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Fetal lung development in surgically induced prolonged gestation
Respiration Physiology (1981) 45, 153-166 Elsevier/North-Holland Biomedical Press
FETAL LUNG DEVELOPMENT IN SURGICALLY INDUCED PROLONGED GESTATION*
EDMUND
E. F A R I D Y
Department of Physiology, University of Manitoba, Winnipeg, Canada
Abstract. In our previous study we prolonged gestation in rats with daily subcutaneous injections of progesterone starting on gestation day 20 (P20). To determine whether exogenous progesterone affects the growth of post-term fetal lungs, we utilized surgical techniques to prolong gestation by retaining 4 living fetuses 1, 2, or 3 days beyond the date of spontaneous delivery of the rest of the litter. The retained fetuses were harvested by caesarean section on gestation days 21 through 25. In spite of an improved general appearance in the retained fetuses, there were many similarities between the retained and the post-term P20 fetuses such as: increase in fetal body, kidney and spleen weights; reduction in lung and liver weights; progressive lung atelectasis; loss of lung and liver glycogen; hypoglycemia; and reduction in placenta DNA content. The significant differences between the two were in the amount of lung disaturated phosphatidyl choline (DSPC) and lung DNA (cell number), expressed per lung or per body weight. In contrast to the P20 post-term fetuses, which exhibited a biphasic phenomenon in DSPC and DNA contents, the retained fetuses showed no change in lung DSPC and DNA contents after term. The P20 fetuses had larger adrenals, which increased with prolongation of gestation. The results suggest that exogenous progesterone does not affect the fetal lung growth post-term. It is speculated that the cellularity and the DSPC content of the post-term fetal lung is adversely affected by the degree of fetal distress. Fetal growth Fetal lung phospholipids in postmaturity Fetal tissue glycogen in postmaturity
Lung atelectasis in postmaturity Postmaturity
I n a previous study (Morris et al., 1980) we e x a m i n e d the biochemical a n d histological features o f fetal rat lungs d u r i n g p r o l o n g e d gestation. The p r o l o n g a t i o n o f gestation was i n d u c e d by daily s u b c u t a n e o u s injections o f progesterone starting o n gestation day 20. This m o d e l o f artificial p r o l o n g e d gestation has been used by n u m e r o u s investigators (Vorherr, 1975). However, o n the basis o f a n u m b e r of o b s e r v a t i o n s which we m a d e in this model, it became evident that there was a need
Accepted for publication 2 May 1981 * This study was supported by a grant from the Medical Research Council of Canada (¢MT-3981).
0034-5687/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press 153
154
E.E. FARIDY
for re-evaluating the effect of prolonged gestation on fetal lungs without the use of exogenous progesterone. These observations were as follows: (1) The general condition of the pregnant rat deteriorated as pregnancy extended beyond term, in that some rats became less active, pale (due to loss of blood : intrauterine homorrhage, vaginal bleeding) and their food consumption decreased about 50 °/',,, (2) The fetal movement noted on abdominal wall, increased in the first part of the post-term period. (3) Placenta weight at gestation day 22 decreased if rats received daily progesterone from gestation day 14. Since the placenta weight also decreased in post-term fetuses of progesterone treated rats, this raised the question whether daily injections of progesterone post-term were responsible for reduction in placenta size. Finally, the direct effect of exogenous progesterone on fetal lung post-term is not known, although we had shown that the daily injections of progesterone pre-term (GD 14 to 21) had no effect on the fetal lung at term (GD 22), With these uncertainties in mind, and in order to preclude exogenous progesterone, we utilized surgical techniques to prolong gestation by retaining 4 living fetuses for 1, 2, or 3 days beyond the date of spontaneous delivery of the rest of the litter.
Methods One hundred forty seven pregnant rats and 501 fetuses were used for this study. Virgin female Sprague-Dawley rats (Canadian Breeding Farm and Laboratories Ltd., Quebec) weighing 195-225 g, when in proesterus phase, were placed with male rats for four hours. Gestation day (GD) one was designated as commencing 24 hours after detection of a positive vaginal smear. Females, if allowed to deliver, did so on day 22 of gestation. Pregnant rats were divided into 3 groups. (1) Control, sacrificed on G D 21. (2) Prolonged pregnancy. This group of rats were anesthetized with ether on gestation day 16. The abdominal cavity was opened at mid line; the uterus was inspected and the fetuses counted. If the litter size was between 9-14, a ligature was made around each uterine cornu caudal to the two cranial gestation sacs on that side. Care was taken to place the suture thread snugly about the circumference of the cornu and inside the vascular supply to the uterus and sac, i.e. between the uterine blood vessels and the perimetrium. The uterus was then replaced into the abdominal cavity and the abdominal wall closed. These rats were sacrificed on G D 21 or allowed to deliver the unimpeded fetuses and nurse the pups for 1, 2, or 3 days following parturition, and then sacrificed on G D 22, 23, 24, or 25 to study the 4 post term ('retained') fetuses. Occasionally, the maternal rat lactated poorly, resulting in the death of the pups. Such rats were discarded from the study. From the group of rats subjected to surgery, 12 pregnant rats were randomly selected. Within an hour after parturition (GD 22), six maternal rats were given subcutaneous injections of 5 mg progesterone in sesame oil (Pregn-4-ene-3, 20-dione, Proluton, Schering Corp,, New Jersey) (P22 rats), and the other six were given sesame oil,
FETAL L U N G IN PROLONGED GESTATION
155
on G D 22 and on day prior to sacrifice. These rats were allowed to nurse their pups and were sacrificed on G D 23 or G D 24. (3) Sham operated. This group was subjected to a surgical procedure similar to that mentioned above except that the uterus was not ligated. These rats were sacrificed on G D 21. Only the two cranial-most fetuses from each uterine cornu were used from this and the non-operated controls. Daily food intake and body weight were measured. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (5 mg/100 g body wt.) on the day of sacrifice. Fetuses were harvested by caesarean section, dried with gauze, and weighed. Only live, normal littermates were used. The fetuses were then decapitated, the lungs were removed, separated from the extrapulmonary airways and weighed. The lungs of littermates were pooled for one of the following determinations: (1) DNA, R N A and total lung protein content (0.2-0.25 g sample); (2) total lung phospholipids (0.2-0.25 g sample). Dry weight, and light microscopic studies were done on individual lungs. Lungs that appeared to contain blood or meconium due to aspiration, or air due to fetal gasping were discarded. In addition, liver, spleen, and kidneys were dissected from the fetuses and weighed. Placentae collected at the time of caesarean section were dissected free of the umbilical cord, blotted and weighed. Placenta and liver dry weights were also collected. The uterus and its contents was removed from some rats and subjected to microwave irradiation to inactivate enzymes responsible for glycogenolysis. Lungs and liver were dissected and used for glycogen determination. For blood glucose measurements, the fetus was delivered through the uterine incision and decapitated while its umbilical cord remained attached at the placenta. Blood was then collected in capillary tubes. The maternal blood was taken from the abdominal aorta. Fetuses obtained from pregnant rats at G D 23, 24 and 25 were compared to fetuses obtained from pregnant rats at G D 22. This comparison was made to determine any differences in fetal development post-term to that of term. Comparisons were also made between the post-term 'retained' fetuses of the present study and the post-term fetuses of progesterone treated (P20) rats of our previous study (Morris et aL, 1980). However, since the animals for the present study were obtained from a laboratory other than that of our previous study, it was necessary to test if the parameters measured were similar in fetuses of rats obtained from both laboratories. Measurements made on both groups of term fetuses demonstrated that all parameters were similar with the exception of fetal lung D N A content when expressed per lung unit. This appears to be due to species differences. Because of this observation we felt it necessary to repeat our previous experiments to obtain not only P20 fetal lung D N A content to compare with the lung D N A content of surgically retained fetuses, but other measurements which are specific for the present study. For this a total of 46 pregnant rats and 143 fetuses were used. Pregnancy was prolonged by daily subcutaneous injections of progesterone, starting on G D 20 (P20 rats) (as described in our previous study, Morris et al., 1980). The following measurements were made on fetuses at G D 21-25:
156
E.E. FARIDY
lung D N A and RNA contents, placenta DNA, and adrenal weight. Fetal and maternal blood glucose were also measured. In spite of the fact that we had found no significant differences in parameters measured between the four cranial-most fetuses and the rest of the litter, we felt it was more appropriate to conduct the above measurements, on P20 rats, on the two cranial-most fetuses from each uterine cornu. Dry weight. Dry weight measurements were made by placing the tissues on preweighed pieces of tin-foil which were then kept in an oven at 60 °C for one week. Phospholipids. Pooled samples of lung tissue (0.25 g or less) 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 (25 # 1 ) o f the lipid extract was used to determine lipid phosphorus according to Brante's modification (1949) of the method of Fiske and Subbarow (1925). A second aliquot (50 /~1) 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 phosphatidyl choline spot was identified and then aspirated into a test tube for measurements of lipid phosphorus as described above. A third aliquot (50 #1) of the original lipid extract was plated on an activated silica gel-H plate and the phosphatidyl choline spot was identified and aspirated into a test tube to isolate the disaturated phosphatidyl choline (DSPC) by mercuric acetate adduction (Mangold, 1961) and the lipid phosphorus determined. DNA, RNA and total protein. Pooled samples of lung tissue (0.25 g or less) were homogenized in 1.25-2.5 ml of normal saline in a Kontes all glass homogenizer. One hundred microliters of this suspension was diluted 1/20 in normal saline and 25 or 50 #1 of the dilution were added to 1 N NaOH and digested for 18-20 hours. The total protein content was then determined by the Lowry method (Lowry et al., 1951). From the above homogenized sample, 1 ml aliquot was used for the extraction and determination of lung deoxyribonucleic acid (DNA) and RNA by the method of Schneider (1957). The number of cells per lung was calculated using the following formula: number of nuclei (in millions)= (total lung D N A (rag) x 10~)/ 6.2 pg, where 6.2 is the amount of D N A in picograms in a single diploid rat nucleus (Enesco and LeBlonde, 1962). Glycogen. Viability of fetuses, while in utero, was determined by pinching the tail with forceps. A fetus was considered dead if fetal movement did not occur and was marked by passing a needle through the body. The uterus en bloc with the fetuses in utero was then removed and placed in a microwave oven and irradiated for 10 to 15 sec. Lung and liver samples 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-e 1,4-e 1,6-glucosidase according to the method of Passoneau and Lauderdale
FETAL LUNG IN PROLONGEDGESTATION
157
(1974) and tissue glycogen calculated using the equation AA/6.22 x volume of cuvette (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. Light microscopy. Fetal lungs were removed, the left bronchus was ligated and the left lung was fixed in 10% buffered formalin, processed and embedded in paraffin. Sections (7/~m) were stained with hematoxylin and eosin, viewed and photographed in a Zeiss photomicroscope. Statistics. Statistical analysis of the data was carried out using a t-test of unpaired variates and a multiple range test for analysis of variance by Duncan's method, when applicable.
Results
The data obtained from retained fetuses are presented in tables 1-3, which compare the post-term with term fetuses, and in tables 4 and 5, which compare the retained fetuses with post-term P20 fetuses of present study. The retained fetuses of present study are also compared with the post-term P:0 fetuses of our previous study (Morris et al., 1980). The results of such comparisons are described in the text without presentation of previous data to avoid repetition. Maternal measurements. There was no significant weight loss from the surgical procedures during pregnancy when compared to controls. Food intake decreased 50% on the day of operation and reached normal level in 24 hours. During post partum the daily food intake was about 75% of that during pregnancy. With the exception of a few (see methods), the maternal rat showed no signs of distress during post-partum in spite of retaining four fetuses in utero. They nursed their pups in much the same way as control rats. On examination of abdominal wall, fetal movements were rarely observed. Maternal blood serum glucose averaged 140.3 4- 6.4 SE, mg/100 ml (N = 8) on GD 23-25 which was significantly greater (P < 0.01) than the progesterone treated rats (111.7 + 4.7 SE, mg/100 ml, N --9). Fetal death was not observed on GD 21 and 22, but on GD 23, 24, and 25 it was 8.8%, 35% and 59%, respectively. Fetal measurements. Since there were no differences in any of the parameters measured on GD 21 between the fetuses of sham operated rats and the uterine ligated rats, the data on fetuses of sham operated rats are omitted from tables. The physical appearance of the retained fetuses, when compared to the post-term fetuses of progesterone treated rats (Morris et al., 1980), gave the impression that they were somewhat less 'postmature'. That is, they were more active and their skin was less cracked and wrinkled. Meconium staining was not as frequently observed. Fetal amniotic fluid decreased on GD 24 and GD 25, similar to that seen in post-term fetuses of P:0 rats. Fetal body weight (table 1) increased progressively from GD 21 to GD 25, much
138.7 ± 2.71 (32)
18.77 ± 0.41 (12)
13.6_+_0.18 (12)
526.5 + 16.95 (24)
340.4 ± 5.84 (24)
33.9 _+ 0.89 (24)
6.3 ± 0.41 (24)
Lung wet wt. (rag)
Lung dry wt. (mg)
(Lung dry/wet wt.) x 100
Placenta wet wt. (mg)
Liver wet wt. (mg)
Kidney wet wt. (mg)
Spleen wet wt. (mg)
5.9 + 0.48 (18)
34.0 ± 0.99 (18)
343.8 ± 6.64 (20)
516.2 ± 16.68 (20)
13.6+0.16 (8)
18.39 + 0.57 (8)
137.9 ± 2.34 (28)
4.63 +_ 0.09 (28) 134.3 _+ 4.66 t (32) 18.74 ± 0.91 (14) 14.1 + 0.26 ~ (14) 470.2 ± 13.52 (29)
145.9 + 2.26 (32) 18.98 + 0.45 (12) 13.2 +_ 0.22 (12) 506.0 ± 16.42 (26)
65.8 +0.98* (29) 19.4+_0.79" (29)
46.8 ± 1.07 (24) 10.1 _+0,35 (24)
327.9 ± 8.31" (29)
6.60 _+ 0.08* (37)
5.94 +_ 0.09 (32)
391.1 ± 5.98 (23)
23
22
33,0 ± 1,66" (27)
77.1 ± 1.98" (27)
279.9 _+ 6.20* (27)
464.5 ± 16.22 (28)
15.6 + 0.77 '~ (10)
15.74 ± 0.79 ~ (10)
106.3 ± 2.80* (28)
6.85 ± 0.09* (33)
24
44.4 ± 2.41" (14)
80.1 ± 2 . 9 3 * (14)
244.2 ± 6.58* (14)
480.6 ± 24.11 (14)
17.0 ± 0.84* (7)
16.03 ± 0.75 e (7)
93.9 ± 5.45* (21)
6.74 ± 0.09* t21)
25
Data are expressed as means _+ 1 SE; numbers in parentheses indicate number of fetal rats studied. The data from sham operated group ( G D 21) are not included in the table because of the similarities between this group and control and 'retained' groups. Comparisons are made as follows: A m o n g groups on G D 2l: no difference. Between post-term and term ( G D 22, retained) fetuses: *P < 0.001; +P < 0.05; " P < 0.1)1 and ~P < 0.02.
4.46 ± 0.07 (32)
21
21
Body wt. (g)
'Retained'
Control
TABLE 1 Measurements in fetal rats at gestation days 21 to 25
,.<
U, OC
FETAL L U N G IN PROLONGED GESTATION
159
the same way as P20 fetuses. Placental weight (wet and dry) decreased slightly on GD 23 (not statistically significant from GD 22) and remained unchanged during prolonged gestation, in contrast to P20 placentae which decreased in weight p r o gressively with prolongation of gestation (Morris et al., 1980). Placenta DNA content, however, progressively decreased from 1.06 (mg) + 0.03 SE (N = 13) on GD 22 to 0.84 ___0.03 (N = 10) on GD 25 (P < 0.001) and were not significantly different from those treated with progesterone (P20 rats). Lung weight decreased post-term and resulted in a smaller lung in proportion to body weight. Reduction in lung dry weight appeared to be greater in P20 fetuses (GD 24, 25) than in retained fetuses. When lung weight was expressed per body weight, the retained fetuses, at GD 25, in comparison to GD 22, had a reduction of 43.3~o and 22.4~o in lung wet and dry weight, respectively, while these values in P20 fetuses were 50~o and 38.5~, respectively. Liver wet and dry weights decreased while kidney and spleen weight increased significantly during prolonged gestation much the same as in the P20 post-term fetuses. Protein, DNA, RNA and phospholipid analysis of fetal lungs at GD 21 to GD 25 are shown in table 2. As gestation was prolonged from GD 22 to 25, total lung protein, DNA, and phospholipid increased when expressed per gram lung. This resulted from the fact that the lung weight progressively decreased, mainly because of loss of pulmonary fluid. When expressed per mg lung DNA, there was a progressive reduction in total protein and RNA but no change in phospholipid content. These findings in retained fetuses resemble those observed in P20 post-term fetuses. In table 3 the total lung protein, lung phospholipid, lung DNA and the number of cells are expressed per lung. Since measurements were made on pooled samples from 2 to 4 fetuses, protein, phospholipid and DNA per lung were based on the average lung weights in that particular sample. With the exception of a small reduction in total protein with prolongation of gestation, the other parameters, such as phospholipids and DNA (cell number) were constant from term (GD 22) through GD 25. Both lung and liver glycogen contents were reduced from term to GD 25 in much the same way as previously reported for progesterone treated rats (Morris et al., 1980). Fetal blood serum glucose levels averaged 87.8 + 4.9 SD, 66.4 + 6.3 and 54.9 + 7.6, mg/100 ml on GD 23, 24 and 25, respectively. These values were slightly (about 4--8 mg) but not significantly higher than the post-term fetuses of progesterone treated (P:0) rats. In table 4 is shown the lung cell number of retained post-term fetuses from maternal rats receiving daily injections of sesame oil (control) or progesterone (P22) starting one hour after parturition on GD 22. The values for cell number, expressed per lung or per body weight, were similar in both conditions. Light microscopic studies revealed a gradual reduction in the numbers of distended terminal sacs, and atelectasis with prolongation of gestation. These findings were similar to those observed previously on post-term fetuses of progesterone treated rats (Morris et al., 1980).
7.80 _+0.17
3.97 _+ 0.08
Disaturated phosphatidyl choline
0.564 _+ 0.01
_+2.57
0.579 _+ 0.01
8.69 _+ 0.31
0.504 _+ 0.01
0.99 _+ 0.02
2.09 _+ 0.03
3.96 _+ 0.12
7.75 _+0.14
16.44 _+0.23
7.86 _+0.16
68.3
_+2.82
0.463 _+ 0.01
9.22 _+ 0.48
0.506 _+ 0.02
1.02 _+ 0.04
2.03 _+ 0.05
4.55 _+ 0.12
9.16 _+0.23
18.30 _+0.28
9.02 + 0 . 2 2
82.2
22
+2.29
_+ 0.08*
0.340 _+ 0.02 t
8.36 _+ 0.36
0.518 _+ 0.02
1.06 _+0.05
2.20 _+ 0.10
5.11
10.45 _+0.12"
21.69 _+0.22*
9.93 _+0.46
82.4
23
+2.34*
0.311 _+ 0.01*
7.79 _+0.28 ¢
0.504 _+ 0.02
1.02 _+ 0.03
2.07 _+ 0.06
6.23 _+0.13*
12.64 _+0.23'
25.68 _+0.50'
12.42 +_0.30 t
96.5
24
+_4.32 t
(6)
0.239 _+ 0.01 t
+_0.17"
0.508 _+ 0.03
1.03 _+ 0.07
2.05 _+ 0.15
7.51
(4)
(6)
(6)
(4)
(4)
(4)
(4)
_+ 0.44 + (4) 7.34 _+0.20 t
15.01
29.59 _+0.51'
15.39 _+ 0.85 + (6)
115.3
25
Data are expressed as means _+ 1 SE: each is the mean of 5 measurements except when indicated in parentheses. The data from sham operated group (GD 21) are not included in the table because of the similarities between this group and control and "retained" groups. Comparisons are made as follows: A m o n g groups on G D 21 : no difference. Between post term and term (GD 22, retained) fetuses: *P < 0.01 ; +P < 0.001 and c P < 0.05.
8.59 _+ 0.48
Lung RNA, m g / m g D N A
0.506 _+ 0.01
Disaturated phosphatidyl choline
Lung total protein, mg/mg D N A
0.99 + 0.03
Phosphatidyl choline
2.04 _+ 0.03
16.03 _+0.20
Phosphatidylcholine
Lung phospholipid mg/mg lung D N A : Total
7.85 _+0.15
_+2.80
Lung phospholipid, mg/g lung: Total
67.2
21
21
LungDNA, mg/glung
Lung total protein, mg/g tung
'Retained'
Control
TABLE 2 Biochemical measurements in fetal rats at gestation days 21 to 25
Total D N A per lung (mg) 216.9
+ 10.9
1.34 ___ 0.07
0.697 + 0.04
1.43 + 0.09
2.96 + 0.20
11.28 + 0.87
23
218.9
-1- 5.8
1.36 + 0 . 0 4
0.682 + 0.02
1.38 + 0 . 0 5
2.81 + 0 . 1 0
10.54 + 0 . 1 7
24
221.6
+ 11.2
1.37 + 0.07
0.690 + 0.02
1.41 + 0.05
2.79 + 0.04
(6)
(6)
(4)
(4)
(4)
10.30 + 0.47*(6)
25
Data are obtained from retained fetuses and are expressed as m e a n s + 1 SE; each is the m e a n of 5 measurements except when indicated in parentheses. Pre- and post-term fetuses are compared with term ( G D 22) fetuses. *P < 0.02; t p < 0.01 and * P < 0.001.
-1- 6.3
1.33 + 0 . 0 4
1.09 _ 0 . 0 3 t
DSPC per lung (mg) 214.0
0.668 + 0.01
0.551 + 0.02 t
Phosphatidyl choline per lung (mg)
+4.9 t
1.35 + 0 . 0 4
1.08 + 0 . 0 3 ~'
176.3
2.70 + 0 . 0 9
2.29 + 0 . 0 5 t
Total phospholipid per lung (mg)
Cell no., in millions per lung
12.23 + 0 . 7 2
9.50 + 0 . 4 3 *
22
Total protein per lung (mg)
21
TABLE 3 Biochemical measurements of fetal rats lungs at gestation days 21 to 25
O~
7~
> -]
O Z 0
©
7~
Z
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162
E.E. FARIDY TABLE4 Lung cell number of retained fetal rats at gestation days 23 and 24
Maternal condition
Control Progesterone treated (P22)
Per l u n g
Per g b o d y weight
23
24
23
24
218.8 ± 7.4 221.6 + 9.4
217.7 ± 10.7
34.2 + 0.82
32.8 ± 1.75
220.9 ± 6.6
33.9 + 1.06
33.4 ± 0.73
D a t a are expressed as m i l l i o n s a n d p r e s e n t e d as m e a n s _+ I SE; each is the mean of 6 m e a s u r e m e n t s . P22 = M a t e r n a l rats injected with p r o g e s t e r o n e on g e s t a t i o n day 22 (within an h o u r after p a r t u r i t i o n ) a n d o n d a y before sacrifice. C o n t r o l = M a t e r n a l rats injected with sesame oil in the s a m e m a n n e r as P22 rats.
Discussion
Surgically induced model of prolonged gestation appears to have advantages over that induced by exogenous progesterone because of the following: The procedure is well tolerated by the pregnant rat; there is no maternal mortality; the food intake and the blood glucose levels are higher; and the overall general condition of the rat is more satisfactory. Moreover, post-term placentae of progesterone treated rats (Thliveris, 1976) undergo significant morphological changes indicative of decreasing placental function. In contrast, the post-term placentae of surgically induced prolonged gestation (Jollie, 1976) show little evidence of placental damage. These could be some of the factors which have contributed in improving the general appearance of the retained fetuses. In spite of this, the retained and the post-term P20 fetuses are simil&r in many aspects, such as: increase in fetal body, kidney and spleen weights; reduction in lung and liver weights; progressive lung atelectasis; loss of lung and liver glycogen; and reduction in placenta DNA content. The main difference between the two are in the contents of lung DSPC and lung DNA (cell number), when expressed per lung or per body weight (fig. 1). The retained fetuses do not exhibit the biphasic phenomenon in lung DSPC and DNA content. While the lung of the P20 fetuses continued its growth beyond term for an additional day (GD 23, gain of 35 million cells) and subsequently regressed with further prolongation of gestation (GD 25, loss of 51 million cells), the lungs of the retained fetuses appeared to have a complete arrest of growth at term (no change in cell numbers post-term). These differing behaviours in lung growth are interesting and need further analysis. Grota and Eik-Nes (1967) analysed plasma progesterone levels in rats during normal pregnancy and lactation and showed that there was a gradual decrease in the concentration of the hormone as term of pregnancy approached followed by an increase during lactation. In the 24 h preceding parturition, the concentration of progesterone decreased significantly from 114 ng/ml of plasma (GD 21) to 10 ng/ml
FETAL LUNG IN PROLONGED GESTATION
~=
163
40[ ",,,
=" 220f ~,
""
~= ~s° t 140 ~
,
*
20"
L
0.14
I
0.8
"~. 0.6 =m
I~l........r,, " •
f
~ o.lo
U
,~ 0.4
0
0
0.08 0.2
f
21
22
2', 2'5 o s , ,oN D,Y
!
22 2'3 2',
Fig. 1. Comparison of lung cell number and dissaturated phosphatidyl choline content of retained fetuses (surgically induced prolonged gestation broken line) and P20 fetuses (post-term fetuses of rats treated with daily injections of progesterone beginning on gestation day 20 - solid line). The data for P20 fetuses are taken from our previous study (Morris eta/., 1980). In addition, the data for P20 fetuses repeated in the present study is shown by the upper solid line in left upper graph and is used to compare with retained fetuses (see text). The vertical lines represent 1SE to either side of the mean, each being the mean of 5-11 measurements. *Significantly different (P < 0 . 0 5 - P <0.001) from the P20 fetuses of identical age. The arrow indicates gestation at term.
on GD 22. Within 6 h post-partum, it rose to 43 ng/ml and remained at this level until 2 days after delivery and then rose to 123 ng/ml of plasma on Day 4 of lactation. It is possible that the retained fetuses in the present study were maintained post-term in utero under the influence of endogenous progesterone secreted during lactation. If progesterone influences the rate of growth of the fetal lung and if the rate of growth depends on the concentration of the hormone in the maternal plasma, then it follows that there would be different rates of fetal lung growth in the two models of prolonged gestation. Although there were no differences seen on GD 22, in lung development, between the retained fetuses (low levels of maternal plasma progesterone?) and the fetuses of rats receiving daily progesterone (higher levels of maternal plasma progesterone ?), and although this appeared to have ruled out the effect of exogenous progesterone p e r s e on fetal lung development, it was decided to further examine the effect of exogenous progesterone on retained fetuses post-term. The results, as shown in table 4 indicated the lung DNA content (cell number) of retained fetuses, expressed either per lung or per body weight was similar in both whether the rat received progesterone or not. These results perhaps further support the notion that, in our studies, the exogenous progesterone p e r s e did not affect the development of the fetal lung, either pre- or post-term. In view of the fact that fetal distress occurs post-term, as suggested by the release of meconium (Clifford, 1957); that cortisol levels are elevated in the umbilical cord blood of postmature infants (Nwosu e t al., 1975); that signs of increased activity
164
E. E. F A R I D Y TABLE 5 Adrenal weight of fetal rats at gestation days 22 to 25 22
23
24
25
Retained
0 . 3 5 7 + 0 . 0 1 " (18)
0.331_+0.01 t (14)
0 . 3 6 2 + 0 . 0 1 " (6)
0.359_+_0.03 "~ (7)
P20
0.406 + 0.01
0.413 + 0.01
0.444 + 0.02
0.463 + 0.020 .(6)
(18)
(11)
(11)
Adrenal weight is expressed as mg/g body weight. Data are expressed as m e a n s + 1 SE; n u m b e r s in parentheses indicate n u m b e r of fetal rats studied. Retained = fetuses in tied off uterus. P20 = fetuses obtained from rats injected with progesterone on gestation day 20 and daily thereafter until day before sacrifice. Comparisons are made as follows: Between retained and P20 fetuses at each G D : *P < 0.01 ; *P < 0.001 a n d ¢ P < 0.02. Between post-term and term ( G D 22) fetuses of P20 rats: ~P < 0.05.
are noted in the adrenal glands of post-term fetuses of progesterone treated rats (Thliveris and Connell, 1973); and that our 'clinical' observations implied that the level of fetal distress was greater in the fetuses of P20 rats than the retained fetuses, we made a preliminary study of measuring the weight of the adrenal glands in the retained and P20 fetuses. As shown in table 5, the 1}20fetuses have larger adrenals than the retained fetuses. Furthermore, in P20 fetuses, the adrenal size, in proportion to body weight, increases with prolongation of gestation in contrast to the constant value observed for the retained fetuses. If adrenal weight is indicative of its activity then the fetal blood steroids level should be higher in P20 fetuses than in retained fetuses. Is it possible that corticosteroids initially stimulated lung growth post-term (GD 23) and subsequently induced slowing of cell mitosis (Carson et al., 1973), disturbing the cell renewal rate of the lung in favour of a cell loss? An interesting phenomenon is the arrest in lung growth in retained fetuses post-term. It seems that the stimulus for lung growth has been removed. It is shown that distension increases the metabolic rate of the lung (Faridy and Naimark, 1971) and perhaps stimulates the lung growth (Thurlbeck, 1975). The terminal sacs of fetal lung contain fluid which is secreted by the lung tissue. If one of the functions of the pulmonary fluid is to distend the terminal sacs and stimulate the lung growth, it is conceivable that a reduction in the rate of secretion of pulmonary fluid, leading to lung atelectasis, would abolish the necessary stimulus for lung growth. Our observations indicate that the post-term fetal lungs do become atelectatic. Kitterman et al. (1979) have shown that the tracheal fluid production in fetal lamb decreases before spontaneous term. They have postulated that the decrease in production may be related to increased secretion of cortisol. On the basis of our results it is tempting to speculate that the cellularity and the DSPC content of the post-term fetal lung is adversely affected by the degree of fetal distress. However, irrespective of fetal condition post-term, the lungs become atelectatic and might require greater inflation pressures at birth. On the other hand, the post-term lungs which contain comparable amounts of DSPC and cells to term lungs, may not necessarily possess the functional capabilities of the normal lungs at
FETAL LUNG IN PROLONGED GESTATION
165
term. For example, the rate of synthesis and release of surfactant in these lungs at birth may be jeopardized because of a severe loss of lung glycogen and a concomittant hypoglycemia.
Acknowledgement The author expresses his appreciation to Mrs. Saniata Bucher for her excellent technical assistance.
References Brante, G. (1949). Studies on lipids in the nervous system: total phosphorus determination. Acta Physiol. Scand. Suppl. 63: 39-40. Carson, S.H., H.W. Taeusch, Jr., and M. E. Avery (1973). Inhibition of lung cell division after hydrocortisone injection in fetal rabbits. J. Appl. Physiol. 34: 660-662. Clifford, S.H. (1957). Postmaturity. Adv. Pediatr. 9: 13~3. Enesco, M. and C.P. LeBlonde (1962). Increase in cell number as a factor in the growth of the organs and tissues of the young male rat. J. Embryol. Exp. Morphol. 10: 530-562. Faridy, E. E. and A. Naimark (1971). Effect of distension on metabolism of excised dog lung. J. Appl. Physiol. 31: 31-37. 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. Grota, L.J. and K.B. Eik-Nes (1967). Plasma progesterone concentrations during pregnancy and lactation in the rat. J. Reprod. Fert. 13: 83-91. JoUie, W. P. (1976). The fine structure of the interhemal membrane of the rat chorioallantoic placenta during prolonged pregnancy. Anat. Rec. 184: 73-90. Kitterman, J. A., P.L. Ballard, J.A. Clements, E.J. Mescher and W.H. Tooley (1979). Tracheal fluid in fetal lambs: spontaneous decrease prior to birth. J. Appl. Physiol. 47: 985-989. 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. Marigold, H.K. (1961). Thin-layer chromatography of lipids. J. Oil Chem. Soc. 38: 708-727. Morris, G.S., J.A. Thliveris and E.E. Faridy (1980). Development of fetal rat lung during prolonged gestation. Respir. Physiol. 42: 263-285. Nwosu, U., E. E. Wallach, T. R. Boggs, R. L. Nemiroff and A.M. Bongiovanni (1975). Possible role of the fetal adrenals in the etiology of post-maturity. Am. J. Obstet. Gynaecol. 121: 366-370. 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. Schneider, W. C. (1957). Determination of nucleic acids in tissue by pentose analysis. Methods Enzymol. 3 : 680-684. Thliveris, J.A. and R.S. Connell, Jr. (1973). Ultrastructure of the fetal rat adrenal gland at full-term and during prolonged gestation. Anat. Rec. 175: 607-624. Thliveris, J. A. (1976). Fine structure of the placental labyrinth in the rat at term and during prolonged gestation. Virchows Arch. B. Cell Pathol. 21 : 169-178.
166 Thurlbeck, W.M. (1975). Postnatal growth and development of the lung. A m . Rev. Re,~p. Dis. 1 l I : 803 844. Vorherr, H. (1975). Placental insufficiency in relation to post term pregnancy and fetal postmaturity. A m . J. Obstet. Gynaecol. 123:67 103.
FETAL LUNG DEVELOPMENT IN SURGICALLY INDUCED PROLONGED GESTATION*
EDMUND
E. F A R I D Y
Department of Physiology, University of Manitoba, Winnipeg, Canada
Abstract. In our previous study we prolonged gestation in rats with daily subcutaneous injections of progesterone starting on gestation day 20 (P20). To determine whether exogenous progesterone affects the growth of post-term fetal lungs, we utilized surgical techniques to prolong gestation by retaining 4 living fetuses 1, 2, or 3 days beyond the date of spontaneous delivery of the rest of the litter. The retained fetuses were harvested by caesarean section on gestation days 21 through 25. In spite of an improved general appearance in the retained fetuses, there were many similarities between the retained and the post-term P20 fetuses such as: increase in fetal body, kidney and spleen weights; reduction in lung and liver weights; progressive lung atelectasis; loss of lung and liver glycogen; hypoglycemia; and reduction in placenta DNA content. The significant differences between the two were in the amount of lung disaturated phosphatidyl choline (DSPC) and lung DNA (cell number), expressed per lung or per body weight. In contrast to the P20 post-term fetuses, which exhibited a biphasic phenomenon in DSPC and DNA contents, the retained fetuses showed no change in lung DSPC and DNA contents after term. The P20 fetuses had larger adrenals, which increased with prolongation of gestation. The results suggest that exogenous progesterone does not affect the fetal lung growth post-term. It is speculated that the cellularity and the DSPC content of the post-term fetal lung is adversely affected by the degree of fetal distress. Fetal growth Fetal lung phospholipids in postmaturity Fetal tissue glycogen in postmaturity
Lung atelectasis in postmaturity Postmaturity
I n a previous study (Morris et al., 1980) we e x a m i n e d the biochemical a n d histological features o f fetal rat lungs d u r i n g p r o l o n g e d gestation. The p r o l o n g a t i o n o f gestation was i n d u c e d by daily s u b c u t a n e o u s injections o f progesterone starting o n gestation day 20. This m o d e l o f artificial p r o l o n g e d gestation has been used by n u m e r o u s investigators (Vorherr, 1975). However, o n the basis o f a n u m b e r of o b s e r v a t i o n s which we m a d e in this model, it became evident that there was a need
Accepted for publication 2 May 1981 * This study was supported by a grant from the Medical Research Council of Canada (¢MT-3981).
0034-5687/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press 153
154
E.E. FARIDY
for re-evaluating the effect of prolonged gestation on fetal lungs without the use of exogenous progesterone. These observations were as follows: (1) The general condition of the pregnant rat deteriorated as pregnancy extended beyond term, in that some rats became less active, pale (due to loss of blood : intrauterine homorrhage, vaginal bleeding) and their food consumption decreased about 50 °/',,, (2) The fetal movement noted on abdominal wall, increased in the first part of the post-term period. (3) Placenta weight at gestation day 22 decreased if rats received daily progesterone from gestation day 14. Since the placenta weight also decreased in post-term fetuses of progesterone treated rats, this raised the question whether daily injections of progesterone post-term were responsible for reduction in placenta size. Finally, the direct effect of exogenous progesterone on fetal lung post-term is not known, although we had shown that the daily injections of progesterone pre-term (GD 14 to 21) had no effect on the fetal lung at term (GD 22), With these uncertainties in mind, and in order to preclude exogenous progesterone, we utilized surgical techniques to prolong gestation by retaining 4 living fetuses for 1, 2, or 3 days beyond the date of spontaneous delivery of the rest of the litter.
Methods One hundred forty seven pregnant rats and 501 fetuses were used for this study. Virgin female Sprague-Dawley rats (Canadian Breeding Farm and Laboratories Ltd., Quebec) weighing 195-225 g, when in proesterus phase, were placed with male rats for four hours. Gestation day (GD) one was designated as commencing 24 hours after detection of a positive vaginal smear. Females, if allowed to deliver, did so on day 22 of gestation. Pregnant rats were divided into 3 groups. (1) Control, sacrificed on G D 21. (2) Prolonged pregnancy. This group of rats were anesthetized with ether on gestation day 16. The abdominal cavity was opened at mid line; the uterus was inspected and the fetuses counted. If the litter size was between 9-14, a ligature was made around each uterine cornu caudal to the two cranial gestation sacs on that side. Care was taken to place the suture thread snugly about the circumference of the cornu and inside the vascular supply to the uterus and sac, i.e. between the uterine blood vessels and the perimetrium. The uterus was then replaced into the abdominal cavity and the abdominal wall closed. These rats were sacrificed on G D 21 or allowed to deliver the unimpeded fetuses and nurse the pups for 1, 2, or 3 days following parturition, and then sacrificed on G D 22, 23, 24, or 25 to study the 4 post term ('retained') fetuses. Occasionally, the maternal rat lactated poorly, resulting in the death of the pups. Such rats were discarded from the study. From the group of rats subjected to surgery, 12 pregnant rats were randomly selected. Within an hour after parturition (GD 22), six maternal rats were given subcutaneous injections of 5 mg progesterone in sesame oil (Pregn-4-ene-3, 20-dione, Proluton, Schering Corp,, New Jersey) (P22 rats), and the other six were given sesame oil,
FETAL L U N G IN PROLONGED GESTATION
155
on G D 22 and on day prior to sacrifice. These rats were allowed to nurse their pups and were sacrificed on G D 23 or G D 24. (3) Sham operated. This group was subjected to a surgical procedure similar to that mentioned above except that the uterus was not ligated. These rats were sacrificed on G D 21. Only the two cranial-most fetuses from each uterine cornu were used from this and the non-operated controls. Daily food intake and body weight were measured. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (5 mg/100 g body wt.) on the day of sacrifice. Fetuses were harvested by caesarean section, dried with gauze, and weighed. Only live, normal littermates were used. The fetuses were then decapitated, the lungs were removed, separated from the extrapulmonary airways and weighed. The lungs of littermates were pooled for one of the following determinations: (1) DNA, R N A and total lung protein content (0.2-0.25 g sample); (2) total lung phospholipids (0.2-0.25 g sample). Dry weight, and light microscopic studies were done on individual lungs. Lungs that appeared to contain blood or meconium due to aspiration, or air due to fetal gasping were discarded. In addition, liver, spleen, and kidneys were dissected from the fetuses and weighed. Placentae collected at the time of caesarean section were dissected free of the umbilical cord, blotted and weighed. Placenta and liver dry weights were also collected. The uterus and its contents was removed from some rats and subjected to microwave irradiation to inactivate enzymes responsible for glycogenolysis. Lungs and liver were dissected and used for glycogen determination. For blood glucose measurements, the fetus was delivered through the uterine incision and decapitated while its umbilical cord remained attached at the placenta. Blood was then collected in capillary tubes. The maternal blood was taken from the abdominal aorta. Fetuses obtained from pregnant rats at G D 23, 24 and 25 were compared to fetuses obtained from pregnant rats at G D 22. This comparison was made to determine any differences in fetal development post-term to that of term. Comparisons were also made between the post-term 'retained' fetuses of the present study and the post-term fetuses of progesterone treated (P20) rats of our previous study (Morris et aL, 1980). However, since the animals for the present study were obtained from a laboratory other than that of our previous study, it was necessary to test if the parameters measured were similar in fetuses of rats obtained from both laboratories. Measurements made on both groups of term fetuses demonstrated that all parameters were similar with the exception of fetal lung D N A content when expressed per lung unit. This appears to be due to species differences. Because of this observation we felt it necessary to repeat our previous experiments to obtain not only P20 fetal lung D N A content to compare with the lung D N A content of surgically retained fetuses, but other measurements which are specific for the present study. For this a total of 46 pregnant rats and 143 fetuses were used. Pregnancy was prolonged by daily subcutaneous injections of progesterone, starting on G D 20 (P20 rats) (as described in our previous study, Morris et al., 1980). The following measurements were made on fetuses at G D 21-25:
156
E.E. FARIDY
lung D N A and RNA contents, placenta DNA, and adrenal weight. Fetal and maternal blood glucose were also measured. In spite of the fact that we had found no significant differences in parameters measured between the four cranial-most fetuses and the rest of the litter, we felt it was more appropriate to conduct the above measurements, on P20 rats, on the two cranial-most fetuses from each uterine cornu. Dry weight. Dry weight measurements were made by placing the tissues on preweighed pieces of tin-foil which were then kept in an oven at 60 °C for one week. Phospholipids. Pooled samples of lung tissue (0.25 g or less) 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 (25 # 1 ) o f the lipid extract was used to determine lipid phosphorus according to Brante's modification (1949) of the method of Fiske and Subbarow (1925). A second aliquot (50 /~1) 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 phosphatidyl choline spot was identified and then aspirated into a test tube for measurements of lipid phosphorus as described above. A third aliquot (50 #1) of the original lipid extract was plated on an activated silica gel-H plate and the phosphatidyl choline spot was identified and aspirated into a test tube to isolate the disaturated phosphatidyl choline (DSPC) by mercuric acetate adduction (Mangold, 1961) and the lipid phosphorus determined. DNA, RNA and total protein. Pooled samples of lung tissue (0.25 g or less) were homogenized in 1.25-2.5 ml of normal saline in a Kontes all glass homogenizer. One hundred microliters of this suspension was diluted 1/20 in normal saline and 25 or 50 #1 of the dilution were added to 1 N NaOH and digested for 18-20 hours. The total protein content was then determined by the Lowry method (Lowry et al., 1951). From the above homogenized sample, 1 ml aliquot was used for the extraction and determination of lung deoxyribonucleic acid (DNA) and RNA by the method of Schneider (1957). The number of cells per lung was calculated using the following formula: number of nuclei (in millions)= (total lung D N A (rag) x 10~)/ 6.2 pg, where 6.2 is the amount of D N A in picograms in a single diploid rat nucleus (Enesco and LeBlonde, 1962). Glycogen. Viability of fetuses, while in utero, was determined by pinching the tail with forceps. A fetus was considered dead if fetal movement did not occur and was marked by passing a needle through the body. The uterus en bloc with the fetuses in utero was then removed and placed in a microwave oven and irradiated for 10 to 15 sec. Lung and liver samples 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-e 1,4-e 1,6-glucosidase according to the method of Passoneau and Lauderdale
FETAL LUNG IN PROLONGEDGESTATION
157
(1974) and tissue glycogen calculated using the equation AA/6.22 x volume of cuvette (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. Light microscopy. Fetal lungs were removed, the left bronchus was ligated and the left lung was fixed in 10% buffered formalin, processed and embedded in paraffin. Sections (7/~m) were stained with hematoxylin and eosin, viewed and photographed in a Zeiss photomicroscope. Statistics. Statistical analysis of the data was carried out using a t-test of unpaired variates and a multiple range test for analysis of variance by Duncan's method, when applicable.
Results
The data obtained from retained fetuses are presented in tables 1-3, which compare the post-term with term fetuses, and in tables 4 and 5, which compare the retained fetuses with post-term P20 fetuses of present study. The retained fetuses of present study are also compared with the post-term P:0 fetuses of our previous study (Morris et al., 1980). The results of such comparisons are described in the text without presentation of previous data to avoid repetition. Maternal measurements. There was no significant weight loss from the surgical procedures during pregnancy when compared to controls. Food intake decreased 50% on the day of operation and reached normal level in 24 hours. During post partum the daily food intake was about 75% of that during pregnancy. With the exception of a few (see methods), the maternal rat showed no signs of distress during post-partum in spite of retaining four fetuses in utero. They nursed their pups in much the same way as control rats. On examination of abdominal wall, fetal movements were rarely observed. Maternal blood serum glucose averaged 140.3 4- 6.4 SE, mg/100 ml (N = 8) on GD 23-25 which was significantly greater (P < 0.01) than the progesterone treated rats (111.7 + 4.7 SE, mg/100 ml, N --9). Fetal death was not observed on GD 21 and 22, but on GD 23, 24, and 25 it was 8.8%, 35% and 59%, respectively. Fetal measurements. Since there were no differences in any of the parameters measured on GD 21 between the fetuses of sham operated rats and the uterine ligated rats, the data on fetuses of sham operated rats are omitted from tables. The physical appearance of the retained fetuses, when compared to the post-term fetuses of progesterone treated rats (Morris et al., 1980), gave the impression that they were somewhat less 'postmature'. That is, they were more active and their skin was less cracked and wrinkled. Meconium staining was not as frequently observed. Fetal amniotic fluid decreased on GD 24 and GD 25, similar to that seen in post-term fetuses of P:0 rats. Fetal body weight (table 1) increased progressively from GD 21 to GD 25, much
138.7 ± 2.71 (32)
18.77 ± 0.41 (12)
13.6_+_0.18 (12)
526.5 + 16.95 (24)
340.4 ± 5.84 (24)
33.9 _+ 0.89 (24)
6.3 ± 0.41 (24)
Lung wet wt. (rag)
Lung dry wt. (mg)
(Lung dry/wet wt.) x 100
Placenta wet wt. (mg)
Liver wet wt. (mg)
Kidney wet wt. (mg)
Spleen wet wt. (mg)
5.9 + 0.48 (18)
34.0 ± 0.99 (18)
343.8 ± 6.64 (20)
516.2 ± 16.68 (20)
13.6+0.16 (8)
18.39 + 0.57 (8)
137.9 ± 2.34 (28)
4.63 +_ 0.09 (28) 134.3 _+ 4.66 t (32) 18.74 ± 0.91 (14) 14.1 + 0.26 ~ (14) 470.2 ± 13.52 (29)
145.9 + 2.26 (32) 18.98 + 0.45 (12) 13.2 +_ 0.22 (12) 506.0 ± 16.42 (26)
65.8 +0.98* (29) 19.4+_0.79" (29)
46.8 ± 1.07 (24) 10.1 _+0,35 (24)
327.9 ± 8.31" (29)
6.60 _+ 0.08* (37)
5.94 +_ 0.09 (32)
391.1 ± 5.98 (23)
23
22
33,0 ± 1,66" (27)
77.1 ± 1.98" (27)
279.9 _+ 6.20* (27)
464.5 ± 16.22 (28)
15.6 + 0.77 '~ (10)
15.74 ± 0.79 ~ (10)
106.3 ± 2.80* (28)
6.85 ± 0.09* (33)
24
44.4 ± 2.41" (14)
80.1 ± 2 . 9 3 * (14)
244.2 ± 6.58* (14)
480.6 ± 24.11 (14)
17.0 ± 0.84* (7)
16.03 ± 0.75 e (7)
93.9 ± 5.45* (21)
6.74 ± 0.09* t21)
25
Data are expressed as means _+ 1 SE; numbers in parentheses indicate number of fetal rats studied. The data from sham operated group ( G D 21) are not included in the table because of the similarities between this group and control and 'retained' groups. Comparisons are made as follows: A m o n g groups on G D 2l: no difference. Between post-term and term ( G D 22, retained) fetuses: *P < 0.001; +P < 0.05; " P < 0.1)1 and ~P < 0.02.
4.46 ± 0.07 (32)
21
21
Body wt. (g)
'Retained'
Control
TABLE 1 Measurements in fetal rats at gestation days 21 to 25
,.<
U, OC
FETAL L U N G IN PROLONGED GESTATION
159
the same way as P20 fetuses. Placental weight (wet and dry) decreased slightly on GD 23 (not statistically significant from GD 22) and remained unchanged during prolonged gestation, in contrast to P20 placentae which decreased in weight p r o gressively with prolongation of gestation (Morris et al., 1980). Placenta DNA content, however, progressively decreased from 1.06 (mg) + 0.03 SE (N = 13) on GD 22 to 0.84 ___0.03 (N = 10) on GD 25 (P < 0.001) and were not significantly different from those treated with progesterone (P20 rats). Lung weight decreased post-term and resulted in a smaller lung in proportion to body weight. Reduction in lung dry weight appeared to be greater in P20 fetuses (GD 24, 25) than in retained fetuses. When lung weight was expressed per body weight, the retained fetuses, at GD 25, in comparison to GD 22, had a reduction of 43.3~o and 22.4~o in lung wet and dry weight, respectively, while these values in P20 fetuses were 50~o and 38.5~, respectively. Liver wet and dry weights decreased while kidney and spleen weight increased significantly during prolonged gestation much the same as in the P20 post-term fetuses. Protein, DNA, RNA and phospholipid analysis of fetal lungs at GD 21 to GD 25 are shown in table 2. As gestation was prolonged from GD 22 to 25, total lung protein, DNA, and phospholipid increased when expressed per gram lung. This resulted from the fact that the lung weight progressively decreased, mainly because of loss of pulmonary fluid. When expressed per mg lung DNA, there was a progressive reduction in total protein and RNA but no change in phospholipid content. These findings in retained fetuses resemble those observed in P20 post-term fetuses. In table 3 the total lung protein, lung phospholipid, lung DNA and the number of cells are expressed per lung. Since measurements were made on pooled samples from 2 to 4 fetuses, protein, phospholipid and DNA per lung were based on the average lung weights in that particular sample. With the exception of a small reduction in total protein with prolongation of gestation, the other parameters, such as phospholipids and DNA (cell number) were constant from term (GD 22) through GD 25. Both lung and liver glycogen contents were reduced from term to GD 25 in much the same way as previously reported for progesterone treated rats (Morris et al., 1980). Fetal blood serum glucose levels averaged 87.8 + 4.9 SD, 66.4 + 6.3 and 54.9 + 7.6, mg/100 ml on GD 23, 24 and 25, respectively. These values were slightly (about 4--8 mg) but not significantly higher than the post-term fetuses of progesterone treated (P:0) rats. In table 4 is shown the lung cell number of retained post-term fetuses from maternal rats receiving daily injections of sesame oil (control) or progesterone (P22) starting one hour after parturition on GD 22. The values for cell number, expressed per lung or per body weight, were similar in both conditions. Light microscopic studies revealed a gradual reduction in the numbers of distended terminal sacs, and atelectasis with prolongation of gestation. These findings were similar to those observed previously on post-term fetuses of progesterone treated rats (Morris et al., 1980).
7.80 _+0.17
3.97 _+ 0.08
Disaturated phosphatidyl choline
0.564 _+ 0.01
_+2.57
0.579 _+ 0.01
8.69 _+ 0.31
0.504 _+ 0.01
0.99 _+ 0.02
2.09 _+ 0.03
3.96 _+ 0.12
7.75 _+0.14
16.44 _+0.23
7.86 _+0.16
68.3
_+2.82
0.463 _+ 0.01
9.22 _+ 0.48
0.506 _+ 0.02
1.02 _+ 0.04
2.03 _+ 0.05
4.55 _+ 0.12
9.16 _+0.23
18.30 _+0.28
9.02 + 0 . 2 2
82.2
22
+2.29
_+ 0.08*
0.340 _+ 0.02 t
8.36 _+ 0.36
0.518 _+ 0.02
1.06 _+0.05
2.20 _+ 0.10
5.11
10.45 _+0.12"
21.69 _+0.22*
9.93 _+0.46
82.4
23
+2.34*
0.311 _+ 0.01*
7.79 _+0.28 ¢
0.504 _+ 0.02
1.02 _+ 0.03
2.07 _+ 0.06
6.23 _+0.13*
12.64 _+0.23'
25.68 _+0.50'
12.42 +_0.30 t
96.5
24
+_4.32 t
(6)
0.239 _+ 0.01 t
+_0.17"
0.508 _+ 0.03
1.03 _+ 0.07
2.05 _+ 0.15
7.51
(4)
(6)
(6)
(4)
(4)
(4)
(4)
_+ 0.44 + (4) 7.34 _+0.20 t
15.01
29.59 _+0.51'
15.39 _+ 0.85 + (6)
115.3
25
Data are expressed as means _+ 1 SE: each is the mean of 5 measurements except when indicated in parentheses. The data from sham operated group (GD 21) are not included in the table because of the similarities between this group and control and "retained" groups. Comparisons are made as follows: A m o n g groups on G D 21 : no difference. Between post term and term (GD 22, retained) fetuses: *P < 0.01 ; +P < 0.001 and c P < 0.05.
8.59 _+ 0.48
Lung RNA, m g / m g D N A
0.506 _+ 0.01
Disaturated phosphatidyl choline
Lung total protein, mg/mg D N A
0.99 + 0.03
Phosphatidyl choline
2.04 _+ 0.03
16.03 _+0.20
Phosphatidylcholine
Lung phospholipid mg/mg lung D N A : Total
7.85 _+0.15
_+2.80
Lung phospholipid, mg/g lung: Total
67.2
21
21
LungDNA, mg/glung
Lung total protein, mg/g tung
'Retained'
Control
TABLE 2 Biochemical measurements in fetal rats at gestation days 21 to 25
Total D N A per lung (mg) 216.9
+ 10.9
1.34 ___ 0.07
0.697 + 0.04
1.43 + 0.09
2.96 + 0.20
11.28 + 0.87
23
218.9
-1- 5.8
1.36 + 0 . 0 4
0.682 + 0.02
1.38 + 0 . 0 5
2.81 + 0 . 1 0
10.54 + 0 . 1 7
24
221.6
+ 11.2
1.37 + 0.07
0.690 + 0.02
1.41 + 0.05
2.79 + 0.04
(6)
(6)
(4)
(4)
(4)
10.30 + 0.47*(6)
25
Data are obtained from retained fetuses and are expressed as m e a n s + 1 SE; each is the m e a n of 5 measurements except when indicated in parentheses. Pre- and post-term fetuses are compared with term ( G D 22) fetuses. *P < 0.02; t p < 0.01 and * P < 0.001.
-1- 6.3
1.33 + 0 . 0 4
1.09 _ 0 . 0 3 t
DSPC per lung (mg) 214.0
0.668 + 0.01
0.551 + 0.02 t
Phosphatidyl choline per lung (mg)
+4.9 t
1.35 + 0 . 0 4
1.08 + 0 . 0 3 ~'
176.3
2.70 + 0 . 0 9
2.29 + 0 . 0 5 t
Total phospholipid per lung (mg)
Cell no., in millions per lung
12.23 + 0 . 7 2
9.50 + 0 . 4 3 *
22
Total protein per lung (mg)
21
TABLE 3 Biochemical measurements of fetal rats lungs at gestation days 21 to 25
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162
E.E. FARIDY TABLE4 Lung cell number of retained fetal rats at gestation days 23 and 24
Maternal condition
Control Progesterone treated (P22)
Per l u n g
Per g b o d y weight
23
24
23
24
218.8 ± 7.4 221.6 + 9.4
217.7 ± 10.7
34.2 + 0.82
32.8 ± 1.75
220.9 ± 6.6
33.9 + 1.06
33.4 ± 0.73
D a t a are expressed as m i l l i o n s a n d p r e s e n t e d as m e a n s _+ I SE; each is the mean of 6 m e a s u r e m e n t s . P22 = M a t e r n a l rats injected with p r o g e s t e r o n e on g e s t a t i o n day 22 (within an h o u r after p a r t u r i t i o n ) a n d o n d a y before sacrifice. C o n t r o l = M a t e r n a l rats injected with sesame oil in the s a m e m a n n e r as P22 rats.
Discussion
Surgically induced model of prolonged gestation appears to have advantages over that induced by exogenous progesterone because of the following: The procedure is well tolerated by the pregnant rat; there is no maternal mortality; the food intake and the blood glucose levels are higher; and the overall general condition of the rat is more satisfactory. Moreover, post-term placentae of progesterone treated rats (Thliveris, 1976) undergo significant morphological changes indicative of decreasing placental function. In contrast, the post-term placentae of surgically induced prolonged gestation (Jollie, 1976) show little evidence of placental damage. These could be some of the factors which have contributed in improving the general appearance of the retained fetuses. In spite of this, the retained and the post-term P20 fetuses are simil&r in many aspects, such as: increase in fetal body, kidney and spleen weights; reduction in lung and liver weights; progressive lung atelectasis; loss of lung and liver glycogen; and reduction in placenta DNA content. The main difference between the two are in the contents of lung DSPC and lung DNA (cell number), when expressed per lung or per body weight (fig. 1). The retained fetuses do not exhibit the biphasic phenomenon in lung DSPC and DNA content. While the lung of the P20 fetuses continued its growth beyond term for an additional day (GD 23, gain of 35 million cells) and subsequently regressed with further prolongation of gestation (GD 25, loss of 51 million cells), the lungs of the retained fetuses appeared to have a complete arrest of growth at term (no change in cell numbers post-term). These differing behaviours in lung growth are interesting and need further analysis. Grota and Eik-Nes (1967) analysed plasma progesterone levels in rats during normal pregnancy and lactation and showed that there was a gradual decrease in the concentration of the hormone as term of pregnancy approached followed by an increase during lactation. In the 24 h preceding parturition, the concentration of progesterone decreased significantly from 114 ng/ml of plasma (GD 21) to 10 ng/ml
FETAL LUNG IN PROLONGED GESTATION
~=
163
40[ ",,,
=" 220f ~,
""
~= ~s° t 140 ~
,
*
20"
L
0.14
I
0.8
"~. 0.6 =m
I~l........r,, " •
f
~ o.lo
U
,~ 0.4
0
0
0.08 0.2
f
21
22
2', 2'5 o s , ,oN D,Y
!
22 2'3 2',
Fig. 1. Comparison of lung cell number and dissaturated phosphatidyl choline content of retained fetuses (surgically induced prolonged gestation broken line) and P20 fetuses (post-term fetuses of rats treated with daily injections of progesterone beginning on gestation day 20 - solid line). The data for P20 fetuses are taken from our previous study (Morris eta/., 1980). In addition, the data for P20 fetuses repeated in the present study is shown by the upper solid line in left upper graph and is used to compare with retained fetuses (see text). The vertical lines represent 1SE to either side of the mean, each being the mean of 5-11 measurements. *Significantly different (P < 0 . 0 5 - P <0.001) from the P20 fetuses of identical age. The arrow indicates gestation at term.
on GD 22. Within 6 h post-partum, it rose to 43 ng/ml and remained at this level until 2 days after delivery and then rose to 123 ng/ml of plasma on Day 4 of lactation. It is possible that the retained fetuses in the present study were maintained post-term in utero under the influence of endogenous progesterone secreted during lactation. If progesterone influences the rate of growth of the fetal lung and if the rate of growth depends on the concentration of the hormone in the maternal plasma, then it follows that there would be different rates of fetal lung growth in the two models of prolonged gestation. Although there were no differences seen on GD 22, in lung development, between the retained fetuses (low levels of maternal plasma progesterone?) and the fetuses of rats receiving daily progesterone (higher levels of maternal plasma progesterone ?), and although this appeared to have ruled out the effect of exogenous progesterone p e r s e on fetal lung development, it was decided to further examine the effect of exogenous progesterone on retained fetuses post-term. The results, as shown in table 4 indicated the lung DNA content (cell number) of retained fetuses, expressed either per lung or per body weight was similar in both whether the rat received progesterone or not. These results perhaps further support the notion that, in our studies, the exogenous progesterone p e r s e did not affect the development of the fetal lung, either pre- or post-term. In view of the fact that fetal distress occurs post-term, as suggested by the release of meconium (Clifford, 1957); that cortisol levels are elevated in the umbilical cord blood of postmature infants (Nwosu e t al., 1975); that signs of increased activity
164
E. E. F A R I D Y TABLE 5 Adrenal weight of fetal rats at gestation days 22 to 25 22
23
24
25
Retained
0 . 3 5 7 + 0 . 0 1 " (18)
0.331_+0.01 t (14)
0 . 3 6 2 + 0 . 0 1 " (6)
0.359_+_0.03 "~ (7)
P20
0.406 + 0.01
0.413 + 0.01
0.444 + 0.02
0.463 + 0.020 .(6)
(18)
(11)
(11)
Adrenal weight is expressed as mg/g body weight. Data are expressed as m e a n s + 1 SE; n u m b e r s in parentheses indicate n u m b e r of fetal rats studied. Retained = fetuses in tied off uterus. P20 = fetuses obtained from rats injected with progesterone on gestation day 20 and daily thereafter until day before sacrifice. Comparisons are made as follows: Between retained and P20 fetuses at each G D : *P < 0.01 ; *P < 0.001 a n d ¢ P < 0.02. Between post-term and term ( G D 22) fetuses of P20 rats: ~P < 0.05.
are noted in the adrenal glands of post-term fetuses of progesterone treated rats (Thliveris and Connell, 1973); and that our 'clinical' observations implied that the level of fetal distress was greater in the fetuses of P20 rats than the retained fetuses, we made a preliminary study of measuring the weight of the adrenal glands in the retained and P20 fetuses. As shown in table 5, the 1}20fetuses have larger adrenals than the retained fetuses. Furthermore, in P20 fetuses, the adrenal size, in proportion to body weight, increases with prolongation of gestation in contrast to the constant value observed for the retained fetuses. If adrenal weight is indicative of its activity then the fetal blood steroids level should be higher in P20 fetuses than in retained fetuses. Is it possible that corticosteroids initially stimulated lung growth post-term (GD 23) and subsequently induced slowing of cell mitosis (Carson et al., 1973), disturbing the cell renewal rate of the lung in favour of a cell loss? An interesting phenomenon is the arrest in lung growth in retained fetuses post-term. It seems that the stimulus for lung growth has been removed. It is shown that distension increases the metabolic rate of the lung (Faridy and Naimark, 1971) and perhaps stimulates the lung growth (Thurlbeck, 1975). The terminal sacs of fetal lung contain fluid which is secreted by the lung tissue. If one of the functions of the pulmonary fluid is to distend the terminal sacs and stimulate the lung growth, it is conceivable that a reduction in the rate of secretion of pulmonary fluid, leading to lung atelectasis, would abolish the necessary stimulus for lung growth. Our observations indicate that the post-term fetal lungs do become atelectatic. Kitterman et al. (1979) have shown that the tracheal fluid production in fetal lamb decreases before spontaneous term. They have postulated that the decrease in production may be related to increased secretion of cortisol. On the basis of our results it is tempting to speculate that the cellularity and the DSPC content of the post-term fetal lung is adversely affected by the degree of fetal distress. However, irrespective of fetal condition post-term, the lungs become atelectatic and might require greater inflation pressures at birth. On the other hand, the post-term lungs which contain comparable amounts of DSPC and cells to term lungs, may not necessarily possess the functional capabilities of the normal lungs at
FETAL LUNG IN PROLONGED GESTATION
165
term. For example, the rate of synthesis and release of surfactant in these lungs at birth may be jeopardized because of a severe loss of lung glycogen and a concomittant hypoglycemia.
Acknowledgement The author expresses his appreciation to Mrs. Saniata Bucher for her excellent technical assistance.
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166 Thurlbeck, W.M. (1975). Postnatal growth and development of the lung. A m . Rev. Re,~p. Dis. 1 l I : 803 844. Vorherr, H. (1975). Placental insufficiency in relation to post term pregnancy and fetal postmaturity. A m . J. Obstet. Gynaecol. 123:67 103.