Molecular Human Reproduction, Vol. 6, No. 12, 1147-1153,
December 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
The expression of glutaredoxin is increased in the human cervix in term pregnancy and immediately post-partum, particularly after prostaglandin-induced delivery
1 Division for Reproductive Endocrinology, 2 Division for Obstetrics and Gynecology, Department of Woman and Child Health and 3 Medical Nobel Institute for Biochemistry, Department of Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
Abstract
Glutaredoxins are glutathione disulphide oxidoreductases catalysing disulphide reductions via a redox active disulphide. We have examined the presence of glutaredoxin in the human cervix, and its differential expression during cervical remodelling in term pregnancy and immediately post-partum as compared to the non-pregnant state. Cervical biopsies were obtained from 24 term-pregnant and 24 post-partal women, of which 10 were taken after spontaneous delivery, 10 after prostaglandin-induced delivery and four after mifepristone-induced delivery, all obtained within 15 min after delivery. Six non-pregnant women served as controls. The tissues were analysed for the glutaredoxin mRNA levels using a solution hybridization method. Glutaredoxin mRNA was expressed in the human cervix, the level increased 
2-fold at term pregnancy and immediately post-partum. The level of cervical glutaredoxin mRNA from prostaglandin E2-treated women was 3-fold higher than after spontaneous ripening and delivery. Localization of glutaredoxin was visualized with immunohistochemistry in cervices from two post-partal women, and was compared to that of thioredoxin. We conclude that glutaredoxin may be involved in the regulation of cervical ripening in humans, particularly in the inflammatory reaction seen during this process. Glutaredoxin mRNA levels are up-regulated after prostaglandin treatment, which is effective and the most commonly used substance for cervical priming and induction of labour.
cervix/glutaredoxin/pregnancy/redox regulation/thioredoxin
Introduction
The precise mechanisms of cervical ripening in humans are not known. In term pregnancy, neutrophils and macrophages have been shown to be present in the human cervix in significantly higher numbers than during the first trimester, indicating a role for these cells during cervical ripening (Bokström et al., 1997
). The final ripening of the human cervix has been compared to an inflammatory reaction (Liggins, 1981), and in accordance with this an infiltration of cervical tissue with inflammatory cells has been shown (Junqueira et al., 1980).
Increased levels of collagenase and leukocyte elastase have been found during the process of final ripening (Uldbjerg et al., 1983; Osmers et al., 1992). Neutrophils, eosinophils and fibroblasts are known to secrete collagenase (Osmers et al., 1992; Jeziorska et al., 1996).
In previous studies we have shown a decrease in the levels of oestrogen receptor 
(ER) and the progesterone receptor (PR) in cervical biopsies from term-pregnant and post-partal women, as compared to non-pregnant women (Stjernholm et al., 1996, 1997). The level of cervical insulin-like growth factor (IGF)-I mRNA was increased 4-fold in term-pregnant women as compared to non-pregnant women. In post-partal women the level decreases again to 50% of the level seen in term pregnancy (Stjernholm et al., 1996, 1997). In addition, the expression of thioredoxin is increased in cervical biopsies from term-pregnant and post-partal women as compared to non-pregnant, whereas no differences are seen between pharmacologically induced and spontaneous deliveries (Sahlin et al., 1997a).
Glutaredoxin is a member of the thioredoxin superfamily, originally discovered as a glutathione (GSH)-dependent hydrogen donor for ribonucleotide reductase and a general GSH-disulphide oxidoreductase (Holmgren, 1976; Holmgren and Åslund, 1995; Holmgren et al., 1998). The localization of bovine glutaredoxin has been mapped by immunohistochemical methods and shown to overlap with the distribution of thioredoxin but also to have striking differences. Thus, strong bovine glutaredoxin activity is present in oocytes in the ovary and in epithelial tissue of the skin, reflecting differential expression during cell differentiation (Rozell et al., 1993). The immunoreactivity of the protein in the human ovary also shows parallel changes related to the functional activity of the corpus luteum (CL) (Garcia-Pardo et al., 1999). Human glutaredoxin has been purified and cloned from human placenta (Padilla et al., 1995).
Thioredoxin is a multifunctional protein disulphide reductase, which plays a key role in redox regulation and defence against oxidative stress as well as in the supply of electrons to ribonucleotide reductase, which is essential for DNA synthesis (Holmgren, 1985, 1989; Holmgren and Björnstedt, 1995). Recently thioredoxin and glutaredoxin have been shown to be of increasing importance in clinical medicine (Holmgren, 1999). The present study was performed to determine the expression of glutaredoxin mRNA in the human cervix, and its possible relation to the ripening process. The localization of glutaredoxin was studied with immunohistochemistry and compared to that of thioredoxin, to disclose the relationship between the two redox enzymes in pregnancy and during delivery. Correlation analyses were also performed, for patient samples where mRNA levels of both thioredoxin and glutaredoxin had been determined, to evaluate possible similarities in the expression pattern of the two enzymes.
Materials and methods
Patients
The non-pregnant group consisted of six regularly menstruating women with a mean age of 46 years (range 42–51), and a mean parity of 1 (range 0–3). All underwent hysterectomy due to benign disorders not involving the cervix.
The term-pregnant group consisted of 24 healthy women with a mean age of 33 years (range 23–41), a mean gestational age of 38 weeks (range 37–40) and a mean parity of 2 (range 1–4). All women had unripe cervices with a Bishop score of <5 points and none of the women were in labour. Elective Caesarean sections on medical indications were carried out in all women. Biopsies were obtained during operation.
The post-partal group consisted of 10 women from which biopsies were taken within 15 min of spontaneous vaginal delivery. They had a mean age of 29 years (range 26–33), a mean gestational age of 40 weeks (range 39–41) and a mean parity of 1.4 (range 1–2).
The prostaglandin-treated group consisted of 10 women with unripe cervices (Bishop score of <5 points) who were treated with 0.5 mg prostaglandin E2 (PGE2) in a viscous gel (dinoproston, Cerviprost®; Organon International, OSS, The Netherlands) intracervically for cervical priming and initiation of labour. This was due to medical reasons such as oligohydramniosis, post maturity and accelerated fetal growth. The biopsies were taken within 15 min of vaginal delivery. These women had a mean age of 31 years (range 21–37), a mean gestational age of 41 weeks (range 40–42) and a mean parity of 1.3 (range 1–2). Four women needed an additional application of 0.5 mg PGE2 intracervically or 2 mg PGE2 gel vaginally (dinoproston, Minprostin®; Upjohn, Pharmacia AB, Stockholm, Sweden) due to insufficient ripening at examination after 24 h.
The mifepristone group consisted of four post-term women treated with 400 mg antiprogestin (Mifepristone®; Roussel-Uclaf, Paris, France) orally, to induce cervical ripening and induction of labour. The biopsies were taken within 15 min of vaginal delivery. These women had a mean age of 26 years (range 21–34), a mean gestational age of 42 weeks (range 42–42) and a mean parity of 1.0 (range 1–1).
Some of these patients were also part of previous published studies on ER, PR, IGF-I and thioredoxin expression in cervical biopsies (Stjernholm et al., 1996, 1997, 1999; Sahlin et al., 1997a).
All women gave their informed consent and the study was approved by the local Ethics Committee of the Karolinska Hospital (96-187; 99-099).
Sampling procedure
The biopsies were taken transvaginally from the anterior cervical lip at the 12 o'clock position, from 10 to 20 mm depth. The mucosa was carefully removed and the biopsies were placed on dry ice and stored at –70°C until analysed. Serum samples were drawn simultaneously, centrifuged within 30 min at 3000 g for 10 min and stored at –20°C. The serum levels of oestradiol and progesterone were quantified by radioimmunoassay (Sufi et al., 1995) (Table I).
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Preparation of total nucleic acids
Total nucleic acids (TNA) were prepared by digestion of homogenized tissue with proteinase K in a sodium dodecyl sulphate-containing buffer, followed by subsequent extraction with phenol-chloroform as described before (Stjernholm et al., 1996
). The concentration of DNA in the samples was measured fluorometrically at the wavelength 458 nm with Hoechst Dye 33258.
Hybridization probes
The probe used for the glutaredoxin mRNA determinations was derived from a clone of human glutaredoxin cDNA (Padilla et al., 1995). Sense and antisense constructs of the cDNA (fragments of 320 base pairs representing 106 amino acids) were generated by polymerase chain reaction amplification of the full-length human glutaredoxin cDNA using oligonucleotides (sense: 5'-GAAAGCTTGCATGGCTCAAGAGTTTGTG-3'; 5'-GAGGATCCTTACTGCAGAGCTCCAATCTG-3' and antisense: 5'-GAGGATCCGC-ATGGCTCAAGAGTTTGTG-3'; 5'-ATGAAGCTTGTGGTTACTGCAGAGCTCC-3') containing HindIII and BamHI restriction sites (underlined sequences). The amplified products were gel-purified and directionally cloned in a pDNA3 vector (Invitrogen, San Diego, CA, USA). Cleavage with BamHI allowed the synthesis of either antisense or sense RNA probes depending on the orientation of the insert. For solution hybridization analysis the probe was labelled with [35S]UTP.
Hybridization analysis of mRNA
A solution hybridization assay of specific mRNA was used and performed as presented before (Sahlin, 1995; Sahlin et al., 1997a). Intra- and inter-assay variations were controlled by specific internal controls used in all hybridizations. Variations <10% were considered acceptable.
Immunohistochemistry
Paraffin sections (5 µm) from cervices after spontaneous delivery were used to study the distribution of thioredoxin and glutaredoxin in the tissue. A standard immunohistochemical technique (avidin-biotin-peroxidase) was used to visualize the immunostaining using antibodies to glutaredoxin and thioredoxin from IMCO (Stockholm, Sweden; www.imcocorp.se). Human recombinant thioredoxin was expressed and purified to homogeneity as described previously (Ren et al., 1993). The polyclonal antisera were obtained by immunization of a goat with oxidized thioredoxin and pure antibodies were obtained by affinity chromatography on immobilized human thioredoxin. The antibody was used at a concentration of 5 µg/ml.
The glutaredoxin antibody was obtained by immunization of a goat with wild type recombinant human glutaredoxin (Padilla et al., 1996). The antibodies were purified by affinity chromatography and used at a concentration of 4.7 µg/ml. The antibodies inhibit glutaredoxin activity and produce a single band of the expected size by Western blotting (data not shown).
Immunohistochemistry procedures were similar to those previously described (Wang et al., 1999). A Leica microscope connected to a video camera (Sony) and computer was used to assess immunostained images.
Statistics
Values are given as median and range. Statistical calculations were done with analysis of variance on ranks (Kruskal-Wallis' test); evaluation of significance was done with Dunn's test and P < 0.05 was considered significant. Correlation was determined by Spearman's rank correlation test.
Results
The glutaredoxin mRNA levels were significantly increased in cervical biopsies from women in term pregnancy and after pharmacologically induced deliveries (Figure 1). The glutaredoxin mRNA levels were particularly high in the cervices from women with prostaglandin-induced deliveries compared with those from spontaneous vaginal deliveries (Figure 1). The cervical biopsies from women who had mifepristone-induced deliveries showed glutaredoxin mRNA levels which were not significantly different from those from spontaneous vaginal deliveries (Figure 1).
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The immunohistochemical localization of glutaredoxin (Figure 2A,B,D,E
) in paraffin sections of cervices from spontaneous vaginal deliveries was different from the localization of thioredoxin (Figure 2G,H,J,K) in the same patients. The positive immunostaining (both cytosolic and nuclear) for thioredoxin (Figure 2G,H) in the epithelial cells was stronger than that for glutaredoxin (Figure 2A,B). In the stroma, glutaredoxin was found mainly in the cell nuclei. Within the vessels the white blood cells were seen strongly positive, and the erythrocytes were also positively stained (Figure 2E). A similar pattern of immunostaining was found also for thioredoxin in the stroma, although it seemed to be relatively stronger (Figure 2J,K). Negative controls (where the antibodies were preincubated with the respective purified enzyme) are shown for glutaredoxin in Figure 2C (epithelium) and 2F (stroma) and for thioredoxin in Figure 2I (epithelium) and 2L (stroma).
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Correlation analyses showed a positive correlation between the levels of glutaredoxin mRNA and thioredoxin mRNA in the post-partal group (P = 0.0012) (Figure 3A
), but this correlation was not seen in any of the other groups. The thioredoxin mRNA levels in these samples were taken from a previous study (Sahlin et al., 1997a). In the term-pregnant group there was a negative correlation between the levels of glutaredoxin mRNA and PR mRNA. The PR mRNA levels were also determined in a previous study (Stjernholm et al., 1996) (P = 0.012) (Figure 3B).
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Discussion
We have found that the expression of glutaredoxin mRNA is increased in cervical biopsies from women in term pregnancy and immediately after delivery (pharmacologically induced) as compared to the non-pregnant state. After PGE2-induced deliveries, the levels of glutaredoxin mRNA were even higher than after spontaneous deliveries. This prostaglandin is effective and the most commonly used substance for cervical priming and induction of labour at term (Ekman et al., 1983; Calder, 1990). We have previously shown that PGE2-induced cervical ripening mimicked spontaneous ripening with regard to cervical concentrations of ER, PR, their mRNA and IGF-I mRNA (Stjernholm et al., 1999).
Why is glutaredoxin particularly up-regulated by PGE2 and what is the role of thioredoxin and glutaredoxin in the delivery process? In a recent paper by Denison et al. (1999), the action of PGE2 in the human cervix was studied, resulting in a hypothetical model for cervical ripening at term (Figure 4). Central in this scheme are the effects of PGE2 to stimulate vasodilatation and the release of inflammatory mediators resulting in oxidative stress.
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Interleukin-8 (IL-8), a chemotactic cytokine for neutrophils, has been shown to induce cervical ripening in pregnant guinea-pigs, both by morphological and biochemical criteria (Chwalisz et al., 1994
). IL-8, which is up-regulated by PGE2, is also a strong candidate for mediating the final step in cervical ripening in humans (Barclay et al., 1993; Sennström et al., 1997, 2000; Luo et al., 2000). Upstream of the effects of IL-8, activation of NF
B and release of nitric oxide (NO) are involved in the synthesis of IL-8 and vasodilatation, respectively. PGE2 has been shown to decrease synthesis of glutathione and the transport of cystine into cells (Yu et al., 1993; Rishikof et al., 1998). This will be sensed as oxidative stress and may induce the synthesis of glutaredoxin, as has been observed in bacteria (Prieto-Álamo et al., 2000). Lower synthesis of GSH and oxidative stress by inflammatory mediators will affect the redox potential in the cytosol given by the 2GSH (reduced)/GSSG (oxidized glutathione) ratio and may lead to an increase in mixed disulphides with GSH. Such oxidatively damaged proteins are repaired by glutaredoxin-catalysed disulphide reduction by GSH (Gravina and Mieyal, 1993; Holmgren and Åslund, 1995; Jung and Thomas, 1996; Davis et al., 1997; Bandyopadhyay et al., 1998).
Immunostaining showed that glutaredoxin was mainly localized to the cytosol, but in many cells, especially in the stroma, glutaredoxin was mainly found in the nuclei. The glutaredoxin gene does not code for known signals for nuclear import (Padilla et al., 1995) consistent with a cytoplasmatic localization. However, immunohistochemical analysis of glutaredoxin in various calf tissues also revealed a significant number of cells with a strong nuclear staining (Rozell et al., 1993). Interestingly, thioredoxin also lacks any known signals for nuclear transport, but has been shown to translocate into the nucleus after exposure to irradiation (Hirota et al., 1999) or phorbol-12-myristate-13-acetate (PMA) (Hirota et al., 1997). Both glutaredoxin and thioredoxin may therefore have the capacity to translocate to the nucleus by as yet unknown mechanisms.
Thioredoxin is secreted from many cell types and acts as a co-cytokine (Ericson et al., 1992; Rosén et al., 1995). Thioredoxin is also recently identified as a chemo-attractant for lymphocytes, monocytes and neutrophiles (Bertini et al., 1999). Thioredoxin has been shown to enhance the mRNA expression of IL-8 in several cell lines (Schenk et al., 1996; Chang et al., 2000). From the immunohistochemistry pictures it could be seen that there are more epithelial cells, both in cytoplasm and nuclei, staining positive for thioredoxin than for glutaredoxin (Figure 2H
compared to 2B), and this seems to be true also for the stroma (Figure 2J compared to 2D). The white blood cells in the vessels have a strong immunopositive staining for both thioredoxin and glutaredoxin, showing that these enzymes are co-localized within cells that produce some of the inflammatory lipid hydroperoxide mediators (Jacobsson et al., 1999) in cervical ripening.
Thiol redox control is important for regulating the binding of transcription factors to DNA (Holmgren, 1985; Schenk et al., 1994; Sen and Packer, 1996). Increased concentrations of oestrogens increase the expression of thioredoxin which enhances the activity of NFB and the liberation of NO (Nikitovic and Holmgren, 1996; Sahlin et al., 1997a,b). Furthermore, 17ß-oestradiol up-regulates thioredoxin and glutaredoxin in bovine aortic endothelial cells and protects them against oxidative stress (Ejima et al., 1999). Regulation of NFB activity has also been shown to be dependent on the GSSG/GSH ratio and the activity of intracellular antioxidant enzymes (Renard et al., 1997). Glutaredoxin could be the prime candidate to transmit these changes to protein function, although the exact mechanism of this action is yet unknown. Glutathione has been implicated in the redox regulation of AP-1 DNA binding through S-glutathiolation (Klatt et al., 1999), but a role for glutaredoxin has not been shown. Glutaredoxin has been shown to increase NFB activity in reporter gene (pNFB-Luc)-transfected HEK293 cells after either tumour necrosis factor - or PMA-induced NFB activity; whereas thioredoxin decreased the activity (Hirota et al., 2000). Glutaredoxin and thioredoxin have also been shown to enhance the activity of PMA-induced AP-1 activation, as well as c-Jun activity, in reporter gene (pAP-1-Luc and pGAL4-cJun respectively)-transfected HEK293 cells (Hirota et al., 2000). The promoter of the collagenase gene contains an AP-1 site (Jonat et al., 1992). Thus, regulation of transcription factor activity may be an important general mechanism by which the redox enzymes participate in cervical ripening. Furthermore, the promoter of glutaredoxin contains an AP-1 regulatory element (Park and Levine, 1977).
Another role of thioredoxin and glutaredoxin in downstream effects (Figure 4) concern their role as general disulphide reductants (Holmgren, 1989) in tissue remodelling. Furthermore, inactivation of tissue inhibitors (TIMP-1 and TIMP-2) of metalloproteinases (MMP) by disulphide reduction may promote MMP-2 and MMP-9 activity involved in collagen breakdown (unpublished data).
In conclusion, increased levels of cervical glutaredoxin mRNA was found in biopsies at term pregnancy and immediately post-partum. The levels were higher after treatment with PGE2 for cervical priming and labour induction than in women after spontaneous vaginal delivery. Thus, glutaredoxin is up-regulated after PGE2 treatment linking this redox enzyme to the activity of this particular member of the prostaglandin family.
Acknowledgments
We are grateful for excellent technical assistance from Britt Masironi and Sonja Åkerberg. This study was supported by grants from the Swedish Medical Research Council, grants 03972 (H.E., L.S.), 09508 (G.E.) and 03529 (A.H.), The Swedish Society of Medicine (L.S.) and Karolinska Institutet.
Notes
4 To whom correspondence should be addressed at: Division for Reproductive Endocrinology, Karolinska Hospital, L5:01, S-171 76 Stockholm, Sweden. E-mail: Lena.Sahlin{at}kbh.ki.se 
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Submitted on May 10, 2000; accepted on August 31, 2000.
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