|Gaffield jr, William|
Submitted to: American Society of Animal Science Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 2/2/2002
Publication Date: 10/1/2002
Citation: Panter, K.E., Wang, S., Gaffield Jr, W.P., James, L.F., Evans, R.C., Bunch, T.D. 2002. Effects of cyclopamine, a veratrum alkaloid, and its synthetic analog cyclopamine-4-ene-3-one on in vitro bovine oocyte maturation and subsequent embryo development. American Society of Animal Science Proceedings. Interpretive Summary: Veratrum californicum (skunk cabbage) caused significant losses from birth defects to the sheep industry in central Idaho in the 1950's. Occasional reports of Veratrum induced malformations and toxicity in animals continue. The toxins in Veratrum responsible for the cyclopic-type birth defects and other craniofacial, skeletal and tracheal malformations were of the steroidal alkaloid class and included cyclopamine. The Veratrum-induced craniofacial defects result from the blocking of Sonic hedgehog (gene) signaling preventing normal cellular movement during early embryo development. Cyclopamine was isolated from Veratrum and was demonstrated to be a potent toxin causing the birth defects. A synthetic analog of cyclopamine, cyclopamine-4-ene-3-one, was produced and determined to be a more potent inhibitor of Sonic hedgehog signaling. Both alkaloids were added separately to the culture media in petri dishes containing cow oocytes collected from ovaries obtained from a slaughterhouse. Both alkaloids affected oocyte maturation and prevented subsequent normal growth and development of the early cow embryo in culture. Therefore, interference of oocyte maturation by these two alkaloids also inhibited subsequent embryo development.
Technical Abstract: Cyclopamine (CPA), from the poisonous plant Veratrum californicum, and cyclopamine-4-ene-3-one (CPA4-3), a synthetic analog of cyclopamine, are steroidal alkaloids that interrupt Sonic hedgehoc (Shh)-mediated dorsoventral patterning of the neural tube and somites resulting in cyclopia and holoprosencephaly when administered to gastrulation-stage embryos. The objective of this study was to investigate the effects of CPA and (CPA4-3 on oocyte maturation using in vitro fertilization procedures. A randomized complete block (5 replications) design with three in vitro maturation treatments was used. Bovine oocytes(n = 799) were aspirated from abattoir ovaries and in vitro matured (IVM) in medium supplemented with 12 FMCPA4-3 (TRT), 12 FMCPA (TRT 2) or IVM medium only (TRT 3, Control). The in vitro matured oocytes were then subjected to in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rates were determined at 48 h after IVF and preimplantation embryo development was evaluated at d 6, 8 and 10 of IVC for TRT 1, TRT 2 andTRT 3, respectively. Percentage data were angularly transformed and analyzed by the general linear model ANOVA. Cleavage rates were 60.7%, 65.0% and 84.1%; percentage of morulae at d 6 of IVC 20.1, 31.8. and 58.3; percentage of blastocysts at d 8 of IVC was 6.3, 8.1 and 28.1; and percentage of expanded and hatching blastocysts at d 10 was 3.8, 9.2 and 21.5 for TRTs 1, 2 and 3, respectively. Development of preimplantation embryos derived from oocytes matured in media containing either steroidal alkaloid was inhibited compared to those of control (P<0.05). Cleavage rates were lower and numbers of embryos developing to morula blastocyst and expanded and hatching blastocyst stages were significantly reduced (P<0.05). Also, the adverse effects of CPA4-3 on oocyte maturation was greater than CPA (P<0.05). We conclude that exposure of bovine oocytes to the Veratrum alkaloid, cyclopamine and its synthetic analog cyclopamine-4-ene-3-one during oocyte maturation will inhibit subsequent preimplantation embryo development in vitro.