Matrix metalloproteinase type-2 and type-9 immunoexpression in bovine caruncular tissue at the time of calving, 2 hours post-calving, and 4 hours post-calving

As bovine parturition nears, the placentome undergoes changes that allow placental delivery within 12 hours after calving. These changes include the expression of enzymatic proteins (e.g. MMPs) within the placentome that are capable of breaking down collagen, allowing for the separation of the cotyledon from the caruncle. Dysregulation of either MMP can reduce collagen breakdown and result in placental retention. The objective of the study was to use immunohistochemistry to semiquantify MMP2 and MMP9 expression in bovine caruncles following a normal delivery. We hypothesized that MMP2 and MMP9 immunoexpression peak at calving and decrease within 4 hours after calving. Caruncles were collected from five cows as previously described]. Tissues were formalin-fixed, paraffin-embedded, and sectioned (6 μm) onto charged slides. All slides were deparaffinized, rehydrated, and subjected to either Proteinase-K (#S3020, Dako) or heat-induced epitope retrieval (#S1700, Dako) for MMP2 and MMP9, respectively. Endogenous peroxidase activity was inactivated with 3% hydrogen peroxide and nonspecific binding was blocked with Protein Block serum (X0909, Dako). MMP2 (LS-B2799) was applied at a 1:1000 dilution and MMP9 (LS-A9461) at a 1:100 dilution. Negative controls from each tissue were treated with a universal negative antibody (#N1699, Dako). Slides were then reacted with One Step Horse Radish Peroxidase-Conjugated Polymer Anti-Rabbit IgG (ImmunoBioScience) followed by Nova Red Peroxidase substrate (#SK4800, Vector Laboratories). Slides were counter-stained with hematoxylin, dehydrated, and mounted. Utilizing bright-field microscopy at 50X magnification, immunoexpression of MMP2 and MMP9 was scored (0-3) across four quadrants of the caruncle. The average score for each time point in each individual was used to calculate the average ± SEM. These preliminary results show that there was a trend for MMP2 expression to be lower at two hours post calving (p=0.07) but this difference did not persist at 4 hours (p=0.11). For MMP9, there was a trend for decreased expression by 4 hours post calving (p=0.06). These results differ from that of Takagi and coworkers who found that MMP2 and MMP9 mRNA expression was lower at parturition compared to six hours postpartum, although protein expression was not determined in their study. Additional studies are underway to determine if acupuncture administered at the time of calving increases MMP expression