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New Strategy of Functional Analysis of PHGPx Knockout Mice Model Using Transgenic Rescue Method and Cre-LoxP System.

Imai H - J Clin Biochem Nutr (2009)

Bottom Line: Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids.PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription.All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane Minato-ku Tokyo 108-8641, Japan.

ABSTRACT
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription. In this review, we focus on our recent experiments on the regulation of promoter activity of the types of PHGPx and on the novel strategy of functional analysis of a PHGPx knockout mice model using the transgenic rescue method and Cre-LoxP system. PHGPx is especially high in testis and spermatozoa. A deficiency is implicated in human infertility. We established spermatocyte-specific PHGPx knockout (KO) mice using a Cre-loxP system. Targeted disruption of all exons of the PHGPx gene in mice by homologous recombination caused embryonic lethality at 7.5 days post coitum. The PHGPx-loxP transgene rescued PHGPx KO mice from embryonic lethality. These rescued floxed PHGPx mice were mated with spermatocyte specific Cre expressing mice. All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa. These results demonstrate that depletion of PHGPx in spermatozoa may be one of the causes of male infertility in mice and humans.

No MeSH data available.


Related in: MedlinePlus

C/EBPε regulates the up-regulation of PHGPx in HL60 cells and neutrophils stimulated by TNFα. C/EBPε is expressed primarily in neutrophils and myeloid and lymphoid cells, but not in differentiated macrophages. The promoter activity of PHGPx and expression of PHGPx mRNA was specifically up-regulated by stimulation with TNFα in C/EBPε expressing neutrophils and HL60 cells. This up-regulation of expression of PHGPx mRNA and promoter activities of PHGPx were inhibited by treatment with the anti-oxidants PDTC and NAC, and by inhibitors of NFκB and Bay 11-7082. The up-regulated promoter activity was effectively abrogated by a mutation in C/EBP-binding sequence (GACGTC). C/EBPε is a critical transcription factor in TNFa-induced up regulation of PHGPx expression.
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Figure 3: C/EBPε regulates the up-regulation of PHGPx in HL60 cells and neutrophils stimulated by TNFα. C/EBPε is expressed primarily in neutrophils and myeloid and lymphoid cells, but not in differentiated macrophages. The promoter activity of PHGPx and expression of PHGPx mRNA was specifically up-regulated by stimulation with TNFα in C/EBPε expressing neutrophils and HL60 cells. This up-regulation of expression of PHGPx mRNA and promoter activities of PHGPx were inhibited by treatment with the anti-oxidants PDTC and NAC, and by inhibitors of NFκB and Bay 11-7082. The up-regulated promoter activity was effectively abrogated by a mutation in C/EBP-binding sequence (GACGTC). C/EBPε is a critical transcription factor in TNFa-induced up regulation of PHGPx expression.

Mentions: We and other groups have investigated the regulation of promoter activity by deletion and mutational analysis of the promoter region of PHGPx gene [3, 22, 23]. We reported a possible construction of the positive regulatory region and the core promoter regions of PHGPx in several cell lines using promoter analysis with luciferase as the reporter gene and electrophoretical mobility shift analysis as shown in Fig. 2 [3]. By comparison of sequences in the 5'-flanking regions of pig, human and mouse PHGPx, we determined 29 high homology domains (H1 to H29) as shown by the gray and black circles in Fig. 2. Twelve regions of the 29 homology domains contained the putative consensus binding sequences for several transcriptional factors, as shown in black circles. Deletion analysis of promoter activity in the mouse PHGPx gene demonstrated that the 5'-flanking regions from −60 to −9 bp, −233 to −158 bp and +342 to +375 bp are critical for the basal transcription of non-mitochondrial, mitochondrial and nucleolar PHGPx mRNA respectively. The core promoter activity in L929 cells was high for non-mitochondrial PHGPx, but relatively low for mitochondrial and nucleolar PHGPx and for the expression levels of each type of PHGPx mRNA.


New Strategy of Functional Analysis of PHGPx Knockout Mice Model Using Transgenic Rescue Method and Cre-LoxP System.

Imai H - J Clin Biochem Nutr (2009)

C/EBPε regulates the up-regulation of PHGPx in HL60 cells and neutrophils stimulated by TNFα. C/EBPε is expressed primarily in neutrophils and myeloid and lymphoid cells, but not in differentiated macrophages. The promoter activity of PHGPx and expression of PHGPx mRNA was specifically up-regulated by stimulation with TNFα in C/EBPε expressing neutrophils and HL60 cells. This up-regulation of expression of PHGPx mRNA and promoter activities of PHGPx were inhibited by treatment with the anti-oxidants PDTC and NAC, and by inhibitors of NFκB and Bay 11-7082. The up-regulated promoter activity was effectively abrogated by a mutation in C/EBP-binding sequence (GACGTC). C/EBPε is a critical transcription factor in TNFa-induced up regulation of PHGPx expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2803127&req=5

Figure 3: C/EBPε regulates the up-regulation of PHGPx in HL60 cells and neutrophils stimulated by TNFα. C/EBPε is expressed primarily in neutrophils and myeloid and lymphoid cells, but not in differentiated macrophages. The promoter activity of PHGPx and expression of PHGPx mRNA was specifically up-regulated by stimulation with TNFα in C/EBPε expressing neutrophils and HL60 cells. This up-regulation of expression of PHGPx mRNA and promoter activities of PHGPx were inhibited by treatment with the anti-oxidants PDTC and NAC, and by inhibitors of NFκB and Bay 11-7082. The up-regulated promoter activity was effectively abrogated by a mutation in C/EBP-binding sequence (GACGTC). C/EBPε is a critical transcription factor in TNFa-induced up regulation of PHGPx expression.
Mentions: We and other groups have investigated the regulation of promoter activity by deletion and mutational analysis of the promoter region of PHGPx gene [3, 22, 23]. We reported a possible construction of the positive regulatory region and the core promoter regions of PHGPx in several cell lines using promoter analysis with luciferase as the reporter gene and electrophoretical mobility shift analysis as shown in Fig. 2 [3]. By comparison of sequences in the 5'-flanking regions of pig, human and mouse PHGPx, we determined 29 high homology domains (H1 to H29) as shown by the gray and black circles in Fig. 2. Twelve regions of the 29 homology domains contained the putative consensus binding sequences for several transcriptional factors, as shown in black circles. Deletion analysis of promoter activity in the mouse PHGPx gene demonstrated that the 5'-flanking regions from −60 to −9 bp, −233 to −158 bp and +342 to +375 bp are critical for the basal transcription of non-mitochondrial, mitochondrial and nucleolar PHGPx mRNA respectively. The core promoter activity in L929 cells was high for non-mitochondrial PHGPx, but relatively low for mitochondrial and nucleolar PHGPx and for the expression levels of each type of PHGPx mRNA.

Bottom Line: Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids.PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription.All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane Minato-ku Tokyo 108-8641, Japan.

ABSTRACT
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription. In this review, we focus on our recent experiments on the regulation of promoter activity of the types of PHGPx and on the novel strategy of functional analysis of a PHGPx knockout mice model using the transgenic rescue method and Cre-LoxP system. PHGPx is especially high in testis and spermatozoa. A deficiency is implicated in human infertility. We established spermatocyte-specific PHGPx knockout (KO) mice using a Cre-loxP system. Targeted disruption of all exons of the PHGPx gene in mice by homologous recombination caused embryonic lethality at 7.5 days post coitum. The PHGPx-loxP transgene rescued PHGPx KO mice from embryonic lethality. These rescued floxed PHGPx mice were mated with spermatocyte specific Cre expressing mice. All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa. These results demonstrate that depletion of PHGPx in spermatozoa may be one of the causes of male infertility in mice and humans.

No MeSH data available.


Related in: MedlinePlus