The non-pregnant (NP) group consisted of healthy, regularly menstruating women (n = 6) without medication, undergoing hysterectomy due to benign disorders not involving the cervix. Hysterectomies were carried out during the follicular phase of the menstrual cycle within ten days from the latest menstrual period, to avoid the risk for an unknown pregnancy. The study subjects had a mean age of 42 years (range 32–49), and a mean parity of 2 (1–3). The term pregnant (TP) group consisted of healthy women (n = 8) with a mean age of 32 years (range 28–38), a mean gestational age of 38 weeks (range 37–39), and a mean parity of 1 (range 1–2). All had unripe cervices as determined by a Bishop score of < 5 points and none of them was in labor. Elective caesareans on medical indications were carried out in all women. The postpartum (PP) group consisted of primiparous, healthy women (n = 9) with uncomplicated pregnancies, from whom biopsies were obtained within 20 minutes after uncomplicated vaginal parturition. They had a mean age of 30 years (range 23–37), and a mean gestational age of 40 weeks (range 39–41).

Every woman had given her informed consent before entering the study. The study was approved by the Karolinska Hospital Ethics Committee (96–187, 99–099).

The biopsies were obtained transvaginally from the anterior part of the uterine cervix at the 12 o'clock position, from 10–20 mm depth. The cervical biopsies were divided and one half was immersion-fixed in 4 % formaldehyde at 4°C overnight, stored at 4°C in 70% ethanol and thereafter embedded in paraffin. One half was immediately frozen at -70°C.

Paraffin sections (5 μm) were used as a standard immunohistochemical technique (avidin-biotin-peroxidase) which was carried out as described before to visualize MAPK (p42/44-ERK1/2, p38 and JNK) immunostaining intensity and distribution 23. Primary antibodies used in this study are listed in Table 1. All antibodies were incubated at 4°C overnight. Replacement of the primary antibody with an equivalent concentration of non-immune mouse IgG (for monoclonal antibody) and rabbit IgG (for polyclonal antibody) was used for negative controls.

A Leica microscope and Sony video camera (Park Ridge, NJ, USA) connected to a computer (Leica Imaging System Ltd, Cambridge, UK) was used to assess immunohistochemistry results. Two sections of each sample were analysed and ten areas of cell compartment from each section were randomly chosen for quantification of immunostaining intensity. The staining intensity was semiquantitatively graded (H.W.) by manual scoring as very strong (4) when 80–100% cells were positive, strong (3) when 60–80% of cells were positive, moderate (2) when 30–60% of cells were positive, weak (1) when less than 30% of cells were positive or as no staining (0).

Frozen pieces of cervical tissue were processed for isolation of total RNA according to the manual for the SV total RNA isolation system (Promega, Madison, WI, USA). One μg of total RNA were reversely transcribed at 42°C for 45 min in a final volume of 40 μl with a reaction mixture containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂, 7.5 mM DTT, 0.5 mM dNTPs, 1 μg random hexamers and 400 IU of ML-MTV reverse transcriptase (GIBO, BRL, Paisley, UK). Primer sequences and the predicted product size were for human c-fos gene: 5'-GGATAGCCTCTCTTACTACCAC-3'; 5'-TCCTGTCATGGTCTTCACAACG-3' and 280 bp; the human c-jun gene: 5'-GGAAACGACCTTCTATGACGATGCCCTCAA-3'; 5'-GAACCCCTCCTGCTCATCTGTCACGTTCTT-3' which amplify a 325 bp fragment and the human ribosomal protein S28 which was used as an internal control: 5'-GTGCAGATCTTGGTGGTAGTAGC-3'; 5'-AGAGCCAATCCTTATCCCGAAGTT-3' and 552 bp. For PCR amplification, the cDNA (corresponding to 50 ng RNA from the RT-PCR reaction) was added to the reaction mixture containing 20 mM Tris-HCl pH 8.4. 50 mM KCl, 2.5 IU Tag DNA polymerase (GIBCO, BRL, Paisley, UK), 0.2 mM dNTPs, 1.5 mM MgCl₂, and oligonucleotide primer pairs (50 pmol/pair), with a final volume of 50 μl. PCR was performed for 30 cycles using 94°C (30 s) for denaturing, 58°C (45 s) for annealing, 72°C (45 s) for extension and a final incubation at 72°C for 3 min on a thermal cycler. The products were subjected to electrophoresis in 2.5 % agarose gels; data were analyzed using a Fujifilm Las-1000 system (Fujifilm, Japan). Statistical analyses were not performed on this data.

Statistical calculations for the immunohistochemistry results by image analysis of pMAPK were performed by ANOVA on ranks (Kruskal-Wallis test) and significances were evaluated by Dunn's test. One-way parametric ANOVA was used for analyses of immunohistochemistry results for c-Jun and c-Fos proteins.

ERK immunostaining was observed in different compartments of the cervix by using non-phosphorylated and phosphorylated (pERK1/2) antibodies. The staining intensity was slightly higher by the non-phosphorylated antibody (data not shown), although the staining patterns did not differ significantly by these two types of antibodies. In cervical stroma, pERK1/2 expression was the lowest in the NP group, and increased in TP and PP groups as compared to NP (P < 0.05) (Figure 1A–D and Figure 3A). In the subepithelial mucosa area, intensive immunostaining was present in the stromal cells in all study groups. In smooth muscle, the pERK1/2 expression was unchanged between the groups (data not shown). The endothelial cells of blood vessels (vein) displayed high pERK1/2 expression in NP group, but weak or absent in TP and PP groups as compared to NP (P < 0.01) (Figure 1A,C and Figure 3A). No immunostaining for pERK 1/2 was found in glandular epithelium (Figure 1B,D).

The phosphorylated JNK (pJNK) immunostaining was mainly localized to the cell nuclei, whereas non-phosphorylated JNK staining was found in both cytosol and nuclei (data not shown). In cervical stroma, pJNK expression was the lowest in the NP group, but significantly increased and high in TP and PP groups as compared to NP (P < 0.05) (Figure 1E–H and Figure 3B). In the subepithelial mucosa area, pJNK staining was present in some stromal cells in all study groups. High intensity pJNK staining was generally found in glandular epithelium but without significant changes between the study groups (Figure 1F,H). Vascular endothelial pJNK expression did not differ between the groups (Figure 1E,G and Figure 3B).

Only the phosphorylated-specific antibody for p38MAPK was used. A reciprocal pattern för p38MAPK expression was observed in cervical stroma in comparison to vascular endothelium between NP and TP groups (Figure 3C). High intensity p38MAPK immunostaining was present in the vascular endothelium in NP and TP groups and absent in PP group (P < 0.01) (Figure 2A,C (magnification), E (arrow head), and Figure 3C). p38MAPK expression in stroma was significantly increased in TP as compared to NP group (P < 0.05) (Figure 2 and Figure 3C). In the subepithelial mucosa area, p38MAPK staining was present in stroma cells in all study groups, but less intensive in the PP group (Figure 2B,D,F).

Immunoreactivity for c-Jun protein was present in all cellular compartments in NP, TP and PP groups (Figure 4). c-Jun expression was significantly increased in cervical stroma (P < 0.01) and smooth muscle (P < 0.001) in TP as compared to NP group (Figure 5). The changes in vascular endothelium, squamous epithelium and glandular epithelium did not differ between the groups.

Immunoreactivity for c-Fos protein was present in all cellular compartments in NP, TP and PP groups, as shown in Figure 4. c-Fos expression was increased in cervical stroma (P < 0.01), squamous epithelium (P < 0.01) and glandular epithelium (P < 0.05) in PP as compared to TP group (Figure 6). The changes in vascular endothelium and smooth muscle did not differ between the groups.

Figure 7. illustrates the presence of c-jun and c-fos mRNAs in human uterine cervix in term pregnancy and immediately after parturition. S28 was used as a standard to enable comparison of the amount of RNA loaded to each well.