Identification of the prolactin-releasing peptide-producing cell in the rat adrenal gland

Prolactin-releasing peptide (PrRP) is a novel peptide found in bovine hypothalamus as an endogenous ligand of an orphan G-protein-coupled receptor (hGR3). It is known that PrRP is widely distributed and plays roles in the central nervous system (CNS). In particular, PrRP acts as a neurotransmitter that mediates stress and activates the hypothalamo-pituitary-adrenal axis. On the other hand, only a few studies have so far been performed on PrRP in peripheral tissues. Among peripheral tissues, appreciable levels of PrRP are found only in the adrenal gland; however, the PrRP-producing cells in the adrenal gland have not been identified. In this study, we detected PrRP mRNA in the rat adrenal medulla. So, we tried to identify the PrRP-producing cells in primary culture cells of the adrenal medulla. We found immunopositive PrRP cells among the cultured cells from the adrenal gland, but not in the adrenal gland tissue, by means of immunocytochemistry. The PrRP immunopositive cells were double positive for tyrosine hydroxylase (TH) and for phenylethanolamine N-methyltransferase (PNMT), which indicates that PrRP may be produced in a part of the adrenaline cells in the adrenal gland. This is the first report that PrRP is produced in the adrenaline-containing cells of the adrenal gland.     

The distribution of cGMP in the adrenal gland in the rat, guinea pig and mouse after stimulation with sodium nitroprusside

Nitric oxide (NO) acts as an intercellular messenger molecule in the nervous system. In the adrenal gland sympathetic preganglionic fibers innervating the medulla, as well as intrinsic neural ganglion cells, contain nitric oxide synthase (NOS). Nitric oxide stimulates the soluble enzyme guanylate cyclase forming cyclic GMP (cGMP). Using sodium nitroprusside (SNP) as nitric oxide donor we have studied the putative target cells for nitric oxide in the rat adrenal gland, both in vivo and in vitro. The guinea pig and a few mouse adrenal glands were studied after SNP perfusion for comparison. Our results show that after vascular perfusion with a high concentration (3 mM) of SNP both noradrenaline and adrenaline chromaffin cells express cGMP-like immunoreactivity in all three species. After incubation of rat adrenal slices with SNP primarily the noradrenaline chromaffin cells are cGMP-positive. In contrast, detectable levels of cGMP-like immunoreactivity were not found in neuronal ganglion cells. In the adrenal cortex cGMP-like immunoreactivity was seen in blood vessel walls, in small cells with processes forming a reticular network, at least partly presumably representing endothelial cells, as well as in some presumable nerve terminals. These findings support the view that chromaffin cells, especially the noradrenergic ones and blood vessels, are targets for nitric oxide in the adrenal gland.     

Native α6β4* nicotinic receptors control exocytosis in human chromaffin cells of the adrenal gland

In the present study, we have electrophysiologically characterized native nicotinic acetylcholine receptors (nAChRs) in human chromaffin cells of the adrenal gland as well as their contribution to the exocytotic process. α-Conotoxin AuIB blocked by 14 ± 1% the acetylcholine (ACh)-induced nicotinic current. α-Conotoxin MII (α-Ctx MII) exhibited an almost full blockade of the nicotinic current at nanomolar concentrations (IC(50)=21.6 nM). The α6*-preferring α-Ctx MII mutant analogs, α-Ctx MII[H9A,L15A] and α-Ctx MII[S4A,E11A,L15A], blocked nAChR currents with an IC(50) of 217.8 and 33 nM, respectively. These data reveal that nAChRs in these cells include the α6* subtype. The washout of the blockade exerted by α-conotoxin BuIA (α-Ctx BuIA; 1 μM) on ACh-evoked currents was slight and slow, arguing in favor of the presence of a β4 subunit in the nAChR composition. Exocytosis was almost fully blocked by 1 μM α-Ctx MII, its mutant analogs, or α-Ctx BuIA. Finally, the fluorescent analog Alexa Fluor 546-BuIA showed distinct staining in these cells. Our results reveal that α6β4* nAChRs are expressed and contribute to exocytosis in human chromaffin cells of the adrenal gland, the main source of adrenaline under stressful situations.     

Active cell movements and embryonic cholinesterase in the postmetamorphic completion of the adrenal morphogenesis in frogs

The adrenal gland in Rana esculenta complex, as in other advanced anurans, is not yet in its definitive position at the end of the metamorphosis and reaches it subsequently, before sexual maturity. The displacement takes place by various means, among which active cellular movements prevail. These are demonstrated by the presence of acetylcholinesterase (AChE) in noninnervated cells (embryonic AChE). The disintegration of the connective tissues which delimit the cords of the adrenal gland, the passive transport of cells by the blood vessels and the movements of adjacent renal and mesenchymal cells collaborate with the active movements of the adrenal cells.     

Distribution and morphology of ghrelin immunostained cells in the adrenal gland of the African ostrich

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor. We investigated the distribution and morphological characteristics of ghrelin-immunopositive (ghrelin-ip) cells in the African ostrich adrenal gland. We found that the adrenal gland of the African ostrich consisted of three parts: capsule, inter-renal tissue and chromaffin cells. The inter-renal tissue and chromaffin cells interdigitated irregularly. The inter-renal tissue consisted of a peripheral zone and a central inner zone. The peripheral zone could be divided into an outer subcapsular zone and an inner zone. The subcapsular zone cells were arranged as a bow, while the inner area cells formed cords that were perpendicular to the capsule. The central inner zone exhibited irregular clumps and the cells were morphologically similar to chromaffin cells. Ghrelin-ip cells were located throughout the adrenal gland except the capsule. The majority of ghrelin-ip cells were found among the chromaffin cells. The number of ghrelin-ip cells in the inter-renal tissue decreased gradually from the central inner zone, to the inner zone to the subcapsular zone. The ghrelin-ip cells were oval or irregular in shape and exhibited cytoplasmic staining. Our findings suggest that ghrelin may play a role in regulating adrenal hormone secretion in the African ostrich.     

Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla

Neuropeptide W (NPW) is an endogenous ligand for GPR7, a member of the G-protein-coupled receptor family. NPW plays an important role in the regulation of both feeding and energy metabolism, and is also implicated in modulating responses to an acute inflammatory pain through activation of the hypothalamus-pituitary-adrenal axis. GPR7 mRNA has been shown to be expressed in the hypothalamus, pituitary gland and adrenal cortex. Similarly, NPW expression has been demonstrated in the brain and pituitary gland. However, the precise distribution of NPW-producing cells in the adrenal gland remains unknown. The aim of this study was to explore the distribution and localization of NPW immunoreactivity in the rat adrenal gland. Total RNA was prepared from the hypothalamus, pituitary gland and adrenal gland. RT-PCR revealed the expression of NPW mRNA in these tissues, while in situ hybridization demonstrated the presence of NPW mRNA in the adrenal medulla. When immunohistochemistry was performed on sections of adrenal gland, NPW-like immunoreactivity (NPW-LI) was observed in the medulla but not in the cortex. Moreover, NPW-LI was found to be co-localized in cells which expressed dopamine beta hydroxylase but not phenylethanolamine-N-methyltransferase. The finding that NPW is expressed in noradrenalin-containing cells in the adrenal medulla suggests that it may play an important role in endocrine function in the adrenal gland.     

Amidated joining peptide in the human pituitary, gut, adrenal gland and bronchial carcinoids. Immunocytochemical and immunochemical evidence

The distribution of the proopiomelanocortin-derivated amidated joining peptide (JP-N) was examined in the human pituitary gland, adrenal gland, gut and in three bronchial carcinoids. Double immunostaining showed coexistence of immunoreactive JP-N and other proopiomelanocortin derivatives, e.g., ACTH, beta-endorphin, Pro-tau-MSH, in the pituitary gland and adrenal medulla. The JP-N immunoreactive cells in the adrenal medulla were identified as a subpopulation of adrenaline-producing cells by means of an antiserum against phenylethanolamine N-methyltransferase. In the gut immunoreactive JP-N was costored with somatostatin in endocrine cells. Using radioimmunoassay, JP-N was found in higher concentrations than ACTH and alpha-MSH in the gut but not in the adrenal gland. Gel chromatography of gastric antrum and adrenal gland extracts showed three and two dominating components of immunoreactive JP-N, respectively, but under reduced conditions most of the immunoreactive material appeared as of low molecular weight in both extracts. In conclusion, immunoreactive JP-N is a major product from the processing of proopiomelanocortin in human extrapituitary tissues. The molecular forms of immunoreactive JP-N correspond to previous findings in the human pituitary gland.     

Tissue-type plasminogen activator in rat adrenal medulla

Summary Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenalin. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Tissue-type plasminogen activator in rat adrenal medulla

Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenaline. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Respiratory motion of adrenal gland metastases: Analyses using four-dimensional computed tomography images

PurposeTo evaluate the respiratory motion of adrenal gland metastases in three-dimensional directions using four-dimensional computed tomography (4DCT) images.MethodsFrom January 2013 to May 2016, 12 patients with adrenal gland metastases were included in this study. They all underwent 4DCT scans to assess respiratory motion of adrenal gland metastases in free breathing state. The 4DCT images were sorted into 10 image series according to the respiratory phase from the end inspiration to the end expiration, and then transferred to FocalSim workstation. All gross tumor volumes (GTVs) of adrenal gland metastases were drawn by a single physician and confirmed by a second. Relative coordinates of adrenal gland metastases were automatically generated to calculate adrenal gland metastases motion in different axial directions.ResultsThe average respiratory motion of adrenal gland metastases in left-right (LR), cranial-caudal (CC), anterior-posterior (AP), 3-dimensional (3D) vector directions was 3.4 ± 2.2 mm, 9.5 ± 5.5 mm, 3.8 ± 2.0 mm and 11.3 ± 5.3 mm, respectively. The ratios were 58.6% ± 11.4% and 63.2% ± 12.5% when the volumes of GTV_In0% and GTV _In100% were compared with volume of IGTV_10phase. The volume ratio of IGTV_10phase to GTV_3D was 1.73 ± 0.48.ConclusionsAdrenal gland metastasis is a respiration-induced moving target, and an internal target volume boundary should be provided when designing the treatment plan. The CC motion of adrenal gland metastasis is predominant and >5 mm, thus motion management strategies are recommended for patients undergoing external radiotherapy for adrenal gland metastasis.     

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.     

Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

alpha-Melanocyte-stimulating-hormone (alpha-MSH) is an agonist at the melanocortin 3 receptor (MC3-R) and melanocortin 4 receptor (MC4-R). alpha-MSH stimulates corticosterone release from rat adrenal glomerulosa cells in vitro. Agouti-related protein (AgRP) an endogenous antagonist at the MC3-R and MC4-R, is expressed in the adrenal gland. We investigated the expression of the MC3-R and MC4-R and the role of AgRP in the adrenal gland. MC3-R and MC4-R expression was detected in rat adrenal gland using RT-PCR. The effect of AgRP on alpha-MSH-induced corticosterone release was investigated using dispersed rat adrenal glomerulosa cells. AgRP administered alone did not affect corticosterone release, but co-administration of AgRP and alpha-MSH attenuated alpha-MSH-induced corticosterone release. To investigate glucocorticoid feedback, adrenal AgRP expression was compared in rats treated with dexamethasone to controls. AgRP mRNA was increased in rats treated with dexamethasone treatment compared to controls. Our findings demonstrate that adrenal AgRP mRNA is regulated by glucocorticoids. AgRP acting via the MC3-R or MC4-R may have an inhibitory paracrine role, blocking alpha-MSH-induced corticosterone secretion.     

Sensitivity of the fetal rat pituitary-adrenal system to corticotropin-releasing factor in organ culture.

Six groups of adrenal glands from 17-day fetal rats were explanted to organ culture for 2 days. In one group, adrenal gland was cultured alone, and in the remaining five groups adrenal gland was cultured with pituitaries from fetuses ranging in age from 14 to 18 days. In each of the groups, half of the cultures had corticotropin-releasing factor (CRF) added to the medium. A histometric parameter utilized the size of adrenocortical cells as an indicator of sensitivity of the pituitary-adrenal system. When 17-day adrenal gland was cultured alone, addition of CRF did not cause any enlargement of cortical cells. When the adrenal gland was cultured with two 14-day pituitaries, cortical cells were enlarged. Addition of CRF to this culture induced no further change. With two 15-day pituitaries in the presence of CRF, cortical cells were slightly larger than those in the absence of CRF. With 16- to 18-day pituitaries, a marked hypertrophy of cortical cells was induced, and the addition of CRF caused further acceleration in their enlargement. These results suggest that, in organ culture, 14-day pituitary can release some adrenocorticotropic hormone (ACTH) with or without additional CRF. Older pituitaries (16- to 18-day) can apparently release an amount of ACTH in the presence of CRF that is greater than their own spontaneous ACTH secretion.     

The effects of new steroidal anti-androgen, TZP-4238, and chlormadinone acetate on the pituitary, prostate and adrenal gland of the rat: histopathological and immunocytochemical studies.

The atrophic effects of a synthetic steroidal anti-androgen, TZP-4238, on the pituitary, prostate and adrenal gland of rats were investigated. Male Sprague-Dawley rats were divided into three experimental groups. Group 1 consisted of controls. Groups 2 and 3 received chlormadinone acetate (CMA) 50 mg/kg/day and TZP-4238 10 mg/kg/day p.o., respectively, for 3 weeks. CMA (Group 2) produced marked atrophy of the prostate. Furthermore, CMA caused marked atrophy of the adrenal gland. Histopathologically, the remarkable atrophy was observed in the adrenal cortical cells of zonae fasciculata and reticularis. The most striking ultrastructural alterations were noted in the mitochondria. In addition, intramitochondrial localization of glutathione-peroxidase (GSH-PO) which effectively reduces the lipid peroxides, was less than that in the controls. In the anterior pituitary gland, CMA induced a reduction in the size of ACTH cells. TZP-4238 (Group 3) produced marked atrophy of the prostate. However, TZP-4238 exerted no effect on the adrenal gland or anterior pituitary ACTH cells. In addition, the present histopathological study showed that TZP-4238 or CMA exerted no effect on the testes or anterior pituitary LH cells. Therefore, it is suggested that TZP-4238 causes atrophy of the prostate without any significant histopathological changes in the adrenal glands or anterior pituitary ACTH cells under the present experimental conditions. We further speculated that TZP-4238 had a more potent anti-prostatic effect than CMA and TZP-4238 had a less inhibitory influence than CMA on the pituitary-adrenal axis.     

Localization, transport, and uptake of D-aspartate in the rat adrenal and pituitary glands

Large amounts of D-aspartate (D-Asp) are present in the rat adrenal and pituitary glands. D-Asp is thought to be synthesized in the mammalian body and also accumulates in various tissues following intraperitoneal or intravenous administration. This report examines the origins of D-Asp in the adrenal and pituitary glands. We administered D-Asp to male rats intraperitoneally and immunolocalized this exogenous D-Asp in adrenal and pituitary tissue, using an anti-D-Asp antiserum which was previously developed in our laboratory. D-Asp levels in the rat adrenal gland have been shown to undergo a transient increase at 3 weeks of age and to decrease rapidly thereafter. We found that in the adrenal gland, exogenous D-Asp administered intraperitoneally was incorporated into the same region of the adrenal cortex in which endogenous D-Asp was present. By Northern and Western blot analysis and immunohistochemistry of glutamate (Glu) transporter, we also found that expression of the Glu transporter (GLAST), which has an affinity for D-Asp, transiently increased at 3 weeks of age and that localization patterns of the Glu transporter within the tissue were almost coincident with those of endogenous D-Asp. These observations suggest that D-Asp in the adrenal cortex of 3-week-old male rats is primarily acquired by uptake from the vascular system. We have previously shown that D-Asp is specifically localized in prolactin (PRL)-containing cells in the anterior lobe of the adult rat pituitary gland. Here we report that in the pituitary gland, exogenous D-Asp accumulated in endothelial cells, but not in PRL-containing cells. Northern and Western blot analysis and immunohistochemistry of Glu transporter revealed that developmental changes in the Glu transporter (GLAST) expression did not correlate with tissue levels of D-Asp and that the Glu transporter was not expressed in PRL-containing cells. These observations suggest that, in contrast to the adrenal gland, most of the D-Asp in the pituitary gland of adult male rats originates inside the gland itself.     

Rates of cholesterol synthesis and low-density lipoprotein uptake in the adrenal glands of the rat, hamster and rabbit in vivo

The absolute rate of cholesterol acquisition from de novo synthesis and from receptor-dependent and receptor-independent low-density lipoprotein (LDL) uptake was determined in the adrenal glands of the rat, hamster and rabbit under in vivo conditions. The rate of incorporation of [3H]water into cholesterol in the adrenal gland was much higher in the hamster (1727 nmol/h per g) and rabbit (853 nmol/h per g) than in the rat (71 nmol/h per g). Assuming that 23 atoms of 3H are incorporated into the cholesterol molecule during its biosynthesis, the absolute rates of cholesterol synthesis were then calculated to equal 59, 29 and 2.4 micrograms/h per g of adrenal gland in the hamster, rabbit and rat, respectively. Rates of LDL-cholesterol uptake were measured using a primed continuous infusion of [14C]sucrose-labeled homologous LDL (total LDL transport) and methylated human LDL (receptor-independent LDL transport). The rate of total LDL-cholesterol uptake in the adrenal gland was much higher in the rabbit (227 micrograms/h per g) than in the rat (18 micrograms/h per g) or hamster (6 micrograms/h per g). In all three species LDL uptake was mediated largely (greater than 93%) by receptor-dependent mechanisms. In terms of total cholesterol acquisition, the hamster adrenal gland derived 10-times more cholesterol from de novo synthesis than from LDL uptake, whereas the converse was true in the rabbit. Rates of de novo synthesis and LDL-cholesterol uptake were both low in the rat adrenal gland, which is known to derive cholesterol mainly from circulating high-density lipoproteins. Thus, the adrenal gland acquires cholesterol for hormone synthesis from at least three different sources and the quantitative importance of these sources varies markedly in different animal species, including man.     

Neurons and neuroendocrine cells contain chromogranin: detection of the molecule in normal bovine tissues by immunochemical and immunohistochemical methods

Gel-eluted bovine chromogranin (CG), the 75,000 dalton acidic protein abundantly present in adrenal chromaffin granules, was used as immunogen to prepare anti-CG serum. The specificity of the antiserum was demonstrated in immunoblots of electrophoresed bovine CG and in immunohistochemical studies of bovine adrenal medulla. In the immunoblots, the predominant immunoreactive band had a molecular weight of 75,000 daltons. Bands with a higher or lower molecular weight were also immunoreactive and may represent CG precursors or breakdown products. In the adrenal gland, only adrenal chromaffin cells contained CG immunoreactivity. Immunoblots and immunohistochemistry were also used to characterize the distribution of CG in bovine tissues. CG was expressed by cells of the diffuse neuroendocrine system (DNS) including: adrenal chromaffin cells, enterochromaffin cells, pancreatic islet cells, cells of the adenohypophysis, thyroid C cells, parathyroid cells, and submandibular gland. CG was also seen in four locations not previously recognized to express this antigen: thymic epithelial cells, neurons, the inner segment of rods and cones, and the submandibular gland. We demonstrate a wider distribution of CG than previously recognized and that the molecule detected in tissue by immunohistochemistry is indeed CG. We conclude that CG is expressed by neurons, cells of the DNS, and by a few other cells that may or may not be related to the DNS. The antiserum described here should prove valuable in developing an understanding of the function(s) of CG.     

Effects of calcium hopantenate on the release of thyrotropin-releasing hormone from the rat adrenal gland in vitro

The effects of calcium hopantenate (HOPA), a GABA agonist, on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of HOPA to the medium. HOPA's effects on TRH release from the adrenal gland were blocked with the addition of bicuculline, a GABA receptor inhibitor. The elution profile of methanol-extracted rat adrenal gland TRH was identical to that for synthetic TRH. The findings suggest that HOPA inhibits TRH release from the rat adrenal gland, and that its effects are mediated via the GABA receptor.     

Postnatal uterine development in the rat: estrogen and antiestrogen effects on luminal epithelium

We examined the effects of the synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), and the triphenylethylene antiestrogen, clomiphene citrate (CC), on uterine growth and development in the rat. These compounds, unlike estradiol, do not bind significantly to rat serum alphafetoprotein (AFP). Administration of DES or EE during the period of normal uterine gland genesis (postnatal days 10-14) induced luminal epithelium hypertrophy and increased uterine wet weight. The durations of these responses were dose-related. By day 26, luminal epithelium cell numbers were significantly depressed, compared to controls. Uterine gland development was delayed 6 to 9 days, depending upon estrogen dose, and the numbers of uterine glands ultimately achieved were generally less than in untreated control animals. While a daily dose of 0.1 micrograms CC/rat did not alter uterine development, 10 micrograms CC/rat caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis without inducing the large increases in uterine weight or the decreases in luminal epithelium cell number seen after estrogen exposure. The number of stromal cells was significantly increased on day 26 after CC exposure. Together with previous studies, these data demonstrate the greater potency and developmental stage specificity of non-AFP-bound estrogens with respect to altering uterine gland development. In addition, these data suggest that the disruptive influence of antiestrogens on gland genesis may be mediated through an indirect influence on the uterine stroma.