Fetal lung growth: influence of maternal hypoxia and hyperoxia in rats

RespirationPhysiology(1988) 73, 225-242

225

Elsevier RSP 01430

Fetal lung growth: influence of maternal hypoxia and hyperoxia in rats E.E. Faridy, M . R . Sanii and J.A. Thliveris Departments of Physiology and Anatomy, Universityof Manitoba, Winnipeg, Manitoba, Canada R3E OW3 (Accepted for publication 12 March 1988) Abstract. The consequences of maternal hypoxia and hyperoxia on maternal and fetal lung growth and in

particular on the relationship between the three gas exchange organs (lungs and placenta) were studied in albino rats. Pregnant rats were exposed to one of the following: (1) 10Yo 02 in N 2 or 100~o O 2 for 2 days beginning at day 7, 11, 14, or 18 of pregnancy; (2) 10~o 02 in N 2 or 100~o 02 for 10 h/day beginning at day 7; or (3) 14-11 ~o 02 in N 2 continuously beginning at day 14 till day 21 when they were sacrificed. Maternal lung growth was assessed by measuring the lung weight, lung air volume and lung DNA content, and the fetal lung growth by lung DNA content. Hypoxia and hyperoxia of short duration (2 days) had no significant effect on maternal and fetal lungs and placenta. The major findings with intermittent hypoxia and hyperoxia, and with 1 week continuous hypoxia were as follows: (1)hypoxia initiated enlargement in maternal lung, liver, kidney and heart, and growth retardation in the fetus; (2) the direct relationships which exist in normal pregnancy between placental weight or DNA content and fetal body weight were abolished by maternal hypoxia, and that which exists between maternal and fetal lung DNA content, by hypoxia and hyperoxia; (3) both hypoxia and hyperoxia, applied at early pregnancy, caused small for body weight placenta and lung; and (4)neither maternal hypoxia nor hyperoxia influenced fetal lung maturation. It is speculated that reduction in fetal lung DNA content with maternal hypoxia may result from the direct and indirect effects of hypoxia on fetal lung, namely inhibition of cell multiplication and reduced pulmonary blood flow; and that a small fetal lung with maternal exposure to 100~o 02 , may result from redistribution of blood flow and nutrient supplies to fetal organs with lungs receiving a smaller proportion of it.

Fetal growth; Fetal lung phospholipids; Lung DNA; Placenta; Pregnancy

In a recent study (Faridy etal., 1988a) we examined the relationship between the maternal and fetal lung growth in pregnant rats. We found that pregnant rats with large litter size have larger lungs than rats with small litter size; that there is a direct relationship between DNA content of the fetal lung and maternal lung when the latter undergoes a growth change during pregnancy, i.e. the larger the maternal lung, the larger the fetal lung; and that no relationship in DNA content is found between the maternal lung and placenta and between the fetal lung and placenta. This led us to postulate that the maternal lung influences the growth of the fetal lung. To test this hypothesis we Correspondence address: Dr. E.E. Faridy, Department of Physiology, Basic Medical Sciences Building, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W3. 0034-5687/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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subjected the pregnant rats to unilateral pneumonectomy at various days during pregnancy in order to stimulate the growth of the remaining lung and to determine the influence of this on fetal lung (Faridy etal., 1988b). We found that the fetuses of pneumonectomized rats have larger lungs for body weight when pneumonectomy is performed in the first half of gestation, and that there is a direct relationship between DNA content of the fetal lung and maternal lung. This substantiated our notion in regard to the influence of maternal lung on fetal lung growth. Since in the first day postpneumonectomy the animal experiences a mild and transient hypoxia (Nattie et aL, 1974), we thought it was relevant to investigate the influence that short periods (2 days) ofhypoxia may have on the growth of the fetal lung. Furthermore, since low concentrations of 02 in environment stimulate lung growth in rats (Cunningham et aL, 1974) and cause fetal growth retardation in pregnant animals (Van Geijn et aL, 1980; Robinson et al., 1983; Chang et al., 1984), we extended the exposure period beyond 2 days to determine whether the relationship between the maternal and fetal lungs is maintained when the growth of the maternal lung is enhanced while that of the fetal body is suppressed. We also tested the effects of 100~o 0 2 because, in contrast to hypoxia, it suppresses the growth of the lung in rats (Bartlett, 1970). The pregnant rats were exposed to low and high concentrations of 02 at different periods during pregnancy between gestation day 7 and 21, i.e. before and after the development of the respiratory system in the fetus which begins by the appearance of the first pharyngeal pouch at gestation day 10 and paired lung buds at day 11.5 (Altman and Dittmer, 1962).

Materials and methods

A total of 169 virgin female Sprague-Dawley rats (Charles River Breeding Laboratories, Quebec) were mated when their body weight was between 195 and 230 g. When in their proestrous phase they were placed with male rats for 4 h. The presence of spermatozoa in vaginal smear indicated that mating had occurred. Gestation day (GD) one was designated as commencing 24 h after successful mating. The rats were then kept singly in wire cages until gestation day 7, 11, 14 or 18 when they were transferred into an environmental chamber. The chamber was made of Plexiglas and had a volume of 98 L. A wire cage within the chamber had sufficient space to accommodate 12 pregnant rats. By closing or opening the cover of the chamber, the rats were exposed either to the gas within the chamber or to the outside air. The chamber gas was maintained at predetermined concentrations of 02 in N2. A continuous flow of gas from air, or 02 and N 2 cylinders washed out the CO2 and excess humidity from the chamber. The gas flow ranged between 5-7 L/min depending on the number of rats in the chamber. The chamber gas was continuously mixed by a noiseless fan, and passed over a temperature controlled copper coil. The inflow and the outflow gases were monitored by 02 and CO2 analyzers (Beckman). A temperature of 23 °C + 0.5 °C and a CO2 concentration of less than 0.2~o were maintained over the duration of the experiment.

MATERNAL-FETAL LUNG RELATIONSHIP

227

Pregnant rats were divided into 6 groups. (1) Two-day hypoxia: 19 rats were placed in environmental chamber for 2 days, 5 rats at gestation day (GD) 7; 5 rats at GD 11; 5 at GD 14 and 4 at GD 18. The concentration of 02 within the chamber was maintained at 11-12~o. (2) Two-day hyperoxia: 14 rats were exposed to 100~o 0 2 for 2 days beginning either at GD 7 (4 rats), GD 11 (5 rats) or GD 18 (5 rats). (3) Hypoxia 7 (Hpo7): 27 rats were placed in the environmental chamber at gestation day 7, for 10 h/day during lighting period, until the day of sacrifice (GD 21). The concentration of O: within the chamber was maintained at 10-11 ~o. (4) Hypoxia 14 (Hpo14): five rats were transferred from wire cages to the environmental chamber at gestation day 14. They were exposed to hypoxic gas 24 h/day, with a 15-20 min daily exposure to room air when food was changed and the cage cleaned. The 0 2 concentration of gas within the chamber was kept at 14~ on GD 14 and 15, at 13~o on GD 15, 12~o on GD 17 and thereafter at 11 ~o until the day of sacrifice (GD 21). (5) Hyperoxia 7 (HprT): 32 rats were exposed to 100~ 0 2 for 10 h/day starting at GD 7. (6) Control: 12 rats were transferred into the chamber on GD 7, exposed to 21.0~ 02 for 10 h/day. Since the data obtained from this group of 12 rats were similar to those kept in single cages (reported previously) (Faridy et al., 1988a) the results were combined (72 rats) to be used as control for all experimental conditions. The rats were allowed food (Purina rat chow) and water ad libitum. The body weights were measured at day zero, at least twice a week, and at the time of sacrifice. Food intake was measured during pregnancy. At gestation day 21 the rats were anesthetized with a subcutaneous injection of sodium pentobarbital (10 rag/100 g BW). Laparotomy was performed, and the fetuses were then harvested by hysterotomy. The umbilical cord was ligated and cut. Fetuses were dried with gauze, weighed and decapitated. The fetal lungs were excised, separated from the extrapulmonary airways and weighed. Lungs that appeared to contain blood were discarded. Determinations of DNA, dry weight, and electron microscopic studies were done on 5 individual lungs, randomly selected, from litter-mates. The rest of the lungs were pooled and used for determinations of DNA, total protein, total lung lipids, phospholipids (lecithin and disaturated phosphatidylcholine (DSPC)). In addition, liver, spleen and kidneys were removed from some fetuses of litter size of 9-14, weighed and the DNA measured on liver and kidneys. Placentae collected at the time of hysterotomy were dissected free of the umbilical cord, blotted on a paper towel and weighed. Five placentae from each mother, belonging to the fetuses whose lungs were analyzed for DNA content, were used for DNA determination. Pacentai dry weights were also collected. In some rats, the uterus and its contents were removed en bloc and subjected to microwave irradiation to inactivate the enzymes responsible for glycogenolysis. Lungs and liver were dissected from these irradiated fetuses and used for glycogen determination. Blood was also collected from some rats and fetuses (while the umbilical cord was intact) for measurement of blood hemoglobin content, hematocrit, and serum glucose. After completion of removal of fetuses and placentae, the rats were exsanguinated by cutting the abdominal aorta. A pneumothorax was produced by opening the diaphragm. The lungs were inspected under a magnifying glass. If the lungs were

228

E.E. FARIDY et al.

uniformly pink in color and showed no signs of possible infection, they were used for air pressure-volume and DNA measurements. Otherwise, the rat and its fetuses were excluded from the study. Liver, kidneys and heart were also removed, cleaned of extra tissues, blotted on a paper towel and weighed (wet and dry). The lungs were left intact in the chest. The trachea was cannulated and the lungs were degassed by placing the animal in a vacuum jar. The chest was then opened by bisecting the sternum. The ribs were separated by forceps. The lungs were again inspected under a magnifying glass. If signs of infection, such as grey spots, were noted the rat and its fetuses were excluded from the study. Otherwise, the large vessels were ligated at the base of the heart, and the cannulated degassed lungs were then attached to a pressure-volume apparatus similar to that described by Gribetz et al. (1959). The lungs were inflated with air to a pressure of 30 cm H20. This inflation pressure was maintained until the lungs were fully inflated and the air volume remained constant for 15 sec. The air volume observed at this transpulmonary pressure, considered as maximal lung air volume (MLV), was designated as 100~o and each volume subsequently observed after deflation to a predetermined transpulmonary pressure (20, 15, 10, 5 and 0 cm H20) was expressed as a percentage of MLV. These pressures were maintained for 20 sec before the volumes were read at each pressure. If, during the procedure, the lung air volume did not remain constant at high pressures, air leaks were assumed to be present, and such lungs were excluded from the pressure-volume study. All pressure-volume measurements were performed at room temperature. At the end of air pressure-volume measurements, the lungs were separated from the extrapulmonary airways, weighed and used to measure DNA and dry weight. Preliminary studies indicated that pressure-volume measurement (the procedure and the time lapse) had no influence on lung D N A content. Air deflationpressure-volume curve.

The tissue was placed on preweighed pieces of tinfoil and dried in an oven at 60 °C for one week.

Dry weight.

Samples of lung tissue were homogenized in 1.25 or 2.5 ml of normal saline in a Kontes all glass homogenizer. One-ml aliquots were used for the extraction and determination of lung deoxyribonucleic acid by the method of Schneider (1957). One-hundred #1 of the sample was diluted 1/20 in normal saline and 25 or 50 #1 of the dilution (in duplicate) were added to 1 N N a O H and digested foir 18-20 h. The total protein content was then determined by the method of Lowry et aL (1951). DNA and totalprotein.

Samples of lung tissue were homogenized in chloroform/methanol (2: 1) in a Kontes all glass homogenizer. The lipid extract was washed according to the method of Folch et al. (1957). The samples were dried in a waterbath at 47 °C under nitrogen and the dried extract reconstituted to 1 ml with chloroform/methanol (2: 1). An aliquot (200/A) of the lipid extract was dried on a hot plate to determine total lipids. Phospholipids.

MATERNAL-FETALLUNG RELATIONSHIP

229

A second aliquot (25/al) of the lipid extract was used to determine lipid phosphorus according to Brante's modification (1949) of the method of Fiske and Subbarow (1925). A third aliquot (50/zl) of the original lipid extract was plated on an activated silica gel-H plate and the lipid fractions separated using a solvent system containing chloroform/methanol/acetic acid/water (25:15:4:2) (Parker and Peterson, 1965). The plate was then exposed to iodine vapor. The phosphatidylcholine spot was identified and aspirated into test tubes for measurements of lipid phosphorus as described above. A fourth aliquot (75/A) of the original lipid extract was used to isolate the disaturated lecithin (DSPC) by osmium tetroxide method (Mason et al., 1976) and the lipid phosphorus determined.

Glycogen. The uterus with the fetuses en bloc was removed, placed in a microwave oven and irradiated for 10-15 sec. Lungs and liver were dissected from the fetuses and lyophilized overnight. Tissues were then weighed and homogenized in 0.05 M acetate buffer (pH 4.7) to extract the glycogen. Glycogen was measured using amylo-a-l,4-ct1,6-glucosidase according to the method of Passonneau and Lauderdale (1974) and tissue glycogen calculated using the equation: AA/6.22 x volume ofcuvette (ml) x [total extraction volume (ml)]/[volume of extract assayed (ml)] x 1/[tissue dry weight (mg)], where AA = change of absorbance at 340 nm and 6.22 = extinction coefficient of NADPH at 340 nm. Electron microscopy. Fetal left lungs were divided into upper, and lower portions. Each portion was cut into 6-8 blocks, and fixed for 2 h at 4 ° C in 3 ~o glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Tissues were rinsed for 24 h at 4 °C in 0.1 M phosphate buffer (pH 7.4) containing 0.2 M sucrose. Tissues were then post-fixed for 2 h at 4 ° C in 2 ~ osmium tetroxide in 0.1 M phosphate buffer (pH 7.4). After rapid dehydration in ascending concentrations of ethanol, the tissues were embeded in Epon 812. Thin sections were made from 4 blocks of each portion (3 grids per block), stained with uranyl acetate and lead citrate. From a total of 24 grids prepared from each lung, 6 grids were randomly selected, viewed and photographed in a Philips EM 201 electron microscope. In order to eliminate observer bias, tissues were examined using coded grids without foreknowledge of their source. Thirty-six random exposures were taken from each grid. Four fetal lungs were studied from each experimental condition. Electron micrographs of lungs (magnification x 14 300) were used to estimate the ratio of Type II cells to total number of lung cells. Only cells containing a nucleus were counted. Cells were considered Type II pneumocytes if they contained lamellar inclusion bodies. Cells such as neutrophils, found in capillaries, were not included in the cell count. Micrographs from each lung were counted 3 times and the average used as the cell count. Lamellar bodies were also counted in Type II cells and the average number of observed lameUar bodies per Type II cell was calculated. Statistics. Statistical analysis of the data was carried out using a t-test of paired or unpaired variates, a multiple range test for analysis of variance by Duncan's method,

230

E.E. FARIDY et al.

and a simple or a multiple linear regression analysis, where applicable. The slope of the regression line was tested to determine if it was significantly different from zero. P values for slope (Ps) are given in the text. When comparing different experimental conditions, only the data from rats with litter size of 9-14 were used to reduce litter size dependent variations. When assessing relationships between maternal, placental and fetal measurements in a given experimental condition, data from all pregnancies (1-18 litter size) were analyzed. Fetuses were considered of equal body weights when the difference in BW did not exceed 50 mg. When fetuses of equal body weights from different conditions were compared and more than one fetus, from a condition, were of the same weight, their average was taken for such comparisons.

Results

Since maternal hypoxia and hyperoxia of short duration (2 days), starting at GD 7, 11, 14 or 18, had no significant effects on either maternal lung weight, DNA content and air volume, or litter size and incidence of fetal resorption, or fetal lung weight, DNA and phospholipid contents, only the results of Hpo7, HpOl4 and Hpr 7 rats are given below in detail. The data on relationships between the three gas exchange organs (maternal and fetal lungs and placenta) in normal pregnancy (control pregnant rats) which will be referred to for comparison were discussed throroughly in a separate publication (Faridy et aL, 1988a). The data on control pregnant rats with 9-14 litter size are given in tables to facilitate comparisons. Rats tolerated the low and high concentrations of 0 2 , but appeared to be less active when in a hypoxic environment. The daily food intake was not affected with intermittent hypoxia or hyperoxia but was decreased by about 5-10 ~o with continuous hypoxia. The body weights of pregnant rats exposed to room air, hypoxia (GD 7, GD 14) and hyperoxia (GD 7) are shown in table 1. Only Hpo 7 rats had lower body weights at GD 21. Neither hypoxia nor hyperoxia affected the litter size. The average number of fetuses per pregnant rat and the percentage of occurrence of 9-14 litter size were as follows: for hypoxia, 11.50 + 2.68 SD, N = 32, and 78.1~o; for hyperoxia, 11.88 + 3.60 SD, N = 32, and 75.0~o; and for 186 control pregnants rats sacrificed during that same period, 11.28 + 3.70 SD, and 69.9~o, respectively. The incidence of fetal resorption did not significantly increase with continuous hypoxia for one week (1.3 ~o) or with intermittent hyperoxia for two weeks (1.9 ~o) but significantly increased with intermittent hypoxia for two weeks (9.1 ~/o), in comparison to controls (0.96~). Lung weight, DNA content and lung air volume (MLV) for the above rats are shown in table 2. Intermittent hyperoxia had no significant effect on maternal lungs while

Mother.

MATERNAL-FETAL LUNG RELATIONSHIP

231

TABLE 1 Measurements in pregnant rats (litter size of 9-14). BWo and BW2~ = body weight at gestation day 0 and 21, respectively. BW _fp = body weight at GD 21 excluding fetus and placenta weights. Data are expressed as mean + 1 SE; numbers in parentheses indicate the number of animals studied. Different from control: *P < 0.001; *P < 0.01; *P < 0.02; gP < 0.05. Different from Hypoxia 7: ap < 0.001; bp < 0.01. Control

Hypoxia 7

Hypoxia 14

Hyperoxia 7

BWo (g)

207.4 + 1.9 (42)

209.8 _+2.9 (18)

203.2 + 4.1 (5)

209.0 _+2.7 (24)

BW21/BWo × 100

161.5 _+ 1.3 (41)

148.9 + 1.8' (18)

165.9 + 3.7a (5)

158.7 + 2.1b (24)

BW~fp/BWo × 100

134.2 =l=1.2 (32)

128.4 + 1.9' (11)

140.4 + 4.2 b (5)

134.3 + 2.8 (11)

Liver wt (g)

10.87 + 0.26 (20)

12.27 + 0.53 § (5)

Liver wt (g)/ 100 g BWo

5.37 _+0.12 (20)

6.05 __ 0.27* (5)

Liver wt (g)/ 100 g BW fp

4.01 _+0.06 (19)

4.31 _+0.13 s (5)

Kidney wt (g)

1.57 _+0.03 (20)

1.85 _+0.06 t (5)

Kidney wt (g)/ 100 g BWo

0.773 + 0.015 (20)

0.910 + 0.032* (5)

Kidney wt (g)/ 100 g BW_rp

0.578 + 0.009 (19)

0.648 + 0.014* (5)

Heart weight (g)

0.710 +_0.015 (20)

0.854 _+0.031" (5)

Heart wt (g)/ 100 g BWo

0.349 + 0.005 (20)

0.420 + 0.012" (5)

Heart wt (g)/ 100 g BW_fp

0.262 + 0.003 (19)

0.299 + 0.007* (5)

i n t e r m i t t e n t h y p o x i a for t w o w e e k s c a u s e d an i n c r e a s e in m a t e r n a l lung w e i g h t a n d lung air v o l u m e . C o n t i n u o u s h y p o x i a for o n e w e e k , h o w e v e r , h a d greater influence, in t h a t the lung weight, D N A c o n t e n t a n d M L V w e r e significantly i n c r e a s e d . N o differences w e r e o b s e r v e d in t h e d e f l a t i o n p r e s s u r e - v o l u m e c u r v e a m o n g t h e f o u r g r o u p s o f rats in this study. O r g a n s o t h e r t h a n lungs s u c h as liver, k i d n e y a n d h e a r t (table 1) w e r e also significantly larger t h a n c o n t r o l s in Hpo14 rats. D a t a o n t h e s e o r g a n s are n o t available for Hpo7 a n d H p r 7 rats. Blood hemoglobin content and hematocrit were c o m p a r e d to c o n t r o l s (table 3).

significantly h i g h e r

in H p o 7 rats

232

E.E. FARIDY etal. TABLE 2

Lung measurements of pregnant rats (litter size of 9-14). Hypoxia 7 and hyperoxia 7 = pregnant rats exposed to intermittent (10h/day) hypoxia or hyperoxia, respectively, starting at gestation day7. Hypoxia 14 = pregnant rats exposed to continuous hypoxia as of gestation day 14. BW0 = body weight at gestation day zero. BW fv = body weight at gestation day 21 excluding fetus and placenta weights. MLV = lung air volume at 30 cm H20 inflation pressure. Data are expressed as mean _+ 1 SE; numbers in parentheses indicate the number of animals studied. Different from controls: * P < 0.001; *P < 0.01; *P < 0.02; 3p < 0.05. Different from hypoxia 7: ~P < 0.001; bp < 0.01; cp < 0.05. Control

Hypoxia 7

Hypoxia 14

Hyperoxia 7

Lung wt (g)

0.942 + 0.013 (29)

1.084 + 0.028* (14)

1.302 _+0.057 *'a (5)

0.994 (21)

Lung wt (g)/ 100 g BWo

0.456 _+0.007 (29)

0.514 _+0.011" (14)

0.642 _+ 0.032 *'a (5)

0.474 _3-0.007 b (21)

Lung wt (g)/ 100gBW fp

0.344 + 0.005 (27)

0.399 _+0.009* (11)

0.456 + 0.011 *'b (5)

0.360 _+ 0.0053,~ (11)

Lung DNA (mg)

7.76 + 0.34 (12)

8.07 + 0.29 (9)

8.99 + 0.393 (5)

7.62 _+0.21 (14)

Lung DNA (mg)/ g lung

8.27 + 0.26 (12)

7.47 _+ 0.233 (9)

6.92 _+ 0.20* (5)

7.75 + 0.17 (14)

Lung DNA (mg)/ 100 g BWo

3.74 + 0.13 (12)

3.78 _+0.14 (9)

4.44 + 0.26* (5)

3.68 _+ 0.11 (14)

Lung DNA (mg)/ 100 g BW_fp

2.82 _+0.08 (12)

2.97 + 0.10 (9)

3.16 _+ 0.123 (5)

2.77 + 0.06 (11)

MLV (ml)

13.27 _+ 0.27 (25)

14.46 + 0.32* (14)

15.52 + 0.80* (5)

13.55 + 0.29 ~ (17)

MLV (ml)/ g lung

14.17 + 0.26 (24)

13.39 + 0.33 (14)

11.94 + 0.48 *'c (5)

13.74 + 0.26 (17)

MLV (ml)/ 100 g BWo

6.47 + 0.13 (25)

6.87 _+0.20 (14)

7.66 + 0.44* (5)

6.49 + 0.17 (17)

MLV (ml)/ 100 g BW rp

4.81 _+ 0.10 (24)

5.26 _+0.16' (11)

5.44 _+0.21' (5)

4.98 + 0.15 (10)

+ 0.016 *'b

Placenta. A s s h o w n in table 4, H p o 7 a n d H p r 7 rats h a d s m a l l e r p l a c e n t a l weight a n d / o r p l a c e n t a D N A c o n t e n t t h a n c o n t r o l s . N o r e d u c t i o n in the p l a c e n t a size w a s n o t e d in HpOl4 rats. P l a c e n t a l w e i g h t a n d D N A c o n t e n t e x p r e s s e d p e r b o d y w e i g h t w a s larger t h a n c o n t r o l s in h y p o x i c rats a n d smaller t h a n c o n t r o l s in h y p e r o x i c rats m a i n l y d u e to l o w a n d high fetal b o d y weights, respectively. C o m p a r i s o n s o f p l a c e n t a D N A c o n t e n t a m o n g fetuses o f e q u a l ( c o m p a r a b l e ) b o d y weights r e v e a l e d t h a t H p o 7 p l a c e n t a D N A c o n t e n t w a s significantly l o w e r t h a n b o t h H p O l 4 a n d c o n t r o l s (fig. 1). T h e b o d y w e i g h t o f the 8 pairs o f fetuses in fig. 1 r a n g e d f r o m 3.16 to 4.27 g, a n d the difference b e t w e e n e a c h equal pair f r o m 0.023 to 0.001 g. Similar c o m p a r i s o n s i n d i c a t e d t h a t H p r 7

233

MATERNAL-FETAL LUNG RELATIONSHIP TABLE 3

Glycogen, glucose, hemoglobin and hematocrit at gestation day 21. Data are expressed as mean + 1 SE; numbers in parentheses indicate the number of rats studied. Different from controls: *P < 0.001; t p < 0.05. Different from Hypoxia 7: ~P < 0.001; bp < 0.02. Control

Hypoxia 7

Hyperoxia 7

Fetus: Lung glycogen (mg/g dry wt)

86.5 + 4.67 (10)

92.8 _+8.97 (8)

77.1 + 4.88 (9)

Liver glycogen (mg/g dry wt)

267.1 + 8.3 (10)

162.3 + 9.9* (8)

231.3 + 12.41 *'a (9)

Serum glucose (mg/100 ml)

39.2 + 2.73 (8)

25.3 + 2.04* (12)

54.7 + 1.80*'a (7)

Blood hemoglobin (g/100 ml)

10.01 + 0.28 (21)

9.47 + 0.27 (13)

10.42 + 0.25 b (12)

Hematocrit Mother: Blood hemoglobin (g/100 ml) Hematocrit

37.8 _+0.61

37.5 + 0.74

37.7 _+0.72

(24)

(27)

(14)

11.85 + 0.74 (5)

16.35 + 0.51 * (8)

11.33 + 0.65 (4)

37.9 + 1.03 (5)

51.7 +_0.92* (7)

37.1 + 0.75 (4)

1.2

1.1

E

v < Z Q

1.0

¢0 Q 0

0.9

0.

0.8

0.7 Control

Hypoxia 7

Hypoxia 14

Fig. 1. Comparison of placenta DNA content of fetuses of equal body weight from control, hypoxia 7 and hypoxia 14 rats. Each point is the average of 1-9 (control), 1-6 (hypoxia 7) or 1-3 (hypoxia 14) fetuses of equal body weights. Lines connect the fetuses of equal body weight. Hypoxia 7 placenta DNA is significantly lower than both control and hypoxia 14 (paired t-test: P < 0.01 and P < 0.01, respectively; unpaired t-test: P < 0.01 and P < 0.001, respectively). Hypoxia 14 is not different from control.

234

E.E. F A R I D Y et al. TABLE 4

Placenta measurements (litter size of 9-14). Hypoxia 7 and byperpoxia 7 = placentae obtained from pregnant rats exposed to intermittent hypoxia, or hyperoxia, respectively, beginning on gestation day 7. Hypoxia 14 = placentae obtained from pregnant rats exposed to continuous hypoxia as of gestation day 14. Data are expressed as mean _+ 1 SE; numbers in parentheses indicate the number of placentae studied. Different from control: * P < 0 . 0 0 1 ; t P < 0 . 0 1 ; * P < 0 . 0 5 . Different from hypoxia 7: a p < 0.001; b p < 0.01. Control

Hypoxia 7

Hypoxia 14

Hyperoxia 7

Fetal body wt (g)

4.09 + 0.017 (406)

3.14 _+ 0.037* (136)

3.86 + 0.046 *'a (60)

4.19 + 0.028 t'a (175)

Placenta wt (mg)

452.4 + 3.29 (385)

403.8 + 6.42* (] 38)

460.4 + 7.57 a (60)

423.6 ± 6.35* (161)

Placenta wt (mg)/ g body wt

111.1 + 0.83 (373)

129.9 + 2.25* (136)

120.0 + 2.12 *'b (60)

101.1 ± 1.28 *'a (161)

Placenta dry wt (mg)

73.87 _+ 2.57 (21)

66.5 + 2.21 ~ (26)

-

69.12 ± 1.99 (50)

Placenta dry wt/ wet wt × 100

16.84 + 0.12 (21)

17.18 + 0.16 (26)

-

16.74 ± 0.08 b (50)

Placenta D N A (mg)

0.947 _+ 0.02 (35)

0.852 + 0.02 t (40)

0.983 + 0.02 ~ (17)

0.898 ± 0.01 (34)

Placenta D N A (mg)/ g tissue

1.97 _+ 0.05 (35)

2.05 _+ 0.05 (40)

2.15 + 0.05* (17)

2.02 + 0.04 (34)

Placenta D N A (mg)/ g body wt

0.239 ± 0.007 (35)

0.269 ± 0.007 ~: (40)

0.253 ± 0.006 (17)

0.207 ± 0.004 *'a (34)

1.4

1.4

1.3

1.3

1.2 E ,< 1.1 z o 1.0

~

1.2

aZ

1.1

c

_~

1.o

0

E

0.9

0.9

0.8

0.8

0.7

,

Control

,

Hpr 7

0.7

,

Control

,

Hpr 7

Fig. 2. Comparisons of placenta and fetal lung D N A contents of fetuses of equal body weight from control and hyperoxia 7 (HprT) rats. Each point is the average of 1-9 (control) and 1-6 (hyperoxia 7) fetuses of equal body weights. Hyperoxia 7 placenta and fetal lung D N A contents are significantly lower than controls (paired t-test: P < 0.001 and P < 0.001, respectively; unpaired t-test: P < 0.01 and P < 0.001, respectively).

MATERNAL-FETALLUNG RELATIONSHIP

235

placenta DNA content was not different from Hpo7, but significantly lower than controls (fig. 2). The body weight of the 22 pairs of fetuses in fig. 2 ranged from 3.26 to 5.18 g, and the difference between each equal pair from 0.036 to 0.002 g. Hyperoxia did not alter the relationship between placenta and fetal body weight, in that, similar to controls (Faridy et al., 1988a) placental weight and DNA content increased with an increase in fetal body weight (n --- 69, r = 0.392, Ps < 0.001; and r = 0.289, Ps < 0.02, for placental weight and DNA content, respectively). This relationship, however, was distorted by hypoxia, either with continuous or with intermittent exposure. The only significant relationship observed was between placenta DNA content and fetal body weight in Hpo 7 (n = 61, r = 0.335, P~ < 0.01). Similar to controls (Faridy et al., 1988a), placenta DNA content of both hypoxic and hyperoxic rats increased with increase in placental weight (Hpo7: n = 61, r = 0.691, Ps < 0.001; Hpol4: n = 17, r = 0.504, P~ < 0.05; and HprT: n = 69, r = 0.703, P~ < 0.001). As in controls (Faridy etal., 1988a) no significant relationship was found between maternal lung DNA, (expressed per lung, or per body weight at gestation day zero, i.e. BWo) and placenta DNA content (expressed per placenta, or per fetal body weight, both averaged for a litter), in either hypoxic or hyperoxic rats. The relationship between fetal lung DNA and placenta DNA was analyzed in two ways: (1) average fetal lung DNA vs average placenta DNA of a litter; and (2) individual fetal lung DNA vs individual placenta DNA. The two methods of analysis did not reveal a relationship between fetal lung DNA and placenta DNA in either hypoxic, hyperoxic or control (Faridy et al., 1988a) rats. Fetal body weight of controls, Hpo7, Hpox4 and Hpr 7 rats ranged between 2.69-5.28 g, 1.65-4.25 g, 3.15-4.53 g and 2.90-5.19 g, respectively. Table 4 shows that hypoxic fetuses are smaller than controls ( P < 0.001) (Hpo 7 the smallest) while hyperoxic fetuses are the largest (P < 0.01) of the four groups. Blood hemoglobin content and hematocrit (table 3) were similar in control, Hpo 7 and Hpr 7 fetuses. The serum glucose level was the lowest in Hpo7 and the highest in Hpr7 (table 3). Although table 5 indicates that the fetal lung weight and DNA content are lower than controls in Hpo 7 and Hpr 7 fetuses, since these values are a function of fetal body weight (Faridy e t a / . , 1988a), a more reliable comparison should be made in fetuses of equal body weights as seen in fig. 3 (the same fetuses as in fig. 1). This figure shows that the DNA content of Hpo 7 fetal lungs is less than both Hpo~4 and control lungs while the latter two are not different from each other. A similar analysis for Hpr 7 fetuses revealed no significant difference between Hpr7 and Hpo 7 fetal lung DNA content but a significant (P < 0.001) reduction in DNA content of Hpr 7 compared to controls (fig. 2). Analysis of maternal and fetal lung DNA content in hypoxic and hyperoxic rats showed that the normal direct relationship which exists between the maternal and fetal lung DNA content in control rats (Faridy et al., 1988a) is distorted by hypoxia and hyperoxia. Such a relationship could not be found in Hpo 7 and Hpr 7 rats and was rather poor for Hpol4. The regression coefficient for Hpol4 maternal lung DNA per BW o vs fetal lung DNA per body weight was 0.907 (n = 5, Ps < 0.05). Fetus.

236

E.E. FARIDY et al. TABLE 5

Fetal lung measurements (litter size of 9-14). Hypoxia 7 and hyperoxia 7 = fetuses obtained from pregnant rats exposed to intermittent hypoxia and hyperoxia, respectively, starting gestation day 7. Hypoxia 14 = fetuses obtained from pregnant rats exposed to continuous hypoxia starting at gestation day 14. DSPC = disaturated phosphatidyl choline. Data are expressed as mean ± 1 SE; numbers in parentheses indicate the number of fetuses studied. Different from control: *P < 0.001; t p < 0.01; *P < 0.02; ~P < 0.05. Different from hypoxia 7: ap < 0.001; bp < 0.01. Control

Hypoxia 7

Hypoxia 14

Hyperoxia 7

Lung wt (mg)

123.0 _+ 1.13 (190)

86.6 ± 1.30" (112)

111.6 + 1.32*'a (56)

118.6 ± 1.01*'a (161)

Lung wt (mg)/ g body wt

29.7 ± 0.20 (190)

27.2 ± 0.33* (112)

29.1 ± 0.40 a (56)

28.2 ± 0.18 *'b (161)

Lung dry wt (mg)

18.15 ± 0.52 (21)

10.59 ± 0.32* (24)

-

16.88 ± 0.38 §'a (33)

Lung dry wt (mg)/ g body wt

4.23 ± 0.10 (21)

3.44 ± 0.12" (24)

-

3.96 + 0.06 *'a (33)

Lung DNA (mg)

1.098 ± 0.018 (57)

0.822 ± 0.014' (47)

1.033 + 0.018 a (22)

1.008 + 0.018 *'a (42)

Lung DNA (mg)/ g lung

9.18 ± 0.14 (57)

9.44 ± 0.11 (47)

9.25 ± 0.07 (22)

8.23 ± 0.09 *'~ (42)

Lung DNA (mg)/ g body wt

0.269 ± 0.004 (57)

0.260 ± 0.004 (47)

0.267 ± 0.006 (22)

0.230 ± 0.003 *,a (42)

Lung total protein (mg)/mg lung DNA

7.79 + 0.29 (15)

7.98 ± 0.17 (16)

9.20 ± 0.11 *'a (21)

9.50 ± 0.27 *'~ (12)

Lung total lipids (mg)/mg lung DNA

2.40 ± 0.09 (5)

2.00 + 0.03 t (5)

1.90 ± 0.05* (5)

2.16 ± 0.03 §'b (5)

Lung phospholipids (mg)/mg lung DNA: Total

1.64 ± 0.07 (5)

1.45 + 0.06 (5)

1.52 + 0.04 (5)

1.81 + 0.05 b (5)

Lecithin

0.821 ± 0.04 (5)

0.708 ± 0.03 (5)

0.730 ± 0.02 (5)

0.895 ± 0.02 a (5)

DSPC

0.413 ± 0.02 (5)

0.348 ± 0.01' (5)

0.364 ± 0.02 (5)

0.443 ± 0.01 a (5)

L u n g t o t a l p r o t e i n , e x p r e s s e d p e r D N A , w a s i n c r e a s e d in H p o l 4 a n d H p r 7 f e t u s e s (P < 0.001) compared to Hpo 7 and controls, while lung total lipids were significantly decreased

( P < 0 . 0 0 1 - P < 0 . 0 5 ) in b o t h h y p o x i c a n d h y p e r o x i c f e t u s e s . A l t h o u g h

h y p o x i a a p p e a r e d t o h a v e r e d u c e d fetal l u n g p h o s p h o l i p i d s , a s i g n i f i c a n t r e d u c t i o n ( P < 0 . 0 2 ) w a s o n l y o b s e r v e d in D S P C in H p o 7 f e t u s e s ( t a b l e 5). H y p e r o x i a in s p i t e o f r e d u c i n g fetal l u n g size h a d n o effect o n fetal l u n g p h o s p h o l i p i d s (total, l e c i t h i n a n d DSPC). HpOT.

Therefore, the values were significantly (P < 0.001-P < 0.01) higher than

MATERNAL-FETAL LUNG RELATIONSHIP

237

1.2 1.1 z~ a

1.0

~'

o.9

,7 0.8

0.7

Control

Hypoxia 7

Hypoxia 14

Fig. 3. Fetal lung DNA compared in the same manner as that for placenta DNA in fig. 1. Each point is the average of 1-9 (control), 1-6 (hypoxia 7), or 1-3 (hypoxia 14) fetuses of equal body weights. Lines connect the fetuses of equal body weight. Hypoxia 7 fetal lung DNA is significantly lower than control (P < 0.001) and hypoxia 14 (P < 0.01), with either paired or unpaired t-test. Hypoxia 14 is not different from control.

Electron micrographs did not reveal any visible structural changes in fetal lungs of hypoxic and hyperoxic rats compared to controls. Furthermore the percentage of Type II cells and the number of lamellar inclusion bodies per Type II cell were similar in all. The weight and DNA content of liver and kidney and the weight of spleen of fetuses of HpOl4 and Hpr 7 were compared with controls in fetuses of equal body weights. Paired t-test revealed a significant (P < 0.05) reduction of 24.5~ and 17.8~ in the liver DNA content of Hpol4 and Hpr 7 rats, respectively. Because of the generally lower weights ofHpo 7 fetuses, data on liver, kidneys and spleen from Hpo7 fetuses with body weights equal to those of control, Hpo~4 and Hpr 7 were not available for comparison. Table 3 indicates that neither hypoxia nor hyperoxia had a profound effect on glycogen content of the fetal lungs, but caused a significant reduction (P < 0.001-P < 0.05) in liver glycogen.

Discussion

The outline of results for hypoxia and hyperoxia of 1 or 2 weeks are as follows: (1) maternal hypoxia causes growth stimulation in maternal lung, liver, kidney and heart, and growth retardation in the fetus; (2) it abolishes the direct relationships which exist in normal pregnancy between placental weight or DNA content and fetal body weight, and between maternal and fetal lung DNA content; (3) both hypoxia and hyperoxia, applied at early pregnancy, suppress the placental and fetal lung growth causing small

238

E.E. FARIDY et al.

for body weight placenta and lung; and (4) neither maternal hypoxia nor hyperoxia influences the maturation of the fetal lung. We were faced with 3 immediate problems when designing the modes of application of low and high concentrations of O 2 to the pregnant rat, namely fetal death and resorption, reduction in food and water intake, and maternal pulmonary injury. Fetal mortality rate increases as the concentration of environmental 0 2 decreases, such that the rate is nearly 100~o after 24 h of maternal exposure to 02 concentrations below 9~o. Fetuses do survive O 2 concentrations between 9 and 10~o. However, the mortality rate is quite high (61 ~o) with continuous maternal exposure to 10 ~o 02 beginning at gestation day 10 (Van Geijn et al., 1980). Furthermore food and water intake is notably reduced in a hypoxic environment which may cause reduction in fetal lung weight and cell number in proportion to body weight (Faridy, 1975). Therefore intermittent hypoxia was chosen to minimize fetal death and to maintain normal food intake. This regimen of intermittent hypoxia appears to have been effective as evidenced by increased maternal lung size, elevated maternal blood hemoglobin and hematocrit, and small body weight for age fetuses (Van Geijn et al., 1980). We also managed to minimize fetal death rate with continuous hypoxia at late gestation by gradually reducing the level of hypoxia from 14~o at GD 14 to 11 ~o at GD 17. In spite of these measures Hpol4 rats consumed about 5-10~o less food than the controls, which is not severe enough to jeopardize the fetal growth at late gestation (Faridy, 1975). We were not confident to expose the rats to continuous hyperoxia because of pulmonary injury which occurs even with 60~o O2 for 7 days (Hayatdavoudi etal., 1981). With intermittent hyperoxia lung injury was prevented and normal food intake was maintained in the pregnant rat. We preselected 9-14 litter size because from experience in our laboratory we were aware that the frequency of 9-14 litter size pregnancy in control rats was about 70~o. Since neither hypoxia nor hyperoxia was subsequently found to have an effect on litter size, we felt comparisons of results of the different experimental conditions were justified. We believe, therefore, that the preselection of litter size range should not have altered the outcome of our results and the conclusions arrived at on the basis of these results. Blood gas tensions were not measured in the fetus during episodes of maternal hypoxia and hyperoxia; however, there are sufficient data in the literature to indicate that maternal hypoxia results in a comparable fetal hypoxia (from about 20 mm Hg Po2 to about 6-7 mm Hg) despite an increased placental diffusing capacity of about 63 ~o, as shown in guinea pigs (Gilbert et al., 1979). Maternal hyperoxia, conversely causes only a small rise in fetal Po2 (Towell et al., 1984). These investigators have shown a rapid increase of tissue Po2 by 46~o within 7 min of exposure of pregnant animal to 100~o 02 , followed by a more gradual rise to a peak of over 50~o at 12 min. However, continued administration of oxygen beyond 15 min did not lead to any further increase in fetal tissue Po2- The mechanisms triggered by maternal hyperoxia which limit the supply of 02 to fetal tissues are not fully investigated. It is well documented that in the pregnant animal, hypoxia causes fetal growth retardation (Van Geijn et al., 1980; Robinson et al., 1983 ; Chang et al., 1984). Because

MATERNAL-FETAL LUNG RELATIONSHIP

239

partial placentectomy results in small fetus (Robinson et al., 1979) one may conceive that the low fetal body weights in hypoxic rats may be the consequence of small placentae incapable of supplying sufficient nutrients to the fetus. Although this can not be ruled out, it appears unlikely since in control rats the placentae vary in size (weight and DNA content) by 10-20 ~o without having any effect on fetal body weight; in Hpo14 rats the placenta size is larger than that ofcontrols while the fetal body weight is smaller; and conversely in Hprv rats the placenta size is smaller while the fetus is larger. Furthermore the analysis of placenta in relation to concentration of 0 2 in the environment of fetuses of equal body weight (figs. 1 and 2) reveals that small placentae are capable of providing adequate supply of nutrients to the fetus. The reduction in DNA content of placenta and fetal organs in response to maternal hypoxia may result from the direct effect of hypoxia which causes inhibition of cell multiplication. Whether fetal organs differ in their response to these effects of hypoxia and whether this response is more pronounced in fetal lungs is not clear. In addition to tissue hypoxia, the distribution of fetal circulation is also altered with maternal hypoxia such that certain organs namely brain and heart receive a greater proportion of blood flow, in order to maintain as high a tissue Po2 as possible, while the lungs receive a smaller proportion (Behrman et al., 1970). Peeters et al. (1979) have clearly shown that fetal pulmonary blood flow decreases with a decrease in fetal fight ventricle 02 content. These measurements were all made in ovine fetus at late gestation. It is most likely that in some of the present experiments with hypoxia the embryo and the fetus were subjected to hypoxia and the fetal lung in particular to both hypoxia and perhaps malnutrition as a consequence of reduced pulmonary blood flow. One has to bear in mind that the lung of fetal rat is highly vascularized by day 15.5 of gestation (Altman and Dittmer, 1962). Although both Hpo7 and HpOl4 fetuses experienced hypoxemia and low pulmonary blood flow, only Hpo7 fetuses had reduced lung DNA content. There is no clear cut explanation for these discrepancies. Some of the possibilities to consider are the following. Firstly the mode of application and the duration of hypoxia were different but more importantly the level of hypoxia was less severe in Hpo14 rats. Secondly it is possible that the embryonic cells in general and the precursors of respiratory system in particular are more susceptible to the adverse effects of hypoxia than the fetal cells. The earlier the inhibition of cell division in the lung buds the smaller the lungs at late gestation (GD 21). Thirdly, hypoxia at early gestation may cause a delay in the appearance of lung buds resulting in a discrepancy between the fetal lung age and the fetal age, hence small lung for body weight. Although lung DSPC content of Hpo 7 fetus is less than controls, which may suggest that the lung age is not comparable with that of the fetus, neither the lung glycogen content nor the percentage of Type II cells and the number of lamenar bodies per Type II cell, which are similar to controls, support this hypothesis. Of interest is the similarity between the effects of maternal hyperoxia and hypoxia on fetal lung DNA content. Oxygen toxicity which is shown to retard lung growth in the newborn mouse by inhibiting DNA synthesis and cell replication (Northway et al., 1972), may be ruled out as a cause for reduction in fetal lung DNA content since fetal

240

E.E. FARIDY et al.

tissue Po2 does not reach the toxic levels (Towell et al., 1984). A possible explanation for reduced lung size in H p r 7 fetus is the impedance in the flow of nutrients to the fetus as a consequence of constriction of uterine arteries in response to high Po2. Maternal malnutrition (protein and calorie) not only gives rise to a smaller (hypocellular) lung in the fetus (Faridy, 1975), but causes a symmetrical reduction in organ growth. In contrast, interference with uterine artery blood flow consistently demonstrates asymmetric effects on organ growth (Lafeber et al., 1984). Apparently the fetal lung and liver, in these circumstances, receive a smaller proportion of the nutrient supply and therefore suffer growth retardation. Although the H p r 7 fetus is larger than control, it has lower liver glycogen and D N A contents. In large litter size pregnancy and in pneumonectomized rats there is a direct relationship between the maternal and fetal lung D N A content, i.e. the larger the maternal lung, the larger the fetal lung. In both situations the maternal lung undergoes a growth change. Such a relationship is not observed in hypoxic rats albeit there is a compensatory growth in maternal lung. This should not be taken as a contradiction to our previous notion that the maternal lung influences the fetal lung growth. It should be noted that hypoxia has opposing effects on maternal and fetal lungs. It enhances the growth of the former, while suppressing that of the latter. The varying effects of hypoxia, therefore, can distort the normal relationship between the two organs, and mask the influence of the maternal lung on fetal lung growth. Perhaps the experiments in which the fetus is subjected to hypoxia but not the pregnant rat may provide an answer. It is feasible to produce hypoxemia in the fetus by surgical procedures such as ligation of uterine artery or partial placentectomy. These, however, are not appropriate measures since they severely jeopardize the nutritional status of the fetus (Robinson et al., 1979).

Acknowledgements. The excellent technical assistance of Mrs. Saniata Bucher and Mrs. Donna Love is greatly appreciated. This study was supported by a grant from the Medical Research Council of Canada.

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Faridy, E.E. (1975). Effect of maternal malnutrition on surface activity of fetal lungs in rats. J. Appl. Physiol. 39: 535-540. Faridy, E.E., S. Bucher and M.R. Sanii (1988a). Relationship between maternal and fetal lung growth. Respir. Physiol. 72: 171-186. Faridy, E.E., M.R. Sanii and J.A. Thliveris (1988b). Influence of maternal pneumonectomy on fetal lung growth. Respir. Physiol. 72: 195-210. Fiske, C.H. and Y. Subbarow (1925). The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400. Folch, J., M. Lees and G.H. Sloane-Stanley (1957). A simple method for the isolation and purification of total lipids from animal tissue. J. Biol, Chem. 226: 497-509. Gilbert, R. D., L. A. Cummings, M.R. Juchau and L.D. Longo (1979). Placental diffusing capacity and fetal development in exercising or hypoxic guinea pigs. J. Appl. Physiol. 46: 828-834. Gribetz, I., N.R. Frank and M. E. Avery (1959). Static volume-pressure relations of excised lungs of infants with hyaline membrane disease, newborn and stillborn infants. J. Clin. Invest. 38: 2168-2175. Hayatdavoudi, G., J.J. O'Neil, B.E. Barry, B.A. Freeman and J.D. Crapo (1981). Pulmonary injury in rats following continuous exposure to 60% 0 2 for 7 days. J. Appl. Physiol. 51: 1220-1231. Lafeber, H.N., T. P. Rolph and C.T. Jones (1984). Studies on the growth of the fetal guinea pig. The effects of ligation of the uterine artery on organ growth and development. J. Dev. Physiol. 6: 441-459. Lowry, O. H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951). Protein measurements with the folin phenol reagent. J. Biol. Chem. 193: 265-275. Mason, J. R., J. Nellenbogen and J.A. Clements (1976). Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17: 281-284. Nattie, E. E., C.W. Wiley and D. Bartlett, Jr. (1974). Adaptive growth of the lung following pneumonectomy in rats. J. Appl. Physiol. 37: 491-495. Northway, W. H., Jr., R. Petriceks and L. Shahinian (1972). Quantitative aspects of oxygen toxicity in the newborn: inhibition of lung DNA synthesis in the mouse. Pediatrics 50: 67-72. Parker, F. and N. F. Peterson (1965). Quantitative analysis of phospholipids and phospholipids fatty acids from silica gel thin-layer chromatograms. J. Lipid Res. 6: 455-460. Passonneau, J.V. and V.R. Lauderdale (1974). A comparison of three methods of glycogen measurement in tissues. Anal. Biochem. 60: 405-412. Peeters, L.L., R.E. Sheldon, M.D. Jones, Jr., E.L. Makowski and G. Meschia (1979). Blood flow to fetal organs as a function of arterial oxygen content. Am. J. Obstet. Gynec. 135: 637-646. Robinson, J.S., E.J. Kingston, C.T. Jones and G.D. Thornburn (1979). Studies on experimental growth retardation in sheep. The effects of removal of endometrial caruncle on fetal size and metabolism. J. Dev. Physiol. 1: 379-398. Robinson, J. S., C.T. Jones and E.J. Kingston (1983). Studies on experimental growth retardation in sheep. The effects of maternal hypoxaemia. J. Dev. Physiol. 5: 89-100. Schneider, W.C. (1957). Determination of nucleic acids in tissue by pentose analysis. Methods Enzymol. 3: 680-684. Towell, M.E., J. Johnson, K. Smedstad, M. Andrew and T.-L Vu (1984). Fetal blood and tissue Po2 during maternal oxygen breathing. J. Dev. Physiol. 6: 177-185. Van Geijn, H.P., W.M. Kaylor, K.R. Nicola and F.P. Zuspan (1980). Induction of severe intrauterine growth retardation in the Sprague-Dawley rat. Am. J. Obstet. Gynec. 137: 43-47.