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School of Molecular Biosciences, Center of 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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T is an androgen that generates direct genomic actions as a result of binding to the androgen receptor (AR). The AR belongs to the family of nuclear receptors that act as ligand-responsive transcription factors (4). In the testis, AR has been localized to Leydig cells, peritubular cells, and Sertoli cells (5, 6). T freely diffuses through the plasma membrane and binds AR, forming a complex that subsequently interacts with the androgen response element in the promoter region of targeted genes. The transcription of targeted genes can be either induced or repressed depending on the factors that associate with the ligand-receptor complex bound to the androgen response element (7, 8). According to evidence collected from many sources, the concentration of T in the testes of the adult rat ranges from 25–100 ng/ml (9). At puberty, there is an increase in T levels (10), suggesting that higher intratesticular concentrations of T might be required to initiate spermatogenesis in mammals. In the adult mammalian male, T is probably the most important factor in the maintenance of normal qualitative spermatogenesis, i.e. T withdrawal leads to incomplete spermatogenesis and infertility in the male. To demonstrate the importance of T in spermatogenesis, various experimental protocols, including ethane dimethane sulfonate treatments (11), administration of antiandrogens (12, 13), immunoneutralization of LH (14), and hypophysectomy (15, 16), have been used to generate T depletion in the rat testes.
The GnRH-deficient or hypogonadal (hpg) mouse is a naturally occurring mutant mouse in which a major deletion in the GnRH gene results in hypogonadism (17). Because this mouse strain lacks significant secretion of FSH and LH, the circulating levels of androgens are very low or nonexistent. These mice exhibit infantile testes with spermatogenesis halted at the first meiosis, rendering the mice infertile (18). Nonetheless, the hpg mice possess a hormonally responsive reproductive tract, and it has been shown that androgen replacement is capable of initiating qualitatively complete spermatogenesis (19, 20) and fertility (21) in the GnRH-deficient mouse strain. Even though it is well established that T is crucial in the initiation and maintenance of spermatogenesis, most of the molecular targets for the action of T in the testis remain unknown. To understand the effects and actions of T in spermatogenesis, it is very important to know what genes are involved in the targeted cells of the testis. One example of a T-regulated gene is placentae and embryos oncofetal gene (Pem) that codes for homeobox protein. The Pem gene has previously been reported to be regulated by androgens in both the testis and in the epididymis (22, 23). In addition, the expression of Pem transcript has been shown to increase after androgen administration to hpg mice (24).
The purpose of this study was to identify genes that are regulated by T in the mouse testis and to monitor their expression over 24 h. We have taken advantage of Affymetrix Murine oligonucleotide arrays (Affymetrix, Santa Clara, CA) to study the expression pattern of T-regulated genes in testes of hpg mice at various time points from 0–24 h after T treatment of whole hpg mice. The Affymetrix Murine Genome U74 version 2 set comprises three different GeneChips (A, B, C) with a total of about 36,700 transcripts, making it a powerful tool to analyze the expression of thousands of genes simultaneously. As a result of this study, we have identified a significant number of transcripts the expressions of which were altered after testosterone proprionate (TP) treatment of hpg mice.
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RESULTS |
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Data generated for each of the experiments have been posted as supplemental data on The Endocrine Society’s Journals Online web site (http://mend. endojournals.org). The public can also access these data on other web sites including Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and the Griswold laboratory web page (www.wsu.edu/
griswold/ microarray/).
Short-Time Point Treatments (4, 8, 12, and 24 h)
The overall expression of genes in the testes of hpg mice was examined after the different TP treatments. The absolute analysis generated by Microarray Suite version 5.0 (MAS 5.0; Affymetrix) revealed that about 42% of the transcripts on chip A, 35% of the transcripts on chip B, and 18% of the transcripts on chip C were present in the testes of hpg mice. In summary, 32% of the 36,899 transcripts contained on these three chips were identified as being present in the testes of hpg mice.
To verify that hpg mice responded to the diverse TP treatments, we monitored the expression of Pem transcripts at each time point after the TP treatments. The experimental design of long-term TP treatment is shown in Fig. 1
. Pem was not detected in testes of control hpg mice; however, its signal increased in testes of hpg mice after TP treatments and started to be detected after 8-h TP treatments (Fig. 2). In the testis of an adult wild-type mouse, Pem has a signal of about 243. A signal of 100 represents approximately 1 pM mRNA, a mRNA present in low abundance.
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Percentage wise, 34, 33, 23, and 63% of the genes regulated by TP were up-regulated at 4, 8, 12, and 24 h, respectively, whereas 66, 67, 77, and 47% of the genes affected were down-regulated at those same time points, respectively. The actual numbers of genes regulated at each time point, for chips A, B, and C, combined, are shown in Fig. 3.
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. Names, GenBank accession numbers, and expression fold change of representative transcripts, selected arbitrarily, are listed in Tables 2 and 3. These transcripts were increased (Table 2) or decreased (Table 3) according to the criteria that were set to select candidates from comparison analyses between samples from the different experimental TP treatment time points and the samples from untreated controls. The expression patterns of both whey acidic protein (WAP) and histocompatibility 2, region locus D 1 (H2-D1) were altered after TP treatment. These two transcripts were selected because of the function of the proteins they encode and were confirmed by real-time PCR for the different experimental time points (Fig. 4).
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These analyses revealed that, according to our candidate selection criteria, a total of 81 transcripts were differentially expressed between the T+T arrays and the T+O array, 96% of which were decreased and 4% were increased (Fig. 6).
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lists the accession number, name, and expression fold change from MAS 5.0 for some of the genes that were regulated in testes of hpg mice after the T+T treatment compared with the T+O treatment. All three transcripts the expression of which increased in T+T compared with T+O are listed, whereas transcripts that were decreased and that are listed were selected mainly because of the interesting function of the proteins they encode. Real-time PCR has confirmed the induction of glucose transporter member 3 and the repression of nuclear protein 220 in T+T groups compared with the T+O group (Table 5).
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Venn diagrams were generated to detect transcripts that were commonly regulated between various time points. Figure 7 shows a Venn diagram of three different comparison analyses including T 4 h vs. C, T 24 h vs. C, and T+T vs. T+O. This analysis shows that four transcripts, including H2-D1, acidic nuclear protein 32 (ANP32), histocompatibility 2 locus K2, and procollagen type 1 
1 were commonly regulated in testes of hpg mice after TP treatment for 4 and 24 h. T commonly regulated three transcripts, including, DAZ-l, gene-encoding gag protein, and an expressed sequence tag (AA716963), after 24-h treatment and in longer term T+T treatment. However, no transcripts were commonly detected as regulated after 4-h short-term treatment and T long-term treatment followed by T depletion with subsequent T treatment for 4 h (T+T).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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This study has shown that, when treated with T for 4, 8, and 12 h, murine testicular mRNAs that were affected by the treatment were mostly decreased. Perhaps due to the lack of stimulation by gonadotropins, which are absent in these mice, somatic cells remain immature in the testes of hpg mice despite their adult age. These speculations are partly supported by a recent investigation by Haywood et al. (26), which demonstrated that 21-d-old hpg mice possess immature Sertoli cells. In normal mice, both circulating and testicular T concentrations start rising around postnatal d 20 (10) after Sertoli cells are believed to have ceased proliferating (27). Initially, T might play a role in the maturation of Sertoli cells and/or peritubular cells by decreasing or repressing the expression of those genes required by the cells for growth and proliferation. After 24 h of T treatment, most changes involved an increase in transcripts levels, possibly as a result of activation of gene transcription in germ cells.
The overall decrease in abundance of mRNAs that was observed in testes of hpg mice in earlier time points of TP treatment might have been due either to the fact that these mice had cryptorchid testes or that these mice had no previous exposure to T. Implementing the experimental strategy of pretreating the mice with TP over 2 wk resulted in germ cells completing meiosis and circumvented these potential problems. It is well documented that murine models that exhibit cryptorchid testes have spermatogenesis halted before meiotic division (18, 28, 29). The T+T/T+O treatment resulted in testes that contained postmeiotic germ cells. Microarray analysis of RNA from testes of T+T-treated hpg mice compared with that of T+O-treated animals showed an overall decrease in the abundance of testicular mRNAs, consistent with the results observed in the short single 4-h T treatments. The transcripts that were affected were not the same in both treatment regimens even though mice in both groups were similarly given the final TP injection 4 h before they were killed. Nevertheless, these results are not surprising because in both experiments, the environments of the testes were quite different. In the long-term T treatment, a subset of genes that were repressed during spermatogenesis by T might have resurfaced when the hpg mice remained for 2 wk without TP treatments. The last T booster would then suppress such genes before spermatogenesis could resume its course.
Nuclear protein 220 and matrin 3 encode for nuclear proteins that have RNA binding motifs and have been speculated to play a role in pre-mRNA splicing (30). We have found the levels of both transcripts to be reduced in the T+T treatment regimen compared with the T+O treatment. Expression of RNA binding proteins is critical during translational control of mRNA such as protamines that are involved with germ cell differentiation (31). It is possible that the regulation of RNA binding proteins subsequently plays a role in controlling the translation of testicular proteins.
A group of transcripts that were up-regulated as a result of 4-h TP treatment in testes of hpg mice are from genes that encode for major milk proteins. These transcripts are expressed in the mammary glands and have not been previously described in the mouse testis. By 8 h after TP treatment, these transcripts disappeared and were not detected again during the course of the experiment. It is possible that this phenomenon is inherent in the hpg mouse model. Nonetheless, another possibility is that those transcripts are involved in very specific events at a specific time during testis development. That would explain the transient appearance of these transcripts after a specific time point of TP treatment. In the testes of normal mice, we detected WAP and the group of casein transcripts transiently between d 5 and d 10 postpartum (data not shown). It is also known that estrogen will up-regulate prolactin (PRL) mRNA in the anterior pituitary (32), and PRL, in turn, can induce the expression of classical mammary gland genes (33). Therefore, TP administered to hpg mice might get aromatized to estrogen, which could have resulted in up-regulation of PRL in the pituitary. Systemic PRL could have subsequently led to the induction of WAP and caseins in testicular cells of hpg mice. However, neither PRL nor its receptor transcripts, which are both present on U74Av2 GeneChip, were shown to be increased at 4 h after TP treatment. More experiments are needed to provide an explanation for the presence of WAP and caseins in testes of hpg mice after 4 h of TP treatment.
Another group of transcripts noticeably regulated by T was that of class I major histocompatibility complex (MHC) to which H2-D1 belongs. MHC class I molecules are involved in self/non-self recognition and have previously been shown to be present on Sertoli cells (34). H2-D1 was also detected as present in cultured primary Sertoli cells. In addition, the role of Sertoli cells in immunosuppression has been reported (34, 35). We have shown that the expression of H2-D1 and other MHC class I genes such as ß2-microglobulins were decreased in murine testis after TP treatment at different time points. These results suggest a potential role of T in immunosuppression in the murine testis. A role for T in systemic immunosuppression has previously been documented in male mice (36).
In summary, genes that are regulated by T at 4, 8, 12, and 24 h in testes of hpg mice were identified. After 24 h, most of the transcripts affected were up-regulated, whereas at the earlier time points, most of the transcripts affected by the TP treatments were down-regulated. Some transcripts that represent proteins that could play a major role in the T regulation of spermatogenesis could have been omitted if they did not meet the criteria that were set for stringency purposes, i.e. if they did not show a change of 2-fold or more with signal strength of 100 in all pairwise comparison analyses. It is also possible that a set of transcription factors were induced as soon as TP was administered, i.e. earlier than 4 h. These unknown transcription factors might have acted as switches to suppress the expression of genes that are not needed during spermatogenesis or that are not needed at a specific period during the testis development. Along the same line, among the genes regulated by T in the testes of hpg mice, those that were directly regulated were not discernable from those that were indirectly regulated. These studies have established a foundation for additional investigations that could lead to in-depth understanding of the action of T in the development of the testis and subsequently in spermatogenesis.
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MATERIALS AND METHODS |
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hpg Mice have undescended testicles (25), and to ensure that any effects of T on gene expression were not due to cryptorchidism, hpg mice were given TP for longer periods of time. This experimental strategy was as follows (see Fig. 1
): for 5 d, hpg mice received a single injection of 5 mg TP every other day, and these mice were left for 14 d without any other treatment. On the 14th day after the last TP injection, the mice were separated into groups of T+T (three groups) or T+O (one group). The mice in the T+T groups were injected with 25 mg TP whereas the mice in the T+O group instead received the vehicle and were considered as the control group for this experimental section. The animals in all the groups were killed 4 h later, their blood was collected by cardiac puncture, and their testes were excised and immediately homogenized in Trizol reagent for RNA extraction. Each group consisted of a single mouse.
T RIA
T levels in the serum from TP-treated and control hpg mice were determined using a Coat-a-Count kit following the manufacturer’s instructions (DPC Laboratories, Los Angeles, CA). This kit has a sensitivity of 0.05–25 ng/ml and a cross-reactivity of less than 6.6%.
RNA Extraction and GeneChip Target Preparation
Total RNA was extracted from whole testes that were excised from our experimental mice using the Trizol reagent (Invitrogen, San Diego, CA) according to the manufacturer’s recommendations. RNA concentration, purity, and integrity were assessed by measuring the 260:280 nm ratios and by fractionation in 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). Total RNA (10 µg) was reverse transcribed into single-stranded cDNA, and the single-stranded cDNA was converted to a double-stranded cDNA. The double-stranded cDNA was extracted using a phaseLock gel (Eppendorf, Hamburg, Germany) and precipitated with ethanol and ammonium acetate. All of these procedures were carried out as described by Affymetrix. 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 UTP 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. The hybridization cocktail consisted of 15 µg fragmented biotin-labeled cRNA spiked with eukaryotic hybridization control. The Murine Genome U74 A, B, or C version 2 microarrays were directly loaded with 200 µl of the hybridization cocktail and 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
Affymetrix Murine Genome U74 version 2 (MG U74v2) set, which consists of three different arrays (denoted A, B, and C), was used for each of the different time points (0 h, 4 h, 8 h, 12 h, and 24 h), and each time point was done in duplicate. For T+T and T+O experiments, only GeneChip A was used. According to Affymetrix, MG U74v2 set comprises about 96% of the mouse genome. The total probe sets in each of the arrays was 12,488 for GeneChip A, 12,477 for GeneChip B, and 11,934 for GeneChip C. After the arrays were scanned, the signals generated were determined and analyzed by MAS 5.0 software. The absolute and comparison analyses were also performed using MAS 5.0.
The absolute analysis includes output such as the signal strength, or the abundance of a specific transcript, and its detection, i.e. whether that transcript is present or absent in the sample analyzed. We have included some data from absolute analyses of RNA from normal wild-type mice testis.
The comparison analysis compares an experimental array to a baseline array so as to monitor changes in the expression of transcripts across the samples targeted to different arrays (refer to www.Affymetrix.com for details on the statistics of these analyses).
To determine which transcripts were significantly regulated by T at 4, 8, 12, or 24 h post treatment, pairwise comparison analyses were performed by MAS 5.0 using the control hpg mice expression levels as time point zero. Time point zero served as the baseline data in all the comparisons done for the short-time points treatments.
GeneSpring Analysis
To monitor the expression of chosen genes over the different experimental time points, data obtained from MAS 5.0 absolute analyses of all the individual arrays were analyzed and clustered using GeneSpring according to the supplier’s recommendations (Silicon Genetics, Redwood City, CA). To identify transcripts the expression of which was affected in two or more experimental time points, we generated Venn diagrams. For this purpose, transcripts that were significantly and consistently regulated across all the pairwise comparisons were imported from MAS 5.0 into GeneSpring where different combinations of Venn diagrams were constructed.
Real-Time PCR
A two-step real-time PCR was carried out to analyze and confirm the expression of candidate genes. RT-PCR was used to generate cDNA from TP-treated and untreated hpg mice testes. Total RNA (1 µg) was reverse-transcribed into cDNA in a reaction primed by oligo deoxynucleotide T (dT)12–15 primer using Superscript II reverse transcriptase (Invitrogen) 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 1 lists the sequences 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, Foster City, CA). The previously synthesized cDNA was used as template. Samples from each of the time points were plated in triplicate PCR reactions. The PCR reaction contained, about 5–10 ng of cDNA, 1 X 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 candidate genes expression fold change was calculated for the different T treatment time points using the formula 2-
CT as described in the SYBR Green user manual: CT = [(CT of gene of interest - CT of S2)A - (CT of gene of interest - CT of S2)B.] in which A = T-treated sample at a particular time point and B = control sample.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AR, Androgen receptor; H2-D1, histocompatibility 2 region, locus D1; MAS 5.0, Microarray Suite version 5.0; MHC, major histocompatability complex; PRL, prolactin; T, testosterone; TP, testosterone proprionate; WAP, whey acidic protein.
Received for publication May 22, 2003. Accepted for publication October 30, 2003.
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