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Biology of Reproduction 62, 1-7 (2000)
©Copyright 2000 Society for the Study of Reproduction, Inc.

Articles

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

Anne Navarrete Santosa, Sabine Körbera, Gerd Küllertzb, Gunter Fischerb, and Bernd Fischera

a Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, D-06097 Halle (Saale), Germany b Max-Planck-Research Unit "Enzymology of Protein Folding", D-06120 Halle (Saale), Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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% O2, 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% O2) resulted in a 1.5-fold increase in rabCyP18 transcription and an increased PPIase activity compared to that of blastocysts cultured with 5% O2. Increase in transcription rate and PPIase activity by oxygen stress suggests an involvement of CyP18 in oxygen defense in rabbit preimplantation embryos.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peptidyl-prolyl cis/trans isomerases (PPIases) are ubiquitous and abundant proteins conserved from bacteria and unicellular eukaryotes to humans. They catalyze the cis/trans isomerization of proline-containing peptides and proteins. Members of several PPIase families are specifically expressed in various cells and cell compartments. PPIases can be classified into three families according to amino acid sequence similarities. Two families can be further characterized by inhibition of their enzyme activity by immunosuppressant drugs. Cyclosporin A (CsA) inhibits the PPIase activity of cyclophilins, and both FK506 and rapamycin inhibit the activity of FK506-binding proteins (FKBPs).

The members of the cyclophilin family differ in their molecular mass, ranging from 18 to 150 kDa. The first PPIase identified was CyP18 [1], an 18-kDa protein originally described as the cytosolic binding protein for CsA [2]. Besides the cis/trans catalysis of peptidyl-prolyl bonds in oligopeptides [3], CyP18 catalyzes the folding of proteins [4]. The CyP18 homologue in yeast and CyP18 mRNA in beans are induced by heat shock [5–7]. The expression of CyP18 protein was found to be oxygen sensitive, as indicated by oxygen stress or by hypoxia treatment in human endothelial [8] and myogenic cells [9]. Human CyP18 binds to the thiol-specific antioxidant protein Aop1 and stimulates its reductase activity [10]. Aop1 belongs to a family of proteins supposed to be involved in the defense of oxygen stress. The interaction of both proteins seems to be specific, since other PPIases do not exert any stimulatory effect on Aop1 activity.

CyP18 expression is described as being higher in mitotic active and developing tissues, as shown in beans [7] and in tissues from reproductive organs of insects [11]. Gene expression in embryos of Blattella germanica is developmentally regulated [11]. Up until now, possible functions of CyP18 during mammalian embryogenesis have not been known.

Rabbit preimplantation embryos cultured with oxygen concentration in air, i.e., 20%, showed an impaired development as compared to that in culture with 5% O2 [12,13]. An increased generation of reactive oxygen species (ROS) [14] and changes in gene expression [15] during 20% O2 culture most likely contribute to the poorer development. ROS are known to induce various cell lesions, e.g., by DNA damage, lipid peroxidation, and oxidative modification of proteins [16]. ROS also modulate the activity of transcription factors, such as the activator protein-1 [17], growth factors such as transforming growth factor-{alpha} [18], heat shock proteins [19,20], mitogen-activated protein kinases [21], antioxidant enzyme systems [22], and various other genes (COX-2) [23]. An involvement of PPIases in ROS and oxygen defense is likely, but this has not yet been investigated in mammalian embryos.

In the present study we report on CyP18 expression and PPIase activity in preimplantation rabbit embryos. A possible involvement of rabCyP18 in oxygen defense was studied by exposing the embryos to different oxygen concentrations during culture.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Trizol reagent, Superscript II RT kit, dNTPs, and Taq polymerase were from Life Technologies (Karlsruhe, Germany); restriction enzymes from Biolabs New England (Schwalbach, Germany); T7 RNA polymerase, RNase inhibitor, and DNase I from Boehringer Mannheim (Mannheim, Germany); and the pPCR-Script SK(+) vector from Stratagene (Heidelberg, Germany). Human recombinant cyclophilin (Cyp18cy; nomenclature of PPIases according to Fischer [3]) was obtained from Boehringer Mannheim. Bovine {alpha}-chymotrypsin was obtained from Merck (Darmstadt, Germany) and succinyl-Ala-Phe-Pro-Phe-4-nitroanilide from Bachem Biochemica GmbH (Heidelberg, Germany). The (amino-tetramethylen-carboxamidomethyl)-D-Ser8-cyclosporin A was received from M. Schutkowski, Max-Planck-Research Unit, Halle.

Embryo Recovery and In Vitro Culture

In total, 208 rabbit blastocysts were used in the present study. Embryos were collected from sexually mature rabbits (hybrid strain Zika) that had been stimulated by FSH (Ovagen; Immuno-Chemical Products Ltd., Auckland, New Zealand). Matings and embryo recovery were performed as described previously [24]. On Day 6 postcoitum, blastocysts were flushed from uteri, washed 3 times, pooled, and randomly divided among the experimental groups.

To study the effects of oxygen concentrations on CyP expression, blastocysts (n = 4 per well) were cultured in 500 µl BSM II medium at 37°C for 6 h in a saturated humidity under 5% O2, 5% CO2, 90% N2 (control, physiological condition of 5% O2) [25] or in 5% CO2 in air (approximately 20% O2, increased oxygen concentration) [13] in water-jacketed incubators (BB 6060; Heraeus, Hanau, Germany). After culture, blastocysts were homogenized in Trizol reagent or in cold PBS buffer and stored at -80°C.

RNA Extraction

Preparation of total RNA was performed by using 1 ml Trizol reagent for 4 embryos and according to the previously described protocol [15]. The amount of total RNA was determined spectrophotometrically at 260 nm. For quantification of mRNA by competitive reverse transcription-polymerase chain reaction (cRT-PCR), the concentration of total RNA was determined twice, and an aliquot of the RNA was checked in a denaturing agarose gel.

RT-PCR of CyP18 mRNA

Total RNA (1 µg) was reverse transcribed in a volume of 20 µl containing 0.5 mM dNTPs, 10 mM dithiothreitol, 200 units Superscript II, 20 units RNase inhibitor, 0.5 µg oligo dT(12–18) primer, and 2 µl reverse transcriptase buffer at 42°C for 1 h, followed by an incubation at 90°C for 5 min. As a control for DNA contamination, 1 µg RNA was PCR amplified without RT reaction. This control reaction was performed for each primer combination and in all PCR amplifications. PCR amplification was carried out with 1 µl of cDNA in a 50-µl volume containing 200 mM dNTP and 2.5 units Taq polymerase, with the primer combinations listed in Table 1. The specific primer sequences were designed from the cDNA clone PRABOC079 (EMBL accession no. R86496) [26]. Resulting PCR products were separated by electrophoresis on 1.8% agarose gel and stained with ethidium bromide. To obtain the full-length coding sequence, 5'-and 3'-rapid amplification of cDNA ends-PCR was performed on reverse-transcribed mRNA [27].


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  		TABLE 1. Primer used in RT-PCR and in competitive RT-PCR for CyP18

Cloning of PCR Products, Sequencing, and GenBank Search

The amplified PCR products were purified by separation in a preparative 1.8% agarose gel and extracted by Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Fragments were cloned into the pCR-Script SK(+) vector and transformed in competent Escherichia coli XL1 blue cells. Recombinant plasmid DNA was examined by restriction analysis and sequenced using the ABI Prism Ready Reaction Dyedeoxy Terminator Sequencing Kit (Perkin Elmer, Weiterstadt, Germany) and T3 and T7 sequencing primers in an ABI Analytical (Ramsey, NJ) 373 automated sequencer. The sequenced cDNA was screened for homology in the GenBank EMBL using the BLASTN search modus and for amino acids using the BLASTP search modus.

Construction of an Internal Standard RNA (cRNA) for the Quantification of CyP18 mRNA by cRT-PCR

An internal standard plasmid for rabbit CyP18 was constructed by cloning the CyP18 PCR fragment (length 302 base pairs [bp] amplified with primers CyP18–2 and CyP18–4) into pCR-Script SK(+).

The construct was digested with the restriction enzyme NcoI, dephosphorylated, and ligated to a 105-bp BspHI fragment from the plasmid pUC18. The resulting plasmid (pCyP18rab-internal standard) was analyzed by restriction analysis and sequencing.

The pCyP18rab-internal standard for the generation of cRNA was linearized by digestion with NotI. Approximately 10–15 µg of cRNA (standard cRNA) was synthesized using this linearized plasmid as a template and T7 RNA polymerase according to the manufacturer's protocol. A single band of cRNA with the expected size was observed in a 1.8% agarose gel. The absolute amount of cRNA was calculated using spectrophotometric absorbance at 260 nm. The cRNA solution was diluted into aliquots of a 5-fold (1000, 200, 40, 8, 1.6, 0.32 pg) or 10-fold dilution (1000, 100, 10, 1, 0.1, 0.01 pg) in diethyl pyrocarbonate-treated H2O with 1 U/µl RNase inhibitor and stored at -80°C. The size difference between the PCR products permitted easy electrophoretic separation of the cRNA product (407 bp) from the target mRNA (302 bp).

Competitive RT-PCR for rabCyP18 mRNA

For quantification of CyP18 mRNA molecules, 20 or 50 ng of total RNA was prepared from a pool of 4 blastocysts as described above. It was mixed with an aliquot of a given dilution of standard cRNA for CyP18. Reverse transcription was performed using the Superscript II RT kit and 10 pmol 3' antisense primer CyP18–4 in a total volume of 20 µl at 42°C for 50 min. The RT reaction was terminated by heating at 90°C for 10 min. The samples were quickly chilled on ice and brought to a 60-µl volume with H2O. Then 5 µl of the resulting cDNAs was amplified by PCR (Trio Thermoblock; Biometra, Göttingen, Germany) (200 µM dNTPs; 20 pmol primers CyP18–2, CyP18–4; 1.5 mM MgCl2; 5 µl PCR buffer; and 0.75 U Taq polymerase) employing the following conditions: 3 min at 94°C denaturation, 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C for 30 cycles followed by a final extension step of 5 min at 72°C. The following controls were performed to monitor DNA contamination: 1) complete assay with Taq polymerase but without template and RT and 2) complete assay with Taq polymerase and RNA template, but without RT. Amplification products from both the target mRNA and the cRNA template were resolved by electrophoresis in 1.8% agarose gels and stained with ethidium bromide. Photographs of gels were subjected to scanning densitometry, and the data were analyzed with ScanPack Software (Biometra). The amount of amplified products was calculated by densitometric measurement of DNA band intensity with correction of the size difference between standard and CyP18 (optical density [OD]rabCyP18 x 1.33/ODstandard). The amount of CyP18 mRNA present in a particular mRNA sample was determined by the cRNA amount corresponding to a standard/target ratio of 1 (equivalence point) [28].

Determination of PPIase Activity

For protein and PPIase activity determinations, the blastocysts were homogenized in cold PBS buffer. PPIase activity was measured spectrophotometrically at 390 nm at 6°C as described previously [29], using bovine {alpha}-chymotrypsin as an isomer-specific protease. In a 96-well (flat bottom) microtiter plate (Bibby Sterilin Ltd., Stone, Staffordshire, UK), a total volume of 140 µl solution per well was added, with 10 µl blastocyst homogenate, 50 µl substrate solution (125 µg/ml succinyl-Ala-Phe-Pro-Phe-4-nitroanilide in 35 mM Hepes buffer, pH 7.8) and 80 µl chymotrypsin solution (1 mg/ml chymotrypsin in 35 mM Hepes buffer, pH 7.8). Bovine {alpha}-chymotrypsin was found to contain 58% active enzyme as determined by active site titration using 4-nitrophenyl acetate [30]. The reaction was started by adding the chymotrypsin solution with the microplate reader dispenser followed by rapid and intense mixing (Plat-Mix; Garching Innovation GmbH, Munich, Germany) of the reaction mixture. The time course of the hydrolysis of the cis conformer in the absence and presence of PPIase was used to calculate enzyme activity as K = (kobs - ko)/ko in arbitrary units (K) with kobs as the first-order rate constant of the PPIase-catalyzed reaction and ko as the uncatalyzed cis to trans interconversion. Human recombinant cyclophilin (Cyp18cy; nomenclature of PPIases according to Fischer) [3] was used as PPIase standard.

Isolation of a 17.9-kDa Protein from Rabbit Blastocysts

A CsA affinity column was prepared with 5 mg (amino-tetramethylen-carboxamidomethyl)-D-Ser8-cyclosporin A. The CsA derivative was dissolved in ethanol and coupled to 1 ml Affi-Gel 10 (BioRad Laboratories, Munich, Germany) according to the manufacturer's instructions. The procedure was stopped by addition of 500 µl ethanolamine (Sigma, Deisenhofen, Germany). The binding capacity of the gel was 80 µg human recombinant cyclophilin as determined by titration with hrCyP18 and measurement of the remaining cyclophilin activity in the supernatant. Cyclophilin extractions from crude blastocyst homogenates were performed in three steps (1–3) at a flow rate of approximately 100 µl/min at 5°C as follows: 1) application of the sample to the column (80 x 8-mm i.d.; disposable polystyrene column; Pierce, Rockford, IL); 2) washing of the column with 10 ml 100 mM ammonium acetate, pH 7.4, to remove nonspecifically bound substances; and 3) cyclophilin elution with 2 ml of a 400 mM methanolic solution containing 120 µmol CsA and fractionation in 4 times 500-µl aliquots. The eluates either were desalted by reversed-phase HPLC on a Nucleosil 500–5 C3-PPN column (125 x 4 mm; Machery Nagel, Düren, Germany) and identified applying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or were analyzed by SDS-PAGE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PPIase Activity and Molecular Mass of the CsA-Binding Protein

A 20% increased PPIase activity was found in homogenates of blastocysts cultured with 20% O2 (Fig. 1). The enzyme activity of the homogenate was completely inhibited by 20 µM CsA. In contrast, rapamycin did not exert any inhibitory effect (data not shown). Therefore, the enzymatic activity of cis/trans isomerization and the increased activity in the 20% O2 groups were exclusively caused by cyclophilins and not by FKBPs.



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  		FIG. 1. PPIase activity in D6 rabbit blastocysts cultured with 5% O2 (white bars) or 20% O2 (gray bars). Activity was determined in two independent experiments (M1, M2). In M1, embryos were tested in 2 groups; in M2 they were tested in 10 groups of 4 pooled blastocysts each. The PPIase activity was measured at least three times in each group (mean ± SEM, PM1 = 0.0006; PM2 = 0.0009).

Blastocyst cyclophilin-like PPIases were isolated by CsA affinity chromatography. Bound proteins were eluted with CsA solution (50 µM CsA, 100 mM NH4-acetate buffer, pH 7.4). A single protein band was found on silver-stained SDS-PAGE that moved with an electrophoretic mobility corresponding to a molecular mass of 17.8 kDa (Fig. 2). This mass corresponds closely to the molecular weight of 17.839 kDa deduced from the amino acid sequence of rabCyP18 (see below).



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  		FIG. 2. Separation of affinity chromatography fractions by SDS-PAGE. a) Silver-stained 10% gel; lanes 1–7: wash steps of the column with 1 ml NH4 acetate buffer (pH 7.4). Lanes 8–11: wash steps with 1 ml 50 µM CsA solution. Lane 12: wash step with 50% acetonitrile/H2O. M lanes: molecular weight markers (Boehringer Ingelheim); Mr (x 10-3) indicated on right. b) Molecular weight determination of the rabbit CsA-binding protein by plotting against marker proteins

Cloning and Sequencing of the Complete Coding Region of Rabbit CyP18 mRNA

A complete coding region of 164 amino acids (aa) was cloned. Sequence data are shown in Figure 3. Analysis of a 682-bp cDNA revealed an open reading frame coding for a polypeptide of 164 aa residues with a molecular weight of 17839. The cDNA sequence of rabCyP18 showed 90% sequence homology on the nucleotide and 96% homology on the protein sequence level to the human cytosolic hCyP18 (Fig. 4, a and b). The protein sequence between human and rabbit CyP18 differed only in 6 aa. The region of aa 48 to 69, corresponding to the PPIase consensus sequence, was identical to the human pattern (Fig. 4b). This identity suggests that the rabCyP18 gene product is a member of the cyclophilin family and represents the cytosolic CyP18 from Oryctolagus cuniculus. The 5'- and 3'-flanking regions did not show any homology and were specific for rabCyP18. The putative start of translation could be identified by homology to other cytosolic cyclophilin isoforms and the sequence surrounding this ATG codon, which is identical to the consensus sequence for translation initiation by eukaryotic ribosomes -GNNATGG-. The cDNA contained an open reading frame of 492 nucleotides encoding a protein of 164 aa with a calculated molecular weight of 17839 and an estimated pI of 7.32. The 5' untranslated region extended 44 bases upstream from the ATG codon. The TAA stop codon was followed by 107 bases of 3' noncoding region. Searches in the GenBank and EMBL data banks identified cyclophilins or cyclophilin-like sequences with high degrees of homology to the coding region of rabCyP18. Computer-assisted analysis of the deduced amino acid sequence indicated that O. cuniculus cyclophilin exhibited no internal homology and did not have significant sequence similarity to any protein other than cyclophilins. The amino acid identity of rabbit CyP18 with CyP18 isoforms was higher than with other cyclophilins such as CyPB, -C, or -D.



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  		FIG. 3. Nucleotide sequence of rabCyP18 cDNA and its deduced amino acid (prot) sequence. The localization of PCR primers and the cleavage sites of the restriction enzymes are underlined. The single open reading frame is translated into a 164 aa protein in prot line. The start and termination codons are indicated by Start and Stop. The rabCyP18 and the deduced amino acid sequence described in this paper have been deposited in GenBank/EMBL under accession no. AF139893



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  		FIG. 4. Alignment of the rabbit (rabCyP18) and human (hCyP18) CyP18 sequence. a) Complementary DNA sequence of rabCyP18 compared to the most homologous cDNA of hCyP18 (GenBank accession Y00052). The coding sequence of hCyP18 is underlined. A gap has been introduced, where necessary, to align the sequences. b) Comparison of the deduced amino acid sequence of rabCyP18 with human CyP18 sequence (sp|P05092|CYPH, EC 5.2.1.8). The amino acids are presented as single-letter codes. Vertical lines denote identity of the sequences; lack of symbols indicates divergence in sequence; + indicate divergence of amino acids with positive homologue features. The PPIase consensus pattern in the protein sequence is boxed. * Indicate residues that define the CsA-binding site

Quantification of rabCyP18 Transcripts in Rabbit Blastocysts Cultured Under Different Oxygen Concentrations

Noncultured rabbit blastocysts contained 8.3 ± 0.6 pg rabCyP18 mRNA in 100 ng total RNA (Fig. 5; Table 2). CyP18 expression was statistically significantly increased by about 50% (~1.5-fold) (P = 0.009) in blastocysts cultured with 20% O2 as compared to 5% O2 (Fig. 6).



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  		FIG. 5. Quantification of rabCyp18 mRNA in rabbit blastocysts by cRT-PCR. The mRNA was measured in three independent replicates of 4 pooled noncultured blastocysts each (Exp.I, Exp.II, and Exp.III). The amount of tested total RNA was varied, using 50 ng of total RNA per cRT-PCR reaction for Exp.I and Exp.III and 20 ng for Exp.II. In Exp.I and Exp.II, a 10-fold dilution was used; in Exp.III, a 5-fold dilution was used. The diagrams show the separation of the cRT-PCR reactions in 1.8% agarose gel. The log of the ratio ODrabCyp18 x 1.33 to the ODstandard was plotted as a function of the amount of rabCyP18standard added to the reaction. The amounts of the standard RNA are indicated: A, 103 pg; B, 102 pg; C, 101 pg; D, 1 pg; E, 10-1 pg; F, 10-2 pg; G, 10-3 pg; H, 200 pg; I, 40 pg; J, 8 pg; K, 1.6 pg; L, 0.32 pg. Results of individual experiments I, II, and III and the amount of rabCyP18 mRNA in 100 ng of total RNA are summarized in Table 2


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  		TABLE 2. Amount of CyP18 mRNA in D6 rabbit blastocysts



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  		FIG. 6. Transcription of rabCyP18 mRNA in rabbit blastocysts after 6-h culture with 5% O2 and 20% O2, respectively, with groups of 4 pooled blastocysts each (n = 3 replicates). The amount of rabCyP18 mRNA was determined by cRT-PCR in three independent culture experiments. In a the mean values (mean ± SD) of CyP18 mRNA in both culture groups are shown; b shows a representative separation of the cRT-PCR reactions in 1.8% agarose gel


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tissue-specific CyP expression has been reported for several eukaryotic and prokaryotic organisms. The present study is the first report on CyP expression in mammalian embryos. In 6-day-old rabbit blastocysts, the major PPIase activity originates from a single cyclophilin, the cytosolic CyP18. Both mRNA level and PPIase activity are altered by oxygen stress in mammalian preimplantation embryos.

The sequence analysis of the new rabbit gene suggests that the rabCyP18-encoded protein is a member of the cytosolic CyP18 family. The amino acid sequence responsible for CsA binding is identical to the human CyP18 sequence (Fig. 4b).

The amount of 8.3 pg rabCyP18 mRNA per 100 ng total RNA is approximately 1–2.5 x 103 rabCyP18 transcripts per embryonic cell or with 0.2–0.4% of the embryonic mRNA. In rat tissues and in S. cerevisiae, CyP18 mRNA levels were about 0.4% of total cellular mRNA [31,32]. Other studies have shown that bovine CyP accounts for 0.1–0.4% of total cytosolic protein [33]. In B. germanica, highest CyP18 mRNA tissue concentrations were found in fully developed ovaries and in early embryos (Days 0–4) [11]. In these embryonic stages, CyP mRNA levels were approximately 5-fold higher than in adult cells. Although CyP18 is a ubiquitous enzyme, it is difficult to explain why higher levels are expressed during embryogenesis. An increased level of cyclophilin might be necessary to assist in rapid protein synthesis. This function would be consistent with the role of CyP18 in the trafficking and folding of proteins in adult cells.

We have shown that the levels of rabCyP18 mRNA and PPIase activity based on CyPs were increased by oxygen stress (Figs. 1 and 6). An induction of hCyP18 protein by oxygen stress has been described using a xanthine oxidase/hypoxanthine system to generate ROS in human umbilical vein endothelial cells. In rabbit embryos, 20% oxygen during culture increased the generation of hydroperoxide in cultured embryos [34] and changed gene expression as compared to 5% O2 [15]. It is known that ROS can modify gene expression and the enzymatic activity of several proteins [35]. It can be hypothesized that the alterations in rabCyP18 gene expression and activity are also linked to ROS generation during 20% O2 culture. In oxygen-stressed blastocysts, higher amounts of CyP18 might be required to accelerate folding and maturation of newly synthesized proteins with O2 protective functions. An increased expression of CyP18 may be a part of a general cellular answer to oxygen stress. A specific interaction partner of hCyP18, Aop1 [10], is described as an oxygen protective protein belonging to a large family of bacterial and eukaryotic enzymes. They facilitate cellular defense mechanism, for example, by eliminating thiol radicals [36–38]. The exact interaction of hCyP18 and Aop1 is still unknown. Both proteins are abundant cytosolic proteins and are induced by oxidative stress [8,39]. The hCyP18 not only binds to Aop1 but also stimulates its enzymatic activity [10].

In conclusion, present results suggest that CyP18 may be part of the oxygen stress response in mammalian embryos. CyPs may have an important physiological role during embryogenesis. During normal development, PPIase activity might be needed to accelerate the folding of newly synthesized polypeptides. In stressed embryos this general function might be broadened to the stimulation of folding of denatured proteins and/or proteins that function in oxygen stress defense.


   FOOTNOTES
 
First decision: 21 July 1999.

1 Correspondence: Bernd Fischer, Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Grosse Steinstr. 52, D-06097 Halle (Saale), Germany. FAX: 49 345 5571700; bernd.fischer{at}medizin.uni-halle.de Back

Accepted: August 18, 1999.

Received: May 24, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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