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Whole Rat Embryos Require Methionine for Neural Tube Closure when Cultured on Cow Serum^1,2,3,4,

Caroline N. D. Coelho^*,,,5, James A. Weber^*,,6, Norman W. Klein^*,,,7, Willard G. Daniels and Thomas A Hoagland

^* Center for Environmental Health Department of Animal Science Department of Molecular and Cell Biology Department of Pathobiology, University of Connecticut, Storrs, CT 06269

Headfold-stage rat embryos, when cultured on cow serum without supplemental methionine, failed to close their neural tubes, lacked eyes and branchial arches, were abnormally shaped and were reduced in protein content compared to methionine-supplemented embryos. Methionine was essential during the first 18 h of culture, a period in which neural tube closure was initiated in supplemented cultures. All cow serum samples tested were found to require methionine addition, and the methionine was not replaced by other amino acids or vitamins, including folate. Methionine was not toxic to cultured rat embryos at concentrations up to at least 500 µg/ml. Analyses of serum free amino acids revealed lower levels of free methionine in cow serum compared to rat serum, and cow serum proteins contained less methionine relative to other amino acids than did rat serum proteins. Dialysis of cow serum reduced but did not eliminate the requirement for methionine. This suggested either that the free amino acids of cow serum were imbalanced or that a dialyzable component in serum interfered with the availability/utilization of methionine. Dietary supplementation of cows with rumenprotected DL-methionine increased the serum methionine level, and serum drawn from supplemented cows supported normal rat embryo development without additional methionine.

KEY WORDS: • methionine • neural tube defects • cow serum • serum amino acids • rat embryo cultures • teratogenicity

¹ This article is dedicated to the memory of Lucille Hurley and for her many contributions to the fields of developmental nutrition and teratology. For one of the authors (N.W.K.) she will always be remembered and appreciated as a teacher, colleague, and friend.

² Supported by U.S. Department of Energy Contract EV03139, Office of Health and Environmental Research (to N.W.K.), and by National Institute of Environmental Health Sciences NIEHS grant ES04312 (to N.W.K.).

³ This report is scientific contribution No. 1289, Storrs Agricultural Experiment Station, The University of Connecticut, Storrs, CT 06269.

⁴ A preliminary report was presented at the 1986 meeting of the American Teratology Society in Boston, MA, and was published as an abstract (1).

⁵ Present address: Department of Anatomy, Room 4006, University of Connecticut Health Center, Farmington, CT 06032.

⁶ Present address: Department of Veterinary Science, University of Idaho, Moscow, ID 83843.

⁷ To whom correspondence and reprint requests should be addressed at: Center for Environmental Health, Box U-39, University of Connecticut, Storrs, CT 06269.

Manuscript received 4 May 1989. Revision accepted 4 July 1989.

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