Actions of tumor necrosis factor-alpha on oocyte maturation and embryonic development in cattle

PROBLEM: Infertility can accompany mastitis in cattle. Involvement of tumor necrosis factor-alpha (TNF-alpha) in this phenomenon is suggested by observations that circulating concentrations of TNF-alpha are elevated after intramammary infection or infusion of endotoxin. It was hypothesized that (1) TNF-alpha acts on the oocyte during maturation to decrease the percent of oocytes that cleave and develop following fertilization; (2) exposure of embryos to TNF-alpha after fertilization reduces development to the blastocyst stage; and (3) TNF-alpha increases the proportion of blastomeres that undergo apoptosis in a stage-of-development dependent manner. METHOD OF STUDY: In one experiment, oocytes were matured with various concentrations of TNF-alpha and then fertilized and cultured without TNF-alpha. In another study, embryos were cultured with TNF-alpha for 8 days beginning after fertilization. Finally, embryos were collected at the two or four-cell stage (at 28-30 hr after insemination) or when greater than or equal to9-cells (at day 4 after insemination) and cultured +/- TNF-alpha for 24 hr. The proportion of blastomeres undergoing apoptosis was then determined by the TUNEL procedure. RESULTS: Addition of TNF-alpha to maturation medium did not affect the proportion of oocytes that cleaved. However, the percent of oocytes that developed to the blastocyst stage at day 8 after insemination was reduced (P = 0.05) at all TNF-alpha concentrations tested (0.1-100 ng/mL). When added during embryo culture, there was no significant effect of TNF-alpha on the proportion of oocytes that became blastocysts. In addition, TNF-alpha did not induce apoptosis in two and four-cell embryos. For embryos greater than or equal to9-cells, however, 10 and 100 ng/mL TNF-alpha increased (P < 0.05) the percent of blastomeres labeling as TUNEL-positive. CONCLUSION: TNF-alpha can have deleterious actions on oocyte maturation that compromise development of the resultant embryo. While exposure of fertilized embryos to TNF-alpha did not inhibit development to the blastocyst stage, TNF-alpha increased the percentage of blastomeres undergoing apoptosis when exposure occurred for embryos greater than or equal to9-cells. Increased blastomere apoptosis could conceivably compromise subsequent embryo survival.