Oxygen Stress Increases Prolyl cis/trans Isomerase Activity and Expression of Cyclophilin 18 in Rabbit Blastocysts

Abstract

The peptidyl-prolyl cis/trans isomerase (PPIase) activity and the expression of cyclophilins were studied in 6-day-old rabbit preimplantation embryos cultured under physiological and increased oxygen concentrations of 5% and 20% O(2), respectively. The PPIase activity was completely inhibited by cyclosporin A (CsA). The inhibitor of FK506-binding proteins, rapamycin, had no effect on the PPIase activity, indicating that the PPIase activity in rabbit blastocysts originates from cyclophilins. Using CsA affinity chromatography, only one cyclophilin with a molecular mass of about 17.8 kDa was separated. The cDNA of rabbit cyclophilin was cloned and sequenced. Analysis of the 682-base pair cDNA revealed an open reading frame coding for a polypeptide of 164 amino acid residues with a molecular weight of 17.83 kDa. Homologies of 90% and 96% for the cDNA and amino acid sequence, respectively, to the human CyP18 were found, suggesting that the novel rabbit cyclophilin is a member of the CyP18 family (rabCyP18). The transcription level of rabCyP18 mRNA was 8.3 +/- 0.6 pg in 100 ng total RNA in noncultured blastocysts. In vitro culture with moderate oxygen stress (20% O(2)) resulted in a 1.5-fold increase in rabCyP18 transcription and an increased PPIase activity compared to that of blastocysts cultured with 5% O(2). Increase in transcription rate and PPIase activity by oxygen stress suggests an involvement of CyP18 in oxygen defense in rabbit preimplantation embryos.

Mitochondrial Targeted Cyclophilin D Protects Cells from Cell Death by Peptidyl Prolyl Isomerization

“…Interestingly, suppression of cyclophilin A, the cytosolic isoform of the family of PPIases that bind cyclosporin A, has been reported in markedly sensitize cells to oxidative stress (50). An involvement of PPIase activity in oxygen defense has been reported for cyclophilin 18 in rabbit blastocysts (51). The thiol-specific antioxidant protein Aop1 has also been reported to bind human cyclophilin 18 (52).…”

Identification and Characterization of Moca-cyp

“…From bacteria to humans, they are ubiquitously, abundantly expressed and show a very strong conservation of their PPIase domain (1,11). Cyclophilins have been localized in different cell compartments, and their poorly understood functions have been so far linked to multiple cellular processes, such as protein folding (12), trafficking (13), chaperone activity (14), oxidative stress response (15), and functional arrangement of components within receptor complexes (16). The abundance and ubiquitous expression of these proteins have suggested that their function might be pleiotropic, but a growing body of evidence points toward the fact that different cyclophilins function in a restricted and specific manner.…”

Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts

“…In adipocytes and myocytes GLUT4 translocation is the rate-limiting step of insulinstimulated glucose uptake (Saltiel 2001). In preimplantation embryos GLUT4 expression has been shown for bovine (Navarrete Santos et al 2000, Augustin et al 2001, rat (Korgun et al 2001) and rabbit (Navarrete blastocysts. The expression data from mouse preimplantation embryos are controversial.…”

“…follicle-stimulating hormone (Ovagen, Immuno-Chemical Products, Auckland, New Zealand, or Folligon, Intervet, Boxmeer, The Netherlands). Mating, embryo recovery (Navarrete Santos et al 2000) and embryo culture (Kietz & Fischer 2003) were performed as described. On days 3, 4 and 6 post coitum (p.c.)…”

“…Semiquantitative RT-PCR of IR, GLUT4 and GLUT8 in rabbit blastocysts Semiquantitative RT-PCR was performed essentially as previously described (Navarrete Santos et al 2000, Kietz & Fischer 2003) with oligonucleotide primers specific for rabbit IR, GLUT4, GLUT8, b-actin and GAPDH. The IR, GLUT4 and GLUT8 primer sequences were derived from published rabbit (rab) sequences (Navarrete .…”