Many genes are known to be involved in gonadal differentiation in vertebrates. Dmrt1, a gene that encodes a transcription factor with a DM-domain, is considered to be one of the essential genes controlling testicular differentiation in mammals, birds, reptiles, amphibians and fish. However, it still remains unknown which testicular cells of animals other than mice and chicks express Dmrt1 protein. For an explanation of its role(s) in testicular differentiation in vertebrates, the expression of the Dmrt1 protein needs to be studied. For this purpose, we conducted an immunohistochemical study of this protein in an amphibian by using an antibody specific for Dmrt1. No positive signal was found in the indifferent gonad of tadpoles of Rana rugosa at early stages. However, in the testis of tadpoles at later stages (XV–XXV) and in frogs one month after metamorphosis, this protein was expressed in interstitial cells and Sertoli cells. In the testis of adult frogs, germ cells also expressed Dmrt1 protein. RT-PCR analysis revealed that the gene for this protein was not transcribed at any time during ovarian development, but was expressed in the female to male sex-reversed gonad. This was true when immunohistological studies were performed. In addition, Southern blot analysis showed Dmrt1 to be an autosomal gene. Taken together, our findings indicate that Dmrt1 protein is expressed by interstitial cells, Seroli cells and germ cells in the testis of R. rugosa. Dmrt1 may thus be very involved in the testicular differentiation of amphibians.   

1.
Burtis KC, Baker BS: Drosophila doublesex gene conrols somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell 56:997–1010 (1989).
2.
Carninci P, Shibata Y, Hayatsu N, Sugahara Y, Shibata K, Itoh M, Konno H, Okazaki Y, Muramatsu M, Hayashizaki Y: Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res 10:1617–1630 (2000).
3.
Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159 (1987).
4.
Cox KH, DeLeon DV, Angerer LM, Angerer RC: Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101:485–502 (1984).
5.
Guan G, Kobayashi T, Nagahama Y: Sexually dimorphic expression of two types of DM (Doublesex/Mab-3)-domain genes in a teleost fish, the Tilapia (Oreochromis niloticus). Biochem biophys Res Commun 272:662–666 (2000).
6.
Honada S, Morohashi K, Nomura M, Takeya H, Kitajima M, Omura T: Ad4BP regulating steroidogenic P-450 gene is a member of steroid hormone receptor superfamily. J biol Chem 268:7494–7502 (1993).
7.
Kashiwag A, Kashiwagi K: Artificially produced chimeras between haploids and diploids in Japanese anurans. Sci Rep Lab Amphibian Biol Horoshima Univ 12:37–53 (1993).
8.
Kawano K, Furusawa S, Matsuda H, Takase M, Nakamura M: Expression of steroidogenic factor-1 in frog embryo and developing gonad. Gen Com Endocrinol 123:13–22 (2001).
9.
Kent J, Wheatley SC, Andrews JE, Sinclai, AH, Koopman P: A male-specific role for SOX9 in vertebrate sex determination. Development 122:2813–2822 (1996).
10.
Kettlewell JR, Raymond CS, Zarkower D: Temperature-dependent expression of turtle Dmrt1 prior to sexual differentiation. Genesis 2:174–178 (2000).
11.
Kim S, Kettlewell J, Anderson RC, Bardwell VJ, Zarkower D: Sexually dimorphic expression of multiple doublesex-related genes in the embryonic gonad. Unpublished (Accession No. AAN77235) (2002).
12.
Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletler J, Housman D, Jaenisch R: WT-1 is required for early kidney development. Cell 74:679–691 (1993).
13.
Kreig PA, Varnum SM, Wormington WM, Melton DA: The mRNA encoding elongation factor 1-α (EF1-α) is a major transcript at the midblastula transition in Xenopus. Dev Biol 133:93–100 (1989).
14.
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 (1970).
15.
Luo X, Ikeda Y, Parker KL: A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490 (1994).
16.
Marchand O, Govoroun M, D’Cotta H, McMeel O, Lareyre J, Bernot A, Laudet V, Guiguen Y: DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss. Biochim biophys Acta 1493:180–187 (2000).
17.
Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey CE, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S, Sakaizumi M: DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417:559–563 (2002).
18.
Morais de Silva S, Hacker A, Harley V, Goodfellow P, Swain A, Lovell-Badge R: Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nature Genet 14:62–68 (1990).
19.
Nakajima T, Takase M, Miura I, Nakamura M: Two isoforms of FTZ-F1 messenger RNA: molecular cloning and their expression in the frog testis. Gene 248: 203–212 (2000).
20.
Nanda I, Shan Z, Schartl M, Burt DW, Koehler M, Nothwang H, Grutzner F, Paton IR, Windsor D, Dunn I, Engel W, Staeheli P, Mizuno S, Haaf T, Schmid M: 300 million years of conserved synteny between chicken Z and human chromosome 9. Nature Genet 21:258–259 (1999).
21.
Nanda I, Kondo M, Hornung U, Asakawa S, Winkler C, Shimizu A, Shan Z, Haaf T, Shimizu N, Shima A, Schmid M, Schartl M: A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. Proc natl Acad Sci, USA 99:11778–11783 (2002).
22.
Nishioka M, Miura I, Saitoh K: Sex chromosomes of Rana rugosa with special reference to local differences in sex-determining mechanism. Sci Rep Lab Amphibian Bio Horoshima Univ 12:55–81 (1993).
23.
Peterson GL: A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:345–356 (1977).
24.
Raymond CS, Shamu CE, Shen MM, Seifert KJ, Hirsch B, Hodgkin J, Zarkower D: Evidence for evolutionary conservation of sex-determining gene. Nature 391:691–695 (1998).
25.
Raymond CS, Kettlewell JR, Hirsch B, Bardwell VJ, Zarkower D: Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development. Dev Biol 215:208–220 (1999a).
26.
Raymond CS, Parker ED, Kettlewell JR, Brown LG, Page DC, Kusz K, Jaruzelska J, Reinberg Y, Flejter WL, Bardwell VJ, Hirsch B, Zarkower D: A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators. Hum molec Genet 8:989–996 (1999b).
27.
Raymond CS, Murphy MW, O’Sullivan MG, Bardwell VJ, Zarkower D: Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev 14:2587–2595 (2000).
28.
Sambrook J, Fritsch EF, Maniatis T: Isolation of high-molecular-weight DNA from mammalian cells, in: Molecular Cloning: A Laboratory Manual, 9.16–9.23, 2nd Ed (Cold Spring Harbor Laboratory, Cold Spring Harbor 1989).
29.
Sato K, Yasug S: Chicken keratin-19: cloning of cDNA and analysis of expression in the chicken embryonic gut. Dev Growth Differ 39:751–761 (1997).
30.
Shan Z, Nanda I, Wang Y, Schmid M, Vortkamp A, Haaf T: Sex-specific expression of an evolutionarily conserved male regulatory gene, DMRT1, in birds. Cytogenet Cell Genet 89:252–257 (2000).
31.
Shen MM, Hodgkin J: Mab-3, a gene required for sex-specific yolk protein expression and a male-specific lineage in C. elegans. Cell 23:1019–1031 (1988).
32.
Shibata K, Itoh M, Aizawa K, Nagaoka S, Sasaki N, Carninci P, Konno H, Akiyama J, Nishi K, Kitsunai T, Tashiro H, Itoh M, Sumi N, Ishii Y, Nakamura S, Hazama M, Nishine T, Harada A, Yamamoto R, Matsumoto H, Sakaguchi S, Ikegami T, Kashiwagi K, Fujiwake S, Inoue K, Togawa Y: RIKEN integrated sequence analysis (RISA) system-384 format sequencing pipeline with 384 multicapillary sequencer. Genome Res 10:1757–1771 (2000).
33.
Shibata K, Takase M, Nakamura M: The expression pattern of Dmrt1 during gonadal development of amphibians. Gen Comp Endocrinol 127:232–240 (2002).
34.
Shumway W: Stages in the normal development of Rana pipiens. I. External form. Anat Rec 78:139–147 (1940).
35.
Smith CA, McClive PJ, Western PS, Reed KJ, Sinclair AH: Conservation of a sex-determining gene. Nature 402:601–602 (1999).
36.
Smith CA, Katz M, Sinclair AH: DMRT1 is upregulated in the gonads during female-to-male sex reversal in ZW chicken embryos. Biol Reprod 68:560–570 (2003).
37.
Sugita J, Takase M, Nakamura M: Expression of Dax1 during gonadal development of the frog. Gene 280:67–74 (2001).
38.
Swain A, Lovell-Badge R: Mammalian sex determination: a molecular drama. Genes Dev 13:755–767 (1999).
39.
Takase M, Nakajima T, Nakamura M: FTZ-F1α is expressed in the developing gonad of frogs. Biochim biophys Acta 1494:195–200 (2000).
40.
Taylor AC, Kollros JJ: Stages in the normal development of Rana pipiens larvae. Anat Rec 94:7–23 (1946).
41.
Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheet: procedure and applications. Proc natl Acad Sci, USA 76:4350–4354 (1979).
42.
Vainio S, Heikkila M, Kispert A, Chinn N, McMahon AP: Female development in mammals is regulated by Wnt-4 signalling. Nature 397:405–409 (1999).
43.
Wagner T, Wirth I, Meyer I, Zabel B, Held M, Zimmer J, Pananyes J, Bricaelli FD, Husteri E, Wolf U, Tommerup N, Schempp W, Scherer G: Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79:1111–1120 (1994).
44.
Yu RN, Ito M, Saunders TL, Camper SA, Jameson JL: Role of Ahch in gonadal development and gametogenesis. Nature Genet 20:353–357 (1998).
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.