Freeman S J, Lloyd J B
J Embryol Exp Morphol. 1983 Dec;78:183-93.
Conceptuses from 9.5-day pregnant rats were cultured for 48 h in heat-inactivated homologous serum to which leupeptin, a specific inhibitor of the lysosomal cysteine-proteinases, was added for the final or the penultimate 6 h. The presence of leupeptin (25 micrograms/ml or above) increased the protein content of yolk sacs at harvesting to approximately twice the control value. The protein content of the embryo at harvesting was lower than that of controls. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. The presence of leupeptin did not affect the rate of uptake of the radiolabelled macromolecule by the yolk sac, nor facilitate its entry into the embryo. When formaldehyde-denatured 125I-labelled bovine serum albumin was added to the culture medium for the final 6 h of culture, little radioactivity was found in the yolk sac at harvesting, and barely any was found in the embryo. Trichloroacetic acid-soluble radioactivity was found in the culture serum. The presence of leupeptin sharply increased the levels of radioactivity in the yolk sac (but not the embryo) and sharply decreased the acid-soluble radioactivity of the culture medium. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in both yolk sac and embryo at harvesting. The presence of leupeptin increased the amount found in the yolk sac and decreased that found in the embryo. These results are interpreted as follows. Leupeptin enters the lysosomes of the yolk sac, inhibiting their cysteine proteinases. The digestion of proteins pinocytosed by the yolk sac is consequently inhibited, resulting in the accumulation of protein by the yolk sac and a decreased flow of amino acids to the embryo. Leupeptin (50 mg/kg), injected into pregnant rats at either 8.5 days or 9.5 days of gestation, induced congenital malformation in the offspring. It is proposed that leupeptin exerts its teratogenic action by inhibiting proteolysis in the lysosomes of the yolk sac, and so depriving the developing embryo of its supply of amino acids at a critical stage of development.
将妊娠9.5天大鼠的胚胎在热灭活的同源血清中培养48小时，在培养的最后6小时或倒数第6小时加入溶酶体半胱氨酸蛋白酶的特异性抑制剂亮抑蛋白酶肽。亮抑蛋白酶肽（25微克/毫升及以上）的存在使收获时卵黄囊的蛋白质含量增加到对照值的约两倍。收获时胚胎的蛋白质含量低于对照组。当在培养的最后6小时向培养液中加入125I标记的聚乙烯吡咯烷酮时，收获时在卵黄囊中发现放射性，但在胚胎中未发现。亮抑蛋白酶肽的存在不影响卵黄囊对放射性标记大分子的摄取速率，也不促进其进入胚胎。当在培养的最后6小时向培养基中加入甲醛变性的125I标记牛血清白蛋白时，收获时在卵黄囊中几乎未发现放射性，在胚胎中几乎也未发现。在培养液中发现了三氯乙酸可溶性放射性。亮抑蛋白酶肽的存在使卵黄囊中的放射性水平急剧增加（但胚胎中未增加），并使培养基中的酸溶性放射性急剧降低。当使用用[3H]亮氨酸标记蛋白质的大鼠血清作为培养基时，收获时在卵黄囊和胚胎中均发现放射性。亮抑蛋白酶肽的存在增加了卵黄囊中发现的放射性量，减少了胚胎中发现的放射性量。这些结果的解释如下。亮抑蛋白酶肽进入卵黄囊的溶酶体，抑制其半胱氨酸蛋白酶。因此，卵黄囊吞噬的蛋白质的消化受到抑制，导致卵黄囊蛋白质积累，以及流向胚胎的氨基酸减少。在妊娠8.5天或9.5天向妊娠大鼠注射亮抑蛋白酶肽（50毫克/千克）可导致后代先天性畸形。有人提出，亮抑蛋白酶肽通过抑制卵黄囊溶酶体中的蛋白水解作用发挥致畸作用，从而在发育的关键阶段剥夺发育中的胚胎的氨基酸供应。
