Human Placental Lactogen Administration in the Pregnant Rat: Acceleration of Fetal Growth
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003 1-399818812406-0663$02.00/0 PEDIATRIC RESEARCH Vol. 24, No. 6, 1988 Copyright O 1988 International Pediatric Research Foundation, Inc. Printed in U.S.A. Human Placental Lactogen Administration in the Pregnant Rat: Acceleration of Fetal Growth JAMES W. COLLINS, JR., SANDRA L. FINLEY, DANIEL MERRICK, AND EDWARD S. OGATA Departments of Pediatrics, Obstetrics and Gynecology, Northwestern University Medical School and Prentice Women's Hospital of Northwestern Memorial Hospital and Children's Memorial Hospital, Chicago, Illinois 60611 ABSTRACT. To determine whether administration of hu- servations suggest that placental lactogen may also directly stim- man placental lactogen (hPL) to pregnant rats during late ulate fetal growth. Ovine placental lactogen stimulates glycoge- gestation might enhance fetal growth, we implanted os- nesis in hepatocytes of the fetal rat (4) and sheep (5) and motically driven minipumps to provide 75 pg h PL/24 h on aminoisobutyric acid uptake in diaphragmatic muscle of the fetal day 14 of the rat's 21.5-day gestation. This substantially rat (6). It also stimulates ornithine decarboxylase activity in fetal increased maternal and fetal plasma hPL concentrations. rat liver (7) and somatomedin secretion in fetal and adult tissue By day 18, hPL fetuses were significantly heavier and had (8-10). Handwerger (11) has suggested that these and other larger placentas than controls. From this point until term, observations indicate a critical role for placental lactogen in not their rate of growth (1.20 g/24 h) significantly exceeded only indirectly but also directly stimulating normal fetal growth that of controls (0.95 g/24 h). Birth weights differed sig- and development. No study, however, has addressed the possi- nificantly (hPL 5.86 f 0.08 g; controls 5.20 f 0.08 g, p < bility that excess placental lactogen might actually accelerate fetal 0.001). This increase was due primarily to significant in- growth. creases in the growth of the liver and carcass. Enhanced For this reason, we determined the effect of administering hPL glucose availability was in part responsible for this phe- to maternal rats upon fetal growth, and maternal, fetal, and nomenon inasmuch as plasma glucose concentrations were neonatal glucose homeostasis. Our data indicate that hPL ad- significantly increased in hPL maternal rats from days 15 ministration in the maternal rat significantly increases fetal to 19. This resulted on days 18 and 19 in significantly growth. The mechanisms for this include enhancement of ma- increased plasma glucose and insulin concentrations in hPL ternal glucose availabiltiy and a significant but transient increase fetuses. Fetal/maternal glucose ratios did not differ be- in fetal insulin secretion. These observations also allow the tween hPL and control fetuses. Fetal hepatic glycogen speculation that hPL might directly stimulate fetal growth. concentrations were significantly increased on day 18 and 19 but were similar to controls from day 20 until birth. These observations suggest that increased maternal glu- MATERIALS AND METHODS cose availability with consequent stimulation of fetal insu- Maternal. We purchased pregnant Sprague-Dawley rats (Har- lin secretion accelerated the growth of hPL fetuses. How- lan Laboratories, Madison, WI) that weighed 180-200 g before ever, maternal and fetal plasma glucose concentrations and mating. Gestation was timed to within 6 h of mating. We fetal plasma insulin and hepatic glycogen concentrations anesthetized rats on day 14 (term, 21.5 days) of gestation with on days 20 and 21 were normal, suggesting that other ketamine (5 mg/100 g body weight) and, after administration of factors also were responsible for sustaining the accelerated local anesthetic (1 % xylocaine), implanted osmotic minipumps fetal growth on these days. These observations and the (Alzet Corp., Palo Alto, CA) into the subcutaneous tissue of the many reports of the stimulatory effect of placental lactogen dorsum of the neck. The minipumps were loaded with hPL upon numerous fetal metabolic functions suggest that hPL supplied by the National Hormone and Pituitary Program at a might have directly stimulated growth. (Pediatr Res concentration to deliver 75 pg in 24 h. Others had reported that 24:663-667,1988) this dose significantly increases maternal and fetal plasma hPL concentrations (9). Control rats received pumps filled with 154 Abbreviations mM NaCl. The volume infused was 12 pl/day in both experi- mental and control animals. Rats had ad libitum access to a hPL, human placental lactogen standard diet (Purina, St. Louis, MO) and water in a temperature- controlled environment with a 12-h light/dark cycle. Serial meas- urements of maternal weight, and plasma concentrations of glucose, insulin, glucagon, and hPL were performed from day 14 Placental lactogen is important for fetal growth. During the until term. Blood was obtained from the warmed cut tail tip. We second half of pregnancy, placental lactogen is one of several used only rats with 8-13 fetuses in a litter to control for the factors that counter the effect of insulin in the maternal circula- potential effect of litter number upon intrauterine growth. tion, thereby facilitating glucose and amino acid availability to Fetal. We characterized the growth and metabolic variables of the fetus (1-3). In this manner, the placental secretion of this fetuses of mothers who had received hPL or NaC1. As in previous hormone indirectly stimulates intrauterine growth. Recent ob- studies, we determined wet and dry weight of liver, spleen, brain, kidneys, and carcass (skeletal muscle, bone, connective and Received May 23, 1988; accepted July 27, 1988. adipose tissue, and skin) and measured plasma glucose, insulin, Correspondence Edward S. Ogata, M.D., Division of Neonatology, Children's Memorial Hospita1,,2300 Children's Plaza, Chicago, IL 606 14. glucagon, and hepatic glycogen concentrations (12). For fetal Supported in part by grants from the NIH (IPO 119070) and the Juvenile Diabetes blood sampling, we used the method of Girard et al. (13) in Foundation. which blood is obtained from the fetus with the fetal-placental 664 COLLINS ET AL. circulation intact. The mother was anesthetized with intraperi- man insulin and porcine glucagon as standards (12, 13). Plasma toneal pentobarbital (5 mg/100 g), and the uterus was exposed samples of 25 p1 were used for each insulin and 50-pl samples so that individual fetuses could be carefully removed without for each glucagon determination. For the insulin assay, the disturbing the fetal-placental circulation. The fetuses were dried minimal detectable mass was 0.78 pU/tube; for glucagon it was and maintained at 37" C with a heat lamp and warm moist 7.7 k 2.0 pgltube. We modified a double antibody 1251-radio- gauze. Blood was obtained from axillary vessels. This sampling immunoassay (Corning Immunophase, Medfield, MA) by dilut- was accomplished within 10- 15 s. Maternal blood was obtained ing the antibody (1:4 v/v) and tracer (15 v/v) solutions with from the cut tail tip at the time of sampling from each fetus. In standard borate buffer. This allowed determination of individual the previous use of this technique, we found maternal blood gas hPL values in 50-p1 aliquots of standard and plasma samples in tensions, pH, and blood pressure to be stable during the sampling the concentration range of the fetuses and mothers in this study. period (12). Pentobarbital was used because it does not alter In preliminary studies, we added hPL to adult male rat plasma glucose concentrations early during anesthesia (14). Adequate and determined hPL concentrations in 50-p1 aliquots. Measure- blood sampling from individual fetuses could only be performed ment of hPL at 50, 100, 150, and 200 ng/ml concentrations were from day 18 onward. After blood sampling, the fetuses were comparable to values for hPL in standard assay solution. When killed by decapitation and the organs quickly removed and frozen hPL was added to plasma from pregnant rats, measured values with liquid nitrogen and stored at -70" C. We sampled from were 15 to 25 ng/ml were than expected. This assay is sensitive only 2-3 fetuses in a litter to avoid any potential effect of to 10.0 pg/ml. Hepatic glycogen concentrations and tissue DNA prolonged maternal anesthesia. Each point represents data from and protein concentrations were determined as established in at least 30 fetuses. Fifteen to 20 litters are represented in each our laboratory. group for each day of gestation. Statisticalanalysis was performed using the Student's unpaired Neonatal. On day 21.5, we delivered rat pups by cesarean t test to determine differences between groups. This protocol was section after stunning and cervical dislocation of the mother. approved by the animal Care Committee of Northwestern Uni- Pups were quickly dried, separated from their placentas, and versity. placed in chambers which provided humidified room air at 37" C. As in previous studies, we sampled blood from individual RESULTS littermates to determine sequential changes in plasma glucose, insulin, and glucagon concentrations at birth and at 20,60, 120, Maternal. Implantation of hPGloaded pumps on day 14 sig- and 240 min of life (15). Blood was collected from severed nificantly increased maternal hPL concentrations by day 15. axillary vessels. After blood sampling, the pups were decapitated Maternal plasma concentrations from day 15 to 21 were 250.2 and their livers obtained for determination of sequential glycogen + 22.5, 265.1 k 23.8, 249.8 -1- 30.1, 255.6 k20.9, 263.8 -1- 23.6, concentrations. Data for each metabolic variable are from at 270.1 -1- 30.3, and 260.5 + 25.7 ng/ml. On days 18-21, fetal least 25 pups at each point. A minimum of 20 litters is repre- plasam concentrations were 120.2 + 18.3, 107.9 k 13.9, 133.0 sented in each group.