Molecular Cloning and Expression Characterization of Dmrt2 in Akoya Pearl Oysters, Pinctada martensii

Abstract

Dmrt genes encode a large family of transcription factors involved in sexual development. These genes have been well studied in various species. However, their expression profiles and functions in bivalves are still unclear. As an important member of the Dmrt gene family. Dmrt2 is controversial because of its role in sex determination and differentiation. In the current study, pmDmrt2 (Dmrt2 from Pinctada martensii) was screened from the male gonads cDNA library. The full length of pmDmrt2 cDNA is 966 bp, with an open reading frame of 836 bp (58-893), which encodes a peptide of 278 amino acids. This gene shows 36.2%, 35.9%, 34%, 33%, 32.7%, and 21.9% identity to Dmrt2 of zebrafish, clawed frog, chicken, house mouse, human, and sea urchin, respectively. Despite the low sequence identity, the highly conserved double sex and mab-3 domain was predicted to exist in pmDmrt2. Results from the reverse transcription polymerase chain reaction indicate that pmDmrt2 is transcribed mainly in the male gonad, slightly in the gill, but not in other tissues. The gene is first transcribed in the early male gonads, and peaks in the mature male gonads. During transition from male to female, pmDmrt2 is gradually downregulated until it eventually becomes nonexistent in the mature female gonads. in situ hybridization analysis reveals that pmDmrt2 m RNA is localized specifically in the spermatogonia, spermatocytes, and spermatids in the male gonads. Our investigation indicates that pmDmrt2 might play a functional role during spermatogenic cell differentiation from spermatocytes and spermatids into sperm. Bivalves and mammals use at least several similar mechanisms to control sexual development.

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Molecular Cloning and Expression Characterization of Dmrt2 in Akoya Pearl
Oysters, Pinctada martensii
Author(s) :Fei-Fei Yu, Mei-Fang Wang, Li Zhou, Jian-Fang Gui and Xiang-Yong Yu
Source: Journal of Shellfish Research, 30(2):247-254. 2011.
Published By: National Shellfisheries Association
DOI: 10.2983/035.030.0208
URL: http://www.bioone.org/doi/full/10.2983/035.030.0208
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MOLECULAR CLONING AND EXPRESSION CHARACTERIZATION OF DMRT2 IN
AKOYA PEARL OYSTERS, PINCTADA MARTENSII
FEI-FEI YU,
1,2
MEI-FANG WANG,
1
LI ZHOU,
2
JIAN-FANG GUI
2
AND XIANG-YONG YU
1,
*
1
Fisheries College, Guangdong Ocean University, Zhanjiang, China 524025;
2
State Key Laboratory of
Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences,
Wuhan, China 430072
ABSTRACT Dmrt genes encode a large family of transcription factors involved in sexual development. These genes have been
well studied in various species. However, their expression profiles and functions in bivalves are still unclear. As an important
member of the Dmrt gene family, Dmrt2 is controversial because of its role in sex determination and differentiation. In the current
study, pmDmrt2 (Dmrt2 from Pinctada martensii) was screened from the male gonads cDNA library. The full length of pmDmrt2
cDNA is 966 bp, with an open reading frame of 836 bp (58–893), which encodes a peptide of 278 amino acids. This gene shows
36.2%, 35.9%, 34%, 33%, 32.7%, and 21.9% identity to Dmrt2 of zebrafish, clawed frog, chicken, house mouse, human, and sea
urchin, respectively. Despite the low sequence identity, the highly conserved double sex and mab-3 domain was predicted to exist
in pmDmrt2. Results from the reverse transcription–polymerase chain reaction indicate that pmDmrt2 is transcribed mainly in the
male gonad, slightly in the gill, but not in other tissues. The gene is first transcribed in the early male gonads, and peaks in the
mature male gonads. During transition from male to female, pmDmrt2 is gradually downregulated until it eventually becomes
nonexistent in the mature female gonads. In situ hybridization analysis reveals that pmDmrt2 mRNA is localized specifically in the
spermatogonia, spermatocytes, and spermatids in the male gonads. Our investigation indicates that pmDmrt2 might play
a functional role during spermatogenic cell differentiation from spermatocytes and spermatids into sperm. Bivalves and mammals
use at least several similar mechanisms to control sexual development.
KEY WORDS: pearl oysters, Pinctada martensii Dunker, Dmrt2, sex determination, sex differentiation, spatiotemporal expression
INTRODUCTION
Bivalves belong to the second largest phylum in the animal
kingdom and are important to humans. The sexual phenotypes
of bivalves are highly diverse. Some exhibit hermaphroditism,
but the majority are gonochoric, whereas some display sex
reversal under certain conditions (Pouvreau & Gangnery 2000).
Several environmental factors, such as nutrition, water temper-
ature, and proportion of gender, have been reported to play
important roles in sex determination and differentiation in
bivalves (Yu et al. 2007a), although the function of genetic
factors in the development of bivalves remains unclear.
The Dmrt (double-sex and mab-3-related transcription factor)
gene family, with a highly conserved domain, is the only common
molecule among the various phyla. Dmrt encodes several tran-
scription factors that share a highly conserved DNA-binding
motif (DM domain) (Kondo et al. 2002, Winkler et al. 2004).
Several members of this family, especially Dmrt1,maybe
involved in sex determination and differentiation (Smith et al.
1999, Veith et al. 2003, Osawa et al. 2005).
Dmrt2, named the terra gene in zebrafish (Danio rerio), is
considered a key factor in the somite formation of vertebrates
(Kim et al. 2003, Sau´ de et al. 2005) and is also required for
skeletal patterning (Jorune et al. 2006). However, whether Dmrt2
is involved in sex determination and differentiation is inconclu-
sive. Seo et al. (2006) reported a case study of Dmrt2 null mutant
mice that had defects in segmentation and died prenatally oflung
defects. However, defects in gonadal development or signs of
incomplete sexual differentiation were uncommon at the time of
death. This implies that Dmrt2 does not participate in gonadal
development or sexual differentiation. However, Raymond et al.
(1999) proposed that the 9p-associated sex reversal in humans
might be a deletion syndrome that requires the inactivation of
both Dmrt1 and Dmrt2. Kim et al. (2003) showed that Dmrt2 is
apparently expressed at higher levels in the testis than in the
ovary in mice.
As a highly conserved gene family, Dmrt has been cloned and
studied in various vertebrates (Ottolenghi et al. 2000, Brunner
et al. 2001, Winkler et al. 2004) and several invertebrates,
including fruit flies (Drosophila) (Cremazy et al. 2001), nema-
todes (Caenorhabditis elegans) (Volff et al. 2003), freshwater
prawn (Macrobrachium rosenbergii) (Peng et al. 2005), and sea
squirt (Ascidian) (Leveugle et al. 2004). However, to date, only
the Dmrt-like protein from the Pacific oyster (Crassostrea gigas)
has been cloned and studied in the Protostomia Lophotrocho-
zoa phylum (Naimi et al. 2009).
The Akoya pearl oyster, Pinctada martensii Dunker is
one of the most important aquaculture marine bivalves for
pearl production in China. Similar to several other oysters,
P. martensii has a tendency toward protandry (Shen 1990).
Most 1-y-old P. martensii are male, of which 98% remain male
during their lifetime, whereas 2% gradually convert themselves
to female at age 2 y or 3 y. This phenomenon implies that sex
reversal occurs in P. martensii development, and that hermaph-
roditic individuals can be found during the period of sex reversal
(Yu et al. 2007a). Therefore, the study of sex-related genes of
P. martensii might elucidate the mechanism of sex reversal and
may provide valuable information for the breeding of bivalves.
We previously cloned three different DM sequences approx-
imately 141 bp from the genomic DNA of P. martensii, which
had 95%, 93%, and 85% identity with Dmrt2,Dmrt3,andDmrt4
of medaka (Oryzias latipes), respectively (Yu et al. 2007b). In the
current study, we report the molecular characterization of
*Corresponding author. E-mail: yuxyong@tom.com
DOI: 10.2983/035.030.0208
Journal of Shellfish Research, Vol. 30, No. 2, 247–254, 2011.
247

Dmrt2 in P. martensii, termed ‘‘pmDmrt2.’’ We also examined
the spatiotemporal expression of pmDmrt2 and discussed its
potential role in P. martensii.
MATERIALS AND METHODS
Experimental Animals
Samples of P. martensii were obtained from Liusha Bay in
the Zhanjiang District, Guangdong, China, where the species is
grown commercially.
Total RNA Isolation and SMART cDNA Synthesis
Total RNA from the early male gonads of P. martensii was
isolated using RNeasy Mini Kit according to the manufacturer’s
instructions (Qiagen, Gaithersburg, MD). The quality of RNA
was measured by electrophoresis on 1% agarose gels. SMART
cDNA was synthesized using the SMART cDNA Library
Construction Kit following commercial protocol (Clontech).
Cloning of Dmrt cDNA and Sequence Analysis
Cloning of pmDmrt2 cDNA by Rapid Amplification of cDNA Ends–
Polymerase Chain Reaction (RACE-PCR)
pmDmrt23#-RACE was performed using common SMART
3#(5#-GTATCAACGCAGAGTACTTTTTTTT-3#)and
pmDmrt-2F primers (5#-GAATTGTCTCCTGGTGGTG-3#).
The PCR conditions were as follows: 94°C for 30 sec, 51°C for
30 sec, and 72°C for 1 min for 38 cycles. For the pmDmrt25#-
RACE, the primers were common SMART 5#(5#-AACGCA-
GAGTACGCGGG-3#) and pmDmrt-2R primers (5#-GGCA-
CAACGGCTTACTGG -3#). The PCR conditions were as
follows: 94°C for 30 sec, 53°C for 30 sec, and 72°C for 40 sec
for 38 cycles. PCR was performed in a 25-mL reaction mix
containing 1 mL cDNA as template DNA, 0.2 mM of each
primer, 0.5 U Taq polymerase (MBI, Fermentas), 0.1 mMof
each dNTP, and 13buffer for Taq polymerase (MBI, Fermen-
tas). All PCR products of the expected size were directly
subcloned into a PMD18-T vector (Takara, Japan) and se-
quenced using an ABI377 autosequencer.
Special sense and antisense primers were designed and
synthesized according to the linked nucleotide sequence to de-
tect the correctness of sequence. The sequence for the forward
primers was 5#-GGATTTCAGTAAGACATAAGGAGG-3#,
whereas that for the reverse forward primers was 5#-GCTAT-
TATGAATTCACATTGCCAG-3#. The PCR conditions were
as follows: 94°C for 30 sec, 51°C for 30 sec, and 72°C for 1 min
30 sec for 38 cycles. The PCR reaction mixture and subsequent
steps were similar to the aforementioned steps.
Sequence Analysis
A range of vertebrate and invertebrate protein sequences that
encode Dmrt2 were aligned using the Clustal W software. A
phylogenetic tree was constructed using Molecular Evolutionary
Genetics Analysis (MEGA) version 3.0 (Tamura et al. 2007).
Semiquantitative Reverse Transcription–Polymerase Chain Reaction
(RT-PCR)
Total RNA was extracted from the gills, mantle, adductor
muscle, digestive diverticulum, and gonads of a male and
a mature female oyster. The RNA of juvenile male, mature male,
Figure 1. Nucleotide sequence of P. martensii Dmrt2 cDNA and its deduced amino acid sequence (GU594226). The DM domain is underlined. The
highly conserved cysteine and histidine residues are circled. The polyadenylation signal is boxed.
YUETAL.248

transitional, early female, and mature female gonads from
various oysters were isolated using the SV Total RNA Isolation
System following the manufacturer’s protocol (Promega). cDNA
was synthesized using a Superscript II polymerase kit (Invitro-
gen) as a template.
One pair of primers was synthesized (Sangon, Shanghai)
according to the obtained nucleotide sequences, and was used to
identify temporal and spatial expression. The forward primer
was 5#-ATGGTGTGGTATCGTGCCTGA-3#, whereas the
reverse primer was 5#-TTCTTAACCCTGAAA TCACGTC-
ACG-3#. Amplification reactions were performed in a volume
of 25 mL containing 1 mL cDNA as template DNA, 0.5 mMof
each primer, 0.5 U Taq polymerase (MBI, Fermentas), 0.1 mM
of each dNTP, and 133 buffer for Taq polymerase (MBI,
Fermentas). Each PCR cycle included denaturation at 95°C for
40 sec, annealing at 56°C for 30 sec, and extension at 72°C for 40
sec. Thirty-six cycles were performed, followed by a final
extension at 72°C for 5 min.
As a positive control for the RT-PCR analysis, b-actin was
amplified with the primers actin-F and actin-R (forward: 5#-GTG-
CACTGGTCTTCAGGGGTT-3#; reversed: 5#-GGGAAGTG-
GATGCGTGGGTAT-3#) to determine the template concentra-
tion and to provide a semiquantitative external control for PCR
efficiency under the same reaction conditions as Dmrt2.Semi-
quantitative RT-PCR was performed as previously described
(Ji et al. 2006). Briefly, 10 duplicate reactions were performed
by alternate cycle numbers from 20–38 to ensure that the
semiquantitative RT-PCR products were in a linear range
of accumulation. After the cycle number was optimized, the
temporal and spatial expression analyses of Dmrt2 were
completed by semiquantitative RT-PCR from the different
samples. About 25% of each PCR product was separated by
electrophoresis on 1.2% agarose gel with 0.5 mg/mL ethidium
bromide in trisborate ethylenediaminetetraacetic acid buffer,
and the separated PCR products were visualized under UV
light.
In situ Hybridization
Dmrt-2F (5#-ATGGTGTGGTATCGTGCCTGA-3#) and
Dmrt-2R primers (5#-TTCTTAACCCTGAAATCACGT-
CACG-3#) were used to generate a 256-nucleotide fragment.
The antisense and the negative control sense probes were
synthesized with a DIG labeling kit (Roche Molecular Bio-
chemicals) using T7 and Sp6 polymerases, respectively.
In situ hybridization of the gonadal sections was performed
following standard protocols (Wang et al. 2004). The gonads
were sectioned to a thickness of 7 mm using a freezing microtome
(Leica), and hybridized with the Dmrt2 sense or antisense
probes at a final concentration of 3 ng/mL. Hybridization was
performed overnight at 59°C. Detection and color reaction were
performed with 5,000-fold dilution of anti-DIG antibodies (Roche
Figure 2. (A) Alignment of amino acid sequences of the DM domains of P. martensii Dmrt (pmDMRT), zebrafish Dmrt1 (DrDMRT1), zebrafish Dmrt2
(DrDMRT2), zebrafish Dmrt3 (DrDMRT3), mouse Dmrt4 (MmDMRT4), zebrafish Dmrt5 (DrDMRT5), mouse Dmrt6 (MmDMRT6), mouse Dmrt7
(MmDMRT7), and Pacific oyster Dmrt-like (CgDMRT-like). Identical amino acids are indicated by asterisks. Amino acids with conserved similarities
are indicated by dots or colons. The zinc module consisting of intertwined CCHC and HCCC Zn
2+
-binding sites are indicated with black boxes and
signed with Site I and Site II, respectively. The alignment was generated using Clustal X. (B) The phylogenetic tree was generated with the Mega 3.0
program using the DM domain of various members of the Dmrt family via the neighbor-joining method (Saitou & Nei 1987). Numbers in the branches
represent the bootstrap values (as a percentage) from 100 replicates. Their GenBank accession numbers are as follows: DrDMRT1, (AAU04562);
DrDMRT2, (NP_571027); DrDMRT3, (AAU89440); MmDMRT4, (AAN77234); DrDMRT5, (AAU85258); MmDMRT6, (AAN77235);
MmDMRT7, (AAN77233); and CgDMRT-like, (EU046234).
MOLECULAR CLONING AND EXPRESSION CHARACTERIZATION OF DMRT2IN P. MARTENSII 249

Molecular Biochemicals) coupled with alkaline phosphatase and
with fresh color reaction buffer containing nitroblue tetrazo-
lium and bromochloroindolyl phosphate.
Hematoxylin–eosin staining was performed following stan-
dard protocols (Gabe 1968) to illustrate the different develop-
mental phases of the gonads and various germ cells.
RESULTS
Cloning and Sequence Analysis of Dmrt cDNA in P. martensii
A complete Dmrt gene (Genbank accession no. GU594226)
from the male gonad cDNA library of P. martensii was cloned
using RACE-PCR. The P. martensii Dmrt cDNA, with a full
length of 966 bp, has an open reading frame of 836 bp (58–893),
a 57-bp 5#-untranslated region (UTR), and a 73-bp 3#-UTR,
including a typical polyadenylation signal sequence (AATAAT)
located 20 bp upstream of the poly(A) tail (Fig. 1). The deduced
amino acid sequence is 278 amino acids long and contains the
DM domain consensus sequence (from 12–67 aa) with con-
served cysteines and histidines, which are characteristic of the
DMRT protein family.
To investigate the relationship between P. martensii Dmrt
and other members of the Dmrt family, sequence comparison of
conserved DM domain among various members of this family
in several animal models was performed. The highest identity
rate of P. martensii Dmrt was 94.5% with zebrafish Dmrt2,
followed by mouse Dmrt4 (83.6%), Pacific oyster Dmrt-like
(81.8%), zebrafish Dmrt5 (80.0%), zebrafish Dmrt1 (78.2%),
zebrafish Dmrt3 (76.4%), mouse Dmrt7 (63.6%), and mouse
Dmrt6 (54.6%). The sequence comparison reveals a conserved
zinc module consisting of intertwined CCHC and HCCC
Zn
2+
-binding sites (Naimi et al. 2009) (Fig. 2A (Saitou & Nei
1987)). The phylogenetic tree generated using the compared
DM domains shows that P. martensii Dmrt is grouped with
zebrafish Dmrt2 according to its high bootstrap support (99%;
Fig. 2B).
Amino acid alignments and comparison of the complete
Dmrt2 of P. martensii and other species were performed. The
complete P. martensii Dmrt2 has the highest identity with
zebrafish (D. rerio)Dmrt2 at 36.2%. The obtained identities
with clawed frog (Xenopus tropicalis), chicken (Gallus gallus),
house mouse (Rattus norvegicus), and human (Homo sapiens)
are 35.9%, 34%, 33%, and 32.7%, respectively (Fig. 3A). The
homology of the N-terminal among different species is obvi-
ously higher than that of the C-terminal. The phylogenetic tree
generated by the complete Dmrt genes of various species
was used to identify and classify the P. martensii Dmrt gene
further (Fig. 3B). All Dmrt2 of the referred species, including
P. martensii, grouped into a close cluster, implying that P.
martensii Dmrt may be a member of the Dmrt2 subfamily.
Therefore, we designated the P. martensii Dmrt gene as
pmDmrt2. The classification of these Dmrt2 genes in the big
cluster basically agrees with known taxonomic relationships
among these species. Outside the big cluster, the other sub-
families of Dmrt genes exhibit farther distances. In accor-
dance with the report by Naimi et al. (2009), the only report
about DM-like protein coding gene from molluscs, we found
that the Pacific oyster Dmrt-like gene is closer to Dmrt4 and
Dmrt5 than the other members of the Dmrt family, grouped
together with a bootstrap support between 99% and 96%.
Figure 3. (A) Amino acid alignment of complete Dmrt2 cDNA among P.
martensii (pmDMRT), zebrafish (DrDMRT2), clawed frog (XtDMRT2),
chicken (GgDMRT2), mouse (MmDMRT2), and human (HsDMRT2),
and DMRT-like of the Pacific oyster (CgDMRT-like). Black shading
corresponds to the conserved amino acid positions. The alignment was gen-
erated using Clustal X. The GenBank accession numbers are as follows:
pmDMRT, GU594226; DrDMRT2, (NP_571027.1); XtDMRT2,
(NP_001093726.1); GgDMRT2, (AAZ03502.1); MmDMRT2,
(NP_001101067.1); HsDMRT2, (NP870987.2); and CgDMRT-like,
(EU046234). (B) The phylogenetic tree was generated with the Mega
(3.0) program using the Dmrt genes of different species via the neighbor-
joining method. Numbers in the branches represent the bootstrap values
(as a percentage) from 100 replicates.
YUETAL.250

Expression Patterns of pmDmrt2 by RT-PCR
High Expression of pmDmrt2 in Male Gonad
The pmDmrt2 expression patterns among the various tissues
of male and female oysters were analyzed by RT-PCR. The
expression profiles in the somatic tissues of both individualswere
similar. Except for the gills, pmDmrt2 mRNA was not tran-
scribed in the mantle, digestive diverticulum, adductor muscle,
and female gonad. Numerous transcripts were detected in the
male gonads in contrast to the female gonads (Figs. 4 and 5).
Differential Expression of pmDmrt2 in Gonads of Different Phases
The pmDmrt2 expression patterns among the gonads in
different phases were analyzed by RT-PCR. The pmDmrt2 gene
was not expressed in the juvenile gonads, but was initially
transcribed in the early male gonads and peaked in the mature
male gonads. During transition from male to female, pmDmrt2
was gradually downregulated until no expression was detected in
the mature female gonads (Fig. 6). According to the expression
patterns, pmDmrt2 was expressed mostly in early male and
mature male gonads.
Expression Localization of pmDmrt2 by in Situ Hybridization
In situ hybridization analyses were performed to confirm
further the localization of the pmDmrt2 transcripts in the male
and female gonads. Strong signals were detected by the
pmDmrt2 antisense probe in the spermatogonia, spermatocytes,
and spermatids of male gonad tissue, but the signal observed in
the sperm of the same male gonad is very weak (Fig. 7A–E). No
signal was detected in any of the germ cells of the female gonads
using the antisense and sense probes (Fig. 7F, G). This result
indicates that pmDmrt2 transcripts were mostly localized in the
early germ cells of the male gonads.
DISCUSSION
The complete P. martensii Dmrt gene was cloned and
characterized. The deduced amino acid sequence presents
a highly conserved DM domain that contains the putative NLS
located in the zinc module, which consists of intertwined CCHC
and HCCC Zn
2+
-binding sites. This structural characteristic
implies the important functions of the DM domain—namely,
DNA binding and nuclear import (Zhu et al. 2000, Naimi et al.
2009). Similar to other transcription factors with a conserved
domain, the Dmrt family mainly acts as transcription regulators
through the DM domain. The DM domain is responsible for the
chelation of zinc, conferring sequence-specific DNA binding
(Ren et al. 2001), as well as playing a varied role. Therefore, we
compared the DM domains of various members of the Dmrt
family to identify and classify P. martensii Dmrt accurately.
Although members of this family have highly conserved DM
domains, the highest identity of the P. martensii DM domain was
with zebrafish DM2. The phylogenetic tree, generated using the
tested DM domains, indicates that the P. martensii DM domain
is grouped with zebrafish DM2 with high bootstrap support.
Furthermore, a phylogenetic tree was constructed using the
complete Dmrt genes of various species. P. martensii Dmrt was
grouped together with all Dmrt2 genes of referred species,
indicating that all referred Dmrt2 genes have a close genetic
distance and may have evolved from the same ancestor. These
results strongly suggest that P. martensii Dmrt is a member of the
Dmrt2 subfamily and is thus designated as pmDmrt2.
Figure 4. Expression patterns of pmDmrt2 in various tissues of male
oysters. (A) RT-PCR analysis of pmDmrt2 expression in a male individual
(b-actin was used as the control). Total RNA was extracted from the gills,
mantle, adductor muscle, digestive diverticulum, and gonads of a male P.
martensii. (B) The pmDmrt2 mRNA intensities shown in view A were
analyzed with Band Leader Amplification Software (ver. 3.0). The values
represent the mean %SD of three separate experiments. AM, adductor
muscle; DD, digestive diverticulum; G, gills; M, mantle; MG, male gonads.
Figure 5. Expression patterns of pmDmrt2 in various tissues of female
oysters. (A) RT-PCR analysis of pmDmrt2 expression (b-actin was used
as the control). Total RNA was extracted from the gills, mantle, adductor
muscle, digestive diverticulum, and gonads of a female P. martensii. (B)
The pmDmrt2 mRNA intensities shown in view A were analyzed by Band
Leader Amplification Software (ver. 3.0). The values represent the mean %
SD of three separate experiments. AM, adductor muscle; DD, digestive
diverticulum; FG, female gonads; G, gills; M, mantle.
MOLECULAR CLONING AND EXPRESSION CHARACTERIZATION OF DMRT2IN P. MARTENSII 251

Dmrt2, a member of the highly conserved Dmrt gene
family and widely reported to participate in somitogenesis,
plays an important role in the establishment of left–right
asymmetry and regulation of muscle development in many
species (Meng et al. 1999, Hong et al. 2007). The Dmrt2 gene is
expressed in gonadal tissues with species difference, as well as
in some nongonadal tissues such as somites (Meng et al. 1999,
Seo et al. 2006, Mogharbel et al. 2007). Some studies have
shown that Dmrt2 is expressed only in the testis, aside from
some nongonadal tissues in humans and mice (Ottolenghi &
McElreavery 2000, Kim et al. 2003). Mogharbel et al. (2007)
showed that Dmrt2 has a wide expression profile, including both
testes and ovaries in platyfish (Platypoecilus maculatus). How-
ever, Winkler et al. (2004) revealed that Dmrt2 expression
initially appears in undifferentiated gonads and remains in the
testis of medaka. Analysis of P. martensii, with no evident
somitogenesis in the development, was performed by RT-PCR
in various tissues of males and females. The pmDmrt2 gene is
mostly expressed in the male gonads, slightly in the gills, and not
in other tissues, indicating that this gene might be involved in
P. martensii sex determination and differentiation.
The DM-like protein coding gene from molluscs was first
reported by Naimi et al. (2009). They indicated that the C. gigas
DMRT-like gene exhibits significantly higher expression in the
male gonads than in the female gonads using RT-PCR, and
suggested that the gene participates in gonadal development,
which is in agreement with our results. However, they also
concluded that the Cg-DMl gene has a more ubiquitous expres-
sion in other tissues, including gills, labial palps, mantle, adductor
muscle, digestive gland, and gonads. Our study shows that
pmDmrt2 is mainly transcribed in the male gonads, slightly in
the gills, but not in other tissues. The variation in expression may
be attributed to the homology of the Cg-DMl gene to Dmrt4 and
Dmrt5, whereas pmDmrt2 is more homologous to the Dmrt2
subfamily. Moreover, the Cg-DMl gene, an ancestral DM factor,
might be involved in various biological processes (Naimi et al.
2009), which is in accordance with its more ubiquitous expres-
sion, whereas pmDmrt2 might have a more specific function in
animal development with its more specific expression.
Dmrt1, the most well-known member of the Dmrt family,
which is involved in sex determination and differentiation, has
been widely reported (Kettlewell et al. 2000, Sreenivasulu et al.
2002, Guo et al. 2005). Although our results show that the
expression pattern and the function of pmDmrt2 are similar to
mammalian Dmrt1, the sequence comparison and phylogenetic
tree analysis of the DM domain and Dmrt genes clearly
classified pmDmrt as Dmrt2 rather than Dmrt1. Some studies
have suggested that Dmrt2 might be ectopically expressed in the
gonadal ridge when Dmrt1 is inactivated, and has a similar
function to Dmrt1 (Raymond et al. 1999). Both Dmrt1 and
Dmrt2 are considered excellent candidate regulators of sexual
development (Ottolenghi & McElreavery 2000, Ottolenghi et al.
2000). Consequently, these genes may cooperate in sex de-
termination and development.
In situ hybridization analyses show that pmDmrt2 expres-
sion is restricted to specific cells during spermatogenesis, in-
cluding spermatogonia, spermatocytes, and spermatids. This
behavior indicates that Dmrt2 expression in spermatogenic
Figure 6. Expression patterns of pmDmrt2 in the gonads during various phases. (A) RT-PCR analysis of pmDmrt2 expression (b-actin was used as
control). The RNA of the early male, mature male, transitional, early female, and mature female gonads from various oysters were isolated, and cDNA
was synthesized. (B) The pmDmrt2 mRNA intensities shown in view A were analyzed with Band Leader Amplification Software (ver. 3.0). The values
represent the mean %SD of three separate experiments. EFG, early female gonads; EMG, early male gonads; JG, juvenile gonads; MFG, mature
female gonads; MMG, mature male gonads; TG, transitional gonads.
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cells is stage specific. Considering that pmDmrt2 is expressed in
spermatogonia, spermatocytes and spermatids, its highest
expression level should be observed in early male gonads.
However, the RT-PCR results imply that pmDmrt2 expres-
sion peaked in the mature male gonads. The disagreement
between in situ hybridization and RT-PCR analysis may be
attributed to the increase in the total amount of male germ cells,
as well as in the gonadic volume during the development of
P. martensii.
The genes that are specifically transcribed during spermato-
genesis are often those that are necessary for the maturation of
sperm. This tendency suggests that pmDmrt2 might play
functional roles during spermatogenic cell differentiation from
spermatocytes and spermatids into sperm. The mechanisms of
Dmrt2 in sex determination and differentiation are therefore
probably more complex. A novel method should be developed
to elucidate the mechanisms of Dmrt2 in sex determination and
differentiation.
ACKNOWLEDGMENTS
This study was supported by the Freshwater Biology and
Biotechnology Key Laboratory Open Foundation of China
(grant no. 06FB02).
Figure 7. Localization of the pmDmrt2 gene in the male and female gonads by in situ hybridization. (A) Hematoxylin–eosin staining of a follicle in the
male gonads of P. martensii. (B) Localization of the pmDmrt2 gene in the male gonad follicle with antisense probe via in situ hybridization. (C)
Hematoxylin–eosin staining of the male gonad follicle configuration in P. martensii. SC, spermatocytes; SD, spermatid; SG, spermatogonia; SP,
spermatozoa. (D) Localization of the pmDmrt2 gene in the male gonad follicle with antisense probe by in situ hybridization. (E) Hematoxylin–eosin
staining of the female gonads of P. martensii. O, ovum; Oo, oocyte. (F) Localization of the pmDmrt2 gene in the female gonad with antisense probe by in
situ hybridization. (G) Localization of the pmDmrt2 gene in the male gonad follicle with sense probe by in situ hybridization.
MOLECULAR CLONING AND EXPRESSION CHARACTERIZATION OF DMRT2IN P. MARTENSII 253

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