Two DM-domain genes, DMY (sex-determining gene) and DMRT1, have been reported to be expressed in the testis of medaka. In this study, a specific RT-PCR assay was used to determine the expression patterns of DMY and DMRT1 in various tissues of medaka during different stages of development. The results show that the transcripts of both DMY and DMRT1 are present not only in testes but also in several other tissues from fry and adults of medaka.
Sex-determining genes (SRY/Sry) have been identified in mammals (Sinclair et al., 1990; Koopman et al., 1991). However, no comparable genes have been found in non-mammalian vertebrates. Using positional cloning, we recently identified the sex-determining gene of medaka (Matsuda et al., 2002). This gene encodes a protein that contains a DNA-binding motif called a DM-domain, which was originally described as a DNA-binding motif shared between doublesex (dsx) in Drosophila melanogaster and mab-3 in Caenorhabditis elegans (Raymond et al., 1998). We thus named the sex-determining gene of madaka DMY (DM-domain gene on the Y-chromosome). Another DM-domain gene, DMRT1, has also been reported to be present in the testis of medaka (Brunner et al., 2001; Nanda et al., 2002). Despite the great diversity of sex-determining mechanisms involved, DMRT1 is one of well conserved genes related to sexual differentiation from invertebrates to vertebrates irrespective of whether the initiation signal of sex determination comes from genetic or environmental elements (Zarkower, 2001). In this study, we used a specific RT-PCR assay to examine the expression of two DM-domain genes, DMY and DMRT1, in various tissues of fry and adults as well as embryos of madaka just before and after hatching. We showed that DMY and DMRT1 transcripts were present not only in testes but also in several other tissues of fry and adults.
MATERIALS AND METHDS
PCR for Genotyping
Medaka fry's tails were incubated in the buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.0, 0.5% NP40, 0.5% Tween20, 0.5% proteinase K) for 2 hr at 50–60°C and were boiled for 10 min. DMY and DMRT1 were amplified from the sample using one primer pair; PG17-5 (CCGGGTGCCCAAGTGCTCCCGCTG) and PG17-6 (GATCGTCCCTCCACAGAGAAGAGA).
Total RNA Extract
Total RNA was isolated from embryos and various tissues from fry and adults of medaka using the RNeasy Mini Kit (QIAGEN).
With the Onestep RT-PCR Kit (QIAGEN), DMY and DMRT1 were amplified from the total RNA using one primer pair: PG17-25 (CCCACCAGATCCTATACAAGTGAC) and PG17-24 (GTAAACACGGGCCGCAGCCGATGGGC). To distinguish DMY and DMRT1, DMY was digested by Nhe I. The PCR conditions and specific primers for PG04 were described previously (Matsuda et al., 2002).
RESULTS AND DISCUSSION
The similarity between DMY and DMRT1 in both amino acid and nucleotide sequences is very high (Matsuda et al., 2002). For this reason, the primers were designed to amplify both of DMY and DMRT1. To distinguish DMY and DMRT1, DMY was digested by Nhe I. The present study using RTPCR combined with enzyme digestion revealed that DMY was expressed in XY males throughout the developmental stages, but not in XX females, confirming that DMY is a Y-specific gene.
Expression of DMY was evident in male embryos just before (Fig. 1A) and after hatching (Fig. 1B). Two separate portions (the head portion which includes brain and the body portion which includes gonads) were used for the analyses of embryos and fries till 20 days after hatching (dah). As shown in Fig. 1B, DMY transcripts were already present in the body portion of males collected just after hatching. This is consistent with the results of our earlier RT-PCR and in situ hybridization studies which show that DMY mRNA is expressed in testicular somatic cells of embryos and fry of male medaka (Matsuda et al., 2002). In medaka, gonadal sex differentiation becomes evident in embryos before hatching; the number of germ cells in females begin to increase at stages 37 and 38 (Kobayashi et al., unpublished). Thus, DMY expression at these periods may be responsible for the suppression of mitosis of germ cells in males.
DMRT1 expression does not occur in either male or female embryos and fry until 20 dah and becomes evident in testis when tested at 30 dah (data not shown). Thus, there appeared to be no overlapping between expression of DMY and DMRT1 during earlier stages of gonadal sex differentiation. Similar findings were reported in medaka by Nanda et al. (2002). However, the expression pattern of DMRT1 in medaka differs from those of other fishes. For example, in tilapia, Oreochromis niloticus, DMRT1 is expressed in XY gonads prior to the formation of testis, suggesting a significant role for DMRT1 in the earlier stages of gonadal sex differentiation (Kobayashi et al., unpublished). The first appearance of DMRT1 in medaka at 30–40 dah suggests that DMRT1 may function in spermatogenesis in medaka.
Under the conditions used in this study, both male and female medaka become sexually mature at about 80-100 dah. In sexually maturing fish sampled from 40 to 75 dah, DMY expression was observed not only in testis but also in several other tissues (brain, liver, eye and muscle) of XY males, with the highest expression in testis (Fig. 2). Interestingly, DMRT1 is also expressed in various male tissues with the highest expression in testis and also in ovary (Fig. 2). The expression patterns of DMY in sexually mature fish were similar to those of sexually maturing males, except for a high expression in spleen as well. In the case of DMRT1, high expression was evident in testis, ovary and spleen (Fig. 3).
It is of interest that although the sex-determining gene, DMY, is expressed only in males, expression does not restrict to testis. Indeed, its expression occurs in many other tissues including brain, liver, eyes, muscle, pituitary, heart, spleen, and intestine, as well as the head portion of embryos and fry that includes the brain. This expression pattern of DMY is similar to that of SRY in humans, in which SRY transcripts are not confined to the presumptive and mature gonadal tissues in the embryo and the adult but are more widely expressed (Clepet et al., 1993). In contrast, in the mouse embryo, Sry transcripts appear primarily in the genital ridge at a stage corresponding to the onset of testis formation (Koopman et al., 1990). The detection of DMY transcripts in medaka and SRY transcripts in humans, even at low levels, could suggest roles for these sex-determining genes outside testis and genital ridges.
This work was supported in part by Grants-in-Aid for Research from the Ministry of Education, Science, Sport and Culture of Japan, the Ministry of the Environment (Environmental Endocrine Studies), and the Ministry of Agriculture, Forestry and Fisheries (Bio Design).