Fetal Adrenal Cortex Research Articles

Proliferation and apoptosis in the human adrenal cortex during the fetal and perinatal periods: implications for growth and remodeling.

After 10-15 weeks of gestation, the human fetal adrenal cortex undergoes rapid growth due to enlargement of a specialized cortical compartment known as the fetal zone (FZ). Soon after birth, the FZ regresses and the adult zonation pattern develops at least in part from cells derived from the persistent definitive zone (DZ), a thin layer of tightly packed cells surrounding the FZ. We postulated that growth of the fetal adrenal cortex involves zone-specific cellular hyperplasia, whereas the postnatal involution of the FZ is due to apoptosis. Therefore, we investigated the pattern of cellular proliferation and death in the FZ and DZ of the human fetal and postnatal adrenal cortex using immunohistochemical staining for proliferating cell nuclear antigen as a marker of mitosis and in situ detection of DNA fragmentation as a marker of apoptosis. Between 10-14 weeks' gestation, the mitotic indexes (percentage of proliferating cell nuclear antigen-positive cells) in the DZ (26.46 +/- 2.95%) and in the FZ (21.26 +/- 2.57%) were not significantly different. Between 15-20 weeks gestation, the mitotic index increased significantly (P < 0.05) in both zones (FZ, 33.84 +/- 5.21%; DZ, 67.45 +/- 7.58%) relative to levels before 15 weeks. This increase persisted between 21-24 weeks gestation (FZ, 39.5 +/- 4.22%; DZ, 58.63 +/- 6.83%). Interestingly, after 14 weeks, the mitotic index of the DZ was significantly greater (P < 0.05) than that of the FZ. In adrenal specimens obtained from infants born prematurely and treated in utero with glucocorticoid, the mitotic indexes in the FZ and DZ were significantly decreased. At all stages of gestation, no apoptotic nuclei were detected in the DZ. However, scattered apoptotic nuclei were detected in the central portions of the FZ. The number of apoptotic nuclei in the inner FZ increased with advancing gestation and was maximal during the first postnatal month. To identify factors that may regulate apoptosis, primary cultures of midgestation FZ cells were treated with activin A and transforming growth factor-beta (TGFbeta). Activin A and TGFbeta both induced apoptotic cell death, as assessed by internucleosomal DNA cleavage (DNA laddering). Induction of apoptosis by activin A was prevented by concomitant addition of follistatin, an activin-binding protein. Taken together, these data indicate that 1) growth of the human fetal adrenal cortex involves cellular hyperplasia, mainly in the DZ and to a lesser extent in the FZ, which is probably dependent on ACTH; and 2) apoptosis occurs predominantly in the inner cortical compartment and may be responsible for the rapid regression of the FZ after birth, a process that may be regulated by activin A and/or TGFbeta.

Corticotropin regulates vascular endothelial growth factor expression in human fetal adrenal cortical cells.

The human adrenal cortex has a complex vasculature that is essential for growth, organ maintenance, and access of secreted hormones to the circulation. Growth and function of the adrenal cortex are regulated by corticotropin (ACTH), the actions of which are in part mediated by locally produced growth factors. As cortical growth and vascularization must increase in a coordinated manner, we hypothesized that ACTH also influences adrenal cortical angiogenesis by stimulating the local expression of specific angiogenic factors. Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific angiogenic peptide, the expression of which has been detected in adrenal cortical cells. Therefore, we examined the localization of VEGF expression in the midgestation (16-20 weeks) human fetal adrenal cortex and determined whether VEGF expression and secretion by isolated human fetal adrenal cortical cells are regulated by ACTH. By immunohistochemical analysis, strong cytoplasmic staining for VEGF was detected in scattered clusters of fetal zone (inner cortical compartment) cells. In contrast, cells in the outer, definitive zone of the cortex stained only weakly for VEGF. The predominant staining for VEGF in the fetal zone correlated with the extensive vasculature of this zone as detected by immunohistochemical staining for von Willebrand factor, which is specific for endothelial cells. In primary cultures of human fetal adrenal cortical cells, ACTH (1 nmol/L) and forskolin (10 micromol/L) increased the abundance of messenger ribonucleic acid transcripts encoding VEGF, as assessed by Northern and slot blot analyses. The stimulatory effect of ACTH and forskolin on VEGF gene expression occurred within 2 h of agonist exposure and persisted for at least 24 h. ACTH and forskolin also increased VEGF protein secretion by fetal adrenal cortical cells, as assessed by enzyme-linked immunosorbent assay for VEGF in fetal adrenal cortical cell-conditioned medium. A significant (P < 0.05) increase in VEGF secretion was detected as early as 8 h after ACTH or forskolin treatment. By 24 h after the addition of ACTH or forskolin, VEGF secreted from isolated human fetal adrenal cells was increased 5- to 6-fold. These data demonstrate that the human fetal adrenal cortex, particularly the cells of the inner fetal zone, express VEGF and that VEGF expression and secretion by these cells are directly regulated by ACTH and the activation of adenylate cyclase. Thus, VEGF may be a local regulator of adrenal cortical angiogenesis and an important mediator of the tropic action of ACTH, ensuring the coordination of ACTH-stimulated cortical growth and vascularization.

Tissue-specific messenger ribonucleic acid expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 and the glucocorticoid receptor within rat placenta suggests exquisite local control of glucocorticoid action.

Placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD) regulates transplacental passage of maternal glucocorticoids to the fetus and is thus a key determinant of fetal glucocorticoid levels. It has also been proposed that placental 11beta-HSD expression may influence local glucocorticoid actions by regulating access of corticosterone to the glucocorticoid receptor (GR) or mineralocorticoid receptor (MR). Therefore, the present study used a rat model to assess whether the GR or MR are coexpressed with the two forms of 11beta-HSD (types 1 and 2) in the placental labyrinth zone, the major site of maternal-fetal transfer, and in the basal zone, the primary site of placental hormone synthesis. In situ hybridization analysis was used to assess messenger RNA (mRNA) expression for the GR, MR, 11beta-HSD-1, and 11beta-HSD-2 in the two placental zones on days 16, 19 and 22 of pregnancy (term = day 23). Whereas expression of the GR appeared relatively unchanged in both zones at these three stages of pregnancy, that of 11beta-HSD-1 clearly increased in the labyrinth zone but fell in basal zone, whereas the opposite pattern of expression was observed for 11beta-HSD-2. MR expression was not detected at any stage. The pattern of placental 11beta-HSD-2 mRNA expression over days 16, 19, and 22 of pregnancy was paralleled by changes in 11beta-HSD-2-specific bioactivity, but despite clear expression of 11beta-HSD-1 mRNA, no bioactivity attributable to this enzyme was measurable in either placental zone. To assess the role of fetal adrenal maturation on these changes in 11beta-HSD, two experimental models, maternal adrenalectomy and fetectomy, were employed. Maternal adrenalectomy on day 13 advanced maturation of the fetal adrenal cortex but had no effect on 11beta-HSD-2 bioactivity in either of the placental zones at day 19. Placental 11beta-HSD-2 bioactivity on day 22 was also unaffected by fetectomy 3 or 6 days earlier. In conclusion, the consistent expression of the GR in the two placental zones late in pregnancy suggests that concomitant and marked changes in 11beta-HSD-1 and 11beta-HSD-2 expression could have a major influence on glucocorticoid action in the placenta at this time. Moreover, the changes in 11beta-HSD expression appear to be unrelated to development of the fetal adrenal cortex and are likely to reduce the placental glucocorticoid barrier near the end of pregnancy.

Regulation and function of steroid production by mid gestation ovine fetal adrenal cortex in vivo.

By mid-gestation (75-85 days, term=150 days), the ovine fetal adrenal gland is zoned into cortex and medulla. The cortex has an outer layer of cells which have the morphological characteristics of zona glomerulosa cells, containing mitochondria with lamellar cristae. It has been reported that cultured adrenal cells from mid-gestation bovine and ovine fetuses can be stimulated to increase aldosterone production, ten fold, by angiotensin II, and that this can be maintained for at least 3 days. However, the situation in vivo is unknown. In the current report we show that in chronically cannulated ovine fetuses at mid-gestation, angiotensin II (1 μg/h) does not increase aldosterone either in the short term (3 hours) or long term (3 days). However, ACTH (450 ng/h) can increase plasma aldosterone in the short but not long term. ACTH at this dose produces progressive and large increases in cortisol production. Angiotensin II is pressor and produces a modest diuresis without stimulating cortisol.

Developmental and Functional Biology of the Primate Fetal Adrenal Cortex*

The unique characteristics of the primate (particularly human) fetal adrenal were first realized in the early 1900s when its morphology was examined in detail and compared with that of other species. The unusual architecture of the human fetal adrenal cortex, with its unique and disproportionately enlarged fetal zone, its compact definitive zone, and its dramatic remodeling soon after birth captured the interest of developmental anatomists. Many detailed anatomical studies describing the morphology of the developing human fetal adrenal were reported between 1920 and 1960, and these morphological descriptions have not changed significantly. More recently, it has become clear that fetal adrenal cortical growth involves cellular hypertrophy, hyperplasia, apoptosis, and migration and is best described by the migration theory, i.e. cells proliferate in the periphery, migrate centripetally, differentiate during their migration to form the functional cortical zones, and then likely undergo apoptosis in the center of the cortex. Consistent with this model, cells of intermediate phenotype, arranged in columnar cords typical of migration, have been identified between the definitive and fetal zones. This cortical area has been referred to as the transitional zone and, based on the expression of steroidogenic enzymes, we consider it to be a functionally distinct cortical zone. Elegant experiments during the 1950s and 1960s demonstrated the central role of the primate fetal adrenal cortex in establishing the estrogenic milieu of pregnancy. Those findings were among the first indications of the function and physiological role of the human fetal adrenal cortex and led Diczfalusy and co-workers to propose the concept of the feto-placental unit, in which DHEA-S produced by the fetal adrenal cortex is used by the placenta for estrogen synthesis. Tissue and cell culture techniques, together with improved steroid assays, revealed that the fetal zone is the primary source of DHEA-S, and that its steroidogenic activity is regulated by ACTH. In recent years, function of the human and rhesus monkey fetal adrenal cortical zones has been reexamined by assessing the localization and ontogeny of steroidogenic enzyme expression. The primate fetal adrenal cortex is composed of three functionally distinct zones: 1) the fetal zone, which throughout gestation does not express 3 beta HSD but does express P450scc and P450c17 required for DHEA-S synthesis; 2) the transitional zone, which early in gestation is functionally identical to the fetal zone but late in gestation (after 25-30 weeks) expresses 3 beta HSD, P450scc, and P450c17, and therefore is the likely site of glucocorticoid synthesis, and 3) the definitive zone, which lacks P450c17 throughout gestation but late in gestation (after 22-24 weeks) expresses 3 beta HSD and P450scc, and therefore is the likely site of mineralocorticoid synthesis. Indirect evidence, based on effects of P450c21 deficiency and maternal estriol concentrations, indicate that the fetal adrenal cortex produces cortisol and DHEA-S early in gestation (6-12 weeks). However, controversy exists as to whether cortisol is produced de novo or derived from the metabolism of progesterone, as data regarding the expression of 3 beta HSD in the fetal adrenal cortex early in gestation are conflicting. During the 1960s, Liggins and colleagues demonstrated that in the sheep, cortisol secreted by the fetal adrenal cortex late in gestation regulates maturation of the fetus and initiates the cascade of events leading to parturition. Those pioneering discoveries provided insight into the mechanism underlying the timing of parturition and therefore were of particular interest to obstetricians and perinatologists confronted with the problems of preterm labor. However, although cortisol emanating from the fetal adrenal cortex promotes fetal maturation in primates as it does in sheep, its role in the regulation of primate parturition, unlike that in sheep

Immunolocalisation of P450(C17) in the fetal sheep adrenal gland during gestation and in response to ACTH and glucocorticoid administration.

In sheep, increased output of cortisol from the fetal adrenal gland is critical to organ maturation and parturition. Cortisol synthesis is determined in part by the activity of P450(C17) enzyme. We have used immunohistochemistry and Western immunoblotting to examine the distribution of P450(C17) in the ovine fetal adrenal during gestation, and after ACTH or dexamethasone administration to fetuses between Days 125 and 130. The patterns were compared with changes in 3beta-hydroxysteroid dehydrogenase (3beta-HSD) localisation and levels. Adrenal tissue was obtained from four fetuses at each of Days 63-65, 100, 125-130 and term (>140 days). Further animals were chronically catheterised and infused with ACTH, dexamethasone or saline for 96 h beginning on Day 125. Immunohistochemistry for P450(C17), 3beta-HSD, and phenylethanolamine-N-methyl transferase (PNMT) was conducted using standard techniques. At Day 63-65 of pregnancy immunoreactive (ir-)P450(C17) was present in cords of cells throughout the adrenal gland. Ir-P450(C17) was reduced or was undetectable at Day 100, but had increased by Day 125-130, and was present throughout the zona fasciculata of the adrenal cortex of term animals. An increase in P450(C17) protein was also seen between Day 100 and 125 by Western blotting, and after ACTH treatment. Dexamethasone administration led to a marked reduction in ir-P450(C17) levels. In contrast, ir-3beta-HSD was present in the fetal adrenal cortex between Day 100 and term, and was less affected by ACTH or dexamethasone treatment. We conclude that P450(C17) in the fetal sheep adrenal is responsive to regulation by ACTH, and that changes in its levels correlate with previously reported alterations in patterns of cortisol output by the fetal adrenal gland.

Developmental and Functional Biology of the Primate Fetal Adrenal Cortex

Developmental and Functional Biology of the Primate Fetal Adrenal Cortex

Role of growth factors in the developmental regulation of the human fetal adrenal cortex

Development of the human fetal adrenals is characterized by rapid growth and high levels of steroidogenic activity during the latter two-thirds of pregnancy. By midgestation, the human fetal adrenals are composed of two distinct cortical zones: the predominant fetal zone, which occupies 80–90% of the cortical volume and produces large amounts of the Δ 5-steroid dehydroepiandrosterone sulfate, and the narrow definitive zone, which surrounds the fetal zone. Late in gestation, the peripheral portion of the fetal zone develops into a third, functionally distinct compartment, the transitional zone, which is the likely site of cortisol synthesis. Soon after birth, the adrenal cortex is remodeled and the fetal zone disappears. The adult cortical zones are thought to arise from the definitive zone, which persists postnatally. Development of the human fetal adrenals is regulated primarily by corticortropin (ACTH) secreted from the fetal pituitary. However, as ACTH is not a mitogen per se, its proliferative actions on human fetal adrenal cortical cells are thought to be mediated by autocrine/paracrine growth factors produced by adrenal cortical cells in response to ACTH. In addition, these growth factors appear to modulate the functional response of fetal adrenal cortical cells to ACTH. The roles of several growth factors, including the insulin like growth factors I and II (IGF-I and IGF-II), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), activin, inhibin, and the transforming growth factors α and β (TGF-α and TGF-β) have been examined. In cultured human fetal adrenal cortical cells, EGF, bFGF, and IGF-I and -II are mitogenic, whereas activin and TGF-β inhibit proliferation. IGF-II, activin, and TGF-β also modulate ACTH-stimulated steroidogenesis. Human fetal adrenal cortical cells express IGF-II, bFGF, and the activin/inhibin subunits, and the abundance of mRNAs for each of these factors is up-regulated by ACTH, suggesting that these growth factors are autocrine/paracrine mediators of ACTH action. Thus, although human adrenal development is primarily regulated by ACTH, its actions appear to be mediated/modulated by a cohort of locally expressed growth factors, the net effect of which results in the unique growth and steroidogenic activity of the human fetal adrenal cortex.

Pro-Opiomelanocortin and adrenal function

Pro-opiomelanocortin (POMC) is a polyhormone precursor produced predominantly in the pars distalis and pars intermedia of the pituitary gland where it undergoes tissue specific processing to produce a whole array of peptides. We have shown previously that peptides derived from the N-terminal region of POMC are involved in adrenal growth in rats. Using specific two site immunoradiometric assays we have found that the plasma of 17 week old fetal sheep contain a 50 fold excess of pro-gamma-MSH over ACTH. As term approached, the levels of pro-gamma-MSH fell and ACTH rose with evidence of fragmentation of pro-gamma-MSH, suggesting that these peptides act in concert in the development of the fetal adrenal cortex and also provide the necessary drive to bring about parturition. In an attempt to explore the pathophysiology of adrenal function we have cloned human POMC cDNA which led to the discovery of a 9bp addition/deletion mutation in the C-terminus of gamma 3-MSH between positions 67-73. Chinese hamster ovary cells (CHO) cells stably transfected with constructs containing the variant POMC cDNAs have shown a degree of partial processing. Work is currently underway to further investigate the effects of these mutations on the processing by the prohormone converting enzymes PC1 and PC2.

PROLIFERATIVE ACTIVITY OF ADRENAL GLANDS WITH ADRENOCORTICAL CYTOMEGALY MEASURED BY MIB-1 LABELING INDEX

To investigate the proliferative activity of cytomegalic cells in the fetal adrenal cortex, we studied adrenal glands with cytomegaly by immunohistochemistry using the nuclear proliferation marker MIB-1. The percentage of positively stained nuclei was quantified using the SAMBA 4000 image analysis system. Only one case showed occasional positively stained cytomegalic cell nuclei. The permanent cortices showed proliferative activity that decreased with increasing gestational age. No proliferative activity was seen in normal fetal cortices except in one case that received corticosteroid therapy and had a maternal history of diabetes. The near absence of proliferative activity of the cytomegalic cells supports the previously proposed theory of cellular exhaustion following hyperactivity. The high proliferative activity in the fetal cortex of the infant receiving corticosteroid therapy may provide insight into the stimulus causing the hypermetabolic state.

Expression and Regulation of the Lipoprotein Lipase Gene in Human Adrenal Cortex

Lipoprotein lipase (LPL), an enzyme which hydrolyzes triglycerides and participates in the catabolism of remnant lipoproteins, plays a crucial role in energy and lipid metabolism. The goal of this study was to analyze the expression and regulation of the LPL gene in human adrenals. Reverse transcriptase-polymerase chain reaction amplification and sequence analysis demonstrated the presence of LPL mRNA in fetal and adult human adrenal cortex. Furthermore, the human adrenocortical carcinoma cell line, NCI-H295, expresses LPL mRNA and protein, which is localized to the outer cellular membrane as demonstrated by immunofluorescence confocal microscopy and can be released in the medium by heparin addition. To asses whether the LPL gene is regulated by agents regulating adrenal steroidogenesis, NCI-H295 cells were treated with activators of second messenger systems. Whereas the calcium-ionophore A23187 did not affect LPL gene expression, treatment with phorbol 12-myristate 13-acetate decreased LPL mRNA levels in a time- and dose-dependent manner. This decrease after phorbol 12-myristate 13-acetate was associated with diminished heparin-releasable LPL mass and activity in the culture medium. Addition of the cAMP analog 8-Br-cAMP to NCI-H295 cells resulted in a rapid, but transient dose-dependent induction of LPL mRNA. Treatment with the protein synthesis inhibitor cycloheximide gradually induced, whereas simultaneous addition of cAMP and cycloheximide superinduced LPL mRNA levels. Nuclear run-on analysis indicated that the effects of cAMP and cycloheximide occurred at the transcriptional and post-transcriptional level, respectively. Transient co-transfection assays demonstrated that the first 230 base pairs of the proximal LPL promoter contain a cAMP-responsive element activated by protein kinase A and transcription factors belonging to the CREB/CREM family. These data indicate that LPL is expressed in human adrenal cortex and regulated in NCI-H295 adrenocortical carcinoma cells by activators of the protein kinase A and protein kinase C second messenger pathways in a manner comparable to P450scc, which catalyzes the first step in adrenal steroidogenesis. These observations suggest a role for LPL in adrenal energy and/or lipid metabolism and possibly in steroidogenesis.

Glucocorticoid receptors immunoreactivity in tissue of human embryos.

The exact period when glucocorticoid receptors (GR) appear in human embryos is unknown, however their presence is acknowledged in target tissues before the fetal adrenal cortex secretes cortisol. Determining when GR develop could serve as an index of the importance of glucocorticoids in the morphological and functional development of tissues. The aim of this study was to determine time of onset of GR in human tissues using an immunohistochemical method. Results indicate GR are present in tissues of 8-10-week-old human embryos: in most tissues, the immune reaction was only or predominantly nuclear.

Proliferative Activity of Adrenal Glands with Adrenocortical Cytomegaly Measured by MIB-1 Labeling Index

To investigate the proliferative activity of cytomegalic cells in the fetal adrenal cortex, we studied adrenal glands with cytomegaly by immunohistochemistry using the nuclear proliferation maker MIB-1. The percentage of positively stained nuclei was quantified using the SAMBA 4000 image analysis system. Only one case showed occasional positively stained cytomegalic cell nuclei. The permanent cortices showed proliferative activity that decreased with increasing gestational age. No proliferative activity was seen in normal fetal cortices except in one case that received corticosteroid therapy and had a maternal history of diabetes. The near absence of proliferative activity of the cytomegalic cells supports the previously proposed theory of cellular exhaustion following hyperactivity. The high proliferative activity in the fetal cortex of the infant receiving corticosteroid therapy may provide insight into the stimulus causing the hypermetabolic state.

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

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

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.

Expression of DAX-1, the Gene Responsible for X-Linked Adrenal Hypoplasia Congenita and Hypogonadotropic Hypogonadism, in the Hypothalamic-Pituitary-Adrenal/Gonadal Axis

DAX-1, an orphan member of the nuclear hormone receptor superfamily, is responsible for X-linked adrenal hypoplasia congenita (AHC) and the frequently associated hypogonadotropic hypogonadism (HH). The entire DAX-1 genomic region has been sequenced and a putative steroidogenic factor-1 response element has been identified in the promoter region of the gene. The purpose of these investigations was to determine if DAX-1 was expressed in the central nervous system, particularly the hypothalamus and pituitary, in order to better understand the relationship of mutations in this gene to fill associated with AHC. We used Northern blot analysis and reverse transcription PCR to demonstrate that DAX-1 was expressed in the hypothalamus and the pituitary, and to confirm its expression in adrenal cortex and gonads. The expression of DAX-1 in these tissues indicates the involvement of DAX-1 in the development of the reproductive system at multiple levels within the hypothalamic-pituitary-adrenal/gonadal axis. We also observed the expression of DAX-1 in a human adrenocortical carcinoma cell line, NCI-H295, that has features characteristic of the fetal adrenal cortex. Therefore, NCI-H295 cells will be a useful cellular model for investigating the involvement of DAX-1 in the regulation of steroidogenesis.

The prolactin receptor in the fetal rat: cellular localization of messenger ribonucleic acid, immunoreactive protein, and ligand-binding activity and induction of expression in late gestation.

The cellular distribution and developmental expression of the PRL receptor (PRLR) in the late gestational fetal rat were examined by in situ hybridization, immunohistochemistry, and radioligand binding. Antisense and sense strand RNA probes encoding the long and short isoforms of the rat PRLR were hybridized to tissue sections under stringent conditions. Messenger RNA (mRNA) encoding the two isoforms of the receptor was expressed widely in tissues derived from all three germ layers; these included various tissues not known previously to contain lactogenic receptors, such as the olfactory neuronal epithelium and olfactory bulb, trigeminal and dorsal root ganglia, cochlear duct, brown adipose tissue, submandibular glands, whisker follicles, tooth primordia, and proliferative and maturing chondrocytes of developing bones. Prominent expression of PRLR mRNA was also detected in the fetal adrenal cortex, gastrointestinal and bronchial mucosae, renal tubular epithelia, choroid plexus, thymus, liver, pancreas, and epidermis. Immunohistochemical studies using monoclonal anti-PRLR antibodies demonstrated that the distribution of PRLR immunoreactivity was similar to that of PRLR mRNA, suggesting that the PRLR mRNA is translated to receptor protein in the fetus in vivo. The encoding of functional PRL receptor proteins by fetal PRLR mRNA was revealed by the presence of specific rat placental lactogen II-binding sites in fetal adrenal cortex, renal tubules, small intestinal villi, pancreatic ductules and islets, hepatic parenchymal cells, choroid plexus ependymal cells, and microsomal fractions of fetal lung and thymus. Levels of expression of PRLR mRNA and protein increased between days 17.5 and 20.5 of gestation in a number of fetal tissues, including the adrenal, pancreas, small intestine, pituitary, thymus, liver, and submandibular gland. The widespread expression of the PRLR in the fetal rat and the induction of receptor expression in late gestation suggest novel roles for the lactogenic hormones in fetal and neonatal development.

Transplacental toxicity of 3-methylsulphonyl-DDE in the developing adrenal cortex in mice

The transplacental transfer, irreversible binding, and ultrastructural lesions in the fetal adrenal cortex were studied following single injections of the persistent DDT-metabolite 3-methylsulphonyl-DDE (MeSO 2-DDE) in pregnant C57B1 mice. Tape-section autoradiograms of fetuses on gestation days 12 to 17 revealed a high and tissue-specific accumulation of MeSO 2-DDE- 14C-derived radioactivity in the fetal adrenal gland. On gestation day 12 the adrenal radioactivity could be extracted with organic solvents, whereas on days 13 to 17 the radioactivity in the adrenal was irreversibly bound and could not be extracted from the tissue. As determined by computer-assisted image analysis of autoradiograms, the uptake of radioactivity in the fetal adrenals increased continuously with gestational age. Electron microscopy revealed mitochondrial degeneration and vacuolation in fetal adrenal cortex cells following injection of MeSO 2-DDE (25 mg/kg b.w.) to the pregnant dam. The lesions were clearly visible on days 14 to 15 but most pronounced on days 16 to 17. Administration of the cytochrome P450(11β) inhibitor metyrapone to pregnant dams (gestation day 17) reduced the mitochondrial toxicity induced by MeSO 2-DDE in the fetal adrenal cortex. In conclusion, the adrenocorticolytic DDT metabolite MeSO 2-DDE is transformed to a reactive, cytotoxic metabolite in the fetal adrenal cortex from its earliest stage of development. Hence, the activating cytochrome P450 form, previously proposed to be P450(11β), seems to be expressed during gestation days 12 to 13 in the adrenal cortex in the mouse fetus.

Programmed cell death in remodeling of the human fetal adrenal cortex: possible role of activin-A

Programmed cell death in remodeling of the human fetal adrenal cortex: possible role of activin-A

Expression of gap junction genes, connexin40 and connexin43, during fetal mouse development

The expression patterns of the gap junction genes connexin40 and connexin43 have been analyzed during late mouse fetal development, i.e., at embryonic days 14.5 and 16.5, by in situ hybridization and immunofluorescence. Connexin40 was found in endothelial cells of vessels, cardiomyocytes and in developing myoblasts and myotubes. Expression of connexin40 in developing muscle fibers was strong in the back muscles and weaker in the muscles of the limbs. The number of labeled cells in the back muscle decreased with ongoing differentiation of myoblasts, in accordance with the idea that connexin40 is only expressed in the early stages of muscle cell differentiation. Within a muscle bundle, connexin40 expression was predominantly found at the outermost side where myoblasts fuse to multinucleated myotubes. In contrast, connexin43 exhibits a wide and complex pattern of expression in fetal mouse development. It is found in organs originating from all three germ layers, such as epidermis, heart, lung, muscle, kidney and gut. Connexin43 transcript and protein were very abundant in tissues that had been undergoing inductive interactions, e.g., the inner enamel epithelium of the teeth, the glomeruli of the kidneys and the infundibulum forming the neural part of the pituitary gland. Very high connexin43 expression was found in the embryonic meninges (dura mater) and in the fetal adrenal cortex. During keratinocyte differentiation connexin43 mRNA expression decreased, being much stronger in the stratum basale than in stratum granulosum. No obvious discrepancy between the amount of mRNA and protein of either connexin was noticed, suggesting that there is no specific translational regulation at these developmental stages.