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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 30–32% 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, 40–42% of the genes were present. Similarly, 36–43%, 41–43%, and 41–45% 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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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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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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). 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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 20–30%, 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 manufacturer’s 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit gene expression in rat Sertoli cells by 8-bromo-3',5'-cyclic-adenosine monophosphate. Mol Endocrinol 7:469–476[Abstract]
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