Edward W. Gresik

Postnatal developmental changes in submandibular glands of rats and mice.

The submandibular glands of mice and rats are not fully developed at birth. In early postnatal life, differentiation of acini takes place before that of granular convoluted tubule (GCT) cells. The latter develop from striated duct cells, and first appear in both species around 15 days of age. In mice their full development gets under way by 20 days of age and is rapid in males and slow in females, resulting in a clear sexual dimorphism in adults. In rats, GCT development is more protracted, and accelerates around 40 days of age, with no sexual dimorphism seen at any time. The course of postnatal development of several GCT cell products is correlated with the cytodifferentiation of these cells. Reliable data are available for the development of amylase, proteases (including kallikrein), renin, epidermal growth factor, and nerve growth factor. Preliminary information exists for a glucagon-like substance. Cytodifferentiation of GCT cells is under hormonal control. Androgens alone can not precociously induce GCT cells, but thyroid hormones can do so, acting either alone or synergistically with androgens.

The postnatal development of the sexually dimorphic duct system and of amylase activity in the submandibular glands of mice.

SummaryThe submandibular glands of developing and mature Strong A mice were studied by light and electron microscopy. The glands of both sexes show the same cell types during development, but during maturation the glands display a degree of sex-dimorphism. Striated ducts, which differentiate from the larger intralobular ducts present in the neonatal gland, first appear by 5 days of age and reach their mature condition by 20 days of age. Granular convoluted tubule cells, which differentiate from striated duct cells, are first seen at 15 days of age in both sexes. Subsequently, they show a more rapid development in males than in females, and are dimorphically represented by 20 days of age. Intercalated ducts in the neonatal gland contain nongranular and granular cells. With maturation the number of granular cells decreases, apparently due to their conversion into the nongranular type, with their eventual disappearance from the glands of adult males. Their retention in adult females further defines the sexual dimorphism shown by these glands.Amylase activity in gland homogenates is first detectable at 20 days of age in both sexes. During development the male glands show a rapid rise in levels of amylase activity, whereas female glands show a more gradual rise. In mature animals, male glands have higher levels of amylase activity than female glands. The developmental and adult status of amylase activity parallels that of the granular convoluted tubules.

Immunocytochemical localization of renin in kidneys and submandibular glands of SWR/J and C57BL/6J mice.

By using antibodies against highly purified submandibular gland renin, renin was localized immunocytochemically at the light and electron microscopic level in the submandibular glands and kidneys of adult male SWR/J and C57BL/6J mice. In accord with the data of Wilson et al. (Proc Natl Acad Sci USA 75:1185, 1977), renin was demonstrable only in the submandibular glands of SWR/J mice (high strain), where it was confined to the secretory granules of the granular convoluted tubules. In the kidneys of both strains, renin was confined to epithelioid cells of the juxtaglomerular apparatus. Electron microscopically immunostaining was restricted to the granules of the juxtaglomerular epitheliod cells. Morphometric analyses suggested that the kidney of the C57BL/6J mice contained more immunoreactive complexes per unit volume of cortex than SWR/J mice kidney. The data indicate that submandibular gland renin cross-reacts with kidney renin, but that genetic controls of these polypeptides in the two organs are independent.

Transduction of TAT-HA-β-galactosidase Fusion Protein into Salivary Gland-derived Cells and Organ Cultures of the Developing Gland, and into Rat Submandibular Gland in Vivo

We have studied the transduction of TAT-HA-β-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6–21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate β-galactosidase activity. Transduction of the fusion protein into A5 and C6–21 cells was concentration- and time-dependent. Therefore, the intensity of the β-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-β-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for β-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.

Expression of Epidermal Growth Factor Receptor in Fetal Mouse Submandibular Gland Detected by a Biotinyltyramide-based Catalyzed Signal Amplification Method

Branching morphogenesis of the fetal mouse submandibular gland (SMG) can be modulated in vitro by stimulation or inhibition of the epidermal growth factor receptor (EGFR). Because the mRNAs for EGF and EGFR are detectable in RNA of SMG rudiments isolated directly from fetuses, the EGF system probably operates physiologically as a regulator of SMG morphogenesis. However, neither EGFR protein nor its precise cellular localization has been characterized in the fetal SMG. Here we show EGFR protein in fetal mouse SMG by immunoprecipitation, affinity labeling, ligandinduced autophosphorylation, and immunohistochemistry. SMGs from E16 fetuses (day of vaginal plug = EO) were labeled with [35S]-cysteine/methionine and homogenized. After addition of specific antibody to EGFR, the immunoprecipitate was isolated, resolved by polyacrylamide gel electrophoresis, and detected by autoradiography. A single band of 170 kD was detected, corresponding to the EGFR protein. Affinity labeling with [125I]-EGF of the membrane fraction of E18 SMG also revealed a prominent band at 170 kD, showing that this EGFR protein can bind specifically to its ligand. Incubation of SMG membranes from E18 fetuses with EGF in the presence of [γ-32P]-ATP, followed by immunoprecipitation with anti-phosphotyrosine antibody also showed a single band at 170 kD, demonstrating autophosphorylation of the EGFR in response to binding of its ligand. Immunohistochemical localization of the cellular sites of EGFR in the fetal SMG required use of a catalyzed signal amplification procedure, with biotinyltyramide as the amplifying agent. EGFR was localized predominantly, if not exclusively, in cell membranes of epithelial cells of the rudiment, whereas staining of mesenchymal cells was equivocal. Staining was strongest on duct cells, and weak on cells of the end-pieces. These findings clearly show that a functional EGFR protein is expressed in fetal SMG chiefly, if not exclusively, on epithelial cells.

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse.

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.

Immunohistochemical localization of renin in the human kidney.

By using an antiserum to purified human renal renin, renin was localized immunocytochemically in the human kidney under normal and various pathological conditions by the unlabeled antibody enzyme light microscope: (LM) and protein A-gold colloid electron microscope (EM) procedures. In the normal kidney, renin was confined to the epithelioid cells of the afferent arteriole of the juxtaglomerular apparatus (JGA). These cells were small and few, and always in the immediate neighborhood of the glomerulus. Fine structural analysis showed renin only in the secretion granules of the epitheloid cells. All granules within a given cell were stained with comparable intensity. In cases of renal artery stenosis (ischemic kidney) and of Bartters syndrome, renin-positive epithelioid cells were larger, showed increased staining intensity, and were often found along the afferent arteriole at some distance from the glomerulus. Again, by electron microscopic observation, renin was seen only in secretion granules of epithelioid cells. In all of the above pathologic cases, plasma renin activity was very high. However, in the other nephropathies studied, renin staining in the kidney resembled that seen in normal kidneys, even when levels of plasma renin activity were quite high.

Intracellular signalling cascades activated by the EGF receptor and/or by integrins, with potential relevance for branching morphogenesis of the fetal mouse submandibular gland.

The fetal mouse submandibular gland grown in vitro is a valuable model system to study epithelio-mesenchymal interactions vital for branching morphogenesis. This basic developmental process is dependent upon simultaneous engagement of receptors for growth factors and components of the extracellular matrix. Here we review intracellular signalling cascades mediated by activation of the EGFR in the developing mouse submandibular gland.

Epidermal Growth Factor and Renin in Mouse Submandibular Glands

Epon sections of the submandibular gland of SWF/J male mouse were stained immunocytochemically for epidermal growth factor (EGF) and renin. Most cells of the granular convoluted tubules (GCT) contained both EGF and renin. However, examinations of adjacent semithin or thin sections stained for EGF and renin, respectively, revealed a small population of GCT cells that contained EGF but no renin. Within a cell all secretory granules contained both EGF and renin. The renin-negative/EGF-positive cells may represent a subpopulation of tubular cells that do not express, or carry, the renin gene.