Open Access

Microarray-based analysis of fish egg quality after natural or controlled ovulation

BMC Genomics20078:55

DOI: 10.1186/1471-2164-8-55

Received: 21 October 2006

Accepted: 21 February 2007

Published: 21 February 2007

Abstract

Background

The preservation of fish egg quality after ovulation-control protocols is a major issue for the development of specific biotechnological processes (e.g. nuclear transfer). Depending on the species, it is often necessary to control the timing of ovulation or induce the ovulatory process. The hormonal or photoperiodic control of ovulation can induce specific egg quality defects that have been thoroughly studied. In contrast, the impact on the egg transcriptome as a result of these manipulations has received far less attention. Furthermore, the relationship between the mRNA abundance of maternally-inherited mRNAs and the developmental potential of the egg has never benefited from genome-wide studies. Thus, the present study aimed at studying the rainbow trout (Oncorhynchus mykiss) egg transcriptome after natural or controlled ovulation using 9152-cDNA microarrays.

Results

The analysis of egg transcriptome after natural or controlled ovulation led to the identification of 26 genes. The expression patterns of 17 of those genes were monitored by real-time PCR. We observed that the control of ovulation by both hormonal induction and photoperiod manipulation induced significant changes in the egg mRNA abundance of specific genes. A dramatic increase of Apolipoprotein C1 (APOC1) and tyrosine protein kinase HCK was observed in the eggs when a hormonal induction of ovulation was performed. In addition, both microarray and real-time PCR analyses showed that prohibitin 2 (PHB2) egg mRNA abundance was negatively correlated with developmental success.

Conclusion

First, we showed, for the first time in fish, that the control of ovulation using either a hormonal induction or a manipulated photoperiod can induce differences in the egg mRNA abundance of specific genes. While the impact of these modifications on subsequent embryonic development is unknown, our observations clearly show that the egg transcriptome is affected by an artificial induction of ovulation.

Second, we showed that the egg mRNA abundance of prohibitin 2 was reflective of the developmental potential of the egg.

Finally, the identity and ontology of identified genes provided significant hints that could result in a better understanding of the mechanisms associated with each type of ovulation control (i.e. hormonal, photoperiodic), and in the identification of conserved mechanisms triggering the loss of egg developmental potential.

Background

Fish egg quality can be defined as the ability of the egg to be fertilized and subsequently develop into a normal embryo. The egg's potential to produce a viable and normal embryo can be affected by many environmental and biological factors acting at various steps of the oogenetic process (see [1, 2] for review). The determinism of egg quality has also been shown to be under the influence of genetic factors [35]. While the effects of many experimental factors have been studied, the mechanisms by which they trigger egg quality losses are far less documented. Yolk composition as a result of a specific diet has been intensively studied in several fish species in relationship with egg developmental capacities [68]. Hormones of maternal origin supplied to the embryo by the egg also have a significant effect on embryonic development as shown by several studies [9]. In contrast, the putative role of non-yolky cytoplasmic components accumulated during oogenesis, such as structural and regulatory proteins, cortical alveoli content and messenger RNAs (mRNAs), has received far less attention [1]. Nevertheless, maternal mRNAs that accumulate in the oocyte during oogenesis are essential for early embryonic development [10, 11]. Like in other animals, some maternal mRNAs are involved in embryonic germ cells formation in fish [12], but other oocyte mRNAs, such as those involved in growth regulation, could be necessary to ensure a normal early development [13]. Thus, in bovine two-cell embryos, a relationship between embryonic developmental competence, assessed in terms of time of first cleavage, and the expression of IGF1 mRNA was reported [14]. In addition, other studies showed a relationship between variation of maternal RNA polyadenylation levels and developmental competence of mammalian oocytes, thus pointing out a relationship between maternal mRNA stability and embryonic developmental capacities [15]. In fish, the possibility that specific oocyte mRNAs might be affected when egg quality is experimentally decreased has been seriously suggested by a previous work dealing with the effect of egg post-ovulatory ageing on the mRNA levels of many genes (~40) in rainbow trout eggs [16].

In fish, it is often useful or necessary to control the timing of spawning or induce the ovulatory process. These techniques are used for biotechnical, experimental or economical reasons to obtain out of season egg production and/or synchronous egg production within a group of females or, for some species, to obtain eggs from captive fish. The effects of these manipulations on fish egg quality have been thoroughly studied [1, 17]. However, the impact on egg transcriptome as a result of these manipulations has received far less attention despite recent efforts to study the ovarian or follicular transcriptome during oogenesis [1820]. In the present study, we analyzed the transcriptome of unfertilized rainbow trout (Oncorhynchus mykiss) eggs after natural or controlled ovulation. Two different protocols of controlled ovulation that are widely used in laboratories and fish farms were carried out: (i) a hormonal induction of ovulation using intra-peritoneal GnRH-analog injection, and (ii) a specific photoperiod regime designed to advance the spawning period. In addition, a third group was not subjected to any specific manipulation to allow egg collection after natural spontaneous ovulation. For each individual female, egg samples were collected and either subjected to a microarray analysis or transferred in an experimental hatchery after fertilization for monitoring developmental success (e.g. embryonic survival, malformations). Thus, the present study aimed at (i) analyzing the effect of ovulation control processes on egg transcriptome and (ii) analyzing possible links between egg transcriptome and egg developmental potential.

Results

Egg quality

Both hormonal induction and photoperiodic manipulation of ovulation had a negative impact on egg quality. The percentage of normal (i.e. without morphological abnormalities) alevins monitored at yolk-sac resorption (YSR) was used to characterize the egg quality of each individual female. The higher percentage of normal alevins at YSR, 84 ± 5%, was observed after natural (N) ovulation (Figure 1). In contrast, significantly lower percentages were observed after hormonal induction (HI) of ovulation (65 ± 9%) or photoperiodic manipulation (PM) of ovulation (37 ± 16%) (Figure 1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-55/MediaObjects/12864_2006_Article_768_Fig1_HTML.jpg
Figure 1

Percentage of normal alevins at yolk sac resorption (mean ± 95% confidence interval) observed after fertilizing eggs of females subjected to natural ovulation (N, n = 25), hormonal induction of ovulation (HI, n = 33) and photoperiod manipulation of ovulation (PM, n = 17). Significantly different from natural ovulation at p < 0.0001 (***).

Transcriptomic analysis

After signal processing, 8423 clones out of 9152 were kept for further analysis. SAM analysis was performed using the expression data of those 8423 clones. Twenty six genes exhibiting a differential mRNA abundance among at least 2 of the 3 experimental groups were identified (Table 1, Figure 2) with a false discovery rate (FDR) of 3.4%. The ontologies of those genes are presented in Table 2. Thirty one genes putatively linked to egg quality were identified (Table 3, Figure 3) with a FDR of 30%. The ontologies of those genes are presented in Table 4.
Table 1

Genes exhibiting differential egg mRNA abundance among experimental groups identified from the microarray analysis.

Clones

GenBank

Sigenae contig

Symbol

swissprot_hit_description

Score

UniGene

tcac0001.c.18

BX082249

tcac0001c.c.18_5.1.s.om.8

 

YEAST (P53230) Hypothetical 44.2 kDa protein in RME1-TFC4 intergenic region

283

 

1RT159P21_B_H11

CA388269

CA388269.1.s.om.8

    

tcbk0051.e.02

BX878405

tcay0028b.c.19_3.1.s.om.8

    

1RT65F10_D_C05

CA353171

tcab0001c.m.15_5.1.s.om.8

APOC1

MOUSE (P34928) Apolipoprotein C-I precursor (Apo-CI) (ApoC-I)

123

Omy.10219

1RT121B08_D_A04

CA359367

CA359367.1.s.om.8

CTNNBL1

HUMAN (Q8WYA6) Beta-catenin-like protein 1 (Nuclear-associated protein)

950

Omy.23137

1RT56O04_C_H02

CA351228

CA351228.1.s.om.8

DAB2

MOUSE (P98078) Disabled homolog 2 (DOC-2)

215

 

1RT64F24_D_C12

CA358202

CA358202.1.s.om.8

DBNL

MOUSE (Q62418) Drebrin-like protein (SH3 domain-containing protein 7)

720

 

1RT87E10_C_C05

CA345343

tcay0028b.g.03_3.1.s.om.8

DDAH2

MOUSE (Q99LD8) NG, NG-dimethylarginine dimethylaminohydrolase 2

629

Omy.23405

1RT68D18_D_B09

CA34327

tcba0017c.p.21_5.1.s.om.8

HCK

MOUSE (P08103) Tyrosine-protein kinase HCK (EC 2.7.1.112)

324

Omy.9448

tcay0037.m.11

BX319623

tcay0032b.l.02_3.1.s.om.8

HNRPK

RAT (P61980) Heterogeneous nuclear ribonucleoprotein K

1171

Omy.26818

tcay0030.n.02

BX316222

tcav0005c.k.03_3.1.s.om.8

HSPA9B

HUMAN (P38646) Stress-70 protein, mitochondrial precursor

901

Omy.26983

1RT162C23_A_B12

CA382140

tcbk0012c.o.01_5.1.s.om.8

ING1

HUMAN (Q9UK53) Inhibitor of growth protein 1

435

Omy.24666

1RT121G15_A_D08

CA362639

tcay0007b.n.06_3.1.s.om.8

LYPA3

HUMAN (Q8NCC3) 1-O-acylceramide synthase precursor (EC 2.3.1.-)

816

Omy.9525

tcbk0023.o.24

BX875550

tcbk0005c.o.10_5.1.s.om.8

MR-1

ARATH (O24496) Hydroxyacylglutathione hydrolase cytoplasmic (EC 3.1.2.6)

466

Omy.19659

1RT131K20_C_F10

CA383630

CA383630.1.s.om.8

MYO1B

MOUSE (P46735) Myosin Ib (Myosin I alpha) (MMI-alpha) (MMIa)

985

 

tcba0025.n.15

BX866389

tcay0008b.e.10_3.1.s.om.8

NTAN1

HUMAN (Q96AB6) Protein N-terminal asparagine amidohydrolase (EC 3.5.1)

510

 

tcbk0049.m.03

BX884905

tcbk0002c.c.19_5.1.s.om.8

OSBPL5

MOUSE (Q9ER64) Oxysterol binding protein-related protein 5

344

Omy.14649

tcbk0050.a.20

BX886190

tcbk0050c.a.20_5.1.s.om.8

PGH2

CHICK (P27607) Prostaglandin G/H synthase 2 precursor

1636

Omy.20943

tcbk0055.m.20

BX880138

tcbk0055c.m.20_5.1.s.om.8

PKP1

HUMAN Plakophilin 1

337

 

tcay0027.b.13

BX313624

tcay0027b.b.13_5.1.s.om.8

PYC

HUMAN (P11498) Pyruvate carboxylase, mitochondrial precursor (EC 6.4.1.1)

1021

 

1RT67N13_B_G07

CA360456

CA355135.1.s.om.8

RBM5

HUMAN (P52756) RNA-binding protein 5 (Putative tumor suppressor LUCA15)

296

 

tcbk0027.b.05

BX887647

tcbi0025c.k.02_5.1.s.om.8

RL10

HUMAN (P27635) 60S ribosomal protein L10

1039

Omy.4144

1RT139F11_B_C06

CA384643

tcay0034b.h.11_3.1.s.om.8

RPL24

GILMI (Q9DFQ7) 60S ribosomal protein L24

529

Omy.9444

1RT63M02_C_G01

CA343028

tcac0004c.h.08_5.1.s.om.8

RPN2

HUMAN (P04844) Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 63 kDa subunit precursor (Ribophorin II)

643

Omy.24414

tcad0007.p.12

BX078856

tcac0005c.e.12_3.1.s.om.8

SEC22

YARLI (Q6C880) Protein transport protein SEC22

237

Omy.913

1RT56L15_B_F08

CA351681

CA351681.1.s.om.8

TPH

XENLA (Q92142) Tryptophan 5-hydroxylase (EC 1.14.16.4)

725

 

Genes subsequently studied by real time PCR are bolded. For each gene, clone name, GenBank accession number, official human symbol and corresponding UniGene cluster are indicated. The Sigenae contig name [60] used for Blast comparison against the Swiss-Prot database is shown. Resulting best hit and corresponding score are indicated.

Table 2

Ontologies of the genes exhibiting differential egg mRNA abundance among experimental groups identified from the microarray analysis.

Symbol

Biological Process (P)

Cellular component (C)

Molecular Function (F)

APOC1

negative regulation of lipoprotein lipase activity

negative regulation of binding

lipid metabolism

chylomicron

enzyme activator activity

lipid binding

CTNNBL1

induction of apoptosis

nucleus

 

DAB2

cell proliferation

 

protein binding

DBNL

Rac protein signal transduction

activation of JNK activity

Lamellipodium

Cytoplasm

cell cortex

actin binding

enzyme activator activity

protein binding

DDAH2

anti-apoptosis

arginine catabolism

nitric oxide mediated signal transduction

 

hydrolase activity

HCK

protein amino acid phosphorylation

mesoderm development

 

protein-tyrosine kinase activity

protein binding

HNRPK

 

nucleus

protein binding

single-stranded DNA binding

HSPA9B

hemopoiesis

mitochondrial matrix

ATPase activity

ATP binding

ING1

negative regulation of cell proliferation

negative regulation of cell growth

nucleus

DNA binding

LYPA3

fatty acid catabolism

lysosome

phospholipid binding

lysophospholipase activity

MR-1

   

MYO1B

nervous system development

Cytoskeleton

brush border

motor activity

NTAN1

memory

adult locomotory behavior

Nucleus

cytoplasm

protein N-terminal asparagine amidohydrolase activity

OSBPL5

cholesterol metabolism

cholesterol transport

Golgi to plasma membrane transport

integral to membrane

cytosol

oxysterol binding

PGH2

physiological process

keratinocyte differentiation

cyclooxygenase pathway

Nucleus

cytoplasm

Peroxidase activity

prostaglandin-endoperoxide synthase activity

PKP1

signal transduction

cell adhesion

desmosome

nucleus

intermediate filament

structural constituent of epidermis

signal transducer activity

intermediate filament binding

PYC

  

ATP binding

biotin binding

pyruvate carboxylase activity

RBM5

RNA binding

DNA binding

nucleus

RNA processing

RPL10

Spermatogenesis

protein biosynthesis

cytosolic large ribosomal subunit (sensu Eukaryota)

mitochondrial large ribosomal subunit

structural constituent of ribosome

RPL24

translation

mitochondrial large ribosomal subunit

structural constituent of ribosome

RPN2

protein modification

protein amino acid N-linked glycosylation

oligosaccharyl transferase complex

 

SEC22

ER to Golgi vesicle-mediated transport

endoplasmic reticulum membrane

transporter activity

TPH1

serotonin biosynthesis from tryptophan

cytoplasm

tryptophan 5-monooxygenase activity

YG1W

protein import into mitochondrial matrix

mitochondrion

Protein binding

Table 3

Genes exhibiting differential mRNA abundance in eggs of varying quality identified from the microarray analysis

Symbol

Genbank

Sigenae contig

Symbol

swissprot_hit_description

Score

UniGene

tcbr0001.b.08

 

NO CONTIG

    

1RT42N11_B_G06

CA378261

tcba0024c.d.03_5.1.s.om.8

AGM1

HUMAN (O95394) Phosphoacetylglucosamine mutase (EC 5.4.2.3)

1065

Omy.22147

1RT120K08_C_F04

CA362248

CA362248.1.s.om.8

ALG2

HUMAN (Q9H553) Alpha-1,3-mannosyltransferase ALG2 (EC 2.4.1.-)

662

 

1RT85M16_C_G08

CA345100

tcac0001c.c.13_3.1.s.om.8

APOB

HUMAN (P04114) Apolipoprotein B-100 precursor

630

Omy.8599

tcay0008.m.21

BX301016

tcav0001c.l.14_3.1.s.om.8

BMP7

MOUSE (P23359) Bone morphogenetic protein 7 precursor (BMP-7)

1154

Omy.19556

tcbk0001.p.13

BX873334

tcbk0001c.p.13_5.1.s.om.8

CASZ1

HUMAN (Q86V15) Probable transcription factor CST

234

Omy.20281

tcay0007.f.20

BX300279

tcay0007b.f.20_5.1.s.om.8

CF188

RAT (Q5FWS4) Protein C6orf188 homolog

468

Omy.26998

tcba0030.i.17

BX867113

tcay0040b.g.08_5.1.s.om.8

CUL5

RABIT (Q29425) Cullin-5 (CUL-5)

1694

Omy.21358

1RT63D22_D_B11

CA343059

CA343059.1.s.om.8

DCPS

MOUSE (Q9DAR7) Scavenger mRNA decapping enzyme DcpS

195

 

tcba0010.c.10

BX861936

tcba0010c.c.10_5.1.s.om.8

DUSP24

HUMAN (Q9Y6J8) Dual specificity protein phosphatase 24

688

 

1RT104M18_C_G09

CA347317

tcay0009b.d.09_3.1.s.om.8

FGD5

MOUSE (Q80UZ0) FYVE, RhoGEF and PH domain containing protein 5

145

Omy.10646

1RT147H01_B_D01

CA350285

tcad0009a.n.22_3.1.s.om.8

GMCL1

HUMAN (Q96IK5) Germ cell-less protein-like 1

1353

Omy.2306

tcbk0042.i.19

BX879311

tcav0003c.a.13_3.1.s.om.8

GTF2B

RAT (P62916) Transcription initiation factor IIB

1449

 

tcay0006.d.12

BX299916

tcab0003c.m.12_5.1.s.om.8

HCFC1

MOUSE (Q61191) Host cell factor (HCF) (HCF-1)

695

 

tcay0009.k.03

BX302686

tcay0009b.k.03_5.1.s.om.8

KHK

HUMAN (P50053) Ketohexokinase (EC 2.7.1.3)

591

 

1RT68H23_B_D12

CA343227

CA343227.1.s.om.8

KIF4A

XENLA (Q91784) Chromosome-associated kinesin KLP1

477

 

tcbk0045.a.13

BX883207

tcbk0045c.a.13_5.1.s.om.8

LAMB2

HUMAN (P55268) Laminin beta-2 chain precursor (S-laminin)

1097

 

1RT77F09_B_C05

CA354296

CA354296.1.s.om.8

LRTM1

PONPY (Q5R6B1) Leucine-rich repeat transmembrane neuronal protein 1 precursor

410

 

tcbk0030.p.11

BX885788

tcbk0030c.p.11_5.1.s.om.8

MCF2L

MOUSE (Q64096) Guanine nucleotide exchange factor DBS

468

Omy.6690

1RT79A16_C_A08

CA355005

CA355005.1.s.om.8

NEK1

MOUSE (P51954) Serine/threonine-protein kinase Nek1 (EC 2.7.1.37)

359

 

1RT94E22_C_C11

CA347857

CA347857.1.s.om.8

OBSCN

CAEEL (O01761) Muscle M-line assembly protein unc-89

271

Omy.24205

tcab0002.l.03

BX080933

tcab0002c.l.03_5.1.s.om.8

PDCL3

HUMAN (Q9H2J4) Phosducin-like protein 3

305

 

tcbk0044.f.24

BX877936

tcbk0044c.f.24_5.1.s.om.8

PDGFRA

XENLA (P26619) Alpha platelet-derived growth factor receptor precursor

248

 

tcay0023.m.15

BX310740

tcay0002b.p.08_3.1.s.om.8

PHB2

RAT (Q5XIH7) Prohibitin-2 (B-cell receptor-associated protein BAP37) (BAP-37)

1013

Omy.9050

tcay0029.o.17

BX314382

tcay0029b.o.17_3.1.s.om.8

RAB3IP

HUMAN (Q96QF0) RAB3A-interacting protein (Rabin-3)

206

 

1RT149L18_D_F09

CA350883

tcay0003b.j.14_3.1.s.om.8

TF

SALSA (P80426) Serotransferrin I precursor (Siderophilin I) (STF I)

663

Omy.9801

1RT142A22_C_A11

CA349568

CA349568.1.s.om.8

TGFBR2

RAT (P38438) TGF-beta receptor type II precursor (EC 2.7.1.37)

1133

Omy.23150

tcay0018.b.11

BX307666

tcab0003c.i.10_5.1.s.om.8

TLE1

MOUSE (Q62440) Transducin-like enhancer protein 1 (Groucho-related protein 1)

2032

Omy.9672

1RT165F23_B_C12

CA388009

CA388009.1.s.om.8

VWF

HUMAN (P04275) Von Willebrand factor precursor (vWF

579

 

tcay0018.a.17

BX307636

tcay0009b.k.21_3.1.s.om.8

ZNF16

HUMAN (P17020) Zinc finger protein 16 (Zinc finger protein KOX9)

311

Omy.6191

tcbk0009.g.08

BX875276

tcbk0009c.g.08_5.1.s.om.8

ZNF261

MOUSE (Q9JLM4) Zinc finger protein 261 (DXHXS6673E protein)

254

Omy.2759

Genes subsequently studied by real time PCR are bolded. For each gene, clone name, GenBank accession number, official human symbol and corresponding UniGene cluster are indicated. The Sigenae contig name [60] used for Blast comparison against the Swiss-Prot database is shown. Resulting best hit and corresponding score are indicated.

Table 4

Ontologies of the genes exhibiting differential mRNA abundance in eggs of varying quality identified from the microarray analysis.

Symbol

Biological Process (P)

Cellular component (C)

Molecular Function (F)

AGM1

glucosamine metabolism

 

phosphoacetylglucosamine mutase activity

APOB

circulation

lipid transport

signal transduction

extracellular region

endoplasmic reticulum

microsome

lipid transport activity

receptor binding

BMP7

BMP signalling pathway

cell development

organ morphogenesis

mesoderm formation

pattern specification

positive regulation of cell differentiation

Extracellular space

cytokine activity

protein binding

CUL5

cell cycle arrest

cell proliferation

G1/S transition of mitotic cell cycle

induction of apoptosis by intracellular signals

negative regulation of cell proliferation

regulation of progression through cell cycle

 

calcium channel activity

protein binding

receptor activity

DCPS

mRNA catabolism

 

pyrophosphatase activity

FGD5

cytoskeleton organization and biogenesis

regulation of cell shape

cytoplasm

Golgi apparatus

protein binding

small GTPase binding

GMCL1

nuclear membrane organization and biogenesis

regulation of transcription

spermatogenesis

nuclear lamina

nuclear matrix

protein binding

GTF2B

mRNA transcription from RNA polymerase II promoter

transcription initiation from RNA polymerase II promoter

transcription factor complex

general RNA polymerase II transcription factor activity

protein binding

RNA polymerase II transcription factor activity

HCFC1

positive regulation of progression through cell cycle

regulation of transcription

transcription from RNA polymerase II promoter

cytoplasm

nucleus

identical protein binding

transcription coactivator activity

transcription factor activity

KHK

carbohydrate catabolism

 

ketohexokinase activity

KIF4A

organelle organization and biogenesis

anterograde axon cargo transport

cytoplasm

spindle microtubules

microtubule motor activity

LAMB2

synaptic transmission

electron transport

basal lamina

membrane

calcium ion binding

oxidoreductase activity

phospholipase A2 activity

structural molecule activity

MCF2L

Rho protein signal transduction

membrane

lamellipodium

phosphatidylinositol binding

Rho guanyl-nucleotide exchange factor activity

NEK1

response to DNA damage stimulus

response to ionizing radiation

cytoplasm

nucleus

protein binding

protein kinase activity

PDCL3

phototransduction

cytoplasm

protein binding

PDGFRA

cell proliferation

extracellular matrix organization and biogenesis

male genitalia development

morphogenesis

integral to plasma membrane

platelet-derived growth factor binding

protein dimerization activity

protein serine/threonine kinase activity

PHB2

signal transduction

negative regulation of transcription

mitochondrial inner membrane

nucleus

estrogen receptor binding

protein binding

specific transcriptional repressor activity

RAB3IP

 

cytoplasm

nucleus

protein Binding

GTPase binding

TGFBR2

regulation of cell proliferation

Cell fate commitment

protein amino acid phosphorylation

protein amino acid dephosphorylation

integral to membrane

ATP binding

protein binding

protein tyrosine kinase activity

TLE1

signal transduction

regulation of transcription, DnA-dependent

organ morphogenesis

nucleus

 

VWF

cell adhesion

response to wounding

extracellular space

protease binding

protein binding

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-55/MediaObjects/12864_2006_Article_768_Fig2_HTML.jpg
Figure 2

Unsupervised average linkage clustering analysis of the 26 differentially abundant genes in eggs collected after photoperiod-manipulated ovulation (PM), hormonally-induced ovulation (HI) and natural ovulation (N). Each row represents a gene and each column represents an egg RNA sample. For each gene, the expression level within the sample set is indicated using a color intensity scale. Red and green are used for over and under abundance respectively while black is used for median abundance.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-55/MediaObjects/12864_2006_Article_768_Fig3_HTML.jpg
Figure 3

Supervised average linkage clustering analysis of 31 genes significantly linked to egg quality. Each row represents a gene and each column represents an egg RNA sample. The 31 samples are supervised according to the percentage of normal alevins at yolk-sac resorption. For each gene, the expression level within the sample set is indicated using a color intensity scale. Red and green are used for over and under abundance respectively while black is used for median abundance.

Real-time PCR analysis

From the 57 (26+31) genes identified in the transcriptomic analysis, 32 were ultimately kept for real-time PCR analysis (Table 5). Real-time PCR data corresponding to the remaining 25 was not used in the analysis because of methodological reasons (e.g. low expression, poor PCR efficiency, double amplification).
Table 5

Real-time PCR primer sequences. For each target gene, official symbol of the human protein, GenBank accession number of the clone and clone name are indicated

Reverse sequence

Foward sequence

Symbol

GenBank

Clones

ACTTCCTCCTCCTCCACGTT

CCAGCTCATGTACCCGTTCT

 

CA388269

1RT159P21_B_H11

CTGGGTTCAGGAAGTGTGGT

TCGCTGGAGGAGTAGGAGAG

AGM1

CA378261

1RT42N11_B_G06

GCTGTCCCCAATCTTTTCAA

GGCTGAGAAGACCATTGAGG

APOC1

CA353171

1RT65F10_D_C05

GTGTCTGGACCGTCTGACCT

CCTGCACAAGTACGTGGAGA

BMP7

BX301016

tcay0008.m.21

GCAGTGGTAGTGGGTCACCT

CTTCCGCCGTTATGATCTGT

CASZ1

BX873334

tcbk0001.p.13

GATCAGTAGCCAACCCAGGA

GCAGTGCCAGGATGAACTTT

CF188

BX300279

tcay0007.f.20

CAACTTGCGTACGATGCTGT

AGGCCTACATTGTGGAGTGG

CUL5

BX867113

tcba0030.i.17

GGAAAGCTGGTTGCTTGCTG

CTTCAAACTCTGCGCCGGCACA

cyclinA2

BX080925

tcab0003.e.11

GAGCTGCTATGGGAGAGGTG

CTAAGGCTGGACGAGGTCTG

DAB2

CA351228

1RT56O04_C_H02

TCCCCTCGTAGGTGAACAAG

CAAGTGTTTGAGCGAACGAA

DBNL

CA358202

1RT64F24_D_C12

TCTCCAACAGGGTGTCTTCC

ACGGAAAGTTGAACGACCAG

DCPS

CA343059

1RT63D22_D_B11

TGGTCTTTCTCCAGGGTGAG

CCTCGGAGGCATCTAGCATA

GMCL1

CA350285

1RT147H01_B_D01

GTGCAAATTTTTGGGGAAGA

CCCGAGATAAGGACTGATGG

HCFC1

BX299916

tcay0006.d.12

GACAAATGATGACAGTGGCCTA

TGCGATGTGATGTGACATTTT

HCK

CA34327

1RT68D18_D_B09

CAGACTTGCCACTGACCAGA

CAGCATCATTGGTGTGAAGG

HNRPK

BX319623

tcay0037.m.11

CCGTGGTCACATTGCTTATG

GGCATGTTGCAGACTTCGTA

KHK

BX302686

tcay0009.k.03

GTTCCCATACGCACATTCCT

TCCCAGCCATCTTCAAAGTC

LYPA3

CA362639

1RT121G15_A_D08

TCGACTCGTACGTCAACTGG

GCTCTCCAACTCTTCGGATG

MCF2L

BX885788

tcbk0030.p.11

CTGTGGTCCCAGTGTTTGTG

GCAGACCCACAGACAGTTCA

MR-1

BX875550

tcbk0023.o.24

CATGGCCAGGATACCATTCT

TTCATCGAACTGACGCTACG

MYO1B

CA383630

1RT131K20_C_F10

AACTAGGTGGCAGGTGGTTG

GAGAGTTTGCAGCCACAACA

NTAN1

BX866389

tcba0025.n.15

TCCTGGATGTGGAAGGAGTC

AAGCTGAAGTTCGACCCAGA

PGH2

BX886190

tcbk0050.a.20

TCGTCCAGGATGATGTTGAA

GTTCAATGCCTCACAGCTCA

PHB2

BX310740

tcay0023.m.15

CAGCAGGGGAGAGATTTCAG

CCAGCCAGAGAGAAGACACC

PKP1

BX880138

tcbk0055.m.20

GAAGGGGATGTTGGTCTTGA

GCATTCCAAGGAGCAGTCAT

PYC

BX313624

tcay0027.b.13

CTCCGGTGTGCCCTAATAAA

GCTGGGCTTCTACCTCACAG

RAB3I

BX314382

tcay0029.o.17

ACGGAGGAGGAAGAGGAGAG

GGGGCAAGGAGAAGAAAGAC

RBM5

CA360456

1RT67N13_B_G07

CAGGCTTCTGGTTCCTCTTG

CAAGAAGGGCCAGTCTGAAG

RPL24

CA384643

1RT139F11_B_C06

AGCTGTGGTGGAGAAGCAAT

GGGGTGGGGGAGATACTAAA

SEC22

BX078856

tcad0007.p.12

TCGTGGGAGATGTCGATACA

GCCAAAGTCTGCTTCTCCTG

TLE3

BX307666

tcay0018.b.11

GAGGAAGGAGGCAGTCACAG

GCTCCACTGGAAGACCATGT

YG1W

BX082249

tcac0001.c.18

GTGCACTCGTAGGGCTTCTC

AACACCTCCGAAGTCACACC

ZNF16

BX307636

tcay0018.a.17

GAGTCCGAGCACTTGGAAAG

AGAGGAGGTGCTGGAGATGA

ZNF261

BX875276

tcbk0009.g.08

Genes exhibiting a differential egg mRNA abundance among experimental groups

Among the 26 genes exhibiting a differential mRNA abundance between experimental groups, 17 were studied by real-time PCR. Among those 17 genes, 7 were found to be differentially expressed in the real-time PCR study (Figure 4). The identity of those 7 genes is presented below. Only the informative alignments obtained using the full rainbow trout coding sequence (CDS) or a substantial part of the CDS are presented (Figures 3, 4). For clarity reasons, the official human protein symbol was used in the text.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-55/MediaObjects/12864_2006_Article_768_Fig4_HTML.jpg
Figure 4

Real-time PCR analysis of gene mRNA abundance (mean ± SEM) in unfertilized eggs collected after natural ovulation (n = 4), hormonally-induced ovulation (n = 11) and photoperiod-manipulated ovulation (n = 14). Different letters indicate significant differences between groups at p < 0.05. The official human symbol is indicated for all studied genes.

Clone # 1RT65F10_D_C05 exhibited significant sequence similarity with mouse Apolipoprotein C-I precursor (APOC1, Table 1) and was significantly more abundant in eggs of the HI group than in eggs of the N group while intermediate levels were observed in eggs of the PM group. The mRNA abundance in the HI group was 13 times higher than in the N group while it was 2 times higher than in the PM group (Figure 4). After performing a Blast search in the GenBank database, the complete rainbow trout amino acid sequence deduced from the EST sequence exhibited 54% sequence identity at the amino acid level with the zebrafish (Danio rerio) cognate protein (Figure 5A). A sequence identity of 33 and 26% was observed with mouse and human proteins respectively (Figure 5A). The number of amino acids deduced from the trout EST is consistent with the number of amino acids present in mammalian and zebrafish sequences.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-55/MediaObjects/12864_2006_Article_768_Fig5_HTML.jpg
Figure 5

Amino acid sequence alignment of rainbow trout APOC1 (A), MR-1 (B), and RPL24 (C) with cognate vertebrate forms. For each target species, the GenBank accession number of the protein is indicated.

A similar expression pattern was observed for clone # 1RT68D18_D_B09 that exhibited sequence similarity with mouse Hemopoietic cell kinase (HCK, Table 1). The deduced partial amino acid sequence generated from the corresponding UniGene cluster exhibited 40% and 38% identity with mouse and human HCK proteins respectively.

Clone tcbk0023.o.24 exhibited sequence similarity with hydroxyacylglutathione hydrolase cytoplasmic (MR-1, Table 1) and was less abundant in eggs of the HI group than in eggs of the 2 other experimental groups (Figure 4). A contig sequence was generated using all rainbow trout ESTs belonging to the same UniGene cluster (Omy.19659). This contig sequence was then used to perform a blastX search in GenBank. This contig sequence corresponded to a partial CDS of the putative rainbow trout cDNA. The deduced rainbow trout amino acid sequence exhibited 59% identity with the mouse brain protein 17 isoform 1 (Figure 5B). This mouse protein is also known as myofibrillogenesis regulator 1. In addition a 60% identity was observed with human cognate protein (Figure 5B) know as myofibrillogenesis regulator 1 (MR-1).

Clone tcba0025.n.15 exhibited sequence similarity with human N-terminal asparagine amidase (NTAN1, Table 1) and was more abundant in eggs of the HI group than in eggs of N and PM groups (Figure 4). This sequence did not belong to any UniGene cluster and did not include a complete CDS. After performing a Blast search using this partial sequence, a 47% identity with the cognate human form (NTAN1) was observed.

Clone 1RT131K20_C_F10) exhibited sequence similarity with mouse myosin Ib (MYO1B, Table 1) and was more abundant in eggs of the PM group than in eggs of HI and N groups (Figure 4). This sequence did not belong to a UniGene cluster and did not contain a full CDS. The observed identity with predicted zebrafish and chicken cognate forms was 93 and 86% respectively. An 85 and 86% amino acid sequence identity was observed with human and murine proteins respectively.

Clone tcay0027.b.13 exhibited sequence similarity with human pyruvate carboxylase (PYC, Table 1) and was more abundant in eggs of the PM group than in eggs of the N group, while intermediate levels were observed in eggs of the HI group (Figure 4). This sequence did not include a full CDS. After performing a Blast search using this partial coding sequence, the amino acid sequence identity with cognate vertebrate forms was above 80%.

Clone 1RT139F11_B_C06 exhibited sequence similarity with ribosomal protein RPL24 and was more abundant in eggs of the HI group than in eggs of the PM group (Figure 4). This clone included a full CDS and the deduced amino acid sequence exhibited very strong (above 95%) sequence identity with cognate fish proteins (Figure 5C).

For 5 genes (HRNPK, RBM5, DAB2, PGH2 and SEC22, Table 1) similar expression profiles were observed in real-time PCR and microarray analyses. However, no statistical differences between groups were observed in the real-time PCR experiment (Figure 4).

For 3 genes (PKP1, DBNL and LYPA3, Table 1) the consistency between real-time PCR and microarray data was limited to 2 of the 3 experimental groups. In addition, no statistical differences between groups were observed in the real-time PCR analysis (Figure 4).

For the 2 remaining clones (BX082249 and CA388269, Table 1), no correlation was observed between real-time PCR and microarray data (data not shown).

Genes exhibiting a quality-dependent mRNA abundance in the eggs

Among the 31 genes identified as linked to egg quality, 15 were analyzed by real-time PCR. Among those 15 genes, the mRNA abundance of 1 gene was found to be significantly correlated with egg quality. This clone (PHB2) exhibited significant sequence similarity with rat prohibitin 2 (Table 3). Its mRNA abundance in the eggs was negatively correlated (R = -0.47, p < 0.05) with the percentage normal alevins at yolk-sac resorption. In addition the mRNA abundance of this gene was significantly higher in eggs exhibiting the lowest developmental potential (Figure 6). An amino acid sequence was generated from nucleotide sequences of Omy.9050 UniGene cluster. This deduced amino acid sequence exhibited 83% identity with zebrafish sequence and 76% identity with human and rat sequences (Figure 6).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-55/MediaObjects/12864_2006_Article_768_Fig6_HTML.jpg
Figure 6

(A) Amino acid sequence alignment of deduced rainbow trout prohibitin 2 (PHB2) with human, rat and zebrafish forms. For each target species, the GenBank accession number of the protein is indicated. (B) Real-time PCR analysis of PHB2 mRNA abundance (mean ± SEM) in eggs of low (n = 10), intermediate (n = 9) and high (n = 10) developmental potential estimated by the percentage normal alevins at yolk-sac resorption. Different letters indicate significant differences between groups at p < 0.05.

Discussion

Microarray analysis efficiency and reliability

The hybridization of radiolabeled cDNAs with cDNAs deposited onto nylon membranes has been used for several decades. However, the use of nylon cDNA microarrays is not very common in comparison to glass slide microarrays. Nevertheless, this technology has successfully been used for several years [21]. In our laboratory, we have successfully used this technology to identify differentially expressed genes during oocyte maturation and ovulation [18]. In the present study, we have used the same methodology and have identified a group of 26 genes exhibiting differential egg mRNA abundance after natural controlled ovulation with a false discovery rate of 3.4%. Using real-time PCR, the egg mRNA abundance of 17 genes was analyzed. Among those 17 genes, only 2 exhibited expression patterns totally inconsistent with microarray data. In contrast, the expression patterns of the other genes were very similar to microarray data, even though observed differences were not always significant. It is noteworthy that the 2 genes exhibiting inconsistent expression patterns between PCR and microarray experiments correspond to uncharacterized proteins. Indeed, one of the genes (CA388269) had no significant hit in the Swiss-Prot database while the other one (BX082249) had a significant hit with a hypothetical yeast protein (Table 1). To conclude, the overall consistency of PCR and microarray data suggests that the microarray analysis performed in the present study is robust and reliable.

Genes exhibiting a differential mRNA abundance after natural or controlled ovulation

Hormonal induction of ovulation

Among identified genes, APOC1 and HCK were the most affected by a hormonal-induction of ovulation. Thus, the egg mRNA abundance of those 2 genes was dramatically increased after hormonal induction of ovulation in comparison to natural ovulation (Figure 4). Human APOCs are protein constituents of chylomicrons, very low density lipoproteins, and high-density lipoproteins [22]. The human APOC1 protein is predominantly expressed in liver and adipose tissue [23]. APOC1 may modulate the activity of plasma enzymes involved in lipid metabolism. Besides, APOC1 has also been reported to interfere with the APOE-dependent hepatic uptake of lipoprotein remnants by the low density lipoprotein receptor (LDLr) and LDLr-related protein [24]. Interestingly, it was previously shown in rainbow trout that the same clone of the APOC1 gene was significantly up-regulated in the ovary at the time of oocyte maturation [18]. This could be related to the arrest of lipoproteins uptake by the oocyte at the end of vitellogenesis concomitantly with a decrease of the expression of vitellogenin receptor [25]. It is therefore possible that the hormonal induction of ovulation induces an artificial over abundance of some hormonally-dependent genes, such as APOC1, in the eggs. However, the possible consequences of such an over abundance on lipid metabolism of the embryo is so far unknown.

Similarly to APOC1, the egg mRNA abundance of HCK gene was also dramatically increased after hormonal induction of ovulation. HCK, hemopoietic cell kinase, belongs to Src-familly tyrosine kinases and is expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells. It was previously shown that the conventional progesterone receptor could interact, in a progestin-dependent manner, with various signaling molecules, including Src tyrosine kinases [26]. Indeed, these authors used downregulated HCK as a general model of the c-Src family tyrosine kinases to investigate the mechanism of activation by conventional progesterone receptor. In addition, the participation of the conventional progesterone receptor in African clawed frog (Xenopus laevis) oocyte maturation process was seriously suggested by two independent studies [27, 28]. Besides, Src tyrosine kinase activation has been shown to be one of the earliest transcription-independent responses of Xenopus oocytes to progesterone during in vitro induced maturation; a period when oocyte mRNA content remains stable [29]. Interestingly, we observed a dramatic over abundance of HCK mRNA in the eggs after hormonal induction of ovulation. To date, the significance of this over abundance as a result of hormonally-induced ovulation is unknown. However, it further demonstrates that the egg mRNA abundance of specific genes can be dramatically affected by a hormonal induction of ovulation.

In addition to APOC1 and HCK, eggs obtained after hormonal induction of ovulation were also characterized by higher NTAN1 and lower MR-1 mRNA abundance. However, the fold difference observed for those 2 genes was less important. In mice it has been shown that NTAN1 encodes an N-terminal amidohydrolase specific for N-terminal asparagines, which is involved in ubiquitin-proteasome proteolysis termed as the N-end rule pathway [30]. N-end rule pathway determines metabolic instability of different proteins that contain a destabilizing N-terminal residue [31]. More specifically, a recent study suggested that an over expression of NTAN1 using recombinant NTAN1 adenovirus vector resulted in a marked decrease in the microtubule-associated protein 2 (MAP2) expression in hippocampal neurons in rat [32]. Regardless of the specific target of NTAN1 in the oocyte, an increased expression of this enzyme should participate in protein turnover, and its regulation might be important for the normal development of the oocyte. The second gene, MR-1, is a newly identified protein that interacts with contractile proteins and exists in human myocardial myofibrils [33].

Finally, the egg mRNA abundance of RPL24 was higher after hormonal induction of ovulation. However, this difference was only significant in comparison with the PM group. The 60S ribosomal protein L24 (RPL24) is one of the forty seven 60S ribosomal proteins present in eukaryotic organisms and often used as markers for phylogenetic studies and comparative genomics. Those ribosomal proteins have been sequenced recently in catfish (Ictalurus punctarus) and high similarities with mammalian ribosomal protein were found [34]. 60S ribosomal subunit participates in translational initiation in combination with 40S ribosomal subunit [35]. An insertional mutagenesis study carried out in zebrafish (Danio rerio) reported this gene to be essential for early embryonic development. Mutation of this gene resulted in small head/eyes mutants [36]. Interestingly, when monitoring embryonic development in the present study, we noticed that many embryos originating from eggs of hormonally-induced females exhibited small eyes at eyeing stage. Precise quantification of this phenomenon would be necessary to stress its relationship with RPL24 over abundance in the eggs.

Photoperiodic control of ovulation

Four genes exhibited differential egg mRNA abundance after photoperiod treatment in comparison to natural ovulation. Similarly to eggs obtained after hormonal induction of ovulation, eggs of the PM group also exhibited increased levels of APOC1 and HCK. The differential abundance of both genes was high but less pronounced than after hormonally-induced ovulation. In addition eggs obtained after photoperiod manipulation of ovulation were also characterized by higher MYO1B and PYC mRNA abundance. According to the gene ontology analysis, MYO1B is a cytoskeleton protein involved in nervous system development (Table 2). It is also expressed in a wide variety of tissues including rat neonatal tissues [37, 38]. The class I myosin, MYO1B, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues [39]. MYO1B can interact on the dynamic actin filament populations and might play a role in intracellular membrane trafficking [40]. Myosin light chain has been recently suggested to participate in anchoring the 26S proteasome, a 26S multiprotein complex that catalyses the breakdown of polyubiquitylated proteins, to the actin cytoskeleton of goldfish oocyte [41]. Degradation of proteins mediated by ubiquitin-proteasome pathway plays important roles in the regulation of eukaryotic cell cycle [42] and can be involved in oocyte maturation and further embryonic cell cleavages.

Pyruvate carboxylase (PYC) is a mitochondrial biotin-dependent carboxylase. In the adipose tissue and liver PYC participates in the citrate shuttle by which NADPH equivalents are transported out of mitochondria to the cytosol for lipogenesis [43]. Five alternative forms of rat pyruvate carboxylase cDNAs have been identified in liver, kidney, brain, and adipose tissue and these are expressed in a tissue-specific manner [4446]. In red Seabream (Pagrus major), PYC mRNA was detected by Northen blot analysis in heart, liver, muscle and ovary [47]. Interestingly, it was previously shown that a photoperiod manipulation of spawning date was associated with a significantly higher occurrence of yolk-sac resorption defects [48]. Together, these observations suggest a putative link between an abnormal stockpiling of PYC mRNA in the egg and problems in the processing and/or use of yolk-sac lipidic stores. Indeed, it was previously reported that non viable gilthead sea bream eggs have lower pyruvate carboxylase activity than viable eggs [49].

Genes exhibited an egg mRNA abundance correlated with egg's developmental potential

From microarray data, 30 genes were identified as exhibiting an egg mRNA abundance correlated with egg's developmental potential. However, the false discovery rate was elevated and those genes were considered as candidate genes requiring PCR validation. Nevertheless, it is noteworthy that the ontological analysis of this group showed that 5 genes are involved in the regulation of transcription and others in cell proliferation/development and cytoskeleton organization and biogenesis. In addition, the correlation was confirmed for 1 of the 15 genes analyzed by real-time PCR: prohibitin 2 (PHB2). In animals and yeast, prohibitins have been shown to play important roles in cell cycling and senescence. One of prohibitin 2 major role is to be a chaperone-like regulator of the AAA protease in the mitochondrial matrix that assists in the assembly of inner membrane complex [50]. In Caenorhabditis elegans, PHB proteins were showed to be essential during embryonic development and are required for somatic and germ line differentiation in the larval gonad [51]. Moreover, deletions of the Saccharomyces cerevisiae homologues, PHB1 and PHB2, result in a decreased replicative lifespan, and a defect in mitochondrial membrane potential. The prohibitin protein has been immunolocalized in mammalian oocytes and embryos and suggested to have an antiproliferative activity [52]. Besides, a higher immunoreactivity level was found in the nucleus of embryo that failed to develop normally in comparison to morphologically normal ones. In the present study, we observed a higher prohibitin 2 mRNA abundance in eggs exhibiting the lowest developmental potential. This differential abundance in eggs of varying quality suggests that prohibitin 2 plays a role in the developmental potential of the embryo. Further studies are needed to unravel the link between an overabundance of prohibitin 2 mRNA in the eggs and a reduced egg developmental potential. Thus, this overabundance could be the result of a reduced prohibitin 2 synthesis during oogenesis.

Conclusion

In the present study we successfully used rainbow trout cDNA microarrays to analyze egg transcriptome after natural and controlled ovulation and in relationship with the developmental potential of the eggs. We showed that the control of ovulation using either a hormonal induction or a manipulated photoperiod could induce differences in the egg mRNA abundance of specific genes.

In addition, we showed that the egg mRNA abundance of prohibitin 2 (PHB2) was negatively correlated with the developmental potential of the egg.

Furthermore, the identity and ontology of identified genes provided significant hints that could result in a better understanding of the mechanisms associated with each type of ovulation control (e.g hormonal, photoperiodic) or conserved mechanisms triggering a loss of egg developmental potential.

Methods

Animals

Investigations were conducted according to the guiding principles for the use and care of laboratory animals and in compliance with French and European regulations on animal welfare. Three groups of male and female rainbow trout (Oncorhynchus mykiss) were obtained from our experimental fish farm (Sizun, France) and maintained until reproductive season under natural photoperiod and water temperature conditions. A first set of egg samples was collected from females undergoing natural (N) ovulation. Four weeks before expected ovulation fish (25 females) were transferred in a controlled recirculated water system (12°C) under natural photoperiod in INRA experimental facilities (Rennes, France). A second set of egg samples was collected from females subjected to a hormonal induction (HI) of ovulation. Four weeks before expected ovulation fish were transferred in a controlled recirculated water system (12°C) under natural photoperiod in INRA experimental facilities (Rennes, France). Females (n = 33) were given a 250 μL.Kg-1 body weight (b.w) intraperitoneal injection of [Des-Gly10, DArg6, Pro-NHEt9]-GnRH analog (Bachem, Allemagne) at 60 μg.Kg-1 b.w. A third set of egg samples was collected from females subjected to a photoperiod manipulation (PM) of ovulation. After a first reproduction, fish (17 females) were isolated in light-proofed tanks and exposed to an artificial photoperiod. Beginning on January 15th, all fish were held under constant light (24L:0D) for 490°C.day. Then, beginning on March 27th, they were held under short photoperiod (8L:16D) until ovulation (1230°C.day). Light was supplied by 4 neon tubes (58 Watts).

Gamete collection

In order to avoid excessive post-ovulatory ageing, unfertilized eggs were collected by manual stripping 5 days after detected ovulation. Two batches of 5 mL of eggs (approximately 100 to 200 eggs per batch) were used for fertilization. At each egg collection day, fresh sperm samples were collected from 10 mature males originating from the same group in order to fertilize eggs with a pool of sperms. Sperm samples were obtained by manual pressure on the abdomen and kept at 4°C for a short time before use.

Fertilization and early development

Fertilization was performed under previously described standardized conditions [16]. The two batches of 5 mL of eggs were fertilized with 5 μl of pooled semen. Fertilized eggs were transferred into compartmentalized incubation trays supplied by recirculated water (10°C). Water temperature and chemistry were routinely monitored and maintained constant over the entire incubation period. Dead eggs and embryos were periodically removed and survival rates were estimated as percentages of the initial number of eggs used for fertilization. Survival at the completion of yolk sac resorption (YSR, 550°C.day) was monitored. The occurrence of noticeable morphological malformations at YSR was also monitored. Survival and malformation data were used to calculate the proportion of normal alevins at YSR expressed as a percentage of the initial number of eggs.

RNA extraction

Extractions were performed as previously described [53] with minor modifications. Total RNA was extracted from 20 unfertilized eggs using 9 mL of TRizol (Invitrogen) in 13 mL sterile polypropylene tubes. Because of high egg vitellogenic content, each RNA was subsequently repurified using a Nucleospin RNA 2 kit (Macherey Nagel) in order to obtain genomic-grade RNA quality. For each egg sample, three RNA extracts were obtained, pooled and precipitated with sodium acetate (3 M, pH5.2, Prolabo) to increase RNA concentration. Thus, any RNA sample used for transcriptomic analysis originated from 60 unfertilized eggs of an individual female.

cDNA microarrays

Nylon micro-arrays (7.6 × 2.6 cm) were obtained from INRA-GADIE (Jouy-en-Josas, France) resource center [54]. A set of 9152 distinct rainbow trout cDNA clones originating from 2 pooled-tissues library [55, 56] were spotted in duplicates after PCR amplification. PCR products were spotted onto Hybond N+ membranes as previously described [57]. This rainbow trout generic array was deposited in Gene Expression Omnibus (GEO) database (Platform# GPL3650) [58].

Microarray hybridization

Four RNA samples originating from naturally ovulating females, 11 RNA samples originating from hormonally-induced females and 14 RNA samples originating from photoperiod-manipulated females were used for microarray hybridization according to the following procedure. Hybridizations were carried out as previously described [21], with minor modifications, at INRA genomic facility (Rennes). A first hybridization was performed using a 33P-labelled oligonucleotide (TAATACGACTCACTATAGGG which is present at the extremity of each PCR product) to monitor the amount of cDNA in each spot. After stripping (3 hours 68°c, 0.1× SSC, 0.2% SDS), arrays were prehybridized for 1 h at 65°C in hybridization solution (5× Denhardt's, 5× SSC, 0.5% SDS). Complex probes were prepared from 3 μg of RNA by simultaneous reverse transcription and labelling for 1 hour at 42°C in the presence of 50 μCi [alpha-33P] dCTP, 5 μM dCTP, 0.8 mM each dATP, dTTP, dGTP and 200 units M-MLV SuperScript RNase H-reverse transcriptase (GIBCO BRL) in 30 μL final volume. RNA was degraded by treatment at 68°C for 30 min with 1 μl 10% SDS, 1 μl 0.5 M EDTA and 3 μl 3 M NaOH, and then equilibrated at room temperature for 15 min. Neutralization was done by adding 10 μl 1 M Tris-HCl plus 3 μl 2N HCl. Arrays were incubated with the corresponding denatured labeled cDNAs for 18 h at 65°C in hybridization solution. After 3 washes (1 hours 68°C, 0.1× SSC 0.2% SDS), arrays were exposed 65 hours to phosphor-imaging plates before scanning using a FUJI BAS 5000. Signal intensities were quantified using ArrayGauge software (FujifilmMedical Systems, Stanford, CT) and deposited in GEO database (Series# GSE5928) [58].

Microarray signal processing

Spots with low oligonucleotide signal (lower than three times the background level) were excluded from the analysis. After this filtering step, signal processing was performed using the vector oligonucleotide data to correct each spot signal by the actual amount of DNA present in each spot. After correction, signal was normalized by dividing each gene expression value by the median value of the array.

Microarray data analysis

Statistical analysis was performed using Significance Analysis of Microarray (SAM) software [59]. For each comparison, the lowest false discovery rate (FDR) was used to identify differentially abundant genes. A first analysis was performed in order to identify differentially abundant transcripts between N group and the two other experimental groups (HI and PM). A second analysis was performed in order to identify differentially abundant transcripts in relation with egg quality, estimated by percentage of normal alevins at YSR within the complete data set or inside each experimental group (HI and PM).

Identity of mircroarray cDNA clones

Rainbow trout sequences originating from INRA AGENAE [55] and USDA [56] EST sequencing programs were used to generate publicly available contigs [60]. The 8th version (Om.8, released January 2006) was used for BlastX [61] comparison against the Swiss-Prot database (January 2006) [62]. The score of each alignment was retrieved after performing BlastX comparison. This was performed automatically for each EST spotted onto the membrane and used to annotate the 9152 clones of the microarray.

Data mining

For all the clones identified as differentially abundant after a SAM analysis (Table 1, 3) the official human gene symbol was retrieved [63] and used in the text, figures and tables for clarity reasons. In addition, the accession number of the corresponding rainbow trout cluster (UniGene Trout, January 2006), if any, was retrieved from the UniGene database [64]. For all genes identified as differentially abundant in the transcriptomic analysis, ontologies were obtained using the AmiGO tool [65]. Finally, for the differentially abundant genes identified in the real-time PCR analysis, a BlastX search was performed against the GenBank NR database. When possible, this was done using the contig sequence generated from all the ESTs present in the corresponding UniGene cluster. Subsequently, the amino acid sequence deduced from the trout contig sequence was aligned with cognate vertebrate forms.

Real-time PCR analysis

Real-time PCR was performed using all RNA samples used for microarray analysis (N = 29). Reverse transcription and real time PCR were performed as previously described [66]. Briefly, 2 μg of total RNA were reverse transcribed using 200 units of Moloney murine Leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI) and 0.5 μg dT15 Oligonucleotide (Promega) per μg of total RNA according to manufacturer's instruction. RNA and dNTPs were denatured for 6 min at 70°C; then chilled on ice for 5 min before the reverse transcription master mix was added. Reverse transcription was performed at 37°C for 1 hour and 15 min followed by a 15 min incubation step at 70°C. Control reactions were run without MMLV reverse transcriptase and used as negative controls in the real-time PCR study. Real-time PCR experiments were conducted using an I-Cycler IQ (Biorad, Hercules, CA). Reverse transcription products were diluted to 1/25, and 5 μl were used for each real-time PCR reaction. Triplicates were run for each RT product. Real-time PCR was performed using a real-time PCR kit provided with a SYBR Green fluorophore (Eurogentec, Belgium) according to the manufacturer's instructions and using 600 nM of each primer. After a 2 min incubation step at 50°C and a 10 min incubation step at 95°C, the amplification was performed using the following cycle: 95°C, 20 sec; 60°C, 1 min, 40 times. The relative abundance of target cDNA within sample set was calculated from a serially diluted oocyte cDNA pool using the I-Cycler IQ software. After amplification, a fusion curve was obtained using the following protocol: 10 sec holding followed by a 0.5°C increase, repeated 80 times and starting at 55°C. The level of CyclinA2 RNAs was monitored using the same sample set to allow normalization. Cyclin A2 was used for normalization because its mRNA abundance was shown to be elevated and highly stable in rainbow trout eggs collected 5 days after ovulation ([16]). Statistical analyses were performed using Statistica 7.0 software (Statsoft, Tulsa, OK). Differences between groups were analyzed using non parametric U tests.

Declarations

Acknowledgements

This work was funded by an INRA-AGENAE-IFOP grant to JB. The authors thank INRA-GADIE (Jouy en Josas, France) resource center for providing micro-arrays and the INRA-SIGENAE group (Toulouse, France) for bioinformatic support. The authors also thank all INRA experimental facility personnel (Sizun and Rennes) for animal care and the staff of INRA transcriptomic facilities for their help in microarray hybridization and data processing.

Authors’ Affiliations

(1)
INRA, UR1037 SCRIBE, IFR140, Ouest-Genopole

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