Turkish Journal of Zoology Turk J Zool (2019) 43: 319-330 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1808-25

Identification and expression of Dmrt1 and Sox9 during the gonadal differentiation of Rana chensinensis

Xin-Yi LI, Wen-Jie JIN, Lei LEI, Yu-Hui ZHANG* College of Life Science, Shaanxi Normal University, Xi’an, P.R. China

Received: 12.08.2018 Accepted/Published Online: 05.05.2019 Final Version: 01.07.2019

Abstract: Gonadal sex differentiation proceeds by the interplay of various including the transcription factors and secretory factors in a complex network. Dmrt1 and Sox9 were considered to be the essential genes involved in the testicular differentiation in mammals, birds, reptiles, and fishes. Here, we have successfully isolated and characterized cDNAs encoding theRana chensinensis orthologs of Dmrt1 and Sox9, and we have determined patterns of expression during gonadal differentiation and adult tissues. At the molecular level, a clear sexual dimorphism of rcDmrt1 and rcSox9 was observed in the gonad-mesonephros complex or gonads of tadpoles during stages 26–46. The rcDmrt1 transcript was first detected in undifferentiated gonads at stage 30 and became stronger at stages 32–36. Thereafter, the rcDmrt1 transcript was only observed in males and gradually increased from stage 42 to stage 46. By contrast, rcSox9 was observed in the whole gonadal differentiation period (stages 26–46) of males and females. In adult frogs, Dmrt1 was merely detected in testes, while rcSox9 was more highly expressed in the brain, liver, and testes. These results showed that both Dmrt1 and Sox9 participate in gonadal differentiation, and Dmrt1 is more closely implicated in testicular differentiation.

Key words: Dmrt1, Sox9, stage-specific expression, gonadal development, Rana chensinensis

1. Introduction structure of the Dmrt transcription regulation factor binds Sexual development is a fundamental process of life, which to a specific DNA sequence, which could function as a is a regulatory cascade of spatial-temporal expression of regulator in sex determination and differentiation (Zhu multiple genes. In mammals, the sex-determining region of et al., 2000; Kim et al., 2007). Dmrt1 has been reported the Y (SRY) initiates male sex determination, in mouse (Matson et al., 2010); chicks (Smith et al., 2003, which is a testis-determining factor (Koopman et al., 2009; Lambeth et al., 2014); turtles Chelydra serpentina 1990; Polanco and Koopman, 2007). In contrast, Sry does (Rhen et al., 2007), Trachemys scripta (Barske and Capel, not exist in nonmammalian vertebrates (Tiersch et al., 2010), and Lepidochelys olivacea (Torres Maldonado et 1991; Denny et al., 1992). In previous studies, genetic sex al., 2002); frogs Xenopus laevis and Xenopus tropicalis determination and environmental sex determination were (Yoshimoto et al., 2006), Rana temporaria (Rodrigues performed in a range of vertebrate amphibian, reptile, and et al., 2017), and Rana rugosa (Shibata et al., 2002); and fish species, but the molecular mechanism triggering the fishes Oryzias latipes (Kobayashi et al., 2004), Danio in these species remained unclear rerioze (Guo et al., 2005), Xiphophorus maculates (Veith (Czerwinski et al., 2016; Geffroy et al., 2016). Thus, et al., 2006), Oreochromis niloticus (Kobayashi et al., investigation of the molecular mechanism determining 2008), and Acanthopagrus schlegelii (Wu et al., 2012). the sex of nonmammalian vertebrates remains a primary In most species, Dmrt1 is conserved in genetic sex- challenge and will be important for evolutionary biology. determining signal pathways of mammals, birds, reptiles, The double-sex and mab-3-related etc. (Raymond et al., 1999a; Shan et al., 2000; Murdock et (Dmrt) is a homologous gene family to (Dsx) al., 2003, 2006; Rhen et al., 2007). Dmrt1 plays a key role of Drosophila melanogaster and Maleabnormal-3(Mab-3) in initializing testis differentiation and later maintenance of Caenorhabditis elegans, which encodes a conserved of male development; it is first expressed in the genital double-sex and mab-3 (DM) domain, containing a novel ridge of embryos, and then becomes male-specific and zinc module and disordered tail (Shen and Hodgkin, restricted to the seminiferous tubules of the testis (Pask et 1988; Burtis et al., 1989; Zhu et al., 2000). The al., 2003; Abramyan et al., 2009). These results suggested * Correspondence: [email protected] 319

This work is licensed under a Creative Commons Attribution 4.0 International License. LI et al. / Turk J Zool that the upregulation of Dmrt1 might play a role in male cloned and their sequence features were analyzed. In sexual development. Dmrt1, the male sex determination- addition, the tissue expression and distribution of rcDmrt1 related gene, appears to be more directly involved in sex and rcSox9 were examined during gonadal differentiation. differentiation than sex determination. In mice, Dmrt1 is Finally, all data were used to analyze the role of Dmrt1 expressed in all stages of spermatogonia, localized in Sertoli and Sox9 in the regulation of gonadal differentiation and cells and some developing germ cells in testes (Brunner et maintenance of testicular function. al., 2001; Shibata et al., 2002; Kim et al., 2007; Abramyan et al., 2009). In addition, knockout of the Dmrt1 could 2. Materials and methods lead to a reduced number of germ cells, which indicates 2.1. Animals and treatments that Dmrt1 is essential for spermatogenesis (Matson et al., Two-year-old adult Chinese brown frogs (Rana 2010). chensinensis) were obtained from the Qinling Mountains Sex-determining region Y box 9 (Sox9), a Sox of Shaanxi, China, in November and December of 2016. superfamily member, has a conserved high mobility group Males (n = 5; 7.7–8.1 g) and females (n = 5; 9.2–10.0 g) (HMG) DNA-binding motif shared with SRY and several were fed Tenebrio molitor twice a day and cultured at related . Among the vertebrate Sox superfamily 25 ± 2 °C in tanks containing 2 L of dechlorinated tap members, Sox9 is known to be critical for many aspects water. The light/dark cycle was 14 h/10 h. After 10 days of development including sex determination and gonadal of environmental adaption, brains, livers, kidneys, testes, differentiation, nervous system development, heart valve and ovaries were dissected immediately following ether development, and chondrogenesis (Hong and Saint- anesthesia (euthanasia). Samples were frozen in liquid Jeannet, 2005; Lincoln et al., 2007; Akiyama et al., 2011; nitrogen and stored at –80 °C until further analysis. Jakob et al., 2011; Nakamura et al., 2012). Defects of Tadpoles were collected in March and April 2017 from the Sox9 in XY individuals with campomelic dysplasia (CD) same area as the adult frogs, and they were raised in similar showed complete or partial sex reversal (Meyer et al., 1997; conditions as adult frogs while being fed with boiled lettuce Michel-Calemard et al., 2004; Katoh-Fukui et al., 2015). In every other day ad libitum. According to Gosner stages adult individuals, Sox9 was strongly expressed in the onset (Gosner, 1960), the gonad-mesonephros complex (GMC) of differentiation, whereas it was downregulated in the female gonads (Fröjdman et al., 2000). In addition, of tadpoles at stages 26–28, the undifferentiated gonads Sox9 could upregulate the transcription of the anti- at stages 30–40, and the testes and ovaries at stages 42–46 Müllerian hormone gene and maintain spermatogenesis were isolated under a dissecting microscope (Figure 1), and fertility in the testes (Josso et al., 1993; Mackay et and then specimens were stored at –80 °C. All procedures al., 2000; Josso et al., 2001; Ortega et al., 2015), which involving animals were in accordance with the ethical induced the of granulosa cells into standards of the Animal Research Ethics Committee of Sertoli-like cells in the ovary of adult mouse (Couse et al., Shaanxi Normal University, China. 1999; Dupont et al., 2003; Barske and Capel, 2010). These 2.2. Nucleic acid extractions and RT-PCR results suggest that Sox9 may be critical for Sertoli cell and Total RNA was isolated using RNAiso Plus (TaKaRa) as testicular differentiation. described by the manufacturer. All RNA samples were In the present study, the full-length cDNAs of Dmrt1 quantified using the absorption of light at 260 and 280 nm and Sox9 from testes of the frog Rana chensinensis were by Thermo Scientific NanoDrop 2000 (Thermo Fisher).

Figure 1. Images of undifferentiated gonads and differentiated testes and ovaries of R. Chensinensis tadpoles. A, Stage 28; B, stage 46, testis; C, stage 46, ovary. Wd, Wolffian duct; Gmc, gonad-mesonephros complex; Pe, perisome; Ki, kidney; Te, testis; Ov, ovary.

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Nucleic acids of ratio 1.7–1.9 were used for semiquantitative 2.4. rcDmrt1 and rcSox9 expression analysis RT-PCR analysis. In order to clone Dmrt1 and Sox9 in Expressions of rcDmrt1 and rcSox9 were analyzed in adult testes of R. chensinensis, specific primers (Table 1) were tissues including testis, ovary, kidney, liver, and brain by designed referring to the relevant cDNA sequences of Rana RT-PCR using Dmrt1 and Sox9 primers. To standardize rugosa (AB272609.1, AB035887.1), Rana nigromaculata each experiment, the amount of a specific transcript was (EF524050.1), and Bufo marinus (FJ697174.1). PCR divided by the amount of rp18 transcript. PCR reactions was performed in a final volume of 50 µL containing 10 were performed and the amplified products were analyzed µL of cDNA, 1 µL of forward primer (10 µM), 1 µL of as previously described. In tadpoles, expression of rcDmrt1 reverse primer (10 µM), 5 µL of 10X PCR buffer (Mg2+), and rcSox9 in the GMC during stages 26–46 was examined 4 µL of dNTP mix, 0.5 µL of TaKaRa Taq, and 28.5 µL of just like in adult frogs. ddH O. The amplified products were analyzed on a 1.5% 2 2.5. In situ hybridization agarose gel and purified using the MiniBEST Agarose Digoxigenin (DIG)-labeled RNA probes of rcDmrt1 Gel DNA Extraction Kit, Ver. 4.0 (TaKaRa), followed by used for in situ hybridization were synthesized by in ligation with the pGEM-T easy vector (Promega) and vitro transcription with a DIG RNA labeling kit (Roche transformation into DH5α E. coli cells. In order to obtain Diagnostics). The transcriptions were performed from accurate sequences, at least 3 individuals of positive clones linearized plasmids using either T7 (antisense) or SP6 were selected for sequencing. Finally, SeqMan of DNAStar software was used for sequence assembly. (sense) RNA polymerases. The testes were sectioned at a thickness of 8 µm. The in situ hybridization protocol was 2.3. Sequence and phylogenetic analysis performed essentially as described previously (Wilhelm et Full-length amino acid sequences of rcDmrt1 and rcSox9 al., 2007; Abramyan et al., 2009). were deduced from the obtained nucleotide sequences. Potential functional motifs were analyzed by SMART 3. Results (http://smart.embl.de/smart/set_mode.cgi?NORMAL=1) (Letunic et al., 2012). Signal peptide prediction was 3.1. Identification and characterization of rcDmrt1 and performed with SignalP (http://www.cbs.dtu.dk/services/ rcSox9 SignalP/) (Petersen et al., 2011). Secondary structure was Through sequence assembly, we obtained a cDNA of predicted using PSIPRED v3.3 (http://bioinf.cs.ucl.ac.uk/ 1118 bp from R. chensinensis, which was named rcDmrt1 psipred/). Molecular weight and pI were predicted using (GenBank Accession No. KC439687). The rcDmrt1 Compute pI/Mw (http://web.expasy.org/compute_pi/). In sequence was composed of a 5’ untranslated region of 4 order to construct phylogenetic trees, multiple sequence bp, 3’ untranslated region of 109 bp, and ORF of 1005 bp, alignments were performed by ClustalX (Thompson et which encoded 334 amino acids (Figure 2). The predicted al., 1997). Neighbor-joining phylogenetic trees of Dmrt1 amino acid sequence of rcDmrt1 had a conserved DM and Sox9 from different amphibians and reptiles were domain, a male-specific motif, and a P/S-rich element created by the MEGA 5.1 program, in which we applied a (Figure 2), which showed high homology to G. rugosa bootstrap test of 2000 replicates to check the robustness of (93%), X. tropicalis (66%), X. laevis (65%), H. sapiens the phylogram. (53%), and M. musculus (52%) (Table 2). The identities

Table 1. Oligonucleotide primers used for PCR.

Gene name Forward primer (5’ to 3’) Reverse primer (5’ to 3’) Size (bp) Dmrt1-1 AAAATGCCTAACAGCGAGGA TATTCTTCACCGAGGATCCAC 1079 Dmrt1-2 ATGCCTAACAGCGAGGAGCCAT CGCCACGCAGATCATAAAATTGTC 628 Dmrt1-3 GCTCAATCGAAGAAATCC CTCCAGCAGTAGTACTAGTCG 325 Dmrt1-4 ATGCCAAAATCTTCTCGTT ACCTGTAAGGGTCACGGAAGGCA 301 Sox9-1 GCATGAATCTCTTGGATCCCT GGATGGTCCTTCTTGTGCTG 511 Sox9-2 CTCCCCATCTCATTCGCAT TCCATTGACCCTGACTGGC 308 Sox9-3 AAGAACAAACCGCATGTCA CTGTAATAAGAGTTTGAGCCTT 980 Sox9-4 ATGTCAAGAGGCCCATGAATGC TGCTGGACTCTCAGACGTTC 184 Sox9-5 ACACATCAAAACCGAGCAG TTGTGCCATGTCTCAGATAAGGG 433 Sox9-6 CCAGGCCATAGACTTAATGACT TTTACGGGAAGAGTGACACC 140 rp18 GCTGTCGACTTCGCAGAAAGGCA ACCTGTAAGGGTCACGGAAGGCA 115

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Figure 2. Nucleotides and deduced amino acids of rcDmrt1. The DM domain is boxed in shaded rectangles, while the male-specific motifs and P/S-rich element are underlined with a solid and dotted line, respectively. Cysteines and histidines that coordinate Zn2+ are aligned as two intertwined binding sites: site I (circles) and site II (boxes). The stop codon is marked by an asterisk. of amino acid sequences of the DM domain and male- (86%) (Figure 3; Table 2). Amino acid sequences of the specific motif showed 82%–100% and 68%–91% identities, HMG-box domain and PQA-rich element showed 97%– respectively, while the P/S-rich element showed 27%–90% 99% and 37%–85% identities among the tested species. identities among the tested species except B. gargarizans, The start and stop codon positions of theSox9 ORF of C. palustris, C. mydas, and T. scripta (Table 2). The start and R. chensinensis and G. rugosa were similar to each other stop codon positions of Dmrt1 of R. chensinensis and G. as well as no frame shift. There was no N-terminal signal rugosa were similar to each other as well as no frame shifts. sequence of rcSox9, which suggested that rcSox9 was also a There was no N-terminal signal sequence of rcDmrt1, nonsecretory . The molecular weight of rcSox9 was which suggested that rcDmrt1 was a nonsecretory protein. 53.6 kDa with an isoelectric point of 6.32. The secondary The molecular weight of rcDmrt1 was 36.8 kDa with structure of rcSox9 contained 17.12% alpha helix, 2.92% an isoelectric point of 8.45. The secondary structure of extended strand, 0.84% beta turn, and 79.12% random rcDmrt1 contained 14.97% alpha helix, 22.75% extended coil. strand, 0.84% beta turn, and 62.28% random coil. In addition, we also obtained cDNA of 1580 bp for R. 3.2. Multiple alignments and phylogenetic tree chensinensis, which was named rcSox9 (GenBank Accession construction No. KC442293). The rcSox9 sequence was composed of a Multisequence alignments of Dmrt1 and Sox9 of R. 5’ untranslated region of 3 bp, 3’ untranslated region of chensinensis and other tested species were performed 128 bp, and ORF of 1449 bp, which encoded 482 amino by DNAman software. Results showed that the identity acids (Figure 3). The predicted amino acid sequence of of rcDmrt1 with other amphibians, fishes, and reptiles rcSox9 had a conserved HMG-box domain and PQA- was 53%–93%, 39%–41%, and 28%–40% (Table 2), rich element (i.e. rich in proline, glutamine, and alanine), respectively. In addition, the identity of rcSox9 with other which displayed high identities to G. rugosa (97%) and amphibians, fishes, and reptiles was 82%–97%, 74%–77%, some reptiles, such as T. scripta (87%) and C. palustris and 86%–88% (Table 2), respectively.

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Table 2. Comparison of identities among Dmrt1 and Sox9 from R. chensinensis and other species.

Dmrt1 Sox9 Species DM domain Male-specific motif P/S Overall HMG domain PQA Overall Rana chensinensis 100 100 100 100 100 100 100 Glandirana rugosa 100 91 90 93 99 85 97 Pleurodeles waltl 89 82 77 53 99 70 82 Xenopus tropicalis 91 83 64 66 97 69 91 Xenopus laevis 90 83 66 65 99 58 89 Rhinella marina 94 73 47 64 98 65 93 Bufo gargarizans - - - - 98 67 93 Epinephelus coioides 88 68 27 39 98 50 77 Danio rerio 82 68 37 41 99 69 74 Crocodylus palustris - - - - 99 56 86 Chelonia mydas 93 0 0 28 99 72 88 Trachemys scripta 65 63 0 40 99 59 87 Homo sapiens 91 77 80 53 99 37 82 Mus musculus 92 73 77 52 - - -

The values are % aa identities of rcDmrt1 and rcSox9, respectively. Dmrt1 identity was compared againstDanio rerio (AAQ04555.1), Glandirana rugosa (BAF31129.1), Epinephelus coioides (ABK15558.1), Xenopus laevis (NP_001089969.1), Rhinella marina (ACN54528.1), Chelonia mydas (EMP30456.1), Pleurodeles waltl (ABB83370.1), Xenopus tropicalis (XP_012808036.1), and Rana chensinensis (AGH33736.2). Sox9 identity was compared against D. rerio (NP_571718.1), G. rugosa (BAA95427.1), E. coioides (ACZ51153.1), X. laevis (NP_001084276.1), R. marina (ACN54527.1), Trachemys scripta (ACG70782.1), C. mydas (EMP39318.1), P. w altl (ACF95883.1), X. tropicalis (NP_001016853.1), Crocodylus palustris (ACU12296.1), R. chensinensis (AGI56296.2), and Bufo gargarizans (ABI34568.1). Identity was calculated after Cluster X pairwise alignment using the DNAman tool.

Phylogenetic analysis indicated that rcDmrt1 and females. In contrast, the rcSox9 transcript was detected in rcSox9 were grouped in the clades of amphibian Dmrt1 all GMC samples and became stronger at stages 32–38 and and Sox9, respectively (Figure 4). Furthermore, rcDmrt1 42–46. The rcSox9 transcript was detected in both males and rcSox9 respectively constituted a sister group with the and females at stages 42–46, whereas rcDmrt1 was only Dmrt1 and Sox9 of G. rugosa (Anura), showing an identity detected in males (Figure 5). of approximately 93%–97% (Figure 4; Table 2), but a 3.4. Organ and tissue distributions of rcDmrt1 and relatively distant relationship was detected for P. waltl rcSox9 in adults (Caudata), D. rerio (fishes), and Chelonia mydas (reptiles) Expression of rcDmrt1 and rcSox9 was detected in various (Figure 4). tissues including testis, ovary, liver, brain, and kidney of 3.3. Expression of rcDmrt1 and rcSox9 in gonads of adults by RT-PCR while rpl8 was an internal control. The tadpoles results showed that Dmrt1 had a higher expression level As Figure 5 shows, expression of rcDmrt1 and rcSox9 was in the testis, but it was not examined in the ovary or any examined in the GMC of tadpoles during stages 26–28 and other tissues (Figure 6). As Figure 6 also shows, rcSox9 in the gonads at stages 33–46 by RT-PCR. ThercDmrt1 was expressed in adult testes, ovaries, kidneys, livers, and transcript was first detected in undifferentiated gonads brains, while stronger expressions of rcSox9 were detected at stage 30, which became stronger at stage 32, and the in testes and brains, respectively. In addition, we found strongest expression was identified at stages 34–36. At that rcSox9 had higher expression than rcDmrt1 in testes. stage 38, rcDmrt1 had approximately the same expression 3.5. Location of rcDmrt1 expression in testes level as stage 30, and there was no rcDmrt1 transcript Expression of rcDmrt1 was detected in adult testes by in observed at stage 40. From stage 42, rcDmrt1 expression situ hybridization. In the sections, strong positive signals was examined and it became stronger at stage 46 in testes of rcDmrt1 were mainly detected in interstitial cells, Sertoli of the males, but it was not observed in ovaries of the cells, and developing germ cells (Figure 7A). In contrast,

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Figure 3. Nucleotides and deduced amino acids of rcSox9. The HMG-box domain is boxed in shaded rectangles, while the Sox-N domain and PQA-rich element are underlined with a solid and dotted line, respectively. The stop codon is marked by an asterisk. no signals were detected in the negative control under the sequence of rcDmrt1 showed more than 92% identity DIG RNA labeling sense probe (Figure 7B). to Dmrt1 homologs of G. rugosa and P. nigromaculatus in anurans, and 59% identities to Dmrt1 of P. w altl in 4. Discussion Caudata. Dmrtl was reported in human, mouse, chick, 4.1. Characteristic analysis of rcDmrt1 and rcSox9 turtle (Chelydra serpentina, Trachemys scripta), frog The sex determination mechanism of amphibians mainly (Xenopus laevis, Xenopus tropicalis, Rana rugosa), and fish includes genetic sex determination and environmental sex (Raymond et al., 1999; Guan et al., 2000), which contained determination. Dmrt1, Daxl, Sfl, Sox3, Sox9, and Foxl2 are a DM domain, male-specific motif, and P/S-rich element. sex determination-related genes in amphibians (Clarkson The DM domain, a novel DNA binding motif, contained and Harley, 2002; Nakamura, 2009). In the present study, the conserved cysteines, histidines, and chelated zinc, we have obtained and characterized a Dmrt1 cDNA from which combined with the DNA minor groove (Zhu et R. chensinensis, named rcDmrt1. The deduced amino acid al., 2000). Through the zinc finger structure, Dmrt1 as

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Figure 4. The neighbor-joining phylogenetic trees of Dmrt1 and Sox9. Phylogenetic tree was constructed on the basis of alignment of the amino acid sequences of Dmrt1 and Sox9 homologs, showing the evolutionary relationship of rcDmrt1 (A) and rcSox9 (B) with other species of the Dmrt1 and Sox9 family. Numbers at branch nodes are percentages of bootstrap confidence values derived from 2000 replications. GenBank Accession Nos. of various species are shown in Table 2.

a regulator bound to the specific DNA sequence and Members of the Sox superfamily were characterized was involved in sex determination and differentiation with the presence of a Sry-related HMG box, which was (Raymond et al., 1999). Thew male-specific motif, a sex involved in various developmental processes of animals, specific motif near the C-terminus, was well conserved particularly in organogenesis (Bowles et al., 2000; Jakob among numerous Dmrt1 and mainly involved in male sex et al., 2011; Nakamura et al., 2012; Wei et al., 2016). Here, determination (Guan et al., 2000). The P/S-rich element as we also identified and characterized the complete coding a C-terminal domain rich in proline and serine residues sequence of rcSox9. The conserved HMG-box domain was detected in the Dmrt1 of many species. As Hoshi and PQA-rich element were found in rcSox9, respectively, and Nakao (2008) showed, the homology within the P/S- and their relative positions were similar to Sox9 homologs rich region of rcDmrt1 was high throughout the other throughout the investigated species (McDowall et al., vertebrates, except fish. 1999; Clarkson and Harley, 2002; Smith and Koopman,

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Figure 5. Expression of rcDmrt1 and rcSox9 in GMC/gonads of R. chensinensis tadpoles at stages 26–46. M, Male; F, female.

Figure 6. rcDmrt1 and rcSox9 expression in various tissues of adult R. chensinensis. Upper two panels, RT-PCR using rcDmrt1 and rcSox9 gene-specific primers, respectively; lower panel, control RT-PCR using rpl8 gene-specific primers.

Figure 7. The cellular localization of rcDmrt mRNA in adult testis. A, The positive results of rcDmrt1 mRNA in testis by in situ hybridization; B, negative control. IC, Interstitial cells; SC, Sertoli cells; GC, germ cells.

2004). The HMG-box as a DNA binding domain has a and alanine, which contributed to maximal transactivation high affinity with the minor groove of non-B-type DNA (McDowall et al., 1999; Smith and Koopman, 2004). In structures, which was involved in the regulation of DNA- humans, mutations of Sox9 caused CD, and many of these dependent processes such as transcription, replication, mutations could result in truncations and frameshifts in recombination, and DNA repair (Stros et al., 2007). It the C terminus of Sox9 (McDowall et al., 1999). has been confirmed that Sox9 bound to a minimal DNA In phylogenetic trees, Anura formed a cluster by element in intron 1 of Col2a1 and acted as a potent itself, whereas Caudata and reptiles formed another two activator in the differentiation program of chondrocytes separate clusters. The evolutionary relationships were (Lefebvre et al., 1997). The PQA-rich element as a C consistent with the one in traditional taxonomy. All these terminus domain consisted entirely of proline, glutamine, results revealed that rcDmrt1 and rcSox9 were orthologous

326 LI et al. / Turk J Zool to the Dmrt1 and Sox9 of G. rugosa in the evolutionary of testes (Guan et al., 2000; Brunner et al., 2001; Shibata et relationships, respectively. In addition, rcDmrt1 and al., 2002; Veith et al., 2006; Ge et al., 2017, 2018). rcSox9 were well conserved in the living organisms. Individual development was strictly regulated by the 4.2. Expression analysis of rcDmrt1 and rcSox9 during gene regulatory networks, in which the specific genes gonadal differentiation and other related genes were involved, and any obstacle Dmrt1 is essential for male sexual phenotypic would lead to abnormal development. In vertebrates, Sox9 characterization in vertebrates. In amphibians, sex- was expressed in the GMC of both sexes during gonadal determination systems are often highly dynamic, although differentiation (Barske and Capel, 2010; Dumond et al., genetic effects have been documented in common frogs 2011), which indicated that Sox9 might be involved in (Rodrigues et al., 2014). However, the basic mechanism gonadal development. Moreover, Sox9 clearly showed of sex determination and gonadal differentiation is still sexually dimorphic patterns in adults; the defect of Sox9 of XY individuals in CD showed complete or partial sex unknown. In X. laevis, Dmrt1 was transcribed in gonads reversal (Meyer et al., 1997; Michel-Calemard et al., 2004; at stages 53–62, which was expressed exclusively in Katoh-Fukui et al., 2015). Here, we found that the different primordial gonads and continued its expression after sex expressions of rcSox9 were not only related to the sexes but determination rather than in the sex determination period also the development stages. rcSox9 was detected in all the (Osawa et al., 2005; Yoshimoto et al., 2006; Uno et al., 2008). tested tissues, with the highest expression found in the testes In G. rugosa, Dmrt1 was first expressed in differentiating and ovaries. In addition, rcSox9 has also been expressed in testes at stage XXV (unlike Gosner stages), which only the GMC of larvae at stages 32–45. These results suggest existed in spermatogonia cells (Shibata et al., 2002). The that Sox9 plays a role in gonadal differentiation and the role of rcDmrt1 in sex determination of R. chensinensis is maintenance of the functioning of adult sexual glands. still not examined and confirmed. Basing on Gosner stages, Sox9 and forkhead box L2 (Foxl2) could have synergism the development of tadpoles can be divided into 4 periods. and antagonism for each other in regulating the expression There is no sex differentiation during stages 27–30 and the of Cyp19 and the conversion of testosterone to estradiol gonads come into differentiation at stages 31–36, the end of (McElreavey et al., 1993; Lavery et al., 2012; Navarro- gonadal differentiation. At stages 37–42 the disappearance Martín et al., 2012). However, the absence of would of the tail occurs and the formation of the juvenile frog is lead to Sox9 expression, testis development, and XX sex during stages 43–46 (Gosner, 1960; Hogan et al., 2008). In reversal (Veitia, 2010). Therefore, we speculated that this study, we examined the expression of rcDmrt1 in R. Sox9 may indirectly affect steroid hormone synthesis and chensinensis tadpoles. It was specifically expressed in the regulate gonadal development. primordial gonads at stages 30–36. In addition, rcDmrt1 In summary, this study described the cloning and expression was examined and became increased gradually characterization of the R. chensinensis Dmrt1 and Sox9 at stages 42–46 in males, but it was not observed in the genes. Their mRNA transcripts presented a tissue-specific females. These results showed that Dmrt1 transcription manner and natural linear flow in regulation of gonadal was restricted in the gonadal differentiation period of development of R. chensinensis, which indicated their tadpole. Therefore, we supposed that rcDmrt1 played a critical roles in gonadal development and maintenance key role in the early sex differentiation of R. chensinensis. of gonadal function. In addition, RcDmrt1 was only Nevertheless, it will be meaningful to determine whether detected in the testes of adult frogs, and RcDmrt1 mRNA rcDmrt1 is involved in sex determination pathways or was located in interstitial cells, Sertoli cells, and germ directly regulates the synthesis of sex steroids. cells of adult testes. These results show that both Dmrt1 A previous study showed that Dmrt1 was expressed and Sox9 participate in gonadal differentiation and in the gonads of Bufo marinus, and Dmrt1 mRNA was maintain testicular function, and Dmrt1 is more closely restricted to Sertoli cells (Abramyan et al., 2009). In implicated in testicular differentiation. These explorations mammals, Dmrt1 was localized in the germ cells and have provided novel molecular insights to understand Sertoli cells of the testes in Macropus eugenii, but in the mechanism of sex determination of nonmammalian addition it was detected in Leydig cells, peritubular myoid vertebrates. cells, and the acrosome of sperm heads (Pask et al., 2003). In this study, rcDmrt1 was only expressed in testes of adult Acknowledgment frog R. chensinensis, and rcDmrt1 mRNA was localized in This study was funded by the Shaanxi Province Natural interstitial cells, Sertoli cells, and some developing germ Science Foundation Research Project (Grant No: 2019JM- cells in testes. All these results suggest that Dmrt1 has a 391) and the Fundamental Research Funds for the Central key role in spermatogenesis and maintaining the function Universities (Grant No: GK201703039).

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