old. At 52-days and the subsequent stages, in addition to 28-kDa, activities associated with 60-, 32-, and 29-kDa proteins were strong. When the conditioned media were treated with 1,10-phenanthroline and diisopropyl fluorophosphate (DFP), both products inhibited activity of 88-kDa enzyme, indicating that this enzyme is Cls-like enzyme. The 64- and 57-kDa activities were inhibited by 1,10-phemanthroline, but not by DFP; when the conditioned medium of the tissue from 24-day old rats was treated with p-aminophenylmercuric acetate, gelatinolytic activity associated with 64-kDa converted to 57-kDa. Therefore, 64- and 57-kDa activities were concluded to be progelatinase A and gelatinase A, respectively. On the other hand, the gelatinolytic activities associated with 60-, 32-, 29- and 28-kDa proteins were inhibited by DFP but not by 1,10-phenanthroline, indicating that these enzymes belong to the family of serine proteinase, most probably kallikrein-related enzymes. From these findings, it was suggested that gelatinase A, along with Cls-like enzyme, participates in the maturation of the submandibular gland before it becomes active as an exocrine organ.

Selective processing of submandibular rat 1 protein at dibasic cleavage sites

The amino acid sequence of submandibular rat 1 (SMR1) protein, deduced from its cDNA sequence, led to the prediction that the SMR1 gene encodes a hormone-like precursor [Rosinski-Chupin, I., Tronik, D. & Rougeon, F. (1988) Proc. Natl Acad. Sci. USA 85, 8553-8557]. SMR1 contains an N-terminal putative secretory signal sequence and a tetrapeptide (QHNP), located between dibasic amino acids which constitute the most common signal for prohormone processing. We have isolated and characterized from the male rat submandibular gland and its secretions three structurally related peptides, namely an undecapeptide (VRGPRRQHNPR), a hexapeptide (RQHNPR) and a pentapeptide (QHNPR) generated from SMR1 by selective proteolytic cleavages at pairs of arginine residues. The biosynthesis of these peptides is subjected to distinct regulatory pathways depending on the organ, sex and age of the rat. Furthermore, the peptides are differentially distributed in the submandibular gland and in resting or epinephrine-elicited submandibular salivary secretions, suggesting distinct proteolytic pathways for their maturation. The undecapeptide is generated in the gland of both male and female rats, but under basal conditions it is only released into the saliva in male animals. The hexapeptide is produced in large amounts in the gland of adult male rats and released into the saliva in both resting and stimulated conditions. The pentapeptide appears only in the male saliva and is present mostly under stimulated conditions. In addition, administration of epinephrine induces the release of the hexapeptide from the submandibular gland into the bloodstream. The evidence indicates that the rat submandibular gland can function as a dual exocrine and endocrine organ for the SMR1-derived hexapeptide, as has been reported for nerve growth factor, epidermal growth factor, renin and kallikrein. Although the biological activities of the SMR1-derived peptides are not yet known, their high production and adrenergic-induced release only into the saliva and bloodstream of adult male rats, suggest a physiological involvement in some male-specific processes.

Chronoimmunohistochemical localization of epidermal growth factor in the submandibular gland of male rats

Chronobiological oscillation was established for epidermal growth factor (r-EGF) in granular convoluted tubule (GCT) cells of the male rat submandibular gland (SMG) using immunohistochemistry. The high amplitude oscillation with a peak at 9 p.m. and low amplitude oscillation during the day time was noted. The distribution pattern of the r-EGF in the GCT segment was time-dependent, as it was seen at 3 p.m. when many cells with r-EGF staining were detectable. The higher reactivity during the activity phase of the rats may correlate with the intracellular biosynthesis of r-EGF required for cell proliferation and maintenance of ductal integrity. The chronobiological oscillation pattern of r-EGF seems to be important for the maintenance of ductal integrity and wound healing.

Detection of nerve growth factor mRNA in rodent salivary glands with digoxigenin- and 33P-labeled oligonucleotides: effects of castration and sympathectomy.

Nerve growth factor (NGF) is a protein highly expressed in the male mouse submandibular gland. We have applied a non-radioactive in situ hybridization method using digoxigenin-labeled NGF oligonucleotides, and have found the highest amounts of NGF mRNA in the secretory striated ducts of the male mouse submandibular gland. Scattered strongly positive cells were found in male mouse sublingual glands. Weakly labeled cells were seen in female mouse and in male rat submandibular gland striated duct cells. Using 33P as an alternative to 32P and 35S, we demonstrated a 1.3 KB NGF mRNA in salivary glands of male mice by Northern blot hybridization. Using 33P we detected NGF mRNA in male mouse submandibular glands by in situ hybridization but with a signal that, compared with the non-radioactive method, had a very low resolution. Castration of male mice almost abolished both the 1.3 KB NGF mRNA seen with Northern blots and the NGF mRNA labeling in submandibular glands 4 weeks after the operation, whereas levels were increased 6 hr and 2 days after sympathectomy. We conclude that hybridization with digoxigenin-labeled NGF oligonucleotides is a good tool to study the expression and regulation of NGF mRNA in male mouse submandibular glands.

Purification and tissue distribution of rat epidermal growth factor

1. 1. Two forms of rat epidermal growth factor, EGF-I and EGF-II, were purified to homogeneity from male rat submandibular glands. 2. 2. The mol. wts of EGF-I and -II were estimated to be 5200 and 5400, respectively, both of them having an apparent biological activity. 3. 3. The antiserum against EGF-II strongly cross-reacted with EGF-I; however, it did so only slightly with mouse or human EGF. 4. 4. EGF was detected by radioimmunoassay in various tissues of male and female rats, and the concentrations of rat EGF in the submandibular gland, parotid gland, sublingual gland, and liver were significantly higher in the male than in the female.

Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family

Cells expressing mRNA for hippocampus-derived neurotrophic factor (HDNF/NT-3) or brain-derived neurotrophic factor (BDNF) were identified by in situ hybridization. In the rat brain, HDNF mRNA was predominantly found in pyramidal neurons in CA1 and CA2 of the hippocampus. Lower levels of HDNF mRNA were found in granular neurons of the dentate gyrus and in neurons of the taenia tecta and induseum griseum. BDNF mRNA-expressing cells were more widely distributed in the rat brain, with high levels in neurons of CA2, CA3, and the hilar region of the dentate gyrus, in the external and internal pyramidal layers of the cerebral cortex, in the claustrum, and in one brainstem structure. Lower levels were seen in CA1 and in the granular layer of the hippocampus, in the taenia tecta, and in the mammillary complex. In peripheral tissues, HDNF mRNA was found in glomerular cells in the kidney, secretory cells in the male rat submandibular gland, and epithelial cells in secondary and tertiary follicles in the ovary. Cells expressing BDNF mRNA were found in the dorsal root ganglia, where neurons of various sizes were labeled.

Changes in Protein Secretion by Rat Submandibular Glands in Response to Isoproterenol, a-Methylnoradrenaline, and Clonidine during Post-natal Development

We studied developmental changes in salivary volumes and proteins secreted by the submandibular glands of male rats at weekly intervals from two to ten weeks of age in response to the beta 1-, alpha 1-, and alpha 2-adrenoceptor agonists, isoproterenol (IPR), alpha-methylnoradrenaline (alpha-mNA), and clonidine (Clonid). The types of proteins in saliva samples were determined and compared by isoelectric-focusing electrophoresis with the Phast system in both the gradient pH -3.5-to-5 and pH-3.5-to-9 gels by means of silver staining. Salivary volume and protein concentration in saliva samples elicited by IPR and alpha-mNA were positively related to the weight of the submandibular glands up to six or seven weeks of age, whereas in saliva elicited by Clonid, no relation was found in the protein concentration [corrected]. The isoelectric-focusing electrophoretic patterns of proteins secreted by the glands in response to three stimuli were different from each other during post-natal development. Within one stimulation, differences were also observed at two and three weeks of age for Clonid, and from seven weeks of age for the three stimuli, respectively. The alpha-type proteins, but not the beta-type proteins, were very similar to those in extracts from glands of rats at seven weeks of age. Almost all of the alpha-type proteins, but not the beta-type proteins, reacted with antibodies to two proteases. We conclude that functional maturation precedes morphological maturation in the submandibular glands of rats.

Sex dimorphism and inflammatory regulation of T-kininogen and T-kininogenase

Studies were carried out in order to better understand hormonal and inflammatory regulation of the T-kininogen and T-kininogenase system. T-kininogen from rate serum and T-kininogenase from rat submandibular gland were purified to homogeneity, and specific antisera to the purified proteins were generated. Simple, sensitive and specific radioimmunoassays were developed for measuring both T-kininogen and T-kininogenase. The assays incorporated a modified poly(ethylene glycol) technique for separating free from antibody-bound forms. Optimal combinations of poly(ethylene glycol) and γ-globulin were found, yielding low background and high specific binding. The assays can detect a minimum of 160 pg of T-kininogen and 80 pg of T-kininogenase per tube, Serial dilutions of sera from normal and turpenting-treated rats showed complete parallelism with the T-kininogen standard curve. T-kininogen levels in rat serum and rat tissues increased more than 10-fold following turpentive treatment, while T-kininogenase levels in the submandibular gland and other tissues remained unchanged Through used of a kinin-directed kininogen monoclonal antibody. Western blots of two-dimensional gels of serum following acute inflammation showed increased levels of several kininogens which vary in both molecular weight and isoelectric point. Analysis of serum kininogen levels shows sexual dimorphism, with female rats having 3.9-fold higher levels than males. Contrarily, T-kininogenase levels in the submandibular gland of male rats are 2.4-fold higher than those in females. The studies also showed that the T-kininogen and T-kininogenase system is regulated by sex hormones. T-kininogenais an acute-phase protein whose rapid increase and mobilization following inflammation may provide a primary defense against proteolytic damage during trauma.

Age-related changes of the granular intercalated duct cells of male rat submandibular gland

The granular intercalated duct (GID) cells showed a progressive increase in number from two to four months of age ( p < 0.01). Their secretory granules also increased in number. Among all the age groups (2–22 months), the number of the GID cells in submandibular gland was highest of six months; they were then also most conspicuous with many electron-dense secretory granules in the cytoplasm. From 12 to 22 months, they showed regressive changes such a decrease in cell number and in the number of secretory granules. The significance of these age-related changes of the GID cells is unknown.

Isolation of rat epidermal growth factor (r-EGF): Chemical, biological and immunological comparisons with mouse and human EGF

1. 1. Rat epidermal growth factor, (r-EGF), was isolated from adult male rat submandibular glands, with final yields of 4–6 mg r-EGF from 20 to 25 g wet weight of tissue. 2. 2. Amino acid analysis of r-EGF indicated a high degree of homology with murine EGF (m-EGF) and human EGF, (h-EGF). 3. 3. However, r-EGF contains 49 amino acid residues, versus 53 for human and murine EGFs, and lacks two characteristic trytophan residues present in the other two species. 4. 4. The lack of tryptophan residues did not affect cellular binding or mitogenic activity or r-EGF. 5. 5. Polyclonal antisera to each of the three separate species demonstrated crossreactivity with the other species of EGF. 6. 6. A sensitive radioimmunoassay was developed for r-EGF which can detect 25 pg of hormone.