Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 2 3 4 5 6 7 8 9 10 11 12 13 Q1 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Contents lists available at ScienceDirect

Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed

Original Contribution

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae Kristin Hamre n, Samuel Penglase, Josef D. Rasinger, Kaja H. Skjærven, Pål A. Olsvik National Institute of Nutrition and Seafood Research, 5817 Bergen, Norway

art ic l e i nf o

a b s t r a c t

Article history: Received 15 May 2014 Accepted 19 May 2014

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (Eh) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH þGSSG) increased from 610 7100 to 1260 7150 μmol/kg between 7 and 14 dph and was then constant until 49 dph, after which it decreased to 8107100 μmol/kg by 63 dph. The 14- to 49dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing Eh from  239 71 to  262 77 mV between 7 and 14 dph, after which it remained constant until 63 dph. The changes in GSH and Eh were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20 dph (6.8 mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50 dph (10–15 mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH and low reduction potentials. & 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Keywords: Marine fish larvae Cod Glutathione Reduction potential Redox signaling Free radicals

Atlantic cod (Gadus morhua) is an ecologically important apex predator in the North Atlantic ocean [1] and has a high commercial value in both wild fisheries and aquaculture [2]. Cod larvae hatch as immature organisms, still relying on their yolk sac for 3 to 5 days before first feeding, which occurs at 4- to 5-mm standard length (SL) [3,4]. Several organ systems undergo further development during the larval stages. Larval muscle development is divided into a period of stratified hyperplasia, which continues from the late embryonic stage, followed by a period termed mosaic hyperplasia, which extends from later in the larval stage into the adult stage [5–7]. Early larvae have a tube-like digestive tract and lack a stomach. Adult-type digestive enzymes such as amylase, proteases, and lipases appear successively, with all being present by approximately 50 days post-hatch (dph), depending on temperature and feeding conditions [8–11]. In the same period, skin and skin pigmentation, bone, scales, osmoregulatory capacity, gills, and other systems develop into the adult forms, as in fish in general [12–18]. Peak metamorphosis, traditionally defined as the disappearance of

n

Corresponding author. Fax: þ 47 55905299. E-mail address: [email protected] (K. Hamre).

the larval finfold, occurs at 15 mm SL in cod and coincides with the appearance of the stomach and pyloric caeca [19]. The redox environments of cells and organisms are under strict control through regulation of the concentrations and ratios of several redox couples, the major systems being the glutathione (GSH) couple (glutathione disulfide (GSSG)/2GSH), the cystine/ cysteine couple (CysSS/2CysSH), and the thioredoxin couple (Trx (SS)/2Trx(SH)2) [20]. GSSG/2GSH is considered the most important redox couple and is present in all cells in millimolar concentrations, usually at ratios higher than 100:1 [21]. The extracellular CysSS/2CysSH pool is larger and more oxidized than the GSSG/ 2GSH pool [20]. Thioredoxin (Trx) is a protein involved in reduction of inter- and intraprotein disulfides, in contrast to GSH, which reduces small disulfide molecules and reacts directly with reactive oxygen species (ROS). The thioredoxin system is the third most important couple involved in determining cellular reduction potentials. TrxSS is reduced by thioredoxin reductase (Trxrd) at the expense of NADPH. The three couples are not at equilibrium, have different reduction potentials, and appear to be regulated independent of each other [20]. Evidence suggests that the redox environment regulated by these and perhaps other redox couples controls the oxidation states of protein thiols and consequently the

http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017 0891-5849/& 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

2

amount of intra- and extra-S-S bonding, glutathionylation, and phosphorylation of proteins. Changes in the three-dimensional conformation along with the addition of side chains to proteins, mediated by these reactions, change their activity [21,22]. Regulation of the average reduction potential (Eh) can therefore modulate signaling pathways and hence change cell fate. This is in accordance with the finding that proliferating cells have a lower Eh (are more reduced) than differentiating cells [23,24] and that apoptosis occurs under the least reduced, and thus most oxidized, cellular conditions [22,25–27]. ROS in low concentrations act as signaling molecules in normal metabolism. The main ROS generated by external signals and cell metabolism is the superoxide anion (O2  ). Superoxide anion signaling is often mediated by NADPH oxidases (Nox’s), whereas the superoxide dismutases (SODs) are involved in regulating O2  by metabolizing it to hydrogen peroxide (H2O2). In turn, H2O2 is a major redox signaling molecule that can diffuse through biological membranes [20] and is reduced to water by the antioxidant enzymes glutathione peroxidase (GPx) and catalase (Cat). In addition, reactive nitrogen species and lipid oxidation products, such as hydroxyl nonenal, participate in redox signaling [20]. The oxidants all participate in the regulation of the cellular reduction potential, perhaps in local microenvironments, and can, even at low concentrations, have profound effects on metabolism [20]. Larval ontogeny involves cell differentiation when the various organs and tissues form, proliferation when they grow, and apoptosis when larval-type tissue is replaced by adult-type tissue [28,29]. Therefore, one can hypothesize that the redox balance during larval development is spatially and temporally dynamic to allow for these processes to occur, in line with our recent study on the ontogeny of the redox balance in cod embryos [30]. Timme-Laragy et al. [31] investigated glutathione redox dynamics in zebrafish embryos and concluded that ontogeny occurs in distinct phases of development accompanied by changes in organismal reduction potentials, also in line with the above-mentioned hypothesis. To explore this hypothesis for the development of cod larvae, we have measured the concentration and ratio of the GSSG/2GSH couple, the activities of antioxidant enzymes, and the mRNA of genes involved in ROS metabolism and in GSH and protein thiol metabolism, and we have calculated the reduction potential during cod larvae development. To monitor cell cycle progression and cellular stress, we examined the mRNA expression of the cell-cycle-regulating proteins ccne2, cdk2, pcna, p53, and ccar1 and the heat shock proteins hsp90A, hsp70, and stip1; and we analyzed myod, myog, and pax7 as markers of muscle development.

7.1 71.0 1C at 3 dph to 11.270.5 1C by 17 dph, at which it was maintained for the rest of the sampling period. Larval tank oxygen saturation was maintained between 84 and 97% during the trial period by an automated aeration system. Sampling procedures Pooled samples of whole larvae were taken from the three tanks at every sampling point from 3 to 62 dph. The SL (from the tip of the nose to the end of the notochord) of the sampled larvae, together with the dietary regime, is given in Fig. 1. The growth was characterized as good compared to other trials with intensively reared larvae [32] and the survival of the population from fertilized eggs to juveniles ready for sale was 25% (Espen Grøtan, Cod Culture Norway, personal communication), indicating healthy larvae. This series of samples was also used for a study of the ontogeny of lipid digestive enzymes [10,11]. Fish were collected from approximately 40 cm below the surface and from the areas in the tank with the highest fish densities. The larvae were filtered using a plankton net screen, which was patted dry from underneath with a paper towel to remove excess water from the larvae. All larvae were killed by inserting a needle through the brain. The whole larvae were then collected in 1.5-ml Eppendorf tubes and the samples processed immediately. For RNA extraction the whole larvae were pooled (0–13 dph, 15 larvae per tank; 20– 34 dph, 10 larvae per tank; 48 dph, 7 larvae per tank; 62 dph, 1 larva per tank) and homogenized with a micropestle in Eppendorf tubes containing 1 ml Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA), frozen on dry ice, and stored at  80 1C until RNA extraction. For enzyme activities and GSSG/2GSH concentrations, larvae were flash-frozen in liquid nitrogen without any treatment. At every sampling point, standard length (minimum n¼ 10 fish/tank) was measured. All fish sampled for measuring length (SL) were euthanized with an overdose of metacaine (MS222TM; Norsk Medisinaldepot AS, Norway) dissolved in seawater. RNA extraction and qPCR The samples stored in Trizol were thoroughly homogenized using a Precellys 24 (Bertin Technologies, Montigny le Bretonneux, France). Total RNA was extracted using Trizol reagent (Invitrogen, Life Technologies), according to the manufacturer’s instructions. Isolated RNA was diluted in RNase-free double-distilled water 30 24

Materials and methods Cod larvae Atlantic cod (G. morhua) larvae were sampled from three production tanks at a commercial hatchery (Cod Culture Norway, Rong, Norway). The fish were maintained in accordance with the Animal Welfare Act of 20 December 1974, No. 73, Section 20–22, amended 19 June 2009. Eggs were collected from a natural spawning brood stock population containing a large number of individuals (55 females and 39 males) and therefore probably originated from several parents. Eggs were incubated for 15 days at 7 1C, and upon hatching larvae were transferred to three startfeeding tanks (diameter 3 m, depth 1 m, volume 7000 L) and fed rotifers (Brachionus plicatilis) from 3 dph. Formulated diet (Gemma Micro, Skretting, Norway) was introduced to the cod larvae at 18 dph and co-fed with rotifers until 30 dph. From 31 dph on, the larvae were fed solely on the formulated diet. The water temperature in the sampling tanks was raised incrementally from

20

SL (mm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

15 10

10 4.7

5.4

6.8

0 0

20

40

60

80

Age (dph) Rotifers Formulated diet Fig. 1. Standard length (SL mm, mean 7SD; mean values are given above the points) and periods of diet administration for larvae sampled for analyses.

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Q2 65 66

(ddH2O) to 200 ng/μl and treated with DNA-free TM (Ambion) according to the manufacturer’s description. The concentration and purity of the RNA were assessed with the NanoDrop ND-1000 UV/Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The OD260 nm:OD280 nm ratios for total RNA samples ranged between 1.87 and 2.06. Integrity of the RNA was analyzed in 12 randomly chosen samples using the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and the RNA 6000 Nano LabChip kit (Agilent Technologies). RNA integrity numbers [33,34] were between 8.9 and 10.

3

RT-PCR was run in duplicates on a 96-well plate. Twofold serial dilutions of total RNA in triplicates were run for efficiency calculation, ranging from 1000 to 31 ng/μl. Total RNA input for each reaction was 500 ng/well. No-template controls and RT controls (a reaction without RT enzyme) were run for each assay. Plates were run on a GeneAmp PCR 9700 (Applied Biosystems, Foster City, CA, USA) using TaqMan reverse transcription reagent containing Multiscribe reverse transcriptase (50 U/μl; Applied Biosystems). Reverse transcription was performed at 48 1C for 60 min by using oligo(dT) primers (2.5 μM) for all genes in 30 μl

Table 1 Gene abbreviation, GenBank accession number, forward and reverse primer sequences, primer amplicon size, and qRT-PCR efficiency for reference and target genes analyzed. Abbreviation

Accession No.

Forward primer

Reverse primer

Amplicon size

PCR eff.

Reference genes Ubiquitin rpl37 hsp90β

EX735613 EX738140 EX730769

GGCCGCAAAGATGCAGAT CCGAGAAGCGCAAGAGAAAG CGTGGCGTGGTGGACTCT

CTGGGCTCGACCTCAAGAGT GGTGGTACCTTCCCGGAATC GACTATGTTCTTGCGGATGACCTT

69 131 96

2.06 1.82 1.91

EX725875 EX724801 EX721840 EX727686 EX729450 EG641174 ES480178 EX728920 EX727949 GE905819 CO541611 DQ270487 CO542775 EX741726 EX732872 EG632414 EX734107 4 GmE090818r4687 4 isotig18352 gene¼ isogroup11902 EX735558 EX723548 4 GmE090818r6461 JQ582407 JQ619515

CCAAATATGGACGGCATAGGA CGTTCTCGGGTTTCCCTGTA CCCTGTGGAAGTGGCTGAAG GATCTGAATGAGCGCCTGAAG ACCGCAACGTGGTCTTCCT AGACTCCAAACGGTCAGGAGGTA CGAGAACGAGTCCGATCACTT TCACGCTCACCACCAAGGA GCCTATATGATTGGCCTGATGAC ATGTGGCCTCCTCCATTGAA CATGGCTTCCACGTCCATG GCCAAGTTGTTTGAGCACGTT CCTTGCGACTGCACCAAGA CATGACATCGTCCTGGTTGGT GGCTGGACCGCCAACA GCTGGAGCATCTCGTTGAAGA CCGGGATGCTAAGCTCTTCA TGGCACAACATGCTGGAAAA

CAAACGCTACAGCCGGAACT GCTCAAACAGCGGGAACGT CATCCAAGGGTCCGTATCTCTT GAAGCTGCTGAACTGGATGCT ATTCGCCCCAGCAAAGTTATC GAGTTCACCGTGGCCAACA GTCAGTCAGCTGCACCTCCAT GTGTGGAGGCCAGTCGTGTT GCTGTGCTGAGTGGGTCGTA GCATCACGCCACCTATGTCA CGTTTCCCAGGTCTCCAACAT CTGGGATCACGCACCGTATC CAGTTTAGGCAGGTGCATGATG CGTAGGCCACAGCTTCATCA GTGCTTCTTAGCCGCCATGT GGCAAGTTCGACTGCAGCAT CCTTGCGTGTGTACCCTTTGA ACCTCCATGAGCCAATCCAA

101 125 129 144 134 126 133 121 107 129 133 101 62 121 87 121 131 116

1.95 1.95 2.02 1.91 1.99 2.03 2.02 1.86 2.00 1.96 1.94 2.00 1.95 1.95 1.91 2.03 1.95 1.95

ACCCCCAACAACGACATCTG ACTCCTACCGCTGCGACAGA CGCTGCTGCTGAACTTCATG ACTGCTCCGACGGCATGA CGCTGAAGAGGAGCACCCTGATG CGTGTTGAGGGCCCGGTTTGGCA

AGGCCGTTCTTGTCCAGGTT CGAGCGTGTCAGCATTGTCT GGATGGCTCTCCGGTTCAT CTGGAAATGACCGATTTTTTGC TCCTGCTGGTTGAGCGAGGAGAC CCTCGTCTGTGCGGTTGCCTTTA

119 126 63 116 121 131

2.14 1.84 2.04 1.90 1.73 1.81

Target genes gpx1 gpx3 gpx4b glrx3 trx1 trxrd3 gclc gr nox1 Mnsod CuZnsod cat mt hsp70 hsp90a ccar1 stip1 ccne2 cdk2 pcna p53 myog myod1h1 pax7

Fig. 2. PCA plots of unfiltered (A) gene expression and (B) enzyme activity data of cod larvae. Gene expression and enzyme activity data obtained after (A) 3–62 and (B) 11– 63 days post-hatch, respectively, were visualized by PCA without prior statistical filtering. The colored nodes represent days post-hatch as indicated by the key. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

4

total volume. The final concentrations of the other chemicals in each RT reaction were MgCl2, 5.5 mM; dNTPs, 500 mM each; 10  TaqMan RT buffer, 1  ; RNase inhibitor, 0.4 U/μl; and Multiscribe reverse transcriptase, 1.67 U/μl. See Table 1 for a list of the primers use for PCR. The cDNA from each RT reaction was pipetted in duplicates into 384-well plates and qPCR run using TaqMan Universal PCR Master Mix (Applied Biosystems) and 50 μM forward and reverse primers. Real-time PCR was run on the LightCycler 480 Real-Time PCR System (Roche Applied Sciences, Basel, Switzerland), with a 5-min activation and denaturation step at 95 1C, followed by 40 cycles of a 15-s denaturing step at 95 1C, a 60-s annealing step, and a 30-s synthesis step at 72 1C. The annealing temperature was 60 1C for all primer pairs. For the mean normalized expression (MNE) calculations of the target genes, the geNorm VBA applet for Microsoft Excel 97 was

used to calculate a normalization factor based on three reference genes [35]. Ubiquitin, RPL37, and HSP90B were the selected reference genes based on Olsvik et al. [36] and Saele et al. [37], and the normalizing factor calculated from these genes was used to calculate the MNE for each of the target genes.

Enzyme activity and GSSG/2GSH concentration analysis To assess the redox status of the developing cod larvae, the concentrations of total glutathione (tGSH; GSH þGSSG) and GSSG and the enzyme activities of total superoxide dismutase (Mn and CuZn SOD; tSOD), Cat, and GPx were analyzed. Supernatants were prepared and analyzed for enzyme activities in a manner similar to that in [30]. Briefly, frozen samples (128756 mg, mean 7SD, n ¼ 23) were placed in Eppendorf tubes

tGSH

GSSG

2000

*

*

12

Embryo Larvae

ns

10

ns

1500 µmol/kg

µmol/kg

8 1000

6 4

500 2 0

0 0

20

40

60

0

20

40

dph

60

dph

Eh (mV)

GPx 5

-200

ns

nmol/min/mg protein

**

mV

-225

-250

-275

**

4 3 2 1 0

-300 0

20

40

60

0

20

dph

40

60

40

60

dph

tSOD

Cat

3

25

**

*

20 U/min/mg protein

U/min/mg protein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

2

1

15 10 5

0

0 0

20

40 dph

60

0

20 dph

Fig. 3. Total glutathione (tGSH, GSH þGSSG), glutathione disulfide (GSSG), reduction potential (Eh), and activities of glutathione peroxidase (GPx), total superoxide dismutase (tSOD), and catalase (Cat) during ontogeny of cod larvae from 7 or 11 until 63 days post-hatch. Open circles indicate the GSSG/2GSH concentrations and E just before hatching [30]. Data are given as the mean 7SD, n¼3, except for GSSG and Eh at 7 and 14 dph, for which n¼2, owing to values of GSSG below the detection limit of the method. GSSG/ 2GSH and Eh data were analyzed separately with ANOVA for two periods, 0–20 and 20–63 dph, whereas enzyme activities were analyzed as one dataset. Significant differences determined with ANOVA are marked: *po 0.05; **p o 0.01.

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

(2 ml), a 7  volume of ice-cold homogenization buffer (50 mM potassium phosphate, 0.95 mM EGTA, pH 7.2) was added, and the sample was homogenized with a ball mill (30 shakes/s for 1 min;

Retsch MM301 ball mill, Haan, Germany). The homogenate was then centrifuged (5 min, 1500g, 4 1C). For tSOD analysis, a 100-μl aliquot of the clear upper layer was transferred to a new tube and

gpx3

gpx1 2.0

1.5

1.5

***

**

MNE

MNE

1.0

1.0

***

0.5

ns

0.5

0.0

0.0 0

20

40

60

0

20

dph

40

60

40

60

40

60

dph

gpx4b

gr

1.0

1.0

**

ns

0.8

*

0.9

ns

0.8 MNE

MNE

0.6 0.7

0.4 0.6 0.2

0.5

0.0

0.4 0

20

40

60

0

20

dph

dph

gclc

trx1 1.0

1.0

**

ns

*** 0.8

0.8

** 0.6 MNE

MNE

0.6 0.4

0.4

0.2

0.2 0.0

0.0 0

20

40

60

0

20

dph

dph

trxrd

glrx3

1.5

1.0

ns

*

ns

*

0.8

1.0 0.6 MNE

MNE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

5

0.4

0.5 0.2 0.0

0.0 0

20

40 dph

60

0

20

40

60

dph

Fig. 4. Gene expression of glutathione and SH-group metabolic enzymes during ontogeny of cod larvae from 3 until 62 days post-hatch. Data are given as the mean 7SD, n¼ 3, except at 48 dph, for which n ¼2, owing to loss of one replicate during analysis. The data were analyzed with ANOVA for two periods, 0–20 and 20–63 dph. MNE, mean normalized expression. Significant ANOVAs are marked: *p o 0.05; **po 0.01; ***p o 0.001. See Table 2 for explanation of gene abbreviations.

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

100 μl of ice-cold buffer (50 mM potassium phosphate, pH 7.2, 400 mM mannitol, 130 mM sucrose, 0.95 mM EGTA) was added. The remaining homogenate was then centrifuged further (15 min, 10,000g, 4 1C). For Cat analyses the clear supernatant was used without further modification. For GPx analyses, 100 μl of the clear supernatant was added to 50 μl of ice-cold buffer (50 mM potassium phosphate, pH 7.6, 10 mM EDTA, 3 mM dithiothreitol) in a new tube. For GSH analyses, frozen samples (55 7 2 mg) were placed in tubes along with a 1  volume of ice-cold saline buffer (9 g/L NaCl in ddH2O) and homogenized as described above. Metaphosphoric acid solution (5% w/v) was then added to the tubes (600 μl), and the tube was shaken by hand and centrifuged (15 min, 10,000g, 4 1C), and the clear supernatants were transferred to new tubes. Samples for GSSG (52 73 mg) were treated as for tGSH samples, except an additional 50 μl ice-cold scavenger (N-ethylmaleimide pyridine derivative solution, Cat. No. GT40c; Oxford Biomedical Research, Oxford, UK) was included before homogenization. All extracts were frozen on dry ice immediately upon preparation and stored at  80 1C for a maximum of 1 month before analysis. The SOD, GPx, and Cat activities were analyzed with commercial kits (Items 706002 (SOD), 703102 (GPx), 707002 (Cat); Cayman Chemical Co., Ann Arbor, MI, USA). The GSH and GSSG were analyzed with a commercial kit (Product No. GT40; Oxford Biomedical Research). Total protein concentrations of supernatants for enzymes were measured with a Coomassie brilliant blue reagent kit (Bio-Rad Laboratories, Hercules, CA, USA). All samples were analyzed spectrometrically for absorbance in ELISA plates with a microplate reader (iEMS Reader Ms; Labsystems, Finland) measuring tSOD at 450 nm, GPx at 340 nm, Cat at 531 nm, GSSG/ 2GSH at 405 nm, and protein at 531 nm.

Calculations and statistics The two-electron half-cell reduction potential of the GSSG/2GSH redox couple was calculated according to the Nernst equation, Eh ¼ E00 RT=nF ln ð½GSH2 =½GSSGÞ; where the concentrations of GSH and GSSG are given in mol/kg and Eh is given in volts. E00 is the standard reduction potential at pH 7 and 25 1C and was assumed to be 0.240 V [22,25]. The measurements are the average of whole larvae and do not take into account that the reduction potential varies between organs and between organelles within the cells [22,25,38,39]. Principal component analysis (PCA) was performed using Qlucore Omics Explorer 2.3 (Qlucore AB, Lund, Sweden). Based on the outcome of the PCA, the ontogeny data on gene expression, GSSG/2GSH, and Eh were divided into early (0– 20 dph) and late (20–62 dph) development and analyzed separately. The data on enzyme activity were analyzed as one time series. Data that met the assumptions of parametric tests were subjected to ANOVA and Tukey’s post hoc test in Statistica (version 11; Statsoft, Inc., Tulsa, OK, USA). Data that violated the assumptions of parametric tests were analyzed with robust ANOVA and robust post hoc tests [38] using the package WRS (version 0.21) in R (version 3.0.1). Product-moment and partial correlations were performed on 0- to 20-dph gene expression data, using Statistica. Data are presented as means7SD, and differences were considered significant at po0.05.

Results Patterns of gene expression and enzyme activities changed during ontogeny, as shown by PCA (Fig. 2). The results on gene

Table 2 Gene abbreviation, name, and gene product function for the target genes analyzed. Abbreviation Name

Function

Ref.

gpx1 gpx3 gpx4b gr gclc

Reduces hydrogen peroxide to form water, localized in the cytosol and in the mitochondria Reduces hydrogen peroxide, localized in the extracellular fluid, kidneys, and embryonic tissues Reduces phospholipid hydroperoxide, localized in membranes, various isoforms Reduces glutathione disulfide to glutathione (GSH), NADPH dependent Combines Glu and Cys as the first step to produce GSH; the rate-limiting enzyme in GSH synthesis

[56] [56] [53] [56] [56,57]

Small protein that reduces inter- and intramolecular S–S bonds in proteins Reduces oxidized thioredoxins Cytosolic; reduces glutathionylated protein thiols Mitochondrial; transforms superoxide into hydrogen peroxide Cytosolic; transforms superoxide into hydrogen peroxide Reduces hydrogen peroxide to form water, mainly localized in peroxisomes Membrane-bound enzyme that catalyzes superoxide production Cysteine-rich protein that binds metals; involved in Zn metabolism; binds Cu to prevent redox cycling and protects against Cd toxicity; may act as antioxidant and interacts with GSH; prevents apoptosis Involved in binding of coactivators to gene promoters in response to activity of nuclear receptors and transcription factors; implicated in regulation of cell cycle and stimulation of apoptosis Chaperone; forms a complex in which Stip1 acts as a cochaperone; folds newly synthesized proteins and refolds proteins unfolded owing to, e.g., oxidative stress; may stimulate cell proliferation Chaperone; forms a complex in which Stip1 acts as a cochaperone; folds newly synthesized proteins and refolds proteins unfolded owing to, e.g., oxidative stress Chaperone; forms a complex in which Stip1 acts as a cochaperone; folds newly synthesized proteins and refolds proteins unfolded owing to, e.g., oxidative stress Binds and activates cyclin-dependent kinases Cell cycle regulation; necessary for cells to transverse the G1/S transition in mitosis Auxiliary protein of DNA polymerase-δ, essential for mitosis and proliferation

[20,21] [58] [20,24] [27,59] [27,59] [27] [27] [60]

Transcription factor inducing DNA damage-induced transcription, such as inducing reversible cell cycle arrest, apoptosis, and DNA repair Muscle-specific transcription factor; a marker of entry of myoblasts into the differentiation pathway in mammals Muscle-specific transcription factor; in mammals upregulated in proliferating myoblasts concomitant with upregulation of pcna Marker of muscle-specific stem cells such as satellite cells in mammals; involved in lineage specification

[52]

trx1 trxrd glrx3 Mnsod CuZnsod cat nox1 mt ccar1

Glutathione peroxidase 1 Glutathione peroxidase 3 Glutathione peroxidase 4b Glutathione reductase γ-Glutamylcysteine synthetase Thioredoxin 1 Thioredoxin reductase Glutaredoxin 3 Mn superoxide dismutase CuZn superoxide dismutase Catalase NADPH oxidase 1 Metallothionein

hsp70

Cell cycle and apoptosis regulator 1 Stress-induced phosphoprotein 1 Heat shock protein 70

hsp90a

Heat shock protein 90a

ccne2 cdk2 pcna

Cyclin E2 Cyclin-dependent kinase E2 Proliferating cell nuclear antigen Tumor-suppressing protein p53 Myogenin Myogenic differentiation factor 1, homolog 1 Paired box transcription factor

stip1

p53 myog myod1h1 pax7

[49] [50,51] [50,51] [50,51] [47] [47] [48]

[43] [44] [45]

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

expression showed that the replicates were well grouped and separated from one another. There was also a shift in patterns between early (0–20 dph) and late (20–63 dph) larval stages and therefore these periods were treated separately in subsequent statistical analyses. The PCA plot based on enzyme activities was not as defined as for gene expression, as sampling points overlapped to a larger extent. However, there was still a direction of change with larval age. The data on GSH and reduction potential (Eh) were also treated separately in early and late larval periods, whereas enzyme activity data were treated as one dataset for the whole period or as subsets of data according to the visual appearance of the graphs. The concentration of tGSH increased sharply from 7 until 14 dph (Fig. 3 A), from 610 7100 to 1260 7150 μmol/kg (mean7 SD, p ¼0.02). After 20 dph, tGSH was constant until 49 dph, whereafter there was a decrease to 810 7100 μmol/kg by 63 dph (p ¼0.02). In contrast, the concentration of GSSG was constant throughout the larval period at 3.17 1.4 μmol/kg (mean 7SD of all

Mnsod

1.0

CuZnsod

1.0

ns

*

0.8

*

0.8

0.6

*

0.6 MNE

MNE

data, Fig. 3B). This resulted in a decreasing reduction potential from  239 71 to 262 77 mV from 7 to 14 dph (p¼ 0.02, Fig. 3C). From 14 until 63 dph there were no changes in reduction potential. When treating data from the whole period, the activity of GPx (Fig. 3D) was stable from 11 to 28 dph, increased significantly from 28 to 42 dph, and then decreased again from 42 until 63 dph (p ¼0.007). However, when analyzed as data subsets, GPx activity decreased from 11 to 28 dph (p¼ 0.04). Total SOD activity (Fig. 3E) increased from 28 until 35 dph (p ¼0.003), whereas Cat activity (Fig. 3F) was lower during the late (20–63 dph) compared to the early larval stages (p ¼0.03). The GSH and protein–SH-metabolizing enzymes for which gene expression was measured were gclc; gpx1, 3, and 4b; gr; trx; trxrd; and glrx3 (Fig. 4; see Table 2 for gene abbreviations). The expression of gclc, the rate-limiting enzyme in GSH synthesis, had a characteristic profile, similar to several other genes in this study, with high expression at 6 and 10 dph compared to 3 and 13 dph (p ¼0.01). The expression of gclc

0.4 0.2

0.4 0.2

0.0

0.0 0

20

40

60

0

20

40

dph

cat

1.5

nox1

1.5

ns

ns

1.0

1.0

ns

MNE

MNE

*

60

dph

0.5

0.5

0.0

0.0 0

20

40

60

dph

0

20

40

60

dph

mt

1.5

*

*** 1.0 MNE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

7

0.5

0.0 0

20

40

60

dph

Fig. 5. Expression of genes involved in metabolism of reactive oxygen species and in regulation of cell proliferation, differentiation, and apoptosis during ontogeny of cod larvae from 3 until 62 days post-hatch. Data are given as the mean 7 SD, n¼ 3, except at 48 dph, for which n¼ 2, as one replicate was lost during analysis. The data were analyzed with ANOVA for two periods, 0–20 and 20–63 dph. MNE, mean normalized expression. Significant ANOVAs are marked: *p o 0.05; ***p o 0.001. See Table 2 for explanation of gene abbreviations.

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

8

decreased from 20 to 62 dph (p ¼0.002). The early expression profiles of gpx4b and glrx3 were similar to that of gclc, with peak expression at 6–13 dph, compared to 3 and 20 dph. However, because of the large variation, expression of glrx3 at 3 and 13 dph

was not different from that at 6 or 10 dph, and also unlike gclc, both glrx3 and gpx4b had stable expression between 20 and 62 dph. The cytosolic and extracellular gpx1 and gpx3 both showed low expression in the early larval stages (0–20 dph).

ccne2

0.8

***

cdk2

1.0

***

*

ns

0.8

0.6 MNE

MNE

0.6 0.4

0.4 0.2

0.2

0.0

0.0 0

20

40

60

80

0

20

Day

40

60

80

60

80

Day

p53

pcna 1.0

1.0

***

***

ns

0.9

0.8

ns 0.8

MNE

MNE

0.6 0.7

0.4 0.6 0.2

0.5 0.4

0.0 0

20

40

60

0

80

20

stip 1

ccar1

1.5

40 Day

Day

1.0

ns

*

ns

*

0.8

1.0 MNE

MNE

0.6 0.4 0.5 0.2 0.0

0.0 0

20

40

60

0

20

dph

40

60

dph

hsp90

hsp70 1.0

1.0

ns

**

0.8 0.6

ns

***

0.8 0.6 MNE

MNE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

0.4

0.4

0.2

0.2 0.0

0.0 0

20

40 dph

60

0

20

40

60

dph

Fig. 6. Gene expression of the cochaperone stip1 and the chaperones hsp70 and hsp90a and the cell cycle regulators ccar1, ccne2, cdk2, pcna, and p53, during ontogeny of cod larvae from 3 until 62 days post-hatch. Data are given as the mean 7 SD, n¼ 3, except at 48 dph, for which n¼ 2, as one replicate was lost during analysis. The data were analyzed with ANOVA for two periods, 0–20 and 20–63 dph. MNE, mean normalized expression. Significant ANOVAs are marked: *p o 0.05; **po 0.01; ***p o 0.001; ns, not significant. See Table 2 for explanation of gene abbreviations.

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

myog

0.4

MNE

ns

**

0.3

0.2

0.1

0.0

0

20

40

60

80

60

80

60

80

Day

pax7

0.25

ns 0.20

**

0.15

MNE

The expression of gpx1 decreased between 3 and 20 dph (p ¼5  10  5), increased sharply between 20 and 34 dph (p ¼10  4), and remained constant thereafter, whereas gpx3 showed gradually increased expression until 62 dph (p¼ 0.03). Peak expression of gr occurred at 3 dph from which it decreased during the early larval phase (0–20 dph, p ¼0.02) and was stable from 20 until 62 dph. Expression of trx1 increased in the early larval period (p ¼7  10  4) and was constant in late larvae, whereas expression of trxrd was constant until a slight increase starting in the later larval phase from 34 dph (p ¼0.02). Expression of Mnsod, CuZnsod, cat, nox1, and mt was measured to target proteins involved in metabolism of water-soluble ROS (Fig. 5). mt expression was at a minimum at 6 and 10 dph compared to 3, 13, and 20 dph (p¼5  10  4). Furthermore, mt expression decreased slightly between 20 and 48 dph. The cytoplasmic CuZnsod showed an increase in expression between 3 and 20 dph (p¼0.04) and thereafter a decrease between 20 and 62 dph (p¼ 0.004), whereas the mitochondrial Mnsod decreased slightly in the early larval phase (p¼0.01) and was then stable from 20 until 62 dph. cat showed higher expression at 10 dph than at 6 and 20 dph. There were no significant changes in the expression of nox1 during larval development. The genes ccne2, cdk2, pcna, p53, and ccar1 (Fig. 6) are important in cell cycle progression. Early expression of ccne2, cdk2, pcna, and ccar1 was similar to that of gclc with peak expression at 6 and 10 dph (p¼ 5  10  4 for ccne2, 6  10  5 for cdk2, 0.005 for pcna, and 0.01 for ccar1). The peak expression of p53 was delayed compared to gclc and the other cell cycle genes and occurred at 10 and 13 dph (p¼0.0005). After 20 dph there were no significant changes in expression of the cell-cycle-regulating genes. The (co-) chaperones stip1 (p¼ 0.03) and hsp70 (p¼0.003) showed peak expression at 6 and 10 dph compared to 3 and 13 dph (Fig. 6), similar to gclc (Fig. 4). The expression of hsp90a had a profile between 3 and 13 dph that was similar to that of gclc, but the differences were not significant (p¼0.05–0.06). The expression of hsp90a, but not that of the other two chaperone genes, increased between 13 and 20 dph (p¼2  10  5). All three chaperones had stable expression between 20 and 62 dph. The genes myog, pax7, and myod1 are markers of myogenesis and muscle differentiation (Fig. 7). mRNA of myog increased (p ¼0.008) and that of myod1 decreased (p¼ 0.006) from 3 to 6 dph and was then stable until 20 dph, whereas pax7 expression did not change during early development (3–20 dph). From 20 to 63 dph there were no statistically significant changes in expression of myog, possibly because of large variation. pax7 mRNA decreased (p¼ 0.009) and myod1 mRNA increased (p ¼ 4  10  4) during the later larval stages. Correlation analyses on the gene expression data from 0 to 20 dph (Table 3) showed positive correlations of ccne2 with cdk2 and cdk2 with pcna (R2 40.72). The expression of all three of these genes correlated positively with gclc, stip1, and hsp70 and negatively with mt. Furthermore, ccne2 and cdk2 correlated with ccar1, whereas cdk2 and pcna correlated with gpx4. There were also significant correlations of the heat shock proteins stip1 and hsp70 with mt and gclc.

0.10

0.05

0.00

0

20

40

Day

myod1 h1

0.6

*** ** 0.4

MNE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

9

0.2

0.0 0

20

40

Day Fig. 7. Gene expression of the muscle-specific transcription factors myog, pax7, and myod1 h1 during ontogeny of cod larvae from 3 until 62 days post-hatch. Data are given as the mean 7 SD, n ¼3, except at 48 dph, for which n¼ 2, as one replicate was lost during analysis. The data were analyzed with ANOVA for two periods, 0–20 and 20–63 dph. MNE, mean normalized expression. Significant ANOVAs are marked: **p o 0.01; ***p o 0.001; ns, not significant. See Table 2 for explanation of gene abbreviations.

Discussion Glutathione concentrations, reduction potential, and larval ontogeny With regard to redox balance, larval ontogeny seems to occur in distinct phases, as shown earlier for cod and zebrafish embryos [30,31]. This is visualized by PCA of the gene expression data, which separates early (0–20 dph) and late (20–62 dph) development. Furthermore, the whole-body concentration of tGSH increased from approximately 500 to 1200 μmol/kg between 7 and 14 dph. During

gastrulation tGSH starts to increase and before hatching, the concentration of tGSH in cod embryos reaches 250 μmol/kg [30]. From 14 until 49 dph the concentration of tGSH was constant, followed by a decrease until 63 dph in this study. Because GSSG was constant, the change in tGSH led to a change in Eh from a relatively oxidized state at hatching (approximately  200 mV) [30] to a more reduced state between 14 and 49 dph ( 26177 mV, mean7SD of all data). This falls within the normal range of cell potentials, which is  260 to  200 mV [22,25]. The decrease in tGSH after 49 dph did not give a

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Table 3 Correlations in expression of selected genes at 0–20 dph.

ccne2 cdk2 pcna p53 gpx4 gclc mt stip1 hsp90a hsp70 ccar1

ccne2

cdk2

pcna

p53

gpx4

gclc

mt

stip1

hsp90a

hsp70

ccar1

1.00 0.72 0.45  0.16 0.41 0.76  0.55 0.69 0.09 0.63 0.59

0.72 1.00 0.74  0.03 0.64 0.72  0.72 0.81  0.04 0.87 0.79

0.45 0.74 1.00 0.20 0.66 0.53  0.66 0.51  0.12 0.62 0.38

 0.16  0.03 0.20 1.00 0.51  0.51 0.15  0.50  0.59  0.33  0.23

0.41 0.64 0.66 0.51 1.00 0.26  0.39 0.35  0.41 0.27 0.36

0.76 0.72 0.53  0.51 0.26 1.00  0.48 0.89 0.41 0.71 0.63

 0.55  0.72  0.66 0.15  0.39  0.48 1.00  0.55 0.34  0.79  0.46

0.69 0.81 0.51  0.50 0.35 0.89  0.55 1.00 0.25 0.80 0.86

0.09  0.04  0.12  0.59  0.41 0.41 0.34 0.25 1.00 0.03  0.07

0.63 0.87 0.62  0.33 0.27 0.71  0.79 0.80 0.03 1.00 0.77

0.59 0.79 0.38  0.23 0.36 0.63  0.46 0.86  0.07 0.77 1.00

Boldface numerals indicate a significant correlation (Spearman rank, po 0.05).

significant increase in E, probably because of the large variation in GSSG. The reduction potential plays a critical role in controlling the oxidation, reduction, and glutathionylation of protein thiols, which in turn modulates protein phosphorylation. As a consequence, the reduction potential is important in determining the activity of a range of proteins including those within enzyme systems, transcription factors, and DNA methylation and posttranscriptional control mechanisms of gene expression [21–23,25,27]. The magnitude of the cellular changes can be large, because a change in potential from 260 to  200 mV can give a 100-fold change in protein phosphorylation [22]. Many of the analyzed genes had different expression patterns before and after 13 or 20 dph, coinciding with the change in the curve of tGSH concentration. Cod larvae have absorbed most of their yolk sac and begin feeding at 4 dph. Because of their immature digestive tract they are unable to utilize formulated feeds, and in culture they are normally fed rotifers (live feed) for the first 20–30 days [4,32]. In this study, the larvae were offered formulated feed at 18 dph, which was co-fed along with rotifers until 30 dph. After 30 dph, the larvae were fed only formulated feed. However, the shifts in GSH concentrations and Eh did not coincide with change of feed. Metamorphosis in cod is defined as the time of absorption of the larval finfold at 15 mm standard length [40], which occurred when the cod larvae were at 48 dph in this study. However metamorphic changes in organ systems occur during a distended period [19], which is currently not well described. Because the muscle represents 40–60% of fish biomass [7], it has an important contribution to results from measurements in whole larvae. Muscle development has been extensively studied in herring larvae [5,6]. After hatching, there is a period of stratified hyperplasia, in which stem cells in discrete germinal zones at the edge of the myotome undergo asymmetric cleavage. One daughter cell migrates into the myotome to form myogenic progenitor cells. The second phase of myogenesis is called mosaic hyperplasia, in which myogenic progenitor cells scattered in the myotome continue to proliferate in parallel with differentiation, e.g., fusions of cells and hypertrophy of individual muscle fibers. The two types of muscle growth overlap during metamorphosis; however, stratified hyperplasia is exhausted in early juveniles, whereas mosaic hyperplasia continues until the fish has reached 40% of its maximal length [5]. Stratified hyperplasia gives a relatively slow increase in muscle mass, whereas mosaic hyperplasia leads to a large increase in number and cross-sectional area of fibers within the muscle [6,41], manifesting itself in a high larval growth rate. Our hypothesis is that mosaic hyperplasia in cod started around 20 dph in this study and that this was the reason for the apparent two phases of ontogeny. Galloway et al. [42] studied muscle development in Atlantic cod; however, the larvae were kept only until 31 dph. The larval growth was slower than in the present study, and the largest sampled larvae were 8 mm SL. The period of

stratified hyperplasia was confirmed, and there was a large increase in dry weight per larva and total cross-sectional area of muscle fibers after 17 dph, supporting the hypothesis of a shift in muscle metabolism. These authors also marked the beginning of metamorphosis as the appearance of dorsal fin rays at 20 dph. We measured mRNA of the myogenic transcription factors myod and myog and the muscle-specific stem cell marker pax7, to verify the hypothesis of a shift in muscle ontogeny. According to findings in mammals, myod is a marker of proliferating myoblasts, whereas myog is a marker of myoblast differentiation [43,44]. In general, the expression profiles found here were not in line with mammalian results, but fish may express the markers differently. The expression of myod was low before 20 dph and then started to increase, perhaps as a result of beginning mosaic hyperplasia. pax7 was high early in development and decreased with age, in line with findings in mice [45]. The differentiation of the stomach is completed around 40 dph (10 mm SL) in cod [19]. Kamisaka and Rønnestad [46] found that the growth of the digestive tract tissue was slow before the stomach had been formed and increased substantially thereafter. The digestive tract represents only about 5% of body mass, so any changes in gene expression in this organ will be diluted when whole larval bodies are analyzed, as in this study. If the appearance of fin rays and start of mosaic hyperplasia mark the beginning of metamorphosis and the formation of the stomach, and disappearance of the larval finfold at 40–50 dph marks the end of metamorphosis, this correlates with the period of high tGSH in the present study. Early ontogeny of gene expression (0–20 dph) The mRNA expression of the rate-limiting enzyme in GSH synthesis, gclc, was high at 6 and 10 dph compared to 3 and 13 dph. The relatively high expression of gclc at 6 and 10 dph is in agreement with the increase in tGSH concentration between 4 and 14 dph. When tGSH concentration stabilized between 14 and 49 dph, gclc expression was gradually decreasing. An early profile similar to that of gclc was seen for genes coding for proteins involved in cell cycle progression (ccne2, cdk2, pcna, and ccar1) and for the chaperones (stip1, hsp70, and hsp90a). However, hsp90a showed a prominent increase in expression between 13 and 20 dph, in contrast to the other mentioned above. Cyclin E2 (coded by ccne2) in complex with cyclin-dependent kinase 2 (cdk2) peaks during the G1/S phase of mitosis and is thought to be rate limiting in cell cycle progression [47], whereas Pcna is widely used as a marker of proliferating cells [48]. Ccar1 has been implicated in stimulation of apoptosis and cell proliferation [49]. Stip1 is a cochaperone, which is necessary for formation of the chaperone complex between Hsp70 and Hsp90 [50]. The best known function of the chaperones is folding of newly synthesized proteins or

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

refolding of proteins denatured as a result of stress, for example, oxidative stress [50,51]. The similarity in the mRNA expression profiles of these genes with gclc can be interpreted as a link between GSH synthesis and the development of the reduction potential to the general cell cycle machinery. The early mRNA expression profile of mt was approximately the inverse of the gclc profile. The peak expression of p53 was 4 days delayed compared to the other cell-cycle-regulatory genes, the chaperones and mt. Target genes of p53 regulate cell cycle progression, modulate DNA repair, and induce apoptosis [52]. A delayed peak expression was also found for gpx4b, a glutathione peroxidase that reduces lipid hydroperoxides [53]. The expression profiles of these genes may indicate that the cod larvae encountered oxidative stress during the first 20 days of development, causing DNA damage and reactions of DNA repair, cell proliferation, and apoptosis to replace damaged cells. Oxidative stress that is compensated for may happen in parallel with a well-balanced cellular redox environment and the compensation in itself may lead to changes in gene expression. Alternatively, the high expression early in development may be coupled to proliferation of myogenic progenitor cells during stratified hyperplasia. Further studies are needed to elucidate this. The early expression profiles of gpx1, gpx3, and gr were low and decreasing and in agreement with the highly reduced state of the GSSG/2GSH redox couple and the lowering of E. The increasing trx1 expression may be a parallel to the increase in GSH concentration, leading to a more reduced cellular environment. The glrx3 expression was again similar to expression of gclc and the proposed representatives of the cell cycle apparatus, although because of large variation the only significant differences were between 20 and 6–10 dph. An increased expression of CuZnsod may have favored increasing concentrations of H2O2 in the larvae during early development, which may have been part of the redox signaling. Late ontogeny of gene expression (20–62 dph) The redox-related genes that showed changes in expression during late larval stages were gpx1, gpx3, gclc, trxrd, CuZnsod, and mt. Both gpx1 and gpx3 showed increased expression, whereas CuZnsod decreased, perhaps indicating decreased H2O2 signaling. The expression of gclc decreased, corresponding to the decrease in tGSH. Enzyme activities (0–63 dph) Enzyme activities and gene expression did not correlate well in this study, as was the case in the study of redox regulation through embryogenesis by Skjaerven et al. [30]. The half-lives of various proteins can vary from minutes to days, whereas the degradation rate of mRNA would fall within a much tighter range (2–7 h for mammalian mRNAs versus 48 h for proteins [54]). The mammalian CuZnSOD protein has been found to be extremely stable, with a half-life of 25 to 100 h [23,55]. If a similar stability is found in cod, it is not surprising that the activities of this long-lived protein and the short-lived mRNA do not correlate in the current study. GPx activity is a measure of the sum of the activities of various GPx isoforms, which show differences in ontogenetic mRNA expression in this study. However, both GPx and tSOD activities showed an increase after 28 dph, which coincided with the suspension of rotifers from the diet, after which the larvae were fed only formulated feed.

Conclusions This study is a descriptive first approach to investigate the link between redox regulation and cod larval development. The ontogeny series appears to be divided into two main phases, the first of which

11

(0–20 dph) is characterized by a sharp increase in tGSH, decrease in average whole-body Eh, and dynamic changes in gene expression, which indicate a link between redox signaling and cell cycle regulation. The second phase occurs during later development (20– 62 dph), in which there are relatively few changes in gene expression. It is proposed that the increase in tGSH concentration and decrease in Eh between hatching and 20 dph coincide with the period when muscle-stratified hyperplasia is the dominating process of myogenesis. Metamorphosis appears to start with the onset of mosaic hyperplasia and end with differentiation of the stomach and disappearance of the larval finfold and seems to coincide with the period of high and stable tGSH concentration in this study.

Acknowledgments We thank Espen Grøtan from Cod Culture Norway (now Marine Harvest Labrus) for letting us collect the larval samples for this study from their cod hatchery near Bergen in western Norway. Thanks also to Andreas Nordgreen, for performing the sampling, and to Synnøve Wintertun and Hui-Shan Tung for analyzing the samples. References [1] Steneck, R. S. Apex predators and trophic cascades in large marine ecosystems: learning from serendipity. Proc. Natl. Acad. Sci. USA 109:7953–7954; 2012. [2] Food and Agriculture Organization of the United Nations. The state of the world fisheries and aquaculture. Rome: FAO; 2010. [3] Kjorsvik, E.; Vandermeeren, T.; Kryvi, H.; Arnfinnson, J.; Kvenseth, P. G. Early development of the digestive-tract of cod larvae, Gadus-morhua L, during start-feeding and starvation. J. Fish Biol. 38:1–15; 1991. [4] van der Meeren, T.; Mangor-Jensen, A.; Pickova, J. The effect of green water and light intensity on survival, growth and lipid composition in Atlantic cod (Gadus morhua) during intensive larval rearing. Aquaculture 265:206–217; 2007. [5] Johnston, I. A. Environment and plasticity of myogenesis in teleost fish. J. Exp. Biol. 209:2249–2264; 2006. [6] Johnston, I. A.; Cole, N. J.; Abercromby, M.; Vieira, V. L. A. Embryonic temperature modulates muscle growth characteristics in larval and juvenile herring. J. Exp. Biol. 201:623–646; 1998. [7] Valente, L. M. P.; Moutou, K. A.; Conceicao, L. E. C.; Engrola, S.; Fernandes, J. M. O.; Johnston, I. A. What determines growth potential and juvenile quality of farmed fish species? Rev. Aquacult 5:S168–S193; 2013. [8] Kortner, T. M.; Overrein, I.; Oie, G.; Kjorsvik, E.; Bardal, T.; Wold, P. A.; Arukwe, A. Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae. Comp. Biochem. Phys. A 160:190–199; 2011. [9] Kvale, A.; Mangor-Jensen, A.; Moren, M.; Espe, M.; Hamre, K. Development and characterisation of some intestinal enzymes in Atlantic cod (Gadus morhua L.) and Atlantic halibut (Hippoglossus hippoglossus L.) larvae. Aquaculture 264:457–468; 2007. [10] Saele, O.; Nordgreen, A.; Olsvik, P. A.; Hamre, K. Characterization and expression of digestive neutral lipases during ontogeny of Atlantic cod (Gadus morhua). Comp. Biochem. Phys. A 157:252–259; 2010. [11] Saele, O.; Nordgreen, A.; Olsvik, P. A.; Hamre, K. Characterisation and expression of secretory phospholipase A(2) group IB during ontogeny of Atlantic cod (Gadus morhua). Br. J. Nutr. 105:228–237; 2011. [12] Hamre, K.; Opstad, I.; Espe, M.; Solbakken, J.; Hemre, G. I.; Pittman, K. Nutrient composition and metamorphosis success of Atlantic halibut (Hippoglossus hippoglossus, L.) larvae fed natural zooplankton or Artemia. Aquacult. Nutr. 8:139–148; 2002. [13] Le Guellec, D.; Morvan-Dubois, G.; Sire, J. Y. Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio). Int. J. Dev. Biol. 48:217–231; 2004. [14] Saele, O.; Solbakken, J. S.; Watanabe, K.; Hamre, K.; Power, D.; Pittman, K. Staging of Atlantic halibut (Hippoglossus hippoglossus L.) from first feeding through metamorphosis, including cranial ossification independent of eye migration. Aquaculture 239:445–465; 2004. [15] Seikai, T. Process of pigment cell-differentiation in skin on the left and right sides of the Japanese flounder, Paralichthys-olivaceus, during metamorphosis. Jpn. J. Ichthyol. 39:85–92; 1992. [16] Sire, J. Y.; Akimenko, M. A. Scale development in fish: a review, with description of sonic hedgehog (shh) expression in the zebrafish (Danio rerio). Int. J. Dev. Biol. 48:233–247; 2004. [17] Tagawa, M.; Miwa, S.; Inui, Y.; Dejesus, E. G.; Hirano, T. Changes in thyroidhormone concentrations during early development and metamorphosis of the flounder, Paralichthys-olivaceus. Zool. Sci. 7:93–96; 1990. [18] Zapata, A.; Diez, B.; Cejalvo, T.; Frias, C. G.; Cortes, A. Ontogeny of the immune system of fish. Fish Shellfish Immun 20:126–136; 2006.

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

K. Hamre et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

[19] Pedersen, T.; Falk-Pedersen, I. B. Morphological changes during metamorphosis in cod (Gadus morhua L.), with particular reference to the development of the stomach and pyloric caeca. J. Fish Biol. 41:449–461; 1992. [20] Huseby, N. E.; Sundkvist, E.; Svineng, G. Glutathione and sulfur containing amino acids: antioxidant and conjugation activities. In: Masella, R., Mazza, G., editors. Glutathione and Sulfur Containing Amino Acids in Human Health and Disease. Hoboken, NJ: Wiley; 2009. [21] Dalle-Donne, I.; Rossi, R.; Giustarini, D.; Colombo, R.; Milzani, A. S-glutathionylation in protein redox regulation. Free Radic. Biol. Med. 43:883–898; 2007. [22] Kemp, M.; Go, Y. M.; Jones, D. P. Nonequilibrium thermodynamics of thiol/ disulfide redox systems: a perspective on redox systems biology. Free Radic. Biol. Med. 44:921–937; 2008. [23] Hoffman, A.; Spetner, L. M.; Burke, M. Ramifications of a redox switch within a normal cell: its absence in a cancer cell. Free Radic. Biol. Med. 45:265–268; 2008. [24] Lillig, C. H.; Berndt, C.; Holmgren, A. Glutaredoxin systems. Biochim. Biophys. Acta 780:1304–1317; 2008. [25] Schafer, F. Q.; Buettner, G. R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30:1191–1212; 2001. [26] Allen, R. G.; Balin, A. K. Oxidative influence on development and differentiation: an overview of free radical theory of development. Free Radic. Biol. Med. 6:631–661; 1989. [27] Ufer, C.; Wang, C. C.; Borchert, A.; Heydeck, D.; Kuhn, H. Redox control in mammalian embryo development. Antioxid. Redox Signaling 13:833–875; 2010. [28] Cole, L. K.; Ross, L. S. Apoptosis in the developing zebrafish embryo. Dev. Biol. 240:123–142; 2001. [29] Meier, P.; Finch, A.; Evan, G. Apoptosis in development. Nature 407:796–801; 2000. [30] Skjaerven, K. H.; Penglase, S.; Olsvik, P. A.; Hamre, K. Redox regulation in Atlantic cod (Gadus morhua) embryos developing under normal and heatstressed conditions. Free Radic. Biol. Med. 57:29–38; 2013. [31] Timme-Laragy, A. R.; Goldstone, J. V.; Imhoff, B. R.; Stegeman, J. J.; Hahn, M. E.; Hansen, J. M. Glutathione redox dynamics and expression of glutathionerelated genes in the developing embryo. Free Radic. Biol. Med. 65:89–101; 2013. [32] Busch, K. E. T.; Falk-Petersen, I. B.; Peruzzi, S.; Rist, N. A.; Hamre, K. Natural zooplankton as larval feed in intensive rearing systems for juvenile production of Atlantic cod (Gadus morhua L.). Aquacult. Res. 41:1727–1740; 2010. [33] Mueller, O.; Hahnenberger, K.; Dittmann, M.; Yee, H.; Dubrow, R.; Nagle, R.; Ilsley, D. A microfluidic system for high-speed reproducible DNA sizing and quantitation. Electrophoresis 21:128–134; 2000. [34] Imbeaud, S.; Graudens, E.; Boulanger, V.; Barlet, X.; Zaborski, P.; Eveno, E.; Mueller, O.; Schroeder, A.; Auffray, C. Towards standardization of RNA quality assessment using user independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 33; 2005. [35] Vandesompele, J.; De Preter, K.; Pattyn, F.; Poppe, B.; Van Roy, N.; De Paepe, A.; Speleman, F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3: RESEARCH0034; 2002. [36] Olsvik, P. A.; Lie, K. K.; Jordal, A. E. O.; Nilsen, T. O.; Hordvik, I. Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol. Biol. 6; 2005. [37] Saele, O.; Nordgreen, A.; Hamre, K.; Olsvik, P. A. Evaluation of candidate reference genes in Q-PCR studies of Atlantic cod (Gadus morhua) ontogeny, with emphasis on the gastrointestinal tract. Comp. Biochem. Phys. B 152:94–101; 2009. [38] Cherkasov, V.; Haunhorst, P.; Godoy-Berthet, J. R.; Schutte, L. D.; Hudemann, C.; Lonn, M. E.; Lillig, C. H. Redox compartmentalization of the mammalian cell: subcellular localization of human glutaredoxin isoforms. Eur. J. Cell Biol. 87; 2008. (37-37). [39] Hamre, K.; Torstensen, B. E.; Maage, A.; Waagbo, R.; Berge, R. K.; Albrektsen, S. Effects of dietary lipid, vitamins and minerals on total amounts and redox

[40]

[41]

[42]

[43]

[44]

[45]

[46] [47]

[48]

[49]

[50]

[51]

[52]

[53] [54]

[55]

[56] [57]

[58]

[59] [60]

status of glutathione and ubiquinone in tissues of Atlantic salmon (Salmo salar): a multivariate approach. Br. J. Nutr. 104:980–988; 2010. vonHerbing, I. H.; Boutilier, R. G.; Miyake, T.; Hall, B. K. Effects of temperature on morphological landmarks critical to growth and survival in larval Atlantic cod (Gadus morhua). Mar. Biol. 124:593–606; 1996. Campos, C.; Valente, L. M. P.; Conceicao, L. E. C.; Engrola, S.; Sousa, V.; Rocha, E.; Fernandes, J. M. O. Incubation temperature induces changes in muscle cellularity and gene expression in Senegalese sole (Solea senegalensis). Gene 516:209–217; 2013. Galloway, T.; Kjørsvik, E.; Kryvi, H. Muscle development in Atlantic cod larvae (Gadus morhua, L.) related to different somatic growth rates. J. Exp. Biol. 202:2111–2120; 1999. Andres, V.; Walsh, K. Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J. Cell Biol. 132:657–666; 1996. Megeney, L. A.; Kablar, B.; Garrett, K.; Anderson, J. E.; Rudnicki, M. A. MyoD is required for myogenic stem cell function in adult skeletal muscle. Gene Dev 10:1173–1183; 1996. Seale, P.; Sabourin, L. A.; Girgis-Gabardo, A.; Mansouri, A.; Gruss, P.; Rudnicki, M. A. Pax7 is required for the specification of myogenic satellite cells. Cell 102:777–786; 2000. Kamisaka, Y.; Ronnestad, I. Reconstructed 3D models of digestive organs of developing Atlantic cod (Gadus morhua) larvae. Mar. Biol. 158:233–243; 2011. Gudas, J. M.; Payton, M.; Thukral, S.; Chen, E.; Bass, M.; Robinson, M. O.; Coats, S. Cyclin E2, a novel G(1) cyclin that binds Cdk2 and is aberrantly expressed in human cancers. Mol. Cell. Biol. 19:612–622; 1999. Wullimann, M. F.; Knipp, S. Proliferation pattern changes in the zebrafish brain from embryonic through early postembryonic stages. Anat. Embryol. 202:385 400; 2000. Kim, J. H.; Yang, C. K.; Heo, K.; Roeder, R. G.; An, W.; Stallcup, M. R. CCAR1, a key regulator of mediator complex recruitment to nuclear receptor transcription complexes. Mol. Cell 31:510–519; 2008. Tsai, C. L.; Tsai, C. N.; Lin, C. Y.; Chen, H. W.; Lee, Y. S.; Chao, A.; Wang, T. H.; Wang, H. S.; Lai, C. H. Secreted stress-induced phosphoprotein 1 activates the ALK2–SMAD signaling pathways and promotes cell proliferation of ovarian cancer cells. Cell Rep 2:283–293; 2012. Freeman, M. L.; Borrelli, M. J.; Meredith, M. J.; Lepock, J. R. On the path to the heat shock response: destabilization and formation of partially folded protein intermediates, a consequence of protein thiol modification. Free Radic. Biol. Med. 26:737–745; 1999. Brodsky, M. H.; Weinert, B. T.; Tsang, G.; Rong, Y. S.; McGinnis, N. M.; Golic, K. G.; Rio, D. C.; Rubin, G. M. Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol. Cell. Biol. 24:1219–1231; 2004. Brigelius-Flohe, R.; Maiorino, M. Glutathione peroxidases. Biochim. Biophys. Acta 1830:3289–3303; 2013. Vogel, C.; Marcotte, E. M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat. Rev. Genet. 13:227–232; 2012. Borchelt, D. R.; Guarnieri, M.; Wong, P. C.; Lee, M. K.; Slunt, H. S.; Xu, Z. S.; Sisodia, S. S.; Price, D. L.; Cleveland, D. W. Superoxide-dismutase-1 subunits with mutations linked to familial amyotrophic-lateral-sclerosis do not affect wild-type subunit function. J. Biol. Chem. 270:3234–3238; 1995. Ufer, C.; Wang, C. C. The roles of glutathione peroxidases during embryo development. Front. Mol. Neurosci 4:12; 2011. Stover, S. K.; Gushansky, G. A.; Salmen, J. J.; Gardiner, C. S. Regulation of γ-glutamate–cysteine ligase expression by oxidative stress in the mouse preimplantation embryo. Toxicol. Appl. Pharmacol. 168:153–159; 2000. Bondareva, A. A.; Capecchi, M. R.; Iverson, S. V.; Li, Y.; Lopez, N. I.; Lucas, O.; Merrill, G. F.; Prigge, J. R.; Siders, A. M.; Wakamiya, M.; Wallin, S. L.; Schmidt, E. E. Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome. Free Radic. Biol. Med. 43:911–923; 2007. Nordberg, J.; Arner, E. S. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31:1287–1312; 2001. Coyle, P.; Philcox, J. C.; Carey, L. C.; Rofe, A. M. Metallothionein: the multipurpose protein. Cell. Mol. Life Sci. 59:627–647; 2002.

Please cite this article as: Hamre, K; et al. Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.017i

53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae.

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic ...
919KB Sizes 2 Downloads 3 Views