Immunohistochemical analysis of primary sensory neurons latently infected with herpes simplex virus type 1.

We characterized the populations of primary sensory neurons that become latently infected with herpes simplex virus (HSV) following peripheral inoculation. Twenty-one days after ocular inoculation with HSV strain KOS, 81% of latency-associated transcript (LAT)-positive trigeminal ganglion (TG) neurons coexpressed SSEA3, 71% coexpressed Trk(A) (the high-affinity nerve growth factor receptor), and 68% coexpressed antigen recognized by monoclonal antibody (MAb) A5; less than 5% coexpressed antigen recognized by MAb KH10. The distribution of LAT-positive, latently infected TG neurons contrasted sharply with (i) the overall distribution of neuronal phenotypes in latently infected TG and (ii) the neuronal distribution of viral antigen in productively infected TG. Similar results were obtained following ocular and footpad inoculation with KOS/62, a LAT deletion mutant in which the LAT promoter is used to drive expression of the Escherichia coli lacZ gene. Thus, although all neuronal populations within primary sensory ganglia appear to be capable of supporting a productive infection with HSV, some neuronal phenotypes are more permissive for establishment of a latent infection with LAT expression than others. Furthermore, expression of HSV LAT does not appear to play a role in this process. These findings indicate that there are marked differences in the outcome of HSV infection among the different neuronal populations in the TG and highlight the key role that the host neuron may play in regulating the repertoire of viral gene expression during the establishment of HSV latent infection.

Functional maturation of the primate fetal adrenal in vivo. II. Ontogeny of corticosteroid synthesis is dependent upon specific zonal expression of 3 beta-hydroxysteroid dehydrogenase/isomerase.

Cortisol, produced by the primate fetal adrenal, regulates the maturation of organ systems necessary for extrauterine life. During most of primate pregnancy, however, the fetal adrenal lacks the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), which is essential for cortisol synthesis. Therefore, we used immunohistochemistry and in situ hybridization techniques to investigate the developmental expression of 3 beta HSD in the fetal rhesus monkey adrenal from 109 days' gestation until term (165 +/- 5 days) and assessed the role of ACTH in the induction of its expression and localization. We also examined whether ACTH regulates the expression of two other steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase, 17/20-lyase (P450c17), in the fetal rhesus monkey adrenal. To stimulate ACTH secretion from the fetal pituitary in vivo, we administered metyrapone to late gestation fetal rhesus monkeys for 3-7 days. Adrenals were collected from untreated fetuses at 109-125 days (n = 5), 130-148 days (n = 7), 155-172 days (n = 4), and after metyrapone treatment at 135-137 days (n = 4). The cortical width and total amount of 3 beta HSD staining were measured using an image analysis system. 3 beta HSD was localized primarily in the definitive zone cells of the adrenal from fetuses between 109-148 days, whereas at term (155-172 days), 3 beta HSD was localized in both definitive and transitional zone cells. The cortical width and total amount of 3 beta HSD staining in the adrenal increased significantly (P < 0.05) between 148 days (137 +/- 14 microns and 3,689 +/- 522 grains) and 155 days (315 +/- 61 microns and 7,321 +/- 2,008 grains). Interestingly, in metyrapone-treated fetuses at 135-137 days, 3 beta HSD messenger RNA (mRNA) and protein were localized extensively in both the definitive and transitional zones, a pattern seen only in term fetal adrenals in untreated animals. In addition, metyrapone treatment significantly (P < 0.05) increased cortical width (386 +/- 95 microns) and total 3 beta HSD immunostaining (29,063 +/- 13,692 grains) compared with age-matched controls. In contrast to 3 beta HSD, P450scc mRNA was detected in the definitive, transitional, and fetal zones, and its expression was not altered after metyrapone treatment. P450c17 mRNA was detected in the transitional and fetal zones, and the relative abundance was greater in the transitional zone. The relative abundance of P450c17 mRNA was increased in the fetal zone after metyrapone treatment. In summary, at term or after metyrapone treatment, expression of 3 beta HSD is induced in the transitional zone of the fetal rhesus monkey adrenal gland, an indication of functional maturation of the primate adrenal cortex. These data suggest that the ontogenetic increase in fetal pituitary ACTH secretion plays an important role in the induction of 3 beta HSD expression in the transitional zone.

Functional maturation of the primate fetal adrenal in vivo: I. Role of insulin-like growth factors (IGFs), IGF-I receptor, and IGF binding proteins in growth regulation.

The rapid growth of the primate fetal adrenal from midgestation until term is regulated by ACTH secreted by the fetal pituitary. Previous studies suggest that the trophic actions of ACTH are mediated by insulin-like growth factor II (IGF-II) synthesized by fetal adrenal cortical cells. To characterize further the role of IGF-II in the regulation of fetal adrenal growth, we investigated the expression of the messenger RNAs (mRNAs) encoding IGF-I, IGF-II, IGF-I receptor (IGF-IR) and IGF binding protein (IGFBP) 1-6 in the fetal rhesus monkey adrenal in vivo from 109 days of gestation until term (165 +/- 5 days) using in situ hybridization. To assess the role of ACTH in the regulation of expression of the IGF system in vivo, we administered metyrapone (3-7 days) to late gestation fetal rhesus monkeys (n = 4) in utero to increase fetal pituitary ACTH secretion. IGF-II mRNA was abundant in the definitive, transitional and fetal zones of the adrenal cortex from 109 days until term. IGF-IR mRNA was expressed in the definitive, transitional and fetal zones and decreased to nondetectable levels at term. IGFBP-2 and IGFBP-6 mRNAs were expressed in the definitive, transitional, and fetal zones, whereas IGFBP-1, -3, -4, and -5 were not detected in adrenal cells. The effects of increasing ACTH secretion on the growth of the specific zones of the adrenal were determined using morphometric techniques. Metyrapone treatment approximately doubled adrenal weight, which was due to an increase in the area of the definitive, transitional, and fetal zones with decreased cell density of the definitive, transitional, and fetal zones compared with controls and not due to a change in total cell number. Therefore, the increase in adrenal weight after metyrapone treatment was due to hypertrophy of the three cortical zones; there was no effect on adrenal medullary growth. The relative abundance of the mRNAs encoding IGF-II and the IGF-IR was increased after metyrapone treatment, whereas the localization and relative abundance of IGFBP 1-6 mRNAs were not altered by metyrapone treatment. We conclude that the ontogenetic increase in adrenal growth may be regulated, at least in part, by locally synthesized IGF-II, and the cessation of adrenal growth that occurs at term may be mediated by the decrease in the IGF-IR. The adrenal cortical expression of IGFBP-2 and IGFBP-6 suggests that these IGFBPs may modulate the IGF-IGF-IR interaction. Metyrapone treatment, which likely increased fetal pituitary ACTH secretion, causes a coordinated increase in expression of IGF-II and IGF-IR in fetal adrenal cortical cells, which may be an important mechanism of regulation of fetal adrenal cortical growth.

Localization and regulation of corticotropin receptor expression in the midgestation human fetal adrenal cortex: implications for in utero homeostasis.

Developmental changes in the responsiveness of the fetal adrenals to corticotropin (ACTH) play an important role in the regulation of the fetal hypothalamic-pituitary-adrenal axis. Responsiveness of adrenal cortical cells to ACTH is dependent on the extent of ACTH receptor expression. Therefore, we examined the localization and regulation of ACTH receptor expression in the midgestation (16-24 weeks) human fetal adrenal cortex. In situ hybridization analysis was used to localize messenger RNA (mRNA) encoding the ACTH receptor in sections of human fetal adrenal glands. Messenger RNA encoding the ACTH receptor was localized in cells from all cortical zones; abundance was higher in definitive zone than in fetal zone cells and was least abundant in the more central portions of the cortex. Regulation of ACTH receptor expression was studied using Northern blot analysis of total RNA extracted from primary cultures of fetal and definitive zone cells. Two major (1.5 and 3.5 kilobases) and, upon stimulation with ACTH, 3 minor (4.0, 6.0 and 10.0 kb) ACTH receptor mRNA transcripts were detected in RNA from fetal and definitive zone cells. In both cell types, ACTH-(1-24) increased the abundance of mRNA encoding the ACTH receptor 10- to 20-fold compared with untreated cells. The effects of ACTH-(1-24) on ACTH receptor expression in fetal zone cells were time- and dose-dependent. The ED50 for the stimulation of ACTH receptor expression by ACTH-(1-24) was 1-10 pM, and maximal response to 0.1 nm ACTH-(1-24) was detected after 12-16 h. Eight-bromoadenosine cAMP and forskolin also stimulated ACTH receptor expression in fetal zone cells and closely mimicked the effects of ACTH-(1-24). In contrast, stimulation of protein kinase C with 12-O-tetradecanoyl phorbol 13-acetate had no effect on ACTH receptor expression. Changes in ACTH receptor expression in response to ACTH-(1-24), cAMP and forskolin were paralleled by changes in expression of the P450 cholesterol side chain cleavage (P450scc) enzyme. These data demonstrate that expression of the ACTH receptor by the human fetal adrenal cortex is up-regulated by its own ligand and that this effect is mediated by a cAMP-dependent mechanism. In addition, the coordinate stimulation of ACTH receptor and P450scc expression by ACTH indicates that the gene for the ACTH receptor is one of a specific cohort of genes regulated by ACTH that are required to facilitate fetal adrenal cortical response to ACTH. ACTH regulation of its own receptor may represent a mechanism by which fetal adrenal responsiveness to ACTH is maintained and possibly enhanced during fetal development.

Response to hemorrhagic stress in the rhesus monkey fetus in utero: effects on the pituitary-adrenal axis.

In human pregnancy, fetoplacental hemorrhage is often associated with morbidity and potentially mortality. We investigated the effect of acute fetal hemorrhage on the fetal pituitary-adrenal axis in the subhuman primate (Macaca mulatta). We measured fetal and maternal plasma concentrations of ACTH, cortisol, and dehydroepiandrosterone sulfate (DHAS) by RIA in 10 rhesus monkeys. At 133-158 days of gestation (d) the fetuses were subjected to acute hemorrhage (0.5 mL/min for 10 min). Fetal plasma ACTH and cortisol concentrations were significantly increased above baseline in response to fetal hemorrhage. There was a more rapid, but significantly reduced, fetal plasma ACTH and cortisol response in the 133-143 d fetuses than in the 147-158 d fetuses. In 133-143 d fetuses, the maximal changes from baseline in plasma ACTH and cortisol concentrations were +6.5 +/- 2.6 pmol/L at +25 min and +4.55 +/- 1.19 nmol/L at +10 min, respectively. Fetal plasma ACTH and cortisol concentrations remained elevated throughout the 60-min period after hemorrhage (3.06 +/- 1.21 pmol/L and +2.42 +/- 1.05 nmol/L at +70 min). In 147-158 d fetuses, the maximal changes from baseline in plasma ACTH and cortisol concentrations were +13.7 +/- 4.23 pmol/L and +12.92 +/- 3.25 nmol/L at +70 min. In the 133-143 d fetuses, plasma DHAS concentrations did not change in response to hemorrhage, whereas in the 147-158 d fetuses, plasma DHAS concentrations were elevated above baseline during the hemorrhage (+624.2 +/- 269.5 nmol/L, at +9 min) and returned quickly to pre-hemorrhage values. These data suggest that the fetal pituitary-adrenal axis plays a role in the fetal response to intrauterine stress, but that only later in gestation is the fetal cortisol response to fetal hemorrhage mediated via ACTH secreted by the fetal pituitary.