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A method has been developed for obtaining cell-free visceral yolksac membrane using the chelating agent tetrasodium salt of ethylenediaminetetraacetic acid (EDTA) combined with mechanical disruption of cells. Rabbit antiserum to this acellular membrane preparation when injected ip into 8-day pregnant rats produced malformation, growth retardation, and fetal resorption. This yolk-sac membrane was partially solubilized with sodium deoxycholate. The resultant insoluble material was further partially solubilized by 1 M NaCl and fractionated by molecular sieve chromatography on BioGel A-5m to yield two fractions. The insoluble material after NaCl extraction was treated with purified collagenase to obtain a soluble and insoluble fraction. Rabbit antisera to all five soluble and insoluble fractions proved to be teratogenic in rat. Immunodiffusion studies indicated that there was at least one common antigen in all of these fractions; antisera to these fractions also crossreacted with soluble rat kidney. Fluorescent-labeled antibody localization studies revealed that the teratogenic antibodies localized in the maternal renal glomerular basement membrane and the yolk-sac placenta. The findings that ( 1 ) the hydroxyproline-free collagenasetreated residue was capable of stimulating the production of teratogenic antiserum and ( 2 ) rabbit antiserum against undenatured rat-tail collagen was not embryotoxic provide evidence to support the view that antibodies against collagen fibers are not teratogenic. The hypothesis that teratogenesis resulting from yolksac dysfunction induced by heterologous antibodies was discussed.

ExtractRabbit antineuraminidase-treated rat kidney and rabbit antitrypsin-treated rat kidney supernatant antisera were embryotoxic resulting in embryonic growth retardation, malformations, and death when injected into 15 pregnant rats. The same two antisera were also found to be nephrotoxic as manifested by their ability to induce proteinuria in 28 male rats. However, the antiserum against neuraminidasetreated rat kidney homogenate was significantly less nephrotoxic (averaging 46 mg urinary protein/24 hr) than the antiserum against the soluble supernatant obtained from trypsin-digested rat kidney homogenate (averaging 180 mg urinary protein/24 hr). The embryotoxic potency of these two antisera did not seem to correlate with their respective nephrotoxic potency. The less nephrotoxic antiserum, namely the antiserum against neuraminidase-treated rat kidney, is about five times as embryotoxic as that of the more nephrotoxic antiserum.Fluorescent-labeled antibody localization studies showed that the teratogenic antibodies localized in vivo in the maternal glomerular basement membrane, Reichert's membrane, and the endodermal cells of the visceral yolk sac of the developing embryo. There were at least two bands of identity between antiserum against trypsin-treated kidney supernatant and antiserum against neuraminidase-treated kidney homogenate.The possible role played by Reichert's membrane and the visceral yolk sac endodermal cells in teratogenesis induced by heterologous antibodies is discussed along with other relevant hypotheses. Speculation