Mouse Adrenal Gland Research Articles

Molecular genetic analysis of glucocorticoid signalling in development

A null mutation of the glucocorticoid receptor was generated by homologous recombination. Mutant newborn mice showed impaired lung development, hypertrophy of the adrenal cortex and a strongly reduced size of the adrenal medulla. Phenylethanolamine N-methyltransferase (PNMT) was undetectable in the adrenals of the mutant mice. Serum levels of corticosterone were moderately and ACTH levels were strongly elevated in the mutants. A weaker but significant increase of corticosterone and ACTH was observed already in heterozygous animals. This points to a dysregulation of the HPA axis due to defective feedback regulation via the glucocorticoid receptor. Liver gluconeogenetic enzymes were reduced to a variable degree. Whereas survival of heterozygous mutants was not affected, most of the homozygous mutant mice died during the perinatal period.

Corticosterone secretion in response to serotonin and acth by perfused adrenal of normal and athymic nude mice

Adrenocortical secretory responses to chemical mediators and ACTH in GD1 ICR nu/nu (athymic) mice were compared with those in CD1 ICR (normal) mice. The bilateral adrenals of normal or athymic mice were perfused in situ with artificial medium equilibrated by 95% O 2 + 5% CO 2. Infusion of serotonin induced the secretory response of corticosterone significantly at 10 nM and markedly at 100 nM and the response at 1000 or 10000 nM declined as compared with that at 100nM in normal mice. Total corticosterone secretion in response to 100 or 1000 nM serotonin in athymic mice was about one fourth that in normal mice, respectively. Corticosterone responses to ACTH at the range of 10 to 300 pg/ml in athymic mice were comparable to those in normal mice. Infusion of histamine, platelet activating factor(PAF), or compound 48 80 did not induce significant corticosterone response in both normal and athymic mice. The data suggest that the congenital defect of the thymus and/or hair causes the hyporesponsiveness of adrenocortical cells to serotonin although the adrenal cortex of athymic mice is able to perform its function in response to ACTH.

Perspectives in steroid hydroxylase gene expression: novel sites of expression during embryonic development.

CYP17 is not expressed in adult mouse adrenal glands but is expressed in a subset of fetal adrenocortical cells, indicating the potential to produce both corticosterone and cortisol during murine embryogenesis. CYP11A is expressed in the fetal adrenal but also in developing hindgut, which will form the colon, and in cells located beneath the skin of the embryo. Novel sites of expression of CYP17 and CYP11A in the mouse embryo suggest potential physiological roles for local production of steroids in diverse organs systems during development.

Functional expression of the human angiotensinogen gene in transgenic mice.

The renin-angiotensin system is a major determinant of arterial pressure and volume homeostasis in mammals through the actions of angiotensin II, the proteolytic digestion product of angiotensinogen. Molecular genetic studies in several human populations have revealed genetic linkage between the angiotensinogen gene and both hypertension and increased plasma angiotensinogen. Transgenic mice were generated with a human angiotensinogen genomic clone to develop an animal model to examine tissue- and cell-specific expression of the gene and to determine if overexpression of angiotensinogen results in hypertension. Human angiotensinogen mRNA was expressed in transgenic mouse liver, kidney, heart, adrenal gland, ovary, brain, and white and brown adipose tissue and, in kidney, was exclusively localized to epithelial cells of the proximal convoluted tubules. Plasma levels of human angiotensinogen were approximately 150-fold higher in transgenic mice than that found normally in human plasma. The blood pressure of mice bearing the human angiotensinogen gene was normal but infusion of a single bolus dose of purified human renin resulted in a transient increase in blood pressure of approximately 30 mm Hg within 2 min. These results suggest that abnormalities in the angiotensinogen gene resulting in increased circulating levels of angiotensinogen could potentially contribute in part to the pathogenesis of essential hypertension.

Distribution of the adrenocortical inner zone antigen.

Previous studies using a mouse monoclonal antibody (IZAb) have identified inner zone-specific antigens (IZAg 1 and 2) in the rat adrenal cortex. The expression of IZAg1 is enhanced by dibutyryl-cyclic AMP or ACTH in vitro and by in vivo ACTH treatment. These studies also suggest that IZAg may be involved in steriogenesis in rat adrenals. Using sensitive detection methods, the distribution of IZAg in adrenals from other species and in other rat tissues was studied. Pig and rabbit adrenals expressed an IZAb-immunoreactive protein with molecular mass identical with that of IZAg1, but no immunoreactivity was detected in bovine, guinea-pig or mouse adrenals. In the rabbit adrenal, as in the rat, IZAb bound only to zonae fasciculata/reticularis cells. However, the whole adrenal cortex in the pig immunoreacted with IZAb. Although immunofluorescence was observed in human adrenal sections, predominantly in the zona reticularis, no significant immunoreactive protein band was detected by immunoblot analysis. Apart from the zonae fasciculata/reticularis in the adrenal gland, an IZAb-immunoreactive protein with a molecular mass of 26,000 Da, corresponding to IZAg1, was also found in rat hepatocytes, renal tubules, ovary (corpus luteum, interstitial cells, theca interna of mature follicles) and Leydig cells. The observations suggest that IZAg may be involved in the processes of biosynthesis, metabolism and/or excretion of steroid hormones.

The presence of two cytochrome P450 aldosterone synthase mRNAs in the hamster adrenal

We isolated a cDNA from a hamster adrenal cDNA library which was similar in sequence to those of the mouse and rat P450 c18 cDNAs. The hamster P450 c18 cDNA, however, was shorter than the rat and mouse P450 c18 cDNAs at its 5′-end and the peptide leader sequence was absent. From a hamster genomic library we isolated and sequenced the first seven exons and a 5′-flanking region of the first P450 c18 gene exon. With this information we were able to generate a P450 c18 cDNA containing the peptide leader sequence using the polymerase chain reaction. Northern analyses were performed on adrenals from hamsters maintained on low sodium diet for 0, 4, 7 and 10 using a 32P-labeled sequence specific to P450 c18; two mRNA bands were found at 2 and 3.4 kb. The intensity of both bands was increased about 3- to 5-fold under sodium restriction compared to controls. A distinct mRNA band of 2.3 kb hybridized with an oligonucleotide specific to P450 11 β and its intensity did not change following low sodium intake. Immunoblotting analyses were performed using an antibovine adrenal P450 11 β antibody that does not discriminate between P450 11 β and P450 c18 proteins. Three bands were detected at 52, 48 and 45 kDa in homogenate preparations of entire glands. Furthermore, the 45kDa protein band was present in homogenates of the zona glomerulosa and absent in homogenates of the zone fasciculata-reticularis. In conclusion, these results show that the hamster adrenals express P450 c18 as do mouse, rat and human adrenal glands. Furthermore, two P450 c18 mRNAs, which are inducible by a low sodium intake, are present in the hamster adrenal vs one for the rat. The physiological role of these two hamster adrenal mRNA species remains to be elucidated.

Endocrine and Neurogenic Regulation of the Orphan Nuclear Receptors Nur77 and Nurr-1 in the Adrenal Glands

nurr77 and nurr-1 are growth factor-inducible members of the steroid/thyroid hormone receptor gene superfamily. In order to gain insight into the potential roles of nur77 in the living organism, we used pharmacologic treatments to examine the expression of nur77 in the mouse adrenal gland. We found that nur77 and nurr-1 are induced in the adrenal gland upon treatment with pentylene tetrazole (Ptz; Metrazole). This induction is separable into distinct endocrine and neurogenic mechanisms. In situ hybridization analysis demonstrates that nur77 expression upon Ptz treatment in the adrenal cortex is localized primarily to the inner cortical region, the zona fasciculata-reticularis, with minimal induction in the zona glomerulosa. This induction is inhibitable by pretreatment with dexamethasone, indicating involvement of the hypothalamic-pituitary-adrenal axis in the activation of adrenal cortical expression. When mice were injected with adrenocorticotrophic hormone (ACTH), nur77 expression in the adrenal gland spanned all cortical layers including the zona glomerulosa, but medullary expression was not induced. Ptz also induces expression of both nur77 and nurr-1 in the adrenal medulla. Medullary induction is likely to have a neurogenic origin, as nur77 expression was not inhibitable by dexamethasone pretreatment and induction was seen after treatment with the cholinergic neurotransmitter nicotine. nur77 is also inducible by ACTH, forskolin, and the second messenger analog dibutyryl cyclic AMP in the ACTH-responsive adrenal cortical cell line Y-1. Significantly, Nur77 isolated from ACTH-stimulated Y-1 cells bound to its response element whereas Nur77 present in unstimulated cells did not. Moreover, Nur77 in ACTH-treated Y-1 cells was hypophosphorylated at serine 354 compared with that in untreated cells. These results, taken together with the previous observation that dephosphorylation of serine 354 affects DNA binding affinity in vitro, show for the first time that phosphorylation of Nur77 at serine 354 is under hormonal regulation, modulating its DNA binding affinity. Thus, ACTH regulates Nur77 in two ways: activation of its gene and posttranslational modification. A promoter analysis of nur77 induction in Y-1 cells indicates that the regulatory elements mediating ACTH induction differ from those required for induction in the adrenal medullary tumor cell line PC12 and in 3T3 fibroblasts.

Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands.

nurr77 and nurr-1 are growth factor-inducible members of the steroid/thyroid hormone receptor gene superfamily. In order to gain insight into the potential roles of nur77 in the living organism, we used pharmacologic treatments to examine the expression of nur77 in the mouse adrenal gland. We found that nur77 and nurr-1 are induced in the adrenal gland upon treatment with pentylene tetrazole (Ptz; Metrazole). This induction is separable into distinct endocrine and neurogenic mechanisms. In situ hybridization analysis demonstrates that nur77 expression upon Ptz treatment in the adrenal cortex is localized primarily to the inner cortical region, the zona fasciculata-reticularis, with minimal induction in the zona glomerulosa. This induction is inhibitable by pretreatment with dexamethasone, indicating involvement of the hypothalamic-pituitary-adrenal axis in the activation of adrenal cortical expression. When mice were injected with adrenocorticotrophic hormone (ACTH), nur77 expression in the adrenal gland spanned all cortical layers including the zona glomerulosa, but medullary expression was not induced. Ptz also induces expression of both nur77 and nurr-1 in the adrenal medulla. Medullary induction is likely to have a neurogenic origin, as nur77 expression was not inhibitable by dexamethasone pretreatment and induction was seen after treatment with the cholinergic neurotransmitter nicotine. nur77 is also inducible by ACTH, forskolin, and the second messenger analog dibutyryl cyclic AMP in the ACTH-responsive adrenal cortical cell line Y-1. Significantly, Nur77 isolated from ACTH-stimulated Y-1 cells bound to its response element whereas Nur77 present in unstimulated cells did not. Moreover, Nur77 in ACTH-treated Y-1 cells was hypophosphorylated at serine 354 compared with that in untreated cells. These results, taken together with the previous observation that dephosphorylation of serine 354 affects DNA binding affinity in vitro, show for the first time that phosphorylation of Nur77 at serine 354 is under hormonal regulation, modulating its DNA binding affinity. Thus, ACTH regulates Nur77 in two ways: activation of its gene and posttranslational modification. A promoter analysis of nur77 induction in Y-1 cells indicates that the regulatory elements mediating ACTH induction differ from those required for induction in the adrenal medullary tumor cell line PC12 and in 3T3 fibroblasts.

Purification and properties of six aldo-keto reductases from rat adrenal gland.

Six aldo-keto reductases from rat adrenal gland have been highly purified to apparent homogeneity. These enzymes were identified as carbonyl reductases (CR-K1, CR-K2, CR-A, and CR-B), aldehyde reductase (AR-H), and aldose reductase (AR-L) in terms of substrate specificity, molecular weight (33,000-39,000), inhibitor susceptibility, cofactor requirement, and immunochemical properties. Both CR-K1 and CR-K2 were characterized as possessing high affinity towards 13,14-dihydro-15-ketoprostaglandin F2 alpha and were localized immunohistochemically in the zona glomerulosa and the zona reticularis of adrenal cortex, and in the ganglion cell of adrenal medulla. Immunoreactive proteins to anti-CR-K2 antibody were observed in male and female reproductive tissues of rats. Positive immunoreactive protein to anti-CR-A antibody was found in mouse, hamster, and rabbit adrenal gland, whereas that to anti-CR-K2 antibody was present only in rat adrenal gland. AR-H and AR-L mainly reduced aromatic and aliphatic aldehydes. All the aldo-keto reductases from rat adrenal gland were completely inhibited by p-chloromercuribenzoate. Barbiturate and 3,3'-tetramethylene glutarate potently inhibited AR-H, and quercitrin significantly decreased the activity of CR-K1, CR-K2, and AR-L. We propose that these aldo-keto reductases may play important roles in the rat adrenal functions.

Distinct gene expression of the [formula omitted] receptor channel subunit in peripheral neurons of the mouse sensory ganglia and adrenal gland

The distribution of five N- methyl- d-aspartate (NMDA) receptor channel subunit mRNAs was examined in the mouse peripheral nervous tissues by in situ hybridization. The trigeminal and dorsal root ganglion cells showed signals for the ζ1 subunit mRNA from embryonic stages through postnatal day 21, but not for the ε1, ε2, ε3, and ε4 subunit mRNAs at any developmental stages examined. The adrenal medulla also expressed the ζ1 subunit mRNA alone. These findings suggest that molecular organization of the NMDA receptor channel in peripheral neurons are distinct from those in central neurons, which may result in different functional properties of the channel between them.

Evidence for localization of GTP cyclohydrolase I immunoreactivity in the mouse brain, adrenal gland and liver

Evidence for localization of GTP cyclohydrolase I immunoreactivity in the mouse brain, adrenal gland and liver

Sex differences in plasma corticosterone in mouse fetuses are mediated by differential placental transport from the mother and eliminated by maternal adrenalectomy or stress

The effect of changes in maternal corticosterone concentrations, induced by maternal stress, maternal adrenalectomy or both, on concentration of corticosterone in serum and in adrenals of mouse (Mus domesticus) fetuses was examined. Higher baseline serum corticosterone concentrations were found in female fetuses than in male fetuses; however, there was no sex difference in the content of corticosterone in adrenals collected from these fetuses. Sex differences were observed in the fetal response to changes in maternal concentrations of serum corticosterone resulting from stress (bright light and heat) or adrenalectomy, and both factors eliminated the sex difference in corticosterone in fetal serum. When females were injected i.v. with [3H]corticosterone on day 17 of pregnancy, significantly more 3H was recovered from the serum of female than of male fetuses 15 min after the injection, while more 3H was recovered from placentae of male fetuses. This finding suggests that the difference in serum corticosterone in male and female mouse fetuses is due to greater transport of corticosterone from maternal blood across the placenta of female than of male fetuses.

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland.

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.

MS strain of type 2 herpes simplex virus produces necrotizing retinitis in mice

Intracerebral inoculation of mice with the MS strain of type 2 herpes simplex virus (HSV-2) causes a brief encephalitis associated with multifocal central nervous system demyelination. Many of the mice develop unilateral or bilateral impairment of the pupillary light reflex. We have examined the development of ocular disease in inbred NIH mice inoculated intracerebrally with a low dose (10 pfu) of HSV-2 (MS). The resulting acute encephalitis was fatal in 30–50% of the mice. By 1 month after inoculation, the pupillary response to light was absent or impaired in approximately 80% of the surviving mice. Infectious virus could be isolated from the trigeminal ganglia and optic nerves from day 2 and from the eyes by day 4. Viral antigen was first immunohistochemically detectable in the optic nerves on day 5 and in the retinae on day 6. During the second week after inoculation up to half of the mice developed unilateral or bilateral necrotising retinitis associated with high titres of virus in the eyes and abundant viral antigen in the retinae. Electron microscopy confirmed the presence of viral particles in the retinae, in glia and degenerating neurons. No viral antigen was detected in the corneas and only rarely was antigen found in the ciliary body or iris. Infectious virus persisted longer in the eyes than in the trigeminal ganglia or optic nerves and could still be isolated from a few of the animals 2 weeks after inoculation. By 1 month the titres of virus within the eyes had fallen to undetectable levels. At this stage many of the retinae were replaced by glial and collagenous scar tissue with foci of mineralisation. Attempted isolation of infectious virus from the trigeminal ganglia and adrenals of mice given an intravenous inoculum of 10 pfu HSV-2 (MS) was unsuccessful, indicating that haematogenous spread is unlikely to have contributed to the distribution of lesions after intracerebral inoculation of virus. Our findings suggest that the pupillary abnormalities noted in mice with HSV-2 (MS) encephalitis are at least partly due to rapid spread of virus along the optic nerves to the eyes with resulting necrotising retinitis.

Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex

Previous studies demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) regulates steroid biosynthesis. In this study we investigate further PBR action by examining its subcellular localization in mouse adrenal gland using anti-peptide PBR antiserum and employing biotin-strep-tavidin peroxidase immunocytochemistry. Results demonstrated PBR immunostaining exclusively in the cortex. Within this region, however, PBR staining was homogeneously distributed in cells of the zona glomerulosa, whereas in cells of the zona fasciculata both cytoplasmic and prominent plasma membrane immunostaining was evident. Next, PBR distribution was examined using confocal microscopy. Confocal optical sections were obtained, 3-D reconstructions of these sections generated, and vertical, z-sections of the 3-D reconstruction recreated. The immunostaining pattern observed was consistent with a cell surface distribution of PBR. The demonstration of a subset of PBR at the plasma membrane may account for actions of PBR ligands not related to mitochondrial function.

High carbonyl reductase activity in adrenal gland and ovary emphasizes its role in carbonyl compound detoxication

Carbonyl reduction has been studied in liver, kidney, adrenal gland and ovary of female Wistar and Sprague-Dawley rats as well as of female NMRI mice by using metyrapone as a substrate and by means of direct HPLC analysis of the reduced alcohol metabolite metyrapol. Carbonyl reducing activities were found in all tissues examined so far, with that in rat ovary and adrenal gland cytosol exceeding the liver cytosolic specific activity severalfold: 15-fold and 12-fold in the Wistar strain; 12-fold and 7-fold in the Sprague-Dawley strain, respectively. In general, Wistar rat enzyme activities were about four times higher than those of Sprague-Dawley rats in all fractions, which indicates an interesting genetic difference between the two rat strains. Due to the sensitivity towards the diagnostic inhibitor quercitrin, carbonyl reductase (EC 1.1.1.184) seems to be mainly responsible for metyrapone reduction in rat and mouse adrenal gland and ovary cytosol. However, sensitivity towards dicoumarol in microsomal fractions of mouse tissues points to the involvement of further carbonyl reducing enzymes. Western blot experiments revealed immunological differences between metyrapone reductase from liver microsomes and respective enzymes of all other tissues. In conclusion, the difference in tissue and intracellular distribution suggests that several enzymes are involved in carbonyl reduction of metyrapone and the intracellular multiplicity of the enzymes may have some relation to their significance in carbonyl compound detoxification. These results support the hypothesis that carbonyl reductases, besides their participation in the metabolism of physiologically occurring substances, provide the enzymatic basis for the detoxification of xenobiotic carbonyl compounds in adrenal gland and ovary which have escaped their metabolic conversion by the liver.

Detection of tyrosine hydroxylase and phenylethanolamine-N-methyltransferase messenger RNAs in the mouse adrenal gland and the brain by in situ hybridization.

To study the expression of tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) genes in the mouse adrenal gland and the brain, we performed in situ hybridization studies by using several types of complementary DNA probes recognizing coding regions of human TH (THc), the 3'-end of the human region of TH (TH3'), and the coding region of the human PNMT (PNMTc). THc mRNA was detected in the chromaffin cells of the mouse adrenal medulla and brain catecholaminergic neurons including the substantia nigra and ventral tegmental area. TH-immunopositive neurons were located in a similar pattern in adjacent sections. However no positive signals were detected by the TH3' probe. Using the PNMTc probe, the majority of cells in the adrenal medulla demonstrated positive labelling. Although the mouse TH and PNMT genes have not been fully isolated and sequenced, the present study strongly suggests that the sequence of the coding regions of TH and PNMT are similar in human and mouse. The THc and PNMTc probes are particularly useful in investigating the loci of gene transcription in mouse tissues.

Natural mouse IgG reacts with self antigens including molecules involved in the immune response.

IgG isolated on protein A-Sepharose from pools of normal sera from various mouse strains were examined by immunoblotting for reaction with self antigens. Homogenates of the major mouse organs, i.e. brain, skin, spleen, kidney, adrenals, thymus, heart, muscle and liver were used as the source of autoantigens. IgG stained at least 220 bands on the immunoblots. The antigens corresponding to these bands were tentatively identified by molecular mass estimation and referenced to computerized mouse protein data banks. IgG mainly recognized enzymes but it also stained intracellular structural constituents and surface molecules implicated in the functioning of the immune system. The validity of this identification was confirmed by analyzing purified antigens from mouse or other animal species by immunoblotting and enzyme immunoassays. Furthermore, extracts of 125I-surface-labeled cells were immunoprecipitated with IgG in the liquid phase or immobilized on beads. The proteins precipitated migrated to the same positions as those precipitated by specific monoclonal antibodies (mAb), such as class I alpha chain and beta 2-microglobulin, class II alpha and beta chains, CD3, CD4 and CD8 antigens. The results obtained with several enzyme immunoassay procedures using cell membrane extracts, specific mAb and normal IgG further supported the specific interaction of IgG with Ia, CD4 and CD8 molecules. Affinity chromatography indicated that at least 20% of normal mouse IgG possess polyreactive autoantibody function. Dissociation constants of these IgG were calculated for some autoantigens and found to be in the range of 2 x 10(-6)-7 x 10(-6) M. It is concluded that normal mouse IgG exhibit autoreactivities similar to those previously described for IgM.

The in vitro effect of metyrapone on steroid synthesis in mice adrenals at different circadian stages.

The circadian rhythm of the in vitro biosynthesis of cortisol and cortisone in mice adrenals has been documented in the absence and presence of 0.1 mumol metyrapone, an inhibitor of steroid 11 beta-monooxygenase. After 3 weeks of synchronization with 12 h light:12 h darkness, adrenalectomy was performed at eight circadian stages: 0, 4, 9, 10, 13, 16, 21, and 22 h after light onset (HALO). Because it has been shown that mice adrenals could convert exogenous 11-deoxycortisol, the synthesis of 11-oxysteroids (cortisol+cortisone) in adrenal homogenates was studied from tritiated precursor. The pattern of steroid synthesis showed a maximum around the end (10 HALO) and a minimum at the beginning of the resting period (0 HALO); the variation was approximately 10%. A similar pattern was observed in the presence of a approximately 50% inhibiting dose of metyrapone. On the other hand, the percent inhibition of 11-oxysteroids synthesis was greater at the beginning of the resting period (0 HALO) and minimum around the end of the activity span (21 HALO), with an overall variation of 20%. However, the variations were statistically insignificant (unpaired t test).

Adrenal glands of mouse and rat do not synthesize androgens

Human adrenal glands produce considerable amounts of the C-19 steroids dehydroepiandrosterone (DHEA) and androstenedione. To investigate the capability of rodent adrenals to produce these steroids, cell suspensions of mouse and rat adrenal glands were incubated in the absence and presence of adrenocorticotropic hormone (ACTH). Corticosterone levels in the incubation medium increased dramatically in the presence of ACTH, but no significant amounts of 17-hydroxyprogesterone or androstenedione could be detected. This indicates that the adrenals of rat and mouse lack the enzyme 17α-hydroxylase. Absence of plasma cortisol in the presence of high levels of corticosterone confirmed these data. Plasma levels of androstenodione were significantly decreased in castrated male rats as compared to levels observed in intact males, showing the contribution of the testes to the plasma content of androstenedione. Very low levels of androstenedione were observed in female, male and castrated male mice. Plasma concentrations of DHEA were not detectable in intact and castrated male mice and rats. It is concluded that rat and mouse lack the enzyme necessary to synthesize adrenal C-19 steroids and that the adrenals in these animals, therefore, do not contribute to plasma levels of androstenedione and DHEA.