Exp. Path., Ed. 12, S. 100-108 (1976)

Teratology Research Laboratory, Department of Anatomy, University of Manitoba, Winnipeg,Canada

Influence of prostaglandin F2 <> on the development of thechick em.bryo: morphological and biochemical aspects

By G. B. P. MATTHEWS and T. V. N. PERSAUD

With 5 figures

(Received October 9,1975)

Key words: prostaglandin F2<>; chick embryo; development

Summary

Fertile eggs were incubated for 48, 72 or 96 hours. Experimental eggs were treated with 10, 40or 100 Ilg PGF2<> in 10 III of saline while controls received either the saline alone or were left unopened.All embryos were recovered at day 9 of incubation. The number of dead embryos were noted, andall living embryos were staged for growth and examined for external malformations. Treatment ofthe embryos with the prostaglandin did not significantly affect their development. A small numberof embryos incubated for 96 hours and treated with 40llg PGF2<>, received 50llCi of 3H-thymidineor 3H-proline at periods of 3, 6 or 24 hours following exposure to the prostaglandin. The uptake 6fthe radiolabelled precursors. as well as the total protein content, was significantly reduced in thetreated embryos compared to the corresponding controls.

In the past decade, intense and diversified investigations with the prostaglandins haveled not only to an exponential growth of the literature, but also to their implication in alarge number of physiological and pathological processes (BERGSTROM et aI. 1968, OESTER­LING et al. 1972, KARIM and HILLIER 1974, GOLDBERG and RAMWELL 1975). From earlierstudies there were also indications that the prostaglandins may be of importance in severalareas of clinical application. However, other than in the field of human reproduction, manyof the earlier promises of prostaglandins have yet to be fulfilled (ANDERSON and SPEROFF1973, PERSAUD 1974, CRAIG 1975).

For therapeutic mid-term abortions, the prostaglandins have displayed distinct advan­tages over other methods of inducing abortion and have been successfully used in a largenumber of clinical trials. Following their use as a once-a-month contraceptive a significantnumber of pregnancies have occurred (BEHRMAN and ANDERSON 1974).

Surprisingly, the wide-spread clinical use of these substances has preceeded their precli­nical toxicological evaluation (KIRTON and CARLSON 1973). In view of the known adverseaction of certain drugs during pregnancy, the teratogenic risk of the prostaglandins warrantsfurther investigation (PERSAUD 1974, GOLDBERG and RAMWELL 1975).

The chick embryo is considered a highly sensitive and reliable experimental model forteratological studies (MATTHEWS et aI. 1975). The present pa,per describes the effects ofprostaglandin F 2<> (PGF2<» on the developing chick embryo. In addition, the influence ofPGF2<> on the uptake of tritium-labelled thymidine and proline, as well as on the totalprotein content, in the chick embryo was investigated.

Materials and methods

Fertile eggs (N = 307) of a hybrid White Leghorn flock (University of Manitoba) were incubatedon their sides at 37.5 °C and 60 % relative humidity, and treatments were carried out at either 48,72 or 96 hours of incubation.

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The eggs were swabbed with 70 % ethyl alcohol and windowed by means of a dental sanding disc.Both shell membranes were removed and 10 III of Howard's chick saline solution containing 10, 40or lOOl1g of prostaglandin F2" was dropped onto the dorsal surface of the embryo from a microlitersyringe. Rigid control of the experiment involved the use of matched saline and unopened controlsfor each batch of eggs incubated in order to establ ish possible effects of the experimental technique,transportation of the eggs, and incubation.

After trEatment, the eggs were incubated for a further period until total incubation had reachednine d,tys. At this time al1 embryos were recovered, staged according to the method of HAMILTONand HHIRl'RGElt (1951). and examined for external malformations. The number of dead embryoswas also noted.

Statistical analysis of the data was carried ont using the Chisquare test for mortality and mal­fOfll;ation rates and the Student's test and factorial analysis of variance for embryonic growth.

In a further series of experiments, eggs were incubated for 96 hours under the same conditionsas before and treated a,s previollsly described with 10111 Howard's saline containing 40l1g PGF2".

Control eggs were treated with the solvent. After further incubation of 3, 6 or 24 hours the embryosreceived 50 III sterile distilled water containing 50l1Ci L-proline-5-H3 (AmershamjSearle, specificactivity 18 Cijmmol, radioactive concentration 1 mCijml) or 50 flCi tritiated thymidine (methyl-H3)(AmershamjSearle, specific activity 5 Cijmmol, radioactive concentration 1 mCi/ml). The number ofeggs in each series of experiments were 30 and 33 respectively. The substance was dropped onto thedorsal surface of the chick embryo as described above. Radiochemical purity of the compoundswas greater than 99 % as determined by thin-layer chromatography and dilution analysis withL-proline for the labelled proline, and by thin-layer chromatography and paper chromatographyfor the tritiated thymidine.

Thirty minutes later treated and control embryos were recovered. Embryos from a given experi­mental group (N = 4-7) were pooled and kept at -10°C until subsequent determination of theirlevels of radioactivity. The embryos were then thawed, homogenized and weighed (10-20 mg;four samples from each pooled embryonic homogenate).

Each sample was dissolved in 0.6 ml NCS tissue solubilizer and neutralized with 40 A 9 N aceticacid. Ten m!. of a scintillation cocktail (5 g ppo; 0.5g dimethyl POPOP; 250 ml ethylene glycolmonomethyl ether; 750 ml toluene) were added and the samples were counted four times for 30 se­conds in a Beckman LS 150 Liquid Scintillation Counter. The results were expressed as dpm/mgof wet tissue weight.

Results were statistically analyzed using the Student's t-test.In addition, the total protein content in embryonic tissue homogenates following treatment with

40l1g PGF2", at 96 hours incubation, was determined by the method of LOWRY et al. (1951).Relative comparison of the total protein content of experimental and control tissues for all pe­

riods of incubation following treatment with PGF2" was carried out using the Student's t-test.

Results

Morphological Studies

Embryos treated with PGF2" at 48 and 96 hours of incubation showed significantlyhigher mortality rates than the corresponding unopened controls (tables 1 and 2). However,in the saline-treated control embryos the incidence of embryonic deaths was not signifi­cantly different from that of the prostaglandin-treated embryos. In neither of these groupswere there any significant differences in mortality due to treatment with PGF2" at differentdose-levels. Embryos exposed to PGF2" at 72 hours incubation showed no significant dif­ferences in mortality from either the unopened or saline control embryos (table 3).

Table 1. Mortality of chick embryos following PGF2" treatment at 48 hours incubation

Treatment Number of embryos Number of dead Mortality rate (%)incubated embryos

unopened control 70* 4 5.7**saline control 39 9 23

10 fig PGF2" 10 2 2040 fig PGF2" 20 4 20

lOOl1g PGF2'" 10 5 50

* common to all incubation periods** significantly lower than 48 hour saline controls (p < 0.02) and pooled 48 hour experimen­

tal embryos (p < 0.025)

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Table 2. Mortality of chick embryos following PGF2", treatment at 96 hours incubation

Treatment Number of embryos Number of dead Mortality rate (%)incubated embryos

unopened control 70* 4 5.7**saline control 40 11 28

10 fig PGF2", 10 2 2040 fig PGF2", 20 7 35

100 fig PGF2", 10 2 20

* common to all incubation periods** significantly lower than saline controls (p < 0.005) and the three groups of experimental embryos(p < 0.05)

Table 3. Mortality of chick embryos following PGF2", treatment at 72 hours incubation

Treatment Number of embryos Number of dead Mortality rate ('Yo)incubated embryos

unopened control 70* 4 5.7saline control 43 1 2.3**10 fig PGF2", 8 2 2540 fig PGF2", 17 2 12

100 fig PGF2'" 10 1 10

* common to all incubation periods** significantly lower than corresponding 48 and 96 hour values (p < 0.005)

The day of treatment did not significantly influence embryonic mortality at any givendose level, but mortality in the saline-treated embryos varied significantly with the timeof treatment. Significantly fewer deaths occurred in embryos treated with saline at 72 hoursincubation than in embryos treated at 48 or 96 hours (table 3). The incidence of embryonicdeaths in the later two groups did not differ significantly from each other.

Three embryos treated with 40 Itg PGF2'" at 72 hours incubation showed ectopia viscerum.Exencephaly, bilateral anophthalmia and reduced beak as well as ectopia viscerum wereseen in one embryo treated with 40 Itg PGF2'" at 96 hours incubation (figs. 1 and 2). How­ever, similar malformations were also detected in embryos of the control as well as of otherexperimental groups. Statistical evaluation of the incidence of abnormal embryos in allgroups revealed no significant differences with respect to the time of treatment or dose-levelsof the prostaglandin (tables 4, 5 and 6).

The mean growth rate of unopened control embryos was significantly higher than thatof embryos treated with Howard's saline at 48 or 72 hours of incubation (p < 0.05 andp < 0.001 respectively). Saline controls treated at 96 hours developed equally as well astheir unopened controls (table 7).

Embryonic growth was significantly greater in embryos treated with saline at 96 hoursincubation than in the 48 and 72 hours incubation groups of saline-treated embryos (p <0.025 and p < 0.001 respectively). The mean growth rate of 48 hour saline control embryoswas significantly higher than that of the 72 hour saline-treated controls (p < 0.005).

Different dose levels of PGF2'" did not produce significant differences in embryonicgrowth (table 8). Growth in the saline control embryos was not significantly different fromthat of the corrseponding experimental embryos.

The time of treatment however, did correlate with significant variations in embryonicgrowth. Embryos treated with all dose levels at 96 hours incubation showed equal or highergrowth rates compared to the 48 hour (p < 0.01) and 72 hour (p < 0.001) treatment groups.The growth rate of embryos treated with all dose-levels of prostaglandin at 48 hours incu­bation was significantly higher than that of the corresponding embryos treated at 72 hoursincubation (p < 0.01).

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Fig. 1. Normal 9 day old chick embryo (1 a). Severely malformed 9 day embryo displaying exen­cephaly, bilateral anophthalmia and reduced beak as well as ectopic viscerum (1 b).

Fig. 2. Normal 9 day old chick embryo after being cleared with benzyl benzoate for skeletal exa­mination (2a). Malformed embryo from above (2b) following clearance with benzyl benzoate. Notereduced upper beak.

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Table 4. Frequency of abnormal embryos following PGF2" treatment at 48 hours incubation

Treatment Number of liveembryos recovered

Number of malformed Malformation rate (%)embryos

unopened controlsaline control

10 f.tg PG F2"

40 fig PGF2"

100 !!f5 PGF2"

66*308

165

82o2o

126.7o

13o

* common to all incubation periods

Table 5. Frequency of abnormal embryos following PGF2" treatment at 72 hours incubation

Treatment Number of live embryos Number of malformed Malformation rate (%)recovered embryos

unopened controlsal ine control

lOfLg PGF2"

40 fLg PGF2"

100;Ug PGF2"

66*426

159

86141

1214172711

* common to all incubation periods

Table 6. Frequency of abnormal embryos following PGF2" treatment at 96 hours incubation

Treatment Number of live embryos Number of malformed Malformation rate (%)recovered embryos

unopened controlsaline control

10 f.tg PGF2"

40 fLg PGF2"

100 f.tg PGF2"

66*298

138

8113o

123.4

1323o

* common to all incubation periods

Table 7. Growth of chick embryos left unopened or treated with Howard's saline at 48, 72 or 96hours incubation*

Treatment 48 hours 72 hours 96 hours

llnopened** 34.9 ± 0.036 34.9 ± 0.036 34.9 ± 0.036controls N = 65 N = 65 N = 65sal ine treated 34.8 ± 0.092 34.4 ± 0.010 35.0 ± 0.035controls N = 30 N = 42 N = 29

* Growth expressed in terms of mean Hamilton and Hamburger stage on recovery (± S.E.)** common to all incubation periodsN = number of embryos observed

Table 8. Growth of chick embryos following PGF2" treatment - 48, 72 or 96 hours incubation

Treatment 48 hours 72 hours 96 hours

10 f.tg PGF2" 34.8 ± 0.16 34.7 ± 0.21 34.9 ± 0.13N = 8 N = 6 N = 8

40 f.tg PGF2" 34.2 ± 0.19 34.1 ± 0.13 35.0 ± 0.000N = 16 N = 15 N = 13

100 fLg PGF2" 34.8 ± 0.20 34.1 ± 0.11 34.8 ± 0.16N = 5 N = 9 N =8

* Growth expressed in terms of mean Hamilton and Hamburger stage on recovery ± S.E.N = number of embryos observed

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0=Controlp<

.08 • =Experimental

0 ......_ ......-3 hrs. 6 hrs. 24 hrs.

Time after PG-treatment at which HTdR was administered**

100

QI::I

'"'" -- '*- 0QI~~

" 50en QIE ::I...... "0EQ. >

"0 "0c ...-0 CQI 0~ ~

Fig. 3. Effect of PGF2<x on 3H-thymidine uptake in chick embryos at 96 hours incubation. *Signifi­cance of the difference between control and experimental values as determined by the Student'st-test. ** 50 flCi 3H-thymidine (specific activity 5 Cijmmol) in 50 fll sterile distilled water.

140p<O.lo*

o =Control

• =Experimental

O~-~

3 hrs. 6 hrs. 24 hrs.

Time after PG-treatment at which 3H-proline was administered**

G>;:)

::: 100

- *j ~II

01 G>

~~E aQ. >"00c: .::~ & 50~ ~

Fig. 4. Effect of PGF2<x on 3H-proline uptake in chick embryos at 96 hours incubation. * Signifi­cance of the difference between control and experimental values as determined by the Student'st-test. ** 50 flCi 3H-proline (specific activity 18 Cijmmol) in 50 fll sterile distilled water.

105

200 D=Controlp<0.10

• =Experimental...c:~c: *0u 0c: 0

GI "0 GI 100;)...0a. >

GI (5>·z .::c c:

""ii 0a:: u

03 hrs. 6 hrs. 24 hrs.

Recovery of Embryos

Fig. 5. Effect of PGF2<> on total protein content of chick embryos at 96 hours incubation. * Signifi­cance of the difference between control and experimental values as determined by the Student'st-test.

Biochemical Studies

Incorporation of tritium-labelled thymidine in chick embryos was significantly reducedfollowing treatment with 40 f-Lg PGF2<>, compared to that of the controls (fig. 3). Relativeuptake of the labelled precursor was significantly reduced when it was administered 3 hoursafter treatment with prostaglandin, while at 6 hours and 24 hours, the reduction in theincorporation was less significant.

Compared to the controls, the uptake of tritium-labelled proline was significantly reducedin embryos six hours after they were treated with prostaglandin (fig. 4). At 24 hours thisdifference was less significant. After three hours a negative correlation was evident whichwas shown to be of little significance (p < 0.10).

The total protein content of embryonic tissues was significantly reduced following treat­ment with 40 f-Lg PGF2<> and re-incubation for periods of 3 and 24 hours (fig. 5). The proteincontent of the prostaglandin-treated embryos was greater than that of controls in the groupthat was re-incubated for 6 hours following treatment with the prostaglandin. This differen­ce, however, was not significant (p < 0.10).

Discussion

PERSAUD et al. (1973) reported that PGE2 (20-100 f-Lg) produced a relatively high in­cidence of embryonic death in the chick when administered at 48 hours incubation. However,the differences in mortality rates between saline- and prostaglandin-treated embryos werenot significant. Administration of PGE2 at 48 und 72 hours incubation induced a relativelyhigh incidence of malformed embryos compared to the controls, which was significant onlyat the earliest incubation period (48 hours) and at the highest dose level (100 f-Lg PGF2<»'

The growth of the chick embryo was not significantly affected by PGE2 treatment.In contrast, however, the results of the present investigation have shown that PGF2<>

is not deleterious to the developing chick embryo. Neither the growth of the embryo, morta­lity nor the malformation rate was significantly affected following treatment with the pro­staglandin at all dose-levels and at the different periods of incubation.

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The uptake of 3H-thymidine was significantly reduced following treatment of the embryoswith the prostaglandin at two of the three time intervals. Similar observations were madeby THOMAS et al. (1974) who investigated the influence of different concentrations of PGEon the rate of cell replication in human tissue culture. It is found that the amount of pro­staglandin synthesized showed an inverse relationship to the rate of all growth and cellproliferation.

Embryonic incorporation of tritium-labelled proline, a measure of protein synthesis, wassignificantly reduced following treatment with 40,ug PGF2'" in two of three periods. Anestimation of the total protein content in the same tissues revealed a decrease in proteinlevels, also at two of the three observation periods.

KISCHER (1969, 1970) in extensive studies of PGB, effects on chick embryonic skin cul­tures found that it caused rapid maturation of the epidermis and placed a complete organo­genic block on the development of feathers. At the ultrastructural level he observed areas ofdermo-epidermal disjunction continuous with complete perforations of the epidermis anddisruption of the pattern of mesenchymal collagen following PGB1 treatment.

Evidence of such prostaglandin-induced alterations in the essential processes of cellgrowth and differentiation and connective tissue formation add significance to the presentfindings of PGF2", effects on protein and DNA synthesis and total protein content in chickembryonic tissues.

Although PGF2", at the dose-levels used proved to be nonteratogenic when administeredto developing chick embryos, significant reductions in cell replication and protein synthesisa pparently did occur. In view of these findings and previous observations of prostaglandinaction on other developmental systems (PERSAUD 1974) further teratological studies areindicated.

Acknowledgements

This work was supported by the Faculty of Medicine, University of Manitoba and the MedicalResearch Council of Canada. We thank Dr. R. MANN and Mr. P. CHOW for helpful discussions, and:1\'[s. ROBERTA BIEDRON for typing the manuscript.

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Authors' address:

Teratology Research Laboratory, University of Manitoba, Department of Anatomy, Bannatyneand Emily, Winnipeg, Manitoba, Canada R3E OW3.

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