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Protein turnover in rat placenta: Effects of maternal fasting and maternal protein restriction
Placenta(1992), 13, 141-150
Protein Turnover in Rat Placenta: Effects of Maternal Fasting and Maternal Protein Restriction
JOHN D. JOHNSON” GREENBERG
& ROBERT
E.
Department of Pediatric, University (If New Mexico School of Medicine, Albuquerque, NM 87131, USA a To whom all correspondenceshould be addressed Paper accepted24.9.1991
SUMMARY Biochemical compositionand variables of protein turnover were determined in rat placenta at 21 days gestation in control animals and in animals subjectedto 72-h fasting and protein/calorie restriction (6 per cent protein) throughout gestation. Placental protein synthesis was determined following the injection of ‘massive’ amounts of “C-phenylalanine intravenouslyto the pregnant rat. Protein contentwas reduced in placentasfrom rats that werefastedfor 72 h and in those who were protein restrictedthroughout pregnancy. Placental RNA content was signa$cantlyreduced only in theprotein-restrictedanimals. Fractional ratesofprotein synthesis were reduced in placentas from the protein-restricted animals (K, = 17.9 + 0.8per centperday in controlsversus 11.7 f 0.9 in protein restricted, P = 0.002), but not in fasted animals. Fractional protein breakdown was markedly enhanced in placentas following maternal fasting (Kd = 9.9 per cent per day in controlsversus 26Oper cent in fasted), but not affectedbyprotein deprivation. These resultsmimic those previously reportedfor otherfetal tissuesunder theseexperimental conditions.
INTRODUCTION The mammalian placenta is an active metabolic organ. It requires metabolic substrates for its own energy metabolism (Battaglia and Hay, 1984) and is active in synthesizing and secreting specific hormones, e.g., chorionic gonadotropin and placental lactogen, as well as synthesizing constitutive proteins and performing many transport functions (Munro, 1983). Festudies have explored protein turnover in the mammalian placenta, a major aspect of placental metabolism. In this study, we report our determinations of protein turnover in the rat placenta during late gestation (days 19-22) in normal pregnancies, as well as the effects of various maternal 0143-+004/92/020141
+ 10 $05.00/O
@ 1992 Baillike Tindall
I .td
142
Placenta(1992), Vol. 13
and fetal perturbations on placental protein turnover. We find that acute maternal fasting late in gestation and a low protein diet throughout gestation have markedly different effects upon placental protein turnover.
MATERIALS
AND
METHODS
Virgin Sprague-Dawley rats were mated at 100 days of age. Day zero of gestation was defined as the day on which a pan plug was found (Szabo et al, 1969). Animals were maintained in our Animal Resource Facility at 22 + 1°C with a 12 h on-off light cycle. We studied animals under two conditions and have previously reported protein turnover in selected fetal tissues in these experiments (Johnson et al, 1986; Johnson and Dunham, 1988).
Maternal starvation. Pregnant
animals were fed a standard diet (Wayne Lab Blox, Allied Mills, Chicago, IL, 24 per cent protein) ad libitum throughout pregnancy until day 18 gestation, after which they were provided only with water. Concurrent controls were fed the same diet throughout the entirety of gestation (Johnson et al, 1986). Determinations of protein turnover were made at day 2 1 gestation.
Maternal malnutrition. Pregnant
animals were fed a 6 per cent protein diet ad libitum throughout gestation. This diet was isocaloric with a concurrent control group (Johnson and Dunham, 1988). Determinations of placental protein turnover were made at day 21 gestation.
Determination of protein synthesis We employed the method used by Garlick, McNurlan and Preedy (1980) to measure fractional rates of protein synthesis in placenta. We rapidly infused large amounts of 3H-phenylalanine (150 pmol containing 65 ,uCi L-4-PH] phenylalanine [25 ,&i/mmol] per 100 g body weight) into pregnant animals at day 21 gestation intravenously with animals under light ether anesthesia as previously described (Johnson et al, 1986). We have previously reported that this protocol results in a constant specific radioactivity of free 3Hphe in other fetal tissues for 30 min (Johnson et al, 1986). The same is true for placenta. We sacrificed animals 20 min after 3H-phe injection with an overdose of diethyl ether and rapidly submerged placentas in ice-cold 0.9 per cent NaCl. Each whole placenta was sliced 3-4 times to allow blood to escape and rinsed three times in the same iced solution. Subsequently, placentas were frozen in liquid Nz and thereafter processed to determine the specific radioactivities of free and protein-bound 3H-phe by the method of Garlick, McNurland and Preedy (1980). Values for K, (fractional rate of protein synthesis, expressed as per cent of existing protein content of placenta synthesized per day) were calculated from the equation KS = S&S, X t), where &, = specific radioactivity (d/min/nmol) of protein-bound phe, S, = specific radioactivity of free placental phe and t = time in days. Determination of protein accretion and degradation The fractional rate of protein degradation (Kd) was determined by subtracting fractional protein accretion (Kg) from KS. Kg was determined by analysis of protein content of placenta from days 19-22 gestation using a separate group of pregnant animals for each experimental
143
Johnsma, Greenberg: Protein Tumm~er in Rat Placenta
condition. These data were analysed by polynomial regression analyses and quadratic equations were utilized for determination ofabsolute protein accretion. Growth rates (dP/dt) were obtained from derivatives of these equations and fractional growth rates (K,) from the equation, Kp = dP/dt + P(t), where P(t) is the protein content of placenta at day 21 gestation. Biochemical determinations Protein content of placental homogenates was determined by the method (1951) and RNA content as described previously (Johnson et al, 1986).
of Lowry et al
Fetal/placental weight ratios All fetuses and placentas were weighed promptly and individual ratios calculated. While this ratio may vary as a function of location in the uterus, values presented represent the average for the entire litter. Statistical analyses The two-tailed t-test for independent samples was utilized to compare results from control versus experimental animals using starved and malnourished mothers. Polynomial regression analyses were used to construct protein accretion curves for placenta as a function of gestational age. Linear regression analyses by least squares techniques were employed as indicated.
RESULTS Biochemical content of placenta The protein content of placenta for control versus fasted and low-protein maternal diet is shown in Figure 1. Protein content of fasted animals diminishes considerably between 2 l22 days gestation; the protein content of placentas from pregnant animals on a low-protein diet is significantly reduced at all time points (days 19-22). The values for ‘control’ placentas
._ 60??
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F&we 1. Placental protein content. Values for control animals from both experimental conditions were pooled and represent from 21-25 experiments at each gestational age (mean + SE.). Values for fasted animals represent means + SE. of S-10 experiments and for protein/calorie restricted animals means + S.E. of 6 experiments. *P < 0.05 versus controls by unpaired two-tailed t-test. O-O, controls; O-O, fasted; A - A, protein/calorie restricted.
144
Placenta (1992), Vol. 13
_
h.
I
I
I
20
21
22
I
19
Gestational
age (d)
Ft”re2. Placental RNA content. Values for control animals represent pooled results from both experimental conditions. Values for fasted and protein/calorie restricted animals represent means + s.e. for the same number of experiments as depicted in Figure 1. *P < 0.05 versus controls by unpaired two-tailed student t-test. O-O, controls; O-O, fasted; A -A, protein/calorie restricted.
shown in Figure 1 represent those from all experiments combined. However, the results are no different from those in which the controls for individual experimental conditions were compared with their respective experimental values. Placental RNA content decreased from day 19-day 22 in controls (Figure 2). RNA content per placenta diminished in parallel to controls in fasted mothers. Placentas from fasted mothers had significantly lower RNA content at day 22 gestation versus their own controls (p = O.Ol), but not versus the entire group of controls. Placentas from pregnant animals subjected to prolonged protein/calorie malnutrition had markedly lower RNA content versus controls at all gestational ages studied. Placental and fetal weights As can be seen in Figure 3, there was a significant correlation between fetal and placental weights for controls, fasted mothers and protein/calorie restricted mothers. The relationship
r
-5
_.__ n An
.
4.0
5.0
6.0
J 7.0
Fetal weight (g)
Figure 3. Correlation between placental and fetal weight. Individual values for control fetuses (a), fetuses from fasted mothers (0) and fetuses from protein restricted mothers (A) at day 22 gestation are displayed. For all 28 values, fetal weight = 10.48 placental weight + 0.045; R = 0.86, R2 = 0.74.
145
johnsotr. Greenberg: Protein Turnooer in Rat Placenta
GestatIonal
oge (d 1
Figure4. Fetal/placental
weight ratios in control versus fasted animals. Means + s.e. are shown for both control (n = 6-10) and fasted (n = 10) animals. Regression lines were constructed using all data points at each gestational age. For controls, feWplacenta weight ratio = 2.57 gestational age - 44.33, R = 0.932, R* = 0.869; for fasted animals, fetal/placental ratio = 1.64 gestational age - 25.91; R = 0.927, R2 = 0.859. “PC 0.05 versus controls. O-O, controls; O-O, fasted.
in Figure 3 is for fetuses at day 22 gestation. Similar relationships were also found at days 20 and 21 gestation (Z?> 0.72, P < 0.001). Thus, fetal growth retardation paralleled placental weight reduction under these two conditions over a greater than two-fold difference in weights of both fetuses and placentas. Despite this latter correlation, fetal/placental weight ratios increased throughout late gestation at a greater rate in control versus both fasted and protein/calorie restricted animals (Figures 4, 5). These differences were greatest at days 21-22 gestation. Placental protein turnover Figure 6 shows that the specific radioactivity of placental free 3H-phenylalanine remained constant for 30 min following its injection into maternal blood, both in control and fasted animals. Furthermore, during this time period there was a linear increase in the incorporation of 3H-phenylalanine into protein in both groups. Variables of protein turnover in placentas for control and the two experimental conditions are exhibited in Table 1. Protein synthesis, determined at day 21 for fasted mothers, was not different from controls; the loss of placental protein late in gestation in fasted mothers was secondary to accelerated protein degradation (&) versus controls. Placentas from mothers who had received restricted protein diets throughout gestation had significantly reduced protein synthesis (KS) at day 21 gestation, but low rates of protein degradation (&), comparable to controls. Fractional accretion of protein was diminished both in fetuses from fasted and protein-restricted mothers at day 21 gestation versus controls. RNA ‘activity’, i.e., protein synthesized per unit RNA was decreased 37 per cent in
Placenta(1992), Vol. 13
146
19 GestatIonal
Figure 5. Fetal/placental weight ratios in control control (a) and protein-restricted (0) groups (n = as in Figure 4. For controls, fetal/placental weight protein-restricted animals, fetal/placental weight *P < 0.05 versus controls.
21
20 age
(d)
versus protein-restricted animals. Means + se. are shown for 6 at each age for both groups). Regression lines were constructed ratio = 2.14 gestational age - 34.83, R = 0.895, Rz = 0.801; for ratio = 1.45 gestational age ~ 21.43, R = 0.791, R2 = 0.625.
800 600
Time
Figure 6. Placental
(mm1
free and protein-bound specific radioactivity (SRA) of 3H-phenylalanine. Means rt s.e. are shown for control (0) and fasted (0) groups (n = 4 to 6 at each time point). Regression lines were constructed using all data points at each time. For control animals, tissue free SRA = 0.35t + 991.2, where t = time in min (P > O.l), and tissue bound SRA = 0.20t - 0.67 (P < 0.001). For fasted animals, tissue free SRA = -3.22t + 906.3 (P > O.l), and tissue bound SRA = O.llt + 0.13 (P < 0.001).
~ohnsorr,Greenbq: Protein Tumm~erin Rat Placenta
147
Table 1. Protein turnover in rat placenta*
Control Fasted
22.3 k 1.8 (12) 19.6 k 1.1 (16)
Control Low- protein
17.9 + 0.8 11.7 f 0.9
(4) (5)f
12.4 -6.4
0.9 26.0
14.9 7.7
3.0 4.0
“All values are expressed as per cent per day. Values for K, are means + s.e. and values in parenthesis are numbers of experiments. Calculations for Kg and Kd are defined in Methods. tP = 0.002 versus control.
placentas from protein-restricted fasted mothers (Table 2).
animals, but only minimally
(S-10 per cent) in placentas
of
DISCUSSION In these studies, we have described placental chemical composition and protein turnover variables in late gestation in the rat, comparing normal control animals with two experimental conditions, viz, short-term maternal fasting and maternal protein/calorie malnutrition throughout pregnancy. During gestation, the rat fetus exhibits a high rate ofprotein accretion making it potentially susceptible to alterations of maternal nutrition. Placental protein content and weight increased little from 19 to 22 days gestation in controls, similar to results reported previously by others (Williams and McAnulty, 1976; Robinson et al, 1988). Maternal fasting from day 18 gestation resulted in a terminal decrease in placental protein content. Maternal protein/calorie deprivation throughout pregnancy resulted in a highly significant decrease in placental protein content from days 19-22 gestation, consonant with findings in studies by Hastings-Roberts and Zeman (1977). Placental RNA content decreased throughout the last 3 days of gestation in the rat in our controls, as well as in fasted animals. These results are in concert with those reported previously by Winick (1968), Williams and McAnulty (1976), and Robinson et al (1988). By contrast, placental content of RNA was markedly decreased in pregnant animals that were protein/calorie deprived throughout gestation (Figure 2). These results are concordant with results reported by Hastings-Roberts and Zeman (1977). The correlation between fetal and placental weight shown in Figure 3 is consistent with Table 2. Protein synthesis per unit RNA (RNA ‘activity’)
Condition
RNA Activity” (g protein/g RN,4/ &O
Control Fasted
7.26 6.56
(-10%)
Control Protein restricted
5.68 3.56
(-37%)
*RNA RNA.
activity = KS (fractional)
0
protein/
148
Placenta(I 992), Vol. 13
results from many mammalian species under diverse experimental conditions (Jones and Battaglia, 1977). However, the effects of maternal fasting and protein/calorie restriction upon fetal/placental (F/P) weight ratios (Figures 4,5) vary considerably from some reports of other investigators. Our results show decreased F/P weight ratios both in fetuses from fasted mothers and fetuses from protein/calorie restricted mothers. Results for fasted and protein/ calorie restricted conditions suggest the possibility that both of these conditions result not only in reduced placental size but also impaired placental function, e.g., substrate transport, late in gestation with inadequate support of fetal growth. Some previous studies support the results we report here. Lederman and Rosso (1981) reported that when pregnant rats were food restricted prior to fasting on day 17 of gestation, F/P decreased. Hastings-Roberts and Zeman (1977) reported a slight decrease in F/P ratio on day 20 gestation in pregnant rats fed a 4 per cent casein diet versus those fed a 24 per cent casein diet throughout gestation. Young and Widdowson (1975) found that both calorie and protein restricted pregnant guinea pigs had decreased F/P ratios versus controls. Conversely, F/P ratios increased versus controls in studies of calorie restriction in rats by Anderson et al (1980) and in studies of protein deprivation by van Marthens and Shimomaye (1978) and Mazel-Afshar and Grimble (1983). Furthermore, studies both in growthretarded fetal sheep (Alexander, 1964) and in human subjects (Thomson, Billewicz and Hytten, 1969) reveal F/P ratios that are higher than those of normally grown fetuses. The reason(s) for the discrepancies between our results and those of other investigators in this regard are not readily apparent. Differences in the precise experimental conditions utilized to produce fetal growth retardation from fasting or protein-restriction may explain some of these differences. Few previous studies have explored variables of placental protein turnover. We report here that fractional protein synthesis in placenta (K,) was no different from controls for fasted mothers; however, placentas from pregnancies in which mothers were protein/calorie restricted throughout gestation had significantly reduced K, at day 21 gestation. Calculated rates of placental protein degradation on day 21 gestation were accelerated by maternal fasting and unchanged by protein/calorie deprivation. Fractional rates of protein synthesis in rat placenta at day 21 gestation in our control experiments agree closely with those reported previously by Morton and Goldspink (1986) and Robinson et al (1988) utilizing the ‘massive’ phenylalanine injection technique. However, & in placenta in our controls was considerably lower than that reported by Robinson et al (1988). We are unaware of any previous reports on the effect of maternal fasting on placental protein turnover. Mazel-Afshar and Grimble (1983) found that protein restriction in pregnant rats from day zero gestation (5 per cent protein diet) actually enhanced both K, and Kd in placenta at day 21 gestation, in marked contrast to our results. They employed the constant infusion of 14C-tyrosine over 6 h to pregnant rats to assess KS. Their values both for KS and Kd in placenta are significantly greater than ours, both for control and experimental animals. Other than the differences in experimental technique, we are not able to explain these discrepant results. Wunderlich, Baliga and Munro (1979) have reported that placentas from rats fed a 5 per cent protein diet during the last 14 days of pregnancy had reduced RNA and protein at day 20 gestation and reduced amounts of both free and membrane-bound ribosomes versus controls and a reduced capacity per ribosome to incorporate amino acids into peptide chains in vitro, results comparable to our in rino findings. Our results of protein turnover in rat placenta with maternal fasting compare closely with those of fetal heart, diaphragm and liver in which fasting results in reduced protein accretion
Johnson. Greenberg: Protein Turnmer in Rat Placenta
149
with relatively constant KSversus controls, but markedly accelerated Kd (Johnson et al, 1986). In addition, protein turnover variables in placentas from protein/calorie restricted rats late in gestation exhibit similar changes versus controls as we have reported for fetal heart, diaphragm and liver, viz, reduced K, and similar to reduced Kd (Johnson and Dunham, 1988). In summary, placental protein content is decreased in late gestation in the rat both b> maternal protein/calorie restriction throughout gestation and by acute maternal fasting. Both conditions also reduced fetal/placental weight ratios. However, reduced placental protein accretion is accomplished in placentas from fasted animals by enhanced protein breakdown, while in protein/calorie restricted animals reduced placental protein is the result of diminished protein synthesis. The placenta mimics other fetal tissues in terms of protein turnover in response to these models of fetal growth retardation. Thus, protein accretion, in placenta and tissues of the fetus, may be differentially regulated by altering the duration and/ or magnitude of maternal caloric and/or protein intake.
ACKNOWLEDGEMENTS ‘The authors thank Eileen Sever for preparation of the manuscript. This work was supported from the National Institute of Child Health and Human Development, USA.
by Grant HD-I 5243
REFERENCES Alexander, G. (1964) Studies on the placenta of the sheep.looumal ofReproduction and Fertility 7, 289-305. Anderson, G. D., Ahokas, R. A., Lipshitz, J. & Dilts, P. V. Jr (1980) Effect of maternal dietary restriction during pregnancy on maternal weight gain and fetal birth weight in the rat.Jo’oumal ofNutrition, 110, 883-890. Battaglia, F. C. & Hay, W. W. Jr (1984) Energy and substrate requirements for fetal and placental growth and metabolism. In Fetal Physiologyand.Medicine (Eds) Beard, R. W. & Nathanelsz, P. \V. pp. 601-628. New York: Dekker. Garlick, P. J., McNurlan, M. A. & Preedy, V. R. (1980) A rapid and convenient technique for measuring the rate BiochemicalJournal, 142, 719-723. I of protein synthesis in tissues by injection of 3H-phenylalanine. Hastines-Roberts. M. M. & Zeman. F. 1. (1977) Effects of orotein deficiencv. uair-feedine. and diet supp;mentation bn maternal, fetal and’placintai gro\;th in rats.Joimal ofNutrition, ik( 973-982.“’ Johnson, J. D., Dunham, T., Skipper, B. J. & Loftfield, R. B. (1986) Protein turnover in tissues of the rat fetus following maternal starvation. Pediatric Research, 20, 1252-1257. Johnson, J. D. & Dunham, T. (1988) Protein turnover in tissues of the fetal rat after prolonged maternal malnutrition. Pediatric Research, 23, 534-538. Jones, M. D. Jr SK Battaglia, F. C. (1977) Intrauterine growth retardation. An&ran J~umal of Obstetricsand Gynecology,127, 540-549. Lederman, S. A. & Rosso, P. (1981) Effects of fasting during pregnancy on maternal and fetal weight and body composition in well-nourished and undernourished rats..jTuunzal ofNutrition, 111, 1823-1832. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, k. J. (1951) Protein measurement with the Folin phenol reagent. ?ournal ofBioloaica1Chemistrv, 193. 265-275. Maye%Afihar, S. & G&ble, R. F. (1583) dhanges in protein turnover during gestation in the foetuses, placentas, liver, muscle and whole body of rats given a low-protein diet. Biochimica et BiophysicuActa, 756, 182-190. Morton, A. J. & Goldspink, D. F. (1986) Changes in protein turnover in rat uterus during pregnancy. Ammcan Journal ofPhysiology,250, El 14-E120. Munro, H. N. (1983) The placenta in nutrition. Annual Reviews in Nutrition, 3, 97-124. Robinson, J., Canavan, J. P., El Haj, A. J. 8~ Goldspink, D. F. (1988) Maternal diabetes in rats. 1. Effects on placental growth and protein turnover. Diabetes, 37, 1665-1670. Szabo, K. T., Free, S. M., Birkhead, H. A. & Gay, P. E. (1969) Predictability of prebmancp from various signs of mating in mice and rats. LaboratoryAnimals, 19, 822-825. Thomson, A. M., Billewin, W. Z. SKHytten, F. E. (1969) The weight of the placenta in relation to birthweight. BritishJournal of Obstetricsand Gynaecology,76,865-872. van Marthens, E. & Shimomaye, S. Y. (1978) In-utero fetal and placental development following maternal protein repletion in rats.pumal ofNutrition, 108, 959-966.
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Placenta (1992), Vol. 13
Williams, J. P. G. & McAnulty, P. A. (1976) Foetal and placental ornithine decarboxylase activity in the rat- effect of maternal undernutrition. ‘lournal ofEmbrvolow and Eximimental~VIomholow. 35.545-559. Winick, M. (1968) Cellular growth 0; the plac&ta as ah indicator of’abnormal fetal growth. In Diagnosis and Treatment ofFeta Disorders (Ed. ) Adamsons, K. pp. 83-101. New York: Springer-Verlag. Wunderlich, S. M., Baliga, B. S. & Munro, H. N. (1979) Rat placental protein synthesis and peptide hormone secretion in relation to malnutrition from protein deficiency or alcohol administration.3oamal qfl’utrition, 109, 1534-1541. Young, M. & Widdowson, E. M. (1975) The influence of diets deticient in energy, or in protein, on conceptus weight, and the placental transfer of a non-metabolizable amino acid in the guinea pig. Biolon ofthe Neonate, 27, 184-191.
Protein Turnover in Rat Placenta: Effects of Maternal Fasting and Maternal Protein Restriction
JOHN D. JOHNSON” GREENBERG
& ROBERT
E.
Department of Pediatric, University (If New Mexico School of Medicine, Albuquerque, NM 87131, USA a To whom all correspondenceshould be addressed Paper accepted24.9.1991
SUMMARY Biochemical compositionand variables of protein turnover were determined in rat placenta at 21 days gestation in control animals and in animals subjectedto 72-h fasting and protein/calorie restriction (6 per cent protein) throughout gestation. Placental protein synthesis was determined following the injection of ‘massive’ amounts of “C-phenylalanine intravenouslyto the pregnant rat. Protein contentwas reduced in placentasfrom rats that werefastedfor 72 h and in those who were protein restrictedthroughout pregnancy. Placental RNA content was signa$cantlyreduced only in theprotein-restrictedanimals. Fractional ratesofprotein synthesis were reduced in placentas from the protein-restricted animals (K, = 17.9 + 0.8per centperday in controlsversus 11.7 f 0.9 in protein restricted, P = 0.002), but not in fasted animals. Fractional protein breakdown was markedly enhanced in placentas following maternal fasting (Kd = 9.9 per cent per day in controlsversus 26Oper cent in fasted), but not affectedbyprotein deprivation. These resultsmimic those previously reportedfor otherfetal tissuesunder theseexperimental conditions.
INTRODUCTION The mammalian placenta is an active metabolic organ. It requires metabolic substrates for its own energy metabolism (Battaglia and Hay, 1984) and is active in synthesizing and secreting specific hormones, e.g., chorionic gonadotropin and placental lactogen, as well as synthesizing constitutive proteins and performing many transport functions (Munro, 1983). Festudies have explored protein turnover in the mammalian placenta, a major aspect of placental metabolism. In this study, we report our determinations of protein turnover in the rat placenta during late gestation (days 19-22) in normal pregnancies, as well as the effects of various maternal 0143-+004/92/020141
+ 10 $05.00/O
@ 1992 Baillike Tindall
I .td
142
Placenta(1992), Vol. 13
and fetal perturbations on placental protein turnover. We find that acute maternal fasting late in gestation and a low protein diet throughout gestation have markedly different effects upon placental protein turnover.
MATERIALS
AND
METHODS
Virgin Sprague-Dawley rats were mated at 100 days of age. Day zero of gestation was defined as the day on which a pan plug was found (Szabo et al, 1969). Animals were maintained in our Animal Resource Facility at 22 + 1°C with a 12 h on-off light cycle. We studied animals under two conditions and have previously reported protein turnover in selected fetal tissues in these experiments (Johnson et al, 1986; Johnson and Dunham, 1988).
Maternal starvation. Pregnant
animals were fed a standard diet (Wayne Lab Blox, Allied Mills, Chicago, IL, 24 per cent protein) ad libitum throughout pregnancy until day 18 gestation, after which they were provided only with water. Concurrent controls were fed the same diet throughout the entirety of gestation (Johnson et al, 1986). Determinations of protein turnover were made at day 2 1 gestation.
Maternal malnutrition. Pregnant
animals were fed a 6 per cent protein diet ad libitum throughout gestation. This diet was isocaloric with a concurrent control group (Johnson and Dunham, 1988). Determinations of placental protein turnover were made at day 21 gestation.
Determination of protein synthesis We employed the method used by Garlick, McNurlan and Preedy (1980) to measure fractional rates of protein synthesis in placenta. We rapidly infused large amounts of 3H-phenylalanine (150 pmol containing 65 ,uCi L-4-PH] phenylalanine [25 ,&i/mmol] per 100 g body weight) into pregnant animals at day 21 gestation intravenously with animals under light ether anesthesia as previously described (Johnson et al, 1986). We have previously reported that this protocol results in a constant specific radioactivity of free 3Hphe in other fetal tissues for 30 min (Johnson et al, 1986). The same is true for placenta. We sacrificed animals 20 min after 3H-phe injection with an overdose of diethyl ether and rapidly submerged placentas in ice-cold 0.9 per cent NaCl. Each whole placenta was sliced 3-4 times to allow blood to escape and rinsed three times in the same iced solution. Subsequently, placentas were frozen in liquid Nz and thereafter processed to determine the specific radioactivities of free and protein-bound 3H-phe by the method of Garlick, McNurland and Preedy (1980). Values for K, (fractional rate of protein synthesis, expressed as per cent of existing protein content of placenta synthesized per day) were calculated from the equation KS = S&S, X t), where &, = specific radioactivity (d/min/nmol) of protein-bound phe, S, = specific radioactivity of free placental phe and t = time in days. Determination of protein accretion and degradation The fractional rate of protein degradation (Kd) was determined by subtracting fractional protein accretion (Kg) from KS. Kg was determined by analysis of protein content of placenta from days 19-22 gestation using a separate group of pregnant animals for each experimental
143
Johnsma, Greenberg: Protein Tumm~er in Rat Placenta
condition. These data were analysed by polynomial regression analyses and quadratic equations were utilized for determination ofabsolute protein accretion. Growth rates (dP/dt) were obtained from derivatives of these equations and fractional growth rates (K,) from the equation, Kp = dP/dt + P(t), where P(t) is the protein content of placenta at day 21 gestation. Biochemical determinations Protein content of placental homogenates was determined by the method (1951) and RNA content as described previously (Johnson et al, 1986).
of Lowry et al
Fetal/placental weight ratios All fetuses and placentas were weighed promptly and individual ratios calculated. While this ratio may vary as a function of location in the uterus, values presented represent the average for the entire litter. Statistical analyses The two-tailed t-test for independent samples was utilized to compare results from control versus experimental animals using starved and malnourished mothers. Polynomial regression analyses were used to construct protein accretion curves for placenta as a function of gestational age. Linear regression analyses by least squares techniques were employed as indicated.
RESULTS Biochemical content of placenta The protein content of placenta for control versus fasted and low-protein maternal diet is shown in Figure 1. Protein content of fasted animals diminishes considerably between 2 l22 days gestation; the protein content of placentas from pregnant animals on a low-protein diet is significantly reduced at all time points (days 19-22). The values for ‘control’ placentas
._ 60??
I
50 -
x _____/J'_
',
F"
40A*
'.
'. ‘d*
I* -I
30 7
r
21 Gestotlonol
22
age (d)
F&we 1. Placental protein content. Values for control animals from both experimental conditions were pooled and represent from 21-25 experiments at each gestational age (mean + SE.). Values for fasted animals represent means + SE. of S-10 experiments and for protein/calorie restricted animals means + S.E. of 6 experiments. *P < 0.05 versus controls by unpaired two-tailed t-test. O-O, controls; O-O, fasted; A - A, protein/calorie restricted.
144
Placenta (1992), Vol. 13
_
h.
I
I
I
20
21
22
I
19
Gestational
age (d)
Ft”re2. Placental RNA content. Values for control animals represent pooled results from both experimental conditions. Values for fasted and protein/calorie restricted animals represent means + s.e. for the same number of experiments as depicted in Figure 1. *P < 0.05 versus controls by unpaired two-tailed student t-test. O-O, controls; O-O, fasted; A -A, protein/calorie restricted.
shown in Figure 1 represent those from all experiments combined. However, the results are no different from those in which the controls for individual experimental conditions were compared with their respective experimental values. Placental RNA content decreased from day 19-day 22 in controls (Figure 2). RNA content per placenta diminished in parallel to controls in fasted mothers. Placentas from fasted mothers had significantly lower RNA content at day 22 gestation versus their own controls (p = O.Ol), but not versus the entire group of controls. Placentas from pregnant animals subjected to prolonged protein/calorie malnutrition had markedly lower RNA content versus controls at all gestational ages studied. Placental and fetal weights As can be seen in Figure 3, there was a significant correlation between fetal and placental weights for controls, fasted mothers and protein/calorie restricted mothers. The relationship
r
-5
_.__ n An
.
4.0
5.0
6.0
J 7.0
Fetal weight (g)
Figure 3. Correlation between placental and fetal weight. Individual values for control fetuses (a), fetuses from fasted mothers (0) and fetuses from protein restricted mothers (A) at day 22 gestation are displayed. For all 28 values, fetal weight = 10.48 placental weight + 0.045; R = 0.86, R2 = 0.74.
145
johnsotr. Greenberg: Protein Turnooer in Rat Placenta
GestatIonal
oge (d 1
Figure4. Fetal/placental
weight ratios in control versus fasted animals. Means + s.e. are shown for both control (n = 6-10) and fasted (n = 10) animals. Regression lines were constructed using all data points at each gestational age. For controls, feWplacenta weight ratio = 2.57 gestational age - 44.33, R = 0.932, R* = 0.869; for fasted animals, fetal/placental ratio = 1.64 gestational age - 25.91; R = 0.927, R2 = 0.859. “PC 0.05 versus controls. O-O, controls; O-O, fasted.
in Figure 3 is for fetuses at day 22 gestation. Similar relationships were also found at days 20 and 21 gestation (Z?> 0.72, P < 0.001). Thus, fetal growth retardation paralleled placental weight reduction under these two conditions over a greater than two-fold difference in weights of both fetuses and placentas. Despite this latter correlation, fetal/placental weight ratios increased throughout late gestation at a greater rate in control versus both fasted and protein/calorie restricted animals (Figures 4, 5). These differences were greatest at days 21-22 gestation. Placental protein turnover Figure 6 shows that the specific radioactivity of placental free 3H-phenylalanine remained constant for 30 min following its injection into maternal blood, both in control and fasted animals. Furthermore, during this time period there was a linear increase in the incorporation of 3H-phenylalanine into protein in both groups. Variables of protein turnover in placentas for control and the two experimental conditions are exhibited in Table 1. Protein synthesis, determined at day 21 for fasted mothers, was not different from controls; the loss of placental protein late in gestation in fasted mothers was secondary to accelerated protein degradation (&) versus controls. Placentas from mothers who had received restricted protein diets throughout gestation had significantly reduced protein synthesis (KS) at day 21 gestation, but low rates of protein degradation (&), comparable to controls. Fractional accretion of protein was diminished both in fetuses from fasted and protein-restricted mothers at day 21 gestation versus controls. RNA ‘activity’, i.e., protein synthesized per unit RNA was decreased 37 per cent in
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19 GestatIonal
Figure 5. Fetal/placental weight ratios in control control (a) and protein-restricted (0) groups (n = as in Figure 4. For controls, fetal/placental weight protein-restricted animals, fetal/placental weight *P < 0.05 versus controls.
21
20 age
(d)
versus protein-restricted animals. Means + se. are shown for 6 at each age for both groups). Regression lines were constructed ratio = 2.14 gestational age - 34.83, R = 0.895, Rz = 0.801; for ratio = 1.45 gestational age ~ 21.43, R = 0.791, R2 = 0.625.
800 600
Time
Figure 6. Placental
(mm1
free and protein-bound specific radioactivity (SRA) of 3H-phenylalanine. Means rt s.e. are shown for control (0) and fasted (0) groups (n = 4 to 6 at each time point). Regression lines were constructed using all data points at each time. For control animals, tissue free SRA = 0.35t + 991.2, where t = time in min (P > O.l), and tissue bound SRA = 0.20t - 0.67 (P < 0.001). For fasted animals, tissue free SRA = -3.22t + 906.3 (P > O.l), and tissue bound SRA = O.llt + 0.13 (P < 0.001).
~ohnsorr,Greenbq: Protein Tumm~erin Rat Placenta
147
Table 1. Protein turnover in rat placenta*
Control Fasted
22.3 k 1.8 (12) 19.6 k 1.1 (16)
Control Low- protein
17.9 + 0.8 11.7 f 0.9
(4) (5)f
12.4 -6.4
0.9 26.0
14.9 7.7
3.0 4.0
“All values are expressed as per cent per day. Values for K, are means + s.e. and values in parenthesis are numbers of experiments. Calculations for Kg and Kd are defined in Methods. tP = 0.002 versus control.
placentas from protein-restricted fasted mothers (Table 2).
animals, but only minimally
(S-10 per cent) in placentas
of
DISCUSSION In these studies, we have described placental chemical composition and protein turnover variables in late gestation in the rat, comparing normal control animals with two experimental conditions, viz, short-term maternal fasting and maternal protein/calorie malnutrition throughout pregnancy. During gestation, the rat fetus exhibits a high rate ofprotein accretion making it potentially susceptible to alterations of maternal nutrition. Placental protein content and weight increased little from 19 to 22 days gestation in controls, similar to results reported previously by others (Williams and McAnulty, 1976; Robinson et al, 1988). Maternal fasting from day 18 gestation resulted in a terminal decrease in placental protein content. Maternal protein/calorie deprivation throughout pregnancy resulted in a highly significant decrease in placental protein content from days 19-22 gestation, consonant with findings in studies by Hastings-Roberts and Zeman (1977). Placental RNA content decreased throughout the last 3 days of gestation in the rat in our controls, as well as in fasted animals. These results are in concert with those reported previously by Winick (1968), Williams and McAnulty (1976), and Robinson et al (1988). By contrast, placental content of RNA was markedly decreased in pregnant animals that were protein/calorie deprived throughout gestation (Figure 2). These results are concordant with results reported by Hastings-Roberts and Zeman (1977). The correlation between fetal and placental weight shown in Figure 3 is consistent with Table 2. Protein synthesis per unit RNA (RNA ‘activity’)
Condition
RNA Activity” (g protein/g RN,4/ &O
Control Fasted
7.26 6.56
(-10%)
Control Protein restricted
5.68 3.56
(-37%)
*RNA RNA.
activity = KS (fractional)
0
protein/
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Placenta(I 992), Vol. 13
results from many mammalian species under diverse experimental conditions (Jones and Battaglia, 1977). However, the effects of maternal fasting and protein/calorie restriction upon fetal/placental (F/P) weight ratios (Figures 4,5) vary considerably from some reports of other investigators. Our results show decreased F/P weight ratios both in fetuses from fasted mothers and fetuses from protein/calorie restricted mothers. Results for fasted and protein/ calorie restricted conditions suggest the possibility that both of these conditions result not only in reduced placental size but also impaired placental function, e.g., substrate transport, late in gestation with inadequate support of fetal growth. Some previous studies support the results we report here. Lederman and Rosso (1981) reported that when pregnant rats were food restricted prior to fasting on day 17 of gestation, F/P decreased. Hastings-Roberts and Zeman (1977) reported a slight decrease in F/P ratio on day 20 gestation in pregnant rats fed a 4 per cent casein diet versus those fed a 24 per cent casein diet throughout gestation. Young and Widdowson (1975) found that both calorie and protein restricted pregnant guinea pigs had decreased F/P ratios versus controls. Conversely, F/P ratios increased versus controls in studies of calorie restriction in rats by Anderson et al (1980) and in studies of protein deprivation by van Marthens and Shimomaye (1978) and Mazel-Afshar and Grimble (1983). Furthermore, studies both in growthretarded fetal sheep (Alexander, 1964) and in human subjects (Thomson, Billewicz and Hytten, 1969) reveal F/P ratios that are higher than those of normally grown fetuses. The reason(s) for the discrepancies between our results and those of other investigators in this regard are not readily apparent. Differences in the precise experimental conditions utilized to produce fetal growth retardation from fasting or protein-restriction may explain some of these differences. Few previous studies have explored variables of placental protein turnover. We report here that fractional protein synthesis in placenta (K,) was no different from controls for fasted mothers; however, placentas from pregnancies in which mothers were protein/calorie restricted throughout gestation had significantly reduced K, at day 21 gestation. Calculated rates of placental protein degradation on day 21 gestation were accelerated by maternal fasting and unchanged by protein/calorie deprivation. Fractional rates of protein synthesis in rat placenta at day 21 gestation in our control experiments agree closely with those reported previously by Morton and Goldspink (1986) and Robinson et al (1988) utilizing the ‘massive’ phenylalanine injection technique. However, & in placenta in our controls was considerably lower than that reported by Robinson et al (1988). We are unaware of any previous reports on the effect of maternal fasting on placental protein turnover. Mazel-Afshar and Grimble (1983) found that protein restriction in pregnant rats from day zero gestation (5 per cent protein diet) actually enhanced both K, and Kd in placenta at day 21 gestation, in marked contrast to our results. They employed the constant infusion of 14C-tyrosine over 6 h to pregnant rats to assess KS. Their values both for KS and Kd in placenta are significantly greater than ours, both for control and experimental animals. Other than the differences in experimental technique, we are not able to explain these discrepant results. Wunderlich, Baliga and Munro (1979) have reported that placentas from rats fed a 5 per cent protein diet during the last 14 days of pregnancy had reduced RNA and protein at day 20 gestation and reduced amounts of both free and membrane-bound ribosomes versus controls and a reduced capacity per ribosome to incorporate amino acids into peptide chains in vitro, results comparable to our in rino findings. Our results of protein turnover in rat placenta with maternal fasting compare closely with those of fetal heart, diaphragm and liver in which fasting results in reduced protein accretion
Johnson. Greenberg: Protein Turnmer in Rat Placenta
149
with relatively constant KSversus controls, but markedly accelerated Kd (Johnson et al, 1986). In addition, protein turnover variables in placentas from protein/calorie restricted rats late in gestation exhibit similar changes versus controls as we have reported for fetal heart, diaphragm and liver, viz, reduced K, and similar to reduced Kd (Johnson and Dunham, 1988). In summary, placental protein content is decreased in late gestation in the rat both b> maternal protein/calorie restriction throughout gestation and by acute maternal fasting. Both conditions also reduced fetal/placental weight ratios. However, reduced placental protein accretion is accomplished in placentas from fasted animals by enhanced protein breakdown, while in protein/calorie restricted animals reduced placental protein is the result of diminished protein synthesis. The placenta mimics other fetal tissues in terms of protein turnover in response to these models of fetal growth retardation. Thus, protein accretion, in placenta and tissues of the fetus, may be differentially regulated by altering the duration and/ or magnitude of maternal caloric and/or protein intake.
ACKNOWLEDGEMENTS ‘The authors thank Eileen Sever for preparation of the manuscript. This work was supported from the National Institute of Child Health and Human Development, USA.
by Grant HD-I 5243
REFERENCES Alexander, G. (1964) Studies on the placenta of the sheep.looumal ofReproduction and Fertility 7, 289-305. Anderson, G. D., Ahokas, R. A., Lipshitz, J. & Dilts, P. V. Jr (1980) Effect of maternal dietary restriction during pregnancy on maternal weight gain and fetal birth weight in the rat.Jo’oumal ofNutrition, 110, 883-890. Battaglia, F. C. & Hay, W. W. Jr (1984) Energy and substrate requirements for fetal and placental growth and metabolism. In Fetal Physiologyand.Medicine (Eds) Beard, R. W. & Nathanelsz, P. \V. pp. 601-628. New York: Dekker. Garlick, P. J., McNurlan, M. A. & Preedy, V. R. (1980) A rapid and convenient technique for measuring the rate BiochemicalJournal, 142, 719-723. I of protein synthesis in tissues by injection of 3H-phenylalanine. Hastines-Roberts. M. M. & Zeman. F. 1. (1977) Effects of orotein deficiencv. uair-feedine. and diet supp;mentation bn maternal, fetal and’placintai gro\;th in rats.Joimal ofNutrition, ik( 973-982.“’ Johnson, J. D., Dunham, T., Skipper, B. J. & Loftfield, R. B. (1986) Protein turnover in tissues of the rat fetus following maternal starvation. Pediatric Research, 20, 1252-1257. Johnson, J. D. & Dunham, T. (1988) Protein turnover in tissues of the fetal rat after prolonged maternal malnutrition. Pediatric Research, 23, 534-538. Jones, M. D. Jr SK Battaglia, F. C. (1977) Intrauterine growth retardation. An&ran J~umal of Obstetricsand Gynecology,127, 540-549. Lederman, S. A. & Rosso, P. (1981) Effects of fasting during pregnancy on maternal and fetal weight and body composition in well-nourished and undernourished rats..jTuunzal ofNutrition, 111, 1823-1832. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, k. J. (1951) Protein measurement with the Folin phenol reagent. ?ournal ofBioloaica1Chemistrv, 193. 265-275. Maye%Afihar, S. & G&ble, R. F. (1583) dhanges in protein turnover during gestation in the foetuses, placentas, liver, muscle and whole body of rats given a low-protein diet. Biochimica et BiophysicuActa, 756, 182-190. Morton, A. J. & Goldspink, D. F. (1986) Changes in protein turnover in rat uterus during pregnancy. Ammcan Journal ofPhysiology,250, El 14-E120. Munro, H. N. (1983) The placenta in nutrition. Annual Reviews in Nutrition, 3, 97-124. Robinson, J., Canavan, J. P., El Haj, A. J. 8~ Goldspink, D. F. (1988) Maternal diabetes in rats. 1. Effects on placental growth and protein turnover. Diabetes, 37, 1665-1670. Szabo, K. T., Free, S. M., Birkhead, H. A. & Gay, P. E. (1969) Predictability of prebmancp from various signs of mating in mice and rats. LaboratoryAnimals, 19, 822-825. Thomson, A. M., Billewin, W. Z. SKHytten, F. E. (1969) The weight of the placenta in relation to birthweight. BritishJournal of Obstetricsand Gynaecology,76,865-872. van Marthens, E. & Shimomaye, S. Y. (1978) In-utero fetal and placental development following maternal protein repletion in rats.pumal ofNutrition, 108, 959-966.
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Williams, J. P. G. & McAnulty, P. A. (1976) Foetal and placental ornithine decarboxylase activity in the rat- effect of maternal undernutrition. ‘lournal ofEmbrvolow and Eximimental~VIomholow. 35.545-559. Winick, M. (1968) Cellular growth 0; the plac&ta as ah indicator of’abnormal fetal growth. In Diagnosis and Treatment ofFeta Disorders (Ed. ) Adamsons, K. pp. 83-101. New York: Springer-Verlag. Wunderlich, S. M., Baliga, B. S. & Munro, H. N. (1979) Rat placental protein synthesis and peptide hormone secretion in relation to malnutrition from protein deficiency or alcohol administration.3oamal qfl’utrition, 109, 1534-1541. Young, M. & Widdowson, E. M. (1975) The influence of diets deticient in energy, or in protein, on conceptus weight, and the placental transfer of a non-metabolizable amino acid in the guinea pig. Biolon ofthe Neonate, 27, 184-191.