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BOR - Papers in Press, published online ahead of print February 16, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037499
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BIOLOGY OF REPRODUCTION 72, 1405–1415 (2005)
DOI: 10.1095/biolreprod.104.037499
© 2005 by the Society for the Study of Reproduction, Inc.

Gene Expression in Rat Leydig Cells During Development from the Progenitor to Adult Stage: A Cluster Analysis1

Ren-Shan Ge 3, Qiang Dong 3, Chantal M. Sottas 3, Haolin Chen 5, Barry R. Zirkin 5, and Matthew P. Hardy 2 ,3,4 

The Population Council,3 Rockefeller University,4 New York, New York 10021 Division of Reproductive Biology,5 Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The postnatal development of Leydig cells can be divided into three distinct stages: initially they exist as fibroblast-like progenitor Leydig cells (PLCs) appearing in the testis by Days 14–21; subsequently, by Day 35, they become immature Leydig cells (ILCs) acquiring steroidogenic organelle structure and enzyme activities but metabolizing most of the testosterone they produce; finally, as adult Leydig cells (ALCs) by Day 90, they actively produce testosterone. The factors controlling proliferation and differentiation of Leydig cells remain largely unknown, and the aim of the present study was to identify changes in gene expression during development through cDNA array analysis of PLCs, ILCs, and ALCs. By cluster analysis, it was determined that the transitions from PLC to ILC to ALC were associated with downregulation of mRNAs corresponding to 107 genes. The downregulated genes included cell-cycle regulators, e.g., cyclin D1 (Ccnd1); growth factors, e.g., basic fibroblast growth factor (Fgf2); growth-factor-related receptors, e.g., platelet-derived growth factor {alpha} receptor (Pdgfra); oncogenes, e.g., kit oncogene (Kit); and transcription factors, e.g., early growth response 1 (Egr1). Conversely, expression levels of 264 genes were increased by at least twofold. Most of these were related to differentiated function and included steroidogenic enzymes, e.g., 11ß-hydroxysteroid dehydrogenase 2 (Hsd11b2); neurotransmitter receptors, e.g., acetylcholine receptor nicotinic {alpha} 4 (Chrna4); stress response factors, e.g., glutathione transferase 8 (Gsta4); and protein turnover enzymes, e.g., tissue inhibitor of metalloproteinase 2 (Timp2). The detection of Hsd11b2 mRNA in the array was the first indication that this gene is expressed in Leydig cells, and parallel increases in Hsd11b2 mRNA and enzyme activity were recorded. Thus, gene profiling demonstrates that postnatal development is associated with changes in the expression levels of several different clusters of genes consistent with the processes of Leydig cell growth and differentiation.

developmental biology, gene regulation, Leydig cells, luteinizing hormone, steroid hormone receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The levels of testosterone in circulation are a function of both the steroidogenic capacity of individual Leydig cells and their total number per testis. Leydig cell numbers are determined during postnatal development, and their proliferative activity is limited to the prepubertal period [1]. The Leydig cell lineage can be conceptually divided into three stages based on morphological and biochemical criteria. Although the developmental sequence of Leydig cells has been defined only in the rat [1], a similar progression is postulated for other species, including humans [2]. In the rat, progenitor Leydig cells (PLCs), the first recognizable stage in the lineage, are highly proliferative and yet express markers of differentiated function, such as P450 side-chain cleavage enzyme (Cyp11a1), 3ß-hydroxysteroid dehydrogenase (Hsd3b) [3], 17{alpha}-hydroxylase (Cyp17a1), and a truncated form of the luteinizing hormone receptor (Lhcgr) [4, 5]. PLCs contain negligible amounts of smooth endoplasmic reticulum (SER), the membranous organelle that houses several steroidogenic enzymes. Despite the relative absence of SER membranes, these cells are competent to produce steroid, secreting mainly androsterone [6]. PLCs gradually enlarge, become round, and reduce their proliferative capacity. This second transition results in another intermediate, immature Leydig cells (ILCs), most commonly seen in the testis during Days 28–56 postpartum. ILCs have more SER compared with PLCs and, in addition, contain cytoplasmic lipid droplets which support a high level of steroidogenic capacity, primarily 3{alpha}, 5{alpha}-androstanediol [6]. ILCs undergo a final division before the transition to adult Leydig cells (ALCs), which occurs by Day 56 [7]. ALCs are large, with an abundance of smooth endoplasmic reticulum, few lipid droplets, high levels of steroidogenic enzyme activity, and testosterone is the predominant androgen secreted. ALCs comprise the predominant population of Leydig cells in the sexually mature testis.

Leydig cells are the primary target for LH action in the testis [8]. LH is the master control factor for completing and maintaining fully differentiated structure and testosterone biosynthetic function in Leydig cells [9]. LH also plays a critical role in the development of Leydig cells. For example, in Gnrh2hpg mice, which are deficient in circulating LH, Leydig cell numbers are about 10% of control [10]. Moreover, Leydig cells are severely hypoplastic in Lhcgr knockout (LHRKO) mice [11, 12]. However, Leydig cells retain the ability to develop partially and produce testosterone in Gnrh2hpg [10] and LHRKO mice [11, 12], indicating that this hormone is unlikely to be the initial stimulus for Leydig cells to differentiate. Several types of factors, including hormones, locally produced growth factors, such as insulin-like factor I (IGF-I) [13] and platelet-derived growth factor (PDGF) [14], nuclear transcription factors [15], and enzymatic modulators [16] are also thought to be involved in the regulation of mitosis and differentiation in Leydig cells.

To understand which of the above factors are important for Leydig cell proliferation and differentiation, it will be essential to identify a broader spectrum of genes that are changed during postnatal development. Several research groups have approached this issue using serial analysis of gene expression serial analysis of gene expression (SAGE) and real-time polymerase chain reaction (PCR) in whole testis during development [17, 18]. However, because numerous cell types exist in the testis, it is difficult to identify changes that are specific to Leydig cells using these methodologies. Other groups have reported developmental changes in the expression levels of several genes by PCR, real-time PCR, and Northern blotting [6, 17, 19, 20]. In these instances, however, a broader pattern of gene expression in Leydig cells has not been analyzed. To achieve this goal, we purified PLCs, ILCs, and ALCs and used DNA arrays to identify genes having markedly different expression levels over the course of development. The developmental trends of these genes were followed through the transitions of PLCs into ILCs and ILCs into ALCs using clustering analysis. The glucocorticoid-metabolizing enzyme Hsd11b2 was seen to be present as an expressed gene in Leydig cells, with implications for direct mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mediated actions on the testosterone biosynthetic pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

DMEM medium DMEM-Ham F-12, D-2906 was purchased from Sigma Chemical Co., St. Louis, MO. Rat altas I kits were purchased from Clontech, Palo Alto, CA. A kit was used for total RNA extraction (Trireagent, Molecular Research Center Inc., Cincinnati, OH). Avian myeloblastosis virus reverse transcriptase was purchased from Promega (Madison, WI). The [{alpha}-32P] dATP was from Amersham Biosciences Corp., Piscataway, NJ; 10 µCi/µl. The Rat Atlas Nylon Array I, which contains 1176 selected, well-characterized cDNAs, was purchased from Clontech. This type of array was chosen because it is based on Northern blotting chemistry and proved useful for studies of aging and caloric restriction in Leydig cells [21], and adult Leydig cell [22] and epididymal functions [23] obtained from the nylon membrane array studies can be readily compared with other measurements of Leydig cell mRNA [19–23]. [1,2,-3H]Corticosterone, specific activity 40 Ci/mmol, was purchased from Dupont-New England Nuclear (Boston, MA). Cold corticosterone was purchased from Steraloids (Wilton, NH).

Animals

Sprague-Dawley rat dams with litters of male pups, immature males, and adult males were purchased from Charles River Laboratories, Wilmington, MA. Male rats were 21, 35, and 90 days of age on the day of Leydig cell isolation. The animals were killed by asphyxiation with CO2. The animal protocol was approved by the Institutional Animal Care and Use Committee of the Rockefeller University (#01-041).

Cell Isolation

A complete description of the cell-isolation procedure has been published [24, 25]. In brief, testes from forty 21-day-old rats were removed for isolation of PLCs. Decapsulated testes were dispersed with 0.25 mg/ ml collagenase collagenase-D (Boehringer Mannheim Biochemicals, Indianapolis, IN) in medium 199 for 10 min at 34°C with shaking. The separated cells were filtered through two layers of nylon mesh, centrifuged at 250 x g, and resuspended in 55% isotonic Percoll. Following density gradient centrifugation at 25 000 x g for 45 min at 4°C, the PLC fraction was collected between densities of 1.064 and 1.070 g/ml. The cells were washed with HBSS, centrifuged at 250 x g, and resuspended in phenol red-free 1:1 DMEM:F12 supplemented with 1 mg/ml bovine albumin. ILCs were isolated from the testes of twenty 35-day-old rats, with the following modifications to the above procedure. Testes were perfused with 1 mg/ml collagenase in medium 199 via the testicular artery before decapsulation. The ILC fraction was collected from the Percoll gradient between densities of 1.070 and 1.088 g/ml. ALCs were purified from the testes of six 90-day-old rats according to the method of Klinefelter et al. [25]. Before the Percoll density gradient centrifugation, collagenase dispersed interstitial cells were elutriated in a Beckman JE-6B elutriation chamber (Palo Alto, CA) at a flow rate of 16 ml/min at 2000 rpm, after which ALCs were collected from the Percoll gradient between densities of 1.070 and 1.090 g/ml. Purities of Leydig cell fractions were evaluated by histochemical staining for 3ß-hydroxysteroid dehydrogenase (HSD) activity, with 0.4 mm etiocholanolone as the steroid substrate [26]. Leydig cells were typically enriched more than 95% and were stained intensely.

Preparation of RNA

Typically, Leydig cells from several animals in each age group (40 animals for PLCs, 20 for ILCs, and 6 for ALCs) were pooled to isolate RNA. Four pools of each group of Leydig cells were used for the analysis. RNA samples were processed according to the Atlas gene protocol (Clontech). Total RNA was extracted from isolated Leydig cells by a single-step method, using phenol and guanidinium thiocyanate. The purity of isolated mRNAs was evaluated spectrophotometrically, using the A260: A280 ratio. To reduce contamination by genomic DNAs, total RNAs were treated with ribonuclease-free deoxyribonuclease I for 1 h at 37°C as recommended by the manufacturer (Clontech), followed by phenol:chloroform purification. Samples of total RNAs (400 ng) were reverse transcribed with avian myeloblastosis virus reverse transcriptase in the presence of random hexamer plus dNTPs at 42°C for 75 min, and the reaction was terminated by heating at 95°C for 5 min.

Probe Preparation and Hybridization

The three cRNA samples (PLCs, ILCs, and ALCs) were used for hybridization to oligonucleotide arrays corresponding to approximately 1185 known genes, including 9 housekeeping control genes. To generate reproducible gene expression data, four independent replicates of PLCs, ILCs, and ALCs were performed. To generate radiolabeled cDNA probes, total RNAs were transcribed with Moloney murine leukemia virus reverse transcriptase and radiolabeled with [{alpha}-32P] dATP. The radiolabeled cDNA probes were purified from unincorporated nucleotides by gel filtration in Chroma Spin-200 columns (Clontech) and hybridized overnight at 68°C to a rat microarray I, as described by the manufacturer (Clontech).

Phosphor Imaging

After three stringent 20-min washes in 2x saline-sodium citrate (SSC): 1% SDS, followed by two 20-min washes in 0.1x SSC:0.5% SDS at 68°C, the membranes were sealed in plastic (Kapak Corp., Minneapolis, MN) and exposed to phosphorimager plates for 3- to 48-h-dependent hybridization signal. Images of the hybridized filters were obtained after scanning of the plates (Storm, Molecular Dynamics, Inc., Sunnyvale, CA).

Image Analysis

The image intensity of each cDNA was imported into AtlasImage software (Version 2.01; Clontech). The intensity of each spot, reflecting the relative abundance of mRNA in the sample, was analyzed in 12 different membranes. Individual gene intensities were normalized to internal control housekeeping genes, ß-actin and glyceraldehyde-3-phosphate dehydrogenase (Gapd). All data collected were exported into ASCII files and then imported into a relational database Microsoft Access 2000, which was designated as a Leydig cell array database. In the Leydig cell array database, bioinformatic information for each gene was available via links from the accession numbers, locus link, and Swissprot accession.

Reverse Transcriptase-PCR Analysis

To corroborate the array data, reverse transcriptase (RT)-PCR assays were performed as described previously [27]. The conditions for PCR were 94°C, 30 sec for one cycle, followed by 94°C, 30 sec; 65°C, 30 sec; and 72°C, 12 min for 34 cycles. The primers were designed to span at least two exons to eliminate the possible contamination by genomic DNA (Table 1). PCR products were then subjected to agarose gel electrophoresis. Gel pictures were taken and analyzed by Kodak Digital software. The optical densities of bands fell within a linear gray scale on the response curve for PCR cycle. The target genes were normalized to an internal control, ribosomal protein S16.


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  		TABLE 1. Primer sequences

Cluster Analysis

In the rat Atlas array, genes are arranged into 24 groups, from transporters to xenobiotic metabolizing enzymes, according to functional group. K-means clustering was performed using GeneSpring software (Silicon Genetics, CA) on the data set imported from the Leydig cell array database. In brief, the K-means clustering algorithm initially divides genes into a number of equal-sized groups based on a user-defined number K. Centroids were first created representing the average location of each of these groups in expression space. Each gene was reassigned to the centroids that best matched its expression pattern over time. The centroids were then recalculated and the process was repeated until all were transferred into the multiple clusters representing sets of genes that shared a distinct pattern of expression with respect to time. According to the patterns of the clusters, we then assigned the similar trends of clusters to three different groups.

11ß-HSD Enzyme Activity Assay

Leydig cell homogenates were prepared as described previously [28]. The 11ß-HSD (11ßHSD) activity assay tubes contained 25 nM radiolabeled substrate, and the reactions were initiated by the addition of protein with and without NAD+ at a final concentration of 0.5 mM. The reactions were stopped by adding 2 ml ice-cold ethyl acetate. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin-layer plates in chloroform and methanol (90:10), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc., Washington, DC). The percentage conversion of corticosterone to 11-dehydrocorticosterone was calculated by dividing the radioactive counts identified as 11-dehydrocorticosterone by the total counts associated with corticosterone plus 11-dehydrocorticosterone.

Statistics

Hybridizations were carried out four times for each group using a hybridization-stripping-hybridization procedure. The hybridization membranes were arranged randomly for different samples. Data from each repetition of each group were exported into the relational database and mean and SD values were obtained for each data point. In the database, removing spots that fell at or below the mean normalized background intensity plus two SDs of the background filtered the data. The remaining datasets were exported into the SAS program (SAS Institute, Cary, NC) and a two-way ANOVA was used to evaluate statistical differences (P < 0.05) among genes in preparations of PLCs, ILCs, and ALCs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Developmental Changes in Leydig Cell Gene Expression

The purities of Leydig cell preparations PLCs, ILCs, and ALCs were established by the fact that the following genes encoding markers for other testicular cell types were undetectable: CD8 (Cd8) expressed by blood cells and lymphocytes, low-affinity nerve growth factor receptor (Ngfr) in sperm [29, 30], follicle stimulating hormone receptor (Fshr) in Sertoli cells [31], and erythropoietin (Epo) in myoid cells [32].

PLCs, ILCs, and ALCs were sampled in quadruplicate for DNA array analysis. Using the database constructed, the GenBank sequence archive was searched using the accession numbers of the relevant genes on the array. On the array, 1176 genes were present, representing 24 functional groups, including apoptosis and cell cycle proteins, extracellular regulators, heat shock, intracellular signaling proteins, metabolizing enzymes, oncogenes, transporters, and transcription factors. Of the 1176 genes, 513 were identified in PLCs, 423 in ILCs, and 581 in ALCs based on detection signals that were twofold above background. The relative intensity was normalized to ß-actin (Actb). When gene intensities of PLCs were compared with ALCs, expression of mRNAs corresponding to 107 known genes was downregulated by at least twofold. Of these genes, the 30 that were most abundant are listed in Table 2. Among them, RCL (Rcl), a c-myc target gene, was the most abundant in PLCs, and was downregulated by fourfold in ALCs. In contrast, expression levels of mRNAs corresponding to 202 genes were increased by at least twofold from PLC to ALC. Of these genes, the 30 most abundant genes are listed in Table 3. For example, androgen biosynthetic enzymes were highly expressed in ALCs with Cyp17a1 (Accession #M21208) ranked number 1, HSD3b1 (M38178) number 4, and Cyp11a1 (J05156) number 17. Messenger RNA levels for all of these enzymes were increased by four- to eightfold, which was consistent with the increase of Leydig cell steroidogenic capacity during conversion of PLCs into ALCs. The developmental patterns of gene expression are grouped in Figure 1.


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  		TABLE 2. Downregulated genes in the PLC to ALC transitions


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  		TABLE 3. Upregulated genes in the PLC to ALC transitions



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  		FIG. 1. Gene level trends and their categories during Leydig cell development. PLC, Progenitor Leydig cell; ILC, immature Leydig cell; ALC, adult Leydig cell

Global Changes in Gene Expression During Leydig Cell Differentiation

Genes were grouped according to their expression patterns by K-means clustering [33]. Optimal results were obtained for K = 9 (Fig. 2). Several clusters were enriched for genes in similar functions (Table 4). Based on these functions and their category location, three larger groups of clusters were identified in Figure 1.



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  		FIG. 2. Cluster analysis of gene expression in Leydig cells during postnatal development. Abbreviations are as in Figure 1. According to the gene pattern, clusters are divided into three groups, A–C


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  		TABLE 4. Gene cluster trends during Leydig cell development

Group A reflected genes downregulated during development from PLC to ALC (Fig. 1A). This group contained three clusters: in cluster 1, genes significantly declined from PLC to ILC and further from ILC to ALC; in cluster 5, gene trends declined significantly from PLC to ILC and maintained a low level from ILC to ALC; and in cluster 8, genes decreased to reach a significantly lower level in ALC. In group A, the genes changed are mostly involved in cell cycle regulation. The downregulated genes were predominantly cell cycle regulators, including Ccnb1(X64589), cyclin C (Ccnc, D14013), cyclin D2 (Ccnd2, D16308), galactosyltransferase-associated protein kinase (Zfp58, L24388), cyclin-dependent kinase 2 (Cdk2, D28753), cell-division-cycle 25 homolog C (Cdc25c, D16237) and cell-division-cycle 20 homolog (Cdc20), transcription factors, e.g., Rcl (U82591), Egr1 (M18416), DNA-binding protein inhibitor ID1 (Ibd1, D10862); proliferation-related oncogenes, e.g., c-fos (Fos, X06769), c-ets-1 proto-oncogene protein (Zfp54, L20681), N-myc (Nmyc1, X63281), c-raf (Faf1, M15427), trk (Ntrk1, M85214), jun-B (Junb, X54686), rac-ß serine/threonine kinase (Atk2, D30041), Kit (D12524); growth factors, e.g., neurophilin (Rgs19ip1, L27867), platelet-derived growth factor B-chain (Pdgfb, Z14117); and growth factor receptors, e.g., transforming growth factor-beta II receptor (Tgfbr2, L09653), basic fibroblast growth factor receptor 1 (Fgfr2, D12498), mannose-6-phosphate/insulin-like growth factor II receptor (Igf2r, U59809), gp130 (Il6st, M92340), c-ErbA (Thra, X12744), and Pdgfra (M63837) (Fig. 3). Other genes, such as annexin A1 (Anxa1, M19967) and heat shock 70-kDa protein (Hspa1b, Z27118), that are closely associated with cell proliferation in other cell types were dramatically reduced. This indicated that expression of proliferation-related genes was shut down, allowing Leydig cells to switch into the differentiation pathway.



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  		FIG. 3. Downregulation of gene expression in Leydig cells during postnatal development. Abbreviations are as in Figure 1. The mRNA levels are expressed relative to PLC. Gene symbols designated from mouse genome informatics (http://www.informatics.jax.org)

Group B reflected the elevation of gene levels during initial differentiation from PLC to ILC (Fig. 2B). This group contained three clusters: in cluster 4, mRNA levels increased significantly during the transition from PLC to ILC and increased further from ILC to ALC; in cluster 7, the trends were to increase from PLC to ILC and then decline; and in cluster 9, levels increased from PLC to ILC and then remained higher in ALCs. Members of this group were associated with differentiation from PLC into ILC. These upregulated genes encoded steroidogenic enzymes Cyp11a1, Hsd3b, 20{alpha}-hydroxysteroid dehydrogenase (Hsd20a, L32601), and 11ß-hydroxysteroid dehydrogenase (Hsd11b1, J05107). Levels of other genes, such as Timp2 (L31884), microsomal glutathione S-transferase (Gst12, J03752), cathepsin H (Ctsh, M36320), were also significantly increased.

Group C reflected genes that may be involved in later stages of Leydig cell differentiation (Fig. 1C). This group had the following three clusters: in cluster 2, gene trends were unchanged from PLC to ILC and then increased significantly from ILC to ALC; in cluster 3, the trend was to increase from PLC to ILC and then increase further during the transition to ALC; and in cluster 6, to decrease from PLC to ILC, followed by an increase from ILC to ALC. These upregulated genes were involved in developmental functions, including extracellular signaling, e.g., gonadotropin-releasing hormone (Gnrh, M15527), gastric inhibitory polypeptide (Gip, L08831); ion channels, e.g., sodium/calcium exchanger NCX2 (Slc8a2, U08141), brain sodium channel beta 2 (Scn2b, U37026), sodium-glucose cotransporter 1 (Slc2a1, U03120); transporters, e.g., Na+, K+-ATPase ß (Atp1b, J02701), ATP synthase subunit c (Atp5c, D13123); steroid metabolic pathways, e.g., cytochrome P450 IIA3 (Cyp2a3, J02852), cytochrome P450 IIJ3 (Cyp2j3, U39943), cytochrome P450 IIIA1 (Cyp3a1, M10161), cytochrome P450 IVF1 (Cyp4f1, M94548), cytochrome P450 IVF4 (Cyp4f4, U39206), and Hsd11b2 (U22424); general metabolic pathways sterol carrier protein-2 (Scp2, M34728) and receptors, e.g., Lhcgr (M26199), insulin-like growth factor I receptor (Igf1r, L29232), glutamate receptor subunit 3 (Grik3, M85036), vasopressin V2 receptor (Avpr2, Z11932), mineralocorticoid receptor (Nr3c2, M36074), TR4 orphan receptor (Nr2c2, L27513), growth hormone receptor (Ghr, J04811), and insulin receptor (Insr, M29014).

Verification by RT-PCR

To verify the results of the DNA array analysis using an independent method, semiquantitantive RT-PCR was performed on randomly selected cDNAs representative of all gene categories. In Figure 4, the RT-PCR analysis is depicted using total RNA from PLCs, ILCs, and ALCs. Genes with marked increases were observed, whereas other genes decreased during the transitions from PLC to ILC and ILC to ALC, confirming the findings of the array analysis.



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  		FIG. 4. Upregulation of gene expression in Leydig cells during postnatal development. Abbreviations are as in Figure 1. The mRNA levels are expressed relative to PLC. Gene symbols designated from mouse genome informatics (http://www.informatics.jax.org)

Identification of 11ßHSD2

The array analysis showed for the first time that Hsd11b2 is expressed in Leydig cells. It was missed in earlier analyses because the level of expression relative to Hsd11b1 is approximately 1000-fold lower (unpublished results), but the developmental trends for the mRNAs of the two isoforms increased similarly. To further evaluate the development of glucocorticoid metabolizing enzyme activity in Leydig cells, we measured rates of substrate conversion in the presence of pyridine nucleotide cofactor NAD+. The results were in parallel with the array data and showed that 11ßHSD2 activity was significantly higher in ALCs compared with PLCs and ILCs, which had only marginal rates of conversion (Table 5). The oxidative inactivation of glucocorticoids by 11ßHSD2 has been proposed to have two functions: 1) reducing levels of glucocorticoid activity and 2) conferring the selectivity of mineralocorticoid receptor binding to aldosterone, its cognate ligand. Therefore, the developmental trends of glucocorticoid receptor (Nr3c1) and Nr3c2 in the array were also analyzed (Table 5). The levels of both Nr3c1 and Nr3c2 mRNA were higher in ALCs compared with PLCs and ILCs. Therefore, the newly discovered developmental increase in Hsd11b2 expression may have essential involvement in regulating actions of corticosteroids corticosterone and aldosterone on Leydig cells.


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  		TABLE 5. Messenger RNA levels of Hsd11b1, Hsd11b2, Nr3c1, and Nr3c2 and 11ßHSD2 enzyme activity during development


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using cluster analysis, the present study clarifies the identities of genes turning on or off during the transitions from PLC to ILC and ILC to ALC. Although the developmental trends of a few genes have been tracked over the course of Leydig cell differentiation using various methods [6, 17, 19, 20], the present report is the first to systemically describe the gene expression patterns in this lineage. The developmental increases of Hsd11b2 mRNA and activity in Leydig cells occurred in parallel with rises in Nr3c1 and Nr3c2 mRNA levels, supporting the hypothesis that this enzyme plays a critical role in regulating the actions of corticosteroids on the testosterone biosynthetic pathway. This is supported by the fact that 11ßHSD2 oxidatively inactivates corticosterone, thereby protecting Leydig cells from the adverse effects of overactivation of the GR pathway and also allowing for selective binding of aldosterone to the MR [34].

Previously, we had shown that PLCs are steroidogenic and less responsive to LH stimulation [19, 20] compared with ALCs. This was confirmed by the present array analysis in that PLCs expressed steroidogenic enzyme genes, Cyp11a1, Hsd3b1, and Cyp17a1, and had detectable levels of Lhcgr mRNA. In addition, we identified several abundantly expressed genes in PLCs that had significant developmental trends. The most abundantly expressed gene was Rcl, a c-myc target gene that was four times higher relative to Actb. The functions of Rcl in Leydig cells are unknown, but it is thought to stimulate cell proliferation [35]. Decreased Rcl levels during the transition from PLC into ALC may be involved in the loss of Leydig cell proliferative capacity. The second most abundantly expressed gene was Egr1, a transcription factor that was 1.2 times higher compared with Actb. Egr1 has been shown to stimulate Lhcgr expression [36], and in Egr1 and Egr4 double-mutant mice, postnatal Leydig cell development is severely hindered [37]. Expression of Egr1 in PLCs could contribute to the initial differentiation of Leydig cells from stem cells. In addition to Rcl and Egr1, PLCs abundantly expressed Kit, Pdgfra, and Il6st. Because these growth factor receptors are also expressed in many types of stem cells and are used as stem cell markers [38, 39], their presence in PLCs indicated that these cells are newly formed members of the Leydig cell lineage derived from a stem cell pool.

It is well established that PLCs have a high proliferative capacity, which declines during the transition to ILC [7, 16]. Consistent with this developmental change, there was a fourfold decline in the cell proliferation marker PCNA (Pcna) from PLC to ALC (Fig. 3). The high proliferative capacity of PLCs is undoubtedly supported by abundant expression of genes encoding growth factor receptors such as Kit, Pdgfra, and Il6st and their intracellular signaling pathways that act on the cell cycle machinery [40–43]. Our cluster analysis showed that downregulated genes were mainly related to cell cycle regulators Ccnb1, Ccnc, Ccnd1, Ccnd2, Ccnd3, Cdk2, Cdk4, Cdc25b, P55cdc (Cdc20), p58/ GTA (Rad23b), Rb/p105 (Scyl1), and Rb/p130 (Rbl2); transcription factors Idb1, Idb2, Idb3, Egr1, and Rcl; and proliferation-related oncogenes c-Jun (Jun), JunB (Junb), JunD (Jund), c-Fos (Fos), Fra2 (Rabep2), Akt1 (Akt1), and Akt2 (Akt2). These genes were downregulated from 2 to 23 times during the transition of PLCs into ILCs.

Proliferation of PLCs is critical for generation of sufficient Leydig cell numbers to populate the adult testis. PLCs expressed Il6st, the intracellular subunits of LIF and IL-6, Pdgfra, and Kit abundantly, and these proteins are required to confer sensitivity to their respective ligands in Leydig cells. Among the cell signaling ligands that may be acting on Leydig cells, leukemia inhibitory factor (LIF) is a highly pleiotropic cytokine [44], the effects of which are mediated by binding to a heterodimeric receptor composed of the LIF-specific binding subunit gp190 and the transmembrane signal transducing subunit gp130, which is shared by several receptors of cytokines related to IL-6 [45]. LIF was first identified through its ability to maintain murine embryonic stem cells in an undifferentiated totipotent state as it promotes their proliferation [46]. Both LIF binding unit gp190 and the IL-6 binding unit IL6R are present primarily in PLCs [47, 48]. Furthermore, LIF and IL-6 are expressed in Leydig cells in prepubertal and adult animals [47, 48], indicting that LIF and IL-6 act as proliferative factors for PLCs. PDGF is produced in the seminiferous tubule [49], although Leydig cells are also sites of expression [50, 51]. However, the Pdgfra is exclusively present in Leydig cell precursors and Leydig cells [49]. PDGFR{alpha} signaling is thought to be critical for development of Leydig cells postnatally because ALCs are deficient in PDGF-A knockout mice [49] and PDGFR{alpha} activity is implicated in Leydig cell proliferation [50, 51]. C-kit, the receptor for stem cell factor kit ligand produced by Sertoli cells, is known to be expressed in the Leydig cell lineage and blocking c-kit with the ACK-2 antibody inhibits Leydig cell proliferation [52].

Some genes that are abundantly expressed in PLCs are dramatically switched off as a function of age, such as Anxa1, which decreased dramatically by 23-fold. Anxa1 is a glucocorticoid-regulated protein that has been implicated in cell signaling and proliferation [53], and glucocorticoid receptor is also expressed in PLCs [54]. The abundant expression of Anxa1 suggests that glucocorticoid hormone action is necessary for the proliferation and differentiation of Leydig cells, which was consistent with the results described for the 11ßHSD2 enzyme.

Postnatally, proliferative capacity in the Leydig cell lineage is suppressed during the transition from PLC to ILC as differentiation is initiated. The cluster analysis showed the expression levels of several genes that are initially elevated first as PLCs differentiate into ILCs (Fig. 2B). Significant increases in gene expression levels for Cyp11a1, Hsd3b1, and carboxyesterase ES-10 and FABP-H (Fabp3), which transports lipid and cholesterol [19, 20]. These changes were consistent with a sharp increase in ILC steroidogenic capacity.

In the second developmental transition, when Leydig cells progressed from ILC to ALC, 88 mRNAs were further increased in abundance. The cluster analysis showed that upregulated genes during the transition from ILC to ALC are primarily distributed according to the following functional categories: ion channels and transporters, metabolic pathways, protein turnover proteins, and stress response and targeting proteins. Steroid biosynthetic enzymes (Cyp11a1, HSD3b1, Cyp17a1) became the most abundantly expressed genes in ALCs. In addition to the steroidogenic enzymes, antioxidant enzymes were increased as a group, including glutathione-S-transferase: glutathione S-transferase Ya subunit (Gsta-rs1), microsomal glutathione S-transferase (Mgst1), glutathione S-transferase subunit 5 theta (Gstt5), and glutathione S-transferase Yb subunit (Gsta2). Differentiation is correlated with increases in oxidative metabolism [55]. Oxidative damage to proteins, mitochondrial and genomic DNA, and lipids [56–58] increases when exposure to reactive oxygen species increases [59]. Leydig cells have developed a mechanism for antioxidant defense [55]. Antioxidant enzymes may be globally activated during the course of Leydig cell development to protect steroidogenic function. Later in the life cycle, declines in antioxidant enzyme expression levels are a cause of age-related decreases in Leydig cell steroidogenesis [21].

The largest increases in gene expression during progression of PLCs into ALCs are tissue inhibitor Timp, 406-fold; Lip1, 157-fold; Chrna4, 85-fold; Cyp2a1, 65-fold; and Fabp3, 33-fold. The function of Timp2 remains unclear. Because TIMP1 (Timp1) is abundantly expressed in ALCs [60] and Timp1 has been shown to stimulate Leydig cell steroidogenesis, expression of Timp2 may fulfill the same function. Cholesterol esterase (Lip1) has been shown to use lipoproteins as substrates in steroidogenic cells [61], thereby increasing the rate of androgen synthesis. The present report is the first to identify the presence of Chrna4 in Leydig cells. Expression of this receptor suggests that Leydig cells are regulated by the neurotransmitter acetylcholine. Cyp2a1 has been shown to be exclusively present in Leydig cells and is involved in testosterone metabolism by 7{alpha}-hydroxylation [62]. Fabp3 is thought to have a role in fatty acid uptake, trafficking, and metabolism [63], again suggesting possible involvement in steroidogenesis.

In general, the cluster analysis of the array data conformed to established expectations that pubertal development of Leydig cells would be associated with decreased cell division and increased expression of proteins that are needed for onset of steroidogenic function. However, one of the unexpected discoveries of the array approach was detecting the presence of Hsd11b2 in Leydig cells. In comparison with Hsd11b1, which is an oxidoreductase known to be abundantly expressed in Leydig cells, the mRNA levels for Hsd11b2 were lower. However, levels of both Hsd11b2 mRNA and activity increased during the transitions from ILC to ALC. In contrast, Nr3c1 and Nr3c2 mRNA levels reached the threshold of detection only at the ALC stage. Glucocorticoid hormone exerts a suppressive action on testosterone biosynthesis upon binding the GR [64–66]. The presence of Nr3c2 mRNA in ALCs confirmed the results of a previous study employing RT-PCR amplification [28] and is also notable because, unlike glucocorticoid, aldosterone stimulates testosterone biosynthesis (unpublished results). Due to the nonselective binding properties of the mineralocorticoid receptor, the heterologous ligand corticosterone would normally be bound because it is present at concentrations that are up to 1000-fold higher relative to aldosterone [67, 68]. In this context, 11ßHSD2 lowers the intracellular level of corticosterone, allowing aldosterone to bind the MR selectively. The role of 11ßHSD2 in conferring MR binding selectivity is seen in the human condition of apparent mineralocorticoid excess, which is caused by mutations in HSD11b2 resulting in the indiscriminate binding of corticosterone to the MR and leading to hypokalemic hypotension [69]. Based in part on the present findings, we postulate that 11ßHSD2 has a similar role in the regulation of the actions of corticosteroids in Leydig cells.

In summary, analysis of Leydig cell development from PLC to ALC by DNA array highlights the inverse patterns of downregulated proliferation-related genes and induced differentiation-related genes, which together are associated with development of Leydig cell steroidogenesis.



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  		FIG. 5. PCR confirmation of the genes randomly picked to be representative in the cDNA array. Igf2r (Accession #U59809), Nr4a1 (U17254), Pcna (Y00047), Pdgfra (M63837), and Rcl (U82591) are downregulated; HSD11b1(J05107), Lip1 (L46791), Galr2 (U94322), Fabp3 (J02773), Atp1a1 (M28647), and Chrna4 (L31620) are upregulated; and Srd5a1 (S81448) peaked in ILC


   ACKNOWLEDGMENTS
 
We are grateful to Michael Holmes for technical help.


   FOOTNOTES
 
1 Supported, in part, by NIH grant HD-32588 to M.P.H. Back

2 Correspondence: Matthew P. Hardy, The Population Council, 1230 York Ave., New York, NY 10021. FAX: 212 327 7678; m-hardy{at}popcbr.rockefeller.edu Back

Received: 28 October 2004.

First decision: 6 December 2004.

Accepted: 10 February 2005.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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