The effects of progesterone (Pro), 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E2), T3, and dexamethasone (Dex), given alone or in combination, on induction of epidermal growth factor (EGF) and proteinase isozymes in the submandibular glands of hypophysectomized mice were examined. Each hormone, except E2, acting alone had essentially comparable inductive effects on EGF concentrations and on total proteinase activity. E2 alone had no inductive effect at all. Pro acted synergistically with T3, but its inductive effect was diminished when given with Dex. E2 was not synergistic with T2, and it inhibited the effect of Dex; it also partially blocked the action of DHT on induction of proteinase activity but not of EGF. Simultaneous administration of all five hormones restored total proteinase activity completely but EGF levels to only 50% of values for intact males, respectively. Submandibular proteinases were resolved by isoelectric focusing into four isozymes: proteinase F (pI 4.8), proteinase D (pI 5.8), proteinase A (pI 6.2), and proteinase P (pI 10.0). Pro alone slightly increased levels of proteinase F but greatly raised levels of the other three isozymes. This inductive action was augmented when Pro was given with T3 but blocked when it was given with Dex. E2 alone not only failed to induce any of the isozymes, but even further reduced levels of proteinase F. It also decreased the inductive effects of T3 on these isozymes, and with Dex completely blocked induction of proteinases D, A, and P. E2 plus DHT suppressed proteinase F levels and only induced proteinase A. All five hormones together reestablished the isozyme profile seen in intact males. These results show that Pro by itself is as capable as androgens, thyroid hormone, or glucocorticoid in regulating expression of these submandibular polypeptides, and that its action can be modulated by other pituitary-dependent hormones. In addition, they demonstrate that E2 does not regulate their expression, and that it has an inhibitory effect on the inductive action of other hormones. Last, they indicate that these various hormones may regulate expression of EGF and of each of the individual proteinase isozymes differently.

The cells of the granular convoluted tubule (GCT) of the rodent submandibular gland (SMG) are under complex developmental and multihormonal regulation. Recent findings indicate that GCT cells also synthesize transforming growth factor alpha (TGF-alpha), hepatocyte growth factor, erythroid differentiation factor, endothelin, and insulin-like growth factor, as well as several novel androgen-dependent proteins of unknown function. The GCTs of hypophysectomized mice provide a convenient model to study multihormonal regulation of gene expression. The GCT system of the rodent SMG also is a fruitful model for study of hormone receptors.

Abstract-The area of wound holes made in the parietal bone of 4-week-old rats was measured at 1, 2 and 4 weeks after the operation using Alizarin red S staining without the use of histological sections.

The effects of thyroid hormone, androgen, glucocorticoid, and mineralocorticoid on Na+,K+-AT€%e activity and on levels of its a-subunit protein (a1 isoform) in mouse submandibular gland (SMG) were studied by enzyme assay for ouabainsensitive ATP hydrolysis, by quantitative densitometric scanning of Western blots, and by immunohistochemistry. To define the specific regulatory effects of various pituitarydependent hormones on expression of Na',K+-ATPase in the SMG, we treated hypophysectomized (hypox) male mice with triiodo-L-thyronine (T3), 5 a-dihydrotestosterone (DHT) , dexamethasone (Den), and aldosterone (Ald), injected singly or in combination. Na+,K+-ATPase was confined to the duct system of the SMG. In intact mice there was a gender &rence in SMG Na',K+-ATPase, with levels of the enzyme's activity and of its a1-subunit being less in the glands of d e s . In males, hypophysectomy caused a rise in levels of Na+,K+-ATPase activity and in levels of the al-subunit protein of this enzyme, and in intensity of immunocytochemical staining for this subunit but there were no such changes in the SMG of hypox females. Changes caused by hormonal replacement to hypox males in Na+,K+-ATPase activity, lev-

The administration of cortisol acetate and dexamethasone to castrated-adrenalectomized mice increased the submandibular gland weight, the granular duct cell size of the gland and the number of androgen-dependent granules detected with the staining for tryptophan in the duct cell. These changes were not as pronounced as when testosterone propionate was given. Similar effects on the duct cells and an increase in androgen-dependent esteroprotease activity of the submandibular glands were observed in castrated mice, but not in mice with testicular feminization which are genetically deficient in androgen receptors. These results suggest that glucocorticoids exert a certain degree of androgenic action on the granular duct cell through androgen receptors when androgens are absent or deficient.