Inherited congenital adrenal hyperplasia in the rabbit: absent cholesterol side-chain cleavage cytochrome P450 gene expression.

We investigated adrenal steroidogenic enzymes, their activity and mRNA expression, and in vitro biosynthesis of an enzyme in rabbits with congenital adrenal hyperplasia (CAH; weight: CAH, 19 +/- 5 mg/adrenal; normal, 2.7 +/- 1.0 mg/adrenal). Serum pregnenolone (delta 5-P) levels in CAH newborn rabbits (12-36 h) were normal (mean/range, 438/51-2191 ng/dl), but corticosterone levels were low [0.05 +/- 0.05 microgram/dl; P less than 0.001 vs. normal (0.66 +/- 0.57)]. Serum Na+ levels in CAH newborn rabbits were in the normal range (143 +/- 30 meq/liter), but K+ levels were elevated [7 +/- 1.1 meq/liter; P less than 0.05 vs. normal (5.9 +/- 0.6 meq/liter)]. Minced normal adrenal tissue incubated with [3H] cholesterol (30-100 pmol/flask) and ACTH (100 mU/flask) produced [3H]delta 5-P (newborn, 21 and 45 fmol/100 mg; adult, 3 and 5 fmol/100 mg) and [3H]corticosterone (newborn, 23 fmol/100 mg; adult, 11.3 fmol/100 mg), but CAH adrenals produced no product (less than 1.3 fmol/100 mg). Adrenal mitochondria from normal newborn rabbits produced delta 5-P (4.4-7 nmol/mg protein), but CAH adrenals did not, while CAH adrenal mitochondria demonstrated over 4 times greater 11 beta-hydroxylase activity. A Western blot of adrenal homogenate from normal newborn rabbits revealed a cholesterol side-chain cleavage cytochrome P450 (P450scc)-immunoreactive species (mol wt, 53 x 10(3), but this species was absent in CAH adrenals; CAH adrenals had a normal adrenodoxin and intensified 17 alpha-hydroxylase cytochrome P450 (P450(17)alpha) band compared to normal adrenals. In vitro translation of RNA in a cell-free rabbit reticulocyte lysate system containing [35S] methionine yielded a precursor P450scc protein (mol wt, 58.5 x 10(3)) with normal adrenal RNA, but not with CAH adrenal RNA. P450scc mRNA was detected in all normal adrenals, but was not detected in all CAH adrenals. 21-Hydroxylase cytochrome P450 mRNA expression was detected at a similar level in both normal and CAH adrenals. We conclude that CAH in the rabbit is caused by inherited absent P450scc gene expression. The clinical, pathological, and biochemical manifestations of P450scc deficiency in the rabbit are nearly identical to the human disorder. Increased 11 beta-hydroxylase activity and increased P450(17)alpha on Western blot of CAH adrenals indicate altered gene expression of other steroidogenic enzymes due to CAH. Further molecular analysis of the P450scc gene in this animal CAH model will facilitate understanding of P450scc deficiency CAH.

Thyroid hormone antibodies and Hashimoto's thyroiditis in mongrel dogs.

Abnormally elevated serum T3 concentrations measured by RIA were observed in 19 clinically euthyroid or hypothyroid mongrel dogs. The serum T4 concentrations in these sera were low, normal, or high. Measurement of the intensity of thyroid hormone binding to serum proteins was determined by equilibrium dialysis. A marked decrease in the percent free T3 was observed in these abnormal sera. Polyacrylamide gel electrophoresis, pH 7.4, of normal dog serum enriched with tracer 125I-labeled thyroid hormones demonstrated binding of [125I]T4 to transthyretin, thyroid hormone-binding globulin, and albumin and of [125I]T3 primarily to thyroid hormone-binding globulin. In all abnormal sera, polyacrylamide gel electrophoresis demonstrated strikingly higher binding of T3 to immunoglobulin (Ig). Eleven of 16 abnormal sera had minimal to moderate binding of T4 to Ig. The percent free T4 was lower only in dogs whose sera demonstrated markedly increased binding of T4 to Ig. All abnormal sera tested had positive antithyroglobulin antibodies, consistent with the diagnosis of autoimmune lymphocytic thyroiditis. As in humans, antibodies to thyroid hormones in dogs are more common in the presence of Hashimoto's thyroiditis and should be considered when elevated serum thyroid hormone concentrations are observed in the absence of clinical thyrotoxicosis. When an antibody to only one thyroid hormone is present, a marked discrepancy in the serum concentrations of T3 and T4 will be observed.

The differential regulation of the intranuclear progestin in mouse kidney and prostate-seminal vesicles.

This study was designed to confirm the identity of the enzyme involved in the appearance of a nuclear metabolite of 6a-methyl progesterone (6MP) in mouse kidney but not in prostate-seminal vesicle. 6MP and progesterone competed for metabolism by the kidney enzyme. Using progesterone as substrate, the results of recrystallization of the product with 20a-hydroxy progesterone supported the identification of the enzyme as 20a-hydroxysteroid dehydrogenase (20aHSD). The enzyme had a substrate specificity similar to that reported for 20aHSD in other tissues. Renal enzyme activity was higher in female than in male CD-1 mice while activity was generally lower and without a sex difference in a random bred colony. Renal enzyme activity varied slightly with the estrous cycle, being highest at proestrus and lowest on day 2 of diestrus. Little enzyme activity was detected in prostate-seminal vesicles. It appears that 20aHSD is an important factor in regulating the differential presence of the 20a-metabolite of 6MP in kidney and prostate-seminal vesicle nuclei.

Differences in the androgen response between two mouse species.

Renal weight and beta-glucuronidase activity are two of several well-characterized androgen-responsive parameters in Mus musculus. A similar sexual dimorphism was not reported for a second mouse species, Mus caroli, however. Since this was not associated with a general absence of androgen action, we considered whether a localized defect in androgen receptors or a difference in renal androgen-responsive endpoints in the two species existed. Only minor differences in the characteristics of renal androgen receptors from the two species were found when they were analyzed by two different methods. These differences were not thought to be sufficient to account for the apparent renal androgen unresponsiveness. No differences were found in androgen receptors from brain. Subsequently, a third renal endpoint, ornithine decarboxylase activity, was found to respond to androgen stimulation in Mus caroli. Control of renal androgen action in these two mouse species thus differs at the level of genetic regulatory elements.

Weak androgen reduces the rate constant of beta-glucuronidase induction.

Administration of androgen to mice induces kidney beta-glucuronidase. Measuring beta-glucuronidase activity, rate of beta-glucuronidase synthesis, beta-glucuronidase mRNA activity and beta-glucuronidase mRNA concentration, the time course of induction was compared using a strong androgen, dihydrotestosterone (DHT), and a weakly androgenic progestin, medroxyprogesterone acetate (MPA). Using MPA resulted in a longer lag, a 3-4-fold slower rate of induction as defined by the forward rate constant, ka, a lower final extent of induction, and a slightly lower turnover constant, kb. Differences in kinetics of induction were consistent for all 4 measured parameters, and mimicked previously described genetic differences in these rate constants. The coordinate induction of beta-glucuronidase protein and beta-glucuronidase mRNA indicates that the response to androgen is regulated at a pre-translational level. That substitution of MPA for DHT decreases ka, rather than increasing kb, suggests that induction of beta-glucuronidase follows an increased rate of mRNA synthesis rather than a decreased rate of mRNA turnover. Finally, the results are consistent with a model in which the kinetic constants for beta-glucuronidase induction are dependent on the concentration of receptor molecules in the active conformational state.

Receptor binding of 6 alpha-methylprogesterone in mouse kidney.

6 alpha-Methylprogesterone (6MP) is an androgenic progestin that binds to the androgen receptor. However, results from an in vivo study suggested that 6MP was also bound by a second receptor. In the present study, we found that 6MP was bound in kidney cytosol from adrenalectomized/ovariectomized female mice as well as Tfm/Y mice, which lack androgen receptors. 6MP was bound with high affinity (Kd = 1.2 X 10(-8) M) by a binder that was present in 7-8 times greater concentration than the androgen receptor and had the specificity of a glucocorticoid receptor. 6MP was bound with similar specificity in liver cytosol. These data indicate that, despite its androgenic effects, 6MP binds primarily to a glucocorticoid receptor in mouse kidney.

Two forms of ornithine decarboxylase activity in mouse kidney.

Renal ornithine decarboxylase (ODC) activity was evaluated in normal female, male, testosterone-treated female and androgen-insensitive Tfm/Y mice for its heat sensitivity and in vivo half-life. ODC activity in normal female kidney consisted of 2 forms which differed in their heat sensitivity at 46 degrees C. Androgens, either endogenous in normal males or administered exogenously to females, induced primarily the heat-sensitive form. Results from mixing experiments indicated that the heat-sensitive form represented a change in the property of the ODC activity rather than a change in cytoplasmic factors. The in vivo half-life of ODC activity was increased slightly in males and short-term androgen-treated females over normal females and was markedly increased by prolonged androgen treatment. In vivo, the androgen-induced, heat-sensitive form decayed faster than did the heat-resistant form. We conclude that androgens have specific effects on both the amount as well as the biochemical properties of ODC activity in mouse kidney.

Drug metabolism in spontaneously diabetic guinea pigs.

Both sexes of spontaneously diabetic guinea pigs exhibit hyperinsulinemia (greater than 4-fold normal). This diabetic state is associated with the inhibition of hepatic drug metabolism in males but not females.

Androgen and progestin stimulation of ornithine decarboxylase activity in the mouse kidney.

This study was designed to characterize mouse kidney ornithine decarboxylase (ODC) activity as an androgenic end point and to use ODC activity to detect an androgenic effect of antiandrogens. Enzyme activity was not affected by freezing the whole kidney or the 15,000 X g supernatant for up to 7 days. ODC activity in female mice had a diurnal variation that peaked at midday. This diurnal variation did not affect the androgenic response of ODC. Enzyme activity was lower in females than in males and, in both sexes, could be induced further to similar levels with testosterone treatment. A single dose of crystalline testosterone induced a marked increase in activity, which peaked sharply, up to 100-fold above baseline, 12-17 h after treatment. Enzyme activity could be maintained with continued treatment for at least 28 days and reached levels up to 1,000-fold above baseline. The response was specific for androgens and required a functional androgen receptor. Other hormones had permissive effects. The early androgen-stimulated response (less than 24 h) was partially diminished by hypophysectomy. Propylthiouracil reduced both early and chronic responses. Genetic factors were also involved. The testosterone-stimulated response of C57BL/6J mice was consistently approximately half that of DBA/2J mice. Using this very specific and sensitive increase in ODC activity as an end point, we did not detect an androgenic response to treatment with the antiandrogens, cyproterone acetate (6-chloro - 17 alpha - acetoxyl - 1,2 alpha - methylene - 4,6- pregnadiene- 3,20-dione) and flutamide (4'-nitro-3'-trifluoromethylisobutyranilide), despite an increase in RNA polymerase activity. The functionality of the polymerase activity induced by antiandrogens thus remains in question. These data suggest that mouse renal ODC activity can be a useful tool for future study of androgen action at the physiological and molecular level.

Effects of shipping on the immune function in mice.

The effect of shipping stress on immunologic functions was examined in mice. The mice were shipped either by truck or by plane, 2 of the most common modes for transport of animals. While mice were in transit, temperature fluctuations and light intensity were monitored. The foot pad test, hemagglutination assay, and plaque-forming cell assay were used to measure immunologic function. Corticosterone concentrations were quantitated with a competitive protein-binding technique. Regardless of the method of shipment, corticosterone values in the mice were markedly increased at arrival and remained at the high value for a 48-hour period. Immune-function assays were significantly lessened in the mice at arrival, but returned to base line within 48 hours, indicating that a minimum 48-hour stabilization period is required for all new arrivals.

Morphological profiles of cryptorchid XXY mouse testes.

Two mice with an XXY karyotype and cryptorchid testes appeared spontaneously in a colony. The animals were H-Y antigen-positive, and had elevated serum levels of follicle-stimulating (FSH) and luteinizing (LH) hormones. Testes of the affected mice were atrophic, containing a few solid seminiferous cords surrounded by vast amounts of compact interstitial material. The cords were delimited by a broad tunica propria in which the basal lamina was irregularly thickened and stratified into a number of alternating dense and less dense layers. Most sex cords were populated by mature Sertoli cells and small pleomorphic elements resembling monocytic-derived macrophages. Within some cords, the macrophages aggregated into a central mass with which identifiable Sertoli cells and (PAS) periodic acid Schiff-positive fragments of basal lamina were associated. In more severely damaged cords, the basal lamina and peripheral carpet of Sertoli cells were totally missing. Such cords were populated only the the central macrophages with fragments of basal lamina and degenerating Sertoli cells. Finally, a few collapsed remnants of cords contained compact nodules of macrophages surrounded by what appeared to be the outer part of the tunica propria. The interstitial area, as well as the outer walls of the seminiferous cords were also heavily infiltrated by macrophages. Overall, the morphological picture was one of severe immunological injury. We do not know what role, if any, the genetic constitution and/or intra-abdominal environment may play in the expression of these bizarre pathologies. However, such severe changes have not been reported in either Klinefelter's syndrome or the undescended testes of any human or subprimate species.

Brief overview of selected aspects of testicular hormone action.

This paper is designed to give an overview of the mechanism of androgen action and some of the factors that can affect it. The discussion of androgen action includes androgen transport in the blood, metabolism, receptor binding, nuclear activation and selected aspects of biological response. The importance of recognizing interspecies and interstrain differences in specific aspects of androgen action is mentioned. Some examples of the effects of environmental agents on androgen metabolism, receptor binding and biological response are included.