Antibodies and Recombinant Ovine IFN-т
A monoclonal mouse antibody against recombinant bovine GM-CSF, GM-CSF17.2, was purchased from VMRD (Pullman, WA). The negative control, mouse immunoglobulins G1 (IgG1), was acquired from Dako Diagnostics (Mississauga, ON, Canada), and the biotinylated goat-anti-mouse secondary antibody was obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Polyclonal rabbit antisera against ovine COX-1 (COX-1 241) and COX-2 (COX-2 243) were donated by Merck Frosst (Kirkland, QC, Canada). Biotinylated goat-anti-rabbit secondary antibody was obtained from Dako Diagnostics. Recombinant ovine IFN-т was kindly donated by Dr. Fuller W. Bazer (College Station, TX). Its amino acid sequence is 80% homologous to bovine IFN-т and it cross-reacts in cattle.
In experiment 2, six healthy, sexually mature mixed-breed beef heifers (1.5-2 yr of age; 520 ± 31-kg bodyweight) showing a cycle length of 1920 days were randomly assigned to control (n = 3) or IFN-т treatments (n = 3) and treated midcycle with Estrumate (500 mg cloprostenol, Sch-ering Canada Inc., Pointe Claire, QC, Canada) to synchronize estrus. Animals were observed at least twice daily for estrus throughout the experiment. In the morning of Day 14 after estrus (Day 0), each heifer received either vehicle (5 ml, 0.1% BSA in PBS) or recombinant ovine IFN-т (5 ml, 0.25 mg, equivalent to 2.5 X 107 antiviral units) transcervically into the uterine body. Infusions were repeated three times at 12-h intervals.
Animals and Tissue Collection
All procedures performed were in accordance with the guidelines of the Canadian Council on Animal Care and were reviewed and approved by the Nova Scotia Agricultural College Animal Care and Use Committee. In experiment 1, 12 sexually mature, healthy mixed-breed beef heifers were slaughtered at government-inspected abattoirs on Days 0 (estrus), 7, 16, and 18 of the estrous cycle (n = 3 per day). To confirm the stage of the estrous cycle, animals were observed for estrous behavior, and blood samples taken at estrus and slaughter were assayed for progesterone (P4) by RIA with a Coat-A-Count kit (Diagnostic Products Corporation, Los Angeles, CA).
However, COX-2 itself does not discriminate between PGF2a and PGE2 because it catalyzes the transformation of arachidonic acid into the same precursor for both PGF2a and PGE2, PGH2, which is rapidly converted by PGF-synthase or PGE-synthase, among others. It is notable that in many species, endometrial expression of COX-2 is stimulated during the attachment period. Granulocyte-mac-rophage colony-stimulating factor (GM-CSF) is another important factor expressed in the uterine epithelium of the mouse, human, and ruminants because it is known to promote the survival and growth of embryos in these species.
In the endometrium, IFN-т has been shown to stimulate granulocyte chemotactic protein-2 and monocyte che-motactic protein-1 and -2, IFN-7-inducible protein 10 kDa, IFN-stimulated gene product 17, 2’5′-oli-goadenylate-synthetase, 1-8U and Leu-13, major histocompatibility complex class I and beta-2-microglobu-lin, Mx protein, signal transducer and activator of transcription 1 and 2, interferon regulatory factor (IRF) 1 and IRF9. In the ovine uterus, the receptor for type I interferons is predominantly expressed in the luminal epithelium (LE) and shallow glands (SG) but also in the caruncular stroma. However, binding of IFN-т is limited to the LE and SG.
In the bovine species, 25%-40% of all embryos are lost during the first month of pregnancy. During this period, dramatic cellular transformations in both the maternal epithelium and the conceptus are accompanied by the transient expression at the materno-fetal interface of potent molecules ensuring the survival, accommodation, and growth of the conceptus.