The ligand insertion hypothesis in the genomic action of steroid hormones

Gene regulation by steroids is tightly coupled to hormone concentration and stereochemistry. A key step is binding of hormones to receptors which interact with consensus DNA sequences known as hormone response elements (HREs). The specificity and strength of hormone binding do not correlate well with hormonal activity suggesting an additional step involving recognition of ligand by the gene. Stereospecific fit of hormones between base pairs and correlation of fit with hormonal activity led to the proposal that such recognition involves insertion of hormone into DNA. Here, the feasibility of insertion was investigated using computer models of the glucocorticoid receptor DNA binding domain bound to its HRE. The site reported to accommodate glucocorticoids was found in the HRE and was exposed to permit unwinding at this locus. The resulting cavity in the unwound DNA/receptor interface fit cortisol remarkably well; cortisol formed hydrogen bonds to both the receptor and DNA. Current experimental evidence is generally consistent with ligand binding domains of receptors undergoing a conformational change which facilitates transfer of the ligand into the unwound DNA/receptor interface. We propose this step is rate limiting and alterations in receptor, DNA or hormone which attenuate insertion impair hormonal regulation of gene function.     

Correlation of the production of gonadotropic and sex hormones in boys and girls during sex maturation

There is an early period of puberty in boys at the age of 10 to 13, accompanied by an increase in the level of follicle stimulating hormone and a low secretion of luteinizing hormone and testosterone, with no secondary sexual signs. The beginning of an increase in the luteinizing hormone and testosterone concentration in boys coincides with the development of secondary sexual signs. The stabilization of the level of luteinizing hormone takes place in boys already at the 2nd degree of the sexual development. The secretion of luteinizing hormone at this point corresponds already to the normal level in adult men. The level of testosterone in the blood continues to increase and does not reach its definitive level until after the age of 17, or at the 5th degree of sexual development. Like in boys, the increase of the follicle stimulating hormone in girls precedes the increase of the luteinizing hormone secretion. The beginning of the increase in the follicle stimulating hormone coincides with an increase in the level of estrogens and testosterone in the blood, and with the appearance of secondary signs of sexual maturation. The increase of the luteinizing hormone secretion takes place later in girls than in boys and is accompanied by a new and significant increase in the level of estrogens in the blood. At the 2nd degree of sexual development, the level of testosterone in the blood of girls is substantially higher than at the other stages.     

Interaction between triiodothyronine and ovarian steroid hormones on the regulation of the release of thyrotropin and thyrotropin-releasing hormone in vitro

Laparoscopic access to the retroperitoneum without abdominal insufflation has recently been made possible by devices designed to create a dilated laparoscopic cavity maintained by a fixed retraction system. This technique was applied in 10 patients undergoing laparoscopic ligation of the internal spermatic vein via extraperitoneal and intraperitoneal approaches. Gasless laparoscopic varicocelectomy was completed in 7 of 8 men by the extraperitoneal route and in neither of the 2 men approached intraperitoneally. The mean operative time was 170+/-55 min and postoperative pain exceeded the norm for standard insufflative laparoscopic varicocelectomy. Whereas certain theoretical advantages are offered by the gasless extraperitoneal approach to varicocele ligation, exposure through the gasless technique is currently suboptimal. Further development of "retraction" technology is required, prior to its routine application for varicocele ligation.     

Sex cord-stromal and steroid cell tumors of the ovary

Gonadal cell types that derive from the coelomic epithelium (sex cords) or mesenchymal cells of the embryonic gonads include granulosa cells, theca cells, fibroblasts, Leydig cells, and Sertoli cells. Ovarian tumors of these cell types are called sex cord-stromal tumors. This group of tumors represents approximately 8% of ovarian neoplasms and affects all age groups. The more common types are granulosa cell tumors (GCTs), fibrothecomas, and Sertoli-Leydig cell tumors. Sex cord-stromal tumors are of interest partly because of their hormonal effects, which are rare for other ovarian neoplasms. These effects include estrogenic effects (pseudoprecocious puberty, endometrial bleeding, endometrial hyperplasia and carcinoma) and virilization. The variety of gross appearances of these tumors, ranging from large multicystic masses to small solid masses, would appear to preclude a specific radiologic diagnosis. However, in many patients, both clinical and radiologic clues can suggest the diagnosis, including predominantly fibrous content at ultrasound or magnetic resonance imaging (fibrothecoma), large hemorrhagic multicystic mass in a child with pseudoprecocious puberty (juvenile GCT), and associated syndromes such as Peutz-Jeghers syndrome (sex cord tumor with annular tubules) or Ollier disease and Maffucci syndrome (juvenile GCT).     

Restitution of functional neural connections in chick embryos assessed in vitro after spinal cord transection in Ovo

Functional neural reconnection is not common after spinal cord transection in the CNS of adult higher vertebrates but has been demonstrated in embryonic avian and neonatal mammalian CNS. Chick brainstem spinal cord preparations from nontransected controls and embryos transected at the cervical level on embryonic days (E) 8, 9, or 10 in ovo were assessed in vitro between E12 and E20 for their ability to produce and maintain episodic motor activity (EMA) using electrophysiological, voltage sensitive dye and anatomical tract-tracing techniques. After 3 to 4 days recovery, cycle-by-cycle coupling of EMA between segments separated by a transection was absent or inconsistent, although otherwise normal bouts of locally stimulated and spontaneous EMA were routinely observed restricted to segments of a cord separated by a transection site. After 5-7 days recovery in ovo the cross-transection coordination during bouts of EMA approached that of nontransected controls. The delay between the initiation of EMA in cervical segments to its initiation in lumbosacral segments caudal to a transection was an indicator of reconnection strength. The delay shortened from 0.5 to a few seconds after 3 days of recovery to around 150 ms (i.e., normal) after 5 days of recovery. We conclude that the reconnection of spinal central pattern generators for EMA across the transection was served mainly by axons which established connections with local circuits after extending 1-3 segments through a transection. Propriospinal axons that originated within 1-3 segments rostral to the transection then served to serially initiate EMA in distal caudal segments.     

Mesonephric origin of the gonadal primitive medulla in chick embryos

The development of the gonadal primitive medulla in embryonic chick gonads was studied with the light microscope, using serial longitudinal sections from 72 h to 108 h of incubation. The sex of embryos was established from karyotypes. At 72 h, the germinal epithelium in the genital ridges was thickened. The nephrogenic cord was not differentiated into nephrons underneath, although the surrounding mesonephros displayed renal corpuscles and tubules. Clusters and trabeculae of mobilized mesonephric cells piled up under the germinal epithelium, forming the rudiment of the primitive medulla. From 78 h onwards, nephrotome-like structures existed in the mesenchyme underlying the germinal epithelium. Mesonephric cells became detached from their ventral walls and incorporated into the rudiment of the medulla. Finally, at 90 h, when the gonads were constituted, the primitive medulla was definitively formed without any contribution of the germinal epithelium. Adrenal cortical cells, also originating from the mesonephric blastema, showed tight relationships with the gonadal medullarian structures. Our observations support the concept of the mesonephric origin of the gonadal components having male potentialities in birds.     

Inhibition by anandamide of gap junctions and intercellular calcium signalling in striatal astrocytes

Anandamide, an endogenous arachidonic acid derivative that is released from neurons and activates cannabinoid receptors, may act as a transcellular cannabimimetic messenger in the central nervous system. The biological actions of anandamide and the identity of its target cells are, however, still poorly documented. Here we show that anandamide is a potent inhibitor of gap-junction conductance and dye permeability in striatal astrocytes. This inhibitory effect is specific for anandamide as compared to co-released congeners or structural analogues, is sensitive to pertussis toxin and to protein-alkylating agents, and is neither mimicked by cannabinoid-receptor agonists nor prevented by a cannabinoid-receptor antagonist. Glutamate released from neurons evokes calcium waves in astrocytes that propagate via gap junctions, and may, in turn, activate neurons distant from their initiation sites in astrocytes. We find that anandamide blocks the propagation of astrocyte calcium waves generated by either mechanical stimulation or local glutamate application. Thus, by regulating gap-junction permeability, anandamide may control intercellular communication in astrocytes and therefore neuron-glial interactions.     

Effects and metabolism of steroid hormones in human neuroblastoma cells

The development of the central nervous system is influenced by sex steroids and by their metabolites. However, little information on the possible effects of steroid hormones on neuroblastoma cells is available. Human neuroblastoma cell lines have been used as a model of human neuroblasts in vitro to study the metabolism of steroid hormones; in addition, the effects of steroids and steroid antagonists on neuroblastoma cell growth have also been investigated. The results obtained show that SH-SY5Y human neuroblastoma cells may actively metabolize testosterone and progesterone to their respective 5 alpha-reduced metabolites and that differentiation of neuroblastoma cells is paralleled by a significant increase in expression of the type-1 5 alpha-reductase and of the formation of steroid metabolites. All these data are suggestive of a potential role of steroid 5 alpha-reduced metabolites in the biology of neuroblastoma cells. Studies performed to analyze the role of steroid hormones on neuroblastoma cell proliferation show that progesterone at low doses may induce minor stimulation, and at higher doses, a toxic effect on the neuroblastoma cell line SK-N-SH is seen. Moreover, the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) decreases the proliferation of these cells in a dose-dependent manner. The effect of RU486 is not antagonized by either progesterone or dexamethasone, a result that seems to exclude the action of RU486 via classic intracellular steroid hormone receptors.     

Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier I screening battery

Retinal ganglion cell (RGC) axons in lizards (reptiles) were found to regenerate after optic nerve injury. To determine whether regeneration occurs because the visual pathway has growth-supporting glia cells or whether RGC axons regrow despite the presence of neurite growth-inhibitory components, the substrate properties of lizard optic nerve myelin and of oligodendrocytes were analyzed in vitro, using rat dorsal root ganglion (DRG) neurons. In addition, the response of lizard RGC axons upon contact with rat and reptilian oligodendrocytes or with myelin proteins from the mammalian central nervous system (CNS) was monitored. Lizard optic nerve myelin inhibited extension of rat DRG neurites, and lizard oligodendrocytes elicited DRG growth cone collapse. Both effects were partially reversed by antibody IN-1 against mammalian 35/250 kD neurite growth inhibitors, and IN-1 stained myelinated fiber tracts in the lizard CNS. However, lizard RGC growth cones grew freely across oligodendrocytes from the rat and the reptilian CNS. Mammalian CNS myelin proteins reconstituted into liposomes and added to elongating lizard RGC axons caused at most a transient collapse reaction. Growth cones always recovered within an hour and regrew. Thus, lizard CNS myelin and oligodendrocytes possess nonpermissive substrate properties for DRG neurons--like corresponding structures and cells in the mammalian CNS, including mammalian-like neurite growth inhibitors. Lizard RGC axons, however, appear to be far less sensitive to these inhibitory substrate components and therefore may be able to regenerate through the visual pathway despite the presence of myelin and oligodendrocytes that block growth of DRG neurites.     

Placental proteins and steroid hormones in the soluble fraction of human syncytiotrophoblast plasma membrane

Placental protein 5 (PP5), pregnancy-specific glycoprotein (SP1), pregnancy-associated alpha 2-glycoprotein (SP3) and chorionic gonadotrophin could not be demonstrated in appreciable molar quantities in the soluble fraction from microvillous plasma membrane preparations isolated from the syncytiotrophoblast of full-term human placentae. However, progesterone, total oestriol and placental lactogen may have some association with this membrane.     

Mechanism of steroid action in inflammation: inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5-8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 mug/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 - 30 mug/ml) were ineffective. Aldosterone and progesterone (100 mug/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 mug/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 mug/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 mug/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.