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Gene Expression Profile of the Neonatal Female Mouse Brain After Administration of Testosterone Propionate Yutaka Nakachi, PhD,* Mioko Iseki, BSc,* Tomotaka Yokoo, PhD,† Yosuke Mizuno, PhD,‡ and Yasushi Okazaki, MD, PhD*‡ *Division of Translational Research, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan; †Experimental Animal Laboratory, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan; ‡Division of Translational Research, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan DOI: 10.1111/jsm.12802
ABSTRACT
Introduction. Clinical care decisions for peripubertal adolescents with gender dysphoria (GD) should be made carefully. Furthermore, the identification of biomarkers is very important for rapid and accurate diagnosis of GD in young people. Aim. The aim of this study was to investigate gene expression profiles during masculinization of the neonatal female mouse brain by testosterone and to identify biomarkers related to GD. Methods. Microarray analysis was performed using RNAs extracted from the brains of neonatal mice treated by intraperitoneal injection of testosterone propionate during the sexual determination period. Sequence motif enrichment analysis for sex hormone receptor responsive elements was performed for the flanking regions of genes that showed significant expression changes following administration of testosterone propionate. Main Outcome Measures. We revealed a gene set with marked changes in expression during brain masculinization of neonatal female mice following administration of testosterone propionate. Results. We identified 334 genes that showed differential expression in the masculinized neonatal female brain after testosterone propionate treatment. Interestingly, most of these genes are not reported to be expressed in a sexually dimorphic manner. Moreover, sequence motif enrichment analysis suggested that masculinization of the neonatal female brain by testosterone was controlled more by estrogen receptors than androgen receptors. Conclusions. Differences in genes that are expressed differentially following administration of testosterone injection from known sexually dimorphic genes suggest that many GD-related genes are upregulated during female brain masculinization. The gene set identified in this study provides a basis to better understand the mechanisms of GD and delineate its associated biomarkers. Nakachi Y, Iseki M, Yokoo T, Mizuno Y, and Okazaki Y. Gene expression profile of the neonatal female mouse brain after administration of testosterone propionate. J Sex Med 2015;12:887–896. Key Words. Brain; Sexual Differentiation; Gene Expression Profiling; Animal Model; Gender Dysphoria; Testosterone; Cross-Sex Hormone Treatment
Introduction
G
ender Dysphoria (GD), previously known as gender identity disorder, is characterized by dissociation between psychological gender identity and physical sex [1–3]. This condition causes clinically significant distress or impairment in social,
© 2015 International Society for Sexual Medicine
occupational, or other important areas of functioning. GD prevalence has been estimated as 1:11,900 for male-to-female GD and 1:30,400 for female-tomale GD [4]. Most affected individuals experience GD continuously from childhood [5,6]. The mental stress caused by the uneasiness with their assigned sex at birth is significant and often results J Sex Med 2015;12:887–896
888 in self-injury and/or suicidal intent [7,8]. Although puberty suppression using a gonadotropinreleasing hormone agonist may be considered as a valuable contribution to the clinical management of GD in adolescents without the distress of irreversible development of secondary sex characteristics [9–12], clinical decisions for peripubertal adolescents with GD should be made cautiously [10]. Therefore, the identification of biomarkers is very important to provide rapid and accurate diagnosis of GD in young people. Studies of the biological mechanisms in GD have been reported continually. For example, histological findings of sexually dimorphic areas in the brain, such as the bed nucleus of the stria terminalis and limbic nucleus, of male-to-female GD sufferers are more similar to those of the female than male [13,14]. Genetic studies of sexual differentiation of the human brain as well as sexual behavior indicate the contribution of genetic factors to GD [15–19]. However, in terms of steroidogenic enzymes and sex hormone receptors, there are no reports of genetic variants that are strongly associated with GD [20–23]. It has been considered that human GD is associated with sexual differentiation of the brain via sex hormones [10,19,24–26]. However, it is inadequate to consider that only steroidogenic enzymes and sex hormone receptors drive determination of gender identity because these genes are partly responsible for disorders of sexual development [27,28]. Thus, we present a novel hypothesis that genes directly related to GD are target genes of sex hormones and/or downstream genes of sex hormone signaling pathways as well as steroidogenic enzymes or sex hormone receptors. In particular, we believe that genes with a specific function in sexual differentiation of the brain might be related to GD and may be regarded as biomarkers. However, the specific gene regulation and molecular mechanisms during sexual differentiation of the mammalian brain are still unclear [29]. To identify GD-related genes as biomarkers for diagnosis, we examined gene sets regulated by sex hormones during sexual differentiation of the brain. To identify these gene sets, we performed comprehensive gene expression analyses of neonatal mice treated with an androgen reagent (testosterone propionate [TP]) during the period of sexual differentiation in the brain. Recent studies indicate that the mechanisms of brain sex differentiation are conserved among mammals, and TP administration to neonatal female mice induces masculinization of the GD-related sexual dimorJ Sex Med 2015;12:887–896
Nakachi et al. phic area of the brain (the bed nucleus of the stria terminalis) and ensuing masculinized behavior [10,13,30]. Therefore, our expression analyses may lead to the identification of GD biomarkers. Aim
The aim of this study was to investigate gene expression profiles during masculinization of the neonatal female mouse brain by testosterone and to identify biomarkers related to GD. Materials and Methods
Neonatal Mice C57BL/6J (CLEA Japan, Tokyo, Japan) wild-type neonatal mice were used in experiments. All animal studies were approved by the Institutional Animal Care and Use Committee of Saitama Medical University. Sex Hormone Injection into Neonatal Mice Six neonatal mice were subjected to each of the four treatment conditions: TP-injected female mice, vehicle control-injected female mice, TP-injected male mice, and vehicle control-injected male mice (Figure 1A). TP (1 mg in 20 μL of sesame oil per mouse [31]) or the vehicle control (20 μL of sesame oil only per mouse) was intraperitoneally injected into neonatal mice at postnatal day 2 (the early period of mouse brain sex differentiation). We isolated total RNA from whole brains at postnatal day 6, just prior to the start of cell apoptosis in the bed nucleus of the stria terminalis, which is related to sexual dimorphism in the mouse brain [30] (Figure 1A). Total RNA was extracted from homogenized whole brains using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Genomic DNA for sex identification of neonates was extracted from ear tissue using a REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, USA). Sex determination of neonates was performed by PCR amplification of Zfy1/Zfy2 genes using the following primer set: 5′-AAGATAAGCTTACATAATCACATGGA-3′/ 5′-CCTATGAAATCCTTTGCTGCACATGT-3′ (data not shown). Quantitative Reverse Transcription Polymerase Chain Reaction for Confirmation of TP Effects To confirm the influence on gene expression in the brain by intraperitoneal hormone injection, Kiss1 expression was measured in each mouse before
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Figure 1 Gene expression analysis of the neonatal mouse brain treated with testosterone propionate (TP). (A) Schematic protocol of TP injection. TP was injected at 2 days after birth when mouse brain sex differentiation is initiated. Total RNA from whole brains was extracted at 6 days after birth and subjected to microarray analysis. (B) Quantitative RT-PCR analyses of Esr1, Kiss1, Esr2, and Cyp19a1 that are considered to be associated with sexual dimorphism in the brain. The expression level of each gene in the brain treated with or without TP was compared for both male and female mice. Each group included six mice. Error bars indicate the standard deviation. Asterisks indicate the significance value of the t-test between TP-treated samples and the vehicle control (*P < 0.05; **P < 0.01). (C) Venn diagram showing the number of genes whose expression level changed (fold change > three times the standard deviation) in the brain of neonatal mice. For each gene set, the number of genes that changed after TP treatment is shown for female and male mice. Genes with dimorphic expression without TP treatment were considered as genes originally showing sexually dimorphic expression.
microarray analysis (Figure 1B). Kiss1 is known as a sexually dimorphic gene expressed in the brain [15–19,32], and its expression level in the male brain is lower than that in the female brain (P = 0.00737, Figure 1B). Therefore, Kiss1 can be used as a marker to indicate successful hormone injection and masculinization of the female mouse brain (P = 0.00406). In addition, we measured the expression of other genes related to sexual development, including Esr1 (ERα), Esr2 (ERβ), and Cyp19a1 (aromatase). We confirmed that the expression of these genes did not change following TP treatment, although Esr1 expression changed significantly in male mice (Figure 1B). After confirmation of the expression levels of these control genes, the RNAs of TP- and vehicle-treated female and male mice were subjected to microarray analysis (Figure 1A and C). cDNAs
shown in Figure 1B were synthesized from 500 ng total RNA with an oligo-dT primer and Transcriptor Reverse Transcriptase (Roche Diagnostics, Basel, Switzerland) in a total volume of 20 μL. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and the MX3000P Real-time PCR System (Stratagene, La Jolla, CA, USA) according to the manufacturers’ instructions. The expression level of each gene was normalized to the endogenous level of Gapdh. Primer sequences were as follows: • Kiss1: 5′-CTTCCTCCCAGAATGATCTC-3′/ 5′-ATTAACGAGTTCCTGGGGTC-3′, • Esr1: 5′-AGAAGAGTTTGTGTGCCTCA3′/5′-CATCAGGTGGATCAAAGTGT-3′, J Sex Med 2015;12:887–896
890 • Esr2: 5′-AGAGTGGAATCTCTTCCCAG-3′/ 5′-ACATTTTTGCACTTCATGCT-3′, • Cyp19a1: 5′-AACATCATTCTGAACATCGG3′/5′-GGCTGAAAGTACCTGTAGGG-3′, • Gapdh: 5′-CTACACTGAGGACCAGGTTG3′/5′-CTGTTGCTGTAGCCGTATTC-3′.
Gene Expression Analysis by Microarray Gene expression analysis was performed with GeneChip Mouse 430 v2.0 arrays (Affymetrix, Santa Clara, CA, USA) to detect genes that showed expression changes caused by perturbation of sex hormones. To eliminate the variance between samples within treatment conditions, equal amounts of total RNA from the six mice in each group were mixed and applied to the microarray analysis. To confirm the expression levels and variation among mice within groups, the same total RNA samples from individual mice were analyzed by RT-PCR. After quality assessment of total RNA samples using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), biotin-labeled complementary RNA was synthesized using a GeneChip 3′ IVT Express Kit (Affymetrix) and then hybridized to the GeneChip arrays according to the manufacturers’ instructions. Scanning was carried out in a GeneChip scanner 3000 7G (Affymetrix). Microarray data were deposited in the GEO database under accession number GSE59375. Bioinformatics Analysis To assess gene expression levels, r 2.12.0 [33] and Bioconductor 2.18.0 [34] were used for the statistical and bioinformatics analysis environment. The intensity of the microarray data was background-subtracted and normalized using the robust multi-array analysis (RMA) method [35] in the r/Bioconductor affy 1.28.1 package [36]. Next, we calculated each expression ratio between the RMA values of TP-treated samples and the vehicle control. In the differential expression analyses, we used two expression ratio thresholds to estimate whether the expression of a gene showed a significant change. If a gene had an expression ratio over or under either of the thresholds, we considered that the gene showed a significant change in expression. One of the thresholds was three times the standard deviation of the whole expression ratio on a microarray (termed as global ± 3SD). The other threshold was three times the standard deviation of limited microarray probes with RMA values similar to those of the vehicle J Sex Med 2015;12:887–896
Nakachi et al. control (around ±0.5, termed as local ± 3SD; for details, see Figure 4). The reason why the local ± 3SD criterion was also required was that the global ± 3SD threshold underestimated genes with high expression levels because the number of genes with a low expression level was much higher than that of other genes. All calculations were performed with r. Gene sets showing significant differences in expression were extracted by comparing (i) TP- and vehicle-treated females; (ii) TP- and vehicletreated males; and (iii) vehicle-treated females and males (Figure 1C). Gene set enrichment analysis with Gene Ontology (GO) [37] and KEGG [38] were performed using the r/Bioconductor GOstats 2.16.0 package [39] with GO.db 2.4.5 and KEGG.db 2.4.5. Motif sequence prediction to identify transcription factor responsive elements was performed using the prophecy and profit package of EMBOSS 6.5.7.0 [40] with JASPAR release 5.0_ALPHA [41] and TRANSFAC version 11.2 [42] databases (see Figure 5). To test for enrichment of responsive elements, we performed the 2 × 2 Fisher exact test (P < 0.05) with r by comparing the enrichment of observed elements in each gene set with the expected rate calculated from whole genes. Results
Gene Expression Analysis of Brains from Mice Treated with TP Gene expression changes in 394 and 319 genes (corresponding to 518 and 478 probes, respectively) following TP injection were found in newborn female and male mouse brains, respectively (Figure 1C, Supporting Information Table S1). The 17 genes with known sexually dimorphic expression patterns showed expression changes in female brains treated with TP. Moreover, three genes showed changes in female and male brains following TP injection (Figure 1C). The other 374 differentially expressed genes in the female brain were not initially different between males and females, and no expression changes of these genes were detected in the male mouse brain after TP injection (Figure 1C). GO/KEGG Enrichment Analysis GO and KEGG pathway enrichment analysis of the 394 genes showing markedly changed expression in the female neonatal mouse brain indicated that neuron development and differentiation processes were enriched significantly (GO:
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regulation of localization peptidyl−arginine methylation, to asymmetrical−dimethyl arginine regulation of natural killer cell differentiation positive regulation of natural killer cell differentiation peptidyl−arginine omega−N−methylation peptide catabolic process transport regulation of transport establishment of localization biomineral tissue development regulation of B cell differentiation peptidyl−arginine methylation peptidyl−arginine N−methylation cellular component organization mRNA metabolic process localization microtubule−based movement natural killer cell differentiation atrial cardiac muscle tissue development atrial cardiac muscle tissue morphogenesis response to biotic stimulus system development neuron development multicellular organismal development neuron differentiation regulation of melanocyte differentiation regulation of pigment cell differentiation negative regulation of cell development microtubule−based process negative regulation of neuron differentiation
Gene Ontology Figure 2 GO/KEGG enrichment analysis of genes with expression changes in the female mouse brain following TP treatment. (A) GO enrichment analysis of genes with expression changes in the female mouse brain. (B) KEGG pathway enrichment analysis of the genes shown in (A). Each bar indicates the transformed value from the P value (equal to −1 × log10 [P value]) using the R/BIOCONDUCTOR GOstats package.
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0048666; P = 0.0038, GO: 0030182; P = 0.0043, GO: 0045665; P = 0.0048, Figure 2A). Additionally, genes related to the axon guidance pathway (ko04360; P = 0.035, Figure 2B) and cell adhesion molecules (ko04514; P = 0.036, Figure 2B) were enriched in the female mouse brain by TP injection.
Enrichment Analysis of Responsive Element Motifs Responsive elements of the estrogen receptor (ER) and androgen receptor (AR) were searched for computationally in the flanking regions (within 10 kb from each gene body) of the differentially expressed genes. We then evaluated the enrichment of these responsive elements in the flanking region by Fisher’s exact test (Figure 3). As a result, we found significant enrichment of ER-responsive elements (M00191, MA0112, and MA0258) in the female gene set (143–175; P = 8.96 × 10−4), while
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Synthesis and degradation of ketone bodies Spliceosome Chronic myeloid leukemia Butanoate metabolism Acute myeloid leukemia Axon guidance Renin−angiotensin system Cell adhesion molecules (CAMs) Pancreatic cancer Vasopressin−regulated water reabsorption
AR-responsive elements (M00447, M00481, M00953, M00956, and MA0007) showed no significant enrichment in the female gene set (281– 277; P = 7.01 × 10−1). However, intriguingly, we also observed a significant deficiency of both ERand AR-responsive elements in the male gene set (180–116; P = 4.74 × 10−10 and 355–249; P = 7.074.08 × 10−23, respectively). Discussion
In this study, we used neonatal female mice as the animal model for masculinization of the female brain during the early period of development [30]. We identified differentially expressed gene sets after sex hormone injection during the period of sexual differentiation of the brain. We observed marked differential expression changes in the female brain after testosterone treatment. Gene J Sex Med 2015;12:887–896
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function and pathway enrichment analyses showed that the differentially expressed gene set in females was enriched with functions related to neural development. Moreover, responsive elements of ER were significantly enriched in differentially expressed gene sets of females, whereas both responsive elements of ER and AR were significantly deficient in the differentially expressed gene set of males. The gene sets that showed sexually dimorphic expression without hormone injection were not consistent with those showing differential expression following TP injection. This inconsistency indicates that, in addition to known sexually dimorphic genes, other unidentified genes are involved in the mechanism of sexual differentiaJ Sex Med 2015;12:887–896
6,477
230 21,233 21,463
Figure 3 Numbers of ER-/ARresponsive elements in the flanking regions of differentially expressed genes and enrichment analysis. The 2 × 2 tables correspond to those in Figure 1C. Each P value was calculated by Fisher’s exact test using R/BIOCONDUCTOR. Analyses were performed with whole genes on the GeneChip microarray (21,463 genes in total).
tion in the brain, especially masculinization of the fetal brain via sex hormone perturbation. Some of these unidentified genes may be potential GD biomarkers. The differentially expressed gene sets in the neonatal TP-injected female brain showed significant enrichment of genes with functions in neuron development processes and the axon guidance pathway. These results imply that some of the genes showing changes in masculinizing gene expression following TP injection might have functions that influence the neural circuits or brain structures related to gender identity. This inference may be consistent with the reported findings that the microstructure of white matter, which is known to be sexually dimorphic, is correlated with
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Expression Ratio (M−A plot): Female Mice TP vs. Vehicle
1427346_at Ott
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3721 probes exist between 7.44358 and 8.44358 (aronud ± 0.5 from X−axis value of 1427346_at). local±3SD: Threshold of at 7.94358 on X−axis (1427346_at) are calculated from 3721 probes by mean of ratio (TP/Vehicle) ± 3 × SD. ( 0.470508 and −0.517908 )
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Figure 4 Scheme and description of the local ± 3SD threshold for gene expression analysis. This threshold was three times the standard deviation of limited microarray probes with RMA values similar to those of the vehicle control. For example, if the RMA value of the vehicle control was 3.5, the standard deviation using local ±3SD was calculated using the expression ratio from all probes for which the vehicle control RMA value was inside the range from 2.5 to 4.5
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global±3SD: Thresholds are calculated from all 45101 probes on a microarray by mean of ratio (TP/Vehicle) ± 3 × SD. ( 0.411242 and −0.429174 )
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gender identity rather than physical sex [43,44]. We analyzed total RNA from the whole brain including the well-known sexual dimorphic nucleus in the hypothalamus and most of the cortex related to the microstructure of white matter. Further studies of a more limited part of the brain or a focused area are necessary to clarify more detailed functions in specific parts of the brain. Fifty-five genes showed expression changes in the brain of both male and female mice following TP injection (Figure 1C and Supporting Information Table S1). One of these genes, ovary testis transcribed (Ott), is an X-linked multi-copy gene that shows specific expression during meiosis [45]. Although deficiency of Ott expression in spermatogonia causes abnormal spermatogenesis [46], the function of this gene in the brain is yet to be determined. The expression of Ott disappears in the adult mouse brain. However, Ott expression is the highest in NeuroD2 cells (a cell line derived from a mouse neuroblastoma) among all tissues and cell lines in the gene expression database BioGPS [47]. Moreover, the Allen Brain Atlas [48] shows that Ott is expressed in an area close to the sexually dimorphic region, the bed nucleus of the stria terminalis, in the mouse brain. These expres-
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sion data suggest that Ott has a specific function in neural cells during sexual differentiation of the mammalian brain. Testosterone in the brain is transformed to both an active form of androgen, dihydrotestosterone, and estrogen 17 beta-estradiol, which are derived by hydroxylation and aromatization, respectively. Both hormones can influence sexual differentiation of the mammalian brain via their specific nuclear receptors, AR and ER, respectively [24,25]. Enrichment analysis of ER-/AR-responsive element motifs using the promoter regions of the differentially expressed gene sets implies that the gene expression changes during masculinization of the neonatal female brain were mainly affected by ER and the estradiol derived from the injected TP that was transported across the blood–brain barrier. On the other hand, a deficiency of ER-/AR-responsive elements was found in the gene set of males (Figure 3), and the Esr1 expression level was downregulated significantly in the male brain by TP injection (Figure 1B). This observation suggests that the male mice had a sufficient amount of endogenous testosterone, and excessive estradiol derived from aromatized exogenous testosterone in the brain decreased the Esr1 expression level of ERα in the brain by ER autoregulation [49]. In J Sex Med 2015;12:887–896
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addition, an excess of testosterone may indirectly mediate the induction of hormone-sensitive genes that lack conventional hormone response elements by altering the expression of other transcription factors and coupling to second-messenger pathways [50]. Further investigation of gene expression during the critical period of sexual differentiation in the brain is important to identify clinical biomarkers of GD. Although the genes identified as differentially expressed in this study have not been confirmed by molecular analyses, such as chromatin immunoprecipitation assays and/or functional analysis including knockdown assays in vivo/vitro, these genes may be potentially related to GD. J Sex Med 2015;12:887–896
Figure 5 Scheme and description of ER-/AR-responsive motifs in sequence analyses. The sequence logo images show the motif sequences by searching for transcription factorresponsive elements (Figure 3). MA0007, MA0112, and MA0258 were registered in JASPAR, and M00191, M00447, M00481, M00953, and M00956 were registered in TRANSFAC. These motifs of responsive elements were used to search for the responsive elements of ER/AR in the 10-kb flanking region of mouse genes.
Conclusions
The gene sets identified in this study, especially those that show expression changes in the female mouse brain after TP treatment, are significantly involved in neural function and can be controlled by estrogen derived from exogenous testosterone. These genes might provide the basis to better understand the phenotype of GD and lead to the establishment of GD biomarkers. Acknowledgments
We thank Tomoko Hirata, Yumi Mizuno, Takashi Murofushi, Daisuke Muramatsu, Junko Itoh, and all colleagues at the Division of Translational Research, Division of Functional Genomics & Systems Medicine,
Expression of Masculinizing Female Neonatal Brain and Experimental Animal Laboratory, Saitama Medical University for technical assistance and insightful discussions. This study was supported by the Kawano Masanori Memorial Foundation for the Promotion of Pediatrics, a noncommercial foundation. The authors are also supported in part by a grant-in-aid for the Support Project of the Strategic Research Center in Private Universities from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to Saitama Medical University Research Center for Genomic Medicine. Corresponding Author: Yasushi Okazaki, MD, PhD, Division of Translational Research, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan. Tel: +81-42984-0318; Fax: +81-42-984-0349; E-mail: okazaki@ saitama-med.ac.jp,
[email protected] Conflict of Interest: The author(s) report no conflicts of interest. Statement of Authorship
Category 1 (a) Conception and Design Yutaka Nakachi; Tomotaka Yokoo; Yasushi Okazaki (b) Acquisition of Data Yutaka Nakachi; Mioko Iseki; Tomotaka Yokoo; Yosuke Mizuno (c) Analysis and Interpretation of Data Yutaka Nakachi; Yasushi Okazaki
Category 2 (a) Drafting the Article Yutaka Nakachi; Yasushi Okazaki (b) Revising It for Intellectual Content Yutaka Nakachi; Tomotaka Yokoo; Yosuke Mizuno; Yasushi Okazaki
Category 3 (a) Final Approval of the Completed Article Yutaka Nakachi; Mioko Iseki; Tomotaka Yokoo; Yosuke Mizuno; Yasushi Okazaki References 1 American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th edition. Washington, DC: American Psychiatric Association; 2013. 2 Coleman E, Bockting W, Botzer M, Cohen-Kettenis P, DeCuypere G, Feldman J, Fraser L, Green J, Knudson G, Meyer WJ, Monstrey S, Adler KR, Brown GR, Devor AH, Ehrbar R, Ettner R, Eyler E, Garofalo R, Karasic DH, Lev AI, Mayer G, Meyer-Bahlburg H, Hall BP, Pfaefflin F, Rachlin K, Robinson B, Schechter LS, Tangpricha V, van Trotsenburg M, Vitale A, Winter S, Whittle S, Wylie KR, Zucker K. Standards of care for the health of transsexual, transgender, and gendernonconforming people, version 7 [Internet]. Int J Transgend 2014;13:165–232.
895
3 Sohn M, Bosinski HAG. Gender identity disorders: Diagnostic and surgical aspects. J Sex Med 2007;4:1193–207. 4 Bakker A, van Kesteren PJ, Gooren LJ, Bezemer PD. The prevalence of transsexualism in The Netherlands. Acta Psychiatr Scand 1993;87:237–8. 5 Cohen-Kettenis PT, Pfäfflin F. The DSM diagnostic criteria for gender identity disorder in adolescents and adults. Arch Sex Behav 2010;39:499–513. 6 Fisher AD, Ristori J, Bandini E, Giordano S, Mosconi M, Jannini EA, Greggio NA, Godano A, Manieri C, Meriggiola C, Ricca V, Déttore D, Maggi M, Italian GnRH analogs study ONIG group. Medical treatment in gender dysphoric adolescents endorsed by SIAMS-SIE-SIEDP-ONIG. J Endocrinol Invest 2014;37:675–87. 7 Wallien MSC, van Goozen SHM, Cohen-Kettenis PT. Physiological correlates of anxiety in children with gender identity disorder. Eur Child Adolesc Psychiatry 2007;16:309–15. 8 Terada S, Matsumoto Y, Sato T, Okabe N, Kishimoto Y, Uchitomi Y. Suicidal ideation among patients with gender identity disorder. Psychiatry Res 2011;190:159–62. 9 de Vries ALC, Steensma TD, Doreleijers TAH, CohenKettenis PT. Puberty suppression in adolescents with gender identity disorder: A prospective follow-up study. J Sex Med 2010;8:2276–83. 10 Steensma TD, Kreukels BPC, de Vries ALC, Cohen-Kettenis PT. Gender identity development in adolescence. Horm Behav 2013;64:288–97. 11 de Vries ALC, McGuire JK, Steensma TD, Wagenaar ECF, Doreleijers TAH, Cohen-Kettenis PT. Young adult psychological outcome after puberty suppression and gender reassignment. Pediatrics 2014;134:696–704. 12 Giordano S. Lives in a chiaroscuro. Should we suspend the puberty of children with gender identity disorder? J Med Ethics 2008;34:580–4. 13 Zhou J-N, Hofman MA, Gooren LJG, Swaab DF. A sex difference in the human brain and its relation to transsexuality. Nature 1995;378:68–70. 14 Kruijver FP, Zhou JN, Pool CW, Hofman MA, Gooren LJ, Swaab DF. Male-to-female transsexuals have female neuron numbers in a limbic nucleus. J Clin Endocrinol Metab 2000;85:2034–41. 15 Ngun TC, Ghahramani N, Sánchez FJ, Bocklandt S, Vilain E. The genetics of sex differences in brain and behavior. Front Neuroendocrinol 2011;32:227–46. 16 Green R. Family cooccurrence of “gender dysphoria”: Ten sibling or parent-child pairs. Arch Sex Behav 2000;29:499– 507. 17 Coolidge FL, Thede LL, Young SE. The heritability of gender identity disorder in a child and adolescent twin sample. Behav Genet 2002;32:251–7. 18 Gómez-Gil E, Esteva I, Almaraz MC, Pasaro E, Segovia S, Guillamon A. Familiality of gender identity disorder in nontwin siblings. Arch Sex Behav 2010;39:546–52. 19 Heylens G, De Cuypere G, Zucker KJ, Schelfaut C, Elaut E, Vanden Bossche H, De Baere E, T’Sjoen G. Gender identity disorder in twins: A review of the case report literature. J Sex Med 2012;9:751–7. 20 Henningsson S, Westberg L, Nilsson S, Lundström B, Ekselius L, Bodlund O, Lindström E, Hellstrand M, Rosmond R, Eriksson E, Landén M. Sex steroid-related genes and maleto-female transsexualism. Psychoneuroendocrinology 2005; 30:657–64. 21 Bentz E-K, Hefler LA, Kaufmann U, Huber JC, Kolbus A, Tempfer CB. A polymorphism of the CYP17 gene related to sex steroid metabolism is associated with female-to-male but not male-to-female transsexualism. Fertil Steril 2008;90:56–9. 22 Hare L, Bernard P, Sánchez FJ, Baird PN, Vilain E, Kennedy T, Harley VR. Androgen receptor repeat length polymorphism
J Sex Med 2015;12:887–896
896
23
24
25
26
27
28
29 30
31
32
33
34
35
36
37
38 39
associated with male-to-female transsexualism. Biol Psychiatry 2009;65:93–6. Ujike H, Otani K, Nakatsuka M, Ishii K, Sasaki A, Oishi T, Sato T, Okahisa Y, Matsumoto Y, Namba Y, Kimata Y, Kuroda S. Association study of gender identity disorder and sex hormone-related genes. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:1241–4. Swaab DF. Sexual differentiation of the human brain: Relevance for gender identity, transsexualism and sexual orientation. Gynecol Endocrinol 2004;19:301–12. Bao A-M, Swaab DF. Sexual differentiation of the human brain: Relation to gender identity, sexual orientation and neuropsychiatric disorders. Front Neuroendocrinol 2011;32:214– 26. Meyer-Bahlburg HFL. From mental disorder to iatrogenic hypogonadism: Dilemmas in conceptualizing gender identity variants as psychiatric conditions. Arch Sex Behav 2010;39: 461–76. Meyer-Bahlburg HFL. Sex steroids and variants of gender identity. Endocrinol Metab Clin North Am 2013;42:435– 52. Rosenthal SM. Approach to the patient: Transgender youth: endocrine considerations. J Clin Endocrinol Metab 2014;99:4379–89. Yang CF, Shah NM. Representing sex in the brain, one module at a time. Neuron 2014;82:261–78. Wu MV, Manoli DS, Fraser EJ, Coats JK, Tollkuhn J, Honda S-I, Harada N, Shah NM. Estrogen masculinizes neural pathways and sex-specific behaviors. Cell 2009;139:61–72. Vale JR, Ray D, Vale CA. The interaction of genotype and exogenous neonatal androgen and estrogen: Sex behavior in female mice. Dev Psychobiol 1973;6:319–27. Kauffman AS. Sexual differentiation and the Kiss1 system: Hormonal and developmental considerations. Peptides 2009; 30:83–93. R Development Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2010. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JYH, Zhang J. Bioconductor: Open software development for computational biology and bioinformatics. Genome Biol 2004;5:R80. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 2003;31:e15. Gautier L, Cope L, Bolstad BM, Irizarry RA. affy—Analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 2004;20:307–15. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: Tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000;25:25–9. Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000;28:27–30. Falcon S, Gentleman R. Using GOstats to test gene lists for GO term association. Bioinformatics 2007;23:257–8.
J Sex Med 2015;12:887–896
Nakachi et al. 40 Rice P, Longden I, Bleasby A. EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet 2000;16:276–7. 41 Mathelier A, Zhao X, Zhang AW, Parcy F, Worsley-Hunt R, Arenillas DJ, Buchman S, Chen C-Y, Chou A, Ienasescu H, Lim J, Shyr C, Tan G, Zhou M, Lenhard B, Sandelin A, Wasserman WW. JASPAR 2014: An extensively expanded and updated open-access database of transcription factor binding profiles. Nucleic Acids Res 2014;42(Database issue): D142–7. 42 Matys V, Kel-Margoulis OV, Fricke E, Liebich I, Land S, Barre-Dirrie A, Reuter I, Chekmenev D, Krull M, Hornischer K, Voss N, Stegmaier P, Lewicki-Potapov B, Saxel H, Kel AE, Wingender E. TRANSFAC and its module TRANSCompel: Transcriptional gene regulation in eukaryotes. Nucleic Acids Res 2006;34(Database issue):D108–10. 43 Rametti G, Carrillo B, Gómez-Gil E, Junque C, Segovia S, Gomez A, Guillamon A. White matter microstructure in female to male transsexuals before cross-sex hormonal treatment. A diffusion tensor imaging study. J Psychiatr Res 2011;45:199–204. 44 Rametti G, Carrillo B, Gómez-Gil E, Junque C, Zubiarre-Elorza L, Segovia S, Gomez A, Guillamon A. The microstructure of white matter in male to female transsexuals before cross-sex hormonal treatment. A DTI study. J Psychiatr Res 2011;45:949–54. 45 Kerr SM, Taggart MH, Lee M, Cooke HJ. Ott, a mouse X-linked multigene family expressed specifically during meiosis. Hum Mol Genet 1996;5:1139–48. 46 Wang PJ, McCarrey JR, Yang F, Page DC. An abundance of X-linked genes expressed in spermatogonia. Nat Genet 2001;27:422–6. 47 Wu C, Orozco C, Boyer J, Leglise M, Goodale J, Batalov S, Hodge CL, Haase J, Janes J, Huss JW, Su AI. BioGPS: An extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol 2009;10:R130. 48 Sunkin SM, Ng L, Lau C, Dolbeare T, Gilbert TL, Thompson CL, Hawrylycz M, Dang C. Allen Brain Atlas: An integrated spatio-temporal portal for exploring the central nervous system. Nucleic Acids Res 2013;41(Database issue): D996–1008. 49 Castles CG, Oesterreich S, Hansen R, Fuqua SA. Autoregulation of the estrogen receptor promoter. J Steroid Biochem Mol Biol 1997;62:155–63. 50 Simerly RB. Wired for reproduction: Organization and development of sexually dimorphic circuits in the mammalian forebrain. Annu Rev Neurosci 2002;25:507–36.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Table S1 Genes showing changes in expression following administration of testosterone propionate. Female, Male, and Dimorphic indicate expression changes between female mice treated with TP or the vehicle control, between male mice treated with TP or the vehicle control, and between female and male treated with the vehicle control, respectively. (URL: http://www.saitama -med.ac.jp/genome/eng/Div07_TR/staff/pub/jsm12802/Table_1 _SuppInfo.pdf).
