Identification of candidate transcription factors that regulate Sox9 expression through the testis-specific enhancer in the Amami spiny rat Tokudaia osimensis, an XO/XO mammal

ABSTRACT

Testicular differentiation of undifferentiated gonads is triggered by the SRY/Sry (sex-determining region of chromosome Y) gene on the Y chromosome in most mammals. SRY and NR5A1 (nuclear receptor subfamily 5, group A, member 1) proteins regulate transcription of the autosomal SOX9/Sox9 (SRY-box9) gene in XY embryonic gonads, inducing testicular differentiation. One exception, the Amami spiny rat (Tokudaia osimensis), lacks the Y chromosome and Sry. We previously reported that this species has a male-specific duplication upstream of Sox9, and an enhancer (tosEnh14) in the duplication regulates Sox9 transcription without Sry. However, tosEnh14 is not activated by NR5A1 alone, suggesting that another transcription factor(s) which binds to tosEnh14 is necessary. Because this species is endangered and heavily protected, it presents many challenges for genetic studies. Therefore, we explored novel transcription factors that regulate Sox9 via tosEnh14 using mouse samples. To detect proteins that bind to tosEnh14 DNA, Southwestern blotting analysis was performed using mouse embryonic gonad extracts. Bands of a similar molecular weight but prominent in males and faint in females were subjected to mass spectrometry analysis. Peptides derived from 174 genes were identified, and eight genes associated with gene ontology terms such as “DNA binding” and “regulation of transcription by RNA polymerase II” were selected. For further screening, the expression level of each gene was examined using single-cell RNA-sequencing data for mouse progenitor cells, which differentiate into Sertoli cells in mouse embryonic testes and granulosa cells in embryonic ovaries. Finally, five genes (Elf2, Etv6, Fiz1, Gtf2f1 and Trim27) encoding transcription factors, whose expression was confirmed in seminiferous tubules of E13.5 XY embryos by whole-mount in situ hybridization, were selected as candidates. Binding sites for ELF2 and ETV6 are present in the tosEnh14 DNA sequence. Our study contributes to understanding the molecular mechanisms underlying sex determination in mammals.

INTRODUCTION

Testicular differentiation is generally triggered by SRY/Sry (sex-determining region of chromosome Y) in mammals (Gubbay et al., 1990; Sinclair et al., 1990; Koopman et al., 1991). SRY transiently upregulates the expression of SOX9/Sox9 (SRY-box9) in undifferentiated gonads of XY embryos, inducing testicular differentiation (Kent et al., 1996). This sex determination mechanism is conserved in most mammals. However, the genus Tokudaia (Muridae, Rodentia) is one exception that does not use this mechanism. The Amami spiny rat T. osimensis lacks the Y chromosome (2n = 25, XO/XO) and Sry (Honda et al., 1977; Soullier et al., 1998); thus, it has evolved a new sex-determining mechanism that is independent of Sry (Kimura et al., 2014; Otake and Kuroiwa, 2016).

SRY binds to the enhancers of Sox9 and synergically upregulates Sox9 expression with NR5A1 (nuclear receptor subfamily 5, group A, member 1; also known as SF1) (Sekido and Lovell-Badge, 2008). An additional factor, GATA-binding protein 4 (GATA4), interacts with NR5A1 and zinc finger protein FOG family member 2 (FOG2) to regulate expression of Sry and Sox9 (Miyamoto et al., 2008; Viger et al., 2008). After SRY expression disappears, SOX9 binds to the enhancers in order to maintain its expression via self-regulation (Sekido and Lovell-Badge, 2008). Several cis-regulatory regions are spread over a gene desert of >2 Mb upstream of the Sox9 coding sequence (Symon and Harley, 2017). Consequently, multiple enhancers of Sox9 have been identified upstream of Sox9 in humans and mice (Sreenivasan et al., 2022).

In humans, disorders of sex development (DSD) patients who have a complete SOX9 coding sequence but mutations in the upstream region have been reported. Copy number variation (CNV) mapping in the genomes of DSD patients with an SRY-positive 46,XY karyotype identified a 32.5-kb region located 600 kb upstream of SOX9, called XYSR (Kim et al., 2015) (Supplementary Fig. S1). Subsequent analysis of DSD patients carrying CNVs refined this to a small 5.2-kb region (Croft et al., 2018). Additionally, a 68-kb region ~600 kb upstream of SOX9 called XXSR (also known as RevSex) was identified by screening of CNVs in DSD patients with an SRY-negative 46,XX karyotype (Benko et al., 2011; Hyon et al., 2015; Kim et al., 2015), which was further refined to 24 kb (Croft et al., 2018). Therefore, several upstream enhancers are essential for SOX9 activation in early male gonads. By screening of additional patient genomic and bioinformatic data, three putative enhancers were identified: eSR-A in XYSR, eSR-B in RevSex and eALDI (Croft et al., 2018). All three enhancers showed synergistic activity in cell-based reporter assays with different combinations of testis-specific regulators including SRY, NR5A1 and SOX9.

The testis-specific enhancer of SOX9 (termed TES), a 3.2-kb element located 13 kb upstream of Sox9 in mice, was first identified as a testis-specific enhancer to upregulate Sox9 (Sekido and Lovell-Badge, 2008). It has a 1.4-kb core sequence termed TESCO (TES core) that contains binding sites for SRY, NR5A1 and SOX9. Although the Sox9 expression level is decreased in TESCO- or TES-knockout mice, sex reversal is not induced (Gonen et al., 2017). Further analysis based on chromatin accessibility screening methods identified a 557-bp element (termed Enh13) located 565 kb upstream in the region homologous to human XYSR. Enh13-deletion mice showed a remarkable reduction of Sox9 expression and male-to-female sex reversal (Gonen et al., 2018), providing evidence that Enh13 is an essential enhancer to initiate testicular development.

In T. osimensis, the TESCO sequence is highly conserved, but nucleotide substitutions are found in two of three SRY-binding sites and five of six NR5A1-binding sites, resulting in nonfunctional enhancer activity (Kimura et al., 2014). Tokudaia osimensis also has a highly conserved Enh13 sequence (Hirata et al., 2024). The nucleotide sequence identity between T. osimensis and mouse Enh13 is 90%. All binding sites for NR5A1, SOX9 and SRY are conserved in T. osimensis Enh13 (tosEnh13). In our previous study incorporating in vitro reporter gene assays, tosEnh13 was substantially activated in response to co-transfection with Nr5a1 and Sox9; however, activity was not significantly increased after co-transfection with Nr5a1 and mouse Sry. This result indicates that tosEnh13 lacks Sry enhancer activity in Sry-deficient species.

To discover the sex-specific genome region in T. osimensis with XO/XO sex chromosomes, CNV screening was performed using T. osimensis genome sequences. As a result, a single sex-related genomic difference was found in a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on the short arm of chromosome 3 (Terao et al., 2022). The duplicated unit contains a 1,262-bp element homologous to mouse Enh14 (mEnh14). mEnh14 showed robust testis-specific β-Gal activity in an in vivo reporter gene assay, and DNaseI-seq, ATAC-seq and H3K27ac ChIP-seq data all suggest that this enhancer is active and accessible only in Sertoli cells (Gonen et al., 2018). However, Sox9 expression is not altered in embryonic day (E)13.5 XY gonads of mEnh14-knockout mice, indicating that mEnh14 controls Sox9 expression during a different developmental time window and/or functions redundantly with Enh13 and/or TES in mice (Gonen et al., 2018; Ridnik et al., 2021). The sequence identity between mEnh14 (1,288 bp) and tosEnh14 (1,262 bp) is 87.7% (Terao et al., 2022). An in vivo reporter assay showed that tosEnh14 drives Sox9 expression in embryonic gonads of mice, and embryonic gonads of XX mice in which mEnh14 is replaced with duplicated tosEnh14 show increased Sox9 expression and decreased expression of forkhead box L2 (Foxl2), which is a marker gene of ovarian differentiation (Terao et al., 2022). This suggests that the novel sex-determining element in T. osimensis consists of a male-specific duplication of a cis-element that upregulates Sox9 in the absence of Sry.

We previously performed a reporter gene assay to examine whether tosEnh14 can be activated by SRY/SOX9 and NR5A1 in vitro (Hirata et al., 2024). Activity of a single copy of tosEnh14 was low with all combinations. Two copies of tosEnh14 showed a 2.5-fold increase in activity compared with a single copy of tosEnh14 with a combination of NR5A1 and SOX9. However, this was significantly lower than the level required for sex determination in mouse embryos, i.e., the level at which mouse Enh13 is activated in response to SRY/SOX9 and NR5A1. Therefore, we hypothesized that an unknown transcription factor(s) regulates Sox9 through tosEnh14. In this study, we explored unknown transcription factors that bind to the tosEnh14 DNA sequence. We performed Southwestern blotting and subsequently mass spectrometry analysis, which identified 174 candidate genes. Further screening involved gene ontology (GO) analysis and expression analysis using sets of RNA-sequencing (RNA-seq) data for progenitor cells, which differentiate into Sertoli cells in mouse embryonic testes. We confirmed expression of each gene in E13.5 XY mouse gonads by whole-mount in situ hybridization (WISH). Finally, five genes with expression patterns resembling that of Sox9 were identified as candidates for transcription factors that regulate Sox9 through tosEnh14.

RESULTS

Identification of 174 genes as initial candidates for factors that regulate Sox9 through tosEnh14

Gonad extracts of E13.5 mice underwent Southwestern blotting to detect proteins that bind to tosEnh14 (Fig. 1A). An in vivo reporter gene assay was previously performed using transgenic mice carrying two fragments of tosEnh14 with the Sox9 promoter and the reporter gene lacZ (Terao et al., 2022). XY transgenic embryos showed robust β-Gal activity in the testis compared with weak activity in the ovary of XX transgenic embryos. According to this result, among the bands detected by Southwestern blotting, bands of a similar molecular weight but prominent in males and faint in females (indicated by arrows in Fig. 1A) were subjected to liquid chromatography–tandem mass spectrometry analysis (Fig. 1B). In total, 50, 15 and 47 genes were identified from the higher molecular weight band of males, females and both sexes, respectively (Fig. 1C left). In total, 63, 10 and 32 genes were identified from the lower molecular weight band of males, females and both sexes, respectively (Fig. 1C right). Genes counted in both the higher and lower molecular weight bands were counted as one gene, and 174 genes identified in males and both sexes were selected as initial candidates (Supplementary Table S1).

Fig. 1. Screening of proteins that bind to tosEnh14 DNA. (A) Southwestern blotting analysis to detect proteins that bind to tosEnh14. Arrowheads indicate bands of a similar molecular weight that are prominent in males but faint in females. (B) Coomassie Brilliant Blue staining for LC-MS/MS. Each band marked by an arrowhead was excised and subjected to LC-MS/MS. (C) Venn diagram of genes identified in males and/or females.

Identification of seven candidate genes by GO and RNA-seq analyses

For the next step of screening, we investigated the functional characteristics of the 174 candidate genes by GO analysis. The top ten GO terms in each category, namely, cellular component (CC), molecular function (MF) and biological process (BP), are shown in Fig. 2A, 2B and 2C, respectively. We selected genes associated with the GO terms “nucleus” in CC, “DNA binding” in MF, and “regulation of transcription by RNA polymerase II”, “positive regulation of transcription by RNA polymerase II” and “positive regulation of DNA-templated transcription” in BP because these are characteristics of the transcription factors of interest (Fig. 2C). Additionally, genes with the GO term “positive regulation of DNA-binding transcription factor activity”, which was ranked 14th, were selected. This left eight genes as candidates: Elf2, Etv6, Fiz1, Gtf2f1, Rela, Trim27, Trim28 and Zfp58 (Table 1).

Fig. 2. GO analysis of genes identified by mass spectrometry. (A) CC, (B) MF and (C) BP. The top ten GO terms are shown.

Table 1. Candidate genes and their GO terms

Gene symbol	Gene name	Gene ID	GO term in BP
			regulation of transcription by RNA polymerase II	positive regulation of transcription by RNA polymerase II	positive regulation of DNA- templated transcription	positive regulation of DNA- binding transcription factor activity
Elf2	E74-like factor 2	69257	✓		✓
Etv6	ets variant 6	14011	✓	✓
Fiz1	Flt3 interacting zinc finger protein 1	23877	✓	✓
Gtf2f1	general transcription factor IIF, polypeptide 1	98053		✓
Rela	v-rel reticuloendotheliosis viral oncogene homolog A (avian)	19697	✓	✓	✓
Trim27	tripartite motif-containing 27	19720				✓
Trim28	tripartite motif-containing 28	21849			✓
Zfp58	zinc finger protein 58	238693	✓

To determine whether these candidate genes are expressed in embryonic gonad cells at the developmental stage during which Sox9 expression is regulated, the expression levels of the candidate genes were confirmed using single-cell RNA-seq (scRNA-seq) data reported previously (Stévant et al., 2018). The scRNA-seq was performed using NR5A1-GFP-positive cells in XY gonads of transgenic male mice at various embryonic stages: E10.5, E11.5, E12.5, E13.5 and E16.5. A portion of NR5A1-positive cells in embryonic gonads at sex determination differentiate into Sertoli cells by transiently expressing Sry and upregulating Sox9 expression via SRY. NR5A1-positive cells lacking Sry and Sox9 expression differentiate into other cell types such as Leydig cells. Therefore, we extracted the scRNA-seq data of cells expressing Sox9 and calculated the transcripts per million (TPM) value of each candidate gene, as well as those of Sry and Sox9 as controls (Fig. 3A). In mice, expression of Sry starts at E10.5, peaks at E11.5 and disappears after E12.5 (Hacker et al., 1995). We confirmed the same transient pattern in the TPM calculation of Sry (Fig. 3A). Sox9 is expressed in male and female gonads at E10.5. However, it is upregulated only in male gonads at E11.5 because Sry regulates its expression (da Silva et al., 1996; de Santa Barbara et al., 2000). A similar pattern, i.e., upregulation at E11.5, was confirmed in the TPM calculation of Sox9 (Fig. 3A). These results indicate that our TPM calculation was reliable.

Fig. 3. Expression levels of Sry, or of Foxl2 and Sox9, and of eight candidate genes. The TPM value of each gene was calculated using scRNA-seq data for progenitor cells, which differentiate into (pre-)Sertoli cells (A) or (pre-)granulosa cells (B) in mouse embryonic gonads.

In most mammalian species, Sry is essential for initial male sex differentiation. Insufficient Sry expression causes Sox9 reduction and gonadal feminization (Wu et al., 2012). However, the peak expression level is extremely low and the timing of expression is limited (She and Yang, 2017). Therefore, we thought that the candidate transcription factors would need to be expressed at levels equal to or higher than Sry at between E10.5 and E12.5. The expression levels of Zfp58 were lower than that of Sry; therefore, Zfp58 was excluded from candidates.

To elucidate expression levels of the candidate genes in female supporting cells, we also calculated TPM value using scRNA-seq data of XX embryonic gonads (Stévant et al., 2019). We extracted data for cells expressing Foxl2 as an antagonist of Sox9. Values for TPM calculations in XX were obtained from E12.5, E13.5 and E16.5 because Foxl2 expression is initiated around E12.5 (Schmidt et al., 2004). Expression levels of Elf2, Trim27 and Trim28 were higher in XY than in XX at all stages (Fig. 3B). For other genes, the difference in expression levels between XY and XX varied depending on the stage. To further examine the results obtained from these in silico analyses, we performed in vivo analysis using mouse embryonic gonads.

Five candidate genes are clearly expressed in seminiferous tubules

To examine the expression patterns of the candidate genes in male and female embryonic gonads at E13.5, we performed WISH using urogenital tissues (gonad on the mesonephros). The expression pattern of Sox9 is shown in Figure 4 as a positive control. Sox9 was highly expressed in seminiferous tubules of male gonads, but was weakly expressed in female gonads. These patterns are identical to those reported previously (Gregoire et al., 2011). Rela was not clearly expressed in seminiferous tubules of male gonads. Trim28 was highly expressed not only in male but in female. Therefore, Rela and Trim28 were excluded as candidates.

Fig. 4. Expression of Sox9 and candidate genes in E13.5 mouse gonads examined by WISH.

All candidate genes except Rela were clearly expressed in seminiferous tubules, but their expression levels differed. The male expression levels of Etv6, Fiz1, Gtf2f1, Trim27 and Trim28 were identical to the results of the TPM calculation (Fig. 3A). However, while Elf2 was relatively highly expressed according to WISH (Fig. 4), it was weakly expressed according to the TPM calculation (Fig. 3A). Therefore, we may have observed expression in cells other than Sertoli cells in WISH. All genes were also expressed in female gonads. In addition, Fiz1, Gtf2f1 and Trim27 were expressed at lower levels in females than in males, but Elf2, Etv6 and Trim28 were also strongly expressed in females.

Binding sites for three candidates are present in tosEnh14

After these screening processes, five genes were identified as final candidates: Elf2, Etv6, Fiz1, Gtf2f1 and Trim27. Of these candidates, the DNA-binding sites of ELF2 and ETV6 have been reported, and their consensus binding motifs are GGAA and ccGGAAgt, respectively (Karim et al., 1990; Buijs et al., 2000; Potter et al., 2000). According to JASPAR 2024 (Rauluseviciute et al., 2023), the putative binding sites of human ELF2 and ETV6, which share 100% identity with mouse ELF2 and ETV6 in the DNA-binding domain ETS, are present in tosEnh14 (Fig. 5). These results support the possibility that ELF2 and ETV6 are transcription factors that bind to tosEnh14. The binding site of FIZ1 is unknown, but the consensus binding sequence for Sp1, which contains the same DNA-binding motif as FIZ1, is GGGCGG or GAG repeats (Marco et al., 2003), indicating that FIZ1 binds to similar sequences. Of note, tosEnh14 contains a GAGGAG sequence (Fig. 5). Gtf2f1 encodes RAP74 protein, whose binding site is also unknown. RAP74 is a subunit of the general transcription initiation factor TFIIF (Flores et al., 1990). TFIIF binds to the TATA box (Hisatake et al., 1995; Forget et al., 1997), suggesting that RAP74 binds to the TATA box. Indeed, tosEnh14 contains a TATA-like sequence (Fig. 5). As for the remaining gene, Trim27, no binding sites have been reported and therefore we could not confirm them.

Fig. 5. Putative binding sites for ELF2 and ETV6 in tosEnh14. Binding sites for ELF2 and ETV6 are bold and underlined, respectively. A GAG repeat is boxed and a TATA-like sequence is on a black background.

DISCUSSION

We identified five candidate genes as regulators of Sox9. Two of them, Elf2 and Etv6, are members of the ELF and TEL subfamilies of the ETS transcription factor family, respectively, which contain a DNA-binding domain termed the ETS domain (Gutierrez-Hartmann et al., 2007). Members of the ETS family play important roles in various basic biological phenomena such as development, differentiation, cell proliferation, immune responses and apoptosis (Oikawa and Yamada, 2003). In testicular development, Etv2 is upregulated by SRY and ETV2 then regulates Sox9 (DiTacchio et al., 2012). It has been suggested that regulation of Sox9 by ETV2 is conserved in T. osimensis (Otake and Kuroiwa, 2016).

Elf2, also known as Nerf, plays an important role in hemopoietic development (Oettgen et al., 1996). ELF2 contains a transactivation domain at the N-terminus (Gutierrez-Hartmann et al., 2007). It has two major isoforms, ELF2A and ELF2B, which activate and repress expression of their target genes, respectively (Cho et al., 2004). Mouse Elf2a is more highly expressed in the testis than Elf2b, whereas Elf2b is generally expressed at a higher level in the thymus and spleen (Guan et al., 2017). ELF2 transcriptionally regulates target genes involved in lymphocyte development and function, such as B and T cell co-receptor proteins and tyrosine kinases, by binding to promoter or enhancer regions (Oettgen et al., 1996; Ji et al., 2002). It also regulates valosin-containing protein (VCP) expression by binding to the 5-flanking region of VCP in MCF-7 cells (Zhang et al., 2007). However, the function of Elf2 in gonads or testes has not been reported. Our findings indicate that Elf2 is involved in embryonic testicular development.

Etv6, also known as Tel, was first identified by its rearrangement in human chronic myelomonocytic leukemia associated with a chromosomal translocation (Golub et al., 1994). Expression of Etv6 starts from E7.0 at the latest and spreads widely in embryonic and adult mouse tissues, including the testis (Wang et al., 1997). Etv6 homozygous knockout is embryonic lethal in mice and no embryos survive after E13.5, suggesting that Etv6 is essential for normal development, particularly hematopoiesis and vascular development (Wang et al., 1997). In ETV6-overexpressing cells, p53 and its downstream target genes are upregulated, inducing apoptosis (Yamagata et al., 2006). Target genes of ETV6, as well as of ELF2, in embryonic gonads are unknown; hence, it is necessary to validate the function of these candidates at the sex determination stage.

One of the remaining three candidates, Trim27, also known as Rfp, belongs to the tripartite motif (TRIM) family and is classified into the IV subfamily based on the structure of the C-terminal region. TRIM proteins in class IV contain a PRY domain and a SPRY domain in addition to the RING, b-box and coiled-coil domains, similar to other subfamily proteins (Shen et al., 2021). Although RFP is well known as a DNA-binding protein and a transcriptional repressor (Isomura et al., 1992; Shimono et al., 2000; Bloor et al., 2005), it also functions as a positive regulator (Wang et al., 2018; Zhao et al., 2024). Rfp initiates expression at least as early as E8.5, and is expressed in testis and some other tissues in adult mice (Cao et al., 1996; Tezel et al., 2002). In testis, RFP is localized in the nuclei of Sertoli cells and in the cytoplasm of round spermatids (Zhuang et al., 2016), indicating that Rfp is related to embryonic development and spermatogenesis. Our report is the first finding that RFP may be associated with Sox9 in fetal Sertoli cells. FIZ1 was originally isolated as an Flt3 (receptor tyrosine kinase)-interacting zinc finger protein (Wolf and Rohrschneider, 1999). Fiz1 is a widely expressed gene of unknown function. In the retina, FIZ1 acts as a transcriptional regulatory protein complex at the active rod- and cone-specific gene promoter region in mice (Mali et al., 2008). FIZ1 contains 11 C(2)H(2)-type zinc fingers related to DNA binding. The C(2)H(2) zinc finger domain is also found in Sp1, which binds to the promoter region of Wilms’ tumor 1 (Wt1) and Sry and regulates their expression (Cohen et al., 1997; Assumpçao et al., 2005). Interestingly, tosEnh14 contains an Sp1 binding site, suggesting that FIZ1 has the potential to regulate Sox9 in embryonic gonads.

The final candidate gene, Gtf2f1, encodes RAP74, which heterodimerizes with RAP30 to form the general transcription initiation factor TFIIF (Flores et al., 1990). TFIIF binds to RNA polymerase II and helps to recruit it to the initiation complex in collaboration with TFIIB (Roeder, 1996). Subunits of both TFIIF and Pol II crosslink to nucleotides located upstream and downstream of the TATA box (Hisatake et al., 1995; Forget et al., 1997). The Sox9 promoter contains a TATA-like sequence that is conserved in mouse and human (Kanai and Koopman, 1999). It is possible that RAP74 regulates Sox9 by associating with the promoter and tosEnh14.

In this study, we identified candidate transcription factors that regulate the testis-specific enhancer of Sox9. Our study contributes to understanding the molecular mechanisms underlying sex determination in mammals. In the future, it is essential to assess the activities of these candidates against tosEnh14 using in vitro and in vivo experimental systems. The identification of transcription factors will provide new insights into the molecular evolution of sex determination mechanisms.

MATERIALS AND METHODS

Animals and embryo staging

The Amami spiny rat (T. osimensis) is endangered (The IUCN Red List of Threatened Species: DOI, e.T21973A22409638) and has been protected by the Japanese government as a natural monument and a national endangered species of wild fauna and flora since 1972 and 2016, respectively. B6D2F1/Jcl mice were used for protein and total RNA extraction and WISH. All mice were purchased from a local vendor (Hokudo, Sapporo, Japan). Timed mating was used for all embryonic experiments. E0.5 was defined as noon on the day of vaginal plug detection. All animal experiments were approved by the Institutional Animal Care and Use Committee of the National University Corporation, Hokkaido University, and performed following the Guidelines for the Care and Use of Laboratory Animals, Hokkaido University.

Cloning of tosEnh14 and DNA probe synthesis

Genomic DNA of T. osimensis was extracted from tail tissues as reported previously (Kobayashi et al., 2007). A fragment of tosEnh14 amplified by PCR from genomic DNA was cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). Primer sequences are shown in Supplementary Table S2. The nucleotide sequence was confirmed using a SuperDye v3.1 Cycle Sequencing Kit (M&S TechnoSystems, Osaka, Japan) by an ABI Prism 3100 genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA). As the probe, tosEnh14 was labeled with digoxigenin (DIG) using PCR DIG Labeling Mix (Roche, Basel, Switzerland) according to the manufacturer’s instructions. After purification of DIG-labeled PCR products, their concentration was measured by the dsDNA assay using a Qubit 4 fluorometer (Thermo Fisher Scientific).

Southwestern blotting

Proteins were extracted from four gonads and mesonephros of E13.5 mice with RIPA buffer (Merck, Darmstadt, Germany) containing 1 mM sodium orthovanadate prepared in ultrapure water, 2 mM PMSF prepared in DMSO, and a protease inhibitor cocktail prepared in DMSO (NACALAI TESQUE, Kyoto, Japan). Twenty micrograms of protein was subjected to SDS-PAGE with a 10% (w/v) polyacrylamide gel at a constant current of 20 mA for approximately 90 min. Resolved proteins were transferred to a polyvinylidene difluoride membrane (pore size 0.45 μm; Merck) treated with methanol for 1 min in blotting buffers (1: 10% (v/v) methanol and 0.3 M Tris; 2: 10% (v/v) methanol and 25 mM Tris; and 3: 20% (v/v) methanol, 40 mM aminocaproic acid and 25 mM Tris) at a constant current of 60 mA for 60 min. The membrane was blocked in N101 (NOF, Tokyo, Japan) diluted 5-fold with ultrapure water for 1 h at room temperature. After blocking, the probe was added at a final concentration of 50 ng/3 ml to hybridize with proteins overnight at room temperature. The probe and protein were crosslinked by ultraviolet light for 3 min. The membrane was rinsed in TBS-T (Tris-buffered saline containing 0.1% (w/v) Tween-20) and blocked for 1 h in TBS-T containing 5% (w/v) nonfat milk. Anti-Digoxigenin-POD Fab fragments (Roche) were used at a dilution of 1:3,000 and incubated for 1 h at room temperature. After the membrane was washed thrice in TBS-T for 5 min, Immobilon Western Chemiluminescent HRP Substrate (Merck) was used to determine chemiluminescence. Signals were detected with LAS-3000 (FUJIFILM, Tokyo, Japan).

Mass spectrometry analysis

SDS-PAGE was carried out with a 10% (w/v) polyacrylamide gel at a constant current of 20 mA for approximately 90 min. After Coomassie Brilliant Blue staining, protein bands were excised and purified using an In-gel Tryptic Digestion Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. These samples were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and de novo protein sequencing at the Open Facility, Global Facility Center, Creative Research Institution, Hokkaido University. Proteome Discoverer 1.4.0.288 was used to identify proteins.

GO analysis

GO analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, v2024q2) (https://davidbioinformatics.nih.gov/). GO terms were obtained from GOTERM_BP_DIRECT, GOTERM_MF_DIRECT and GOTERM_CC_DIRECT.

scRNA-seq analysis

Data from the NCBI BioProject (accession no. GSE97519) were used for the analysis in XY (Stévant et al., 2018). Each dataset obtained by scRNA-seq (GSE97519) was first trimmed in TrimGalore v0.6.7. Next, using whole mouse genome data (GCF_000001635) in the database as a reference, trimmed reads were mapped using STAR v2.7.9a with the GTF file. TPM values were calculated by RSEM v1.3.3. Likewise, GSE119766 was used for the analysis in XX (Stévant et al., 2019). Data for Sox9-expressing cells and Foxl2-expressing cells were extracted from XY and XX, respectively.

RNA probe design and synthesis

Total RNA was isolated from a gonad of an E13.5 mouse using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Shiga, Japan) following the manufacturer’s instructions. Partial sequences of candidate genes were amplified and cloned into the pGEM-T Easy Vector. Primer sequences are shown in Supplementary Table S2. The nucleotide sequence was confirmed as well as DNA probe synthesis. After linearization of the plasmids by digestion with ApaI, SpeI or SphI, RNA probes were synthesized using a MaxiScript SP6/T7 Transcription Kit (Thermo Fisher Scientific) and labeled with digoxigenin-11-deoxyuridine 5-triphosphate using DIG RNA Labeling Mix (Roche).

WISH

Standard protocols for WISH were used with minor modifications (Liu et al., 2009). Briefly, gonad/mesonephros complexes were dissected and fixed in 4% (w/v) paraformaldehyde (PFA) prepared in PBS-T (DEPC-phosphate-buffered saline containing 0.1% (v/v) Triton X-100) overnight at 4 °C. Samples were gradually dehydrated using ethanol (25%, 50% and 75% (v/v)) diluted in PBS-T and stored in 100% ethanol at –30 °C. After rehydration, samples were treated with 5 μg/ml proteinase K at 37 °C for 10 min and then fixed in 4% PFA prepared in PBS-T at room temperature for 15 min. Samples were pre-hybridized in hybridization buffer (750 mM NaCl and 75 mM trisodium citrate pH 7.0, 50% (v/v) formamide, 2% (w/v) Blocking Reagent (Roche), 0.1% (w/v) CHAPS, 0.1% (v/v) Triton X-100, 1 μg/ml yeast tRNA (Merck), 50 μg/ml heparin sodium and 5 mM EDTA) at 65 °C for 1 h. Subsequently, samples were hybridized with DIG-labeled RNA probes overnight at 65 °C. The next day, samples were incubated in blocking solution (1.5% (w/v) Blocking Reagent, 187.5 mM NaCl, 50 mM Tris, 10 mM KCl, 10 mM maleic acid and 0.1% (v/v) Triton X-100) and then with Anti-Digoxigenin-AP Fab fragments (Roche) diluted 1:2,000 in the solution at 37 °C for 1 h each. Alkaline phosphatase was detected using NBT/BCIP Stock Solution (Roche). At least three urogenital complexes were used for expression analysis of each gene and sex. Antisense and sense probes were tested in parallel, and only signals specific to antisense probes were reported as gene-specific expression.

CONFLICTS OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could appear to influence the work reported in this paper.

ACKNOWLEDGMENTS

This work was supported by JST SPRING (Grant Number JPMJSP2119) and JSPS KAKENHI (Grant Numbers 23K23930, 22H02667 and 19H03267).

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