236 LOCALIZATION OF INTERFERON-TAU IN BOVINE EMBRYOS AND CUMULUS CELLS BY CONFOCAL MICROSCOPY

Abstract

Interferon-tau (IFN-t) is a protein secreted by the conceptus of ruminant species and thought to be the primary signal in maternal recognition of pregnancy. Experiments were conducted to detect IFN-t in bovine oocytes, cumulus cells and embryos by use of immunocytochemistry and laser-scanning confocal microscopy. Embryos were produced by in vitro fertilization of in vitro-matured oocytes. Oocytes and embryos were fixed in formaldehyde at various stages of development, and stored in PBS until staining and microscopy. Cumulus cells were stripped from immature oocytes and cultured in M-199 (10% fetal calf serum) on coverslips treated with poly-D-lysine. They were divided into four treatment groups: (1) without hormones (control), (2) with the addition of FSH, (3) estradiol, or (4) FSH and estradiol. Bovine MBDK cells (ATCC CCL 22) and primary fibroblasts were cultured as controls on coverslips but without addition of hormones. A polyclonal antibody raised against bovine IFN-t was used, followed by a secondary conjugated antibody (AlexaFluor 488, Molecular Probes, Eugene OR). Actin was stained with phalloidin (AlexaFluor 568, Molecular Probes, Eugene OR). Cumulus, MDBK cells and fibroblasts were further stained with propidium iodine to visualize nuclei. Imaging was performed on a Leica laser-scanning confocal microscope. IFN-t was detected in hatched and unhatched Day 7 and Day 9 blastocysts, where its expression was restricted to the trophectoderm. IFN-t was also found in Day 5 and 6 morulae, but not at earlier stages. Furthermore, IFN-t was detected in the cumulus cell masses of oocytes before and after IVM, but not in the oocyte itself. Controls, in which the primary antibody was omitted, were negative regardless of developmental stage. IFN-t was also found in cultured cumulus cells regardless of whether hormones had been added to the medium;; however, the protein was localized in the nuclei of cells only if they had been cultured with FSH, whereas in cells cultured with estrogen alone or without hormones IFN-t was restricted to the cytoplasm. In contrast, no IFN-t was detected in MDBK cells or fibroblasts. These results extend previous findings by showing that IFN-t is expressed as early as the morula stage. Moreover, these results demonstrate that IFN-t is also produced by cumulus cells where FSH appears to initiate a translocation of IFN-t into the nucleus, suggesting a role in regulation of gene expression.