Uridine diphosphate (UDP) glucuronyltransferase activity in chick liver rises at hatching from near zero to adult levels. This rise will occur prematurely in embryo liver during organ culture. Increase in enzyme activity during organ culture differs with embryo age: in liver from 11-day old embryos it ceases at adult values; in liver from 5-day old embryos it continues to much higher-than-adult levels. Phenobarbital added to culture medium accelerates these rises in enzyme activity and elevates the plateau reached in 11-day embryo liver to that observed in 5-day embryo liver. Kinetic analysis of the changes in enzyme activity induced by phenobarbital during culture suggests that the regulatory mechanisms for enzyme activity are different in 5- and 11-day embryo liver and that these differences reflect developmental changes occurring in ovo.

Some enzymes found in adult mammalian liver are absent from fetal liver. These enzymes are present in low activity at the end of gestation, and they increase rapidly to adult levels in a matter of hours or days after birth. This developmental pattern was first demonstrated for glucose-6-phosphatase (1); more recently it has been shown for phenylalanine hydroxylase, tyrosine-oxidizing enzymes, and uridine diphosphoglucuronic acid transferase (2). The questions arise: What mechanisms control the appearance of these enzymes late in gestation? What factors control the dramatic increase in activity which occurs after birth? One suggested mechanism is that of substrate induction. Knox reported that injection of L-tryptophan into normal adult rats or into adrenalectomized animals caused a transient increase in activity of liver tryptophan peroxidase (3). Knox presented evidence that the effect depended upon substrate induction.To assess the possible role of substrate induction in the development of enzymes, we studied liver tryptophan peroxidase in the guinea pig, measuring levels of activity and the effect of L-tryptophan injection in fetal and postnatal stages up to the adult (4). This survey of tryptophan peroxidase revealed that, as with glucose-6-phosphatase, two distinct changes occur during development: an initial appearance of activity late in gestation and a rapid increase to adult levels after birth (Fig. 1). We found that injection of L-tryptophan into the fetus or into the mother before term had no effect on fetal liver tryptophan peroxidase activity. Injection of L-tryptophan in combination with ACTH or ACE also had no effect. Injection of a term fetus, in utero or after delivery by section, resulted in a small increase in fetal liver activity. A newborn guinea pig, after injection, showed a similar small increase. Twenty-four hours after birth, when liver tryptophan peroxidase activity had attained adult levels, the increase in activity following injection was as great as in the adult (Fig. 1).As Knox has shown, the controlling mechanism increasing tryptophan perox-

Abstract.-Very young (5-day-old) chick embryo livers during organ culture on rafts over a chemically defined nutrient medium precociously develop, from zero, adult levels of UDP-glucuronyltransferase activity. Induction of enzyme does not require DNA synthesis or mitosis, or exposure to hormones, serum, or tissue extracts. Thus, this functional differentiation of embryo liver is apparently not dependent on cell proliferation or on extrinsic substances other than simple nutrients.UDP-glucuronyltransferase activity develops rapidly also in embryo liver cells cultured as monolayers or aggregates; specific activity of enzyme on a protein basis increases at the same rate in cells undergoing widely different rates of cell division, and net protein synthesis.These findings suggest that precocious development of UDP-glucuronyltransferase activity in chick embryo liver is due, not to incidents of culture, but to removal from the embryonic environment. Supporting this, chick embryo liver grafted to the chorioallantoic membrane does not develop UDP-glucuronyltransferase activity until subsequently transferred to an in vitro cell culture system. Certain specialized functions of adult mammalian liver, such as production of blood glucose from glycogen,' degradation of amino acids,2 and formation of glucuronides,3 are mediated by enzymes absent from the fetus until about the time of birth. The effect of birth on enzyme formation had been studied previously by varying the gestation period in the rabbit.4 Premature delivery by Cesarean section resulted immediately in a rapid increase of glucose-6-phosphatase, tyrosine aminotransferase, and tryptophan pyrrolase activities; prolongation of gestation prevented appreciable rise of enzyme activities until after delivery. Factors in the uterine environment may, therefore, repress synthesis of these liver enzymes.Induction of enzyme was then studied in cultured cells.5 Chick embryo liver is relatively easy to culture and contains few nonparenchymal cells. In this tissue UDP-glucuronyltransferase activity toward o-aminophenol rises rapidly upon hatching from near zero to high values.6 This rise could be produced precociously during culture of embryo liver segments submerged in a liquid medium.5 Circumstances of culture, however, and not only separation from embryonic environment, might have initiated development of enzyme activity.Therefore, we have tested various other culture systems. Segments of chick embryo liver were maintained on rafts at a gas-liquid interface, or dissociated into cells and incubated in liquid media as aggregates or monolayers. Cells were monitored by microscopy and by estimating generation times and rates of 795

1. The liver of the domestric fowl (Gallus gallus) remains capable of conjugating o-aminophenol with glucuronic acid after 8 days' culture. The pathway of o-aminophenyl glucuronide formation in cultured liver, as in fresh tissue, includes the enzyme UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activity in chick-embryo liver increases on culture from very low to adult values within 6-8 days. 3. The development of UDP-glucuronyltransferase activity in cultured chick-embryo liver requires certain serum factors in the medium. The requirements change with embryo age. Liver from embryos younger than 15 days develops enzyme activity equally well in media containing either foetal or adult serum; liver from embryos older than 16 days develops activity only with adult serum. The development of enzyme activity in liver from the older embryos appears to be stimulated by diffusible factors in adult serum and inhibited by diffusible factors in foetal serum. It is suggested that the stimulation and inhibition of enzyme formation by small, diffusible molecules may be part of the mechanism regulating UDP-glucuronyltransferase activity in vivo. 4. Liver from 19-day-old chick embryos cultured with foetal serum begins to develop UDP-glucuronyltransferase activity if transferred to an adult-serum medium. Its capacity to develop UDP-glucuronyltransferase activity in adult serum survives in a foetal-serum medium for at least 5 days, the longest period tested. 5. The activity of UDP-glucuronyltransferase reached in 19-day chick-embryo liver after 1 or 2 days with adult serum is maintained without further increase after transfer to a foetal-serum medium. After 3 days with adult serum UDP-glucuronyltransferase activity continues to increase when the tissue is transferred to a foetal-serum medium. Thus liver from 19-day-old embryos requires 3 days with adult serum before development of enzyme activity becomes independent of a continuous adult-serum environment.