Download Molecular mechanisms of sex determination and gonadal sex

Fish Physiol Biochem (2005) 31:105–109
DOI 10.1007/s10695-006-7590-2
RESEARCH ARTICLE
Molecular mechanisms of sex determination and gonadal
sex differentiation in fish
Y. Nagahama
Ó Springer Science+Business Media B.V. 2006
Abstract We have used various genetic and
molecular approaches to investigate the mechanisms
of sex determination and gonadal sex differentiation
in fish. DMY was identified as the sex-determining
gene of medaka. In tilapia, endogenous estrogens act
as natural inducers of ovarian differentiation, while
DMRT1 may be important for testicular differentiation. The roles of these regulators in sex determination and gonadal sex differentiation were ascertained
using a gene or hormonal blockade strategy.
Keywords Fish Æ Sex-determining gene Æ
Estrogen Æ DMRT1 Æ Gonadal sex differentiation
Introduction
In vertebrates, sex determination and differentiation
are key events in the development of either the testis
or ovary. SRY/Sry (sex determining region Y chromosome) is the Y chromosome gene responsible for
Y. Nagahama (&)
Laboratory of Reproductive Biology, Department of
Developmental Biology, National Institute for Basic
Biology, Okazaki 444-8585, Japan
Y. Nagahama
CREST, Kawaguchi 332-0012, Japan
e-mail: [email protected]
initiating testicular development in mammals (Sinclair et al. 1990; Koopman et al. 1991). The collection of molecular candidates implicated in the process
of gonadal sex differentiation is now quite extensive.
However, one is not able to fit all of these into simple
testicular and ovarian pathways (Koopman 2001;
Nakagawa 2004; Smith and Sinclair 2004; Yao
2005). In fish, sexual characteristics and gonadal
development vary from gonochoristic species to
several types of hermaphroditism (Devlin and
Nagahama 2002). Thus, a knowledge of sex determination and differentiation in fish will broaden our
understanding of these processes beyond the specific
details found within the group. We have been
investigating the molecular mechanisms of sex
determination and gonadal sex differentiation using
two fish species, the medaka, Oryzias latipes (sex
determination) and tilapia Oreochromis niloticus
(gonadal sex differentiation). We are particularly
interested in identifying genes or hormonal factors
that may regulate the early development of fish
gonads.
Sex determination
In addition to its small size and short generation time,
the medaka has two major advantages for genetic
research: a large interstrain diversity within the species and the existence of several inbred strains
(Schartl 2004; Matsuda 2005). As in mammals, sex
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106
determination in medaka is male heterogametic (a
stable genetic XX/XY sex determining system).
Based on positional cloning and a detailed analysis of
BAC clones (Matsuda et al. 2001) using shotgun
sequencing, we located a unique gene in the short
sex-determining region on the Y chromosome. This
gene consists of six exons and encodes a protein of
267 amino acids including the highly conserved DM
domain. The DM domain was named after a related
DNA-binding motif found in two proteins, doublesex
(dsx) and mab-3, involved in sexual development in
Drosophila and C. elegans, respectively. This Yspecific DM-domain gene was named DMY (DMdomain gene on the Y chromosome) (Matsuda et al.
2002; Matsuda 2005), or Dmrt1b (Nanda et al. 2002;
Volff et al. 2003).
Two naturally occurring XY female medaka
showed different mutations in the DMY gene (Matsuda et al. 2002). One of these mutants was found to
carry a mutation causing a frameshift and premature
termination of the DMY protein. When mated, all XY
offspring with the mutant Y were female (Matsuda
et al. 2002). The other mutant had a severe depression in DMY expression in the embryo and 60% of its
XY offspring with the mutant Y developed as females
(Matsuda et al. 2002). Taken together, the loss-offunction mutant and the depressed expression mutant
strongly indicate that DMY is required for normal
testicular development. More recently, we demonstrated that a genomic DNA fragment carrying DMY,
containing about 56 kb of the coding region, about
60 kb of the upstream non-coding region, and about
1.4 kb of the downstream non-coding region (Matsuda et al., unpublished). Among these transgenic
progeny, we found that 25% of XX fish develop into
males. Furthermore, some males were found to be
fertile. These findings demonstrate that DMY is sufficient to induce male development in XX medaka. It
is important to note that DMY transgenic XX medaka
are fully functional and fertile males, whereas Sry
transgenic mice are sterile (Koopman et al. 1991).
Thus, medaka is the first transgenic vertebrate shown
to undergo a complete sex reversal. Taken together,
these loss- and gain-of-function studies indicate that
DMY is the sex-determining gene of medaka. DMY is
thus the first sex-determining gene to be found in
non-mammalian vertebrates (Swan 2002; Schartl
2004; Matsuda 2005).
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Fish Physiol Biochem (2005) 31:105–109
Y chromosome-linked DMY appears to have originated from a duplicate copy of autosomal DMRT1
(DM-related transcription factor 1), another DM domain gene that is most homologous to DMY (about
80% at the amino-acid level) and is involved in male
development in various vertebrates (Nanda et al.
2002; Lutfalla et al. 2003). Then, the duplicated
DMRT1 in the Y chromosome acquired a new function as a sex-determining gene, DMY (Matsuda 2005).
Further, DMY is also found in Oryzias curvinotus,
which is most closely related to medaka (Matsuda
et al. 2003), but is not found in other Oryzias species
or in other fishes (Kondo et al. 2003) (Table 1).
Importantly, there is no sequence homology between
two known sex-determining genes, SRY/Sry and DMY
(Sinclair et al. 1990; Matsuda et al. 2002; Nanda
et al. 2002). Taken together, these findings suggest
that the top of the cascades leading to males and females, the sex-determining gene, exhibits extensive
diversity among vertebrates.
In medaka, the first apparent morphological sex
difference is in the number of germ cells immediately
before hatching (Kobayashi et al. 2004). In XY fry,
primordial germ cells (PGCs) subsequently enter
mitotic arrest and the proliferation of A-type spermatogonia does not occur until 20–30 days after
hatching (dah). Because of the sequence similarity
between DMY and DMRT1, expression of DMY and
DMRT1 cannot be distinguished by in situ hybridization, but can be distinguished by RT-PCR using
specific primers. DMY mRNA and protein are expressed specifically in the somatic cells (Sertoli cells)
surrounding PGCs in the early gonadal primordium,
before morphological sex differences are apparent
Table 1 SRY/Sry, DMY and DMRT1 in various vertebrate
species
Fishes
Oryzias latipes
Oryzias curvinotus
Oryzias celebensis
Fugu
Zebrafish
Tilapia
Amphibians
Reptiles
Birds
Mammals
SRY/Sry
DMY
DMRT1
)
)
)
)
)
)
)
)
)
+
+
+
)
)
)
)
)
)
)
)
+
+
+
+
+
+
+
+
+
+
Fish Physiol Biochem (2005) 31:105–109
(immediately before hatching). DMY protein is
localized to the nuclei of Sertoli cells, suggesting that
DMY functions as a transcriptional regulator during
early testicular differentiation. RT-PCR analyses
further show that expression of DMY in the testis is
continuous from the embryonic to adult stage. No
specific expression of DMRT1 is observed in either
sex during gonadal sex differentiation. DMRT1 is
expressed in spermatogonium-supporting cells after
testicular differentiation (20–30 dah) and its level is
much higher than that of DMY in mature testes. In
XX sex-reserved testes, DMRT1 is expressed in the
Sertoli cell lineage, similar to the expression of DMY
in XY testes. Taken together, these results strongly
suggest that in medaka, DMY regulates PGC proliferation and testicular differentiation, and that
DMRT1 is a regulator of spermatogenesis.
Gonadal sex differentiation
Unlike in mammals, in non-mammalian vertebrates
sex steroids play important roles in gonadal sex
differentiation. The roles of sex steroids in sex
determination and differentiation in fish have been
comprehensively reviewed (Yamamoto 1969;
Nakamura et al. 1998; Baroiller et al. 1999; Guiguen
2000; Piferrer 2001; Devlin and Nagahama 2002;
Strussmann and Nakamura 2003). The Nile tilapia
provies an excellent example of the precise nature of
the steroidogenic actions during gonadal sex differentiation (Nakamura et al. 1998; Nagahama 2000;
Kobayashi et al. 2003). In this fish, all genetic female
(XX) or male (XY) broods can be obtained from sexreversed, pseudo male sperm (XX), or normal eggs
(XX) and super male sperm (YY), respectively. The
gonadal anlage is not observed in tilapia fry at
hatching and primordial germ cells (PGCs) are located
in the outer layer of the lateral plate mesoderm. At
3 dah, the gonadal anlagen forms and the germ cells in
it are enclosed by somatic cells derived from the
coelomic epithelium (Kobayashi et al. 2000). Using
genetically controlled all-female and all-male tilapia,
we showed that cells immunopositive for four steroidogenic enzymes essential for the biosynthesis of
all major sex steroid hormones including androgens
and estrogens, appeared first in the female gonadal
anlargen of tilapia at 7–10 dah. Immunopositive
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steroid-producing cells increased in number accompanying ovarian differentiation and increased more in
the developed ovaries (Nakamura et al. 1998;
Kobayashi et al. 2003). Real-time PCR analysis further revealed that oP450arom (the ovarian form of
aromatase) mRNA was first detected at 5 dah in female gonads, and then increased rapidly at 7 dah
(S. Ijiri and H. Kaneko, unpublished). We also found
that estrogen receptors a and b are expressed before
the expression of steroidogenic enzymes in XX fry
(T. Kobayashi et al., unpublished). These findings
strongly suggest that estrogen (estradiol-17b) is produced in the gonads around the time of ovarian differentiation and may have an important role in the
differentiation. In tilapia, treatment of genetic females
with a non-steroidal P450arom inhibitor (fadrozole)
before and during gonadal sex differentiation causes
female masculinization (sex reversal from females to
males), suggesting that endogenous estrogens are already synthesized in undifferentiated gonads of genetic females (Nakamura et al. 1998). In contrast, all
fish treated with both fadrozole and estradiol-17b had
normal ovaries. In addition, treatment of genetic females with an estrogen receptor antagonist (tamoxifen) results in their masculinization. These findings
further strengthen the idea that endogenous estrogen
signaling through estrogen receptors plays key roles in
ovarian differentiation in tilapia (Fig. 1).
Medaka
Y
X
DMY
DMY
X
Tilapia
X
?
Foxl2
F
oxl2
oP450arom
oP450arom
X
Y
X
X
?
?
DMR T1
DMRT1
F oxl2
Foxl2
oP450arom
Testis
Ovary
DMR T1
DMRT1
Testis
Ovary
Fig. 1 A potential mechanism of sex determination and
gonadal sex differentiation in medaka and tilapia
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108
In contrast to XX fry, immunopositive reactions to
antibodies against several steroidogenic enzymes
were not observed in undifferentiated gonads of genetic males. Weakly positive cells appeared first after
testicular differentiation and strong reactions, except
for oP450arom, were seen in the testes just before the
onset of spermatogenesis. Thus, it is less likely that
steroid hormones, including androgens, have an
important role in testicular differentiation in tilapia.
A good candidate for a factor regulating testicular
differentiation is DMRT1 (Guan et al. 2000; Devlin
and Nagahama 2002). We found that the DMRT1
transcript was expressed male-specifically in testicular Sertoli cells of tilapia prior to and during sex
differentiation. More recently, we have shown that
genetically female tilapia carrying extra copies of
tilapia DMRT1 as a transgene undergo sex reversal
(D.S. Wang et al., unpublished). These results
strongly suggest an important role for DMRT1 in
testicular differentiation in tilapia (Fig. 1).
Since endogenous estrogens are critical for
directing initial ovarian differentiation, the genes
regulating oP450arom expression are keys to gonadal
sex differentiation in fish. However, the regulatory
factor activating oP450arom during ovarian differentiation is at present unknown. We have previously
shown that Ad4BP/SF-1 acts as an important transcriptional modulator of gonadotropin-induced
oP450arom gene expression in vitellogenic follicles
of tilapia (Yoshiura et al. 2003). Although the
Ad4BP/SF-1 transcript is detected by RT-PCR, no
difference in expression is observed between males
and females during sex determination and differentiation. More recently, we found that the forkhead
transcription factor Foxl2 and oP450arom begin to be
expressed at the same time (5 dah) in genetic females
and their levels increased at 7 dah. Luciferase assays
using mammalian cell lines showed that both Ad4BP/
SF-1 and Foxl2 activate transcription of the
oP450arom gene. Promoter assays and electromobility shift assays revealed that Ad4BP/SF-1 and
Foxl2 most likely act by binding directly to the
oP450arom promoter. We also found that Foxl2 enhances Ad4BP/SF-1-activated transcription of
oP450arom. Our pull-down assays suggest that Foxl2
can heterodimerize with Ad4BP/SF-1. In vivo transgenic over-expression of a dominant negative mutant
of Foxl2 was achieved by injection of these GFP
constructs into fertilized XX eggs. These transgenic
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Fish Physiol Biochem (2005) 31:105–109
XX tilapia showed a reduced expression of
oP450arom, leading to ovarian degeneration, followed by the development of testicular tissue to different degrees and, in some cases, a complete sex
change (D.S. Wang et al., unpublished).
Our recent findings also suggest that Dmrt1 may
be involved in the regulation of oP450arom gene
expression (D.S. Wang et al., unpublished). Luciferase assays revealed that Dmrt1 represses Ad4BP/
SF-1-activated transcription of oP450arom. Our data
suggest that DMRT1 does not bind the oP450arom
promoter, but can heterodimerize with Ad4BP/SF-1.
Over-expression of Dmrt1 in XX gonads resulted in
reduced expression of oP450arom with gonadal
structures similar to those seen in transgenic XX
gonads with a dominant negative mutant of Foxl2.
Taken together, these in vivo and in vitro results
strongly suggest that the transcriptional regulation of
oP450arom by Foxl2 (stimulatory) (Fig. 1) and
Dmrt1 (inhibitory) is critical for gonadal sex differentiation in the Nile tilapia.
Conclusions
In this brief review, three major factors, DMY,
DMRT1 and estrogens (oP450arom), were discussed
in relation to sex determination, testicular differentiation and ovarian differentiation, respectively. Our
findings, together with earlier studies on other vertebrates, suggest that the top of the cascades leading
to males and females, the sex-determining gene,
exhibits extensive diversity among vertebrates
(Table 1), while the molecular cascades leading to
males and females seem to be relatively conserved
(Fig. 1). Preliminary data from our laboratory also
suggest that several transcription factors such as
DMY, Ad4BP/SF-1, Dax1 and Dax2 may be involved
in gonadal sex differentiation in both medaka and
tilapia (D.S. Wang and L.Y. Zhou, unpublished).
Studies on a coordinated interaction between the
transcription and hormonal factors may be important
to understand the mechanisms that lead to testicular
and ovarian differentiation. Among the vertebrates,
fish exhibiting a range of gonadal differentiation
types including gonochoristic species as well as
hermaphroditic species will continue to provide an
excellent model for the study of sex determination
and gonadal sex differentiation.
Fish Physiol Biochem (2005) 31:105–109
Acknowledgements Work from this laboratory presented in
this review was supported, in part, by the CREST and SORST
Research Projects of Japan Science and Technology Corporation,
and Environmental Endocrine Disruptor Studies from the Ministry of the Environment.
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