Embryotoxic effects of three natural occurring Veratrum alkaloids and one synthetic analog using in vitro produced bovine embryos

Authors: Welch, Kevin; WANG, SHIQUAN - Utah State University; Lee, Stephen; GARDNER, DALE - Retired ARS Employee; PANTER, KIP - Retired ARS Employee

Submitted to: Poisonous Plant Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/30/2023
Publication Date: 12/1/2023
Citation: Welch, K.D., Wang, S., Lee, S.T., Gardner, D.R., Panter, K.E. 2023. Embryotoxic effects of three natural occurring Veratrum alkaloids and one synthetic analog using in vitro produced bovine embryos. Poisonous Plant Research. 6:3-12. https://doi.org/10.26077/c087-30ee.
DOI: https://doi.org/10.26077/c087-30ee

Interpretive Summary: Veratrum californicum was responsible for the induction of birth defects in sheep grazing high mountain ranges in central Idaho. The incidence of malformation in some flocks of 5,000-10,000 ewes was 25% or more. When open ewes from presumed Veratrum-induced embryo loss was included, the economic impact was even greater. It was determined that the cyclopic-type craniofacial birth defects occurred when pregnant sheep ingested Veratrum on day 14 of gestation. Other malformations were linked to Veratrum ingestion including skeletal malformations such as carpal and tarsal shortening and tracheal stenosis occur in lambs when ingestion occurred during gestation day 28-33. While cyclopamine is believed to be the putative teratogen in Veratrum, other naturally occurring steroidal alkaloids such as jervine and veratramine are also present in varying concentrations in the plant. The mechanism of cyclopamine-induced birth defects has been shown to result from the inhibition of the Sonic Hedgehog signal transduction pathway. The objective of this research was to determine the effects of selected teratogens and reproductive toxins from Veratrum californicum on oocyte maturation and pre-implantation embryo development using in vitro fertilization techniques (IVF). We believe these IVF techniques provide an economical, rapid through put and effective method to screen natural toxins, especially suspected reproductive toxins for cytotoxicity. Results from this study demonstrate that exposure of bovine oocytes to cyclopamine and its synthetic analog cyclopamine-4-en-3-one during maturation inhibited cleavage rates and subsequent pre-implantation embryo development in vitro. Similarly, exposure of pre-implantation embryos immediately after IVF inhibited cleavage rates and subsequent embryonic development. This research indicates that ingestion of natural toxins such as these steroidal alkaloids can adversely affect oocyte maturation and subsequent pre-implantation embryo development. Furthermore, this research suggests a role of the Hedgehog signaling pathway in the maturation of oocytes as well as the normal development of the pre-implantation embryo. This IVF model provides an alternative to animal testing as a technique to screen potential reproductive and teratogenic toxins.

Technical Abstract: Three natural occurring plant toxins from Veratrum californicum and one related synthetic analog were screened for embryotoxicity using in vitro bovine embryo production techniques. Bovine oocytes were aspirated from ovaries collected from a local abattoir and embryos were generated through in vitro maturation (IVM) and in vitro culture (IVC) procedures. The three natural steroidal alkaloids, cyclopamine, jervine and veratramine and the synthetic steroidal derivative of cyclopamine, cyclopamine-4-en-3-one, were added to IVM and IVC media at 12 µM. Oocytes were exposed to the toxins during maturation (IVM) and pre-implantation embryo during culture (IVC). Cleavage rates and embryo growth (morula and blastocyst production) and development through the hatched blastocyst stage were evaluated. Cyclopamine and cyclopamine-4-en-3-one inhibited cleavage rates and embryo growth and development of morulae and blastocysts in culture. Oocytes that were exposed to cyclopamine and cyclopamine-4-en-3-one during IVM only showed reduced cleavage rates and resulted in lower numbers of embryos that developed to the morula, blastocyst, and hatched blastocyst stages. The effects of these steroidal alkaloids on the oocyte during IVM and on the embryo during all stages of development up to and including the hatched blastocyst stage, demonstrates a dramatic cytotoxic effect on oocytes maturation and early pre-implantation embryos. This research also suggests that the Hedgehog signaling pathway may play a role in the maturing oocyte as well as the pre-implantation embryo.