Identification of testis-relevant genes using in silico analysis from testis ESTs and cDNA microarray in the black tiger shrimp (Penaeus monodon)
BMC Molecular Biology
Identification of testis-relevant genes using in silico analysis from testis ESTs and cDNA microarray in the black tiger shrimp (Penaeus monodon)
Thidathip Wongsurawat 0
Rungnapa Leelatanawit 0
Natechanok Thamniemdee 0
Umaporn Uawisetwathana 0
Nitsara Karoonuthaisiri 0
Sirawut Klinbunga 0
0 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency , Klong 1, Klong Luang, Pathumthani 12120 , Thailand
Background: Poor reproductive maturation of the black tiger shrimp (Penaeus monodon) in captivity is one of the serious threats to sustainability of the shrimp farming industry. Understanding molecular mechanisms governing reproductive maturation processes requires the fundamental knowledge of integrated expression profiles in gonads of this economically important species. In P. monodon, a non-model species for which the genome sequence is not available, expressed sequence tag (EST) and cDNA microarray analyses can help reveal important transcripts relevant to reproduction and facilitate functional characterization of transcripts with important roles in male reproductive development and maturation. Results: In this study, a conventional testis EST library was exploited to reveal novel transcripts. A total of 4,803 ESTs were unidirectionally sequenced and analyzed in silico using a customizable data analysis package, ESTplus. After sequence assembly, 2,702 unique sequences comprised of 424 contigs and 2,278 singletons were identified; of these, 1,133 sequences are homologous to genes with known functions. The sequences were further characterized according to gene ontology categories (41% biological process, 24% molecular function, 35% cellular component). Through comparison with EST libraries of other tissues of P. monodon, 1,579 transcripts found only in the testis cDNA library were identified. A total of 621 ESTs have not been identified in penaeid shrimp. Furthermore, cDNA microarray analysis revealed several ESTs homologous to testis-relevant genes were more preferentially expressed in testis than in ovary. Representatives of these transcripts, homologs of saposin (PmSap) and Dmc1 (PmDmc1), were further characterized by RACE-PCR. The more abundant expression levels in testis than ovary of PmSap and PmDmc1 were verified by quantitative real-time PCR in juveniles and wild broodstock of P. monodon. Conclusions: Without a genome sequence, a combination of EST analysis and high-throughput cDNA microarray technology can be a useful integrated tool as an initial step towards the identification of transcripts with important biological functions. Identification and expression analysis of saposin and Dmc1 homologs demonstrate the power of these methods for characterizing functionally important genes in P. monodon.
The black tiger shrimp (Penaeus monodon) is an aquatic
animal of central importance as it brings an annual
income of over one billion USD in Thailand .
However, domestication of the black tiger shrimp (Penaeus
monodon) is impeded by poor reproductive maturation of
both male and female brooders in captivity. Ovarian
development of penaeid shrimp is induced by a unilateral
eyestalk ablation technique; however, the technique does
not have the same effect in male reproductive maturation
. No molecular markers pinpointing the maturation
stage of testis or sperm quality in penaeid shrimp are
currently known. Domesticated male P. monodon yields
lower fertilization rates of zygotes and lower survival
rates of offspring than wild male P. monodon .
However, the role of genes implicated in the regulation of
spermatogenesis and their patterns of expression in
penaeid shrimp are still poorly understood.
To gain insight on the molecular mechanisms
governing the male reproduction process of this important
species, EST libraries from P. monodon broodstock testis
were previously constructed for gene discovery [4,5]. A
testis-specific transcript, PMTST1 (P. monodon
testisspecific transcript 1), was identified, and expression
levels of 51 additional putative testis-specific genes in
cultured and wild P. monodon were examined by
reverse-transcription (RT)-PCR, semiquantitative
RTPCR, and real-time RT-PCR . Nevertheless, more
exhaustive gene discovery is needed to unravel
testisrelevant genes and their possible functions.
In this study, a total of 4,803 ESTs from the testis
library were sequenced and analyzed in silico using a
customizable data analysis package, ESTplus . Many
transcripts known to be relevant to testicular
development in other organisms were identified. A total of
1,076 of these testis EST sequences were included in the
construction of a new microarray. Gene expression
profiles of testis were simultaneously compared to those of
ovary in both juveniles and broodstock. Among
transcripts with differential expression levels, saposin and
Dmc1 homologs were further examined by quantitative
real-time PCR. Furthermore, full-length sequences of
saposin and Dmc1 cDNAs were obtained by RACE-PCR.
Results and Discussion
Characteristics and functional annotation of testis ESTs of
Previously, we constructed suppression subtractive
hybridization (SSH) libraries comparing cDNA in testis
of wild P. monodon broodstock to juveniles. A total of
365 clones were sequenced from these libraries . In
addition, we established a high quality conventional
cDNA library from testis of wild P. monodon broodstock
and preliminarily sequenced 896 clones . In this
study, additional ESTs were sequenced from the
conventional cDNA library and a total of 4,803 high quality
EST sequences were obtained (Figure 1A). Results of the
in silico analysis are summarized in Figure 2. From
assembling the testis EST sequences, 2,702 unique
sequences comprising of 424 contigs and 2,278
singletons with an average size of 1,130 bp and 584 bp,
respectively, were found. When compared with the
NCBI nucleotide collection (nr/nt) database using
BlastN, 1,181 sequences of the 2,702 unique sequences
were found to be homologs of previously reported
sequences. Similarly, when the unique EST sequences
were compared with the NCBI non-redundant protein
sequence database using BlastX, 1,147 (42%) sequences
were homologs of previously reported sequences,
whereas the remainder (1,555 sequences, 58%) were
unknown (novel) transcripts. Matched sequences (1,147)
from the BlastX analysis were further categorized by
Blast2GO for their potential functions. 1,133 (99%) of
these sequences were similar to genes with putative
functions (Figure 2A).
E-values of most sequences ranged between 1e-30 to
1e-10 (396 sequences accounting for 34% of overall
contigs, Figure 3C). The most frequently matched
transcripts found were Arthropoda (753 sequences, 66%) at
the phylum level and the red flour beetle Tribolium
castaneum (90 sequences) at the species level (Table 1).
This could be due to the limited availability of P.
monodon sequence data in comparison to that of T.
castaneum whose genome was completely sequenced and
published in 2008 .
The 2,702 unique sequences were searched using
BlastN against the P. monodon EST data (PmDB: http://
pmonodon.biotec.or.th) excluding previously identified
testis EST libraries (Figure 2B). A total of 1,579
sequences did not match ESTs from other tissues of
P. monodon making them potential testis-specific
transcripts or rare transcripts in other shrimp tissues.
Examples of genes in this group relevant to testicular
development are phospholipase A2, mago nashi
proliferation-associated protein, actin-depolymerizing factor and
profilin. Phospholipase A2 is required for the acrosome
reaction (AR), a special exocytotic process promoted by
signal transduction pathways, and capacitation, a process
for maturation of spermatozoa [8,9]. Protein mago nashi
originally identified in Drosophila is essential for
germplasm assembly . Actin polymerization is important
for a wide range of cellular functions and properties,
including cell division, cell motility, cell polarity and
cellcell contacts. Profilins are widely expressed small
actinbinding proteins which are functionally involved in the
regulation of actin dynamics [11,12]. On the other hand,
Figure 1 Overview of data analysis of the testis ESTs in this study.
cofilin/actin-depolymerizing protein is an important
factor in spermatogenesis which disassembles actin filaments
when unphosphorylated .
To reveal novel transcripts which are not found in
other Penaeidae ESTs, the testis ESTs from this study
were also compared to Penaeid ESTs (207,852 sequences,
using a keyword of Penaeidae) from the NCBI database
(Figure 2C). A total of 2,081 (77%) sequences were
homologous to those from the NCBIs penaeid shrimp
database. 621 newly identified transcripts (23%) were
found, 475 of which (18%) have not been reported in the
Most of the obtained EST sequences ranged from
2011,100 bp in length (Figure 3A). Apart from a large number
of singletons (2,278 unique sequences, 84% of discovered
sequences), most of the contigs (187 contigs, 7%) were
assembled from two ESTs and only 100 contigs (4%) were
deduced from greater than five ESTs. Only three contigs;
cytochrome C oxidase subunit 1 (COI), elongation factor
1alpha (EF-1a), and cell division cycle 2 (cdc2) were
assembled from over 50 sequences (Figure 3B). EF-1a
functions in protein synthesis by promoting the binding of
aminoacyl tRNA to the 80 S ribosome, and it is one of the
most abundant soluble proteins in eukaryotes .
Mitochondrial COI has been found to be more highly
expressed in well differentiated cells with high activity
than in moderately and poorly differentiated cells [15,16].
Cdc2 is the key regulator of the eukaryotic cell cycle, and
its activity is controlled by interaction with other proteins
such as cyclin A and cyclin B1 . Mitotic cyclins and
Figure 2 Results summary of EST data analysis.
cdc2 are involved in capacitation and/or acrosome
reaction of sperm . During the meiotic cell cycle, the
G2/M phase transition is controlled by the maturation
promoting factor (MPF), a complex of cdc2 (cdk1) and
cyclin B1 . High redundancies of these transcripts in
the ESTs are consistent with their essential cellular
functions during spermatogenesis and testicular development.
The assembled EST sequences with similarity to the
nr/nt database (1,147 from 2,702 sequences; Figure 2A)
were further categorized according to Gene Ontology
(Figure 4). The majority of annotated ESTs (41% of
1,147 sequences) were placed in the Gene Ontology
functional category of biological processes. The
remaining annotated ESTs were placed in the cellular
components (35% of 1,147 sequences) and molecular
function (24% of 1,147 sequences) categories.
Compared to the EST sequences from the P. monodon EST
database, the distribution for the three main categories
was similar (42%, 31%, and 27% for biological processes,
cellular components and molecular function,
In the biological process category, ESTs involved in
the cellular process were predominant (30% of examined
ESTs), followed by those involved in the metabolic
process (25% of examined ESTs). In the cellular component
category, EST functionally involved in the cell (39% of
examined ESTs) predominated followed by those
functionally displayed in the organelles (30% of examined
ESTs) and macromolecular complex (21% of examined
ESTs). In the molecular function category, ESTs
displaying binding function (43% of examined ESTs)
predominated followed by those displaying catalytic activity (28%
of examined ESTs).
The reproduction group, including mago-nashi
proliferation-associated protein, ubiquitin-conjugating enzyme
E2, and small ubiquitin-related modifier precursor
(SUMO), contributes ~2% of the total number of
contigs. This discovery rate is comparable to that identified
from the conventional ovarian cDNA library of P.
The transcripts found in the reproduction group
exhibit relevant functions to male reproduction. For
instance, SUMO plays an important role in diverse
reproductive functions such as spermatogenesis and
modulation of steroid receptor activity . In the
sumoylation pathway, SUMO is transferred to lysine
Figure 3 Distribution of testis ESTs. (A) Length (in bp)
distribution of testis ESTs. (B) Distribution of number of singletons
assembled into a contig. (C) Distribution of E-value.
residues of the protein substrates through the thioester
cascade of ubiquitin activating enzyme E1 and ubiquitin
conjugating enzyme E2 (UBE2), and SUMO ligase E3
. In the kuruma shrimp Marsupenaeus japonicus,
UBE2 was expressed at a higher level in testis than in
ovary. The expression at stage I (GSI = 0.33 0.004,
N = 5) was significantly lower than that of stage II
(GSI = 0.45 0.12, N = 5) but comparable to that of
stage III (GSI = 0.57 0.006, N = 5) of testis . The
analysis of baseline information acquired by this study
addresses the paucity of data and provides a better
understanding of reproductive maturation in male
Differential Gene Expression Patterns by cDNA
To address the functional involvement of various genes
during reproductive development and maturation in
P. monodon, we previously constructed a cDNA
microarray, ReproArrayGTS, and carried out a high-throughput
expression analysis of genes in ovary of wild P. monodon
. A second generation cDNA microarray was
recently fabricated using 12 EST libraries from different
tissues to cover as many and as diverse transcripts as
possible. Expression levels of EST transcripts with gene
ontology related to reproductive functions and those
previously not reported in the NCBI EST penaeid
shrimp database (unique genes) were investigated by
using this new microarray to compare ovary and testis
transcripts in both juvenile and broodstock shrimp
(Figure 5). The expression levels from the microarray
experiments were analyzed, as described in the Methods
section, to obtain transcripts potentially relevant to
Of 5,568 transcripts on the microarray, 2,133
transcripts were present in at least 8 of the 9 microarrays
and had relative expression levels greater than the
median value 1SD in at least one microarray (Figure 5A).
Within this subset, 244 transcripts with higher
abundance (3 fold) in ovary than testis and 239 transcripts
with higher abundance (3 fold) in testis than ovary were
identified (Figure 5B). From these differentially
expressed genes, there were transcripts whose putative
functions are possibly involved in testicular development
When examining the library sources of transcripts
more abundantly expressed in ovaries, we found 51%
were from the ovary, 22% from testis, 9% from hemocyte
and heart each, 8% from gill, 1% from lymphoid organ
and <1% from the remaining tissues (Figure 5D). These
transcripts included peritrophin (also called cortical rod
protein, CRP), thrombospondin (TSP) and nuclear
autoantigenic sperm protein (NASP). In the kuruma shrimp,
CRP and TSP are first scattered throughout the
cytoplasm of oocytes and are subsequently localized in
cortical rods during the cortical rod formation . NASP is
a histone-binding protein. Overexpression of NASP in
mice testis affects progression of cell proliferation
through the cell cycle [26,27]. Expression analysis of
these genes [20,28] and dolichyl diphosphooligocharide
protein glycotransferase, asparagenyl tRNA synthetase and
Table 1 Distribution of species that the testis ESTs were matched to known transcripts in the database using BlastX
3-oxoacid CoA transferase by semiquantitative RT-PCR
(S. Klinbunga, unpublished data) revealed greater
expression levels in ovary than testis of wild P. monodon
broodstock. This validated the accuracy of the microarray
analysis for gene expression analysis in P. monodon.
Transcripts with higher expression in testis were
found mainly from the testis EST library (65%), 8% from
heart, 7% from hemocyte, 5% from gill, 3% from ovary,
2% from hepatopancreas, 1% from intestine, and 1.5%
from the rest (Figure 5E). Examples of these transcripts
include argonaute 1, dynactin 5, and orphan 2.
Argonaute proteins are key mediators for RNA silencing
mechanisms . Cytoplasmic dynactin forms a
complex with dynein and plays a functional role in
spermatid growth in Drosophila .
Based on the distribution of library sources where the
differentially expressed transcripts came from, it is
evident that we can obtain additional information by
including cDNA probes from various EST libraries
beyond testis and ovary libraries. This more
comprehensive coverage of microarray probes demonstrates the
usefulness of the present microarray version in gene
expression analysis of P. monodon.
Within the 1,076 cDNA spots from testis ESTs that
were fabricated onto the present version of cDNA
microarrays, there were 169 transcripts with greater
abundance in testis than ovary. Of these transcripts
saposin-related protein and Dmc1 were consistently
more abundant in testis than in ovary for both juvenile
and broodstock comparisons. Moreover, these genes
have previously been reported to be relevant to male
reproduction in other organisms.
In vertebrates, saposins are a group of four small
glycoproteins (A, B, C, and D), derived from a common
precursor protein called prosaposin, which is reported
to activate glycosphingolipid hydrolysis . Inactivation
of prosaposin gene resulted in accumulation of
lactosylceramide, glucosylceramid, digalactosyl ceramide,
sulfactide, ceramide, and globotriaosylceramide in lysosomes.
A prosaposin -/- mouse model demonstrated that the
mice die at day 35-40 after birth due to neurological
defects . The disruption of the prosaposin gene
resulted in a reduction in size and weight: 30% of the
testis, 37% of the epididymis, 75% of the seminal
vesicles, and 60% of the prostate glands. Moreover, the
smaller testes from the mutant mice were associated
Figure 4 Functional categories by Gene Ontology (GO) analysis of the testis EST sequences with similarity to the nr database (1,147
sequences total from 2,702 sequences). Comparison of different percentages in each GO sub-category between all P. monodon ESTs http://
pmonodon.biotec.or.th (darker colored bars) and the testis ESTs in this study (lighter colored bars) is represented in all categories.
Figure 5 Gene expression analysis by cDNA microarray comparing P. monodon transcripts between testis (TT) and ovary (OV) in
individual juvenile (Ji), pooled juvenile (Jp) and individual wild-caught broodstock (WBi). TT was labeled with Cy5 fluorescent dye (red)
and OV with Cy3 fluorescent dye (green). (A) Hierarchical clustering analysis of the transcripts present in at least 8 of 9 slides and whose
expression differences were at least equal to median value 1SD in at least 1 slide. (B) Clusters I and II of transcripts with higher expression
levels in testis than ovary and vice versa with at least 3-fold difference in at least 6 of 9 slides. (C) Examples of differentially expressed transcripts
with putative functions in reproductive development. Transcripts in blue letters are those which were not found in any EST libraries of other
tissues. Saposin and Dmc1 (in red) were further characterized by RACE-PCR. (D) Library distributions of transcripts expressed higher in ovary than
in testis (244 transcripts) and (E) those expressed higher in testis than in ovary (239 transcripts).
with reduced spermiogenesis, especially in the late
Dmc1 (a RAC A-like recombinase) is known to be a
specific factor for meiotic recombination and has been
identified as a gene product specifically expressed during
the early meiotic prophase . Recently, the full-length
Dmc1 cDNA was cloned from the testis of the Japanese
eel (Anguilla japonica). Dmc1 mRNA was abundantly
expressed in the testis and ovary with lower expressed
in the brain. In situ hybridization revealed that Dmc1 of
A. japonica was localized only in the primary
spermatocytes implying its important role during the initial stages
of spermatogenesis .
Isolation and characterization of the full-length cDNA of
saposin (PmSap) and Dmc1 (PmDmc1) in P. monodon
Based on their differential expression levels seen in the
microarray analysis and their possible involvement in
testicular development from previous studies in other
organisms, saposin-related protein (PmSap) and Dmc1
(PmDmc1) were further characterized by RACE-PCR to
obtain their full-length cDNA sequences.
Full-length PmSap was found to be 3,034 bp in length
containing an open reading frame (ORF) of 2,589 bp
corresponding to a deduced protein of 862 amino acids
with 5 and 3UTRs of 116 and 329 bp (excluding the
poly A tail), respectively (Figure 6A, accession number
GU566728). This sequence significantly matched saposin
isoform 1 of Tribolium castaneum (E-value = 1e-171).
The expected molecular weight (MW) and pI of the
deduced Saposin were 95.63 kDa and 4.65, respectively.
Only one contig of saposin-related protein was
previously found in ESTs from P. monodon.
Most saposins in vertebrate and invertebrate contain a
signal peptide. Typically, vertebrate prosaposins contain
two conserved SapA domains and four conserved SapB
domains in which six equally placed cysteines are found
in each SapB domain. In contrast, the numbers of saposin
domains in invertebrates is less conserved. The absence
of some of SapA domain produced variants of
saposinrelated proteins in various taxa (Figure 6B). Moreover,
there are variable numbers of SapB domains in
invertebrates, from 6 domains present in the pea aphid,
Acyrthosiphon pisum (Homoptera), to up to 9 domains in the
black legged tick Ixodes scapularis (Acari) and Nasonia
vitripennis (Hymenoptera) (Figure 6B). Additional SapB
domains in invertebrate saposins may be explained by
several rounds of tandem internal gene duplication as
previously proposed for the creation of the four domain
saposins in vertebrate .
In P. monodon, the deduced PmSap protein contained a
signal peptide with a cleavage site between Ala21 and
Glu22, two SapA and seven SapB domains (SapA:
positions 25-58 and 823-856 with E-value = 2.74e-12 and
1.56e-07, respectively; SapB: positions 68-144, 178-251,
272-346, 437-512, 531-606, 646-721, and 738-813 with
E-value = 1.32e-22, 5.32e-09, 7.28e-16, 4.34e-23, 4.61e-27,
2.63e-22, and 5.83e-15, respectively). Interestingly, six
fixed positions of cysteine were found in each SapB
domain of PmSap but conserved prolines in identical
positions as previously reported in vertebrate saposins
 were not found (data not shown). Five predicted
glycosylation sites were found in SapB domains 1 (NET,
positions 87-89), 3 (NTT, positions 291-293), 5 (NRT
and NLS, positions 550-552 and 593-595, respectively)
and 6 (NAT, positions 665-667). These predicted SapB
domains significantly matched saposin-related protein
A (E-value = 8e-24; Uniprot No. Q9Y125), BmP109
(E-value = 7.4e-20; Uniprot No. O15997), Saposin
C (E-value = 6.4e-14; Uniprot No. P220097) and BmP109
(E-value = 3.4e-7; Uniprot No. O15997), respectively.
The Dmc1 transcript which was only found in the
testis cDNA library of P. monodon was further
characterized. The full-length cDNA of PmDmc1 (accession
number EU440762) was found to be 1,661 bp in length
containing an ORF of 1,026 bp encoding a polypeptide
of 341 amino acids. The highest similarity to this
transcript was Dmc1 of Ixodes scapularis (E-value = 1e-147).
The predicted pI and MW of the deduced Dmc1 were
5.35 and 37.54 kDa, respectively. The deduced Dmc1
protein contains an HhH motif (positions 46 - 75,
E-value = 2.73e-02) and an AAA domain (positions
118 -308, E-value = 9.73e-06, Figure 7A). The HhH
motif is ~20 amino acid domain present in prokaryotic
and eukaryotic non-sequence-specific DNA binding
proteins involved in enzymatic activity. The gene products
within the superfamily of AAA-ATPase are associated
with diverse cellular activity. A bootstrapped
neighborjoining tree reflects the relationships between the texa
and the sparse representation of invertebrate sequence
for the Dmc1 protein (Figure 7B).
Expression of PmSap and PmDmc1 in testis of juveniles
and domesticated and wild broodstock of P. monodon
Tissue expression analysis is important for providing
basic information needed to prioritize further analysis of
functional genes. Based on the fact that a particular
gene may be expressed in several tissues and possess
different functions, rapid detection of PmSap and
PmDmc1 expression profiles in gonads of P. monodon
by cDNA microarray was further confirmed by
quantitative real-time PCR.
PmSap was more abundantly expressed in testis
than ovary of P. monodon (P < 0.05, Figure 8). The
expression levels of this transcript were not significantly
different across different stages of either testis (WB-TT,
DB- TT and J-TT) or ovary (WB- TT and J-TT). Dmc1
was less abundantly expressed in testis of domesticated
Figure 6 Analysis of Penaeus monodon Saposin (PmSap). (A) The full-length cDNA and deduced protein sequence of PmSap (3034 bp; ORF
2589 bp, 862 aa) of P. monodon. Start (ATG) and stop (TAA) codons are illustrated in boldface and underlined. The signal peptide of the
deduced PmSap protein is italicized and underlined. Putative N-linked glycosylation sites are underlined. Two domains of Saposin A (positions
25-58 and 823-856) and seven domains Saposin B (positions 68-144, 178-251, 272-346, 437-512, 531-606, 646-721, and 738-813 of the deduced
protein) are highlighted. (B) Diagram showing SapA and SapB domains in prosaposin in various vertebrates; Homo sapiens (CAI40836), Bos taurus
(NP_776586), Macaca mulatta (XP_001106592), Canis familiaris (XP_536382 and XP_861441), Gallus gallus (NP_990142), Danio rerio (AAH63994)
and saposin-related protein in invertebrates; Acyrthosiphon pisum (XP_001943244), Apis mellifera (XP_392338), Tribolium castaneum (XP_966852),
Aedes aegypti (XP_001662994), Culex quinquefasciatus (XP_001864689), Drosophila melanogaster (NP_524597 and NP_733408), Drosophila simulans
(XP_002105562), Nasonia vitripennis (XP_001603446) and Ixodes scapularis (XP_002412058). Sequenced were retrieved from the GenBank and
compared with saposin-related protein of P. monodon. Four SapB domains are conserved in vertebrate saposins whereas the number of SapB
domains varies among invertebrate taxa.
Figure 7 Analysis of Penaeus monodon Dmc1 (PmDmc1). (A) The full-length cDNA and deduced protein sequences of Dmc1 (1,661 bp; ORF
1,026 bp, 341aa) of P. monodon. Start (ATG) and stop (TAA) codons are illustrated in boldface and underlined. The deduced DMC1 protein
contains an HhH domain (positions 46-75, E-value = 2.7e-02; highlighted)) and an AAA domain (positions 118-308, E-value = 9.73e-06;
highlighted). (B) A bootstrapped neighbor-joining tree illustrating phylogentic relationships of Dmc1 of various taxa. Values at the node represent
the percentage of times that the particular node occurred in 1000 trees generated by bootstrapping the original aligned protein sequences.
Protein sequences of different isoforms of Dmc1 from Homo sapiens (HS-Dmc1, NM_007068), Equus caballus (EC-Dmc1, XM_001501584), Macaca
mulatta (MM-Dmc1, XM_001094012), Pan troglodytes (PT-Dmc1, XM_515130), Canis lupus familiaris (CLF-Dmc1-1, XM_844891 and CLF-Dmc1-2,
XM_855217), Gallus gallus (GG-Dmc1, XM_425477), Mus musculus (MM-Dmc1, NM_010059), Anguilla japonica (AJ-Dmc1, AB182645), Oreochromis
niloticus (ON-Dmc1, AB182646), Danio rerio (DR-Dmc1, NM_001020782), Carassius auratus (CA-Dmc1, EF545136), Bombyx mori (BM-Dmc1,
NM_001044087), Hydra vulgaris (HV-Dmc1, AB047581) and Strongylocentrotus purpuratus (SP-Dmc1, XM_786187), were retrieved from GenBank and
compared with Dmc1 of P. monodon (PmDmc1).
broodstock than in wild broodstock and cultivated
juvenile P. monodon males (P < 0.05, Figure 8). The
expression level of this gene was also higher in testis than
ovary (P < 0.05). Therefore, PmSap and PmDmc1 should
play an important role in spermatogenesis (and
testicular development) rather than oogenesis (and ovarian
development) of P. monodon. Importantly, lower
expression levels of PmDmc1 in domesticated versus wild
broodstock suggest transcriptional levels of this gene
may be used to indicate possible reduced maturation of
reproduction in domesticated P. monodon.
Molecular mechanisms and expression patterns of
genes controlling different steps of sperm maturation
and testicular development should be examined for a
better understanding of P. monodon reproductive
maturation in captivity. The limited number of known
genes expressed in testis of this economically important
species was partially resolved by EST analysis in the
present study. Typically, molecular studies of the
hormonal and environmental effectors involving shrimp
reproduction have been limited to one or a few genes.
Here, we illustrate that cDNA microarrays are relatively
simple, reliable and cost-effective for examining
integrated interactions among genes in gonads of
P. monodon. This method can help accelerate studies
on reproductive development and maturation of
The testis cDNA library of P. monodon was previously
constructed as described in . The lambda library was
converted to the pBluescript library by in vivo excision.
Colony PCR was performed in a 25 l reaction mixture
containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl,
Enhancer solution, 200 mM of each dNTP, 2 mM MgCl2,
Figure 8 Real-time PCR analysis illustrating the relative
expression levels of saposin in testis (TT) and ovary (OV) of P.
monodon juvenile (J), domesticated (DB) and wild broodstock
(WB). Bars labeled with the same letters do not have significantly
different expression levels (P > 0.05). J-TT (N = 5), DB-TT (N = 5), and
WB-TT (N = 5) are testis of juveniles, domesticated broodstock, and
wild broodstock, respectively. J-OV (N = 4) and WB-OV (N = 4) are
ovary of juvenile and wild broodstock, respectively.
0.2 M each of M13-F (5-ACG TTG TAA AAC GAC
GGC CAG-3) and M13-R (5-ACA GGA AAC AGC TAT
GAC CAT G-3), and 1.25 unit of i-TaqDNA Polymerase
(iNtRON Biotechnology). PCR was carried out in a
thermocycler consisting of predenaturation at 94C for 5 min
followed by 35 cycles of denaturation at 94C for 30 s,
annealing at 58C for 1 min and extension at 72C for 3
min. The final extension was carried out at the same
temperature for 7 min. The colony PCR products were
size-fractionated on 1.5% agarose gel and visualized after
ethidium bromide staining. Clones with >400 bp inserts
were selected for further sequencing. Plasmid was
extracted using 96-well plate format (EZ-10 96-well spin
column, Bio Basic). The concentrations of plasmids were
measured using a NanoDrop 8000. Nucleotide sequence
of each clone was obtained using an automated DNA
sequencer. The 4,803 sequences of the testis ESTs from
this study were deposited in NCBI dbEST http://www.
ncbi.nlm.nih.gov/dbEST/ with accession numbers
of GW992816-GW996323, HO000142-HO000184,
GE613296 - GE614547.
in Figure 1. Briefly, the sequencing data were analyzed by
SeqClean (removal of the polyA/polyT tail, low-quality
ends, short sequences and those containing cloning
vector), RepeatMasker (masking of the sequences when
matched with sequences in the largest repeat library, the
RepBase, that covers a number of organisms including
human, rodent, zebrafish, Drosophila, and Arabidopsis
thaliana) (University of Washington Genome Center,
http://ftp.genome.washington.edu/cgi-bin/RepeatMasker), and CAP3 (assembly and clustering of EST
sequences) . The post-processed sequences were
further annotated for biological activities by comparison
with the NCBI nr database using BlastN (a
nucleotidelevel annotation, E-value < 10-5), BlastX (a protein-level
annotation, E-value < 10-5), BLAST2GO (Gene Ontology
prediction of the annotated proteins from BlastX
program), and InterProScan (protein signature identification).
To identify ESTs found only in the testis cDNA library
of P. monodon, all newly sequenced contigs and
singletons were compared against all sequences (38,429 ESTs)
from the P. monodon database (PmDB:
http://pmonodon.biotec.or.th). Moreover, to identify novel transcripts
that have not been previously reported in shrimp, the
sequenced data were also compared against all available
Penaeidae ESTs (207,852 ESTs) from NCBI http://ncbi.
nlm.nih.gov retrieved on 01/11/09.
Microarray analysis for identification of differential
expression transcripts in testis of P. monodon
A cDNA microarray was constructed from various EST
libraries of P. monodon, consisting of 5,568 features
(1,076 unique cDNA features were amplified from testis
EST libraries). The arrays were post-processed according
to  immediately before hybridization.
Total RNA was extracted from testis and ovary of
juveniles (4 month-old) and broodstock originating from
the Andaman Sea (west of peninsular Thailand).
Contaminating genomic DNA was removed by treatment
with DNase I (0.15 U/g total RNA) at 37C for 30 min.
The first-strand cDNA was synthesized and
fluorescently labeled according to . RNA from ovary was
labeled with Cy3 dye as a reference and RNA from testis
was labeled with Cy5 dye. The Cy3- and Cy5-samples
were mixed together and hybridized onto the arrays
overnight. The hybridized slides were washed according
to Cornings instruction. Nine microarray experiments
were performed and the details of comparison between
gene expression profiles of ovaries and testes are
summarized in Table 2.
In silico analysis of ESTs
Nucleotide sequences were analyzed using ESTplus, an
integrative system for comprehensive and customized EST
analysis and proteomic data matching , as summarized
Microarray imaging and data analysis
The hybridized slides were scanned with a GenePix
4000B (Molecular Devices, Sunnyvale, CA) and
processed using GenePix Pro version 6.1. Only spots with
Table 2 Summary of microarray experiments in this study
I. Comparison of gene expression levels between ovary and testis in individual juveniles
1 Ovary from juvenile 1
2 Ovary from juvenile 2
3 Ovary from juvenile 3
II. Comparison of gene expression levels between ovaries and testes pooled from juveniles
1 Pooled ovaries from juveniles (n = 98)
2 Pooled ovaries from juveniles (n = 98)
3 Pooled ovaries from juveniles (n = 98)
III. Comparison of gene expression levels between ovaries and testes pooled from broodstock
1 Ovary from broodstock 1 (GSI*: 12.4)
2 Ovary from broodstock 2 (GSI: 11.2)
3 Ovary from broodstock 3 (GSI: 12.6)
*Gonadosomatic Index (GSI) = % gonad weight/body weight
Testis from juvenile 1
Testis from juvenile 2
Testis from juvenile 3
Pooled testes from juveniles (n = 114)
Pooled testes from juveniles (n = 114)
Pooled testes from juveniles (n = 114)
Testis from broodstock 1 (GSI: 1.1)
Testis from broodstock 2 (GSI: 1.1)
Testis from broodstock 3 (GSI: 1.1)
intensities greater than one standard deviation above the
background intensity were further analyzed. The
processed data were normalized within each array by the
scaled print-tip (Lowess) method, and across arrays,
using the Aroma package , (available from http://
www.maths.lth.se/help/R/aroma/, run in R project
environment http://cran.r-project.org. The microarray data
have been deposited in NCBIs Gene Expression
Omnibus http://www.ncbi.nlm.nih.gov/geo/ with GEO
accession number GSE19037 at . The average logarithmic
base 2 values of relative intensities between Cy3- and
Cy5- samples (M values) were subjected to hierarchical
clustering analysis and illustrated using the Treeview
software . To identify differential expressed
transcripts, only transcripts with relative expression level
changes more than the median value 1SD in at least 1
array and present in at least 8 of the 9 arrays were
further considered. A 3-fold change in at least 6 of the 9
arrays was considered as criteria for differential
Examination of full-length cDNA sequences of Saposin
and Dmc1 by Rapid Amplification of cDNA
EndsPolymerase Chain Reaction (RACE-PCR)
Gene-specific primers for RACE-PCR of P. monodon
Saposin and Dmc1 were designed (Table 3).
RACEPCRs of Saposin and Dmc1 were carried out using a BD
SMART RACE cDNA Amplification Kit following the
protocol recommended by the manufacturer (BD
Biosciences Clontech). The amplified fragment of each gene
was electrophoretically analyzed and eluted from an
agarose gel before cloning into pGEM-T Easy vector
(Promega) and sequencing . The full-length cDNA
was assembled from EST and RACE-PCR products
Table 3 Primer sequence, melting temperature and the
expected amplification product of PmSap and PmDmc1
F: 5-GCTATGGTTCAGGTTGATGACTTGC-3 74
Real-time PCR F: 5-CCATAAAGTTCTGCCCCCACCAC-3
Real-time PCR F: 5-TTCCGACTCCAAGAACGACC-3
Real-time PCR F: 5-ATGTGCGAGAAGCGAAGGC-3
(hereafter called PmSap and PmDmc1). The sequences
of PmSap and PmDmc1 were deposited with accession
numbers of GU566728 and EU440762, respectively. The
functional domains of the deduced amino acids of these
genes were analyzed using SMART
http://smart.emblheidelberg.de/. The pI and molecular weight were
estimated using ProtParam http://www.expasy.org/tools/
Gene expression analysis of PmSap and PmDmc1 by
quantitative real-time PCR
Gene-specific primers were designed for PmSap,
PmDmc1 and Elongation factor 1 alpha (EF-1a, Table
3). For construction of the standard curve of each
transcript, a plasmid containing the transcript was
constructed by cloning PCR product into pGEM-Teasy
vector and transforming the resulting vector into E. coli
JM109. The plasmid was extracted and used as the
template for construction of the standard curve by 10-fold
serial dilutions (103 - 109 copy numbers). Real-time PCR
reactions were carried out in a 96-well plate in triplicate.
The expression levels of PmSap and PmDmc1 in testis
from juvenile (J-TT, N = 5), domesticated broodstock
(DB-TT, N = 5), and wild broodstock (WB-TT, N = 5)
and ovary from juvenile (J-OV, N = 4) and wild
broodstock (WB-OV, N = 4) were further analyzed by
quantitative real-time PCR analysis. EF-1a was used as an
internal control. Each primer set was designed to
generate 120 to 150 bp amplicons (Table 3). Each reaction
was performed in a 15 l reaction volume containing 2
LightCycler 480 SYBR Green I Master (Roche), 50 ng of
first strand cDNA template, and 0.2 M or 0.4 M of
each primer pair. Cycling parameters were 95C for 10
min, 40 cycles of 95C for 15 sec, 58C for 30 sec, and
72C for 30 sec. The specificity of PCR products was
confirmed by melting curve analysis by heating at 95C
for 15 sec, 65C for 1 min before heating to 97C and
gradually cooling down to 40C within 10 sec. Real-time
PCR of each shrimp was tested in duplicate. Relative
expression levels (copy number of the target gene/that
of EF-1a) of different sample groups were statistically
tested by ANOVA followed by Duncans new multiple
range test or Tukey test (P < 0.05).
This research is supported by the National Center for Genetic Engineering
and Biotechnology (BIOTEC). Part of this research (library construction,
RACEPCR and real-time-PCR) was carried out by RL during the PhD program and
supported by the Royal Golden Jubilee PhD program, the Thailand Research
Funds (TRF), Thailand. We would like to thank Dr. Amy Lum for reading and
improving the manuscript. Also, we would like to thank Prof. Morakot
Tanticharoen for her mentorship of this research program.
1National Center for Genetic Engineering and Biotechnology (BIOTEC),
National Science and Technology Development Agency, Klong 1, Klong
RL and SK constructed the testes EST library. TW, RL and NT carried out
plasmid isolation and sequencing. TW performed in silico analysis of cDNA
sequences. UU, TW and NK constructed cDNA microarray chips and
performed microarray experiments. RL carried out gene expression analysis
experiments and full-length characterization. PM, NK and SK conceived the
work. TW, RL, NK and SK wrote the manuscript. All authors read and
approved this manuscript.
1. Rosenberry B : World Shrimp Farming . San Diego, CA: Shrimp News international 2000 .
2. Browdy CL : A review of the reproductive biology of penaeus species: perspectives on controlled shrimp maturation systems for high quality nauplii production . Proceedings of the Special Session on Shrimp Farming , World Aquaculture Society Baton Rouge, LA, USAWyban J 1992 , 22 - 51 .
3. Withyachumnarnkul B , Boonsaeng V , Flegel TW , Panyim S , Wongteerasupaya C : Domestication and selective breeding of Penaeus monodon in Thailand . Proceedings to the Special Session on Advances in Shrimp Biotechnology, The Fifth Asian Fisheries Forum: International Conference on Fisheries and Food Security Beyond the Year: 11-14 November 1998 ; Chiangmai, Thailand Flegel TW 2000 , 73 - 77 .
4. Leelatanawit R , Klinbunga S , Aoki T , Hirono I , Valyasevi R , Menasveta P : Suppression subtractive hybridization (SSH) for isolation and characterization of genes related to testicular development of the giant tiger shrimp Penaeus monodon . BMB Reports 2008 , 41 ( 11 ): 796 - 802 .
5. Leelatanawit R , Sittikankeaw K , Yocawibun P , Klinbunga S , Roytrakul S , Aoki T , Hirono I , Menasveta P : Identification, characterization and expression of sex-related genes in testes of the giant tiger shrimp Penaeus monodon . Comp Biochem Physiol A 2009 , 152 : 66 - 76 .
6. Pacharawongsakda E , Yokwai S , Karoonuthaisiri N , Wichadakul D , Ingsriswang S : ESTplus: an integrative system for comprehensive and customized EST analysis and proteomic data matching . Proceedings of the 2nd International Conference on Bioinformatics and Biomedical Engineering (iCBBE2008): 16-18 May 2008 ; Shanghai, China 2008 , 29 - 32 .
7. The Tribolium Genome Sequencing Consortium: The genome of the model beetle and pest Tribolium castenuem . Nature 2008 , 452 : 946 - 955 .
8. Shit S , Atreja SK : Phospholipase A2 activation by hydrogen peroxide during in vitro capacitation of buffalo spermatozoa . Indian J Exp Biol 2004 , 42 ( 5 ): 486 - 90 .
9. Pietrobon EO , Soria M , Domnguez L , De Los ngeles Monclus M, Forns MW : Simultaneous activation of PLA2 and PLC are required to promote acrosomal reaction stimulated by progesterone via G-proteins . Mol Reprod Dev 2005 , 70 : 58 - 63 .
10. Newmark PA , Boswell RE : The mago nashi locus encodes an essential product required for germ plasm assembly in Drosophila . Development 1994 , 120 : 1303 - 1313 .
11. Verheyen EM , Colley L : Profilin mutations disrupt multiple actindependent processes during Drosophila development . Development 1994 , 120 : 717 - 728 .
12. Witke W , Sutherland JD , Sharpe A , Arai M , Kwiatkowski DJ : Profilin I is essential for cell survival and cell division in early mouse development . Proc Natl Acad Sci USA 2001 , 98 ( 7 ): 3832 .
13. Ono S , Minami N , Abe H , Obinata T : Characterization of novel A cofilin isoform that is predominantly expressed in mammalian skeletal muscle . J Biol Chem 1994 , 269 : 15280 - 15286 .
14. Cottrelle P , Thiele D , Price VL , Memet S , Micouin J-Y , Marck C , Buhler J-M , Sentenac A , Fromageot P : Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor la . J Biol Chem 1985 , 260 : 3090 - 3096 .
15. Dalla Pavera R , Gelmann EP , Martionotti S , Franchini G , Papa TS , Gallo RC , Wong-Staal F : Cloning and characterization of different human sequences related to the one gene (v-myc) of avian myelocytomatosis virus (MC-29) . Proc Natl Acad Sci USA 1982 , 79 : 6497 - 6501 .
16. Heerdt BG , Halsey HK , Lipkin M , Augenlicht LH : Expression of mitochondrial cytochrome c oxidase in human colonie cell differentiation, transformation, and risk for colonic cancer . Cancer Res 1990 , 50 : 1596 - 1600 .
17. Pines J , Hunter T : p34 cdc2: the S and M kinase? New Biol 1990 , 2 : 389 - 401 .
18. Naz RK , Ahmad K , Kaplan P : Involvement of cyclins and cdc2 serine/ threonine protein kinase in human sperm cell function . Biol Reprod 1993 , 48 : 720 - 728 .
19. Godet M , Damestoy A , Mouradian S , Rudkin BB , Durand P : Key role for cyclin-dependent kinases in the first and second meiotic divisions of rat spermatocytes . Biol Reprod 2004 , 70 : 1147 - 1152 .
20. Preechaphol R , Leelatanawit R , Sittikankeaw K , Klinbunga S , Khamnamtong B , Puanglarp N , Menasveta P : Expressed sequence tag analysis for identification and characterization of sex-related genes in the giant tiger shrimp Penaeus monodon . J Biochem Mol Biol 2007 , 40 : 501 - 510 .
21. Koshiyama A , Hamada FN , Namekawa SH , Iwabata K , Sugawara H , Sakamoto A , Ishizaki T , Sakaguchi K : Sumoylation of a meiosis-specific RecA homolog, Lim15/Dmc1, via interaction with the small ubiquitinrelated modifier (SUMO)-conjugating enzyme Ubc9 . FEBS J 2006 , 273 : 4003 - 4012 .
22. Takahashi Y , Kikuchi Y : Yeast PIAS-type Ull1/Siz1 is composed of SUMO ligase and regulatory domains . J Biol Chem 2005 , 280 : 35822 - 35828 .
23. Shen B , Zhang Z , Wang Y , Wang G , Chen Y , Lin P , Wang S , Zou Z : Differential expression of ubiquitin-conjugating enzyme E2r in the developing ovary and testis of penaeid shrimp Marsupenaeus japonicus . Mol Biol Rep 2008 , 36 : 1149 - 1157 .
24. Karoonuthaisiri N , Sittikankeaw K , Preechaphol R , Kalachikov S , Wongsurawat T , Uawisetwathana U , Russo JJ , Ju J , Klinbunga S , Kirtikara K : ReproArrayGTS: A cDNA microarray for identification of reproductionrelated genes in the giant tiger shrimp Penaeus monodon and characterization of a novel nuclear autoantigenic sperm protein (NASP) gene . Comp Biochem Physiol D 2009 , 4 : 90 - 99 .
25. Okumura T , Kim YK , Kawazoe I , Yamano K , Tsutsui N , Aida K : Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus . Comp Endrocrinol Physiol A 2006 , 143 : 246 - 253 .
26. Richardson RT , Batova IN , Zheng L-X , Whitfield M , Marzluff WF , O'Rand MG : Characterization of the histone H1 binding protein, NASP, as a cell cycle regulated, somatic protein . J Biol Chem 2000 , 275 : 30378 - 30386 .
27. Richardson RT , Alekseev OM , Grossman G , Widgren EE , Thresher R , Wagner EJ , Sullivan KD , Marzluff WF , O'Rand MG : Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation . J Biol Chem 2006 , 281 : 21526 - 21534 .
28. Leelatanawit R , Klinbunga S , Puanglarp N , Tassanakajon A , Jarayabhand P , Hirono I , Aoki T , Menasveta P : Isolation and characterization of differentially expressed genes in ovaries and testes of the giant tiger shrimp (Penaeus monodon) . Mar Biotechnol 2004 , 6 : S506 - S510 .
29. Parker JS , Barford D : Argonaute: a scaffold for the function of short regulatory RNAs . TRENDS Biochem Sci 2006 , 31 : 622 - 630 .
30. Ghosh-Roy A , Kulkarni M , Kumar V , Shirolikar S , Ray K : Cytoplasmic dyneindynactin complex is required for spermatid growth but not axoneme assembly in Drosophila . Mol Biol Cell 2004 , 15 : 2470 - 2483 .
31. Kishimoto Y , Hiraiwa M , O'Brien JS : Saposins: structure, function, distribution, and molecular genetics . J Lipid Res 1992 , 33 : 1255 - 1267 .
32. Fujita N , Suzuki K , Vanier M , Popko B , Maeda N , Klein A , Henseler M , Sandhoff K , Nakayasu H , Suzuki K : Targeted disruption of the mouse sphingolipid activator gene: a comples phenotype, including severe leukodystrophy and wide-spread storage of multiple sphingolipids . Human Mol Gen 1996 , 5 : 711 - 725 .
33. Morales CR , Zhao Q , Lefrancois S , Ham D : Role of prosaposin in the male reproductive system: effect of prosaposin inactivation on the testes , epididymis, prostate, and seminal vesicles. Arch Androl 2000 , 44 ( 3 ): 173 - 186 .
34. Kajiura-Kobayashi H , Kobayashi T , Nagahama Y : Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish . Dev Dynamics 2005 , 232 : 1115 - 1123 .
35. Hazkani-Covo E , Altman N , Horowitz M , Graur D : The Evolutionary History of Prosaposin: Two Successive Tandem-Duplication Events Gave Rise to the Four Saposin Domains in Vertebrates . J Mol Evol 2002 , 54 : 30 - 34 .
36. Huang X , Madan A : CAP3: A DNA sequence assembly program . Genome Res 1999 , 868 - 877 .
37. Bengtsson H : aroma - An R Object-oriented Microarray Analysis environment Preprints in Mathematical Sciences:18, Mathematical Statistics, Lund University 2004 .
38. Edgar R , Domrachev M , Lash AE : Gene Expression Omnibus: NCBI gene expression and hybridization array data repository . Nucleic Acids Res 2002 , 30 : 207 - 210 .
39. Eisen MB , Spellman PT , Brown PO , Botstein D : Cluster analysis and display of genome-wide expression patterns . Proc Natl Acad Sci USA 1998 , 95 : 14863 - 14868 .
40. Sambrook J , Russell DW : Molecular Cloning: A Laboratory Manual New York, USA; Cold Spring Harbor Laboratory Press, 3 2001 .