Abstract
The effect of arachidonic acid (AA) cascade on bovine embryo development in a granulosa cell co-culture system was studied. Arachidonic acid (100 µM) was supplemented from 1-cell to 8-16 cell block stage (first three days of co-culture) and from 1-cell to hatching. Specific cyclooxygenase (indomethacin, 28 µM) and lipoxygenase (nordihydroguaiaretic acid - NDGA, 28 µM) inhibitors were used from 1-cell to 8-16 cell block stage with AA. Embryo development was assessed by cleavage, day 7-day 8 and hatched embryo rates and by measuring growth rates through development stages found in days 7-10 of culture (day 0 = insemination day). Embryo quality was scored at day 8. A 6.5-10.4% increase on cleavage rate after AA supplementation was found. This AA supplementation from 1-cell to hatching delayed embryo growth rate beyond day 7 and a reduction on hatching rate was detected. When AA supplementation was restricted to the first three days of co-culture those negative effects were overcome. Also, indomethacin and NDGA prevented the positive effect of AA and induced a significant reduction on cleavage, respectively. NDGA further decreased day 7 embryo rate and quality. Results suggest that AA has a two-phase action on bovine embryos, promoting early development and impairing embryo growth from day 7 onwards and hatching rates. Both cyclooxygenase and lipoxygenase were found to be important pathways to promote cleavage.
Highlights
Arachidonic acid (AA) is metabolized by cells into prostaglandins, hydroperoxyeicosatetraenoic acid (HPETE) and derivatives such as leukotrienes, through the action of cyclooxygenases (COX) and lipoxygenases (LOX) (SamuelssonCyclooxygenase has been detected as early as two-cell stage of embryonic development (Gurevich & Shemesh, 1994; Van der Weiden et al, 1996) while prostaglandins have been implicated in a number of embryo physiological processes including blastocyst expansion and hatching (Biggers et al, 1978; Lewis, 1986 and 1989)
Our first objective was to study the effect of arachidonic acid (AA) supplementation from 1-cell to 8-16 cell block stage as well from 1-cell to hatching on bovine embryo development
Granulosa cells (1x106 mL-1) collected from refrigerated ovaries were cultured in 100 μl droplets of M199, 10% superovulated oestrus cow serum (SOCS) and antibiotics, placed in 60 mm Petri dishes (Nunc®, Denmark) and covered with paraffin oil
Summary
Arachidonic acid (AA) is metabolized by cells into prostaglandins, hydroperoxyeicosatetraenoic acid (HPETE) and derivatives such as leukotrienes, through the action of cyclooxygenases (COX) and lipoxygenases (LOX) Cyclooxygenase has been detected as early as two-cell stage of embryonic development (Gurevich & Shemesh, 1994; Van der Weiden et al, 1996) while prostaglandins have been implicated in a number of embryo physiological processes including blastocyst expansion and hatching (Biggers et al, 1978; Lewis, 1986 and 1989). Preimplanted bovine embryos are able to produce prostaglandins from 2-cell stage onwards (Lewis et al, 1982; Gurevich & Shemesh, 1994; Nadeau et al, 1994), neither the expression of LOX enzymes nor the synthesis of LOX pathway metabolites have been identified. Our first objective was to study the effect of AA supplementation from 1-cell to 8-16 cell block stage as well from 1-cell to hatching on bovine embryo development. The role of COX and LOX metabolites on all embryonic developmental stages was studied through inhibition of the pathways from 1-cell to 8-16 cell block stage
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