180 EXPRESSION OF TUMOR NECROSIS FACTOR-STIMULATED GENE 6 PROTEIN IN BOVINE CUMULUS–OOCYTE COMPLEXES AT DIFFERENT MATURATION STATUS

Abstract

The TSG-6 is a ~35-kDa protein belonging to hyaluronan binding superfamily proteins. The TSG-6 plays role in inflammation and in inflammation-like processes (i.e. ovulation). The tsg-6 expression is induced in cumulus–oocyte complex (COC) cells just before ovulation. It is involved in the migration of cumulus cells though the formation and stabilisation of the extracellular matrix. Disturbances in secretion of this protein lead to a reduction in the number of ovulated oocytes. Although studies of the prevalence and role of TSG-6 in COC were conducted in several animal models, little is known about TSG-6 in cattle. The aim of this study was to assess tsg-6 mRNA expression and protein localization in bovine cumulus cells from COC at different maturation status. Ovaries were collected from the slaughterhouse. Cumulus cells were isolated from COCs in different stages of maturity. In variant I, COC were isolated from small follicles of Φ 2 to 6 mm. In variant II, COC were also isolated from small follicles and matured in vitro in TCM199 HEPES with 10% FBS and 0.02 IU NIH-pFSH mL^–1, 1 µg mL^–1 β-oestradiol, 0.2 µM sodium pyruvate, 50 µg mL^–1 gentamicin in 5% CO₂, 38.5°C for 24 h. In variant III, COCs were isolated from large follicles of Φ >15 mm. Analysis of tsg-6 expression in cumulus cells was performed using real-time PCR. Expression of Tsg-6 was normalized to that of s18. Statistical analysis of tsg-6 mRNA level in all variants was carried out by 1-way ANOVA, and comparisons of mean values were made with the Tukey honestly significant difference test (Statgraphic 5.1 Centurion); P < 0.05 was considered to reflect the presence of statistical significance. For all variants, paraffin-embedded slices of COC were carried out to localise TSG-6 by indirect method of immunofluorescence. Immunostaining of the TSG-6 protein was performed using primary polyclonal antibody raised against bovine TSG-6. Antigen–antibody complexes were visualised after incubation with secondary IgG conjugated with fluorescein isothiocynanate. Immunolocalization of TSG-6 was performed using fluorescent microscopy. The relative expression of tsg-6 mRNA in variant II was more than 2 times higher (1.887 ± 0.797 a.u.) compared with variant III (0.760 ± 0.130 a.u.). The difference was statistically significant (P < 0.05). No tsg-6 expression in variant I was detected. The presence of TSG-6 protein in the extracellular matrix of cumulus cells from variant II as well as from variant III was detected. The present data suggest that tsg-6 gene expression and its protein presence are stimulated during COC maturation in cattle as previously demonstrated for other species.

Supported by Warsaw University of Life Sciences, Faculty of Veterinary Medicine: 505-10-02330050.