Smooth muscle cells in bovine cervical ripening and dilatation; contractility, degrading enzymes and inflammation

Abstract

Cervical ripening is a complex process of modification of cervical tissue that enables dilation of the cervix at parturition. Cervical smooth muscle tissue might play a role by contracting or by secretion of cytokines or MMPs. To assess a contractile role for the cervical smooth muscle cells in cervical dilatation, we measured cervical diameter with ultrasound cervimetry together with EMG-activity of the myometrium, cervical outer muscular layer (COML) and cervical stromal layer (CSL). This was done in 4-6 cows from the moment a PGF2alpha was injected until 48 hours after expulsion. The COML EMG activity began to increase some12 h before cervical dilatation, and decreased after fetal expulsion. The increase before and decrease after expulsion was low compared to the myometrium and not characterized by an increase and decrease in maximum EMG amplitudes. The COML showed contracture and contraction like EMG activity in unison with in the myometrium, but also irregular EMG activity. After calving, the cervical diameter measured initially 6 cm, increased during the first 15 h and decreased afterwards. Myometrial and cervical EMG burst patterns were accompanied by transient dilatations of the caudal cervix. These data suggest that the activity of the outer muscular layer of the cervix is partly differently regulated compared with the myometrium. Increased activity of the outer muscular layer could have functional relevance in the evacuation of the uterus. The CSL did not exhibit EMG activity and was further characterized. Cervical biopsies were repeatedly taken from 10 pregnant cows at days 185 and 275 of gestation, at calving and 30 days afterwards, and analysed with immuno histochemistry and RT-PCR (n=5). At parturition, the smooth muscle tissue in the CSL remained arranged in bundles but cellular density and the smooth muscle actin alpha (SMA) mRNA expressions were decreased. Additionally, the SMA staining and connexin-43 expression and staining remained constant. This might indicate that stromal smooth muscle cells are not prepared to contract with parturition. Co-expression with vimentin suggests that the stromal smooth muscle cells predominantly have a secretory function in cows. The cervical ripening process is divided in a gradual stage and a final stage which is an inflammatory process. Involvement of Matrix Metallo Proteinases (MMPs), produced by smooth muscle cells and of cytokines and leucocytes in the two stages of bovine cervical ripening were assessed in the series of cervical biopsies. MMP-2 expression had already increased at 5 days before parturition and was present in smooth muscle cells and extra cellular matrix, which suggests involvement of smooth muscle cells in collagen denaturation during gradual cervical ripening. At parturition, IL-8 expression had largely increased enabling the large invasion of neutrophils, and MMP-1 and MMP-9 protein expression were increased. Neutrophils might have contributed to cervical softening by secreting MMPs. At parturition, the levels of IL-1 only slightly increased, of IL-6 remained the same and of TNFalpha decreased, which potentially were the consequences of increased IL-10 expression. This indicates that final ripening at term parturition is a non-infectious inflammatory process influenced by regulatory cytokines.