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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: Dr. Michael Griswold, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660. E-mail: Griswold{at}mail.wsu.edu
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ABSTRACT |
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INTRODUCTION |
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The response of Sertoli cells to FSH signaling varies according to the age of the animal. FSH stimulates Sertoli cell division and differentiation; thus, it is a critical factor in the formation of the environment that leads to the expansion of spermatogenesis (3). Moreover, FSH has been shown to increase the number of spermatogonia in hypogonadal mice (hpg) (4).
Hpg mice are gonadotropin deficient due to a natural mutation in the GnRH gene (5) that results in undetectable levels of LH and FSH in the circulation and hypogonadism in the mice (6). Although the reproductive tracts of hpg mice are immature, they have previously been shown to be responsive to gonadotropin treatments (7, 8).
In a recent study published by our group (9), oligonucleotide microarray technology was used to investigate and monitor changes in gene expression in cultured 20-d-old rat Sertoli cells after FSH treatment. The purpose of the current studies was to identify murine testicular genes that are regulated by FSH in vivo and to analyze the overall pattern of gene expression in the mouse testis as a result of FSH signaling.
We have used the hpg mouse in conjunction with the oligonucleotide array technology to screen for changes in testicular gene expression after hpg mice were treated with FSH (see Table 1
). Thus, we were simultaneously able to monitor the differential expression of thousands of genes after FSH treatment in vivo. We also confirmed changes in the expression of genes that had previously been shown to be affected by FSH in vitro and/or in vivo in Sertoli cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). These changes in the FSHr mRNA were consistent with previously reported studies (10).
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Several transcripts that were found to be regulated by FSH in our experiments and that had also previously been shown by others to be regulated by FSH, either in vitro or in vivo, are listed in Table 1
. The number of transcripts that were differentially regulated in the testes of hpg mice, at 4, 8, 12, and 24 h after FSH treatment, is summarized in Fig. 2. Several representative transcripts that were increased or decreased at 4, 8, 12, and 24 h after FSH treatment, their accession number, and fold change are listed in Tables 2 and 3, respectively. These transcripts were selected depending on the functions of the protein they encode. After FSH treatment, 60, 48, 55, and 60% of the transcripts regulated were increased at 4, 8, 12, and 24 h, respectively.
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). Some of the transcripts that fit that profile are listed in Table 4. This list includes, Kruppel-like factor 4 (Klf4), Nur-related protein 1 (Nurr1), and hairy/enhancer of splits gene 1 (Hey1), none of which had previously shown to be regulated by FSH. Klf4 and Hey1 were both detected in cultures of primary Sertoli cells. These two genes were selected for further studies based on their potential role as transcription factors in the function of Sertoli cells. Their expression was confirmed by real-time PCR using cDNA from testes of hpg mice that were treated with FSH (Fig. 4). The expression pattern of Nurr1 was confirmed by real-time PCR as well (Fig. 5).
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).
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).
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). This diagram revealed that 50 of the transcripts were commonly affected at each of those time points. Several of those transcripts, selected arbitrarily, and their accession numbers are listed in Table 5.
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DISCUSSION |
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Genes that are regulated in the testes of hpg mice after FSH treatment for 4, 8, 12, or 24 h were identified. The experiments were validated by the identification of several genes such as inhibin 
(11), junb (12), cAMP-responsive element modulator (CREM) (13), prolactin-1 (9), and IGF binding protein 3 (14, 15) that had previously been shown by others to be regulated by FSH in vitro and/or in vivo. This indicates that Sertoli cells in the testes of hpg mice were responsive to the FSH treatment despite the use of mice that were more than 21 d old in the experiment. This is partly supported by recent studies in which Sertoli cells from 21-d-old hpg mice were shown to lack the tripartite nucleus characteristic of mature Sertoli cells (4). Taken together, the above observations confirmed that the hpg mouse model is responsive to short-time FSH treatments.
Noteworthy was an overall activation in gene expression observed at 4, 12, and 24 h after FSH treatment, consistent with the results that were observed in gene expression after FSH treatment of rat Sertoli cells in vitro. The correlation between the in vitro and in vivo data is not surprising, considering the fact that the expression of FSHr is highly restricted to Sertoli cells, making them the primary target of FSH in the testis.
It was noticeable that more genes were affected at 4 h than at any other experimental time points. These results suggest that, at early time points, FSH regulates the expression of many genes whose transcripts encode for protein that have long half-lives, enabling them to function even after the message is degraded. This is supported by earlier investigations that have shown that in cultured Sertoli cells treated with FSH, although the transcript of CREM peaks 4 h post treatment and returns to basal levels by 24 h, levels of the CREM protein remain high even after 24 h and can still be detected 36 h later (13).
Several genes that encode for proteins involved with microtubule and cytoskeleton rearrangement were identified as being regulated by FSH. These include actins, tubulins, lamin A, ankrin 3, and catenin 2 (Tables 2 and 3). These findings indicate a role for FSH in cytoskeleton rearrangement or in cell division. This is supported by previous studies that have shown the importance of FSH signaling in Sertoli cell cytoskeleton rearrangement (16). The change in expression of few genes such as junctional adhesion molecule and scinderin whose products are involved in cell adhesion indicated a role for FSH in the adhesion of germ cells to Sertoli cells. This is supported by previous studies that have shown FSH dependence of spermatid binding to Sertoli cells in culture systems (17, 18).
FSH regulated the abundance of Klf4, Hey1, Mad4, and Nurr1 mRNAs. Klf4 is a transcription factor that has been shown to be present in Sertoli cells and in spermatids (19); however, there is no information on how this gene is regulated in the testis. In testes of hpg mice, Klf4 mRNA was increased 4 h after FSH treatment, returned to control levels by 8 h and remained low throughout the experimental time course. A similar pattern of expression was shown in cultured Sertoli cells that were treated with [(BU)2cAMP]. According to our results, the effect of FSH signaling on the expression of Klf4 follows the expression profile of classical immediate-early genes such as junb, suggesting that this zinc finger protein might mediate downstream effects of FSH in Sertoli cells, thus playing a role in spermatogenesis. Likewise, the basic helix-loop-helix (bHLH) Hey1, which has previously been shown to be expressed in testis (20), was found to be increased at 4 h by FSH, and was also regulated by [(BU)2 cAMP] in cultured primary Sertoli cells implying a potential role for this bHLH in the Sertoli cells function. In addition, Hey1 has been associated with the Notch signaling pathway (21) whose importance in development and in spermatogenesis has been reported (22, 23).
Contrary to previous reports that the inhibitor of DNA binding 3 (Idb3) or HLH462 is absent in the mouse testis (24), we have found that Idb3 is present in the testis and that its expression was regulated by FSH. Idb3 contains a putative cAMP response element and serum response element sequences in its promoter (24), supporting the notion that this helix-loop-helix might be controlled by the FSH signaling pathway.
Mad4, also a bHLH, was shown to be decreased after FSH treatments in all the experimental time points. Mad4 has been associated in vitro with cessation of epithelial cell proliferation and was also shown to be induced upon differentiation in some cells (25). Testicular cells of hpg mice that haven’t previously been exposed to FSH would resume proliferation instead of differentiation at the first exposure to FSH; therefore, it would seem logical that factors, like Mad4, that suppress proliferation are repressed. In addition, in the mouse testis, the expression of Mad4 had been associated with completion of meiosis and early development of haploid cells. We speculate that with the completion of meiosis, the more mature Sertoli cells are not as responsive to FSH; thus, there is a halt in the repression of Mad4 expression. The regulation of Mad4 expression is an example of a potential role for FSH signaling in the influence of germ cells differentiation by Sertoli cells.
Nurr1, which encodes for a nuclear orphan receptor, was identified to be regulated in the testes of hpg mice that were given FSH for 4 h. Nurr1 is known to play an important role in the development and maintenance of neurons where it forms a DNA binding complex with RXR/
(26), which are also important during the development of the testis (27). Nurr1 has also been shown to be activated by cAMP regulatory element binding protein in arthritic tissue (28). This suggests a possible connection between Nurr1 and RXR in the development of the testis.
The expression of renin-1 was induced by FSH at all the time points investigated. Unlike most of the genes that were confirmed to be affected by the FSH treatments, renin is reportedly expressed in Leydig cells and not in Sertoli cells (29). Possible explanations for the induction of renin-1 by FSH in testes of hpg mice include a downstream effect from factors secreted from Sertoli cells as a result of FSH signaling. FSH has previously been shown to induce the expression of renin-1 in murine ovary (30).
In summary, transcripts that are regulated in testes of hpg mice 4, 8, 12, and 24 h after FSH treatment were identified. We have used the hpg mouse model along with oligonucleotide microarray analysis to explore the testicular genes expression profile in murine after FSH treatment. FSH primarily increased the levels of mRNAs in the murine testis. The limits we set for stringency purposes during the data analysis, i.e. change of 2-fold or more with signal strength of 100 or higher in all pairwise comparison analyses, could have caused the omission of some transcripts that did not pass the criteria of candidate selection, although their expression were altered by the treatments. It will be of interest to compare the results obtained using the hpg mouse model to another mouse model deficient in FSH such as the FSHß knockout. This study provided a database that will allow more hypothesis based research that will aid in answering questions regarding FSH signaling in the development of the testis and in spermatogenesis. Pressing issues such as molecular mechanisms involved in FSH-mediated action in spermatogonial division as well as in testicular cell-to-cell communication can be investigated by identifying the genes that are involved. The techniques we have used measure steady-state mRNA levels and do not specify whether or not the changes that were observed in gene expression are due to mRNA stability or to gene activation.
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MATERIALS AND METHODS |
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Primary Sertoli Cells Culture and Treatment
Sertoli cells from 16- to 20-d-old Balb/c mice were isolated and cultured as described by Karl and Griswold (31) with modifications. The cells were plated in 100-mm culture dishes coated with 1 mg/ml gelatin (Sigma, St. Louis, MO), in a medium containing 1:1 Ham’s F-12 (Invitrogen Life Technologies) and DMEM (Sigma), and incubated at 32 C in 5% CO2. The medium was replaced on d 2 of culture with fresh medium supplemented with 0.1 mM N,O’-dibutyryl cAMP (Sigma). The cells were harvested at 0, 2, 4, 8, or 24 h post treatment.
RNA Extraction and GeneChip Target Preparation
Total RNA was extracted from whole testes that were excised from our experimental mice using the Trizol reagent according to the manufacturer’s recommendations. RNA concentration, purity, and integrity were assessed by measuring the 260/280 nm ratios and by fractionation 1% denaturing agarose gel (formaldehyde). Total RNA with a 260/280 nm ratio of 1.8 or higher was used to generate biotinylated cRNA target for the Murine GeneChip (Affymetrix, Santa Clara, CA). Ten micrograms of total RNA were reverse-transcribed into single-stranded cDNA, and the single-stranded cDNA converted to a double stranded cDNA. The double-stranded cDNA was extracted using a phaseLock gel (Appendorf, Hamburg, Germany), and precipitated with ethanol and ammonium acetate. To synthesize a biotinylated cRNA, the purified cDNA served as a template in an in vitro transcription reaction using the MEGAscript (Ambion, Austin, TX) high yield transcription kit as described by the manufacturer with the following changes. Biotinylated CTP and uridine triphosphate were added to the reaction mix, and the resulting biotinylated target cRNA was purified using RNeasy columns (QIAGEN, Valencia, CA), ammonium acetate precipitated and quantified spectophotometrically. The purified biotinylated cRNA (15 µg) was then fragmented according to Affymetrix protocols. The hybridization cocktail that was prepared as recommended by Affymetrix consisted of 15-µg fragmented biotin-labeled cRNA spiked with Eukaryotic Hybridization control. The Murine Genome U74A, version 2 microarrays was directly loaded with 200 µl of the hybridization cocktail, then hybridized at 45 C for 16 h in a rotisserie motor that rotated at 50 rpm. After hybridization, the array was washed, stained with streptavidin phycoerythrin using the Affymetrix Genechip Fluidics Workstation 400 as described for the Mini Euk 2v3 protocol, and scanned on a Hewlett-Packard Gene Array Scanner (Hewlett-Packard Co., Palo Alto, CA).
Affymetrix GeneChip Analysis
For this study, we have used the Affymetrix Murine Genome U74version2 A (MG U74Av2). This array contains 12,488 probe sets, each representing a transcript, 6000 of which are from known genes whereas the remainder is expressed sequence tags. After hybridization with the labeled target, the array was scanned, and data generated and analyzed with the Microarray Suite version 5.0 (MAS 5.0) (Affymetrix).
Both an absolute and a comparison analysis were performed using MAS 5.0. Refer to www.Affymetrix.com for the details on the statistics of these analyses. The absolute analysis generates data from a sample that has been hybridized to an array. That data include output such as the signal strength, or the abundance of a specific transcript, and its detection i.e. whether that transcript is expressed or not in the analyzed sample. Some data have included from absolute analyses performed on genechip A that were targeted with RNA from normal wild-type mice testis. In the comparison analysis, an experimental array is compared with a baseline array so as to monitor the expression changes of transcripts across the samples targeted to the arrays.
For our purpose, the arrays were hybridized with targets generated from testes of FSH-treated or untreated hpg mice at different time points (4, 8, 12, and 24 h). The arrays that were targeted with samples from FSH-treated mice were used as experimental arrays, whereas the arrays that were hybridized with samples from control untreated hpg mice were used as the baseline array. All the time points except 12 and 24 h were performed in duplicate. Each experimental array was compared with each of the baseline arrays to generate four pairwise comparison analyses for each of the time points that had a replicate. At 12 and 24 h treatment, the single experiment array was compared with each of the two baseline control arrays to generate two pairwise comparison analyses.
GeneSpring Analysis
Expression data from arrays targeted with testicular RNA from hpg mice treated with FSH for different time points (0, 4, 8, 12, and 24 h) obtained from the MAS 5.0 comparison analyses were analyzed into GeneSpring as described by the supplier (Silicon Genetics, Redwood City, CA). GeneSpring was used for clustering, filtering and other analyses to group genes based on their expression. In addition, GeneSpring Gene Ontology was used to classify the genes differentially regulated into functional biological processes. This analysis allowed us to focus on genes with specific patterns of expression during the time course of FSH treatment. We were also able to select candidate transcripts according to their molecular functions.
Real-Time PCR
A two-step real-time PCR was carried out to quantify and analyze the expression of candidate genes. RT-PCR was used to generate cDNA either from whole testes of untreated and FSH-treated hpg mice RNA or from cAMP-treated and untreated primary cultured Sertoli cells RNA. Total RNA (1 µg) was reverse-transcribed into cDNA in a reaction primed by oligo(deoxythymidine)12–15 primer using Superscript II reverse transcriptase (Invitrogen Life Technologies) according to the manufacturer’s instructions. Reverse and forward oligonucleotide primers, specific to the chosen candidate genes, were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA) as described by the manufacturer. Table 6 lists the sequence of oligonucleotide primers that were used. In the last step, real-time RT-PCR was performed in a 96-well plate using a 7000 ABI prism sequence detection system (Applied Biosystems). The previously synthesized cDNA was used as template. Samples from each of the time points were plated in triplicate PCRs. The PCR contained about 5–10 ng of cDNA, 1x SYBR GREEN master mix (Applied Biosystems), and 600 nM of each reverse and forward primers of the candidate genes. The threshold cycle (CT), which indicates the relative abundance of a particular transcript, was calculated for each reaction by the 7000 ABI prism sequence detection system. Ribosomal protein S2 CT values were used as normalizing endogenous controls. The quantification of the genes expression fold change for each the candidate investigated was calculated for the different FSH or cAMP treatment time points using the formula 2–
CT as described in the SyBR Green protocol.
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A= FSH or cAMP-treated sample at a particular time point
B = control sample
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: bHLH, Basic helix-loop-helix; [(BU)2cAMP], N,O’-dibutyryl cAMP; CREM, cAMP-responsive element modulator; CT, threshold cycle; FSHr, FSH receptor; hyp, hypogonadal; Hey1, hairy/enhancer of splits gene 1; Idb3, inhibitor of DNA binding 3; Klf4, Kruppel-like factor 4; Mad, max dimerization protein; Mad4, max-interacting protein repressor; Nurr1, Nur-related protein 1.
Received for publication June 2, 2003. Accepted for publication July 29, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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