The aim of this study was to establish a link between the highly expressed angiotensin II (Ang II) type 2 receptor (AT2) in human fetal adrenal cells and the proposed apoptotic activity in the center of the gland. There was an important increase in apoptotic DNA fragmentation with age in adrenal glands of fetuses from 15-20 weeks gestation. Adrenal cells showing the characteristic apoptotic internucleosomal DNA fragmentation were localized in the central portion of the fetal zone. In cells cultured for 24 h, Ang II, via the AT2 receptor, induced DNA fragmentation and cleavage of the DNA repair enzyme, poly-(ADP-ribose) polymerase. Furthermore, characteristic membrane blebbing was observed specifically on cells of the fetal zone. Immunofluorescence studies demonstrated that stimulation with Ang II or CGP 42112 (an agonist of the AT2 receptor) strongly modified the actin network, now localized exclusively along the plasma membrane, with a predominance of labeling at the base of the bleb formation. This rearrangement of actin distribution was different in cells from the definitive zone, corroborating the observation that these cells express many more Ang II type 1 receptors (AT1) than AT2 receptors. Taken together, our data indicate that the AT2 receptor is involved in the apoptotic process observed in the human fetal adrenal gland and could participate in the morphological changes occurring after birth, leading to involution of the fetal zone.

Hamster adrenal HMG-CoA reductase activity was enhanced with rat liver cytosolic phosphorylase phosphatase as well as with similarly isolated beef and hamster adrenal cytosolic preparations. HMG-CoA reductase was inactivated when microsomes were incubated in an EDTA-free medium but containing MgCl2 and ATP. The reductase activity of microsomes isolated from adrenals of hamsters sacrificed at 1100 h and 1900 h were (mean ± SEM, pmo1/mg protein/min.) 299.6±62.3 and 588.3 ± 96.6 respectively and could be enhanced by a factor of four when preincubated in the presence of liver phosphatase.

Livers uclei from chick embryos of 17 day incubation are capable of binding T3. This binding was optimal at 37 C and was abolished by the addition of proteolytic enzymes to the incubation medium. Approximately 70% of the nuclear bound [125I]T3 was removed with 0.4 M KC1 at pH 8.0 and was bound to a macromolecule, since 40-60% of the radioactivity in these extracts was excluded from small Sephadex G-50 columns. Glycerol density gradient analysis showed that this macromolecule had a sedimentation coefficient of 4.3S. From these data and the results of Sephadex G-100 gel filtration, the molecular weight of the [125I]T3 protein complex was calculated as 60,000. Binding studies performed on two preparations of purified nuclei and on nuclear extracts revealed a single class of binding sites with the properties of a receptor. In the experiments with purified nuclei, the association constant varied between 1.04– 1.07 × 109 M-1 whereas with nuclear extracts it was 2.17 × 109 M-1 The binding capacity was 363 fmol (0.23 ng) T3 and 836 fmol (0.54 ng) T3 per mg protein in the two preparations of purified nuclei. The nuclear extracts had a capacity of 466 fmol (0.31 ng) T3/mg protein. Expressed as femtomoles per ng DNA, the binding capacities were, respectively, 1.6, 2.61, and 1.06. Serial studies at different periods of embryogenesis showed that the binding capacity and the association constant rose progressively during embryogenesis (binding capacity: 9 days, 0.45 ± 0.08; 19 days, 0.73 ± 0.35 ng T3/mg DNA; Ka: 9 days, 1.04 ± 0.21; 19 days, 3.31 ± 0.32 x 109 M-1. T3 analogs were not very effective in inhibiting [125I]T3 nuclear binding, with the exception of Triac (3,5,3′-triiodothyroacetic acid) and 3,5,3′-triiodo-D-thyronine, but they all displaced the labeled T4. These findings indicate that early in embryogenesis the chick embryo liver possesses a putative T3 receptor similar to that observed in other experimental animals.

The synthesis of hepatic cytosol malate dehydrogenase (MDH) can be stimulated by thyroid hormones under various conditions, and consequently, this enzyme has been considered as a marker of the expression of thyroid hormone action. Since the concentrations of serum thyroid hormones increase considerably during the late phase of chick embryogenesis, we have studied the relationship between this phenomenon and liver MDH activity. A delay of 5 days was observed between the increase in circulating T4 and T3 concentrations and the enhancement of MDH activity. However, treatment with pharmacological doses of T4 (60 microgram/day) resulted in increased MDH activity 48 h after administration of the hormone, while hypothyroidism induced by methimazole decreased the level of the enzyme. Sex-linked differences in MDH activity were found in newborn chickens between 5-9 days of age. No such differences were observed for serum T3 and T4. These findings suggest that under physiological conditions MDH activity is not directly modulated by thyroid hormones in the chick embryo. However, these hormones may well play a permissive role with this enzyme.

A 36-year-old patient with secondary amenorrhea and progressive frank virilization was found by histologic examination to have ovarian hyperthecosis. Baseline androgenic investigation revealed a selective increase in testosterone (T) production rate (3.1 mg/day), which was associated with an increase in both plasma T concentration (total and unbound) and metabolic clearance rate (MCR) of T (2156 liters/day). There was a marked concentration gradient for circulating plasma T between each ovarian vein and the peripheral veins (right ovarian, 50 ng/ml; left ovarian, 21 ng/ml). After bilateral wedge resection there was a rapid decrease in the total and unbound T levels as well as in the MCR of T, which remained normal for at least 6 months. By then the patient had stopped shaving and was menstruating normally. In vitro incubation studies of fragments from each ovary revealed incorporation of [3H]-dehydroepiandrosterone and [3H]-progesterone into [3H]-T in the range reported for normal human ovarian stroma, and short-term cellular culture suggested a normal morphology and steroidogenesis. It is concluded that in this patient with ovarian hyperthecosis, the observed ovarian T production resulted more from an increase in the amount of stromal-thecal tissue than from a functional biochemical defect in the latter.

A technique was standardized to measure nuclear dihydrotestosterone receptors in human prostate. Normal, hyperplastic (BPH), and cancer tissues were homogenized and the homogenate centrifuged at 600 g X 10 min; the pellet was purified on sucrose density gradient. Purified nuclei were extracted with 0.4 M KC1. The nuclear extract was then incubated in the presence of [3H]dihydrostestosterone (DHT) at 4 degrees C for 20 hr. Competition studies with different steroids indicated a high specificity of the receptor towards DHT and no affinity towards progesterone and 17 beta-estradiol. Isolation and purification of [3H]DHT after its incubation in the presence of the KC1 nuclear extract indicated that no significant degradation of the hormone occurred during incubation. The number of binding sites measured was 8.5 +/- 0.77 fmol/mg protein (mean +/- SEM) for normal, 38.6 +/- 5.9 for BPH, and 79.9 +/- 21.9 for cancer. The possible clinical application of these results will require a long-term follow-up of the hormonal responsiveness of the prostatic-cancer patients investigated.