Effects of castration and sex steroids on sexually dimorphic development of the mouse submandibular gland

The aims of this study were to characterize sexual dimorphism in the submandibular glands of young adult mice and to determine how sex differences arise during postnatal development. In the mouse submandibular glands, prominent sexual dimorphism was observed at 30 days of age, when the male gland was superior in both the relative occupied area (ROA) and the mitotic rate of the granular convoluted tubules (GCT) to those of the female. By neonatal castration, this sexual dimorphism was abolished, and the intraglandular structures of castrated males were similar to those of normal females. In castrated mice of both sexes, daily treatment with testosterone and 5 alpha-dihydrotestosterone for 10 days from 20 days induced only the ROA of the GCT to increase to the normal male levels but not those of the other three regions of the glands, the acini, intercalated ducts and excretory striated ducts. Testosterone responsiveness of the glands, considering both the glandular weight gain and the mitotic rate of the GCT, was significantly higher in castrated males than in castrated females. On the other hand, 17 beta-estradiol had no effect on the glands of castrated mice. Therefore, the present study suggests that the testicular hormones are responsible for the masculine development of GCT of the glands, but not the ovarian hormones, and that there is a sex difference in the responsiveness of the glands to testosterone, which is more effective in males than in females.     

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.     

Influence of androgen in the neonatal period on ejaculatory and postejaculatory behavior in the rat

The objective of the present report was to investigate the influence of androgen in the neonatal period on the development of ejaculatory and postejaculatory behavior. At birth, male rats were either castrated (neonatally castrated males), implanted with a Silastic tube of the aromatase inhibitor androsta-1,4,6-triene-3,17-dione for the first 10 days (ATD males), or left untreated (normal males). Female rats were either injected with 0.5 mg testosterone propionate (TP) on Days 1 (day of birth) and 2 (androgenized females) or left untreated (normal females). All gonadally intact animals were castrated at 60 days of age. Following TP administration, all animals were tested for ejaculatory and postejaculatory behavior under both shock and nonshock conditions. All animals were capable of showing the intromission pattern; however, the ejaculatory pattern was exhibited regularly only by those animals exposed to androgen at birth (normal males, androgenized females, and ATD males). The normal males required fewer intromissions to achieve ejaculation than the other two groups exhibiting this reflex. This result is discussed in terms of peripheral genital stimulation deficits and the differentiation of neural tissue responsible for masculine copulatory behavior. Androgenized females and ATD males displayed a refractory period, characterized by 22-kHz vocalizations, equal to or longer than that found in normal males. These results indicate that defeminization is not necessary for the display of normal ejaculatory and postejaculatory behavior.     

Immunocytochemical localization of epidermal growth factor during the postnatal development of the submandibular gland of the mouse.

The time of appearance and the pattern of localization of epidermal growth factor (EGF) in submandibular glands of mice was studied during postnatal development immunocytochemically. EGF was first detectable in the granular convoluted tubule (GCT) cells in the glands of males at 20 days of age and of females at 30 days of age. Development of GCT cells containing EGF was rapid in males, approaching adult conditions by 45 days of age. In females EGF- containing GCTs developed more slowly and irregularly, and did not reach adult status by 45 days of age. It is concluded that EGF is restricted during postnatal development to the GCT cells, and that these cells and the distribution of EGF are represented dimorphically from their first appearance in the submandibular glands of both sexes.     

Immunocytochemical localization of renin in the submandibular gland of the mouse during postnatal development.

The localization of renin in the developing mouse submandibular gland was studied immunocytochemically using the unlabelled antibody-enzyme method of Sternberger ('74). Bouin-fixed submandibular glands of mice of both sexes were examined at 5-day-intervals from birth (day 0) to 50 days of age. At all stages studied, only granular convoluted tubule (GCT) cells stained immunocytochemically for renin; such cells were first seen in glands of 30-day-old males and of 30-day-old females. The size and number of renin-containing GCT cells increased rapidly in males, attaining adult status by 50 days of age. In females, differentiation of GCT cells immunoreactive for renin was slower and less regular than in males, and at 50 days of age the GCT segment had not yet reached adult conditions with respect to the distribution of renin. Renin appears in GCT cells at later ages than other GCT cell products (e.g., EGF and amylase), suggesting the existence of independent developmental control for the expression of various biologically active substances in the GCTs.     

Sex differences,laterality, and hormonal regulation of androgen receptor immunoreactivity in rat hippocampus

Sex differences, laterality, and hormonal regulation of androgen receptor (AR) immunoreactivity in rat hippocampal CA1 pyramidal cells were examined using the PG21 antibody. Adult male rats were either castrated or sham-operated at least 2 weeks prior to sacrifice. Gonadally intact females were sacrificed on the day of proestrus. Animals received an injection of either testosterone propionate (TP) or vehicle 2 h prior to sacrifice. Within CA1, both the intensity of staining and the number of AR+ cells were assessed. AR immunostaining was detected in all the groups with marked variation among them. The overall ranking of staining intensity was: gonadally intact males > females given TP > castrated males given TP > females > castrated males given vehicle. The number of AR+cells within subregions of CA1 showed the same basic pattern: among control-treated animals, gonadally intact males have more than females, but castrated males have the least, and acute TP treatment increases the number in both sexes. The increased level of AR immunoreactivity in CA1 of castrated males following acute TP treatment suggests that testicular androgens in adulthood normally increase AR immunoreactivity there, producing a sex difference favoring males in gonadally intact animals. We also found a higher number of AR+ CA1 cells on the left than on the right, but only in gonadally intact males and in females given TP. These results suggest that a laterality of AR distribution in the rat hippocampus may lead to lateralities in hippocampal structure and function.     

Sexually dimorphic duct system of the submandibular gland in mouse with testicular feminization mutation (Tfm/Y).

The X-linked testicular feminization mutation (Tfm/Y) in the mouse is characterized by androgen insensitivity of the target cells. The aim of this study was to examine sexually dimorphic development of the submandibular gland of Tfm/Y mutant mice in comparison with those of wild-type male, wild-type female and heterozygous Tfm female mice. In either 30- or 90-day-old wild-type male mice, the granular convoluted tubules (GCT) of the glands were more developed, and the relative occupied areas (ROA) of GCT were superior to those of the age-matched wild-type and heterozygous Tfm females. In androgen-insensitive Tfm/Y mice, the glandular structures rather resembled the female glands, showing lower values of the ROA of the GCT. Sex differences in the mitotic rate were observed at 30 days of age, being significantly higher in the wild-type male GCT than in the female GCT. Thereafter, the mitotic rate of the wild-type male GCT declined to the female levels by 90 days of age. The mitotic rate of GCT in Tfm/Y mutants was as low as those of the females during observation periods. An other three regions, the acini, the intercalated ducts and the excretory striated ducts, were not significantly different in either the ROA or the mitotic rate among wild-type males and females, and Tfm/Y. On the other hand, either the ROA or the mitotic activity of GCT of the glands in Tfm/Y mutants was completely unaffected by 5 alpha-dihydrotestosterone (DHT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sex hormones on the onset of diabetic syndrome in WBN/Kob rats]

The effects of sex hormone on diabetic conditions were investigated in WBN/Kob strain rats, i.e., castrated or spayed, hormone-treated, and non-treated rats. The effects of sex hormone on glycosuria, body-weight change, glucose tolerance and histopathology of the pancreas were compared among these animals. There were no abnormal changes in these parameters in the non-treated females and estrogen-treated males. The glycosuria began to be observed from the age of about 30 weeks in the non-treated group and from the age of 52 weeks in the castrated group. In the female animals, this symptom began to appear from the age of 55 weeks in the testosterone-treated group and from the age of 72 weeks in the spayed group. Before the onset of the diabetic symptoms, glucose tolerance was impaired in these animals. Body weights of the castrated and estrogen-treated males were lower than that of the non-treated males, especially in the estrogen-treated males. Those of the spayed and testosterone-treated females were much heavier than that of the non-treated females. Testosterone treatment accelerated body-weight gain in the spayed female animals. Histopathological examination of the pancreas revealed atrophy of the aciner tissue and atrophy and disappearance of the islet cells similar to those of the non-treated WBN/Kob male rats in the castrated males, spayed females and testosterone-treated females. However, these changes were not observed in the non-treated females or estrogen-treated males. These findings suggest that female hormone suppressed the onset of hyperglycemia along with glycosuria and male hormone accelerates the onset of hyperglycemia in the WBN/Kob rats.     

Sexual differentiation of reproductive behavior in pigs: defeminizing effects of prepubertal estradiol

The present study sought to determine (1) whether estrogen by itself can defeminize the behavior of pigs during the late juvenile-early pubertal period, and (2) whether the progressive late defeminization reported for pigs is a true organizational effect, as opposed to an artifact of the time between castration and testing. Male pigs were castrated at 19-22 days or left intact and females were ovariectomized at 3 months. Additional males castrated at 19-22 days and females ovariectomized at 3 months were implanted with estradiol benzoate (EB) from 3 to 5.5 months. After castration of the previously intact males at the age of 5.5 months, all subjects were tested beginning at 6.5 months for proceptivity (choice of a male versus a female in a T-maze) and receptivity (immobilization to a mounting male) following an injection of EB. EB administered during development significantly defeminized proceptivity and receptivity in both sexes. The decrease in proceptivity was more pronounced in males than in females and was more pronounced than the decrease in receptivity, as if differentiation ends earlier for proceptivity than for receptivity; the decrease in receptivity was more pronounced in females. To see whether the capacity to display female-typical behavior is a function of time since castration, we castrated additional males at 4 months and tested for receptivity 9 days later following an injection of EB, then tested again with the other subjects at 6.5 months. The proceptivity and receptivity scores for males castrated at 4 months fell between those for intact males and males castrated at 3 weeks, and thus these animals were not completely defeminized. They were more receptive at 6.5 months than at 4 months, but the difference was not significant. These results indicate that in pigs estradiol defeminizes both receptive and proceptive behavior and that this defeminization can occur relatively late in development.     

An Unusual Sexually Dimorphic Mosaic Distribution of a Subset of Kallikreins in the Granular Convoluted Tubule of the Mouse Submandibular Gland Detected by an Antibody with Restricted Immunoreactivity

The granular convoluted tubule of the mouse submandibular gland contains a wide variety of biologically active proteins, including several kallikreins. The tubule is under multihormonal regulation, and is sexually dimorphic, being larger in males than in females. Correspondingly, levels of its various protein secretory products are more abundant in males than in females. However, isoelectric focussing studies show that the true tissue kallikrein, mK1, is more abundant in the female than in the male submandibular gland. In this study, an antiserum was prepared with restricted immunoreactivity for mouse mK1, and possibly other kallikrein family members of low abundance in the mouse submandibular gland, and used for the immunocytochemical staining of the granular convoluted tubule cells in the submandibular gland of adult male and female mice, by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. The distribution of immunoreactive tubule cells showed an unusual sexual dimorphism. In males only a few scattered slender tubule cells were strongly stained, while the more typical large tubule cells were only occasionally weakly positive, and many of them were not stained. By contrast, in females slender tubule cells were not seen, and about two thirds of the more typical tubule cells showed moderate to strong immunostaining. Immunoelectron microscopy revealed that immunostaining was confined to the secretion granules in granular convoluted tubule cells in both sexes. The slender tubule cells of males had many strongly stained small apical secretion granules and occasional basal infoldings; in the weakly positive larger more typical tubule cells not all secretion granules were positive, and there was intergranular variation in the intensity of staining of positive granules. In females, although more tubule cells were stained, intergranular variations in staining intensity were also noted. In both sexes, many tubule cells did not contain any secretion granules that showed immunogold labeling for kallikreins. These findings establish that, in contrast to the situation for the majority of granular convoluted tubules proteins, mK1 and possibly other minor kallikrein family members are more abundant in the granular convoluted tubules of female mice, and that there is considerable variation in the content of these kallikreins not only between different tubule cells, but also in individual secretion granules in any given tubule cell in either sex.     

Developmental status of sympathetic innervation in relation to calcium accumulation by submandibular gland following reserpine, surgical sympathectomy or cyclocytidine

Sympathectomy (Sx) of the submandibular gland was induced at various postnatal ages by ip administration of a single dose of reserpine or by unilateral excision of a superior cervical ganglion. If animals were 12 days old or less at the time of drug administration, [Ca] of the submandibular gland was not measurably increased 24 hr later; if rats were 14 days of age or older, [Ca] of the gland 24 hr after reserpine injection was nearly double that of untreated controls. Two days after surgical Sx, [Ca] of the denervated submandibular gland was unchanged from that of the innervated member of a pair if animals were less than 14 days of age at the time of denervation; [Ca] was twice that of glands of control rats if animals were older than 14 days of age when the denervation was performed. The anti-tumor agent, cyclocytidine (CC), given daily for 3 days in an ip dose of 500 mg/kg, also caused a two- to threefold increase in [Ca] of the submandibular gland when rats were more than 12 days of age at the time of the initial injection of the drug, but in rats younger than this age, CC caused no change in the [Ca] of the submandibular gland. Present data show that there are age-related differences in the ability of the submandibular gland to accumulate calcium following sympathetic denervation or treatment with a norepinephrine-releasing drug. These differences may be attributed either to incomplete development of calcium transport mechanisms, or incomplete development of the sympathetic innervation before 14 days of age.     

Androgenic regulation of N-acetyl beta-glucosaminidase activity in the submandibular glands of mice.

N-Acetyl beta-glucosaminidase [beta-2-acetamido-2-deoxy-D-glucoside acetylamido-deoxyglucohydrolase; EC 3.2.1.30] in the submandibular gland of mice was found to be androgen-dependent; the specific activities in males, females, and castrated males were 0.25, 0.11, and 0.11 unit/mg protein, respectively. The activities in females and castrated males were increased to the level of normal male mice by testosterone injection. Injections of progesterone and 17 beta-estradiol hardly affected the activity in males. In both males and females, the enzyme activity was detected in the convoluted tubular cells, not in acinous cells. The results of isoelectric focusing have shown that one enzyme having an isoelectric point of 9.0 is present in the glands of both sexes, indicating that the enzyme remains after castration and that the increases caused by testosterone represent the same molecular species. In addition, it was shown that the saliva from both sexes contained significant activity of N-acetyl beta-glucosaminidase, which also changed depending on the androgenic state of the animals. Most of the salivary activity was shown to originate from the submandibular gland, since the extirpation of this gland resulted in a significant decrease of the salivary activity.     

Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland.

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.     

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.     

Gender differences in small intestinal endothelial function: inhibitory role of androgens

Although gender differences exist in cardiovascular endothelial function, it remains unclear whether such differences are also seen in small intestinal endothelial function. To determine this, untreated male, age-matched proestrus female, castrated male, and 17beta-estradiol (E2)-treated noncastrated male rats were studied. Dose response curves to ACh and nitroglycerin (NTG) were determined by measuring changes in perfusion pressure by using an isolated small intestinal perfusion model. Endothelium-derived nitric oxide (NO) production/release was indirectly determined by the ability of intact endothelium to suppress serotonin (10(-5) M)-induced perfusion pressure changes. Intestinal tissue levels of NO were also measured. Moreover, plasma levels of androgen and E2 were determined and correlated with ACh (10(-8) M)-induced perfusion pressure reductions. ACh-induced intestinal perfusion pressure reductions in proestrus females, castrated males, and E2-treated noncastrated males were significantly higher than in untreated males. NTG-induced perfusion pressure reductions were not significantly different among groups. Perfusion pressures after administration of serotonin (10(-5) M) and intestinal tissue levels of NO in proestrus females, castrated males, and E2-treated noncastrated males were also significantly higher than in untreated males. Plasma androgen levels in proestrus females, castrated males, and in E2-treated noncastrated males were significantly lower compared with untreated males. There was a positive correlation between plasma androgen and ACh-reduced perfusion pressure; however, E2 levels did not show a similar relationship. Thus androgens appear to play an inhibitory role in small intestinal endothelial function. These properties in male vessels can be modulated by decreasing the level of circulating androgens or by E2 treatment.     

Postnatal differentiation of sexual preference in male pigs

Attraction to sexually mature males and the immobilization response were evaluated after postpubertal estrogen treatment of ovariectomized females and of males castrated within 48 hr after birth or at 4 or 8 months of age. The time spent in the end of an evaluation pen which housed a mature intact male, the proportion of animals that showed the immobilization response, and the latency to onset and the duration of this response were similar in ovariectomized females and males castrated within 48 hr after birth. These two groups spent more time in the male end of the evaluation pen as opposed to the opposite end which housed an ovariectomized female, showed a shorter latency to the onset of the immobilization response, and expressed this response for a greater number of days than males castrated either at 4 or 8 months of age. Males castrated at 4 or 8 months did not show a strong preference for either a mature male or an ovariectomized female. The immobilization response in estrogen-treated males castrated at 4 or 8 months of age diminished as these animals became older. On the basis of the observations made in this study, attraction to a mature, intact male is a sexually dimorphic behavioral trait in pigs, and defeminization of this trait in male pigs is associated with the pubertal increase in testicular steroid secretion. Presently, pigs are the only mammalian species in which a role has been identified for pubertal, testicular steroid secretion in the defeminization process.     

Comparative image analysis of EGF immunoreaction in rat submandibular gland using 3,3'-diaminobenzidine with metal enhancer substrate

We have shown the efficacy of image analysis using 3,3'-diaminobenzidine (DAB) with a metal enhancer substrate for demonstrating quantitative differences in the amount of epidermal growth factor (EGF) in the submandibular gland from normal, castrated, and testosterone propionate (TP) treated castrated rats. Immunohistochemical determination of EGF visualized by DAB-nickel reagent was performed with image analysis using a computed image analyzer system (ACAS 570). Immunohistochemistry for EGF disclosed positive staining in granular convoluted tubule cells in the tissue sections from each experimental group. Using the tools of a competent data program installed in the ACAS 570 software, we measured quantitative differences among the experimental glands examined. Castration was shown to elicit a significant reduction in the EGF-positive area and staining intensity, and administration of TP to the castrated animals restored these parameters to levels greater than those of normal rats. Our study demonstrates that a simple, inexpensive, commercially available metal enhancer substrate can be applied accurately to the computer assisted quantification of histochemical hormone-induction studies.     

Copulatory behavior of adult tamarins (Saguinus fuscicollis) castrated as neonates or juveniles: Effect of testosterone treatment

The sexual interactions of Saguinus fuscicollis males castrated as neonates, at 37 days of age, or prepubertally with adult intact females were studied. Prepubertally castrated males were observed while receiving testosterone, and while being treated with saline. Males castrated neonatally or at 37 days of age were observed while receiving testosterone. Neonatal castrates had previously been studied without hormone treatment and therefore no control condition was included for these animals. Prepubertally castrated males showed Mounts, Mounts with Thrusts, and Sexual Tongue Flicking when treated with saline only. In three of the four males, all measures of sexual behavior increased with testosterone treatment. Neonatally castrated males had failed to display any mounting or thrusting without testosterone treatment during a previous study. During the present study, three of the four males did not respond to testosterone treatment with sexual behavior. The fourth male and one male castrated at 37 days of age displayed some sexual behavior. These results suggest that most neonatally castrated males are not able to respond to testosterone with the activation of copulatory behavior. The findings are consistent with the hypothesis that in callitrichids the sensitive period for behavioral differentiation is shifted into neonatal life. However, some neonatally castrated males show a weak response to testosterone. This may reflect an extended and perhaps partially prenatal period of sensitivity.     

Androgen receptor in rat Harderian and submandibular glands

Summary Androgens regulate the development and sexual dimorphism of rodent Harderian and submandibular glands. This effect is believed to be mediated by the androgen receptor. Immunohistochemistry and immunoblotting were carried out to study the receptor in normal, castrated and dihydrotestosterone-supplemented rat Harderian and submandibular glands. Immunohistochemically, the most intense nuclear staining was observed in the acinar cells of the submandibular glands, followed by intercalated duct cells. The granular convoluted tubules showed weak immunostaining and the striated ducts were negative. In the Harderian gland, nuclear staining was seen in both type I and II secretory cells. Castration and treatment had no effect on the expression of the androgen receptor protein in either gland. A 110 K androgen receptor signal was detected by immunoblotting in the Harderian gland but not in the submandibular gland. An experiment was designed to explore the possible effect of proteinases on the receptor protein in the homogenate of submandibular gland. Our results demonstrate the cell-specific location of the receptor in Harderian and submandibular glands, and show that the expression of the receptor protein is androgen-independent.     

Progestin and estrogen receptors: characterization and localization in rat submandibular glands, with special reference to epidermal growth factor

By using progestin (P) and estrogen (E), the localization and characterization of both steroid receptors were examined in the submandibular gland (SMG) of 6-week-old immature castrated rats, with special reference to localization of epidermal growth factor (EGF). In the castrated male and female rats, both 3H-estradiol-17 beta (3H-E2 beta) and 3H-promegestone (3H-R5020) bound to SMG cytosol with high affinity and low capacity. These values were similar to those reported for other tissues. However, E-treatment after castration inhibited the specific binding. In sucrose density gradient ultracentrifugation, it was found that P receptors in both castrated males and females had a sedimentation coefficient of 7S, whereas E receptors had sedimentation coefficients of 4S and 7S. A histochemical study of the SMG of castrated male and female rats showed that the E-peroxidase complex (EPC)- and P-peroxidase complex (PPC)-stained cells were predominantly located in the epithelium of the duct system including the excretory duct and the granular convoluted tubules. Few cells were located in the intercalated duct, and none were found in the acinus. EGF-immunoreactive cells were also located in the epithelium of the same tissue region as in PPC- and EPC-stained sections. Moreover, E-treatment after castration inhibited the intensity of staining and immunoreactivity. These results clearly suggest that rat SMG contains specific P and E receptors which are mainly located in the epithelial cells of the duct system in which EGF-containing cells are identified. We discussed the possibility that P and E might affect EGF immunoreactivity, which reflects EGF production, through their receptors in the epithelium of the duct system.