Estrogen-Regulated Genes in Rat Testes and Their Relationship to ...

BIOLOGY OF REPRODUCTION 85, 823–833 (2011) Published online before print 8 June 2011. DOI 10.1095/biolreprod.111.091611

Estrogen-Regulated Genes in Rat Testes and Their Relationship to Recovery of Spermatogenesis after Irradiation1 Wei Zhou,3,6 Olga U. Bolden-Tiller,3,4,6 Shan H. Shao,6 Connie C. Weng,6 Gunapala Shetty,6 Mahmoud AbuElhija,6 Pirjo Pakarinen,8 Ilpo Huhtaniemi,5,8 Amin A. Momin,7 Jing Wang,7 David N. Stivers,7 Zhilin Liu,9 and Marvin L. Meistrich2,6 Departments of Experimental Radiation Oncology6 and Bioinformatics and Computational Biology,7 University of Texas M.D. Anderson Cancer Center, Houston, Texas Department of Physiology,8 University of Turku, Turku, Finland Department of Molecular and Cellular Biology,9 Baylor College of Medicine, Houston, Texas spermatogenesis following gonadotoxic exposures, such as those resulting from cancer therapy.

ABSTRACT

estradiol/estradiol receptor, estrogen, gene expression, irradiation, Leydig cells, microRNA, spermatogenesis, spermatogonia, testis, testosterone

INTRODUCTION During studies of regulation of spermatogenic recovery in rats after gonadotoxic therapy for cancer, we found that estrogen stimulated the recovery of spermatogonial differentiation [1–3], but the mechanism of action was not identified. Estrogens indeed have widespread effects on spermatogenesis and male reproduction, but those effects and the mechanisms involved are quite complex [4, 5]. It is important to understand not only the regulatory effects of endogenous estrogens, primarily 17b-estradiol (E2), but also the harmful effect of exogenous estrogenic compounds to which the human and animal populations are exposed [6]. The deleterious effects of E2 treatment on spermatogenesis in adult mice and rats have long been known [7, 8]. Treatment with E2 also inhibits sperm production in juvenile mice [9] and spermatogonial differentiation in neonatal rats [10]. On the other hand, E2 has beneficial effects on spermatogenesis. Although E2 is not absolutely required for fertility in young mice, spermatogenesis degenerates with time in mice lacking either estrogen receptor a [11] or aromatase [12]. Surprisingly, E2 treatment has been shown to restore spermatogenesis in hypogonadal (hpg) mice that normally have undetectable serum gonadotropin levels [13]. Furthermore, in neonatal rats treated with follicle-stimulating hormone (FSH), E2 actually increases spermatogonial and spermatocyte numbers [10]. Also, E2 as well as an estrogen receptor b-selective ligand, 3b-androstanediol, stimulated spermatogonial DNA synthesis in vitro in tubule segments from adult rat testes [14]. Finally, as we have recently observed, in irradiated rats treated with the gonadotropin-releasing hormone antagonist (GnRH-ant) acyline and the androgen receptor-antagonist flutamide, the addition of E2 to the treatment regimen markedly accelerated and enhanced the recovery of spermatogonial differentiation [3]. Elucidating the molecular mechanisms of direct estrogen action on the testis and seminiferous tubules that might be involved in these effects is generally complicated by estrogen’s multiple indirect actions on other tissues and on areas of the reproductive tract that affect the physiology of the testis and its gene expression [15]. In normal animals, E2 acts on the hypothalamus and pituitary to decrease gonadotropin secretion [16], although in the hpg mouse, it can increase FSH levels

1 Supported by NIH research grant ES-08075 (to M.L.M.), Cancer Center Support Grant CA-16672 (to M.D. Anderson Cancer Center), training grant T32 HD-07324, and research grant HD-16229 (to Joanne Richards, Baylor College of Medicine). 2 Correspondence: M.L. Meistrich, Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77025. FAX: 713 794 5369; e-mail: [email protected] 3 These authors contributed equally to this work. 4 Current address: Department of Agricultural and Environmental Sciences and George Washington Carver Experiment Station, Tuskegee University, Tuskegee, AL 36088. 5 Current address: Department of Reproductive Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, U.K.

Received: 10 February 2011. First decision: 4 March 2011. Accepted: 24 May 2011. Ó 2011 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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Despite numerous observations of the effects of estrogens on spermatogenesis, identification of estrogen-regulated genes in the testis is limited. Using rats in which irradiation had completely blocked spermatogonial differentiation, we previously showed that testosterone suppression with gonadotropinreleasing hormone-antagonist acyline and the antiandrogen flutamide stimulated spermatogenic recovery and that addition of estradiol (E2) to this regimen accelerated this recovery. We report here the global changes in testicular cell gene expression induced by the E2 treatment. By minimizing the changes in other hormones and using concurrent data on regulation of the genes by these hormones, we were able to dissect the effects of estrogen on gene expression, independent of gonadotropin or testosterone changes. Expression of 20 genes, largely in somatic cells, was up- or downregulated between 2- and 5-fold by E2. The unexpected and striking enrichment of transcripts not corresponding to known genes among the E2-downregulated probes suggested that these might represent noncoding mRNAs; indeed, we have identified several as miRNAs and their potential target genes in this system. We propose that genes for which expression levels are altered in one direction by irradiation and in the opposite direction by both testosterone suppression and E2 treatment are candidates for controlling the block in differentiation. Several genes, including insulin-like 3 (Insl3), satisfied those criteria. If they are indeed involved in the inhibition of spermatogonial differentiation, they may be candidate targets for treatments to enhance recovery of

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FIG. 1. Schematic of experimental design. The major end point of the present study was the change in expression of genes in irradiated rat testes after a short period of hormonal suppression (2 wk). Tissues were collected at the 4-wk time point to assess the degree of recovery of spermatogenesis induced by the treatments. Comparison between the XAF and X groups indicates the effects of suppression of testosterone and gonadotropins. Comparison between XAFE and XAF groups indicates the effects of E2.

MATERIALS AND METHODS Animals LBNF1 male rats were obtained from Harlan Sprague Dawley and irradiated at approximately 8 wk of age. Animals were housed under a 12L:12D photoperiod and allowed food and water ad libitum. Animal facilities were approved by the American Association for the Accreditation of Laboratory Animal Care, and all procedures were approved by the M.D. Anderson Cancer Center Institutional Animal Care and Use Committee.

Irradiation Rats were anesthetized and irradiated to the lower part of the body with a single dose of 6 Gy of gamma radiation, as described previously [27].

Animal Groups and Treatments The experimental design is illustrated in Figure 1. All three groups of animals were irradiated. One group received no other agents, just sham treatments (X group). The other two groups were treated, starting 15 wk later, with the GnRH-ant acyline and either flutamide (XAF) or flutamide plus E2 (XAFE) for 2 or 4 wk. Treatments with acyline and flutamide were as described previously [24]; E2 was administered in 2-cm Silastic capsules [3]. Some of the results from the X and XAF groups were reported earlier [24]; for the XAFE group, the animals were treated and testicular RNA extracted, prepared, and analyzed by microarray concurrently with the others. Rats were killed after 2 wk of hormonal treatment, before appreciable differentiation of spermatogonia occurs, for RNA extraction, hormone measurements, and histological analysis. One testis was processed for RNA extraction. A portion of the other testis was fixed in Bouin solution for histological examination, and the remainder was homogenized for ITT measurements. Some rats were killed after 4 wk of hormonal treatment to analyze the extent of recovery of spermatogenesis by histology.

Assessment of Spermatogenesis Spermatogonial numbers were counted in Bouin-fixed, methacrylateembedded tissue stained with periodic acid-Schiff reagent and hematoxylin. Counts were done across the entire testis using the Stereo Investigator software package (MicroBrightField) to superimpose an unbiased counting frame on the video image. Sertoli cell nuclei, which were much more numerous, were counted in every 10th field, but only when their nucleoli were visible [21]. The tubule differentiation index was determined by counting the number of tubules with differentiating germ cells (at least three cells that had reached the B spermatogonial stage or later) in sections [28].

Hormone Measurements Serum testosterone and ITT concentrations were measured using coatedtube radioimmunoassay kits (DSL 4000; Diagnostic Systems Laboratories); the use of this assay for direct measurements of ITT was previously validated [22].


[13]. The reduction in luteinizing hormone (LH), as well as direct estrogen inhibition of the steroidogenic process in Leydig cells, contributes to reduced levels of testosterone within the testis [17]. The resulting changes in LH, FSH, and testosterone levels induce changes in gene expression within the testis. Furthermore, estrogen inhibits testicular descent [18] and is required for absorption of seminiferous tubule fluids in the epididymis [19]; hence, alterations in estrogen levels can exert indirect effects on the testis by these mechanisms. Finally, in most of the studies noted above, changes in estrogen levels usually resulted in major alterations in the germ cell numbers in the testis; any attempts to study E2-induced changes in gene expression were confounded by major changes in germ cell gene levels and changes in somatic cell gene expression in response to the altered germ cell complement. To study the more direct effects of E2 on testicular gene expression, we performed microarray analysis on testes of irradiated LBNF1 rats treated with the GnRH-ant acyline and flutamide, with and without additional E2. The testes of these rats are sensitive to low doses of radiation [20], and although the stem type A spermatogonia survive and remain in the testis, no differentiation of these cells could be observed following irradiation. However, this block was slowly reversed by treatment with GnRH-ant and flutamide, which suppresses intratesticular testosterone (ITT) and gonadotropin levels and inhibits testosterone action [21, 22]. Further studies showed that ITT (and, to a lesser extent, FSH) primarily was responsible for the inhibition of differentiation in this system [23]. The use of this model to study E2-induced changes in gene expression in the somatic cells of the testis overcomes many of the complexities present in other systems. The GnRH-ant treatment reduces gonadotropins and testosterone to very low levels, and flutamide blocks any action of residual androgen, so that changes in these hormones are unlikely to be confounding factors [3]. Any remaining possibility that small changes in these hormones may be involved in the response to E2 treatment can be checked against previous data on the regulation of these genes by testosterone, LH, and FSH in this model system [24]. In addition, we can eliminate major changes in germ cell composition as a significant confounding factor, because type A spermatogonia are the only germ cells present in the irradiated testes and their differentiation does not begin at the time at which we analyze the gene expression. Identifying the genes regulated by both testosterone and E2 will enable us to explore the molecular mechanisms by which

androgen suppression and estrogen treatment can restore spermatogonial differentiation in this irradiated rat model and, thereby, gain insight regarding the mechanisms by which the differentiation is blocked after irradiation. Understanding this block in spermatogenesis after irradiation and other toxic exposures is important to prevent or reverse sterility caused by cancer therapy and environmental gonadotoxic exposures [25]. Previously, we showed that radiation damage to the somatic elements of the LBNF1 rat testes, not the spermatogonia, was responsible for this block [26]. The reversal of this block by suppression of testosterone is further evidence that the somatic cells are responsible [1], because the germ cells do not express androgen receptor. The further restoration of spermatogonial differentiation by additional E2 treatment [3] can also be a result of action on somatic cells, because the Leydig cells and, likely, the Sertoli and peritubular cells contain estrogen receptors [5]. Therefore, we designed the present study to test whether genes or other transcripts were significantly regulated by E2 and could be considered as candidates for blocking spermatogonial differentiation after toxicant exposure.

ESTROGEN-REGULATED GENES IN IRRADIATED RAT TESTES Rat serum FSH and LH levels were measured using immunofluorometric assays (Delfia; PerkinElmer Wallac) [22, 23, 29, 30]. Serum E2 was measured using an anti-E2 antibody, double-antibody radioimmunoassay kit (catalog no. DSL-4800; Diagnostic Systems Laboratories) [3].

RNA Preparation and Microarray Analysis The RNA extraction methods, quality control, microarray hybridization on the Rat 230 2.0 GeneChips (Affymetrix), and data analysis were as previously described [24]. Eleven samples from the XAF group and six samples from the XAFE group, extracted after 2 wk of hormonal treatment, were included in the microarray analysis presented here. The presentation of expression levels, criteria for scoring probes as present, statistical analyses of differences, and probe rankings were as described elsewhere [24]. Note that the expression levels were normalized within each chip by dividing by the median value for all the probes for which expression levels were regarded as being present on the array and then log base 2 transformed. As previously [24], expression levels were considered to have significant changes between the XAF and XAFE treatment groups if the means of the log-transformed expression had a difference of greater than 1/3 (.1.26fold differences on an untransformed scale) and the difference was significant at P , 0.05. Array data sets were deposited in Gene Expression Omnibus as GSE24672. The false-discovery rates (FDRs) of E2-regulated genes based on the differences between the XAF and XAFE groups were calculated using the Significance of Analysis of Microarrays program [31].

Probe set identification numbers of unannotated probes that are differentially expressed by at least 1.4-fold upon estrogen treatment were identified. Genomic coordinates complementary to the selected probes were obtained from the associated annotation file for Rat 230 2.0 GeneChips. During this step, multiple coordinates associated with certain probes were included, whereas ambiguous chromosome locations were discarded. In addition, the coordinates for rat hairpin miRNAs (version 16, Genome Assembly: RGSC3.4) were obtained from mirBase [32]. Both lists were formatted as .BED files, and the coordinates were compared to identify probes within 5000 bp of known miRNAs using BEDTools [33]. To screen potential target genes of each miRNA, the MicroCosm Targets website resources (http://www.ebi.ac.uk/enright-srv/microcosm/cgi-bin/targets/ v5/search.pl) were utilized [34]. Briefly, the potential target gene list of each miRNA was extracted from the database. Then, the genes were selected based on two criteria: first, consistent upregulation in the E2-treatment group by all probes for the gene, and second, significant change (fold-change . 1.4 and P , 0.05).

Quantitative Real-Time PCR Methods used and primer sequences for Pah, Ces, and Rps2 were as described previously [24]. The primers used for Isyna1, Vldlr, Insl3, and the primary and preisoforms of miRNAs are given in Supplemental Table S1 (all Supplemental Data are available at www.biolreprod.org). Relative levels of mRNA concentrations were calculated by normalizing to the mRNA levels for Rps2. The samples used in the real-time PCR were partially overlapping with those used in microarray hybridization.

Transcription Factor-Binding Sites Analysis The occurrence of transcription factor-binding sites in the promoter regions of the E2-upregulated and E2-downregulated genes (.1.5-fold change, P , 0.05, FDR , 10%) was compared with that of the promoter regions of the unregulated genes. The TRANSFAC database was used and analyzed using the MATCH algorithm within the ExPlain 3.0 analysis system software (Biobase Corporation).

RESULTS Cellular Complement and Hormone Levels As observed previously, despite the fact that irradiation produced extensive testicular atrophy, undifferentiated spermatogonia remained in testes but failed to differentiate (Fig. 2, A and D). Suppression of testosterone levels and action with acyline and flutamide resulted in a doubling of the relative

numbers of the type A spermatogonia within 2 wk of treatment (Figs. 2B and 3A), but differentiated cells were rarely observed at this time (Figs. 2E and 3B) [21, 24]. Continuation of the hormonal suppression stimulated the differentiation of spermatogonia in some tubules, which occurred between 2 and 4 wk. Addition of E2 to this regimen did not induce further differentiation beyond type A spermatogonia within the first 2 wk, but it did produce an additional doubling of relative spermatogonial numbers (Figs. 2C and 3A). This additional E2 treatment did initiate the changes, such as those in gene expression, which most likely resulted in a marked stimulation of the differentiation process between 2 and 4 wk of treatment (Figs. 2F and 3B). The changes in hormone levels after 2 wk of treatment with acyline plus flutamide (Fig. 4) have been discussed previously [24]. The addition of E2 to the acyline/flutamide treatment elevated serum E2 from 30 pg/ml (control level) to 110 pg/ml (Fig. 4C). Previously, we demonstrated that the 2-cm E2 capsules raised intratesticular E2 levels from 57 to 136 pg/ml [3]. The present experiments confirmed that the E2 treatment produced a significant further reduction in ITT levels (Fig. 4B). However, this reduction should not affect gene expression, because the action of testosterone is inhibited by flutamide [3]. The 2-wk E2 treatment had no significant effect on LH or FSH levels (Fig. 4, D and E), although in a previous study [3], the addition of E2 to a 4-wk treatment with acyline and flutamide did produce a small, but significant, increase in FSH. Estrogen-Regulated Genes Previously, we described the changes in testicular gene expression following treatment of irradiated rats with acyline combined either with flutamide or testosterone or with flutamide plus FSH [24]. Here, we report the E2-induced changes by comparing the gene-expression levels on microarray analysis of testicular RNA from rats treated using acyline, flutamide, and E2 with those receiving just acyline and flutamide for 2 wk, at which time the only observed change in cell number was the increase in type A spermatogonia (Fig. 3A). Expression levels and changes for all probes are presented in the Supplemental Table S2. In all, 477 probes were upregulated by E2 (using the 1.26fold change criterion). However, this may be an overestimate, because nearly half of the probes were in groups with an FDR of between 10% and 35%. Of these 477 probes, 97% were associated with UniGene entries (Table 1), and 439 of the entries were distinct. Of these 439 genes, 86% were named genes, and the rest were only described as transcribed loci in GenBank. A larger number of probes (n ¼ 1365) were downregulated by E2, all with an FDR of less than 7%. Only 90% of these probes were associated with UniGene entries, and 1155 of the entries were distinct. Of these 1155 genes, only 46% were named genes, and the rest were not identifiable. Both the frequencies of association of probes with UniGene entries and the identification of the UniGene entries with named transcripts were highly significantly lower in the group of downregulated genes than in the upregulated ones. Furthermore, when only the more highly E2-regulated genes (.1.8-fold change) were analyzed, the distinction between the groups was more dramatic: 87% of the upregulated genes were named genes, but only 21% of the downregulated ones were named (Table 1). The genes that were up- and downregulated by E2 more than 2-fold are listed in Table 2. The directions of change for about half of them were confirmed by more than one probe on


MicroRNA Analysis

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FIG. 2. Histology of rat testes after irradiation and with various hormonal treatments. The stimulation of spermatogonial proliferation and differentiation by acyline and flutamide (B and E) compared to controls (A and D) is further enhanced by additional treatment with E2 (C and F). A–C) Undifferentiated spermatogonia (arrows) in tubules of irradiated rats after 2 wk of hormonal treatments. D–F) Differentiated germ cells in tubules of irradiated rats after 4 wk of hormonal treatments. Insets show higher magnifications of the areas marked with rectangles (D, type A spermatogonium; E, leptotene primary spermatocytes; F, pachytene spermatocyte). Bar ¼ 20 lm. (A– C) and 100 lm (D–F).


the microarray. The changes in expression levels for five of these genes (Isyna1, Vldlr, Insl3, Ces, and Igfbp3) were confirmed by real-time PCR (Fig. 5, A–D and F). Because Insl3 and Ces3 are genes in the Leydig cells that are downregulated both by ITT suppression and by addition of E2, we examined other genes that previously were shown to be in the interstitial area and to be strongly downregulated by ITT suppression [24], in particular Pah and Ass1. Microarray analysis showed that Ass1 was indeed downregulated 1.7-fold by E2. Because Pah expression was scored as absent by microarray in both the ITT-suppressed (XAF) and the E2supplemented (XAFE) groups, we analyzed it by real-time PCR, which showed that Pah was also downregulated 2.8-fold by E2 treatment (Fig. 5E).

Because most of the E2-downregulated sequences had not been identified as known genes, we hypothesized that some of these UniGene entries might actually represent miRNAs. Thus, we determined whether known miRNAs (UCSC Genome Browser Baylor 3.4/m4 assembly, November 2004) were within 5 kb of the probes downregulated at least 1.4-fold by E2. For 10 of these probes, miRNAs were within 5 kb on the rat genomic sequence (Table 3). Because 13 of the 14 miRNAs identified were transcribed from the same DNA strands as the transcripts identified by the probes, the probes are most likely detecting the miRNA transcripts. In further support that this is not a random finding, none of the E2-upregulated unannotated probes was in close proximity to miRNAs.

ESTROGEN-REGULATED GENES IN IRRADIATED RAT TESTES

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FIG. 3. Changes in numbers of undifferentiated spermatogonia (A) and spermatogonial differentiation as assessed by tubule differentiation indices (B) in testes of irradiated rats receiving different hormonal treatments for 2 or 4 wk. Tubule differentiation index values for the X and XAF groups at 2 wk are from Zhou et al. [24]. A t-test (with Bonferroni correction for multiple comparisons) was used, with P , 0.05 as the level for significance (n ¼ 4–17 rats/ group); values significantly different between the X and XAF groups (*) and between the XAF and XAFE groups ( ) are indicated. Comparisons were done separately with the 2- and the 4-wk values.


Real-time PCR analysis was first performed for Mir34a, which had the highest fold-change in expression level upon E2 addition, using primers for pri-mir34a (pri34, which does not detect pre-mir34a) and for pre-mir34a (pre34, which also detects pri-mir34a) transcripts. Upon addition of E2 to the treatment regimen, RNA levels with both primer pairs markedly decreased (2.2- to 2.6-fold) (Fig. 5G), consistent with the 3-fold decrease in the expression of the associated microarray probe. Additional real-time PCR analyses were performed on four miRNAs that the microarray results indicated were downregulated 1.5- to 2.1-fold by E2 (Table 3). The real-time PCR results indicated that the pri-miRNAs for all four miRNAs and the pre-miRNAs for two of them (proper primers could not be designed for the other two) showed the same trend of downregulation by E2 of 1.2- to 1.9-fold (Supplemental Table S3). However, because of variability between animals, only the downregulation of pre-mir-103-2 was statistically significant, which was not unexpected due to the relatively low foldchanges. The potential target genes of the E2-downregulated miRNAs were further screened, and the 16 significant candidate genes are listed in Table 3. Among them, Rasd1, Ghr, Homer3, and Fos were potential targets of multiple miRNAs. Transcription Factor-Binding Site Analysis We first analyzed the occurrence of estrogen receptorbinding sites in the promoter regions of the E2-regulated genes. The frequencies of sites were not significantly higher in either the E2-upregulated or E2-downregulated genes than in the promoter regions of the unregulated genes (ratio, 1.1), indicating that most of the genes are not primary targets for estrogen receptor but are indirectly regulated by E2. To determine whether common E2-regulated transcription factors might be responsible for the indirect regulation of these apparently E2-regulated genes, we analyzed the occurrence of known transcription factor-binding sites in the promoter regions. A binding site for CBFB (core-binding factor, beta subunit) and RUNX1,2,3 (runt-related transcription factors 1, 2, and 3) and a site for GFI1 (growth factor independent 1 transcription repressor) were enriched in the promoters of the E2-upregulated genes, and a site for POU1F1 (POU class 1 homeobox 1) and a site for CRX (cone-rod homeobox) and RAX (retina and anterior neural fold homeobox) were enriched in the promoters of the E2-downregulated genes. However,

FIG. 4. Levels of serum testosterone (A), ITT (B), serum estradiol (C), luteinizing hormone (D), and follicle-stimulating hormone (E) in irradiated rats receiving different treatments for 2 wk. The data are presented as the mean 6 SEM (n ¼ 5–21 rats/group) calculated from either untransformed data (FSH) or from log-transformed data (other parameters). Note that the levels of the serum testosterone, ITT, LH, and serum E2 are expressed on log scales. Values significantly different between the X and XAF groups (*) and between the XAF and XAFE groups ( ) are indicated (P , 0.05 by a ttest with Bonferroni correction). L indicates that the concentration was below the detection limit (shown by dashed line) in some or all samples in the group.

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TABLE 1. Summary of numbers of E2-upregulated and -downregulated probes and their association with UniGene entries and the annotation of those entries. Items evaluated

No. of E2-upregulated

No. of E2-downregulated

P value*

477 461 (97%) 439 373 (85%) 23 20 (87%)

1365 1225 (90%) 1155 533 (46%) 214 44 (21%)

3 3 106 2 3 1044 9 3 1012

Probes Probes with UniGene entries (% of probes) Unique UniGene entries Named genes (% of UniGene entries) Unique UniGene entries (.1.8-fold regulation) Named genes (.1.8-fold regulation; % of UniGene entries)

* Significance of the difference in frequency of genes in this category among the E2-upregulated and E2-downregulated genes.

TABLE 2. Genes that are maximally regulated by estradiol (.2-fold).

Gene (only named genes included)

Sulfotransferase family, cytosolic, 1C, member 2 (Sult1c2) Arachidonate 5-lipoxygenase activating protein (Alox5ap) Inhibitor of DNA-binding 1 (Id1) Downregulated by estradiol Myosin, heavy polypeptide 6, cardiac muscle, alpha (Myh6)e ATPase, Caþþ transporting, plasma membrane 1 (Atp2b1)e Insulin-like 3 (Insl3)d Cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1) Carboxylesterase 3 (Ces3) ATPase, Caþþ transporting, plasma membrane 3 (Atp2b3) EMI domain containing 1 (Emid1) Supervillin (Svil)e RAP1 interacting factor homolog (yeast) (Rif1)e Dermatopontin (Dpt)e Insulin-like growth factor binding protein 3 (Igfbp3)d,e Small nuclear ribonucleoprotein 48k (U11/ U12) (Snrnp48)e LUC7-like 3 (S. cerevisiae) (Luc7l3)e Fatty acid desaturase 1 (Fads1) a

Regulation by T, LH, FSHb

False discovery rate (%)

Putative cellular localization Irradiated rat microarrayc

Literature

þ3.9 þ2.8

ITT-up ITT-up

0% 0%

S/G G/S

Various Germ

þ2.3

ITT-up

0%

Somatic

Germ

þ2.3

LH-down

0%

Not calculablef

þ2.1

LH-up or ITT-up

2%

S/G

Spermatogonia, Leydig, Sertoli

þ2.0

LH-up or ITT-up

14%

Somatic

þ2.0

Unchanged

0%

S/G

5.0

ITT-down

0%

S/G

4.6

0%

Somatic

Sertoli, spermatogonia

3.7 3.6

LH-down or ITT-down ITT-up LH-up

0% 0%

Somatic Somatic

Leydig Leydig

2.8 2.5

ITT-up LH-up

0% 0%

Somatic Somatic

Leydig

2.4 2.4 2.3

LH-up Unchanged ITT-down

0% 1% 0%

Somatic Somatic Not calculablef

Spermatogonia, Sertoli Sertoli, spermatogonia Various

2.2

LH-up

0%

Somatic

2.1

Serum T-down

0%

Somatic

Spermatogonia, interstitial, Sertoli Spermatogonia, Sertoli

2.1

LH-down

0%

Uncertain

Spermatids, Sertoli

2.0 2.0

LH-down ITT-up

0% 0%

S/G Somatic

Spermatogonia, Sertoli Sertoli, interstitial, spermatogonia

Spermatogonia, interstitial, myoid Spermatogonia, Sertoli, myoid

For the listed genes, the average standard error of the means of the fold-changes was 17% of the mean fold-changes (XAFE vs. XAF). Determined from concurrent data presented in Supplemental Table S1 in Zhou et al., 2010 [24] by first comparing expression levels in irradiated rats treated with GnRH-ant þ flutamide, which downregulated testosterone (T) and gonadotropins, with those in irradiated-only rats and then comparing the expression levels with GnRH-ant þ testosterone and GnRH-ant þ flutamide þ FSH treatment regimens with GnRH-ant þ flutamide treatment to sort out the effects of ITT, serum T, LH, and FSH. c Determined from concurrent data presented in Supplemental Table S1 in Zhou et al., 2010 [24] by comparison of expression levels in irradiated rats with those in non-irradiated controls. d Confirmed by real-time PCR. e Qualitative results on E2-regulation confirmed by at least one more probe. If other probes expressed did not generally confirm the qualitative results, the gene was not listed. f The cellular localization could not be determined because probe was scored as ‘‘absent’’ in non-irradiated control and irradiated testis. The probe was only significantly expressed after hormonal suppression. b


Upregulated by estradiol Inositol-3-phosphate synthase 1 (Isyna1)d Very low density lipoprotein receptor (Vldlr)d,e SPARC-related modular calcium binding protein (Smoc1)e Stanniocalcin 1 (Stc1)e

Fold change upon E2 additiona


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they were enriched by only approximately 1.6-fold, and the statistical significance was marginal (P ; 0.005–0.01). Also, the microarray results indicated that the expression of these transcription factor genes was either not detectible (Runx1, Runx3, Gfi1, Pou1f1, Crx, and Rax) or unchanged (Cbfb) upon E2-treatment. Thus, this limited analysis did not identify any strongly regulated transcription factors involved in the E2 regulation of testicular genes. DISCUSSION The present results indicate the identity of numerous genes and miRNAs that are regulated by E2 in our irradiated rat testis model. Compared to other in vivo models, the current model has several advantages for identifying genes that are likely to be regulated by E2 action on the target testicular cells. The advantages include 1) the hormonal suppression regimen reduces E2-induced changes in gonadotropins and testosterone that might also affect gene expression, 2) data are available on the regulation of genes by gonadotropins and testosterone, and 3) changes in germ cell numbers resulting from the administration of E2 are minimal. The information obtained

on the E2-regulated genes will be combined with information for those regulated by testosterone to identify more likely candidates for regulating the block in spermatogonial differentiation after exposure to gonadotoxic agents. Because the addition of E2 reduced ITT levels even after suppression with acyline, we first needed to rule out the possibility that these changes attributed to E2 might actually be caused by reduction in ITT and/or possible increases in FSH. Our previous study of the changes in gene expression in irradiated rat testes following treatment with acyline combined with flutamide, with testosterone, or with flutamide plus FSH [24] helped us to dissect the effects of various hormones on gene expression. Five of the E2-regulated genes in Table 2 (Isnya1, Vldlr, Smoc1, Myh6, and Rif1) were regulated in the same direction by increases in ITT levels; hence, changes in them upon E2 addition cannot be due to the decreases in ITT levels. None of the genes listed in Table 2 was regulated by FSH. To further demonstrate that E2 did not indirectly alter gene expression by decreasing ITT levels, we examined the changes in expression of the group of all ITT-regulated genes. Most were not regulated by E2 addition, and of those that were, approximately 70% were changed in the direction opposite that


FIG. 5. Comparison of expression of Isyna1 (A), Vldlr (B), Insl3 (C), Ces3 (D), Pah (E), Igfbp3 (F), and the miRNA Mir34a (microarray probe 1394564_at; G) in the X, XAF, or XAFE groups by real-time PCR and microarray. Real-time PCR results (shaded and filled bars) were normalized to Rps2, and then the transcripts levels in the X group were set as one. Transcript levels on array data (white bars) were also normalized by setting the values in the X group to one. Results are shown as the mean 6 SEM calculated from log-transformed data (n ¼ 3–6 rats/group). Expression levels significantly different between the X and XAF groups (*) and between the XAF and XAFE groups ( ) are indicated (P , 0.05 by a t-test with Bonferroni correction). Ab, absent; LOD, limit of detection.

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TABLE 3. MicroRNAs found within 5 Kb of estrogen-downregulated probes and target genes potentially upregulated by the reduction in the microRNAs. Chromosome (strand) detected

1394564_at 1392675_at

3.0 2.1

chr5 (þ) chr9 (þ)

rno-mir-34a rno-mir-30a

1380041_at 1374290_atd 1374290_atd

1.8 1.6 1.6

chr3 (þ) chr18 () chr18 ()

rno-mir-103-2 rno-mir-145 rno-mir-143

1385205_atd

1.5

chr11 (þ)

rno-mir-99a

1385205_atd 1378867_atd 1378867_atd

1.5 1.5 1.5

chr11 (þ) chrX (þ) chrX (þ)

rno-let-7c-1 rno-mir-222 rno-mir-221

1397745_ate 1375672_at

1.5 1.4

chr18 (þ) chr10 (þ)

rno-mir-3582 rno-mir-22

1385087_at 1395443_at

1.4 1.4

chr11 () chr11 ()

rno-mir-568 rno-mir-21

Probe

miRNA

Potential miRNA target genesa Id3 Rasd1 Cebpb LOC500532 Ghrb c

Myc Rasd1 Cryzb Ccna2 Rasd1 Utrn Srd5a1 Bmp2 Ghrb c

Fos Fos Homer3 c

Homer3 Adam2 c

Wwp1 Lhx9

Fold change of target genes upon E2 addition þ1.7 þ1.6 þ1.6 þ1.4 þ1.4 þ1.9 þ1.6 þ1.8 þ1.7 þ1.6 þ1.5 þ1.8 þ1.5 þ1.4 þ1.5 þ1.5 þ1.4 þ1.4 þ1.4 þ1.4 þ1.4

Genes with complementary 3 0 UTRs and showing at least 1.4-fold increase in mRNA level upon E2 addition. E2 upregulation confirmed by two probes. c Left blank because miRNA is not in MicroCosm database used for identifying target genes. d Probe is within 5 Kb of 2 miRNAs on the same strand. e Probe is within 5 Kb of 2 miRNAs but one of them, rno-mir-133a, is transcribed from the () strand. a

b

expected from the reduction in ITT levels. Thus, the presence of flutamide to block the action of low residual ITT levels remaining after acyline treatment prevented changes in gene expression due to E2-induced reduction in ITT levels. Accordingly, the downregulation by E2 of several ITTupregulated genes (Tables 2 and 4) should be a direct result of E2 action on the target cells and not an indirect effect of the reduction in ITT levels. We have been able to obtain some information regarding the cellular localization of many E2-regulated genes by comparing expression levels between the nonirradiated control and irradiated rat testes (see Supplemental Table S1 in [24]) and from data reported in the literature. Of the more strongly E2downregulated genes (Table 2), approximately 90% were

primarily expressed in the somatic cell compartment, as was Mir34a. Several of them (Insl3, Cyp17a1, and Ces3) are specifically expressed in Leydig cells [35–37]. Among the E2-upregulated genes (Supplemental Table S2), however, were many germ cell genes, as was previously reported for ITT-downregulated genes [24]. Of the 12 genes that were both downregulated by ITT and upregulated by E2, seven (Ret, Ccna2, Egr4, Taf7l, Rps6ka6, Bmp7, and Tmem178) have been reported to be primarily localized to spermatogonia, and four (Cyct, Stmn1, Psip1, and Plk4) have been reported to be highly expressed throughout spermatogenesis [38–41]. Our data showing that the expression of Cyct, Psip1, Plk4, Taf7l, Stmn1, and Bmp7 were reduced between 2and 54-fold by irradiation further supports their localization in

TABLE 4. Genes whose expression levels inversely correlate with tubule differentiation.

Gene Insulin-like 3 (Insl3) Phenylalanine hydroxylase (Pah) Carboxylesterase 3 (Ces3) Argininosuccinate synthetase 1 (Ass1) RGD1308117 b Tubulin beta 3 (Tubb3) Fatty acid desaturase 1 (Fads1) Collagen, type III, alpha 1 (Col3a1) Interleukin 33 (Il33) RGD1304580 RAB6B, member RAS oncogene family (Rab6b) a b

Fold change upon irradiation

Fold change upon hormonal suppression with GnRH-ant and flutamide (.2-fold)

Fold change upon E2 addition

þ11.8 þ22.2 þ13.3 þ13.5 þ6.3 þ3.4 þ11.3 þ9.8 þ31.6 þ5.2 þ6.6

22.4 17.9a 13.5 8.8 3.9 3.1 3.0 3.0 3.0 2.4 2.3

3.7 2.8a 2.8 1.7 1.4 1.3 2.0 1.3 1.3 1.4 1.4

Determined by real-time PCR, as undetectable by microarray after hormone suppression. Change upon FSH addition also correlates with inhibition of tubule differentiation.


Fold change upon E2 addition


numerous noncoding RNAs [50], but except for miRNAs, the annotation of such sequences in rats is lagging far behind their more rapid characterization in mice and humans [51]. Although the identification of other classes of noncoding RNAs corresponding to these unannotated E2-downregualted probes is beyond the scope of the present study, we did investigate their relationship with known miRNAs. Among the unannotated E2-downregulated probes, several were closely linked to miRNAs, and one of them, Mir34a, was indeed shown to be regulated by E2 (Table 3 and Fig. 5E). The fact that no E2-upregulated probe was associated with miRNAs supports previous observations that E2 preferentially downregulates miRNAs [52, 53]. Our data also enabled us to distinguish between alternative hypotheses regarding the step at which the miRNA level was regulated. Because similar decreases in RNA levels were observed with both the pri34 and pre34 primer sets, E2-regulation must occur at the transcriptional level or posttranscriptionally in the stability of the primary transcript, not in the processing of the pri-miRNA to the pre-miRNA by Drosha. This result supports one report of E2 downregulation of miRNAs occurring at the level of transcription of the pri-miRNAs [52] but is inconsistent with another report that the regulation of miRNA levels occurred at the level of processing the pri-miRNAs to pre-miRNAs [53]. Recently, Mir34a has attracted much interest as a tumor suppressor gene that induces apoptosis, cell-cycle arrest, and senescence [54]. The downregulation of Mir34a by E2 could therefore be responsible for the survival and progression of the undifferentiated type A spermatogonia leading to their differentiation. The predicted target genes of these miRNAs are involved in cell cycle (Ccna2), utero embryonic development (Cebpb), oxidation reduction (Cryz and Srd5a1), response to reactive oxygen species and protein dephosphorylation (Dusp1), regulation of transcription (Id3 and Myc), intracellular signaling (Rasd1), and steroid metabolic process (Srd5a1). Further confirmation and investigation of these miRNA target genes will help us understand how E2 controls these processes via miRNAs. The E2-modulated genes are candidates for being involved in the block in spermatogonial differentiation after irradiation and its reversal in irradiated rat testes by testosterone suppression and E2 treatment. If the block and its reversal by hormonal suppression and E2 addition are controlled by the same factor, the expression of this unknown factor should correlate with the tubule differentiation status (Fig. 3). Thus, genes for which the expression patterns either match or are opposite to the tubule differentiation patterns for the various treatment groups may be important candidates for, respectively, stimulating or inhibiting spermatogonial differentiation and subsequent spermatocyte development. Factors supporting spermatogenesis should be downregulated by ITT and upregulated by E2; those inhibiting spermatogenesis should be upregulated by ITT and downregulated by E2. The latter group should also be upregulated in the somatic cells by irradiation. As discussed above, the genes positively correlated with tubule differentiation were germ cell genes, and their changes likely were an indirect consequence of the increase in relative spermatogonial numbers. In contrast, the genes negatively correlated with differentiation (Table 4) were somatic cell genes, and the possibility that they are indeed responsible for the spermatogenesis block/recovery in irradiated rat testis should be examined in future studies. Insl3 has significant roles in spermatogenesis, because it regulates testicular descent and germ cell apoptosis [55]. Pah converts phenylalanine to tyrosine, and its level controls important


the germ cells (see Supplemental Table S1 in [24]). It was noted that these 12 genes showed only a small, 1.3- to 1.7-fold E2-upregulation. One likely explanation for their increases in expression is that ITT suppression stimulates relative spermatogonial numbers by 2.1-fold within 2 wk of treatment and E2 treatment further stimulates the differentiation process by an additional 2.3-fold (Fig. 3A). Therefore, the increase in levels of E2-regulated germ cell genes might be a consequence, rather than a cause, of E2-induced rescue from the spermatogonial block. Three of the genes that were downregulated by E2 (Cyp17a1, Cyp11a1, and Insl3) (Table 2 and Supplemental Table S2) have been previously reported to be E2-downregulated in in vivo studies of specific genes in testes [42–44]. Inhibition of Insl3 expression and the activity of the Insl3 promoter by E2 were also confirmed in vitro [45]. However, in the in vivo studies, changes in gonadotropins, testosterone, and/or testicular cell composition likely occurred, and we have noted that all of these genes are also upregulated by testosterone and/or LH. Hence, the previously reported downregulation of these genes by estrogen, although in agreement with the current results, may have been largely caused by the suppression of testosterone and/or LH. Only recently have a few studies of changes in global testicular gene expression after increased exposure to estrogens been reported [46–48]. One of these studies [48] involved a 10day E2 exposure to normal adult rats, and the results may be compared with those of the present study. However, this treatment induced decreases in ITT and FSH and changes in the germ cell populations [49], and because LH levels were near the limit of sensitivity in all groups [29], expected changes may have gone undetected. Of the 75 probes that Balasinor et al. [48] identified as upregulated at least 2-fold by E2, only four were identified as weakly (,1.8-fold) E2-upregulated in the present study. Of the 146 probes that they reported to be E2downregulated (.2-fold), only six were identified as E2downregulated (all 2.8-fold) in the present study, but we identified 29 of them to be actually downregulated by LH or testosterone suppression [24]. In particular, we found Ces3 to be reduced 13-fold when testosterone was suppressed and reduced only another 2.8-fold when estrogen was given, indicating that the large, 27-fold suppression of this gene previously reported with E2 treatment [48] was a combined effect of ITT suppression and E2 action. Star and Alas2, also reported to be E2-downregulated in the normal rats [48], were not E2-regulated in our model but were LH-regulated, accounting for their decline in normal rats treated with E2. Thus, although differences between normal and irradiated rat testes may produce discrepancies between studies, failure to keep other hormones constant or account for their changes when interpreting data from animals treated with estrogens can lead to incorrect identification of genes as being E2-regulated. The very much higher prevalence of probes that have not been associated with known genes among the E2-downregulated genes (54%) compared to the E2-upregulated genes (14%) or the total probe set (35%) is highly significant (Table 1). Average expression levels of E2-upregulated and E2downregulated probes (Supplemental Table S2) were comparable, so the lack of identification of the E2-downregulated probes is not a result of low levels of expression. We hypothesize that many of the unannotated probes correspond to noncoding RNAs, which in addition the well-known ribosomal, transfer, and small nuclear and nucleolar RNAs include miRNAs, short-interfering RNAs, piwi-interacting RNAs, natural antisense RNAs, and long noncoding RNAs. Previous studies have shown that Affymetrix microarray chips recognize

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ACKNOWLEDGMENTS We thank Mr. Kuriakose Abraham for the histological preparations and Mr. Walter Pagel for editorial advice. We also thank Ms. Tarja Laiho for the skillful assistance in performing gonadotropin assays and Dr. Mini Kapoor and Ms. Sarah Rizvi for performing the microarray procedures. We sincerely thank Drs. R.P. Blye and Hyun K. Kim (Contraceptive Development Branch, National Institute of Child Health and Human Development) and Dr. Sheri Hild (Bioqual, Inc.) for providing the acyline.

12.

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14.

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17.

18. 19.

20.

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22.

23.

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