Characterization of the stress response in 17a-hydroxylase deficient common carp (Cyprinus carpio L.)

M.A. Nematollahi

Research output: Thesisinternal PhD, WU

Abstract

In this thesis we describe interrenal hyperplasia in a teleost fish, the common carp, Cyprinus carpio L, caused by 17α-hydroxylase deficiency. Two homozygous, XX male inbred strains, “E5” and “E7”, consistently show 5-10 fold less plasma cortisol in response to net confinement compared to normal male and female carp strains. Both E5 and E7 fish have enlarged head kidneys, which is caused by an increase in size and number of interrenal cells. The experiments and results described in this thesis aim to provide further insight into the genetic background of the 17α-hydroxylase deficiency, and the expression of genes involved in the stress response in P450c17 deficient common carp during and after stress. First, we describe the sequencing of the 17α-hydroxylase/17, 20 lyase gene, CYP17a1, in a normal, standard strain (“STD”) and one of the 17α-hydroxylase deficient strains (E5) of common carp. Two variants of the CYP17a1 gene were found. The nucleotide coding region of the CYP17a1.I variant contained 8 exons and 7 introns which is identical to the human CYP17. In total 3122 base pairs of the nucleotide sequence were determined. Variant I showed no differences between STD and E5, while variant II is missing exon 7 and part of exon 8 and contains a 3 bp insertion and 8 homozygous SNP’s downstream of exon 6. Next, we describe the stress response and gene expression in 17α-hydroxylase deficient E5 and normal STD common carp. The results show a significant increase of corticosterone levels in E5 carp in response to stress. Normal STD carp produce only small amounts of corticosterone. STD carp show a correlation between cortisol production and 11β-HSD2 mRNA expression during the stress response. In contrast, 11β-HSD2 expression is significantly lower in E5 fish and stays at a constant level during confinement. Real-time PCR analysis of StAR and 3β-HSD show a significantly higher expression of these enzymes in E5 during and after net confinement, but P450c21 not, suggesting that transcription of P450c21 is not a limiting step in corticosterone production. Messenger RNA levels in P450c17a1.I are quite variable with a trend towards lower expression levels in E5, suggesting a dysfunction at the transcriptional level. In the last part of this thesis, we investigate the inheritance of interrenal hyperplasia and low cortisol response using backcross and gynogenetic progeny of heterozygote carriers of common carp. Six-month old backcross (BC5 and BC7) and double haploid (DH) progenies were sacrificed after one hour net-confinement, blood sampled, and dissected to determine sex and head kidney-somatic index. Values for cortisol and head kidney index showed a continuous distribution in BC and DH progeny. Values for corticosterone, on the other hand showed a very clear segregation pattern in high and low responders, consistent with a single gene model. There was a significant difference in mean cortisol level between high (H) and low (L) corticosterone responders in BC and DH progeny groups. Surprisingely, H responders were predominantly male, while L responders were female or intersex. These results confirm that 17α-hydroxylase deficiency is inherited as a recessive mutation, and that 17α-deficiency and sex reversal are either caused by the same mutation (pleiotropy) or by closely linked genes.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Verreth, Johan, Promotor
  • van Arendonk, Johan, Promotor
  • Komen, Hans, Co-promotor
  • van Pelt-Heerschap, Hilde, Co-promotor
Award date5 Mar 2008
Place of Publication[S.l.]
Publisher
Print ISBNs9789085049043
Publication statusPublished - 2008

Fingerprint

Cyprinus carpio
stress response
corticosterone
cortisol
carp
exons
haploidy
kidneys
hyperplasia
genes
fish
mutation
gene expression
pleiotropy
sex reversal
lyases
messenger RNA
genetic background
introns
inheritance (genetics)

Keywords

  • carp
  • cyprinus
  • stress response
  • gene expression
  • deficiency
  • strains
  • dna sequencing
  • polymerase chain reaction
  • kidneys

Cite this

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title = "Characterization of the stress response in 17a-hydroxylase deficient common carp (Cyprinus carpio L.)",
abstract = "In this thesis we describe interrenal hyperplasia in a teleost fish, the common carp, Cyprinus carpio L, caused by 17α-hydroxylase deficiency. Two homozygous, XX male inbred strains, “E5” and “E7”, consistently show 5-10 fold less plasma cortisol in response to net confinement compared to normal male and female carp strains. Both E5 and E7 fish have enlarged head kidneys, which is caused by an increase in size and number of interrenal cells. The experiments and results described in this thesis aim to provide further insight into the genetic background of the 17α-hydroxylase deficiency, and the expression of genes involved in the stress response in P450c17 deficient common carp during and after stress. First, we describe the sequencing of the 17α-hydroxylase/17, 20 lyase gene, CYP17a1, in a normal, standard strain (“STD”) and one of the 17α-hydroxylase deficient strains (E5) of common carp. Two variants of the CYP17a1 gene were found. The nucleotide coding region of the CYP17a1.I variant contained 8 exons and 7 introns which is identical to the human CYP17. In total 3122 base pairs of the nucleotide sequence were determined. Variant I showed no differences between STD and E5, while variant II is missing exon 7 and part of exon 8 and contains a 3 bp insertion and 8 homozygous SNP’s downstream of exon 6. Next, we describe the stress response and gene expression in 17α-hydroxylase deficient E5 and normal STD common carp. The results show a significant increase of corticosterone levels in E5 carp in response to stress. Normal STD carp produce only small amounts of corticosterone. STD carp show a correlation between cortisol production and 11β-HSD2 mRNA expression during the stress response. In contrast, 11β-HSD2 expression is significantly lower in E5 fish and stays at a constant level during confinement. Real-time PCR analysis of StAR and 3β-HSD show a significantly higher expression of these enzymes in E5 during and after net confinement, but P450c21 not, suggesting that transcription of P450c21 is not a limiting step in corticosterone production. Messenger RNA levels in P450c17a1.I are quite variable with a trend towards lower expression levels in E5, suggesting a dysfunction at the transcriptional level. In the last part of this thesis, we investigate the inheritance of interrenal hyperplasia and low cortisol response using backcross and gynogenetic progeny of heterozygote carriers of common carp. Six-month old backcross (BC5 and BC7) and double haploid (DH) progenies were sacrificed after one hour net-confinement, blood sampled, and dissected to determine sex and head kidney-somatic index. Values for cortisol and head kidney index showed a continuous distribution in BC and DH progeny. Values for corticosterone, on the other hand showed a very clear segregation pattern in high and low responders, consistent with a single gene model. There was a significant difference in mean cortisol level between high (H) and low (L) corticosterone responders in BC and DH progeny groups. Surprisingely, H responders were predominantly male, while L responders were female or intersex. These results confirm that 17α-hydroxylase deficiency is inherited as a recessive mutation, and that 17α-deficiency and sex reversal are either caused by the same mutation (pleiotropy) or by closely linked genes.",
keywords = "karper, cyprinus, stressreactie, genexpressie, defici{\"e}ntie, stammen (biologisch), dna-sequencing, polymerase-kettingreactie, nieren, carp, cyprinus, stress response, gene expression, deficiency, strains, dna sequencing, polymerase chain reaction, kidneys",
author = "M.A. Nematollahi",
note = "WU thesis, no. 4400",
year = "2008",
language = "English",
isbn = "9789085049043",
publisher = "S.n.",
school = "Wageningen University",

}

Nematollahi, MA 2008, 'Characterization of the stress response in 17a-hydroxylase deficient common carp (Cyprinus carpio L.)', Doctor of Philosophy, Wageningen University, [S.l.].

Characterization of the stress response in 17a-hydroxylase deficient common carp (Cyprinus carpio L.). / Nematollahi, M.A.

[S.l.] : S.n., 2008. 135 p.

Research output: Thesisinternal PhD, WU

TY - THES

T1 - Characterization of the stress response in 17a-hydroxylase deficient common carp (Cyprinus carpio L.)

AU - Nematollahi, M.A.

N1 - WU thesis, no. 4400

PY - 2008

Y1 - 2008

N2 - In this thesis we describe interrenal hyperplasia in a teleost fish, the common carp, Cyprinus carpio L, caused by 17α-hydroxylase deficiency. Two homozygous, XX male inbred strains, “E5” and “E7”, consistently show 5-10 fold less plasma cortisol in response to net confinement compared to normal male and female carp strains. Both E5 and E7 fish have enlarged head kidneys, which is caused by an increase in size and number of interrenal cells. The experiments and results described in this thesis aim to provide further insight into the genetic background of the 17α-hydroxylase deficiency, and the expression of genes involved in the stress response in P450c17 deficient common carp during and after stress. First, we describe the sequencing of the 17α-hydroxylase/17, 20 lyase gene, CYP17a1, in a normal, standard strain (“STD”) and one of the 17α-hydroxylase deficient strains (E5) of common carp. Two variants of the CYP17a1 gene were found. The nucleotide coding region of the CYP17a1.I variant contained 8 exons and 7 introns which is identical to the human CYP17. In total 3122 base pairs of the nucleotide sequence were determined. Variant I showed no differences between STD and E5, while variant II is missing exon 7 and part of exon 8 and contains a 3 bp insertion and 8 homozygous SNP’s downstream of exon 6. Next, we describe the stress response and gene expression in 17α-hydroxylase deficient E5 and normal STD common carp. The results show a significant increase of corticosterone levels in E5 carp in response to stress. Normal STD carp produce only small amounts of corticosterone. STD carp show a correlation between cortisol production and 11β-HSD2 mRNA expression during the stress response. In contrast, 11β-HSD2 expression is significantly lower in E5 fish and stays at a constant level during confinement. Real-time PCR analysis of StAR and 3β-HSD show a significantly higher expression of these enzymes in E5 during and after net confinement, but P450c21 not, suggesting that transcription of P450c21 is not a limiting step in corticosterone production. Messenger RNA levels in P450c17a1.I are quite variable with a trend towards lower expression levels in E5, suggesting a dysfunction at the transcriptional level. In the last part of this thesis, we investigate the inheritance of interrenal hyperplasia and low cortisol response using backcross and gynogenetic progeny of heterozygote carriers of common carp. Six-month old backcross (BC5 and BC7) and double haploid (DH) progenies were sacrificed after one hour net-confinement, blood sampled, and dissected to determine sex and head kidney-somatic index. Values for cortisol and head kidney index showed a continuous distribution in BC and DH progeny. Values for corticosterone, on the other hand showed a very clear segregation pattern in high and low responders, consistent with a single gene model. There was a significant difference in mean cortisol level between high (H) and low (L) corticosterone responders in BC and DH progeny groups. Surprisingely, H responders were predominantly male, while L responders were female or intersex. These results confirm that 17α-hydroxylase deficiency is inherited as a recessive mutation, and that 17α-deficiency and sex reversal are either caused by the same mutation (pleiotropy) or by closely linked genes.

AB - In this thesis we describe interrenal hyperplasia in a teleost fish, the common carp, Cyprinus carpio L, caused by 17α-hydroxylase deficiency. Two homozygous, XX male inbred strains, “E5” and “E7”, consistently show 5-10 fold less plasma cortisol in response to net confinement compared to normal male and female carp strains. Both E5 and E7 fish have enlarged head kidneys, which is caused by an increase in size and number of interrenal cells. The experiments and results described in this thesis aim to provide further insight into the genetic background of the 17α-hydroxylase deficiency, and the expression of genes involved in the stress response in P450c17 deficient common carp during and after stress. First, we describe the sequencing of the 17α-hydroxylase/17, 20 lyase gene, CYP17a1, in a normal, standard strain (“STD”) and one of the 17α-hydroxylase deficient strains (E5) of common carp. Two variants of the CYP17a1 gene were found. The nucleotide coding region of the CYP17a1.I variant contained 8 exons and 7 introns which is identical to the human CYP17. In total 3122 base pairs of the nucleotide sequence were determined. Variant I showed no differences between STD and E5, while variant II is missing exon 7 and part of exon 8 and contains a 3 bp insertion and 8 homozygous SNP’s downstream of exon 6. Next, we describe the stress response and gene expression in 17α-hydroxylase deficient E5 and normal STD common carp. The results show a significant increase of corticosterone levels in E5 carp in response to stress. Normal STD carp produce only small amounts of corticosterone. STD carp show a correlation between cortisol production and 11β-HSD2 mRNA expression during the stress response. In contrast, 11β-HSD2 expression is significantly lower in E5 fish and stays at a constant level during confinement. Real-time PCR analysis of StAR and 3β-HSD show a significantly higher expression of these enzymes in E5 during and after net confinement, but P450c21 not, suggesting that transcription of P450c21 is not a limiting step in corticosterone production. Messenger RNA levels in P450c17a1.I are quite variable with a trend towards lower expression levels in E5, suggesting a dysfunction at the transcriptional level. In the last part of this thesis, we investigate the inheritance of interrenal hyperplasia and low cortisol response using backcross and gynogenetic progeny of heterozygote carriers of common carp. Six-month old backcross (BC5 and BC7) and double haploid (DH) progenies were sacrificed after one hour net-confinement, blood sampled, and dissected to determine sex and head kidney-somatic index. Values for cortisol and head kidney index showed a continuous distribution in BC and DH progeny. Values for corticosterone, on the other hand showed a very clear segregation pattern in high and low responders, consistent with a single gene model. There was a significant difference in mean cortisol level between high (H) and low (L) corticosterone responders in BC and DH progeny groups. Surprisingely, H responders were predominantly male, while L responders were female or intersex. These results confirm that 17α-hydroxylase deficiency is inherited as a recessive mutation, and that 17α-deficiency and sex reversal are either caused by the same mutation (pleiotropy) or by closely linked genes.

KW - karper

KW - cyprinus

KW - stressreactie

KW - genexpressie

KW - deficiëntie

KW - stammen (biologisch)

KW - dna-sequencing

KW - polymerase-kettingreactie

KW - nieren

KW - carp

KW - cyprinus

KW - stress response

KW - gene expression

KW - deficiency

KW - strains

KW - dna sequencing

KW - polymerase chain reaction

KW - kidneys

M3 - internal PhD, WU

SN - 9789085049043

PB - S.n.

CY - [S.l.]

ER -