Dorothy B. Villee

Ribonucleic acid: control of steroid synthesis in endocrine tissue.

Ribonucleic acid extracted from either adrenal or testis altered the enzyme activity of adrenals and ovaries maintained in organ culture. The pattern of steroid hormone synthesis of the cultured endocrine tissue reflected the origin of the RNA.

An Organ Culture Model for the Study of Metanephric Development

A murine whole organ metanephric culture system was designed to study the developmental aspects of mammalian nephrogenesis. Metanephros and ureteric bud were removed from CFI albino mouse embryos at 13.5 +/- 0.4 days gestation, and grown in Dulbeccos modified Eagles Minimal Essential Medium supplemented with 20 per cent donor bovine serum at 37C in a mixed air--5 per cent CO2 environment. Under the experimental conditions employed, the metanephric explants showed organotypic tubular and glomerular epithelial development. A well-developed proximal tubule with microvilli, and characteristic intracellular organelles and intercellular junctions developed by 72 hours of culture. By 120 hours of culture, unique devascularized glomeruli consisting of parietal and visceral epithelial layers formed. The glomerular visceral epithelial cells formed foot processes and slit pore diaphragms, and produced islands of basement membrane. No endothelial or mesangial elements were present at any stage in organ culture development, indicating that advanced nephrogenesis can occur following initial epithelial-mesenchymal induction despite the absence of vascularization. The whole organ culture model system isolates renal structural development from the influences of perfusion and urine formation. The system thus affords the opportunity to study normal, as well as abnormal mammalian renal development under highly controlled experimental conditions.

Effects of progesterone on enzyme activity of adrenals in organ culture

Abstract Human fetal and mouse adrenals can be maintained for 24 hr or more in a histologically and enzymatically differentiated state in organ culture. Adrenals cultured in the presence of 10 −6 to 10 −4 m progesterone for 24 hr showed enhanced utilization of progesterone and decreased activity of the 3β-hydroxysteroid dehydrogenase-isomerase enzyme systems when subsequently homogenized and incubated with progesterone-4- 14 C and pregnenolone-7α- 3 H, respectively. These findings may represent examples of control of enzyme activity by substrate and product in mammalian cells in organ culture.

Changes in fetal steroid metabolism with age.

As gestation proceeds enzymes develop or are activated. Some of the enzymes catalyze steps in the synthesis of steroid hormones. The fetal adrenal gland has a distinct pattern of steroid metabolism that is due to the relative lack of 3β‐hydroxysteroid dehydrogenase and the very active sulfotransferases and 16α‐hydroxylases. This distinctive pattern is probably confined to the fetal zane af the fetal adrenal cortex. This zone develops during gestatian, passibly under the influence of chorianic and fetal pituitary gonadatrapins, and involutes after birth. The fetal adrenal gland, though unable to form pragesterone, metabalizes it to a variety of hydraxylated steraids. Very early in development only 16α‐hydraxylase and 17‐hydroxylase can be demonstrated in the fetal adrenal gland in vitro. By 7 to 8 weeks 21‐hydroxylase and desmalase are active in vitro, and thereafter 11‐hydraxylase becomes detectable. The cancentrations af steroids in adrenal tissue and fetal blood and urine suggest that there is a preponderance of cartisone aver cortisol and that the corticasterone/cortisol ratio is higher in perinatal life than that found in the child or adult. The activity of the sulfotransferases of the fetus explains the preponderance of sulfurylated steraids in fetal and newborn fluids. Depending on the sex of the fetus, andragen synthesis may rise just before differentiation af the gonaducts and external genitalia. The fetal testis at this period has numerous large Leydig cells, which may also be under the influence of charianic ganadotropin. There is no evidence of impaired 3β‐hydroxysteroid dehydrogenase in the fetal testis, and testasterone is a major product of steroid metabolism in vitro.

Ribonucleic Acid Control of Steroid Synthesis in Human Adrenals and Testes

The pattern of steroid synthesis in human fetal testes and adrenals was altered by prior exposure, in organ culture of the explants, of one gland to the ribonucleic acid extracted from the other gland. The new pattern reflected the origin of the RNA.

285 A Virilizing Adrenocortical Tumor in a Female Infant: Steroidogenesis In Vivo and In Vitro

Normal adrenal and adrenal tumor cells from a female infant with a virilizing adrenal tumor were grown in tissue culture for a period of 7 weeks with and without ACTH (0.1 unit/ml). The cells grew well and continued to produce steroid hormones, as measured by radioimmunoassay of individual steroids in the culture medium. Compared to normal cells, tumor cells with no ACTH added produced equivalent amounts of cortisol, 180H corticosterone, and 180H deoxycorticosterone, but lesser amounts of dehydroepiandosterone (DHA), androstenedione (A), testosterone (T) and progesterone. Normal cells exposed to ACTH showed an increase in all steroids measured whereas ACTH-exposed tumor cells showed an increase principally in DHA, consistent with a deficiency in 3β-hydroxysteroid dehydrogenase. Concentrations of DHA, A and T in the patients serum were elevated before the adrenal tumor was removed. The relative concentration of the three androgens in media of tumor cells in vitro resembled that in patients serum in vivo. These studies demonstrate that both normal and tumor cells of adrenal can be maintained for a long time in vitro, that they retain their ability to respond to ACTH and that they produce their characteristic steroids. The tumor cells appear to have a 3β-hydroxysteroid dehydrogenase deficiency.Supported by the National Foundation, the Grant Foundation, and USPHS Grant RR00128, Div. Research Resources, NIH.

The influence of ACTH on steroid synthesis in normal and abnormal testicular tissues.

Abstract The effects of ACTH on testicular enzymes was studied using human testicular tumors from patients with a genetic deficiency of 21-hydroxylase and a resulting unsuppressed secretion of ACTH from the pituitary. The human tumor cells from patients off glucocorticoid therapy were capable of forming glucocorticoids in vitro , whereas tissue from patients whose ACTH production was suppressed with adequate glucocorticoid therapy (17-ketosteroid excretion maintained at normal values) showed no glucocorticoid formation in vitro . Patients off glucocorticoid therapy had very high 11β-hydroxylase activity in their testicular tumors whereas patients suppressed with glucocorticoids had tumor cells with little or no 11β-hydroxylase activity. Non-tumorous testicular cells from patients on or off therapy formed primarily 17-hydroxyprogesterone and testosterone in vitro with no glucocorticoid formation. The administration of 20 IU ACTH twice daily to rats over a 10-day period altered the kind of RNA formed within certain testicular cells. This RNA enhanced glucocorticoid formation when added to mouse adrenals in vitro . Similar results were obtained using testicular RNA from ACTH-treated adrenalectomized rats, showing that endogenous glucocorticoids could not be mediating the effect. Thus ACTH responsive cells exist in testicular tissues from humans and rats, suggesting that under appropriate circumstances the tropic hormone of the adrenal may influence the biosynthetic processes of certain cells in the testis.