Leucine transport from mother to fetus in rat: Role of the visceral yolk sac

Nutrition Research, Vol. 18. No. 10, pp. 1783-1789.1998 Copyright 0 1998 Elsevia Science Inc. F’rinted in the USA. All tights reserved 0271-5317/98 $19.00+ .oO

PII SO271-5317(98)00139-O

ELSEVIER

LEUCINE

TRANSPORT FROM MOTHER TO FETUS IN RAT: ROLE OF THE VISCERAL YOLK SAC

David A. Beckman, Ph.D., John P. Mullin, Robert L. Brent, M.D., Ph.D., and John B. Lloyd, Ph.D., D.Sc. Division of Developmental Biology, Jefferson Medical College, Philadelphia, Pennsylvania and Alfred I. duPont Hospital for Children, Wilmington, Delaware 19899, USA.

ABSTRACT It is known that intracellular proteolysis of exogenous protein in the visceral yolk sac (VYS) is a significant source of leucine for the fetus from days 8.5 through 17.5 post-conception in the rat. In contrast, little attention has been paid to the potential of the VYS to transport maternal free amino acids to the fetus. We investigated this question using the exteriorized 17.5-day rat fetus, enclosed within its amnion and VYS, but with its placenta attached to the uterus and still functional. We first demonstrated that ['Hlleucine in medium bathing the exteriorized fetus is transported by the VYS to the fetus and then exchanged with the leucine pool in the maternal circulation. This result indicates that the preparation remains viable for the duration of the experiment. We next injected a trace amount of ['Hlleucine intravenously into the 17.5-day pregnant rat and found that the concentration of radioactivity in the plasma of the exteriorized fetus was not much lower than that in plasma of non-exteriorized fetuses in the same litter (8-15 fetuses per litter). We interpret these results to indicate that the VYS transports free leucine to the fetus but that this process makes a minor contribution to the total transfer of free leucine into the 17.5-day rat fetus. 8 1998Elsevierscrzlce Inc. KEY WORDS: Leucine, Rat, Fetus, Placenta, Yolk sac, Amino acid.

INTRODUCTION

It is axiomatic that the growing mammalian net influx of amino acids throughout gestation.

fetus requires a Moreover, it is

Correspondence to: David A. Beckman, Ph.D., Div. Developmental Biology, Nemours Research Programs, P.O. Box 269, Wilmington, DE 19899, USA. E-mail: [email protected], Fax: 302-651-6888. 1783

1784

D.A. BECKMAN etal.

widely assumed that, once the chorioplacenta is functional, amino acid nutrition occurs by the direct placental transfer of free amino acids from the maternal to the fetal bloodstream. While there is no doubt that placental transfer of amino acids occurs and that the transport involves specific porters, little attention has been paid to the possibility that significant amounts of free amino acids might also reach the fetus by other routes. In rodents the visceral yolk sac (VYS) persists until full term. Throughout the placental phase of gestation the VYS is in intimate apposition to the uterine wall, separated only by the highly permeable Reichert's membrane and associated trophoblast giant cell layer, and in the final few days not even by that. Moreover the VYS has a rich blood supply which is continuous with that of the fetus. The VYS has been shown to supply most of the embryo's amino acid needs in early organogenesis (l-81, not by the transfer of free amino acids but by the endocytosis and lysosomal digestion of maternal proteins. There is also evidence that protein uptake and digestion by the VYS contributes significantly to the amino acid nutrition of the fetus in late gestation (9). Because the late-gestation VYS has a large surface area, is in contact with uterine secretions and has a direct vascular connection with the fetus, it is conceivable that amino acid transfer from mother to fetus occurs significantly by this route as well as through the placenta. In search of evidence for or against this conjecture, we used a technique that involves the exteriorization of the 17.5-day rat fetus, enclosed within its amnion and VYS, but with its placenta attached to the uterus and still functional. While a fetus remaining within the uterus can exchange amino acids with the maternal plasma via the placenta and with the uterine fluid via the VYS, an exteriorized fetus can exchange via the placenta only. We injected pregnant rats with L['Hlleucine and investigated the relative contribution of the two routes by comparing the transfer of radioactivity to the exteriorized fetus with that to the in situ fetuses. Leucine was selected because it is an essential amino acid in the rat, and its radiolabel, in the case of the [4,5-jH]leucine used in these experiments, is not transferred to another amino acid to a detectable amount (5). To establish the viability of the preparation, we supplemented the medium bathing the exteriorized fetus with ['Hlleucine and determined the transfer of radioactivity from the medium to the exteriorized fetus, maternal plasma and littermates. MATERIALS AND METHODS Timed oreanancies One female and one male rat of the Wistar strain (Charles River) were housed together overnight. The presence of sperm in the vaginal lavage at 9 a.m. on the following morning was taken to define that time as day 0.5 post-conception.

LEUCINE TRANSPORT TO RAT FETUS

The exteriorized

1785

fetus preparation

The technique for exteriorizing the rat fetus was modified from that described by Mucchielli et al. (10) and Laliberte et al. (11). Pregnant rats were fasted overnight prior to the day of experiment, to minimize the dilution of the radiolabeled amino acid with unlabeled amino acid entering the bloodstream from the portal circulation. On day 17.5, the pregnant rats were anesthetized with 50 mg ketamine HCl and 5 mg xylazine per kg body weight (Parke-Davis), the abdominal area was shaved, and a laparotomy was performed to expose the gravid uterus. A pregnant rat was included in the experiments if the number of implantation sites was adequate (8 to 15) and they were distributed more or less evenly between the two uterine horns. A conceptus centrally located in one uterine horn was isolated from its neighbors by placing a ligature around the entire uterus on each side of the implantation site. The ligature was placed such that it did not enclose any portion of the placenta or cut off the mesometrial blood supply to the implantation site. The laparotomy incisions were closed with sutures to leave the implantation site exteriorized and the mesometrial blood flow unrestricted. The antimesometrial uterine wall was opened by dissection to expose the extraembryonic membranes, allowing the uterine wall to retract on its own to the edge of the chorioallantoic placenta. The parietal yolk sac h;id retracted in every case. The pregnant rat was placed on her abdomen on a sling with a hole large enough to allow the exteriorized fetus to pass through freely. The sling supporting the rat was positioned in a humidified, constant temperature (37°C) chamber. This was done because ketamine anesthesia results in hypothermia unless an external source of heat is applied. The exteriorized fetus, enveloped in its amnion and VYS with intact circulation to the chorioplacenta, was fully immersed in bathing medium within 30 seconds of completing the dissection. Incubation of exteriorized I'Hlleucine

fetus in medium

supplemented

with

The bathing medium was composed of 40 ml medium 199 containing 10% calf serum (GIBCO) to which 20 PCi of ['Hlleucine in 20 ul of aqueous solution (L-[4,5-'Hlleucine; 184 Ci/mmol; Amersham International) was added. The presence of amino acids in the medium 199 and calf serum comprising the medium bathing the VYS of the exteriorized fetus provide an external source of leucine in addition to the trace amount of [ HJleucine. Incubation of exteriorized into oreqnant rat

fetus after in?ection

of

['Hlleucine

Day-17.5 pregnant rats were anesthetized as above, abdominal and inguinal areas were shaved and a femoral vein was exposed and kept moist by the application of sterile saline. A fetus was then exteriorized, covered with gauze soaked in 37.C sterile saline, and the pregnant rat was injected via the exposed femoral vein over 5-10 seconds with 500 ~1 of ['Hlleucine in sterile.

D.A. BECKMAN et al.

1786

phosphate-buffered saline. The ['Hlleucine injection contained 0.05 uCi per g body weight. The gauze covering was then removed and the exteriorized fetus was fully immersed in medium 199 containing 10% calf serum within 30 seconds of the maternal intravenous injection. Collection

and orocessinq

of plasma,

uterine

fluid and medium

A blood sample was taken from the tail at the termination of the experiment, which was at 60 minutes after injection in every case, and collected into a heparinized container. Plasma was prepared by centrifugation. Uterine fluid (loo-150 ul) was obtained by inserting a 27 ga. needle attached to a 1 ml syringe through the uterine wall between two adjacent fetuses centrally located in each uterine horn. Uterine fluid containing blood was discarded. Blood samples (50-75 ul) were taken from the umbilical cord and/or jugular vein of the exteriorized fetus and each in situ fetus and collected into heparinized microhematocrit tubes. After centrifugation, the plasma from all in situ fetuses in a litter was pooled before further processing and the plasma from the exteriorized fetus was processed separately. Samples of the medium (100 ul) were taken immediately after submersion of the exteriorized fetus and at the termination of the experiment. Acid-soluble (low molecular weight) radioactivity was determined after incubation of 50 ul of plasma, uterine fluid or medium with 250 1.11of 12% (w/v) trichloroacetic acid at 4°C for 1 hour. After centrifugation at 2000xg for 10 minutes, a 100 ul portion of the soluble fraction was placed in a scintillation vial, followed by 300 ul of glacial acetic acid and 10 ml of scintillation fluid (Ecoscint, National Diagnostics). Radioactivity was measured with a Beckman LS7500 spectrometer. Statistical

analvsis

Data were analyzed for differences within groups by one-way analysis of variance. Significant findings were followed up with the post-hoc comparisons completed through the Tukey adjustment.

RESULTS

The technique for exteriorization and incubation was evaluated using the following criteria at the end of the experiment: normal appearance of the exteriorized yolk sac and fetus, chorioplacenta not separated from the uterus, and no hemorrhage evident in the uteroplacental unit associated with the exteriorized fetus. By these criteria success was achieved in one out of every two experiments. Experiments in which [jH]leucine was added to the bathing medium of the exteriorized fetus were performed to determine

LEUCINE TRANSPORT TO RAT FETUS

1787

whether transfer of leucine from the medium into the exteriorized fetus and then to the maternal organism could be detected. These experiments (Table 1) resulted in acid-soluble radioactivity appearing within one hour in the exteriorized fetus plasma, maternal plasma, in situ fetus plasma and uterine fluid. Acid-soluble radioactivity in plasma, uterine fluid and bathing medium 60 minutes after intravenous injection of ['Hlleucine into the 17.5-day pregnant rat is presented in Table 2. The acid-soluble radioactivity in the maternal plasma and in situ fetus plasma is similar to that reported previously following intravenous injection of ['Hlleucine in the same amount into the 1?.5-day pregnant rat (9). The acid-soluble radioactivity in the maternal plasma, uterine fluid and in situ fetus plasma are not significantly different from each other

TABLE 1 Acid-soluble Radioactivity in Bathing Medium, Plasma and Uterine Fluid 60 minutes after supplementing Medium bathing the Day 17.5 Exteriorized Fetus with a Trace Amount of ['H]Leucine.

Ave =

Acid-soluble radioactivity, Bathing Fetal Maternal medium plasma, plasma exteriorized 918,420 55,080 12,420 639,300 47,520 11,280 778,860 51,300 11,850

d.p.m./ml Uterine fluid 840 1,020 930

Fetal plasma, in situ* 660 780 720

*Plasma from all in situ fetuses in a litter was pool ed before analysis (litter size was 8-15 fetuses).

TABLE 2 Acid-soluble Radioactivity in Plasma, Uterine Fl uid and Bathing Medium 60 minutes after Intravenous Injection of a Trace Amount of [jH]Leucine into a 17.5-Day Pregnant Rat.

Maternal plasma 31,920 32,820 33,840 25,440 27,660 30,336 i 3,609#

Acid-soluble radioactivity, d.p.m./ml Uterine Fetal plasma, Fetal fluid exteriorized plasma, in situ* 32,760 29,680 39,000 31,620 44,760 45,180 21,240 34,080 35,100 19,380 25,560 36,480 18,360 32,340 30,000 24,672 33,284 37,152 f 7,174# + 5,561# rt 6,952#+

Bathing medium 3,660 5,760 4,680 2.100 2,360 3,712 + 1,546#

*Plasma from all in situ fetuses in a litter was pooled before analysis (litter size was 8-15 fetuses). #Values are mean k SD. 'Significantly different from the value for plasma from in situ fetuses, ~~0.05.

1788

D.A. BECKMAN et al.

(P>O.O5). The acid-soluble radioactivity in plasma from the exteriorized fetus is significantly lower (P
DISCUSSION

The VYS, fetus and chorioplacental-uterine unit of the exteriorized fetus must all remain viable in order for radioactivity to be transferred from the bathing medium to the plasma of the exteriorized fetus and then on to the maternal plasma, the uterine fluid and the plasma of littermates. The results in Table 1 demonstrate that this pathway is intact in the experiments reported. Furthermore, because the chorioplacenta is known to transport leucine to the fetus, an absence or near absence of radioactivity would indicate a failure to maintain a functional uteroplacental unit. This was not seen in any of the experiments reported in Table 2. The results in Table 2 indicate that the concentration of radioactivity in the uterine fluid was similar to that in the maternal plasma. This suggests that, at 60 minutes after injection, the VYS and chorioplacenta associated with in situ fetuses were exposed to a similar concentration of radioactivity. Taken together with our demonstration (Table 1) that the VYS transports leucine from an external source to the fetus, we conclude that there must be some role for the VYS in the transport of free leucine. Table 2 also shows that the ratio of radioactivity in the plasma of exteriorized fetuses to that in plasma of in situ fetuses averages 0.66, with a range of 0.53 to 0.84. We interpret these ratios as follows. First, the wide range of values likely indicates some variation in the functional integrity of the chorioplacental-uterine unit of exteriorized fetuses. Secondly, values as high as 0.84 indicate a minor role at most for the VYS in transporting free leucine from mother to fetus. Taken together with our earlier study (91, the results of this work support a conclusion that leucine is supplied to the rat fetus from two principal sources, free leucine transported chiefly by the chorioplacenta and leucine from protein degradation in extraembryonic tissues such as the VYS.

ACKNOWLEDGMENTS The authors thank Christina Tu for her invaluable technical assistance and Dr. Thomas R. Koszalka for his constructive suggestions. This work was funded by NIH HD 29902.

LEUCINE TRANSPORT TO RAT FETUS

1789

REFERENCES

1. Freeman SJ, Beck F, Lloyd JB. The role of the visceral yolk sac in mediating protein utilization by rat embryos cultured in vitro. J Embryo1 Exp Morph01 1981;66:223-234. 2. Freeman SJ, Lloyd JB. Evidence that protein ingested by the rat visceral yolk sac yields amino acids for synthesis of embryonic protein. J Embryo1 Exp Morph01 1983;73:307-315. 3. Rowe PB, Kalaizis A. Serine metabolism in rat embryos undergoing organogenesis. J Embryo1 Exp Morph01 1985;87:137144. 4. Beckman DA, Pugarelli JE, Jensen M, Koszalka TR, Brent RL, Lloyd JB. Sources of amino acids for protein synthesis during early organogenesis in the rat. 1. Relative contributions of free amino acids and of proteins. Placenta 1990;11:109-121. 5. Beckman DA, Pugarelli JE, Koszalka TR, Brent RL, Lloyd JB. Sources of amino acids for protein synthesis during early organogenesis in the rat. 2. Exchange with amino acid and protein pools in embryo and yolk sac. Placenta 1991;12:37-46. 6. Beckman DA, Brent RL, Lloyd JB. Sources of amino acid for protein synthesis during early organogenesis in the rat, 4. Mechanisms before envelopment of the embryo by the yolk sac. Placenta 1996;17:635-641. 7. Beckman DA, Tu C. Leucine sources for 10.5-day in vivo. Reprod Toxic01 1997;11:875-877.

rat conceptus

8. Lloyd JB, Brent RL, Beckman DA. Sources of amino acid for protein synthesis during early organogenesis in the rat. 3. Methionine incorporation. Placenta 1996;17:629-634. 9. Beckman DA, Brent RL, Lloyd JB. Leucine fetus. Placenta 1997;18:79-82.

sources

for the rat

10. Mucchielli A, Laliberte F, Laliberte M-F. A new experimental method for the dynamic study of the antibody transfer mechanism from mother to fetus in the rat. Placenta 1983;4:175-184. 11. Laliberte F, Mucchielli A, Laliberte M-F. Dynamics of antibody transfer from mother to fetus through the yolk-sac cells in the rat. Biol Cell 1984;50:255-262. Accepted

for

publication

May 20,

1998.