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BMC Genomics
Open Access
Research article
New insights into molecular pathways associated with flatfish
ovarian development and atresia revealed by transcriptional
analysis
Angèle Tingaud-Sequeira1,2, François Chauvigné1, Juanjo Lozano3,
María J Agulleiro1,4, Esther Asensio5 and Joan Cerdà*1
Address: 1Laboratory of Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones
Científicas (CSIC), 08003 Barcelona, Spain, 2Génomique et Physiologie des Poissons, Université Bordeaux 1, UMR NuAGe, 33405 Talence, France,
3Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 08036 Barcelona, Spain, 4Instituto de
Acuicultura Torre de la Sal (CSIC), 12595 Castellón, Spain and 5IFAPA Centro El Toruño, Junta de Andalucía, Cádiz, Spain
Email: Angèle Tingaud-Sequeira - angela.tingaud@u-bordeaux1.fr; François Chauvigné - chauvigne@cmima.csic.es;
Juanjo Lozano - juanjo.lozano@ciberehd.org; María J Agulleiro - mjagulleiro@iats.csic.es;
Esther Asensio - esther.asensio.ext@juntadeandalucia.es; Joan Cerdà* - joan.cerda@irta.es
* Corresponding author
Abstract
Background: The Senegalese sole (Solea senegalensis) is a marine flatfish of increasing commercial interest. However,
the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its
reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian
follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the
present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis), maturation
and ovarian follicle atresia using a recently developed oligonucleotide microarray.
Results: Microarray analysis led to the identification of 118 differentially expressed transcripts, of which 20 and 8 were
monitored by real-time PCR and in situ hybridization, respectively. During vitellogenesis, many up-regulated ovarian
transcripts had putative mitochondrial function/location suggesting high energy production (NADH dehydrogenase
subunits, cytochromes) and increased antioxidant protection (selenoprotein W2a), whereas other regulated transcripts
were related to cytoskeleton and zona radiata organization (zona glycoprotein 3, alpha and beta actin, keratin 8),
intracellular signalling pathways (heat shock protein 90, Ras homolog member G), cell-to-cell and cell-to-matrix
interactions (beta 1 integrin, thrombospondin 4b), and the maternal RNA pool (transducer of ERBB2 1a, neurexin 1a).
Transcripts up-regulated in the ovary during oocyte maturation included ion transporters (Na+-K+-ATPase subunits),
probably required for oocyte hydration, as well as a proteinase inhibitor (alpha-2-macroglobulin) and a vesicle calcium
sensor protein (extended synaptotagmin-2-A). During follicular atresia, few transcripts were found to be up-regulated,
but remarkably most of them were localized in follicular cells of atretic follicles, and they had inferred roles in lipid
transport (apolipoprotein C-I), chemotaxis (leukocyte cell-derived chemotaxin 2,), angiogenesis (thrombospondin), and
prevention of apoptosis (S100a10 calcium binding protein).
Conclusion: This study has identified a number of differentially expressed genes in the ovary that were not previously
found to be regulated during ovarian development in marine fish. Specifically, we found evidence, for the first time in
teleosts, of the activation of chemoattractant, angiogenic and antiapoptotic pathways in hypertrophied follicular cells at
the onset of ovarian atresia.
Published: 15 September 2009
BMC Genomics 2009, 10:434 doi:10.1186/1471-2164-10-434
Received: 11 May 2009
Accepted: 15 September 2009
This article is available from: http://www.biomedcentral.com/1471-2164/10/434
© 2009 Tingaud-Sequeira et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Background
Our understanding of the molecular pathways underlying
reproductive processes and oogenesis in vertebrates is still
limited even in mammalian models. Teleosts show the
most diversified reproductive strategies among vertebrates
making it even more difficult to uncover the underlying
molecular mechanisms. In addition, several factors can
modulate the reproductive processes of teleost fish such as
photoperiod and temperature [1,2], nutrition [3], captiv-
ity [4] and endocrine disruptors [5,6]. Accordingly, most
studies to date on female teleosts have mainly investi-
gated the effect of these conditions on the circulating sex
hormone levels or the reproductive success in terms of
spawning performance (e.g., fecundity, egg and larval sur-
vival). However, the molecular and cellular mechanisms
involved still remain poorly understood.
The development of methods for large-scale gene expres-
sion analysis (e.g., microarrays) in model fish species,
such as the zebrafish (Danio rerio), as well as in salmonids
is improving our knowledge of the molecular basis of
ovarian physiology in teleosts [7]. In the zebrafish, cDNA-
and oligo-based microarrays have been employed to
assess the transcriptome profile of differentiating and
adult gonads. These studies have identified a number of
genes involved in mitochondrial organization and bio-
genesis, cell growth and maintenance, and germ-line dif-
ferentiation, as well as some with sexually dimorphic co-
expression in both the gonads and the brains [8-10]. Mass
sequencing of zebrafish expressed sequence tags (ESTs)
from the ovary [11], or from isolated fully-grown ovarian
follicles through serial analysis of gene expression (SAGE)
[12], has also discovered germ cell-specific genes and
established the complete sequence data set of maternal
mRNA stored in oocytes at the end of oogenesis. In rain-
bow trout (Oncorhynchus mykiss) and coho salmon (O. kis-
utch), cDNA microarrays printed on slides or nylon
membranes, as well as reciprocal suppression subtractive
hybridization (SSH), were used to investigate changes in
the ovarian transcriptome during primary growth and
maturation and bacterial lipopolysaccharide-induced
ovarian apoptosis [13-17]. These studies revealed changes
in the expression of genes involved in cellular organiza-
tion and extracellular matrix (ECM) remodelling, immu-
noregulation, apoptosis, cell cycling, and in different
endocrine and paracrine systems, which might be impor-
tant during ovarian development. The control of ovula-
tion by either hormonal induction or photoperiod
manipulation has also been shown to induce differences
in the egg mRNA abundance of specific genes, which may
affect their developmental competence [18].
However, despite the significant information obtained
from zebrafish and salmonids, no data is currently availa-
ble on the changes of the transcriptome during ovarian
development in other marine teleosts, such as flatfish,
some of which are of economical importance. Particu-
larly, little is known on the molecular pathways involved
in ovarian follicular atresia, a degenerative and resorptive
process of ovarian follicles that determines fecundity in
both natural and captive conditions [19-22]. The use of
functional genomics approaches would contribute with
the identification of molecular signatures associated with
abnormal ovarian development or premature ovarian
regression in cultured fish species, thus providing poten-
tially useful markers to control sexual maturation.
The Senegalese sole, Solea senegalensis, is a marine flatfish
of high commercial value in Southern Europe and Asia
[23]. However, the industrial production of this species is
largely based on wild breeders after long periods of accli-
mation to captivity, and reproduction is not yet controlled
[26]. The F1 generation of fish raised in captivity often fail
to reproduce naturally because egg fertilization is dramat-
ically reduced [24,25]. In some F1 females, an increased
ovarian follicle atresia and/or dysfunctions of the ovula-
tory process might also occur, but no precise studies have
been performed to clarify this phenomenon. In order to
obtain information on the molecular basis of ovarian
development in Senegalese sole, the present study aimed
at performing a transcriptomic analysis of the ovary dur-
ing oocyte growth (vitellogenesis), maturation and follic-
ular atresia using a recently developed oligonucleotide
microarray [27]. The analysis revealed the differential
expression of more than one hundred genes during ovar-
ian development, some of them with yet unknown func-
tions in the fish ovary. In addition, determination of the
cell type-specific expression in the ovary of selected tran-
scripts suggest the activation of genes presumably
involved in chemotaxis, angiogenesis and prevention of
apoptosis in follicular cells of atretic follicles, which have
not been described before in teleosts during ovarian
atresia.
Results
Stages of ovarian development
To identify differentially expressed genes during ovarian
development in Senegalese sole, samples of ovarian tissue
were collected from adult females sacrificed throughout
the annual reproductive cycle [28,29] or after hormonal
treatment (Figure 1). Thus, samples of ovaries at previtel-
logenesis (Figure 1A and 1B), vitellogenesis (Figure 1D
and 1E), maturation (Figure 1G and 1H), and undergoing
follicular atresia (Figure 1J, K and 1L), were used for tran-
scriptome analysis. As many other fractional spawner tel-
eosts, the Senegalese sole has a group-synchronous ovary
in which follicles of all sizes up through vitellogenesis are
present at any time, and populations (or clutches) of fol-
licles are periodically recruited into maturation from a
population of oocytes in late vitellogenic stages [30].
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Developmental stage of the Senegalese sole ovaries used for microarray analysisFigure 1
Developmental stage of the Senegalese sole ovaries used for microarray analysis. Representative light micrographs
of histological sections of the ovary (n = 3 females) stained with hematoxylin-eosin (A, B, D, E, G, H, and J-L), and frequency of
ovarian follicles in the ovary (C, F, I and M) at each ovarian developmental stage, previtellogenic (A and B), vitellogenic (D and
E), mature (G and H) and atretic (J-L). Data on the frequency of ovarian follicles are the mean ± SEM (n = 3 females). (B)
Oocyte at primary growth stage. (D inset) Ovarian follicle containing a cortical alveolus stage oocyte. (G) Follicle at early mat-
uration (note the migration of the germinal vesicle). (H) Follicle containing a mature oocyte (the germinal vesicle is not
observed and yolk is fused). (K) Ovarian follicle at early atresia. (L) Follicle at advanced atresia. gv, germinal vesicle; c, oocyte
cytoplasm; y, yolk; zr, zona radiata; fc, follicular cells; ca, cortical alveolus stage oocyte. P, previtellogenic follicle; E, early vitell-
ogenic follicle; V, vitellogenic follicle; M, mature follicle; A, atretic follicle. Bars, 200 μm (D, J), 100 μm (A, G, H, K), 50 μm (E,
D inset, L), 20 μm (B).
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Therefore, the increased frequency of vitellogenic, mature
or atretic ovarian follicles in the ovary, as determined by
histological analysis (Figure 1C, F, I and 1M), defined the
ovarian developmental stages used in the present study.
The previtellogenic ovary was formed by exclusively ovar-
ian follicles with oocytes at the primary growth stage
(oocyte/follicle diameter up to approximately 150 μm) in
which vitellogenin incorporation and yolk formation did
not yet start (Figure 1A-1C). In the vitellogenic ovary, a
population of follicles were recruited into vitellogenesis,
and consequently the proportion of follicles at the pri-
mary growth stage decreased (Figure 1D-1F). At this stage,
follicles containing oocytes at the cortical alveolus stage
(up to approximately 300 μm), characterized by the pres-
ence of nascent cortical alveoli within the ooplasm, were
more abundant (Figure 1D, inset). Vitellogenic oocytes
surrounded by the zona radiata and the somatic follicular
cells, granulosa and theca cells, increased in size (up to
500 μm in diameter at late vitellogenesis) and their cyto-
plasm was filled with yolk granules where vitellogenin-
derived yolk proteins are stored (Figure 1E). As a result of
this growing phase, the gonadosomatic index (GSI) of
females increased by approximately 7-fold (Figure 1C and
1F).
Maturing ovaries containing follicle-enclosed oocytes
undergoing meiosis resumption, and ovaries carrying
mature oocytes prior to ovulation, were collected 24-48 h
after treatment of vitellogenic females with gonadotropin-
releasing hormone agonist [D-Ala6, Pro9, NEt] (GnRHa)
[24]. In the mature ovary, a population of follicle-
enclosed oocytes at late stages of vitellogenesis was further
recruited into maturation (Figure 1G-1I). In these oocytes,
the germinal vesicle migrates towards the animal pole and
yolk globules fuse one another (Figure 1G), eventually
forming a large mass of yolk (Figure 1H). The mature
oocyte reached 800-900 μm in diameter due to water
uptake (hydration), resulting in a further 2-fold increase
of the GSI (Figure 1F and 1I).
Finally, atretic ovaries were collected from females show-
ing spontaneously occurring ovarian follicle atresia dur-
ing the spawning season, or induced after GnRHa
treatment. In these ovaries, approximately up to 30-40%
of the ovarian follicles showed different levels of atresia
and maturing/mature oocytes were absent (Figure 1J-1M).
In early atretic follicles, vitellogenic oocytes shrank, the
zona radiata folded, and follicles became irregularly
shaped (Figure 1K and 1L). The follicular cells were hyper-
trophied and the theca was poorly developed. Advanced
follicular atresia was characterized by breakdown and
resorption of the zona radiata, and the appearance of
highly columnar follicular cells apparently showing an
intense phagocytic activity as suggested by the presence of
large vacuoles (Figure 2). At this stage, accumulation of
blood cells, erythrocytes and leukocytes in the follicle, as
well as in the oocyte, was also noted (Figure 2B).
Microarray analysis
Differential gene expression in the four ovarian develop-
mental stages was determined using a Senegalese sole-spe-
cific oligonucleotide microarray containing 60-mer
probes representing 5,087 unique genes [27]. This plat-
form was previously designed from a Senegalese sole EST
database derived from a multi-tissue normalized cDNA
library from different adult tissues (including ovaries at
Photomicrographs of ovarian follicles at advanced atresiaFigure 2
Photomicrographs of ovarian follicles at advanced
atresia. Light micrographs of histological sections stained
with hematoxylin-eosin. bc, blood cells; ep, epithelium; tc,
theca cells; v, vacuole; gc, granulosa cells; e, erythrocytes; le,
leukocytes. Bars, 20 μm.
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different developmental stage) and larval and juvenile
stages [27]. Therefore, although this platform was not
ovary-specific and most likely did not contain all the tran-
scripts expressed in the sole ovary, its was useful to obtain
a first insight into the overall changes of gene expression
during ovarian development.
To determine the false discovery rate (FDR) in each of the
differential gene expression experiments (vitellogenic vs.
previtellogenic ovaries, mature vs. vitellogenic ovaries,
and atretic vs. vitellogenic/mature ovaries), an additional
microarray experiment was performed by hybridizing dif-
ferentially labelled (Cy3 and Cy5) aliquots of amplified
RNA (aRNA) from the same sample (previtellogenic
ovary). As expected, there were few differences between
the Cy3 and Cy5 signals for most of the microarray spots
in these experiments giving an estimated overall FDR of
3.0, 3.8 and 5.6% for vitellogenic vs. previtellogenic ova-
ries, mature vs. vitellogenic ovaries, and atretic vs. vitello-
genic/mature ovaries, respectively (see Additional file 1).
Microarray data analysis indicated significant (p < 0.01)
regulation of genes in vitellogenic (46 ESTs), mature (46
ESTs) and atretic ovaries (26 ESTs), which showed fold
change (FC) values from 1.4 up to 5.1. These ESTs, and the
corresponding GenBank accession numbers are listed in
Tables 1, 2 and 3. In Table 3, differential expressed genes
in atretic ovaries relative to vitellogenic or mature ovaries
are pooled together. Some of these ESTs (26%) could not
be annotated, even after sequencing the respective clones
from the 5' end, and are not included in these tables.
Gene ontology annotation
To obtain a first assessment of the more important physi-
ological processes occurring during ovarian development,
gene ontology (GO) analysis was carried out using the
BLAST2GO v1 program [31]. Most of the annotated ESTs
(93%) had GO assignments, and many of those had 3-6
assignments each (49%) and a significant proportion
(34%) had 7 or more assignments.
Figure 3 shows the differentially expressed genes in the
three ovarian stages (vitellogenesis, maturation and follic-
ular atresia) classified according to GO terms biological
process (level 3), cellular component (level 5) and molec-
ular function (level 3). During vitellogenesis, the majority
of regulated ESTs were dedicated to metabolic process,
oxidation reduction, regulation and anatomical structure
development, in the biological process category. A similar
distribution of GO terms was seen within the EST cluster
regulated during maturation, although in this case tran-
scripts related to cell cycle, localization of cell, cellular
component organization, and system process, were also
detected. During ovarian follicle atresia, most regulated
genes fall in the cellular metabolism, establishment of
localization, and cellular component organization
attributes.
During vitellogenesis and maturation, most protein prod-
ucts were mainly inferred to be associated with mitochon-
dria based on the cellular component category, although
some also might be in the cytoskeleton (specially during
vitellogenesis), the nucleus, and intracellularly in
organelles. Interestingly, during atresia, the products of
most of the up-regulated genes showed putative extracel-
lular location, whereas the products of the down-regu-
lated genes had membrane and nucleus locations.
Finally, classification using the molecular function cate-
gory indicated that most of the gene products regulated
during vitellogenesis and maturation were dedicated to
binding and catalytic functions, including nucleotide
binding, protein binding, ion binding, and transferase
and hydrolase activities. However, products involved in
transmembrane transporter activity only appeared during
maturation. In the atretic ovary, the majority of products
were associated with ion and lipid binding.
Vitellogenesis
In the vitellogenic ovary, 34 and 12 transcripts were found
to be up- and down-regulated, respectively, relative to pre-
vitellogenic ovaries; 35 had a significant hit in Swiss-Prot
database (Table 1). The most highly up-regulated tran-
scripts corresponded to selenoprotein W2a (sepw2a),
hypothetical 18K protein from Carassius auratus mito-
chondrion, muscle-specific beta 1 integrin binding pro-
tein 2 (mibp2), zona pellucida protein 3 (zp3),
cytochrome c oxidase subunit I (cox1), cytochrome b
(cytb), cytosolic heat shock protein 90 beta (hsp90b),
NADH dehydrogenase subunit 3 (nd3) and 1 (nd1), and
beta actin 1 (bactin1). The sequence similarity of clone
pgsP0015N21 to tilapia (Oreochromis mossambicus) sepw2a
was low (4E-04) possibly because its nucleotide sequence
only covered the C terminus of tilapia sepw2a.
Other up-regulated genes in the vitellogenic ovary, but at
lower levels, were additional components of the cytoskel-
eton, such as alpha actin (actc1l), keratin 8 (krt8),
tropomyosin1-1 (tpm1-1), myosin (myh11), and transge-
lin (tagln), or of intracellular signaling pathways, such as
Ras homolog member G (rhog), a novel protein similar to
serum/glucocorticoid regulated kinase (sgk) also found in
zebrafish, and inositol monophosphatase 3 (impa3). Pro-
teolytic complexes and enzymes, such as protein arginine
methyltransferase (prmt1), acetyl-coenzyme A acyltrans-
ferase 2 (acaa2), and creatine kinase (ckb), and the puta-
tive homeodomain transcription factor 1 (phtf1),
transducer of ERBB2 (tob1a), neurexin 1a (nrxn1a) and
thrombospondin 4b (thbs4b) were also up-regulated in
vitellogenic ovaries.
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Table 1: Transcripts regulated in vitellogenic ovary relative to previtellogenic ovary
Clone ID GenBank
accession
FCaUniProtKB/
TrEMBL entry
Swiss-Prot hit BLAST
E-value
Length
(% identity)b
Gene Symbol
pgsP0015N21 FF286629 +5.13 Q3ZLC7 Selenoprotein W2a
[Oreochromis mossambicus]
4E-04 27 (70%) sepw2a
pgsP0003P21 FF282633 +4.64 JC1348 Hypothetical 18 K protein,
mitochondrion [Carassius
auratus]
2E-06 40 (70%)
pgsP0005D08 FF283023 +4.49 Unknown
pgsP0017G22 FF287169 +3.94 Q0KJ14 Cytochrome c oxidase
subunit I [Solea senegalensis]
3E-84 230 (92%) cox1
pgsP0029L14 FF291468 +3.67 Q7ZUR6 Muscle-specific beta 1
integrin binding protein 2
[Danio rerio]
2E-83 192 (78%) mibp2
pgsP0018D11 FF287436 +3.56 Q75v54 Cytochrome b [S.
senegalensis]
8E-74 165 (93%) cytb
pgsP0019B22 FF287743 +3.34 A8R7E8 Cytosolic heat shock
protein 90 beta [S.
senegalensis]
6E-70 133 (100%) hsp90b
pgsP0008A11 FF283945 +3.21Q0KJ09NADH dehydrogenase
subunit 3 [S. senegalensis]
1E-43 111 (84%) nd3
pgsP0003A24 FF282359 +3.00 B1B560 Beta actin isoform 1 [S.
senegalensis]
4E-22 51 (100%) bactin1
pgsP0020D03 FF288118 +2.93 P79893 Chorion protein (zona
protein 3) [Sparus aurata]
8E-69 160 (70%) zp3
pgsP0021P08 FF288738 +2.69Q0KJ16NADH dehydrogenase
subunit 1 [S. senegalensis]
2E-124 280 (88%) nd1
pgsP0013B15 FF285673 +2.62 Q7SZR1 Transducer of ERBB2, 1a
[Danio rerio]
1E-57 175 (77%) tob1a
pgsP0007G02 FF283724 +2.59 Q6IQR3 Alpha actin [D. rerio] 7E-123 282 (83%) actc1l
pgsP0025N01 FF290105 +2.47 Q805D1 Tropomyosin1-1 [Takifugu
rubripes]
1E-19 55 (90%) tpm1-1
pgsP0029A20 FF291229 +2.41 Q0P4B4 Transgelin [D. rerio] 4E-60 141 (79%) tagln
pgsP0028P05 FF291191 +2.29 Q6PFN7 Protein arginine
methyltransferase 1 [D.
rerio]
1E-167 308 (95%) prmt1
pgsP0015D24 FF286403 +2.12 Unknown
pgsP0020O05 FF288363 +2.11 Unknown
pgsP0014A08 FF285984 +2.09 Unknown
pgsP0029C16 FF291271 +1.99A1XQX0Neurexin 1a [D. rerio] 3E-132 266 (84%) nrxn1a
pgsP0015H15 FF286488 +1.98 Unknown
pgsP0030I24 FF291759 +1.96 Unknown
pgsP0029L19 FF291472 +1.92Q8JGW0Thrombospondin 4b [D.
rerio]
1E-100 175 (97%) thbs4b
pgsP0004D01 FF282692 +1.93 Q5ZMG8 Ras homolog member G
(rho G) [Gallus gallus]
2E-90 162 (93%) rhog
pgsP0017K07 FF287242 +1.90 Q6NWF6 Keratin, type II cytoskeletal
8 [D. rerio]
4E-33 79 (94%) krt8
pgsP0028F14 FF290985 +1.89 Q9UMS5 Putative homeodomain
transcription factor 1 [Homo
sapiens]
4E-56 144 (70%) phtf1
pgsP0016G07 FF286813 +1.88 Unknown
pgsP0009A17 FF284280 +1.87 Q2YDR3 Inositol monophosphatase 3
[D. rerio]
6E-13 74 (84%) impa3
pgsP0027D14 FF290593 +1.85 Q8AY63 Creatine kinase, brain [D.
rerio]
7E-84 179 (90%) ckb
pgsP0020F22 FF288178 +1.82 Q4U0S2 Smooth muscle myosin
heavy chain [D. rerio]
1E-97 180 (87%) myh11
pgsP0027C08 FF290565 +1.80 Q3T0R7 3-Ketoacyl-CoA thiolase,
mitochondrial [Bos taurus]
1E-30 76 (82%) acaa2
pgsP0028D08 FF290934 +1.76 Unknown
pgsP0008G08 FF284066 +1.74 Unknown
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Two transcripts similar to CDC-like kinase 2 (si:ch211-
81a5.7) and CD53 cell surface glycoprotein (zgc:64051),
as well as alanine-glyoxylate aminotransferase (agxt), were
the most down-regulated genes during vitellogenesis.
Other down-regulated genes included the actin-binding
protein scinderin (scin), mitochondrial enzymes, such as
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex
subunit 11 (ndufa11) and a dihydrodipicolinate synthase-
like enzyme, proteolytic complexes and enzymes, as pro-
teasome subunit beta type-9 precursor (psmb9a) and car-
boxypeptidase H (cph), a hypothetical protein-encoding
gene also found in Xenopus laevis (LOC735233), and a
coiled-coil domain containing 90B novel product
(ccdc90b).
Ovarian maturation
Microarray analysis detected 26 up-regulated and 20
down-regulated transcripts in maturing/mature ovaries
relative to vitellogenic ovaries, and 32 transcripts could be
annotated (Table 2). The most highly up-regulated tran-
script corresponded to an EST showing sequence similar-
ity to the amphioxus (Branchiostoma floridae)
BRAFLDRAFT_128798 gene, which encodes an hypothet-
ical protein with inferred cysteinyl-tRNA aminoacylation
activity. However, the BLAST E-value for the similarity of
Senegalese sole clone pgsP0012B12 to this protein was
relatively low (2E-14), and therefore conclusive annota-
tion will require the cloning of the sole full-length cDNA.
Other highly up-regulated transcripts encoded Na+/K+-
ATPase subunits, such as the beta subunit 1a (atp1b1a),
alpha subunit 1 (atp1a1) and another isoform of the beta
subunit (atpb), and alpha-2-macroglobulin (a2m).
Cytoskeletal proteins, myosin 10 (myo10) and type II ker-
atin E3-like protein, and proteins involved in transcrip-
tional and translational responses, such as makorin RING
zinc finger protein 1a (mkrn1) and ribosomal protein L36
(rpl36), were other up-regulated transcripts in mature ova-
ries. Interestingly, a regulator of vesicular traffic, novel
protein similar to vertebrate ADP-ribosylation factor 4
and extended synaptotagmin-2-A (e-syt2-a), was also up-
regulated. Other transcripts were retinoic acid receptor
responder protein 3 (rarres3), thioredoxin interacting pro-
tein (txnip), mitogen-activated protein kinase p38delta
(mapk13), cytochrome c oxidase subunit I (cox1), UDP-
glucose dehydrogenase (ugdh), and myeloid-associated
differentiation marker homolog (myadm). Some tran-
scripts that were up-regulated during vitellogenesis
showed a further increase during maturation, such as
tob1a and nd1.
During ovarian maturation, more genes appeared to be
down-regulated than during vitellogenesis. Among those
transcripts, we found type II Na/Pi cotransport system
pgsP0001G21 FF281897 +1.74 A3KQ53 Novel protein similar to
vertebrate serum/
glucocorticoid regulated
kinase (SGK) [D. rerio]
2E-45 90 (81%) sgk
pgsP0013L08 FF285880 -5.32 Q1L8U4 Novel protein similar to
vertebrate CDC-like kinase
2 (CLK2) [D. rerio]
4E-91 184 (87%) si:ch211-81a5.7
pgsP0028D18 FF290943 -4.80 Q6PHK4 Alanine-glyoxylate
aminotransferase [D. rerio]
2E-64 150 (76%) agxt
pgsP0020C06 FF288101 -3.58 Unknown
pgsP0028H11 FF291026 -3.17 Q7T376 Novel protein similar to
vertebrate CD53 molecule
[D. rerio]
8E-24 92 (55%) zgc:64051
pgsP0027F18 FF290638 -2.23 Unknown
pgsP0028J05 FF291062 -2.22 Q8HXG6 NADH dehydrogenase
[ubiquinone] 1 alpha
subcomplex subunit 11 [B.
taurus]
2E-23 133 (44%) ndufa11
pgsP0008I12 FF284114 -2.12 Q6NY77 Dihydrodipicolinate
synthase-like, mitochondrial
[D. rerio]
2E-66 172 (77%) zgc:77082
pgsP0007P01 FF283914 -2.12 Q7TQ08 Scinderin [Rattus norvegicus] 3E-79 193 (70%) scin
pgsP0009H07 FF284426 -2.10 Q2TAT1 Hypothetical protein
LOC735233 [Xenopus laevis]
1E-24 166 (43%) MGC130935
pgsP0016E15 FF286777 -2.06 Q8UW64 Proteasome subunit beta
type-9 [Oryzias latipes]
3E-41 98 (84%) psmb9a
pgsP0004A14 FF282647 -2.01 Q5JBI1 Carboxypeptidase H
[Paralichthys olivaceus]
4E-58 133 (94%) cph
pgsP0012M12 FF285564 -1.89 Q6ZM96 Coiled-coil domain
containing 90B [D. rerio]
1E-22 107 (61%) ccdc90b
aFold change; b Extent of BLASTX hit aligned region (in amino acids), and percent identity over the aligned region.
Table 1: Transcripts regulated in vitellogenic ovary relative to previtellogenic ovary (Continued)
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Table 2: Transcripts regulated in mature ovary relative to vitellogenic ovary
Clone ID GenBank
accession
FCaUniProtKB/
TrEMBL entry
Swiss-Prot hit BLAST
E-valueb
Length
(% identity)
Gene Symbol
pgsP0012B12 FF285326 +4.54 B6NTA9 Hypothetical protein
BRAFLDRAFT_128798
[Branchiostoma floridae]
2E-14 107 (41%) BRAFLDRAFT_128
798
pgsP0016M08 FF286940 +2.56Q6NYH5ATPase, Na+\/K+
transporting, beta 1a
polypeptide [Danio rerio]
2E-10 39 (76%) atp1b1a
pgsP0013H16 FF285803 +2.51 Q8AY58 Sodium/potassium ATPase
alpha subunit isoform 1
[Fundulus heteroclitus]
2E-118 213 (95%) atp1a1
pgsP0017B15 FF287055 +2.22Q67EX5Alpha-2-macroglobulin
[Sparus aurata]
4E-81 221 (70%) a2m
pgsP0023I10 FF289288 +1.97 Q56V59 Sodium potassium ATPase
beta subunit [Rhabdosargus
sarba]
1E-79 216 (62%) atpb
pgsP0030L16 FF291822 +1.86 Q694W8 Myosin 10 [Xenopus laevis] 5E-32 78 (83%) myo10
pgsP0027A07 FF290524 +1.75 Q9UL19 Retinoic acid receptor
responder protein 3 [Homo
sapiens]
5E-15 108 (40%) rarres3
pgsP0007C11 FF283652 +1.69 Unknown
pgsP0009C03 FF284311 +1.64 Unknown
pgsP0022M06 FF289024 +1.64 Q7ZWB6 Thioredoxin interacting
protein [D. rerio]
2E-108 296 (68%) txnip
pgsP0013B15 FF285673 +1.61 Q7SZR1 Transducer of ERBB2, 1a
[D. rerio]
1E-57 175 (77%) tob1a
pgsP0022B09 FF288784 +1.60 A9ZTB5 Ribosomal protein L36
[Solea senegalensis]
3E-46 94 (100%) rpl36
pgsP0003B14 FF282371 +1.57 Unknown
pgsP0027G11 FF290655 +1.55 Unknown
pgsP0019D13 FF287779 +1.45 Unknown
pgsP0005H14 FF283112 +1.42 Unknown
pgsP0001N17 FF282035 +1.41 Q5FWL4 Extended synaptotagmin-2-
A [X. laevis]
7E-58 144 (74%) e-syt2-a
pgsP0022B24 FF288799 +1.40 Q5NU14 Makorin RING zinc finger
protein 1a [Takifugu
rubripes]
4E-64 224 (50%) mkrn1
pgsP0022J20 FF288970 +1.40 Q9WTY9 Mitogen-activated protein
kinase p38delta [Rattus
norvegicus]
3E-29 127 (49%) mapk13
pgsP0027B20 FF290555 +1.39 Q6P5M5 Novel protein similar to
vertebrate ADP-
ribosylation factor 4 [D.
rerio]
6E-76 174 (86%) zgc:77650
pgsP0021P08 FF288738 +1.39 Q0KJ16 NADH dehydrogenase
subunit 1 [S. senegalensis]
2E-124 280 (88%) nd1
pgsP0017G22 FF287169 +1.38 Q0KJ14 Cytochrome c oxidase
subunit I [S. senegalensis]
3E-84 230 (92%) cox1
pgsP0007O02 FF283892 +1.37 A8WGP7 UDP-glucose
dehydrogenase [D. rerio]
1E-38 104 (75%) ugdh
pgsP0018P15 FF287693 +1.36 Q63ZU3 Myeloid-associated
differentiation marker
homolog [X. laevis]
3E-15 93 (47%) myadm
pgsP0029B12 FF291245 +1.36 Q4QY72 Type II keratin E3-like
protein [S. aurata]
4E-108 217 (92%)
pgsP0006O02 FF283575 +1.32 Unknown
pgsP0027D07 FF290586 -2.49 Unknown
pgsP0017L02 FF287257 -2.31 Q9PTQ8 Type II Na/Pi cotransport
system protein [D. rerio]
7E-32 128 (73%) slc34a2a
pgsP0023F13 FF289226 -2.11 Q5M901 Myosin binding protein H
[X. tropicalis]
1E-46 139 (64%) mybph
pgsP0002P23 FF282343 -1.80 Unknown
pgsP0019F07 FF287815 -1.72 Q5XWB3 Ubiquitous gelsolin [D. rerio] 2E-81 192 (72%) gsna
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protein (slc34a2a) and enzymes such as succinate dehy-
drogenase complex subunit B (sdhb) and fructose-1,6-
bisphosphatase (fbp1), involved in carbohydrate metabo-
lism, and impa3, which was up-regulated during vitello-
genesis. Transcript abundance was also reduced for some
components and regulators of the cytoskeleton, such as
myosin binding protein H (mybph), gelsolin (gsna), and
centaurin delta 2-like (LOC100005008). Other transcripts
were thrombin (f2), monocytic leukemia zinc finger pro-
tein (myst3), 14-3-3 protein epsilon (ywhae), an apolipo-
protein L-like protein (LOC100150119), zona pellucida
C2 (zpc2) and histone H2B (hist1h2be). These mRNAs are
potentially implicated in proteolysis (f2), transcription
and signal transduction regulation (myst3 and ywhae,
respectively), lipid transport (LOC100150119), formation
of the zona radiata (zpc2), and chromatin compaction
(hist1h2be).
Follicular atresia
The comparison of ovaries undergoing follicular atresia vs.
vitellogenic and mature ovaries revealed the up- and
down-regulation of 10 and 16 transcripts, respectively,
and 18 transcripts could be annotated (Table 3). One of
these transcripts (GenBank accession number FF286365),
which was the most highly up-regulated, had sequence
similarity to two unknown predicted proteins from gilt-
head sea bream (Sparus aurata) and the puffer fish Tetrao-
don nigroviridis (BLAST E-values of 9E-08 and 3E-04,
respectively). This EST apparently encoded a full-length
polypeptide which shared 25% identity with a protein
named gastrula-specific embryonic protein 1 found in the
orange-spotted grouper (Epinephelus coioides). The corre-
sponding cDNA clone (pgsP0015C05) was then
sequenced in full-length, and the presence of conserved
motifs in its deduced amino acid sequence was investi-
gated. These analyses, together with a preliminary phylo-
genetic reconstruction, clearly indicated that sole
FF286365 encoded an ortholog of apolipoprotein C-I
(apoc1) (Additional file 2). The nucleotide and amino acid
sequence of this cDNA was deposited in GenBank with
accession number EU835856.
Other transcripts also significantly up-regulated in atretic
ovaries were leukocyte cell-derived chemotaxin 2 (lect),
thrombospondin (thbs), heme-binding protein 2 (hebp2),
apolipoprotein A-I (apoa1), S100-like calcium binding
protein (s100), and enolase (eno3). The S100-encoding
EST (pgsP0020M08) was a full-length cDNA which
allowed further analysis of its deduced amino acid
sequence. The analysis indicated that this transcript
belongs to the S100a10 subgroup of the EF-Hand cal-
cium-binding proteins superfamily (Additional file 3).
Regarding down-regulated transcripts,
BRAFLDRAFT_128798 and a2m showed the strongest
repression in atretic ovaries, which interestingly were
pgsP0009J15 FF284472 -1.71 Q007T0 Succinate dehydrogenase
[ubiquinone] iron-sulfur
subunit, mitochondrial [Sus
scrofa]
1E-08 30 (93%) sdhb
pgsP0023H12 FF289266 -1.71 A2CEC9 Novel protein similar to
centaurin, delta 2 [D. rerio]
5E-36 272 (36%) LOC100005008
pgsP0009A17 FF284280 -1.70 Q28CL4 Inositol monophosphatase 3
[X. tropicalis]
7E-12 66 (71%) impa3
pgsP0024D08 FF289528 -1.59 Unknown
pgsP0022O08 FF289072 -1.53 Q7SXH8 Coagulation factor II
(thrombin) [D. rerio]
4E-88 140 (76%) f2
pgsP0010A15 FF284617 -1.48 Q6J514 Monocytic leukemia zinc
finger protein [D. rerio]
3E-22 96 (90%) myst3
pgsP0029E16 FF291319 -1.46 Q6P8F7 Fructose-1,6-
bisphosphatase [X.
tropicalis]
1E-132 231 (77%) fbp1
pgsP0019H08 FF287862 -1.43 XP_001920639 Similar to apolipoprotein L,
3 [D. rerio]
9E-60 115 (72%) LOC100150119
pgsP0015B20 FF286358 -1.43 Q8AYL1 Zona pellucida C2 [Oryzias
latipes]
9E-69 161 (67%) zpc2
pgsP0018J23 FF287569 -1.40 B2R4S9 Histone 1, H2bc, isoform
CRA_a [H. sapiens]
3E-35 77 (97%) hist1h2be
pgsP0029D07 FF291286 -1.39 P62260 14-3-3 protein epsilon [R.
norvegicus]
2E-20 65 (76%) ywhae
pgsP0030I24 FF291759 -1.39 Unknown
pgsP0013O20 FF285955 -1.38 Unknown
pgsP0020J11 FF288260 -1.36 Unknown
pgsP0023L15 FF289356 -1.33 Unknown
aFold change; b Extent of BLASTX hit aligned region (in amino acids), and percent identity over the aligned region.
Table 2: Transcripts regulated in mature ovary relative to vitellogenic ovary (Continued)
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highly up-regulated in mature ovaries. Other reduced
transcripts were potentially involved in the organization
of Golgi complex, such as C-terminal binding protein 1
(ctbp1) and Golgi membrane protein 1 (golm1), or the tel-
omeric region, such as a novel protein similar to verte-
brate RAP1 interacting factor homolog (rif1), as well as in
transcription and translation regulation, such as elonga-
tion factor 1 alpha (ef1a) and zinc finger protein 576
(znf576). The identity of sole FF288651 and FF282343 as
ef1a and znf576, respectively, was however not conclusive
since the BLAST E-values were low. Cytoplasmic FMR1
interacting protein 1 homolog (cyfip1) and elongation of
Table 3: Transcripts regulated in atretic ovary relative to vitellogenic/mature ovary
Clone ID GenBank
accession
FCaUniProtKB/
TrEMBL entry
Swiss-Prot hit BLAST
E-valueb
Length
(% identity)
Gene Symbol
pgsP0015C05 FF286365 +5.13 B6DUH2 Apolipoprotein C-I
[Hemibarbus mylodon]
1E-08 64 (46%) apoc1
pgsP0002L08 FF282262 +3.36 Unknown
pgsP0007K07 FF283810 +2.90 A8D3J2 Leukocyte cell-derived
chemotaxin 2 [Lates
calcarifer]
3E-53 129 (77%) lect2
pgsP0010O12 FF284909 +2.35 Q28178 Thrombospondin [Bos
taurus]
1E-27 76 (72%) thbs
pgsP0022K21 FF288994 +2.05 B5X719 Heme-binding protein 2
[Salmo salar]
2E-32 85 (40%) hebp2
pgsP0008C17 FF283994 +1.93 Q5KSU5 Apolipoprotein A-I
[Takifugu rubripes]
7E-53 197 (49%) apoa1
pgsP0020M08 FF288324 +1.84 A8HG28 S100-like calcium binding
protein [Epinephelus
coioides]
6E-38 101 (74%) s100
pgsP0005K16 FF283180 +1.62 Q6TH14 Enolase [Danio rerio] 1E-91 190 (92%) eno3
pgsP0019F17 FF287823 +1.62 Unknown
pgsP0013O20 FF285955 +1.49 Unknown
pgsP0012B12 FF285326 -4.01 B6NTA9 Hypothetical protein
BRAFLDRAFT_128798
[Branchiostoma floridae]
2E-14 107 (41%) BRAFLDRAFT_128
798
pgsP0017B15 FF287055 -2.35 Q67EX5 Alpha-2-macroglobulin
[Sparus aurata]
4E-81 221 (70%) a2m
pgsP0025P21 FF290169 -2.08 XP_001519899 Similar to hCG2006161,
partial [Ornithorhynchus
anatinus]
4E-04 54 (51%) LOC100090881
pgsP0006E10 FF283378 -1.93 Q66KL2 C-terminal binding protein
1 [Xenopus tropicalis]
1E-61 178 (78%) ctbp1
pgsP0013I09 FF285819 -1.88 Unknown
pgsP0021L12 FF288651 -1.83 Q4H447 Elongation factor 1 alpha
[Hyla japonica]
0.64 16 (69%) ef1a
pgsP0002C23 FF282126 -1.74 Q90YM8 Cytoplasmic FMR1
interacting protein 1
homolog [D. rerio]
9E-119 227 (91%) cyfip1
pgsP0013H18 FF285805 -1.68 XP_001340467.2 wu:fb21f05 [D. rerio] 2E-23 112 (50%) wu:fb21f05
pgsP0024C11 FF289508 -1.68 Unknown
pgsP0007E08 FF283689 -1.58 B0UY57 Novel protein similar to
vertebrate RAP1
interacting factor homolog
[D. rerio]
7E-38 112 (76%) rif1
pgsP0009C03 FF284311 -1.58 Unknown
pgsP0013B03 FF285662 -1.58 Unknown
pgsP0014A17 FF285991 -1.56 B5X408 Elongation of very long
chain fatty acids protein 1
[S. salar]
1E-24 60 (60%) elovl1b
pgsP0002P23 FF282343 -1.56 B5X202 Zinc finger protein 576 [S.
salar]
1E-04 65 (41%) znf576
pgsP0006D18 FF283364 -1.55 B5X308 Golgi membrane protein 1
[S. salar]
2E-19 55 (60%) golm1
pgsP0030G11 FF291702 -1.55 Unknown
aFold change; b Extent of BLASTX hit aligned region (in amino acids), and percent identity over the aligned region.
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very long chain fatty acids protein 1 (elovl1b), which may
be involved in the control of cell projections and fatty acid
biosynthesis, respectively, were also down-regulated.
Validation of microarray data by real-time qPCR
A number of differentially expressed ESTs (n = 20) in vitel-
logenic, mature and atretic ovaries were further selected to
verify the changes in expression by real-time quantitative
RT-PCR (qPCR). The expression of all twenty genes fol-
lowed the same pattern whether evaluated by microarray
or qPCR (Figure 4). Two genes, tob1a and LOC100090881,
were however an exception. For tob1a, a significant
increase during maturation observed with the microarray
could not be detected (p = 0.78) by qPCR (Figure 4B and
4C), whereas the significant down-regulation of
LOC100090881 during atresia could not be confirmed (p
= 0.88) by qPCR (Figure 4C). All other genes showed in
general a similar relative expression pattern by both
microarray and qPCR, resulting in an overall success rate
of 91% (2 inconsistencies out of 22 comparisons, since
tob1a and BRAFLDRAFT_128798 were significantly regu-
lated both during vitellogenesis and atresia by microarray
analysis). For a2m, however, the FC determined with the
array (2.35) was about 10 times lower than that measured
by qPCR (18.38), which is a known phenomenon
observed in oligo-arrays when background subtraction is
not performed (as in the present study) [32]. Usually, a
two-fold change is considered as the cut-off around which
microarray and qRT-PCR data begin to loose correlation
[33]. Finally, few transcripts that did not show significant
differences in expression levels with the microarray were
also selected for qPCR. These analyses did not show signif-
icant changes in the expression level consistent with the
array data (data not shown).
Differential expression during follicular atresia
Some regulated transcripts in atretic ovaries relative to
mature/vitellogenic ovaries, such as apoc1, apoa1, thbs,
lect2, s100a10, a2m and BRAFLDRAFT_128798, were fur-
ther analyzed by qPCR to investigate how broadly they
might be expressed during ovarian development (Figure
5). For apoc1, thbs, s100a10, a2m and
BRAFLDRAFT_128798, these analyses were also carried
out on manually isolated ovarian follicles at the stages of
vitellogenesis, maturation and atresia. The results con-
firmed that apoc1, apoa1, thbs, lect2 and s1001a10 tran-
scripts were significantly (p < 0.05) up-regulated in atretic
ovaries, whereas a2m and BRAFLDRAFT_128798 tran-
scripts were accumulated in mature ovaries and strongly
down-regulated in atretic ovaries, thus demonstrating the
same expression pattern as that observed with the micro-
array. The data also revealed that apoa1, thbs and lect2
showed relatively high relative expression levels in pre-
vitellogenic ovaries in addition to during atresia.
Gene ontology (GO) analysis of differentially expressed genes in the Senegalese sole ovaryFigure 3
Gene ontology (GO) analysis of differentially
expressed genes in the Senegalese sole ovary. Genes
regulated during vitellogenesis (V), maturation (M) and
atresia (A) were classified according to GO terms biological
process (level 3), cellular component (level 5) and molecular
function (level 3). For each GO term, the number of differen-
tially expressed transcripts detected in the microarray is indi-
cated using a color intensity scale. Red and green are used
for over and under abundance, respectively, whereas black
indicates no change.
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Cellular localization of differentially expressed genes
To determine the cell type-specific expression of repre-
sentative transcripts in the ovary, in situ hybridization was
carried out on ovarian histological sections using specific
antisense riboprobes. For these experiments, we selected
transcripts that were up-regulated in vitellogenic and
mature ovaries, zp3, tob1a, mapk13 and mkrn1 (Figure 6),
or in atretic ovaries, apoc1, s100a10, thbs and lect2 (Figure
7). The zp3 hybridization signal was weakly detected in
the cytoplasm of previtellogenic oocytes, whereas the sig-
nal increased in early cortical alveolus stage oocytes to
subsequently diminished again at later stages (Figure 6A
and 6B). The staining was absent in vitellogenic oocytes as
well as in the surrounding follicle cell layers. A similar
localization pattern was observed for tob1a (Figure 6D
and 6E) and mkrn1 (Figure 6J and 6K), although their
hybridization signals remained visible, but much weaker,
in the cytoplasm during vitellogenesis. A weak mkrn1
staining was also seen in follicular cells of vitellogenic fol-
licles. mapk13 transcripts were exclusively localized in the
surrounding follicular cells of late vitellogenic oocytes,
whereas expression in ovarian follicles at other stages was
not consistently detected (Figure 6G and 6H). For all these
transcripts, sense probes resulted in no signal (Figure 6C,
F, I and 6L).
Real-time qPCR validation of differential expression in vitellogenic vs. previtellogenic ovaries (A), mature vs. vitellogenic ovaries (B), and atretic vs. vitellogenic/mature ovaries (C)Figure 4
Real-time qPCR validation of differential expression in vitellogenic vs. previtellogenic ovaries (A), mature vs.
vitellogenic ovaries (B), and atretic vs. vitellogenic/mature ovaries (C). Bar graphs represent relative fold change
(FC) of selected transcripts obtained with the microarray (black bars) or by qPCR (with bars). The mean FC ± SEM are shown
for each gene. The qPCR and microarray experiments were both conducted on RNA extracted from the same three female
individuals for each developmental stage examined. See Tables 1, 2 and 3 for transcript abbreviations.
Vitellogenic vs. Previtellogenic
Relative fold change (FC)
-8 -4 0 4 8
clk2
tob1a
hsp90b
sepw2a
acaa2
zp3
Mature vs. Vitellogenic
Relative fold change (FC)
-10 -5 0 5 10
LOC100005008
mkrn1
atp1b1a
mapk13
tob1a
Atretic vs. Vitellogenic/Mature
Relative fold change (FC)
-40-30-20 -5 0 5 101520
ctbp1
cyfip1
a2m
LOC100090881
s100a10
lect
thbs
apoa1
apoc1
qPCR
Array
A
B
C
BRAFLDRAFT
_128798
BRAFLDRAFT
_128798
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Expression profile of selected transcripts during Senegalese sole ovarian developmentFigure 5
Expression profile of selected transcripts during Senegalese sole ovarian development. Histograms represent rel-
ative mean expression values ± SEM (n = 3 females) in ovaries (left panels) or in isolated ovarian follicles (right panels) of apol-
ipoprotein C-I (apoc1; A), thrombospondin (thbs; B), S100A10 calcium binding protein (s100a10; C), alpha-2-macroglobulin
(a2m; D), hypothetical protein BRAFLDRAFT_128798 (BRAFLDRAFT_128798; E), apolipoprotein A-I (apoa1; F), and leukocyte
cell-derived chemotaxin 2 (lect2; G). Data were determined by qPCR and normalized to 18S ribosomal protein (18S) expres-
sion. Data with different superscript are statistically significant (p < 0.05).
apoc1
Relative
expression
0
1
2
3
a
aa
b
A
apoa1
Relative
expression
0
4
8
a
aa
b
thbs
Relative
expression
0
1
2
b
aa
c
lect2
Ovarian stage
PVMA
Relative
expression
0
10
20
a
ab
c
bc
s100a10
Relative
expression
0
5
10
15
aab b
c
B
C
D
E
a2m
Relative
expression
0
2
4
6
a
aa
b
BRAFLDRAFT
_128798
Relative
expression
0
2
4
6
aa
a
b
apoc1
Relative
expression
0,0
0,5
1,0
Ovary Isolated follicles
a
a
b
F
G
thbs
Relative
expression
0
3
6
9
12
aa
b
s100a10
Relative
expression
0
2
4
a2m
Relative
expression
0
5
10
15
aa
b
aa
b
BRAFLDRAFT
_128798
Follicle stage
VMA
Relative
expression
0
2
4
aa
b
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In situ hybridization of zona protein 3 (zp3), transducer of ERBB2 (tob1a), mitogen-activated protein kinase p38delta (mpk13), and makorin RING zinc finger protein 1a (mkrn1) transcripts in the Senegalese sole ovaryFigure 6
In situ hybridization of zona protein 3 (zp3), transducer of ERBB2 (tob1a), mitogen-activated protein kinase
p38delta (mpk13), and makorin RING zinc finger protein 1a (mkrn1) transcripts in the Senegalese sole ovary.
Ovarian histological sections were hybridized with antisense digoxigenin-labeled riboprobes for zp3 (A and B), tob1a (D and E),
mpk13 (G and H) and mkrn1 (J and K). The hybridization signal is colored dark-blue to purple. No staining signal was observed
using sense probes (C, F, I and L). gv, germinal vesicle; p, previtellogenic ovarian follicle; ca, ovarian follicles with oocytes at the
cortical-alveolus stage; c, oocyte cytoplasm; a, atretic ovarian follicle; zr, zona radiata; fc, follicular cells. Bars, 50 μm (B, C, D, E,
F, H, I, and K), 100 μm (A, G, J and L).
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In situ hybridization of apolipoprotein C-I (apoc1), S100A10 calcium binding protein (s100a10), thrombospondin (thbs), and leukocyte cell-derived chemotaxin 2 (lect2) transcripts in the Senegalese sole ovaryFigure 7
In situ hybridization of apolipoprotein C-I (apoc1), S100A10 calcium binding protein (s100a10), thrombospon-
din (thbs), and leukocyte cell-derived chemotaxin 2 (lect2) transcripts in the Senegalese sole ovary. Ovarian his-
tological sections were hybridized with antisense digoxigenin-labeled riboprobes for apoc1 (A and B), s100a10 (D and E), thbs
(G and H), and lect2 (J and K). No staining signal was observed using sense probes (C, F, I and L). gc, granulosa cells; tc, theca
cells; vc, vacuole in granulosa cells; y, yolk granules. Other abbreviations as in Fig. 5. Bars, 20 μm (J, K), 50 μm (B, C, D, E, F, G,
H, I and L), 100 μm (A).
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Regarding the transcripts up-regulated during ovarian
atresia, apoc1-specific antisense probes showed an intense
and specific staining in hypertrophied and vacuolized fol-
licular cells of atretic follicles, which was increasing as fol-
licular atresia progressed (Figure 7A and 7B). The same
staining pattern was found for s100a10 (Figure 7D and
7E) and thbs (Figure 7G and 7H). The lect2 transcripts
were found in theca cells of atretic follicles (Figure 7K
inset) but a weaker and more diffuse staining was also
detected in hypertrophied granulosa cells (Figure 7K). Pri-
mary growth oocytes, including cortical alveolus stage
oocytes, also expressed thbs and lect2 mRNAs (Figure 7J),
in agreement with their increased levels previously found
in previtellogenic ovaries by qPCR (Figure 5B and 5G).
Sense probes for all of these transcripts were negative (Fig-
ure 7C, F, I and 7L).
Discussion
The present work has identified a number of differentially
expressed genes in the Senegalese sole ovary that may play
different roles during ovarian follicle growth and matura-
tion, as well as several genes that were not previously
found to be regulated in the teleost ovary. The expression
of some genes specifically in follicular cells of atretic folli-
cles suggest the role of these cells in the activation of
molecular pathways associated with ovarian follicle
atresia which have not been previously recognized in fish.
Microarray performance
In this study, a first-generation Senegalese sole oligonucle-
otide microarray was employed. This array represents the
second high-density microarray available for commercial
flatfish, in addition to that recently published for Atlantic
halibut (Hippoglossus hippoglossus) [34], and has been pre-
viously shown to perform well to detect differences in
gene expression [27].
Transcriptome analysis during ovarian growth, matura-
tion, and follicular atresia in Senegalese sole showed the
differential expression of 118 genes. This number of regu-
lated genes is lower than that reported in similar studies
on trout ovaries by using cDNA microarrays [13,15] or
SSH [17], where changes in the expression of up to 600
genes have been reported. However, our data are more
similar to the expression profiling obtained from the com-
parison of halibut larval stages not very distant during
development (e.g., mouth opening vs. post-hatch) by
using an oligo microarray (44 differentially expressed
genes in Atlantic halibut [34]). The apparent discrepancy
in the overall number of genes regulated during ovarian
development observed in this work with respect to pub-
lished reports in salmonids using cDNA microarrays may
be related to the limited number of unique genes repre-
sented in our array when compared with the salmonid
platforms, or to the fact that oligo arrays are usually more
stringent than cDNA arrays [35]. Another important
aspect that can be considered is that salmonids have syn-
chronous ovaries, unlike the Senegalese sole that has a
group-synchronous ovary, and therefore ovarian follicles
at different developmental stages are present at any time
during the spawning season.
Folliculogenesis and oocyte growth
The period of ovarian vitellogenesis in fish is mainly reg-
ulated by the follicle-stimulating hormone (FSH) and
involves the differentiation and growth of ovarian follicles
mainly by the incorporation of circulating vitellogenins
and very low-density lipoproteins (VLDL) in the oocyte
[36]. Among the genes regulated in Senegalese sole vitell-
ogenic ovaries, the GO terms overrepresented belong to
the metabolic, cellular, biological and developmental
processes categories, and this is consistent with the rapid
rates of growth and development of the ovarian follicles at
this stage. Thus, transcripts possibly related to mitochon-
drial energy production (cox1, cytb, nd3, nd1, acca2), cys-
toskeleton formation and organization (bactin1, actc1l,
tagln, tpm1-1, krt8, myh11), intracellular signaling path-
ways (rhog, sgk), and cell-to-cell and cell-to-matrix interac-
tions (mibp2, thbs-4b, zgc:64051), that may play different
roles during the formation and growth of the ovarian fol-
licles, were over-expressed relative to previtellogenic ova-
ries. Also, as seen in previous ovarian transcriptome
studies in salmonids and tilapia [15,17,37], as well as in
zebrafish fully-grown ovarian follicles [12], zona radiata
(zp3) and hsp90b transcripts were strongly up-regulated.
Vertebrate members of the heat shock protein 90 family
play a post-translational regulatory role within the cell by
interacting with several important cellular signalling mol-
ecules and transcription factors, such as steroid receptors,
modulating their activity [38]. High abundance of hsp90b
transcripts is a common feature of mammalian, fish and
Drosophila ovaries and eggs [12].
During oocyte growth, meiosis is arrested at prophase I,
and it will not proceed until it is activated by the matura-
tion promoting (MPF). The MPF is a cytoplasmic complex
specifically formed during oocyte maturation consisting
of cyclin B and Cd2, a serine-threonine protein kinase
[39]. Studies in zebrafish have shown that immature
oocytes contain Cd2 proteins but not cyclin B, and there-
fore the absence of cyclin B translation is likely the main
mechanism to maintain meiosis arrest [40]. However, the
roles of other cyclins (e.g., cyclin A) and protein kinases
during this process in growing fish oocytes is poorly
known. It is therefore of interest the strong repression of a
transcript similar to vertebrate CDC-like kinase 2
(si:ch211-81a5.7) in sole vitellogenic ovaries.
The vitellogenic period is also characterized by intense
deposition of RNA and proteins, as well as lipids, vitamins
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and hormones, which are necessary during the earliest
steps of embryonic development [41]. Maternal RNAs are
produced endogenously by the oocyte and stored during
oogenesis, and they become usable for embryogenesis
upon egg activation and fertilization, usually sometimes
after a process of activation involving translation or pro-
tein modification [42]. One of these transcripts found in
the Senegalese sole ovary, and highly expressed in pre-
vitellogenic oocytes of vitellogenic ovaries, was tob1a.
Interestingly, the array and qPCR analyses detected the
up-regulation of tob1a only in vitellogenic ovaries, which
may suggest that this transcript starts to be accumulated in
previtellogenic oocytes at the onset of vitellogenesis. tob1a
is also found in trout ovaries [15] and encodes a transcrip-
tional repressor of the BTG/Tob family of antiproliferative
proteins [43]. Tob1a plays an important role during
embryonic dorsoventral patterning in zebrafish by inhib-
iting transcriptional regulation stimulation by β-catenin, a
factor that is essential for the dorsal development of
amphibian and fish embryos [44]. Other transcripts that
may also be stored in oocytes are nrxn1a and mkrn1.
nrxn1a is a member of a family of adhesion molecules
involved in the formation and function of synapses. In
both zebrafish and amphibians [45,46], neurexins genes
are expressed in the ovary and in embryos before the acti-
vation of zygotic transcription, and interestingly, a paren-
tal origin of some of the embryonic neurexin isoforms has
been suggested [45]. The mkrn1, as tob1a, was detected at
high levels in Senegalese sole previtellogenic oocytes of
vitellogenic ovaries. This transcript encodes a putative
ribonucleoprotein with a distinctive array of zinc finger
domains that may play an important role in embryonic
development and neurogenesis, as reported for the
amphibian makorin-2 [47].
The most highly up-regulated transcript in sole vitello-
genic ovaries showed sequence similarity to tilapia seleno-
protein W2a (sepw2a). Selenoproteins are a diverse group
of proteins, with 25 members in humans [48], that con-
tain selenocysteine (Sec) which is incorporated by a rede-
fined in-frame UGA codon and requires the involvement
of a complex translational machinery [49]. Selenopro-
teins with characterized functions are enzymes involved
in redox reactions, such as glutathione peroxidase, thiore-
doxin reductase, and iodothyronine deiodinase, and thus
they are believed to protect the cells from oxidative dam-
age and apoptosis [49]. Expression of selenoproteins in
fish ovaries has also been reported in salmonids [15] and
tilapia [37]. These coincident findings suggest that selene-
proteins might be accumulated in fish follicles for protec-
tion against oxidative stress during folliculogenesis and
oocyte growth. In mammals, selenium stimulates prolifer-
ation of granulosa cells from small follicles and also
potentiates FSH induction of estradiol secretion [50]. In
addition, accumulation of maternal selenoproteins in
ovarian follicles may have a role for antioxidant protec-
tion of the offspring [50].
Oocyte maturation and hydration
During the maturation of follicle-enclosed oocytes, meio-
sis is reinitiated in response to progestagens produced by
the follicular cells after luteinizing hormone (LH) stimu-
lation [39]. During this process, the germinal vesicle
migrates towards the oocyte periphery, the nuclear enve-
lope breaks down, the first meiotic division occurs, and
the chromosomes proceed to second meiotic metaphase
where they arrest; at this point, the oocyte will ovulate and
becomes an egg [51]. Oocyte maturation is also accompa-
nied by important changes in the cytoarchitecture and
function of the ovarian follicle, since steroidogenic path-
ways in granulosa cells are switched from estrogen to pro-
gestagen production, intercellular communication
oocyte-granulosa cells is resumed, and the zona radiata
becomes more compacted [39,52,53]. At this stage, ovar-
ian transcripts associated with the regulation of intracellu-
lar signalling pathways (such as mapk13 in follicular cells,
and ywhae), and cystoskeleton (mybph, gsna, myo10,
LOC100005008), as well as in the assembly of the nucle-
osome (hist1h2be, myst3), were significantly regulated in
Senegalese sole, which is consistent with the important
nuclear and cytoplasmic changes occurring in the ovarian
follicle during oocyte maturation.
One transcript overexpressed in mature ovaries was e-syt2-
a which is related to the synaptotagmin family of vesicle
proteins that are believed to function as calcium sensors
for vesicle exocytosis at synapsis [54]. It is known that fish
oocytes, as the eggs of most organisms, suffer a transient
elevation of intracellular free Ca2+ following fertilization,
an event that triggers a series of biochemical pathways
required for the block of polyspermy, activation of metab-
olism, re-entry into the cell cycle, and execution of the
developmental program [55]. One of the earliest
responses to the C2+ wave in the oocyte is the cortical alve-
oli exocytosis wich produces an elevation of the chorion
and the separation of the egg surface [55]. Studies in
mammalian eggs suggest that the release of cortical gran-
ules in mature eggs is dependent upon calcium-depend-
ent synaptosome-associated protein 25 (SNAP-25) which
might be regulated by binding to Ca2+-dependent synap-
totagmins as it occurs in neurons [56,57]. These observa-
tions therefore suggest that the induction of e-syst2-a
transcripts in sole mature ovaries might be part of the
molecular pathways activated in the oocyte in preparation
for fertilization.
In marine teleost that produce buoyant (pelagic) eggs,
such as the Senegalese sole, oocytes continue to enlarge
during maturation owing to hydration [58]. The hydroly-
sis of the oocyte yolk proteins by the lysosomal proteases
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cathepsin B (Catb) and/or cathepsin L (Catl) that occurs
during maturation results in the increase of free amino
acids in the ooplasm, incrementing the osmotic pressure
of the oocyte, and hence facilitating water uptake medi-
ated by aquaporin-1b (Aqp1b) [58-60]. Current evidence
in some fish species suggest that both Catb and Aqp1b are
regulated post-translationally rather than transcription-
ally in oocytes during meiotic maturation [61-63].
Accordingly, in the present study, we did not detect
changes in the expression levels of aqp1b and catl, as well
as of other cathepsins and proteases, for which specific
oligos were present in the microarray. However, probes
for catb were lacking, and therefore the regulation of this
transcript during oocyte maturation in Senegalese sole can
not be ruled out. Nevertheless, the up-regulation of
zgc:77650, a transcript showing sequence similarity to a
GTP-binding protein (ADP-ribosylation factor 4)
involved in protein trafficking that may modulate vesicle
budding and uncoating within the Golgi apparatus [64],
it is of interest. This observation may indicate a role of ves-
icle trafficking during oocyte maturation that might be
important, for instance, in the delivery of lysosomal
cathepsin to yolk granules or in the control of Aqp1b
shuttling into the oocyte plasma membrane. These mech-
anisms are however not yet elucidated in fish oocytes and
need to be investigated in the future.
The accumulation of inorganic ions in oocytes undergo-
ing maturation, mainly K+ and Na+, may account for
about 50% of the final osmotic pressure, and therefore it
is considered as an additional mechanism mediating fish
oocyte hydration [59]. Interestingly, we found that three
of the most highly up-regulated transcripts in mature ova-
ries corresponded to Na+-K+-ATPase subunits (atp1b1a,
atp1a1 and atpb), whereas the solute carrier slc34a2a, also
known as the type II Na+/Pi cotransporter, was one of the
most down-regulated transcripts. In trout, slc26 (Na+-
independent chloride/iodide transporter) and aquaporin-
4 (aqp4) were found to be overexpressed in ovarian tissue
at maturation [13]. The causes for the different ion and
water transport-associated transcripts regulated during
oocyte maturation in sole and trout are intriguing,
although the fact that trout oocytes exhibit a much lower
hydration than Senegalese sole oocytes, resulting in the
production of demersal eggs, may be one of the reasons.
In vertebrates, retinoic acid regulates the transcription of
many genes involved in embryonic development and
germ cell differentiation through binding to nuclear
receptors (retinoic acid receptors, RARs and retinoid ×
receptors, RXRs) [65]. In mammals, retinoic acid also
affects the acquisition of developmental competence of
oocytes and the steroidogenesis of ovarian follicle cells
[66,67]. In fish, recent studies in trout suggest that follic-
ular cells express several genes associated with retinoid
and carotenoid metabolism indicating the presence of an
additional pathway to provide retinoids to the oocyte in
addition to the receptor mediated uptake of lipoproteins
[68]. A transcript related to this system was induced in
Senegalese sole mature ovaries, rarres3, which shows
sequence similarity to the tazarotene (synthetic, topical
retinoid)-induced gene 3 (TIG3; Retinoic Acid Receptor
Responder 3). This gene encodes a growth regulator that
possibly mediates some of the growth suppressive effects
of retinoids [69]. Although the cell localization of rarres3
in the ovarian follicle was not determined here its overex-
pression in the mature sole ovary may indicate that retin-
oids could play an additional paracrine role by affecting
the expression of suppressor/growth regulatory pathways
in the ovary.
Another gene that could play a paracrine role in the sole
ovary at the maturation stage is the proteinase inhibitor
a2m. In the mammalian ovary, a2m modulates the actions
of growth factors and cytokines, and recent works suggest
that it may have autocrine or paracrine roles in granulosa
cells potentially important for regulation of estradiol pro-
duction and development of dominant follicles [70].
Interestingly, we observed that the expression of this
mRNA was specifically up-regulated during maturation,
whereas in atretic ovaries its induction was prevented. In
contrast, down-regulation of a2m was reported in trout
precocious mature ovaries [15].
Finally, similarly to that described in trout ovaries at the
time of meiosis resumption, we found high levels of coag-
ulation factor II (thrombin I), f2, in mature ovaries. In
trout, overexpression of the coagulation factor V (cf5) has
been speculated to be related with the prevention of
bleeding from ruptured ovarian follicles at the time of
ovulation [13]. Although in Senegalese sole we found the
induction of an apparently different coagulation factor, a
similar scenario may be considered to occur in the flatfish
ovary.
Follicular atresia
Ovarian atresia is a common phenomenon in teleosts
under both natural and experimental conditions during
which a number of vitellogenic ovarian follicles fail to
complete maturation and ovulation, degenerate and are
eventually reabsorbed [19,20]. Ovarian follicle atresia in
fish seems not to be mediated by apoptosis in the follicu-
lar cells, unlike in mammals, and thus this process
appears to be different than the post-ovulatory follicular
reabsorption mechanism, which is apparently mediated
by apoptosis [22,71,72]. Therefore, apoptosis may not be
relevant at the onset of atresia, although it may contribute
to a more efficient removal of atretic follicles during ovar-
ian follicular regression after spawning [73,74]. The ovar-
ian transcriptome analysis in Senegalese sole might
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support this view since the expression levels of none of the
apoptosis-related genes that were represented in the
microarray, identified by GO annotation, changed in
atretic ovaries related to vitellogenic/mature ovaries.
In Senegalese sole, as in other teleosts [19,20], the process
of ovarian follicle atresia and resorption is preceded by
marked morphological changes in both the oocyte and
follicular cells, such as the disintegration of the oocyte ger-
minal vesicle and of other cytoplasmic organelles, the
fragmentation of the zona radiata, and the hypertrophy of
the granulosa cells. These cells become phagocytic with
digestive vacuoles and incorporate and digest the oocyte
yolk as well as other oocyte components and organelles,
and they may also secrete enzymes which digest the yolk
[19,20,73,75]. In atretic ovaries, two of the up-regulated
genes corresponded to apoa1 and apoc1, which are part of
chylomicrons, very low density lipoproteins (VLDL) and
high density lipoproteins (HDL) involved in lipid trans-
port in the bloodstream [76]. In addition, we found
reduced elovl1b transcripts possibly involved in the con-
trol of the synthesis of very long chain fatty acids and
sphingolipids in the ovary [77]. Studies in rainbow trout
have shown that during the course of follicular atresia
there is a massive transfer of the oocyte yolk proteins, and
possibly lipids, into the bloodstream combined with HDL
[78], as a result of the ingestion and digestion of the yolk
by the follicular cells [19,20]. The finding of high apoa1
and apoc1 transcript levels in hypertrophied follicular cells
(at least for apoc1) of Senegalese sole, together with that
recently reported for the fatty acid-binding protein 11
(fap11) [79], provides additional evidence for this mecha-
nism in teleosts. However, the nature of the numerous
invasive apoc1- and fabp11-expressing cells (theca or gran-
ulosa cells) remains to be clearly established. Neverthe-
less, these data suggest the importance of lipid-metabolic
processes during follicular atresia in fish [79], which may
have evolved to facilitate the redistribution of energy-rich
yolk materials from oocytes that fail to develop properly
[80].
In humans, Apoc-I is primarily expressed in the liver but
also in the lung, skin, spleen, adipose tissue, and brain
[81]. ApoC-I can interact with lipid surfaces and play an
important role in controlling plasma lipoprotein metabo-
lism by the regulation of several enzymes, such as lipopro-
tein lipase or phospholipase A2 [76]. The expression of
ApoC-I in the mammalian ovary has not been reported,
unlike that of ApoA-I and ApoE which are expressed by
luteinizing granulosa cells and theca cells, respectively, of
atretic follicles [82,83]. Intraovarian ApoE controls theca
cell production of androgens as well as limiting the size of
the theca cell compartment [83]. Teleost ovary and
embryos also express an ApoC-I ortholog as it has been
recently shown [18,84,85] and confirmed in the present
study, although its function is largely unknown. In the
embryo, apoc1 is localized in the yolk syncytial layer [85],
along with apoe, apoa1 and apo14 [86,87], suggesting its
role in the nutrition of the developing embryo through
the synthesis and secretion of apolipoproteins and lipo-
proteins. Therefore, the expression of apoc1 in follicular
cells of fish atretic follicles, which has not been previously
reported, may have a similar role for the resorbption of
lipids and lipoproteins stored in the oocyte. Interestingly,
trout eggs obtained by hormonal induction, which result
in alevins with a high percentage of morphological abnor-
malities at the yolk-sac resorption stage, also show a dra-
matic increase of apoc1 [18]. Altogether, these findings
suggest that ApoC-I could be a useful marker to identify
factors involved in premature ovarian regression and
abnormal embryo development in cultured fish. It is
worth noting that in humans it has been recently pro-
posed that serum ApoC-I may be useful for early demon-
stration of metabolic abnormality in women with
polycystic ovary syndrome [88].
In histological sections of Senegalese sole ovarian follicles
at advanced atresia, we observed the presence of blood
cells such as erythrocytes and leukocytes, possibly derived
from the ovarian stroma and/or the theca, which invaded
the degenerating oocyte. The presence of granulocytes
(polymorphonuclear leukocytes) in atretic follicles is
reported in other fish species [21,89,90], and suggest a
relationship between follicular regression and immune
cells [73]. The specific function of immune cells (eosi-
nophilic granulocytes and macrophages) during follicular
atresia in fish is not well known, although it has been pro-
posed that they may act synergistically with follicular cells
in the resorption of the oocyte by releasing their granules
containing lytic enzymes [89]. In the mammalian ovary,
the leukocyte-ovarian cell interactions through the release
of chemokines is believed to play an important role for
leukocyte recruitment and activation during follicular
atresia, ovulation and luteal function [91]. In teleosts,
however, the molecular mechanisms mediating the inva-
sion of immune cells in atretic follicles are largely
unknown. In the present study, we noted high lect2
expression levels in theca cells of atretic follicles, a tran-
script related to mammalian Lect2 which encodes a pro-
tein with chemotactic properties for human neutrophils
[92]. This observation may provide evidence for the pres-
ence of a chemotaxin-mediated mechanism for leukocyte
accumulation in fish follicles at advanced atresia, simi-
larly to that occurs during the formation of the corpus
luteum in the mammalian ovary [91]. However, whereas
a number of different chemokines have been found in the
mammalian ovary [91], ovarian expression of Lect2 has
not been yet reported, and therefore the structural and
functional relationships of Senegalese sole lect2 with
other ovarian chemokines requires further investigation.
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In addition to lect2, we also found high expression levels
of the calcium binding protein-encoding gene s100a10 in
atretic follicles. In mammals, S100 proteins are localized
in the cytoplasm and/or nucleus of a wide range of cells
and are involved in the regulation of a number of cellular
processes such as cell cycle progression and differentiation
[93]. In the ovary, S100a10 plays an antiapoptotic func-
tion by binding the Bcl-xL/Bcl-2-associated death pro-
moter and its expression in granulosa cells is stimulated
by gonadotropins and follicle survival factors, including
the epidermal growth factor, the basic fibroblast growth
factor, and interleukin-1β [94]. It is known, however, that
some S100 proteins can also be released into the extracel-
lular space and act as chemoattractants for leukocytes or
activators of macrophages [93]. Therefore, the high
expression levels of s100a10 in sole atretic follicles may
play a dual function, to protect follicular cells from apop-
tosis during atresia and to act as chemoattractant for leu-
kocytes and macrophages. In support of this hypothesis is
the reported down-regulation of an S100 homologue in
trout post-ovulatory follicles [95], which based on our
phylogenetic analysis is in fact an s100a10 ortholog.
The mammalian ovary is distinctive in that it is a tissue
that undergoes physiological angiogenesis, in which
blood vessels are programmed to develop and regress in a
cyclic manner [96]. This mechanism is tightly regulated by
pro- and antiangiogenic factors such as the members of
the thrombospondin (TSP) family TSP-1 and TSP-2,
which are among the naturally occurring inhibitors of
angiogenesis. These secreted proteins are expressed by
granulosa cells of atretic follicles and in the corpus luteum
after ovulation in rats and primates [96,97], suggesting
that they may be involved in the cessation of angiogenesis
in follicles undergoing atresia [96]. Our microarray analy-
sis revealed that a thrombospondin isoform (thbs), dis-
tinct from thbs4b which was overexpressed in vitellogenic
ovaries, was up-regulated in Senegalese sole follicular cells
during follicular atresia. thbs was similar to mammalian
TSP-1 and TSP-2, which indicates that the inhibition of
angiogenesis may be an important mechanism regulating
atresia in both mammalian and fish ovarian follicles.
However, primary growth oocytes also expressed thbs
which may point to an additional role of this protein dur-
ing folliculogenesis and/or as a maternal molecule for
early embryonic development.
In contrast to apoptosis, Wood and Van Der Kraak [22,80]
proposed that proteolytic degradation of yolk proteins,
mediated by the differential activation of Catl in the
oocyte, may be the initial event leading to follicular atresia
in fish. In the present study we did not detect changes in
the expression of catl transcripts in atretic ovaries, which
may suggest a potential post-transcriptional regulation of
this protease during ovarian follicle atresia as discussed
earlier. In any event, the specific oocyte mechanisms
involved in the regulation of protease activity during
atresia in fish, as well as the origin of the signals that pre-
sumably activate this system, are still largely unknown
and warrant further investigation.
Conclusion
The present study has contributed to identify differences
in gene expression during ovarian development in Sen-
egalese sole despite that, at this point, the microarray plat-
form employed was not ovary-specific and contained a
limited number of represented genes. Some of the genes
identified were not described before in the teleost ovary,
and therefore the present data provide a basis for future
studies on their regulation and function. Particularly,
determination of the cell-type specific localization of
some transcripts suggest the involvement of follicular cells
in yolk resorbption, chemotaxin-mediated leukocyte
migration, angiogenesis and prevention of apoptosis dur-
ing ovarian atresia. These observations also indicate that
some of the transcripts, such as e-syt2-a, apoc1 and
s100a10, may be useful markers contributing to the iden-
tification of factors involved in the acquisition of egg fer-
tilization competence and follicular regression. Clearly,
however, further experimental studies will be necessary to
determine when these mRNAs are translated as well as the
physiological functions of the encoded proteins. The
future increase in the sequencing of the Senegalese sole
ovarian transcriptome will enhance our knowledge on the
molecular pathways involved in oogenesis, and will facil-
itate the comparative genomic analysis of gonad develop-
ment in teleosts.
Methods
Animals and biological samples
Adult Senegalese sole (1219 ± 90g) F1 generation were
maintained as previously described [24]. Females (n = 3-
5) were sacrificed at different times during the annual
reproductive cycle, corresponding to different folliculo-
genesis stages [24,28]. Thus, females with previtellogenic
and vitellogenic ovaries were collected during July and
November, respectively. Maturing and mature ovaries
were collected from females treated with intramuscular
injection of 5 μg/kg GnRHa and killed 24-48 h later as
described [24]. Finally, atretic ovaries were obtained in
April-May, as well as from females treated with GnRHa. At
all sampling times, fish were sedated with 500 ppm phe-
noxyethanol, killed by decapitation, and the body and
gonads of each animal weighed to calculate the GSI
(gonad weight/body weight × 100). Pieces of the ovaries
were immediately removed and placed in Petri dishes
containing 10 ml of 75% Leibovitz L-15 medium with L-
glutamine (Sigma) and 100 mg gentamicin/ml, pH 7.5
[60]. Follicle-enclosed oocytes at vitellogenesis, matura-
tion and atresia were manually isolated from the rest of
the ovary using watchmaker forceps. One piece of the
ovary, as well as the isolated ovarian follicles, were deep-
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frozen in liquid nitrogen and stored at -80°C until RNA
extraction. Two additional pieces of the gonad, adjacent
to the piece sampled for RNA extraction, were fixed in
modified Bouin solution (75% picric acid and 25% for-
malin) for histological analysis, or in 4% paraformalde-
hyde (PFA) for in situ hybridization. Procedures relating
to the care and use of animals were approved by the Ethics
Committee from Institut de Recerca i Tecnologia Agroali-
mentàries (IRTA, Spain) in accordance with the Guiding
Principles for the Care and Use of Laboratory Animals.
Histological analysis
Ovaries fixed in Bouin solution for 3-4 h were dehydrated,
embedded in paraplast, sectioned at 5 μm, and stained
with hematoxilin-eosin. Alternatively, fixed gonads were
embedded in glycol methacrylate resin (Technovit 7100,
Heraeus Kultzer), sectioned at 3 μm and stained with
methylene blue/azure II/basic fuchsin. The percentages of
previtellogenic, early vitellogenic, vitellogenic, mature
and atretic oocytes were calculated by counting 100-150
total ovarian follicles in at least three different histological
sections from the same ovary, as previously described
[24].
RNA extraction, microarray hybridization and analysis
Total RNA was extracted from ovaries at previtellogenesis,
vitellogenesis, maturation and atresia, determined by his-
tological examination, using the RNeasy extraction kit
(Qiagen) and treated with DNAse following the manufac-
turer's instructions. For each ovarian developmental stage,
RNA was extracted from the ovary of three different
females. The quality and concentration of the RNA was
analyzed using the Agilent 2100 bioanalyzer and Nano-
Drop™ ND-1000 (Thermo Scientific). Samples with RNA
integrity number (RIN; [98]) < 6.0 were discarded. Total
RNA (0.5 μg) from each of the twelve samples was ampli-
fied and labelled with fluorescent cyanine dyes, Cy3 or
Cy5, using the Eberwein mRNA amplification procedure
[99] employing the MessageAmp™ aRNA amplification kit
from Ambion (Applied Biosystems) following the manu-
facturer's instructions with minor modifications. The Cy3-
and Cy5-labelled aRNAs synthesized from RNAs origi-
nated from two different ovarian stages were mixed in
equal amounts and hybridized to an oligonucleotide
microarray representing 5,087 Senegalese sole unigenes
[27]. These 60-mer probes were designed against the
3'end sequences of Senegalese sole expressed sequence
tags (ESTs) [27]. Each hybridization was done in triplicate
with aRNA from three different females, so three different
biological replicates per ovarian stage were analyzed. To
estimate the rate of false-positive expression, a self-to-self
hybridization was carried out, in which total RNA from
two different aliquots of previtellogenic ovaries were used
to produce either Cy3 or Cy5 labelled aRNA. Hybridiza-
tions were carried out for 17 h at 60°C using Agilent's gas-
kets G2534-60002, G2534A hybridization chambers, and
DNA Hybridization Oven G2545A, according to the man-
ufacturer's instructions.
Microarray raw data were obtained using Agilent's DNA
Microarray Scanner G2505B and Feature Extraction soft-
ware (v10.1). The raw fluorescence intensity data were
processed using the Polyphemus™ software (Oryzon
Genomics), which includes spatial data compensation,
non-significant expressed data filtering, and data normal-
ization. Data normalization was carried out by an
improved version of the nonlinear Q-splines normaliza-
tion method [100], enhanced with robust regression tech-
niques. Normalized and log-transformed data were used
to calculate the FC values. Differential expression was
assessed with Polyphemus™ analyzing biological repli-
cates based on repeated experiments using robust statistics
on average technical replicates removing the outlier
points (caused by dust or array imperfection). The p-values
were calculated based on the absolute value of the regular-
ized t-statistic [101], which uses a Bayesian framework to
derive the algorithm, using internal replicated controls to
assess the minimum technical variability of the process. A
p-value < 0.01 was considered significant. Cut-offs for sig-
nificant changes were always greater than the inherent
experimental variation as assessed by the FC of internal
controls and/or self-to-self hybridizations. The microarray
data have been deposited in NCBI's [102] Gene Expres-
sion Omnibus (GEO) [103] and are accessible through
GEO Series accession number GSE17337 [104].
EST Annotation, gene ontology and amino acid sequence
analysis
Annotation of ESTs and extraction of GO terms to each
obtained hit using existing annotations was done using
the BLAST2GO v1 program [31] as previously described
[27]. ESTs that were considered significantly expressed
after microarray analysis, but that they could not be ini-
tially annotated, were sequenced from the 5' and 3' ends
using an ABI PRISM 377 DNA analyzer (Applied Biosys-
tems). Basic local alignment search tool (BLAST) software
[102], employing the 5'end nucleotide sequences or the
consensus 5' and 3' end sequence, was used to re-annotate
these ESTs. For some of these ESTs, further annotation
required the search for conserved motifs using PROSITE
[105] and multiple amino acid sequence alignments
using ClustalW [106]. The neighbor-joining (NJ) phyloge-
netic analysis [107] of the amino acid alignments was also
carried out based on mean character distances. The
robustness of the phylogenetic tree was tested by boot-
strap analysis [108] with 1000 repetitions.
Real-time quantitative RT-PCR
The relative mRNA levels of selected ESTs during ovarian
development were determined by qPCR on the same RNA
samples than those employed for the microarray. Total RNA
from the ovary was extracted with the RNeasy Mini kit (Qia-
BMC Genomics 2009, 10:434 http://www.biomedcentral.com/1471-2164/10/434
Page 22 of 25
(page number not for citation purposes)
gen), and treated with DNase I using the RNase-Free DNase
kit (Qiagen). An aliquot of the RNA (0.5 μg) was reverse-
transcribed using 20 IU of AMV RT (Stratagene), 0.5 μM
oligo(dT)12-18 (Invitrogen) and 1 mM dNTPs for 1.5 h at
50°C. Real-time qPCR amplifications were performed in a
final volume of 20 μl with 10 μl SYBR® Green qPCR master
mix (Applied Biosystems), 2 μl diluted (1:5) cDNA, and 0.5
μM of each primer (see Additional file 4). The sequences
were amplified in duplicate for each sample on 384-well
plates using the ABI PRISM 7900HT sequence detection sys-
tem (Applied Biosystems). The amplification protocol was as
follows: an initial denaturation and activation step at 50°C
for 2 min, and 95°C for 10 min, followed by 40 cycles of
95°C for 15 s and 63°C for 1 min. After the amplification
phase, a temperature-determinating dissociation step was
carried out at 95°C for 15 s, 60°C for 15 s, and 95°C for 15
s. For normalization of cDNA loading, all samples were run
in parallel using 18S ribosomal protein (18S) as reference
gene, since its expression between experimental samples did
not show significant differences (data not shown). Negative
control samples were also run in which the template was not
added. To estimate efficiencies, a standard curve was gener-
ated for each primer pair from 10-fold serial dilutions (from
100 to 0.01 ng) of a pool of first-stranded cDNA template
from all samples. Standard curves represented the cycle
threshold (Ct) value as a function of the logarithm of the
number of copies generated, defined arbitrarily as one copy
for the most diluted standard. All calibration curves exhib-
ited correlation coefficients higher than 0.97, and the corre-
sponding real-time PCR efficiencies were above 99%. Values
of relative expression in ovaries at different developmental
stage were statistically analyzed by one-way ANOVA. The sig-
nificance level was set at 0.05.
In situ hybridization
Samples of ovaries were fixed in 4% paraformaldehyde for
16-20 h at 4°C, and subsequently dehydrated and embed-
ded in Paraplast (Sigma). In situ hybridization on 7-μm
sections was carried out with digoxigenin-alkaline phos-
phatase (DIG-AP) incorporated cRNA probes as previ-
ously described [109]. DIG-AP riboprobes were
synthesized with T3 and T7 RNA polymerases using the
DIG RNA Labeling Kit (Roche). The DIG-labeled probes
were detected as previously described [105], and the
resulting dark blue to purple color indicated localization
of the transcripts. Sections were examined and photo-
graphed with a Leica DMLB light microscope.
Authors' contributions
ATS performed RNA sample preparation and quality control
for microarray hibridization, real-time PCR, bioinformatic
analyses together with JL, and drafted the manuscript. MJA
and EA led the hormonal induction experiments and mor-
phometric data analysis, and carried out the histological
studies. FC performed the in situ hybridization experiments.
JC conceived and coordinated the study, participated in the
design of the experiments and analyses of data, and was in
charge of writing the final version of the manuscript. All
authors read and approved the final manuscript.
Additional material
Additional file 1
Microarray hybridizations. Scatter plot of the signal intensities in the
Cy3 and Cy5 channel of replica analysis of self-to-self and differential
gene expression experiments (vitellogenic vs. previtellogenic ovaries,
mature vs. vitellogenic ovaries, and atretic vs. vitellogenic/mature ova-
ries). In each scatter plot, the green and blue lines parallel to the black
diagonal line represent the ± 3
σ
limits of the data from control and Solea
senegalensis specific oligos, respectively. The histograms of the distribu-
tion of fold changes (FC) as log2(S/C) for control and S. senegalensis
specific oligos in each experiment are shown on the right. In these panels,
the green and blue curves represent the ± 3
σ
limits on the data from con-
trol and S. senegalensis specific oligos, respectively.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2164-10-434-S1.pdf]
Additional file 2
Identification of clone pgsP0015C05 (GenBank accession number
FF286365) as Senegalese sole apolipoprotein C-I (ApoC-I). (A)
Amino acid sequence alignment of human, zebrafish and trout ApoC-I
with Senegalese sole FF286365 and sea bream AAT45249 (putative
ApoC-I). In the upper line, the protein family (pfam) database domain
no. 04691 (ApoC-I) is shown in bold letters. In red color, the consensus
(cons) sequence between pfam04691 and each of the amino acid
sequences is indicated. The asterisks at the bottom indicate fully-conserved
residues. (B) Fifty percent majority-rule bootstrap consensus phylogenetic
tree of teleost ApoC-I reconstructed with the NJ method (1000 replica-
tions) based on a mean (uncorrected) character distance matrix. Only
nodes with > 50% bootstrap support are indicated. The GenBank acces-
sion number of the amino acid sequences is indicated for each species.
Scale bar indicates the number of amino acid substitution per site.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2164-10-434-S2.pdf]
Additional file 3
Amino acid sequence analysis of Senegalese sole S100 calcium-bind-
ing protein. (A) Amino acid alignment of sole S100 with S100 proteins
of the A1, A10 and B subgroups from human and other teleosts. In the
upper part is indicated the typical structure of mammalian S100 proteins
formed by four helices (Helix I-IV) separated by a S100 EF hand calcium-
binding domain, a hinge and a canonical EF hand calcium-binding
domain. Conserved residues in most of the sequences are shaded in bold,
identical residues are indicated by asterisks at the bottom, and conserved
amino acid substitutions and substitutions with similar amino acids are
indicated by double or single dots, respectively. Conserved residues in most
of the members of the S100A10 subgroup are shaded in magenta. (B)
Phylogenetic tree of human and teleost S100 proteins reconstructed with
the NJ method (1000 replications) based on a mean (uncorrected) char-
acter distance matrix. Only sequences belonging to the A1, A10 and B
subgroups are employed for the analysis. Nodes with > 60% bootstrap sup-
port are indicated. The GenBank accession number of the amino acid
sequences is indicated for each species. Scale bar indicates the number of
amino acid substitution per site.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2164-10-434-S3.pdf]
BMC Genomics 2009, 10:434 http://www.biomedcentral.com/1471-2164/10/434
Page 23 of 25
(page number not for citation purposes)
Acknowledgements
We thank staff of Oryzon Genomics (Dr. Elisabet Rosell, Dr. Tamara Maes,
Olga Durany and Francesc Subirada) for their assistance during microarray
hybridization and bioinformatic analysis. This work was supported by
Genome Spain and Genome Canada within the framework of the interna-
tional consortium Pleurogene™ coordinated by JC. MJA was supported by
a predoctoral fellowship from the Instituto Nacional de Investigación y Tec-
nología Agraria y Alimentaria (INIA, Spain), and by a postdoctoral fellow-
ship from Juan de la Cierva Programme (Spanish Ministry of Education and
Science).
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