Thomas O. Fox

Animal Cells: Noncorrelation of Length of G1 Phase with Size after Mitosis

The interval between mitosis and initiation of DNA synthesis (G1) varied over a fourfold range for Chinese hamster ovary cells, an established line. This was not because of size differences. Synchlronous cells of different sizes began DNA synthesis at similar times after mitosis. A novel technique of centrifugation for separating cells according to size is described.

Localization of aromatase and 5α-reductase to neuronal and non-neuronal cells in the fetal rat hypothalamus

Experiments were designed to identify the neural cell type(s) responsible for the aromatization and 5 alpha-reduction of androgens in the rat hypothalamus. Primary cultures of fetal rat hypothalamic cells, which had enhanced neuronal morphology, were treated at various times after plating with kainic acid (KA), a neurotoxic agent which selectively destroys neuronal cells. Neuronal morphology was disrupted in a time (0-6 days)- and dose (10(-4)-10(-2) M)-dependent fashion after KA treatment, with no apparent change in the appearance of the flattened, underlying non-neuronal cells. KA treatment for 4 days decreased aromatization by 94% in a dose-dependent fashion (10(-4)-10(-2) M KA), while 5 alpha-reduction declined by no more than 25%. A 6-day time course with 10(-3) M KA showed a dramatic decline in aromatization and no alteration in 5 alpha-reduction. In control experiments, substance P, a neuronal peptide, declined after KA treatment while the activity of glutamine synthetase, a glial enzyme, did not change. We conclude from these results that aromatase is localized primarily to neuronal cells in the hypothalamus while 5 alpha-reductase is confined primarily to non-neuronal cells.

Sex differences in the shape of the sexually dimorphic nucleus of the preoptic area and suprachiasmatic nucleus of the rat: 3-D computer reconstructions and morphometrics.

Three-dimensional images of nuclei facilitate morphological comparisons between differing animal groups. As revealed by computer-assisted techniques, the greater volume in male rats of the sexually dimorphic nucleus of the preoptic area was not proportional in all directions. The nucleus was more elongated in males than females, indicating a sex-dependent shape. This study also indicated that the volume of the suprachiasmatic nucleus was larger in males.

Studies on the role of catecholamines in the regulation of the developmental pattern of hypothalamic aromatase

Experiments were conducted to study the regulation of the developmental pattern of aromatase in the forebrain of the perinatal rat. Two experimental designs were used: aromatase measured in primary cultures of fetal hypothalamic cells and in cell-free preparations of forebrain tissue excised at varying ages. In cultured cells, aromatase decreased logarithmically at a slow rate (t1/2 = 7.8 days). Norepinephrine caused a pronounced dose (4 x 10(-6) M) and time-dependent (2-6 days) drop in aromatase without affecting the levels of 5 alpha-reductase or substance P. In isolated tissue, aromatase activity was compared with the concentrations of norepinephrine and dopamine in the forebrain of males vs females at different perinatal ages and in discrete forebrain areas at postnatal day 4. In no case was a sex difference in catecholamines seen. An overall developmental decline in aromatase was associated with developmental increases in catecholamine levels. Acute treatment with the beta-agonist, isoproterenol, had no effect on brain aromatase activity.

Estrogen binding in nuclear and cytosolic extracts from brain and pituitary of middle-aged female rats.

Estrogen binding was compared in brain and pituitary of long-term ovariectomized young and middle-aged (MA) female rats. Binding was quantified in both cytosolic and nuclear extracts to ascertain whether fractions of estrogen binding are altered in MA females. Estrogen binding detected in nuclear extracts from hypothalamus/preoptic area and anterior pituitary of MA females was significantly lower than levels detected in young females. In each case where an age-related decrease in nuclear estrogen binding was observed, an increased number of putative estrogen receptors was detected in the cytosolic extract. Therefore, the age-related decrease in nuclear estrogen binding did not appear to result from a simple decrease in total available cellular estrogen receptors. Rather these results suggest a decrease in the ability of putative estrogen receptors in aging females to remain tightly bound to nuclei after their isolation. The ability of estrogen receptor complexes from aging animals to bind to DNA was evaluated by DNA-cellulose chromatography in order to examine possible quantitative or qualitative differences in estrogen binding proteins with age. The data did not indicate that the properties of estrogen receptors themselves changed with age. It is possible, therefore, that age-related alterations may interfere with the interaction between the estrogen receptor complex and the nucleus.

Sex and Regional Differences in Intracellular Localization of Estrogen Receptor Immunoreactivity in Adult Ferret Forebrain

Estrogen receptors were visualized in adult ferret brains using the H222 estrogen receptor antibody and immunocytochemical techniques. H222 immunoreactive (H222ir) cell nuclei were present in many forebrain regions in gonadectomized ferrets of both sexes. In many instances, H222ir cells also had immunoreaction product in their processes. All cells with H222ir processes also contained H222ir nuclei. More H222ir processes were observed in females in the medial and lateral preoptic area/anterior hypothalamus, and at the level of the descending fornix and caudal anterior commissure. Quantitative image analysis confirmed that females had significantly more (approximately 50%) extranuclear H222 immunoreaction product than males in cells in the magnocellular or preoptic subnuclei of the bed nucleus of the stria terminalis. Cells in the principal subnucleus of the bed nucleus of the stria terminalis and ventrolateral septum were notable for the relative paucity of H222ir processes. Sex differences in the intracellular extranuclear distribution of estrogen receptor protein in particular brain regions might contribute to the differential regulation of estrogen-dependent functions in the two sexes.

Androgen receptors from rat kidney and brain; DNA-binding properties of wild-type and tfm mutant

Abstract Androgen receptors from rat kidney and hypothalamus-preoptic area have been demonstrated and partially characterized by their binding properties on DNA-cellulose. For both tissues the receptors eluted from DNA-cellulose with characteristic patterns, which distinguished the two extracts from one another; in addition, estrogen receptors from kidney were eluted separately from the androgen receptors. The androgen receptors from rat tissues resembled those from mouse tissues in exhibiting limited capacity and high affinity for testosterone and for dihydrotestosterone, in their chromatographic characteristics on DNA-cellulose, and in the distinctive elution patterns between kidney and brain extracts. Tissues from the androgen-resistant rat mutant, testicular feminization ( tfm ). had reduced levels of androgen receptors in both kidney and hypothalamus-preoptic area. The residual androgen-binding activities in each tissue adhered to DNA-cellulose and eluted with the same patterns as obtained for receptors of the wild-type rats.

A Genetic Variant in the Morphology of the Medial Preoptic Area in Mice

Inbred mice of the DBA/2J and C57BL/6J strains are known to differ in physiological and behavioral characteristics that are partially controlled by nuclei in the preoptic area/anterior hypothalamus. We describe a distinguishing nucleus of darkly staining, densely packed cells, which we term the medioventral pars compacta (MVPC), within the medial preoptic nucleus of DBA/2J, but not C57BL/6J mice. The analysis also indicates that this nucleus is nearly 80% larger in volume in females vs males of the DBA/2J strain. The strain difference may be used to define genetic influences on this neuroanatomical and functional property.

RESIDUAL ANDROGEN BINDING IN TESTICULAR FEMINIZATION (TFM)

Most mutants with genetic androgen-resistance possess some level of androgen binding which exhibits properties of receptors. The present studies aim to determine whether the androgen binding activities in mutants are, or are related to, receptors. This binding portion is termed residual androgen receptors. We have examined several androgen-resistance mutants with testicular feminization (TFM). Putative androgen receptors from mice, rats, and humans with TFM have been compared, and at least three different types of residual receptors have been observed. They are discussed in relation to possible receptor defects and to differences in the nature of androgen-resistance associated with each of them.

Age-dependent expression of microtubule-associated protein 2 in the ventromedial nucleus of the hypothalamus.

A distinctive developmental pattern of microtubule-associated protein 2 (MAP2) was detected in the ventromedial hypothalamus of rats. A region of more intense MAP2 immunoreactivity in this nucleus was present at birth, became prominent in a ringed appearance by postnatal day 4 and disappeared in the third postnatal week. This period of selective MAP2 expression in particular subcortical regions may signify important functions of MAP2 in the stabilization of developing dendritic structure.