Kinetics of placental ovine fetuses P. A.

lactogen in mid- and late-gestation

Schoknecht, W. B. Currie and A. W. Bell

Department of Animal Science, 262 Morrison Hall, Cornell University, Ithaca, New York 14853-4801, U.S.A. received 29 July 1991 ABSTRACT

Placental

lactogen (PL) is throughout gestation, and

found in fetal plasma PL receptors occur on many types of fetal cells. In this study, the entry rate of PL into the fetal circulation was estimated by injection of 125I-labelled ovine PL into two mid- and four late-gestation fetuses. At both ages, PL appears to be distributed into two body pools. One pool has a rapid half-life (approximately 9 min) and a volume of distribution approximately 8% of body weight, while the second pool has a longer half-life (approximately 45 min) and a distribution volume only 4% of body weight. The first pool is presumably blood plasma,

but the physiological identity of the second pool is unknown. The effective half-life of PL is approximately 15 min, and the liver is suggested as a probable major site of degradation. These estimates were confirmed in late gestation by measuring fetal plasma concentrations of PL in response to a continuous infusion of unlabelled PL. The kinetic parameters estimated in this study can be used to determine the quantity of exogenous hormone required to alter PL concentration in fetal plasma in a predictable manner. Journal of Endocrinology (1992) 133, 95\p=n-\100

INTRODUCTION

oPL in both mid and late gestation and confirmed, in late gestation, by use of a continuous infusion of non-labelled oPL.

Placental lactogen (PL) is present in ovine fetal plasma throughout gestation (Gluckman, Kaplan, Rudolph & Grumbach, 1979). It is first detected in the embryo at about the time of implantation (Martal & Djiane, 1977) and is localized to the binucleate cells of the chorion (Martal, Djiane & Dubois, 1977), but its rate

of entry into

or

clearance from the fetus is ill-

defined. Receptors for ovine PL (oPL) have been found in several fetal tissues (Chan, Robertson & Friesen, 1978) and cross-linking studies clearly demonstrate that these oPL receptors are distinct from receptors for growth hormone and prolactin (Freemark, Comer, Körner & Handwerger, 1987). Binding of human PL has been demonstrated in myocytes and fibroblasts (Hill, Crace & Milner,

1985). However, despite speculation concerning a role for oPL in the developing fetus, the function of PL remains unknown. As a first component of studies aimed at chronically manipulating fetal concentrations of oPL, this report describes the entry rate of PL into the fetal circu¬ lation, its volume of distribution within the conceptus and the half time (/i) of elimination from fetal plasma. These parameters were estimated from the disappear¬ ance of immunoreactive, radioisotopically labelled

MATERIALS AND METHODS

Animals and

husbandry

Multiparous Dorset ewes from the Cornell University

flock

were

bred to Dorset

rams

and scanned ultra-

sonically (Johnson and Johnson Technicare 210DX, 3-5 MHz, East Brunswick, NJ, U.S.A.) at day 45 of pregnancy to determine litter size. Only monotocous ewes were used. On day 50 or 100, animals were moved into raised crates (1-4 0-75 m) in a room held at 18 °C and with a constant photoperiod of 16 h light and 8 h darkness. They were fed once daily with 25 g/kg body weight of a total mixed ration (10-5 MJ metabolizable energy and 150 g crude protein per kg dry matter) to meet predicted requirements (National Research Council, 1985). Feed was withheld for 1 day before surgery.

Surgery and post-operative care Surgery was performed at either day 75 or 115 of pregnancy. After premedication with 2 mg

xylazine (Rompun, Miles Laboratories, Shawnee, KS, U.S.A.) given i.m., anaesthesia

induced with 10 mg ketamine hydrochloride/kg (Ketaset, Bristol Laboratories, Syracuse, NY, U.S.A.) given i.v. and maintained with 0-1-0-5 mg/kg per min, as needed, by continuous infusion. On day 75, polyvinyl chloride catheters (0-58 and 0-96 mm, inside and outside diameter respectively) were placed in the umbilical artery and vein via cotyledonary branches (Bell, Battaglia & Meschia, 1987a). On day 115, the fetal abdominal aorta and vena cava were catheterized via the pedal artery and lateral saphenous vein respectively (Rudolph & Heymann, 1980) using larger diameter tubing (0-86 and 1-27 mm, inside and out¬ side diameter respectively). Additional catheters were placed in the amniotic cavity and a maternal iliac artery. All were flushed daily with heparinized saline (100 U heparin/ml sodium chloride; 9 g/1). Ewes were given 20 mg ampicillin/kg (Polyflex; Aveco, Fort Dodge, IA, U.S.A.) and 2 mg gentamycin was

sulphate/kg (Gentocin; Schering Corp., Kenilworth, NJ, U.S.A.) by s.c. injection twice daily for 4 days. Fetuses at mid gestation received 250 mg ampicillin (Omnipen-N; Wyeth, Philadelphia, PA, U.S.A.) into the amniotic fluid for 4 days, while those in late gestation were given 250 mg ampicillin and 7 mg gentamycin sulphate i.v. at surgery and 250 mg ampicillin into the amniotic fluid every 3 days

thereafter. Glucose was measured (YSI Industrial Analyzer; Yellow Springs Instruments, Yellow Springs, OH, U.S.A.) in whole blood taken from the ewes, and haemoglobin and oxygen saturation (OSM2

Hemoximeter; Radiometer America, Westlake, OH,

U.S.A.) were measured second day.

in fetal arterial blood every

oPL kinetics using l25I-labelled oPL A single dose of approximately 0-35 MBq of l25Ilabelled oPL was given to each of six fetuses: one each at day 79 and 81 and four at day 130. The hormone was purified from ovine placentomes by slight modifi¬ cation of methods developed for caprine PL (Currie, Card, Michel & Ignotz, 1990) and was of comparable purity, as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, isoelectric focus¬ ing and relative binding activity in radioreceptor assays. The protein was iodinated (l25I) using IodoGen (Pierce, Rockford, IL, U.S.A.) to a specific radioactivity of 46 GBq/pmol and was used within 5

days.

Arterial blood

was

collected from fetuses immedi¬

ately before injecting l25I-labelled oPL and used to quantify oPL by radioimmunoassay (Bell, McBride, Slepetis et al. 1989) and 0-5-1 ml was taken to mix

with the l25I-labelled oPL for injection into the venous catheter. The dose was washed in with 0-7-1-5 ml of a mixture of saline and blood from the arterial catheter, timing was begun and then saline injected to fill the lumen of the venous catheter. Radioactivity remain¬ ing in the syringe, needle, catheter and on a gauze used during injection was subsequently measured to determine the net dose with precision. Fetal arterial blood (0-4-1-5 ml) was collected 2-5, 5, 7-5, 10, 15, 20, 25 and 30 min after injection, then at 30 min intervals for the next 150 min. Heparinized plasma (200 pi), collected after centrifugation of blood at 8000 £ for 5 min at 4 °C, was held at 4 °C for less than 4 h then incubated with excess rabbit antioPL in phosphate-buffered saline-gelatin (PBS-G) containing 10 mmol EDTA/1 and 1:1200 non-immune rabbit serum. After 16 h at 4 °C, a pretitred quantity of ovine anti-rabbit -globulin was added and incubated for approximately 24 h at 4 °C. The contents of tubes were diluted with PBS-G and centrifuged, and precipitated l25I-labelled oPL was quantified. A suitable dilution of the l25I-labelled oPL used for injection was added to 200 µ fetal ovine plasma and subjected to the same procedure. All data analyses were made on immunoprecipitable radioactivity. Kinetic

analysis relationship

The between the disappearance of 125Ilabelled oPL and time was analysed by an iterative multiexponential curve-peeling program (Brown & Manno, 1978). The data were optimally explained (coefficient of determination (Ä2)>0-99) by a twocomponent model: y

=

Ac~k' + Be'Jt

Equation

1

where y is the concentration of l25I-labelled oPL (c.p.m./ml), A and represent radioactivity of l25Ilabelled oPL (c.p.m./ml) in two pools, a and b, respectively, k and j are rate constants for the exponential disappearance of radioactivity from pools a and b respectively and t is time (min). The volume of distribution was obtained from the sum of the A and coefficients and the net dose injected; the half-life ( ) in each compartment was obtained from k and j; pool size and entry rate for each pool were calculated from these estimates and the endogenous concen¬ tration of oPL (Shipley & Clark, 1972), using the

equation:

0-693 VDC E=t\1

Equation 2

where E is entry rate (pg/h), VD is volume of distri¬ bution (litre/kg fetal weight), C is concentration (pg/1) and fi is half-life (h).

An estimate of the overall < of the injected substance, the effective /,, can be made provided two assumptions are accepted: (1) the injection was made into pool a; (2) total pool size is the sum of pool a and pool b. The actual ?i must lie between estimates based on entry rates into the fetus equalling the sum of exit rates from the two pools or, if complete recycling is assumed, equal to the exit rate from pool a alone. In all cases, these estimates were similar, so the effective t, was calculated using an entry rate equal to the sum of the exit rates. Infusion of radioinert oPL

Purified oPL was administered by continuous infusion (165pg/h for 3 h) via the venous catheter to two fetuses on day 129. The dose was estimated to be 3-7 times the endogenous entry rate, determined by radioisotope kinetics in the first two fetuses studied in late gestation. Fetal arterial blood (0-6 ml) was sampled at 0, 5, 10, 15, 20, 30,40, 60, 80, 100, 120, 150 and 180 min during the infusion, and maternal blood (3 ml) was sampled every 30 min. Samples were col¬ lected using heparinized syringes and plasma was obtained after centrifugation at 2450 # for 15 min at 4 °C. Concentrations of oPL were measured by radioimmunoassay, as described by Bell et al. (1989). On day 130, the same fetuses were used in radioisotope kinetics studies, as described above. Concentrations of oPL were analysed (Shipley & Clark, 1972) using the equation:

removed and the fetus and Placental weighed. weight equalled the placenta of all dissected from weight placentomes, aggregate the allantochorion and uterus. The pregnant uterus

RESULTS

Animals

of similar body weight (51-4+3-1 kg, 6) and were stable metabolically when kinetic determinations were made. Maternal blood glucose averaged 207 + 007mmol/1 (n 6) in samples collected in mid and late pregnancy. Maternal plasma oPL was 378+ 27 pg/1 in late pregnancy and was unchanged by infusion of oPL into the fetuses; no radioactivity was detected in maternal plasma dur¬ ing any of the kinetic experiments using 125I-labelled oPL. Fetuses in late gestation had haemoglobin con¬ centrations of 117 ±2 g/1 and oxygen saturation of 53-3 + 1 -4% in arterial samples collected between days 116 and 130; the averages for the two fetuses sampled from days 76 to 80 were 81 g/1 and 69-6% respectively. Blood sampling removed 27 ml from older fetuses which was approximately 7% of total blood volume (110 mg/kg; Brace, 1983). At mid gestation, when blood volume is about 160 ml/kg (Barcroft & Kennedy, 1939), the 5-6 ml removed during experiments was equivalent to about 11% of the total. The

ewes were

mean

pools.

Dissections

after the radioisotope kinetic study, animals were killed with i.v. sodium pentobarbital and sodium phenytoin (Beuthanasia-D; Schering Corp.).

Immediately

+ s.e.m.,

m

=

=

Re~k' R AC=-Equation 3 where AC is the measured increment in oPL concen¬ trations, R is the known infusion rate of oPL, A" is the fractional rate constant, derived by solution, VO is the sum of the volumes of distribution for pools a and b measured in the radioisotope kinetics study and t is time. Infusion of exogenous oPL will increase plasma concentrations of the hormone until a steady state is reached, provided endogenous entry is unaffected. At steady state, the exponential term tends towards zero. This equation is strictly true only when the hormone is distributed in a single pool, so the value of derived in this manner gives an estimate of the effective t¡ (from which total entry rate may be calculated) without partitioning the characteristics of multiple

was

were

1008060-

4020•

8

()-

30

60 90 120 150 Time after injection (min)

180

1. Rate of disappearance of immunoprecipitable radioactivity from ovine fetal plasma after single i.v. injec¬ tion of fetuses with 125I-labelled ovine placental lactogen at mid gestation ( O ; 2) and late gestation ( · ; 4). figure

=

=

using 125I-labelled oPL Disappearance curves for immunoprecipitable l25I-

Kinetics

labelled oPL, normalized to the 'instantaneous'

1. Equations describing disappearance of 125I-labelled ovine placental lactogen from ovine fetal plasma in mid and late gestation

table

Equations

Infusion of radioinert oPL

Mid gestation Fetus 1: Fetus 2:

4521 -4e 4483-0e

Late gestation Fetus 1: Fetus 2: Fetus 3: Fetus4:

3562-2e »°«>" + 2134-6e-00077' 4055·8ß