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Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, California 94143-0556
Address all correspondence and requests for reprints to: Robert B. Jaffe, M.D., Center for Reproductive Sciences, 1695 HSW, University of California San Francisco, San Francisco, California 94143-0556. E-mail: jaffer{at}obgyn.ucsf.edu.
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Abstract |
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Identification of cellular markers unique to each zone of the fetal adrenal gland will enhance the ability to characterize the proliferative potential of definitive zone cells and assess their capacity to differentiate into cells of the transitional and fetal zones. Purified cells also will permit detailed molecular and mechanistic studies of regulation of human fetal adrenal development.
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Introduction |
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The human fetal adrenal gland (HFA) has unique morphological characteristics (3, 4). The large inner fetal zone (FZ) (>80% by volume) is the site of synthesis of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), precursors of placental estrogen (3). Its large vacuolated cells lack 3ß-hydroxysteroid dehydrogenase and have high concentrations of P450c17, which has both 17
-hydroxylase and lyase activities. The FZ is surrounded by the outer definitive zone (DZ), which is comprised of a thin rim of tightly packed cells with a proliferative phenotype that persists throughout gestation and does not acquire a mineralocorticoid-synthesizing phenotype until late in gestation (3, 4). A third zone, the transitional zone (TZ), develops between the DZ and FZ after about 22 wk of pregnancy and expresses enzymes necessary for cortisol synthesis (3, 4).
The role of the DZ is largely unknown. We have suggested that its cells may comprise a progenitor population, some of which migrate centripetally to populate the TZ and FZ (3, 4). The HFA undergoes remarkable reorganization soon after birth. The FZ undergoes remodeling and involution, primarily by apoptosis (5), and is replaced by the adult zona fasciculata and zona reticularis. Some apoptotic figures are also evident in the DZ, but to a lesser extent than in the FZ (5). Thus, it is possible that DZ cells persist into neonatal life, providing precursor cells for the adult zones.
Consistent with this thesis, the adult rat has a population of cells between the zonae glomerulosa and the fasiculata/reticularis that is nonsteroidogenic and is thought to play a progenitor role (6). A transgenic rat was generated that was chimeric for p450c21, the gene encoding 21-hydroxylase. The adrenal gland of this rat had cords of cells from the outer rim subjacent to the capsule extending into the cortex that were clonal in their expression of the chimeric gene. All the cells of a given cord either expressed the transgene or did not (7, 8). These and other data provide strong evidence that cells from the outer zones move inward to populate the inner zones of the rat adrenal gland and lend credence to the possibility of a comparable role during fetal life (9, 10).
Although the accumulated data point strongly to there being progenitor cells in the adrenal gland, the evidence is not direct. The strongest indication that a cell population is comprised of progenitor cells is to demonstrate, either in vitro and/or in vivo, that the proposed cell type can develop into the differentiated cells of interest. Clearly such a study requires purified cells. The purification of DZ cells has proven difficult. As with most precursor or stem cells, DZ cells make up only a small portion of the HFA. This precludes most physical separation methods because of contamination by the more numerous FZ cells. Furthermore, cell markers unique to the DZ have not been described previously.
Previous attempts at separation failed to demonstrate conclusively that the appropriate cells had been isolated and characterized. Physical separation of DZ cells, involving gentle separation of the capsule from the FZ, followed by scraping DZ cells free, has been used in the past (11, 12, 13, 14, 15). Those groups that characterized the yield, including ours, indicated that by morphological criteria alone, 5–20% of FZ cells could contaminate the detached cell population (11, 12, 13, 14, 15). A more perplexing problem was the contamination by capsular fibroblasts that rapidly overgrew the cells in culture. Thus, we occasionally isolated adequate DZ cells to initiate experiments; however, by the end of the culture period there often was cell heterogeneity, and we could not be confident that our results reflected the activity of the DZ cells exclusively. An additional difficulty in those experiments was quantification. Once separated in culture, the DZ cells could not be positively identified. Size and morphology have been relied upon in the past, but without unique cellular markers, these methods remain subjective. Thus, we set out to identify cellular markers unique to different cell types, focusing particularly on DZ cells, in the HFA gland.
The advent of laser capture microdissection (LCM) allowed us to isolate DZ cells in a relatively pure fashion for the first time (16) (Fig. 1
). Laser microdissection permits selection of a single cell or a small group of cells from a lightly stained cryosection with reproducibility and precision. We used LCM to isolate approximately 15,000 DZ cells with minimal contamination by capsular fibroblasts or FZ cells. RNA isolated from this cell population was used for subtractive hybridization and compared with RNA from microdissected FZ cells. We then constructed a library of markers expressed primarily on DZ cells. Screening of this set of markers was accomplished using in situ hybridization and RT PCR from other cell RNA preparations isolated by laser microdissection. Because of the similar nature of the two starting populations, we anticipated few true positive markers. Indeed, screening of over 800 clones revealed approximately 30 useful sequences. The sequences were searched for similarity using BLAST and the GenBank database. Many sequences were unknowns, established sequence tags of unknown or ribosomal RNA. We focused on known sequences for our initial studies.
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Materials and Methods |
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Ten HFA glands between 8–23 wk gestation were removed after voluntary pregnancy terminations. There were no known fetal abnormalities. The study was approved by the Committee on Human Research, University of California (San Francisco, CA).
LCM
LCM for subtractive hybridization was preformed using a 17-wk-old fetal adrenal, snap-frozen in liquid nitrogen after being placed in OCT (Tissue-Tek, Torrance, CA). Slides either were sectioned that day or used immediately after removal from the freezer. Slides were fixed in ethanol, lightly stained with hematoxylin and eosin, and dehydrated stepwise with increasing ethanol concentrations, followed by xylene, before air-drying in the hood.
Cells were captured using a Pixcell II (Arcturus, Mountain View, CA) laser capture system in groups of approximately 15 cells. A representative photomicrograph illustrating the zones of the HFA before and after LCM of the DZ is presented in Fig. 1
. Particular care was taken to avoid capture of cells from the capsule, TZ or FZ. Often this meant selecting only a thin row of cells in the DZ, leaving a rim of DZ cells on each side to ensure purity. After capture, the cells were immediately dissolved using a standard guanidinium hydrochloride buffer for the preparation of RNA. The cell solution was stored at -80 C until final preparation of RNA.
Preparation of RNA
In initial studies a standard guanidinium hydrochloride buffer to dissolve the cells was used. RNA was extracted and precipitated with ethanol. This was followed by RT and subtractive hybridization.
Subtractive hybridization
Subtractive hybridization was carried out using a PCR-Select cDNA Subtraction Kit (BD CLONTECH Laboratories, Inc., Palo Alto, CA) in which the tester cDNA used was prepared from the laser-microdissected DZ cells, and the driver cDNA was prepared from the FZ cells. In addition, to use the PCR-Select Differential Screening Kit (BD CLONTECH Laboratories, Inc.), a reverse subtraction was performed in which the DZ cDNA was the driver, and the FZ cDNA was the tester. After PCR amplification to enrich for differentially expressed sequences and to suppress background, the cDNAs were inserted into a T/A cloning vector using an AdvanTAge PCR Cloning kit (BD CLONTECH Laboratories, Inc.) and plated. The colonies were grown slightly in 96-well plates and PCR-amplified. The PCR products were spotted in duplicate on Nytran nylon membranes (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH) and differentially screened with the forward-subtracted cDNA probe as well as with the reverse-subtracted cDNA probe. Only colonies hybridizing to the forward-subtracted, and not the reverse-subtracted, probes were prepared for sequencing.
In situ hybridization
In situ hybridization was performed with digoxigenin-labeled probes (Roche Molecular Biochemicals, Indianapolis, IN), using the protocol described by the manufacturer, after subcloning sequences of interest into pBluescript II KS (Stratagene, La Jolla, CA). RNA products of T7 and T3 RNA polymerases (Promega Corp., Madison, WI) were quantified by gel using an RNA standard determined by DIG Quantification Test Strips (Roche Molecular Biochemicals, Indianapolis, IN).
PCR
PCR was carried out using Ready-To-Go Beads for 35 cycles as described by the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ) using a GeneAmp PCR System 9600 (Perkin-Elmer Corp., Norwalk, CT).
Immunofluorescence
HFAs were placed in ice-cold 4% paraformaldehyde in PBS. After immersion for 16 h in paraformaldehyde, the tissue was equilibrated in sucrose/PBS solutions using a gradient from 5–15%, embedded, and frozen in dry ice/ethanol nitrogen. Eight-micron sections were cut and stored at -80 C until use. After brief rehydration in PBS, slides were blocked in BSA and goat serum. Primary antibodies were applied in varying concentrations in PBS/goat serum. Secondary antibody was added at 1:200 dilution after thorough washing in PBS. Antibody incubations were performed at room temperature for 1–2 h each. Images were obtained with a Kodak DCS 420 Digital Camera (Eastman Kodak Co., Rochester, NY) using a DMRB fluorescent microscope (Leica Corp., Wetzlar, Germany). The primary antibodies used were mouse antihuman P-glycoprotein (Immunotech, Marseilles, France) and rabbit antihuman low density lipoprotein (LDL) receptor (Research Diagnostics, Inc., Flanders, NJ). The secondary antibody was Cy3-conjugated goat antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
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Results |
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Nephroblastoma-overexpressing gene (nov).
We found novH, a growth regulatory protein, in the HFA using LCM by subtraction hybridization and in situ hybridization predominantly in the DZ (Fig. 2) at all gestational ages studied, with low expression in some FZ samples. Cells were isolated again from the DZ and FZ by LCM, RNA was prepared, and RT-PCR performed for 35 cycles. This also confirmed that expression was confined primarily to the DZ (Fig. 3).
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). There was little change in expression noted between the 17- and 19-wk tissue examined. The differential expression of MPS-1 between the DZ and FZ was then confirmed by RT-PCR on laser-microdissected tissue (Fig. 4).
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We also performed immunofluorescent histochemistry screens to define the localization of markers of either the DZ or FZ. A cell surface marker of particular interest was P-glycoprotein in the DZ and the low density lipoprotein receptor in the FZ.
P-Glycoprotein.
P-Glycoprotein, the product of the multidrug resistance 1 gene, is expressed ubiquitously on cortical cells of the adult human adrenal gland. We examined adrenal glands from 10–22 wk gestational age to determine the zonal and ontogenic expression of P-glycoprotein. Examination of 10, 13, 17, 18, and 19 wk gestational age adrenals failed to show any expression of P-glycoprotein. However, a 22-wk gestational age HFA revealed bright staining exclusively in the DZ (Fig 5). Thus, the HFA DZ cells begin to express P-glycoprotein only after 20 wk. Subtraction hybridization was performed on 17-wk-old fetal adrenal glands, earlier than the stage at which P-glycoprotein is first expressed and when the enzymes for aldosterone synthesis are first observed.
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LDL receptor.
Search for a cell surface marker for FZ cells led to the LDL receptor, as circulating LDL cholesterol is a major precursor for adrenal steroid synthesis. As the cells of the FZ are steroidogenic beginning in early gestation, whereas those of the DZ are not, we predicted that the LDL receptor might exhibit zonal expression in the HFA. When immunofluorescence was performed, there was far greater expression of the LDL receptor on cells of the FZ than the DZ, although rare LDL receptor-positive cells were noted on the DZ (Fig. 6). This pattern is similar to that seen for P450c17 (4).
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Discussion |
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Our initial approach involved subtractive hybridization using DZ and FZ cells obtained by laser dissection from a 17-wk gestational age HFA gland. This is the first demonstration of the utility of laser microdissection to explore adrenal zonation. Two previously described growth factors were expressed almost exclusively in the DZ. Both of these growth regulatory proteins, novH and MPS-1, have been described and studied regarding their roles in tumorigenesis. It is not unusual to find gene products that are expressed in adult tumors at high levels in fetal tissue. In many cases these proteins have restricted or absent expression in normal adult tissue.
Nov (nephroblastoma overexpressed gene) is a recently described member of the CCN (cyr61, ctgf, nov) family of growth regulatory proteins (18, 19, 20, 21, 22). It was first described as a site of myeloblastoma-associated virus integration in a chicken nephroblastoma model of Wilm’s tumor (23). It was the third member of a family of structurally related proteins, each with four modules sharing identity with IGF-binding proteins, von Willebrand factor, thrombospondin, and an unusual cystein knot (20, 22). The proteins in this family have described roles in differentiation, proliferation, migration, and tumorigenesis (24). We found novH in the HFA at all ages studied. Using LCM and subtraction hybridization, we observed novH expression almost entirely in the DZ and minimally, if at all, in the FZ.
NovH expression has been studied previously in fetal and adult tissues (18, 19, 24, 25, 26, 27). An extensive analysis of adult tissue expression revealed the highest mRNA levels in aorta, followed by lung and brain (18). The adrenal gland was not included in that study. In a limited exploration of human fetal tissue expression, the highest levels of novH mRNA were seen in the adrenal gland (25). NovH was limited to the DZ in that study of 8 and 10 wk gestation tissue. When fetal adrenal tissues were examined by another group, immunohistochemical staining of both FZ and DZ cells of 12 and 20 wk gestation adrenal glands was seen (19). However, the staining was markedly less in the FZ cells. Our in situ hybridization and PCR data are in agreement with the results at 8 and 10 wk gestation; however, at 12 and 20 wk gestation we do not find nov mRNA in the FZ. It is possible that the protein is made in DZ cells, secreted, and taken up by FZ cells, as novH has been noted to be secreted in some instances. It might also reflect a difference in the antibodies used in the two studies, as we have observed high background staining of HFA tissue with rabbit polyclonal antibodies.
The role of novH in adrenal development is unclear. Several lines of evidence support the concept that novH inhibits growth while promoting differentiation. In both Wilm’s tumors and adrenal cortical carcinomas, the level of nov appears to decrease as the tumors take on a more dedifferentiated, aggressive phenotype (19, 26). NovH expression is negatively regulated by WT-1 (30). Interestingly, mice lacking WT-1 fail to develop adrenal glands (29, 30).
Less is known about MPS-1, which was noted to be cross-linked to ribosomal complexes. This growth regulator has been described in many primary neoplasms and cancer cell lines (31, 32, 33). Normal adult cells express little or no MPS-1. It is a zinc finger phosphoprotein with DNA-binding properties. The subsequent demonstration of localization to the nucleus established the protein as a regulator of gene expression. The expression of MPS-1 in developing tissues has not been examined to date. MPS-1 is similar to prokaryotic ribosomal proteins and may be an integral part of the increased translational apparatus necessary for rapidly growing and differentiating tissue. It is unclear whether MPS-1 is fulfilling this general role or a more specific one.
In contrast, P-glycoprotein is probably a marker of differentiation of DZ cells rather than proliferation. P-Glycoprotein is expressed abundantly in the adult and fetal adrenal gland in many species, including human (34, 35, 36). It is a membrane-bound, efflux pump protein and plays a role in drug resistance of cancer cells (37). It is found largely on the cell surface (38, 39) Its normal physiological role currently is under investigation. Several reports indicate that it is involved in active efflux of steroids, particularly aldosterone and cortisol, from steroidogenic cells (40, 41, 42). When transfected into renal epithelial cells, the P-glycoprotein cDNA confers the ability to transport aldosterone from the basolateral to the apical side of the cells. The adrenal carcinoma cell line NCI-H295, when treated with inhibitors of P-glycoprotein, demonstrated considerably reduced efflux of fluorescently labeled aldosterone (40). The expression of steroidogenic enzymes necessary for aldosterone synthesis begins after approximately 24 wk gestation in DZ cells (3, 4). This is consistent with our data indicating that the expression of P-glycoprotein in the HFA occurred after 20 wk. As our subtraction hybridization studies were performed in 17 wk gestational age HFA tissue, this probably explains its lack of detection in the HFA at 17 wk. We believe that P-glycoprotein will be a useful cell surface marker to distinguish between those cells that have left the progenitor pool and differentiated into steroid-producing cells and the truly undifferentiated progenitor cells. It may play a role in aldosterone transport in the DZ late in gestation when DZ cells begin to produce aldosterone. Furthermore, as the TZ acquires enzymes necessary for cortisol production after about 22 wk, it also may function as a cortisol transporter in the TZ.
The FZ is a highly specialized tissue that is the major site of DHEA and DHEAS synthesis. This zone has no exact adult counterpart, although its location and extensive production of DHEA and DHEAS are similar to those of the zona reticularis. The developmental signals leading to such a specialized cell merit further investigation. Thus, although our primary focus is on DZ cell markers, both from the perspective of cell sorting and investigation, it will be useful to have a cell surface marker unique to FZ cells. The LDL receptor will be one such marker. Staining for the LDL receptor was specific for the cell membranes of FZ cells, except for rare staining of cells in the DZ. The reason that the LDL receptor was not found in by subtraction hybridization is unclear. DZ cells would not be expected to express the LDL receptor early in gestation given their lack of steroidogenic activity until close to term (4). However, the ages examined are those before the evolution to a steroid-producing phenotype in the DZ leading to aldosterone synthesis in some DZ cells. It is possible that the cells noted with positive staining are those that have already begun to differentiate either into an aldosterone-secreting phenotype characteristic of the late gestation DZ or the cortisol-synthesizing phenotype of the TZ, located between the DZ and FZ and possessing enzymes necessary for cortisol synthesis (3, 4). Regardless, this marker will be useful for eliminating differentiated cells in our future attempts to isolate homogenous fetal adrenal progenitor cells of the DZ.
In summary, these studies provide a strong foundation on which to build purification strategies for the progenitor cells of the DZ in the HFA. The description of novel new markers and growth factors associated with specific zones of the HFA should aid in the further definition of the role of these cells and their products in the developmental processes extant in the fetal adrenal gland.
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Footnotes |
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J.R. is a recipient of a fellowship from the Endocrine Fellows Foundation.
Abbreviations: DHEA, Dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; DZ, definitive zone; FZ, fetal zone; HFA, human fetal adrenal gland; LCM, laser capture microdissection; LDL, low density lipoprotein; MPS-1, metallopanstimulin-1; nov, nephroblastoma overexpressing; TZ, transitional zone.
Received January 2, 2003.
Accepted March 25, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/17,20-lyase, and 3ß-hydroxysteroid dehydrogenase A isomerase steroidogenic enzymes in human and rhesus monkey fetal adrenal glands: reappraisal of functional zonation. J Clin Endocrinol Metab 77:1184–1189[Abstract]
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