The effects of two proteinase inhibitors, leupeptin and pepstatin on the development of 9·5-day rat conceptuses in vitro has been studied. All cultures were of 48 h duration and the inhibitors were present throughout the entire period. When pepstatin was added to the culture medium (5–25 μg/ml) conceptuses developed and grew to an extent that did not differ from untreated controls. However, leupeptin (l–4 μg/ml) caused severe growth retardation and abnormal development of conceptuses.
The effects of the two inhibitors on the hydrolysis of 125I-labelled BSA and haemoglobin by homogenates of 10·5-day yolk sac indicated the biochemical basis for the differential toxic effects of the two inhibitors on development. Leupeptin was highly inhibitory of the degration of both substrates whereas pepstatin caused no inhibition of 125I-labelled BSA hydrolysis, and only a slight inhibition of haemoglobin hydrolysis.
These observations demonstrate that cathepsin D, a lysosomal aspartic proteinase that is specifically inhibited by pepstatin is not involved in yolk-sac-mediated protein utilization by early organogenesis-phase conceptuses and that lysosomal cysteine proteinases, specifically inhibited by leupeptin, are of paramount importance in this yolk sac function.