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Increases in apoptosis, caspase activity and expression of p53 and bax, and the transition between two types of mitochondrion-rich cells, in the gills of the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater.

Ching B, Chen XL, Yong JH, Wilson JM, Hiong KC, Sim EW, Wong WP, Lam SH, Chew SF, Ip YK - Front Physiol (2013)

Bottom Line: Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities.The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater).Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, National University of Singapore Kent Ridge, Singapore, Singapore.

ABSTRACT
This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na(+):K(+):2Cl(-) cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl(-) channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater acclimation.

No MeSH data available.


Related in: MedlinePlus

Absolute quantification of Bcl-2 associated X protein (bax) mRNA (copies of transcript per ng cDNA) in the gills of Anabas testudineus kept in freshwater (FW) for 1 or 11 days (controls), or exposed to a 5-day progressive increase in salinity (S) from FW (day 1) to S 10 on day 2, S 20 on day 4, S 25 on day 5 and S 30 (seawater) on day 6, and then kept in seawater for another 6 days (day 11). Results are presented as means ± S.E.M (N = 5). Means not sharing the same letter are significantly different (P < 0.05).
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Figure 7: Absolute quantification of Bcl-2 associated X protein (bax) mRNA (copies of transcript per ng cDNA) in the gills of Anabas testudineus kept in freshwater (FW) for 1 or 11 days (controls), or exposed to a 5-day progressive increase in salinity (S) from FW (day 1) to S 10 on day 2, S 20 on day 4, S 25 on day 5 and S 30 (seawater) on day 6, and then kept in seawater for another 6 days (day 11). Results are presented as means ± S.E.M (N = 5). Means not sharing the same letter are significantly different (P < 0.05).

Mentions: The complete coding cDNA sequence of bax obtained from the gills A. testudineus contained 579 bp (Genbank accession number KC513734). It putatively coded for 192 amino acids with an estimated molecular mass of 21.5 kDa (Figure 6). Bax of A. testudineus has Bcl-2 Homology 1, Bcl-2 Homology 2, Bcl-2 Homology 3 and transmembrane regions. The mRNA expression of bax in the gills of A. testudineus kept in freshwater for 1 day was comparable to that of fish kept in freshwater for 11 days (Figure 7). During seawater acclimation, there were significant increases in the mRNA expression of bax in the gills of fish exposed to salinity 20 (day 4), salinity 25 (day 5), and seawater (day 6; Figure 7). However, unlike p53, a significant increase in the mRNA expression of bax was not observed in the gills of fish exposed to salinity 10 (day 2). After 6 days of acclimation to seawater (day 11), the mRNA expression of bax in the gills of A. testudineus was comparable to that of the day 1 freshwater control (Figure 7).


Increases in apoptosis, caspase activity and expression of p53 and bax, and the transition between two types of mitochondrion-rich cells, in the gills of the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater.

Ching B, Chen XL, Yong JH, Wilson JM, Hiong KC, Sim EW, Wong WP, Lam SH, Chew SF, Ip YK - Front Physiol (2013)

Absolute quantification of Bcl-2 associated X protein (bax) mRNA (copies of transcript per ng cDNA) in the gills of Anabas testudineus kept in freshwater (FW) for 1 or 11 days (controls), or exposed to a 5-day progressive increase in salinity (S) from FW (day 1) to S 10 on day 2, S 20 on day 4, S 25 on day 5 and S 30 (seawater) on day 6, and then kept in seawater for another 6 days (day 11). Results are presented as means ± S.E.M (N = 5). Means not sharing the same letter are significantly different (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675322&req=5

Figure 7: Absolute quantification of Bcl-2 associated X protein (bax) mRNA (copies of transcript per ng cDNA) in the gills of Anabas testudineus kept in freshwater (FW) for 1 or 11 days (controls), or exposed to a 5-day progressive increase in salinity (S) from FW (day 1) to S 10 on day 2, S 20 on day 4, S 25 on day 5 and S 30 (seawater) on day 6, and then kept in seawater for another 6 days (day 11). Results are presented as means ± S.E.M (N = 5). Means not sharing the same letter are significantly different (P < 0.05).
Mentions: The complete coding cDNA sequence of bax obtained from the gills A. testudineus contained 579 bp (Genbank accession number KC513734). It putatively coded for 192 amino acids with an estimated molecular mass of 21.5 kDa (Figure 6). Bax of A. testudineus has Bcl-2 Homology 1, Bcl-2 Homology 2, Bcl-2 Homology 3 and transmembrane regions. The mRNA expression of bax in the gills of A. testudineus kept in freshwater for 1 day was comparable to that of fish kept in freshwater for 11 days (Figure 7). During seawater acclimation, there were significant increases in the mRNA expression of bax in the gills of fish exposed to salinity 20 (day 4), salinity 25 (day 5), and seawater (day 6; Figure 7). However, unlike p53, a significant increase in the mRNA expression of bax was not observed in the gills of fish exposed to salinity 10 (day 2). After 6 days of acclimation to seawater (day 11), the mRNA expression of bax in the gills of A. testudineus was comparable to that of the day 1 freshwater control (Figure 7).

Bottom Line: Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities.The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater).Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, National University of Singapore Kent Ridge, Singapore, Singapore.

ABSTRACT
This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na(+):K(+):2Cl(-) cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl(-) channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater acclimation.

No MeSH data available.


Related in: MedlinePlus