Oligonucleotide Microarray Analysis of Gene Expression in Follicle-Stimulating Hormone-Treated Rat Sertoli Cells
Derek J. McLean,
Patrick J. Friel,
Derek Pouchnik and
Michael D. Griswold
School of Molecular Biosciences, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
Address all correspondence and requests for reprints to: Michael D. Griswold, School of Molecular Biosciences, Box 644660, Washington State University, Pullman, Washington 99164-4660. E-mail: griswold{at}mail.wsu.edu.
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ABSTRACT
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Spermatogenesis requires the presence of functional somatic Sertoli cells in the seminiferous tubules of the testis. Sertoli cells provide support and factors necessary for the successful progression of germ cells into spermatozoa. Sertoli cells are regulated to a large degree by the glycoprotein hormone FSH, which is required for the testis to acquire full size and spermatogenic capacity. Signaling events initiated by the binding of FSH to its receptor lead to an alteration of Sertoli cell gene expression. To characterize the changes in gene expression in FSH-treated Sertoli cells, we used the mRNA from these cells to screen Affymetrix U34A rat GeneChip oligonucleotide microarrays. Sertoli cells from 20-d-old rats were cultured in the presence of 25 ng/ml ovine FSH. At 0, 2, 4, 8, and 24 h after the addition of FSH, total RNA was purified and used to prepare biotinylated target, which was hybridized to the U34A rat microarray containing approximately 9000 rat genes. Analysis identified 100300 transcripts at each time point that were up-regulated or down-regulated by 2-fold or greater. Genes previously reported to be FSH or cAMP regulated in rat Sertoli cells were identified, in addition to numerous genes not reported to be expressed or FSH regulated in Sertoli cells. The expression patterns of five of these genes, encoding nerve growth factor inducible gene B, PRL-1, PC3 nerve growth factor-inducible antiproliferative putative secreted protein, diacylglycerol acyltransferase, and an expressed sequence tag, in FSH- and N,O'-dibutyryl cAMP-treated rat Sertoli cells were confirmed and characterized by Northern blot analysis. Thus, we have begun to define the transcriptome induced and repressed by FSH in rat Sertoli cells, and we have generated datasets of genes available for further analysis in regard to spermatogenesis and Sertoli cell signaling.
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INTRODUCTION
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SPERMATOGENESIS REQUIRES COMPLEX cell interactions between somatic Sertoli cells and germ cells in the seminiferous tubule of the testis. These interactions, which begin early in testis development and continue throughout life, are necessary for the production of spermatozoa (1). The entire process of spermatogenesis is regulated by gonadotropins released from the pituitary gland and sex steroids produced within the testisthe pituitary-testicular axis (2). The components of this axis include LH and FSH produced by the pituitary and testosterone secreted by Leydig cells of the testis in response to LH. FSH and testosterone regulate spermatogenesis by interacting with receptors located in or on the plasma membrane of Sertoli cells (3, 4). Alteration of this regulation results in profound changes in testicular morphology and sperm production.
The binding of the glycoprotein FSH to receptors on Sertoli cells stimulates adenylyl cyclase, causing an increase in intracellular levels of cAMP (5). The signaling cascade resulting from the production of cAMP has been studied extensively, and targets affecting cell function have been identified. An increase of intracellular cAMP results in the activation of cAMP-dependent protein kinase A, which phosphorylates the cAMP regulatory element-binding protein (CREB). The nuclear transcription factor CREB and the coregulatory CREB-binding proteins (CBP/p300) have been shown to mediate some effects of cAMP (6, 7, 8, 9). In addition, new signaling mechanisms have been identified after hormonal treatment in endocrine cells. Alternative pathways, such as the pathway regulated by the cAMP-guanine nucleotide exchange factors, appear to be involved in cAMP-dependent signaling without activating protein kinase A (5).
A number of genes that contribute to germ cell survival and are regulated by FSH or cAMP have been identified (10, 11, 12, 13, 14, 15, 16). These genes have been identified with the use of various techniques such as Northern blot hybridization, subtractive hybridization, and differential display RT-PCR (17). Advances in gene array technology now allow scientists to obtain expression data for thousands of genes and generate a global expression profile for specific cell types. In addition, the microarray-based approach allows for discovery and functional annotation of novel genes. We have initiated a genomic approach to identify genes regulated by FSH in rat Sertoli cells. The level of thousands of mRNA transcripts in cultured rat Sertoli cells was determined with the use of rat U34A GeneChip microarray (Affymetrix, Santa Clara, CA), which contains approximately 9000 transcripts. Using this approach, we characterized the partial transcriptome of rat Sertoli cells in vitro and confirmed the expression pattern of several known FSH-regulated genes in rat Sertoli cells. In addition, we identified a number of additional genes regulated by FSH previously characterized in other tissues or cells. The overall goal of this research is to initiate the characterization of the transcriptome of rat Sertoli cells and evaluate FSH signaling in these cells at the molecular level.
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RESULTS
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Oliognucleotide Microarray Hybridization
FSH-treated rat Sertoli cell RNA was used to synthesize targets to screen oligonucleotide microarrays. The term target refers to the biotinylated cRNA hybridized to oligonucleotide microarrays. Quantification of target hybridization allows the user to determine the relative amount of a particular gene in a cell or tissue and the increase or decrease in the expression of that gene when comparing data from multiple microarrays. In each array-screening experiment, two separate preparations of rat Sertoli cells were treated with FSH. Target generated from each experiment was hybridized to an array generating replicate datasets. The rat U34A oligonucleotide microarray contains approximately 9000 genes.
Analysis of the oligonucleotide microarrays with Microarray Suite (MAS) 5.0 (Affymetrix) provides a statistical means to determine the presence or absence of a gene in a sample. This test is based on the comparison of the hybridization efficiency or signal of the target to its complementary sequence, taking into account cross-hybridization of the target for that gene to a mismatch sequence that is exactly the same as the complementary sequence except for one base. Each gene is represented on the array with 16 complementary probes and paired noncomplementary probes, selected from distinct regions of the gene. The signal values from these probes are used to determine the presence of a gene in the target and a P value calculated from this data. A P value less than 0.05 indicated that the message was present in the sample. Similarly, in comparison analyses using the data from one microarray hybridization as the baseline (control) of expression, the signal is used to determine whether an increase or decrease in expression of a gene has occurred after treatment. A two-tailed t test is used to calculate the P value for an increase or decrease call. A P value of 0.025 was considered significantly different.
Absolute Analysis
Absolute analysis with MAS 5.0 uses the signal intensity for each gene to determine whether a gene is present or absent in the sample. On the basis of this analysis, untreated cultured rat Sertoli cells expressed 3032% of the genes on the rat U34A oligonucleotide microarray. This represents approximately 3100 genes. Sertoli cells were treated with FSH for 2, 4, 8, and 24 h. After treatment, the percentage expression was slightly higher than control. At 2 h, 4042% of the genes were present. Similarly, 3643%, 4143%, and 4145% of the genes were present at 4, 8, and 24 h, respectively. The Venn diagram in Fig. 1
graphically represents the number of genes expressed in the control, and in 2-h and 8-h treatment and shows the genes expressed only at each time point.

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Figure 1. Venn Diagram Representing Expression Patterns of Genes in Rat Sertoli Cells after FSH Treatment at 0 (Control), 2, and 8 h
The circles represent a time point with the numbers representing the genes present in each sample (P < 0.05). Numbers in the outer portion of each circle are specific to that sample. The number in the innermost portion of the diagram represents genes expressed in all samples. Likewise, the numbers in the portions of the diagram shared by two circles represent genes expressed in those samples but not in the exclusive sample.
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Data from each experiment are posted on multiple web sites, including The Endocrine Society Journals Online web site (http://mend.endojournals.org) as supplemental data, Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), and the Griswold laboratory web page (www.wsu.edu/
griswold/microarray/), for public access and comparison to other Rat U34A datasets. The Griswold laboratory web site contains raw image files for each of the 10 microarrays, text files containing absolute analyses (MAS 5.0) of each chip, and comparison analyses between different chips.
Comparison Analysis
Oligonucleotide microarray gene expression data from rat Sertoli cells after FSH treatment were compared using two methods. First, comparison analysis of gene expression at each time point after FSH treatment was generated with MAS 5.0 (Affymetrix) using the untreated control as the baseline expression data. Expression data from each time point (2, 4, 8, and 24 h) were compared with gene expression in the untreated control, and the change in expression of each gene was monitored over time. The number of genes that were consistently induced in both experiments more than 2-fold, compared with the untreated control, were as follows: 2 h, 224 genes; 4 h, 246 genes; 8 h, 271 genes; and 24 h, 98 genes. The number of genes repressed more than 2-fold in both experiments were as follows: 2 h, 40 genes; 4 h, 42 genes; 8 h, 66 genes; and 24 h, 41 genes. Four comparisons were made with MAS 5.0 by comparing gene expression of each time point with baseline gene expression at the two 0-h control samples (18). This four pairwise comparison was performed to generate lists of genes induced and repressed at each time point compared with the time zero controls. Comparison of gene expression between time points (e.g. 2 h vs. 4 h) is not reported here for simplicity purposes, although these comparisons can be performed and may provide useful information.
Expression data were further analyzed with the use of cluster analysis. The self-organizing map (SOM) method of clustering (19) was used to segregate genes according to their expression after FSH treatment. This technique distributes genes into groups on the basis of their expression profile, minimizing variability within clusters while maximizing variability between clusters. The SOM clustering method was chosen because of the exploratory nature of the data analysis, the straightforward nature of interpretation it provides, and the scalability to large datasets. A geometry of nodes consisting of a 4 x 4 grid (Fig. 2
) was selected for clustering based on visual interpretation of the output. The neighboring nodes of the SOM (e.g. 1,1; 1,2; 1,3; etc.; Fig. 2
) are more closely related than more distant nodes (e.g. 4,4; Fig. 2
). Lists of genes from each cluster were generated, and several genes from nodes 1,1; 1,2; and 1,3 were generated. These nodes represented genes with an early immediate induction to FSH (node 1,1) and a more delayed induction pattern (nodes 1,2 and 1,3). The expression of several genes known to be induced in rat Sertoli cells after FSH treatment were confirmed (Table 1
). Tables 1
and 2
list a small number of the genes induced or repressed, respectively, at each time point after FSH treatment. Gene lists generated from the four pairwise comparisons generated with MAS 5.0 and from the SOM clustering were compared, and genes were selected for further study on the basis of their consistent expression profile and potential biological role in Sertoli cells.

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Figure 2. FSH-Treated Rat Sertoli Cell SOM Used for Cluster Analysis
A 4 x 4 SOM of rat Sertoli cell genes merged from two independent cell preparations treated with FSH. Expression levels are shown on y-axis, and time points on x-axis. Clusters are grouped in columns according to similarity, and adjacent clusters have similar gene expression patterns. The number of genes within each cluster is listed at the top, along with the cluster number (1,1; 1,2, etc.).
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Three genes that were selected for expression verification and further study with the use of Northern blots showed rapid induction at the 2-h treatment, similar to the expression pattern of c-fos and junB. The expression patterns of these genes, nerve growth factor-inducible gene B (NGFI-B), PC3 NGF-inducible antiproliferative putative secreted protein (PC3), and the tyrosine phosphatase PRL-1 were verified by Northern blot using total RNA from three replicate rat Sertoli cell preparations.
The level of NGFI-B transcript was 13.5 ± 2.1-fold higher 2 h after FSH treatment (Table 3
) and was one of the highest up-regulated genes at this time point on the microarray. NGFI-B transcript level continued to be induced at 4 h after FSH treatment (8.1 ± 1.9-fold) and then returned to baseline at 8- and 24-h treatment. Northern blot analysis confirmed the increase in NGFI-B message (Fig. 3A
and Table 3
) showing an 11.13 ± 2.2-fold (P < 0.05) induction 2 h after FSH treatment and a 5.1 ± 0.98-fold (P < 0.05) induction 4 h after FSH treatment (Fig. 3A
). No change of NGFI-B transcript level was observed at 8 and 24 h after FSH treatment on the Northern blots.
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Table 3. Induction of FSH-Regulated Genes Identified by Affymetrix Microarray Hybridizations and Verified by Northern Blot
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Figure 3. Induction of NGFI-B (A and B), PC3 (C and D), and PRL-1 (E and F) Message in FSH-Treated Rat Sertoli Cells
These genes were selected for rapid induction after FSH treatment. A, C, and E, Northern analysis of NGFI-B (A), PRL-1 (C), and PC3 (E) mRNA from treated Sertoli cells. Total RNA (10 µg) was extracted from Sertoli cells after 0, 2, 4, 8, and 24 h of FSH treatment and run on a denaturing agarose gel. The approximate sizes of 28S and 18S rRNA are shown in kilobases. Lower panels, The same blot stripped and reprobed with ribosomal protein S2 for a loading control. B, D, and F, Graphs showing the effect of FSH on NGFI-B (B), PRL-1 (D), and PC3 (F) expression. Quantification of the mean ± SEM for three independent FSH treatments for each gene at each time point. Images were analyzed using ImageQuant (Molecular Dynamics, Inc.) and normalized to the ribosomal protein S2 signal. Data are expressed as n-fold induction over the 0 h control set as 1. A 24-h control (24C) not treated with FSH indicates gene expression did not change through the course of the experiment. Asterisks indicate significant change in gene expression (P < 0.05).
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Two additional genes that had a similar expression pattern as NGFI-B on the oligonucleotide microarray were PC3 and PRL-1. The level PC3 transcript was 11.9 ± 2.0-fold higher 2 h after FSH treatment and 8.6 ± 0.6-fold higher 4 h after FSH treatment (Table 3
). At 8 and 24 h after FSH treatment, the level of PC3 message was not significantly different than the untreated control. Analysis of the level of PC3 transcript by Northern blot hybridization after treatment of rat Sertoli cells with FSH for 2 h indicated a 4.67 ± 0.6-fold (P < 0.05) induction. Likewise, the PC3 transcript level was 3.71 ± 0.6-fold (P < 0.05) higher 4 h after FSH treatment (Fig. 3B
and Table 3
) than control. The level of PC3 transcript was not affected at 8 or 24 h after FSH treatment.
The level of the tyrosine phosphatase PRL-1 transcript was 7.8 ± 2.3-fold higher 2 h after FSH treatment according to hybridization to the oligonucleotide microarray (Table 3
). Further induction to 14.7 ± 1.8-fold occurred 4 h after FSH treatment. At 8 h after FSH treatment, PRL-1 was induced 8.6 ± 1.21-fold, and the transcript returned to the level of the untreated control 24 h after FSH treatment. The results of the Northern blot analysis indicated that the up-regulation of PRL-1 was not as strong as the oligonucleotide microarray predicted. Northern blot analysis indicated that the level of PRL-1 transcript was induced 2.2 ± 0.7-fold (P < 0.05), 6.1 ± 1.1-fold (P < 0.05), and 1.8 ± 0.25-fold at 2, 4, and 8 h after FSH treatment, respectively (Fig. 3C
and Table 3
). FSH did not affect the level of PRL-1 message at 24 h.
The expression of two genes induced with a delayed pattern at 4 and 8 h according to the oligonucleotide microarray data (Table 3
) was also verified by Northern blot. The level of diacylglycerol acyltransferase (DGAT) transcript was 3.1 ± 0.1-fold and 7.95 ± 0.5-fold higher 4 and 8 h after FSH treatment, respectively. In Northern blot experiments, the level of DGAT was induced 2.3 ± 0.32-fold (P < 0.05) 4 h after FSH treatment and 4.1 ± 0.7-fold (P < 0.05) 8 h after FSH treatment (Fig. 4A
and Table 3
). FSH treatment for 2 or 24 h did not affect the level of DGAT message in rat Sertoli cells. An expressed sequence tag (EST) named RSCF-1 (rat Sertoli cell FSH-regulated gene 1) with weak similarity to calcium/calmodulin-dependent kinase (KCCD) rat calcium/calmodulin-dependent protein kinase type II
-chain was also elevated at 4 and 8 h (5.1 ± 1.9-fold and 7.2 ± 2.5-fold, respectively) after FSH treatment according to the oligonucleotide arrays (Table 3
). Northern blot studies confirmed that the level of this message was higher at 4 h (5 ± 0.8-fold; P < 0.05) and 8 h (6.5 ± 1.1-fold; P < 0.05) after FSH treatment (Fig. 4B
and Table 3
). The transcript level of this EST was higher at 2 h (2.4 ± 0.97-fold; P < 0.05) using Northern analysis but not on the microarray (1.6 ± 0.4-fold). The level of RSCF-1 transcript returned to baseline levels 24 h after FSH treatment.

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Figure 4. Induction of DGAT (A and B) and RSCF-1 (C and D) Message in FSH-Treated Rat Sertoli Cells
These genes were selected for delayed induction (48 h) after FSH treatment. A and C, Northern analysis of DGAT (A) and RSCF-1 (C) mRNA from treated Sertoli cells. Total RNA (10 µg) was extracted from Sertoli cells after 0, 2, 4, 8, and 24 h of FSH treatment and run on a denaturing agarose gel. The approximate sizes of 28S and 18S rRNA are shown in kilobases. Lower panels, The same blot stripped and reprobed with ribosomal protein S2 for a loading control. B and D, Graphs showing the effect of FSH on DGAT (B) and RSCR-1 (D) expression. Quantification of the mean ± SEM for three independent FSH treatments for each gene at each time point. Images were analyzed using ImageQuant (Molecular Dynamics, Inc.) and normalized to the ribosomal protein S2 signal. Data are expressed as n-fold induction over the 0 h control set as 1. A 24-h control (24C) not treated with FSH indicates gene expression did not change through the course of the experiment. Asterisks indicate significant change in gene expression (P < 0.05).
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To ensure that the expression of NGFI-B, PC3, PRL-1, DGAT, and RSCF-1 at 2, 4, 8, and 24 h did not change in cultured cells due to time or medium change, we analyzed expression with Northern blots. The expression of these genes in control rat Sertoli cells at each time point (2, 4, 8, and 24 h) was not different than the expression of each gene at time zero control (data not shown).
To determine whether FSH regulation of the transcript level of these genes was consistent with cAMP signaling, primary rat Sertoli cells were treated with N,O'-dibutyryl cAMP [(Bu)2cAMP]. Northern blot analysis indicated that the pattern of induction was the same for each gene after (Bu)2cAMP treatment, as was observed after FSH treatment. NGFI-B was induced 11.4 ± 1.9-fold (P < 0.05) 2 h and 9.0 ± 1.4-fold (P < 0.05) 4 h after (Bu)2cAMP treatment, whereas PC3 was induced 7.4 ± 1.2-fold (P < 0.05) and 8.3 ± 1.7-fold (P < 0.05) at 2 and 4 h, respectively, after (Bu)2cAMP treatment (Fig. 5
). The level of PRL-1 message was 4.3 ± 0.9-fold (P < 0.05), 14.4 ± 2.2-fold (P < 0 05), and 6.7 ± 1.7-fold (P < 0.05) higher at 2, 4, and 8 h, respectively, after (Bu)2cAMP treatment (Fig. 5
). DGAT and RSCF-1 transcript levels were also elevated by (Bu)2cAMP treatment. The level of DGAT transcript was induced 8.5 ± 1.88-fold (P < 0.05) and 16.6 ± 3.4-fold (P < 0.05) at 4 and 8 h, respectively (Fig. 6
). Likewise, RSCF-1 transcript level increased 4.7 ± 1.1-fold (P < 0.05) and 7.0 ± 2.3-fold (P < 0.05) at 4 and 8 h, respectively, after (Bu)2cAMP treatment (Fig. 6
). The induction of PC3, PRL-1, and DGAT was higher after (Bu)2cAMP treatment than the induction observed after FSH treatment.

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Figure 5. Induction of NGFI-B (A and B), PC3 (C and D), and PRL-1 (E and F) Message in (Bu)2cAMP-Treated Rat Sertoli Cells
A, C, and E, Northern analysis of NGFI-B (A), PC3 (C), and PRL-1 (E) mRNA from treated Sertoli cells. Total RNA (10 µg) was extracted from Sertoli cells after 0, 2, 4, 8, and 24 h of FSH treatment and run on a denaturing agarose gel. The approximate sizes of 28S and 18S rRNA are shown in kilobases. Lower panels, The same blot stripped and reprobed with ribosomal protein S2 for a loading control. B, D, and F, Graphs showing the effect of (Bu)2cAMP on NGFI-B (B), PC3 (D), and PRL-1 (F) expression. Quantification of the mean ± SEM for three independent (Bu)2cAMP treatments for each gene at each time point. Images were analyzed using ImageQuant (Molecular Dynamics, Inc.) and normalized to the ribosomal protein S2 signal. Data are expressed as n-fold induction over the 0 h control set as 1. A 24-h control (24C) not treated with FSH indicates gene expression did not change through the course of the experiment. Asterisks indicate significant change in gene expression (P < 0.05).
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Figure 6. Induction of DGAT (A and B) and RSCF-1 (C and D) Message in (Bu)2cAMP-Treated Rat Sertoli Cells
A and C, Northern analysis of DGAT (A) and RSCF-1 (C) mRNA from treated Sertoli cells. Total RNA (10 µg) was extracted from Sertoli cells after 0, 2, 4, 8, and 24 h of (Bu)2cAMP treatment and run on a denaturing agarose gel. The approximate sizes of 28S and 18S rRNA are shown in kilobases. Lower panels, The same blot stripped and reprobed with ribosomal protein S2 for a loading control. B and D, Graphs showing the effect of (Bu)2cAMP on DGAT (B) and RSCF-1 (D) expression. Quantification of the mean ± SEM for three independent (Bu)2cAMP treatments for each gene at each time point. Images were analyzed using ImageQuant (Molecular Dynamics, Inc.) and normalized to the ribosomal protein S2 signal. Data are expressed as n-fold induction over the 0 h control set as 1. A 24-h control (24C) not treated with FSH indicates gene expression did not change through the course of the experiment. Asterisks indicate significant change in gene expression (P < 0.05).
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DISCUSSION
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In this study, oligonucleotide microarrays were used to provide information on the gene expression of Sertoli cells after FSH treatment. Information obtained from this analysis will aid in identifying genes regulated by FSH through cAMP signaling and may provide insight in the identification of signaling pathways active in these cells.
The overall trend in cellular activity after FSH treatment was an activation of gene transcription and possibly an increase of mRNA stability for select genes. The ratio of induced to repressed genes after FSH treatment was 5.6 at 2 h, 5.8 at 4 h, 4.1 at 8 h, and 2.4 at 24 h. This result was not surprising, because it has been reported that Sertoli cells respond to FSH with increased levels of cAMP, increased protein synthesis, and up-regulation of several genes (20, 21, 22, 23, 24).
The expression pattern of several genes previously described to be FSH regulated in rat Sertoli cells (10, 14) was verified by the microarray data (Table 2
). In addition, five genes that had not been reported as FSH regulated were identified by analysis of the gene array data and verified by Northern blot hybridization. NGFI-B was strongly induced 2 h after FSH treatment similar to c-fos and junB (10, 14). As with c-fos and junB, NGFI-B presumably regulates the expression of other genes resulting in phenotypic changes in cells. NFGI-B encodes for a member of the steroid thyroid hormone superfamily and is known to regulate the expression of genes involved in steroidogenesis and cell death (25). In the rat ovary, NGFI-B expression gradually decreases after pregnant mare serum gonadotrophin treatment. However, expression is induced in theca and granulosa cells of the ovary of animals primed with pregnant mare serum gonadotrophin followed by human chorionic gonadotropin treatment (26). In the mouse, the NGFI-B murine homolog, nur77, is first expressed at a low level in the testis of d 5 animals with expression increasing at d 30 and remaining constant in the adult (27). In addition, the expression of nur77 increased in the testis after human chorionic gonadotropin treatment of peripubertal animals (27). Taken together, these data suggest that expression NGFI-B is regulated in reproductive tissues by LH. NGFI-B expression, possibly regulated by FSH, may occur in Sertoli cells of prepubertal animals followed by expression in Leydig cells of the adult testis. FSH acts as a mitogen during testis development. NGFI-B may regulate genes important during this critical developmental time period.
This is the first report of the expression of NGF-inducible antiproliferative putative secreted protein PC3 in rat Sertoli cells in which it was significantly up-regulated by FSH at 2 and 4 h after treatment. PC3 expression is induced after NGF-dependent differentiation of neural crest cells and appears to be important for neuronal differentiation (28). The function of PC3 is transcriptional coregulation that prevents the entry of cells into the G1 phase of the cell cycle (29). PC3 may function in Sertoli cells by stimulating differentiation of these cells at puberty. In this study, Sertoli cells from 20-d-old rats were treated with FSH. This is the approximate age in rat development in which Sertoli cell mitosis stops.
The tyrosine phosphatase PRL-1 was originally identified as an immediate early gene whose expression is induced in mitogen-stimulated cells and regenerating liver (30). PRL-1 is also significantly expressed in intestinal epithelia, and in contrast to the expression of PRL in liver, its expression is associated with cellular differentiation in the intestine (31). Similarly, PRL-1 is expressed during development in several differentiating epithelial tissues. In rat Sertoli cells treated with FSH, PRL-1 message was higher at 2, 4, and 8 h after treatment and may play a role in Sertoli cell differentiation. The expression of PRL-1 in reproductive tissues has not been previously reported.
The immediate early induction of PRL-1, NGFI-B, and PC3 in rat Sertoli cells is also regulated by cAMP signaling. To investigate the intracellular signaling pathways controlling PRL-1, NGFI-B, and PC3 transcript regulation in rat Sertoli cells, we examined the effect of treating the cells with (Bu)2cAMP. The message level of PRL-1, NGFI-B, and PC3 in rat Sertoli cells after (Bu)2cAMP treatment showed the same pattern of induction as with FSH treatment (Fig. 5
). Genes induced at later time points are also of interest because these may be regulated by early response genes or through alternative signaling pathways or transcription factors. The expression of two genes, DGAT (32, 33) and an EST named RSCF-1, which were induced at 8 h in Sertoli cells according to the oligonucleotide microarray, was verified by Northern blot analysis. As with PRL-1, NGFI-B, and PC3, the level of DGAT and RSCF-1 message was regulated in Sertoli cells by (Bu)2cAMP signaling (Fig. 6
). The pattern of induction of each gene with (Bu)2cAMP was very similar to that of FSH, and in the case of NGFI-B and RSCF-1 the fold inductions were similar values. The fold induction of PC3, PRL-1, and DGAT was higher and more persistent after (Bu)2cAMP treatment than was observed with FSH treatment. Additional determination of the signaling pathways involved in regulating these transcripts is ongoing and may provide insight into how factors not previously identified in Sertoli cells can affect cellular function.
The ability to identify genes regulated by hormonal treatment with the use of gene arrays has been well documented (34, 35, 36). We are using this technology to obtain a global expression profile of cells essential for spermatogenesis and how these cells are affected by hormone treatment. Although FSH is not essential for qualitatively normal spermatogenesis or fertility, it is critical for the ultimate spermatogenic capability of the testis by stimulating Sertoli cell mitosis during testis development (37). The expression profile of FSH-treated Sertoli cells may enable scientists to generate new hypotheses about proliferation and differentiation of these cells.
These data provide multiple avenues for further investigation. Experiments designed to determine the in vivo expression of the genes described here are essential to determine whether the proteins encoded by these genes are important for testicular development or maintenance of spermatogenesis. Likewise, animal models in which FSH expression can be controlled could be used to evaluate gonadotropin regulation of these genes. A particularly useful model is the line of mice deficient in GnRH or hpg mice. Administration of FSH and testosterone to these mice resulted in quantitatively normal spermatogenesis and testes of normal size (38, 39). The expression of genes believed to be FSH regulated could be directly measured in this model. Likewise, mice null for the FSHß gene could be used in a similar manner (37).
The microarray data do not provide information on the mechanism responsible for the up- or down-regulation of a particular transcript. Changes in the level of transcript present may be due to transcriptional activation or mRNA stability. Similarly, the pathway responsible for the regulation of a particular transcript could be determined with the use of specific kinase inhibitors. This approach would be useful in identifying novel pathways or factors responsible for gene expression in Sertoli cells or other endocrine cells. In addition, a limitation of microarray research is the number of genes represented on the array. The arrays used in this study had 9000 transcripts. This number represents only 2030%, depending on the current estimate, of the genes expressed in mammals. Thus, there may be another 300 genes regulated by FSH that were not represented on the array used in this study. Further characterization of the transcriptome of Sertoli cells would require screening additional arrays. In conclusion, these and other approaches will aid in better understanding of the mechanisms involved in regulating spermatogenesis.
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MATERIALS AND METHODS
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Cell Culture and RNA Preparation
Protocols for the use of animals in these experiments were approved by the Washington State University Animal Care and Use Committee and were in accord with National Institutes of Health standards established by the Guidelines for the Care and Use of Experimental Animals. Sertoli cells from 20-d-old Sprague Dawley rats were isolated and cultured as described by Karl and Griswold (40). The cells were plated in 150-mm culture dishes in Hams F-12 medium (Invitrogen, Carlsbad, CA) and incubated at 32 C in 5% CO2. The medium was replaced on d 3 of culture with fresh medium containing FSH treatment or vehicle for controls. The ovine FSH (National Institute of Diabetes and Digestive and Kidney Diseases) was suspended in PBS solution and added to cultures at a final concentration of 25 ng/ml. Treatments and controls were harvested at 0, 2, 4, 8, and 24 h. Sertoli cells were also treated with (Bu)2cAMP (0.1 mM) for 0, 2, 4, 8, and 24 h. For RNA purification, media was removed, and 1.5 ml per plate of cold Trizol reagent (Invitrogen) was immediately added. RNA was extracted according to the manufacturers instructions. RNA concentration and purity were determined by measuring 260/280-nm ratios. RNA with a 260/280 ratio of 1.8 or higher was used for DNA microarray analysis.
DNA Microarray Analysis
Total cellular RNA (10 µg) was used to synthesize the microarray target. Target synthesized from RNA from two independent FSH-treated Sertoli cell preparations hybridized on the microarrays. RNA was reverse-transcribed into double-stranded cDNA with a T7 promoter-containing primer using Superscript II, RNase H, and DNA polymerase (Invitrogen). After extraction with phenol-chloroform and ethanol precipitation with ammonium acetate, the cDNA was used as a template in a biotin-labeled in vitro transcription reaction (Enzo BioArray, Affymetrix). Resulting target cRNA was collected on Rneasy columns (QIAGEN, Valencia, CA) and then fragmented for hybridization to the microarrays.
The rat U34A microarray from Affymetrix was used in all hybridizations. This array contains approximately 9000 genes from Rattus norvegicus. Probes consist of 16 pairs of 25-mer oligonucleotides for each gene. One member of each pair contains a single base point mutation, and the signals of the pairs are compared with assess specificity of hybridization. Biotinylated target cRNA (15 µg) was hybridized to the array and then processed using the Affymetrix GeneChip Fluidics Workstation 400, after the Mini Euk 2v3 protocol. After binding with phycoerythrin-coupled avidin, microarrays were scanned on a Hewlett-Packard Gene Array Scanner (Hewlett-Packard Co., Palo Alto, CA). Results were analyzed using Affymetrix MAS 5.0 software. Individual microarrays were scaled to produce a mean signal intensity of 125. Iterative comparisons of different microarray datasets were done with MAS 5.0 comparison analysis as previously described with modifications (18). Each FSH-treated Sertoli cell microarray dataset (n = 8) was compared with each time zero (control) microarray dataset (n = 2) to determine the expression difference between treatment time and control. This comparison strategy allowed statistical comparison of fold-change values (converted from signal log ratio obtained from MAS 5.0) for genes that survived the four pairwise comparisons with a fold change
±2.0.
Replicate Affymetrix datasets for each experiment were analyzed in GeneSpring 4.2.1 (Silicon Genetics, Redwood City, CA). Gene expression was evaluated with parallel coordinate axis plots and used to generate Venn diagrams and for cluster analysis of the Affymetrix data. Genes with consistent expression patterns as evaluated with MAS 5.0 and GeneSpring were used to generate lists for further study.
Confirmation of RNA Changes
RT-PCR was used to generate Sertoli cell cDNA. Dnase-treated total RNA (1 µg) was reverse-transcribed into cDNA with an oligo(deoxythymidine)12-18 primer using Superscript II reverse transcriptase (Invitrogen) according to the manufacturers instructions. Oligonucleotide primers (Table 4
) were used to generate gene-specific PCR products for Northern blot probe templates. Products were made using standard PCR techniques for 30 cycles of 95 C for 30 sec, 56 C for 30 sec, 72 C for 1 min; final extension at 72 C for 10 min. Products were gel purified before use as probe templates.
Total RNA was fractionated at 10 µg per sample on 1.2% denaturing agarose gels and transferred to Hybond N membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK) via capillary action with 10x SSC (sodium chloride-sodium citrate buffer) and cross-linked to membranes with UV light. Radiolabeled probes were generated with [32P]dATP using the Rad Prime DNA labeling kit (Invitrogen). Hybridization was performed overnight at 65 C using hybridization buffer containing 0.5 M sodium phosphate buffer, 7% sodium dodecyl sulfate (SDS), 1% BSA, and 1 mM EDTA. Blots were rinsed with 2x SSC/0.1% SDS for 10 min at room temperature, then twice with 0.2x SSC/0.1% SDS for 15 min at 65 C. Blots were exposed to phosphorscreens (Molecular Dynamics, Inc., Sunnyvale, CA) for 1648 h. Images were analyzed using a Molecular Dynamics, Inc. PhosphorImager 445 SI and ImageQuant software (Molecular Dynamics, Inc.). Blots were normalized for RNA loading by probing with ribosomal protein S2 cDNA.
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ACKNOWLEDGMENTS
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We thank Alice Karl and Debra Mitchell for preparations of Sertoli cell cultures and James Shima for technical assistance.
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FOOTNOTES
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This work was supported by Grant HD-10808-22 (to M.D.G.) from the National Institute of Child Health and Human Development.
Abbreviations: (Bu)2cAMP, N,O'-Dibutyryl cAMP; CREB, cAMP regulatory element-binding protein; DGAT, diacylglycerol acyltransferase; EST, expressed sequence tag; MAS, Microarray Suite; NGFI-B, nerve growth factor-inducible gene B; RSCF-1, rat Sertoli cell FSH-regulated gene 1; SDS, sodium dodecyl sulfate; SOM, self-organizing map; SSC, sodium chloride-sodium citrate buffer.
Received for publication February 4, 2002.
Accepted for publication August 14, 2002.
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