Serum variants causing the formation of double hearts and other abnormalities in explanted rat embryos

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1 /. Embryol. exp. Morph. Vol. 3, 3, pp. -9, 9 Printed in Great Britain Serum variants causing the formation of double hearts and other abnormalities in explanted rat embryos By C. E. STEELE AND D. A. T. NEW From the Physiological Laboratory, University of Cambridge SUMMARY Rat embryos explanted before organogenesis (8 days gestation) were grown in culture in homologous serum. When the serum was prepared from blood centrifuged after clotting, the embryos developed double hearts. In serum prepared from blood centrifuged before clotting had occurred, and in plasma, the embryos developed normal single hearts. The delayed-centrifuged (D.C.) serum also supported less growth of older embryos than the immediately-centrifuged (i.e.) serum. The harmful properties of appeared rapidly in contact with a normal blood clot but did not develop in contact with separated blood cells and fibrin clot. Mixtures of D.C. and r.c. sera gave results intermediate between those from the two sera alone. No significant differences were found between D.C. and in calcium or complement content but both supported better embryonic development after pre-heating. INTRODUCTION In a study of rat embryos explanted at the egg-cylinder stage and grown in culture in homologous serum, New & Daniel (99) found that most of the explanted embryos developed abnormal double hearts as a result of failure of fusion of the heart primordia. The serum used in their cultures was prepared from freshly extracted blood that was allowed to clot and stand for 8- h before centrifuging and decanting. Steele (9) obtained much improved embryonic development, with the formation of normal single hearts and a functional blood circulation, in serum decanted from blood centrifuged immediately after extraction. It is interesting that such an apparently small variation in the preparation of the serum should have such a large developmental effect and suggests that some abnormalities in vivo might result from minor variations in blood composition. At present the factors in the serum responsible for the single and double hearts are unknown and the experiments described in this paper have been aimed at obtaining further information about serum variations and their effects on embryonic development. Authors' Address: Physiological Laboratory, Cambridge CB 3EG, U.K.

2 8 Immediate C. E. STEELE AND D. A. T. NEW Fibrin clot removed YJ rev min. Freshly ^ min extracted blood Not cen trifu ned cen tri fillet Standard re\ min. blood clot Retracted min standard ck)t Fig.. Preparation of i.e. and D.C. sera. MATERIALS AND METHODS Explanation and culture methods The methods used for explantation and culture of the embryos were as described by New (9) and New, Coppola & Terry (93). Rat embryos of the CFHB strain were explanted in Tyrode saline during the afternoon of the 9th or th days of gestation, i.e. the embryos were 8[ or 9\ days post-coitum. Reichert's membrane was torn open but the visceral yolk sac and ectoplacental cone left intact. In comparing different culture media, embryos from each litter were divided between the different media to eliminate any effects of inter-litter variation. Except where stated otherwise, egg-cylinders (8^-day embryos) were incubated for 8 h in watchglass cultures with a gas phase containing % O, % CO and % N. Each watchglass contained ml medium (serum or plasma) and up to eight egg-cylinders, which attached to the floor of the watchglass by the ectoplacental cone. At the end of the culture period, heart development was classified as single, double, uncertain/intermediate, or none. Headfold (9^-day) embryos were cultured in rotating tubes for periods of 8- h in medium equilibrated with % CO in air. These embryos invariably formed normal single hearts, and development in culture was assessed by the number of new somites formed, the condition of the blood circulation, the adoption by the embryo of the foetal (ventrally curved) position and the expansion of the yolk sac. Growth was assessed by determining the protein content of the embryos by the colorimetric method of Lowry, Rosebrough, Farr & Randall (9).

3 Serum variants and double hearts 9, and plasma The sera (Fig. ) and plasma used as nutrient media were prepared from blood withdrawn from the dorsal aorta of rats anaesthetized with ether. Except where stated otherwise, the media compared in any one experiment were made from the same blood samples. Immediately-centrifuged (i.e.) serum. The blood was centrifuged at rev/ min for min immediately after withdrawal from the rat. This precipitated the cells and a white fibrin clot rapidly formed in the supernatant. The clot was carefully removed with forceps, the fluid being allowed to drain from it back into the centrifuge tube. The contents of the tube were then recentrifuged and the decanted. In some samples the recentrifuging and decanting were delayed for 8 h; these are designated '-(d)'. Delayed-centrifuged (D.C.) serum. The blood was not centrifuged at the time of withdrawal but was allowed to clot and stand overnight (approx. 8 h) at C. The clot was then broken up with a sterile glass rod, the blood centrifuged at rev/min for - min and the decanted. Plasma. Heparin (-%) or sodium oxalate (-3%) was added to the extracted blood which was immediately centrifuged at rev/min for min. No clot formed and the plasma was decanted. The and plasma were pale yellow fluids but the was sometimes pink, presumably as the result of slight haemolysis. Streptomycin was added to all media to a final concentration of /tg/ml and the media were stored at C if they were to be used within h or at - C for longer periods. Some samples of serum were pre-heated to C for 3 min before culturing, and the concentration of complement determined by the method of Demant, Capkova, Hinzova & Voracova (93). Seven series of experiments were made, with media prepared as follows: () Immediately-centrifuged (i.e.) serum, delayed-centrifuged (D.C.) serum, and plasma (8-]-, 9^-day embryos). () Reduced storage periods of clotted blood for preparation (8^-day embryos). (3) Addition of cells or clot to (8^-day embryos). () Delayed decanting of (8 - and 9^-day embryos). () Mixtures of and (8^-day embryos). () Calcium content of i.e. and D.C. sera (8^-day embryos). () Pre-heating and complement assay of i.e. and D.C. sera (8^-day embryos).

4 C. E. STEELE AND D. A. T. NEW Single heart Double heart (Often the hearts are completely separated and not touch as in this diagram.) Uncertain (heart only) Fig..'Single','double' and' uncertain' hearts, ys, Yolk sac; a,amnion; /;, headfold; ht, heart;/, foregut; s, somite. RESULTS Series. Immediately-centrifuged (i.e.) serum, delayed-centrifuged (D.C.) serum and plasma (8-j- and 9j-day embryos) After h in culture, the 8^-day embryos in all three media were at the headfold stage and were very similar in size and appearance. After 8 h, however, a difference in heart development was clearly visible, the embryos in i.e. serum and plasma tending to form single hearts and those in D.c. serum, double hearts (Figs., 3). The average width of the embryo measured across the heart region was greater in explants with a double heart (-9 mm) than in those with a single heart (- mm), but in both the yolk sac was spherical with an average diameter of -9-mm. Table shows the number of 8^-day embryos developing double or single hearts in each of the three media (data previously reported by Steele, 9). Six experiments were made to examine the growth of 9-^-day embryos in the three media. Growth was assessed by determining the final protein content of each embryo, after removal of the membranes. The results are summarized in Table. Each entry in the table gives the mean and standard error of the - embryos in one culture tube. All the culture tubes in each experiment

5 B D FIGURE 3 Rat embryos with single (A, B) and double (C, D) hearts. The embryos were explanted at 8^ days gestation and grown in culture for 8 h. Whole embryos magnified x 3, transverse sections x. Abbreviations as in Fig.. lit

6 C. E. STEELE AND D. A. T. NEW Table. Heart development of 8\-day rat embryos grown in immediatelycentrifuged (i.e.) serum, delayed-centrifuged (D.C.) serum, and plasma Heart No. of embryos Single Double Uncertain None st experiment nd experiment Plasma Table. Final protein (//g) of9\-day embryos cultured in different sera {described in the text) and plasma (Each entry gives the mean and standard error of the - embryos in one culture tube. Letters refer to the blood samples from which the sera and plasma were taken. The embryos in experiments - were grown for 8- h in culture, in experiment for h. Significant differences (P < ) between and from the same blood sample are indicated by >.) Experiment no. -(d) Plasma 3 A 3 ± C8 + B ±\ B 8±/ D 8 ± > F ± G ±3 H8±3 > A IB IB C D E F ±8 ±9 ± ±9 9 ± 9± ± G H 9 + C 8± D ±8 F ± G 3 ± contained embryos from the same litter or litters. The letters in the Table refer to the blood samples from which the sera and plasma were taken. Table shows that mean final protein of the embryos in the eight tubes of and the four tubes of plasma ranged from 3 to 8 /ig. The values for were more variable and usually lower, in six out of the nine tubes being /eg or less. In three experiments (,, ) the protein of the embryos in was significantly higher than in the from the same blood samples (A, B, H). In none of the experiments was embryo protein in significantly higher than in the corresponding. In blood samples C, D, F and G there were no significant differences between, D.C. serum or plasma.

7 Serum variants and double hearts 3 Table 3. Heart development of 8\-day embryos in D.c. serum prepared from clotted blood stored for different periods I h 3 h 8 h No. of embryos 8 9 Single Double Heart Uncertain The sera were heated at C for 3 min before culturing. None Table. Heart development of 8\-day rat embryos in i.e. added blood cells or protein clot serum pre-treated with No. of embryos Single Double Heart Uncertain None + cells -t- clot Mixed i.e. + cells and i.e. - clot Control The amount of development shown by the 9^-day embryos in culture varied with the amount of protein synthesis. For example, in Expt 3 most of the embryos in all three media attained - somites, rotated into the foetal position and developed a yolk sac 3--- mm diameter with a good blood circulation; similar development was shown by the embryos of Expt. in i.e. serum, but those in averaged only 9 somites, failed to rotate, developed smaller yolk sacs and lacked a blood circulation. Series. Reduced storage periods of clotted blood for D.c. serum preparation (8\-day embryos) Freshly extracted blood was allowed to clot (without centrifuging) and stored at C. The clot was broken up, centrifuged and the serum decanted after shorter periods (\ h an^ 3 h) than normally used for the preparation of. The effects of these sera on the heart development of 8^-day embryos were examined and compared with those of standard and. The results, summarized in Table 3, suggest that serum prepared from blood stored for as little as y h before centrifuging has already acquired some tendency to cause the formation of abnormal double hearts, and that by 3 h this effect has become accentuated almost to that of the standard D.c. serum prepared from blood stored 8 h.

8 C. E. STEELE AND D. A. T. NEW Table. Heart development of 8^-day rat embryos in -{d) prepared by delaying decanting for 8 h, compared with controls Heart st Experiment -(d) nd experiment -(d)* * embryos Heated at C Single 9 Double for 3 min before culturing. Uncertain None Series 3. Addition of cells or clot to (S J -day embryos) The cells and clot removed during the preparation of some were washed in Tyrode saline and then each recombined with the serum. The serum + cells and the serum + clot were allowed to stand for about 8 h at C and then centrifuged and decanted. The serum removed from the cells was usually pink, similar to, but the serum removed from the clot remained pale yellow. Development of 8^-day embryos in each of these two sera alone was compared with development in a mixture (in equal proportions) of the two together, and in control i.e. and D.C. sera. The results are shown in Table. All the seven embryos grown in control formed single hearts. The that had been stored with the protein clot gave similar results ( single hearts; uncertain). In each of the other two i.e. sera, one embryo developed a double heart but in the six of the embryos formed double hearts. Evidently storage in contact with blood cells or fibrin clot does not give i.e. serum the double heart-forming character of. Series. Delayed decanting of (8\- and 9\-day embryos) Samples of blood, centrifuged immediately after extraction, were left to stand in the centrifuge tubes at C for 8 h and then centrifuged again and the serum decanted. In Tables and this serum is designated '-(d)'. Table shows the results of two experiments with 8^-day embryos. The i.e. serum-(d) in the first experiment was stirred with the cells and clot between the first and second centrifugations, that in the second experiment was left unstirred. Both these sera supported development of single hearts in most of the explanted embryos. In this they resembled control and differed from. Table shows the effect of -(d) prepared from five different samples of blood (B, D, F, G, H) on protein synthesis by 9 -day embryos. In two of the

9 Serum variants and double hearts Table. Heart development of 8-\-day embryos grown in and mixed in various proportions % 8?/ o % % % % 8% % No. of embryos Single 8 3 Double Heart The sera were pre-heated at Cfor 3 min. Uncertain None Table. Calcium content of different samples of and D.c. serum; heart development of 8\-day embryos in these media (Embryos cultured in, or 3 were from the same litter as those in,, or 3, respectively.) Ca + (mg%) No. of embryos Single Double Heart Uncertain None i.e. Serum Serum Serum D.C. Serum Serum Serum samples (B, H) protein synthesis was significantly higher in -(d) than in D.c. serum. (Of the two tubes of -(d) prepared from blood sample B, one was stirred with the clot and cells during preparation, the other left unstirred; both gave similar results.) Although no trials were made with i.e. serum-(d) and control from the same blood sample, growth in these two sera appeared to be similar. Series. Mixtures of and D.c. serum {8\-day embryos) Table shows the heart development of 8^-day embryos grown in mixtures of and, compared with development in whole or whole. The addition of up to % appears to have little effect on the frequency of double hearts formed in D.c. serum, nor does the addition of up to % D.c. serum affect the frequency of single hearts in i.e. serum. In a mixture of i.e. and in equal proportions, five out of nine embryos formed double hearts and only two single hearts.

10 C. E. STEELE AND D. A. T. NEW Table 8. Effect ofpre-heating on development of explanted 8\-day embryos No. of embryos Embryonic protein (fig) mean ± S.E. Mean yolk-sac diameter (mm) Embryos with heartbeat after h Embryos with yolk sac circulation after h pre-heated 3 ± ± -3 3 Table 9. The effect of pre-heating the culture serum on development of 8\-day rat embryos heart No. of embryos Single Double Heart Uncertain None pre-heated pre-heated Series. Calcium content of i.e. and D.c. sera (8%-day embryos) The calcium ion content of three samples of and three samples of was determined by absorption spectrophotometry. Embryos of 8 days were grown in each of the six media but the type of heart development obtained appeared to be unrelated to the small differences in calcium content (Table ). Series. Pre-heating and complement assay of i.e. and D.C. sera {8\-day embryos) The protein content of embryos explanted at 8^- days and cultured in preheated was significantly higher (P < -) than in from the same batch which had not been pre-heated (Table 8). (The embryos in this experiment were cultured for h in watchglasses and then transferred to rotating tubes for a further 8 h.) Pre-heating also improved development in D.c. serum, resulting in more single hearts and fewer double hearts than in control D.c. serum (Table 9 - and see also Tables 3, and ). The pre-heating inactivated most of the complement in the sera but no significant difference could be detected between the complement content of i.e. and (Table ).

11 Serum variants and double hearts Table. Complement content of i.e. and D.C. rat sera expressed as percentage of content in standard guinea-pig serum (Assay method of Demant et al. 93.) Rat no. Serum Complement i.e. D.C. D.C. pre-heated i.e. D.C. i.e. pre-heated D.C. pre-heated i.e. D.C. i.e. D.C. i.e. pre-heated D.C. pre-heated DISCUSSION The results that have been described show that serum (D.C.) as usually prepared from clotted blood causes the formation of double hearts in rat embryos explanted before organogenesis; but if serum (i.e.) is prepared from blood centrifuged before clotting has occurred, or if plasma is used instead of serum, the embryos develop normal single hearts (series ). D.c. serum also supports less growth of older embryos than, though different samples of D.C. serum vary widely in this respect (Table ). The harmful properties of D.C. serum develop rapidly in contact with a normal blood clot (series ) but do not appear after storage in contact with separated blood cells and fibrin clot (series 3 and ). Mixtures of D.C. and i.e. sera give results intermediate between those from the two sera alone (series ). No significant differences were found between D.C. and in calcium (series ) or complement content (series ) but both supported better embryonic development after pre-heating. The factor(s) in that cause double heart development appear to be a product of normal blood clotting in which the blood cells are trapped in the fibrin coagulum; they are not produced by a cell-free clot nor by cells in suspension, even when both are present in the same serum. An example of a difference between cells held in a fibrin clot as compared with those suspended in plasma was demonstrated by Bernstein (93), who found more variability in potassium exchange by erythrocytes in the clot compared with those in suspension. But differences in potassium concentration do not seem to be the cause of the heart abnormalities. Although Steele (9) found that D.c. serum contains more than twice as much potassium as either or plasma, the addition of extra

12 8 C. E. STEELE AND D. A. T. NEW potassium to did not result in the development of double hearts. DeHaan (98a, b) showed that acetylcholine, sodium acetate and EDTA all produced double hearts in chick embryos and he suggested that these agents disrupted normal intercellular relationships by binding calcium. But in the present study, calcium concentrations in i.e. and D.C. sera were found to be similar and unlikely to be the cause of differences of heart development in the cultured rat embryos. At present it remains unknown how the formation of a normal blood clot renders teratogenic. The pre-heating of the sera in series inactivated most of the complement, as confirmed by a subsequent assay (Table ). Pre-heating both D.C. and i.e. sera gave improved development of the 8^-day embryos and suggests that in untreated sera development might be hindered by a complement-dependent immune reaction. Some of the embryos grown in pre-heated formed single hearts, suggesting that the heat treatment may remove some of the harmful factor(s) acquired from the blood clot, but further data are needed to confirm this. The double hearts result from a failure of the two heart primordia to fuse and it is interesting that Severn & Holyoke (93) have recently described failures of heart formation in human development which they attribute to a similar mechanism ('primary cardia bifida'). The cause of these failures remains unknown but in view of the present findings the possibility seems worth examining that such congenital defects might arise in vivo as a result of any small haemorrhage and clotting of maternal blood in the uterus. This work was supported by the Medical Research Council. We are grateful to C. W. S. Howe for performing the complement assay. REFERENCES BERNSTEIN, R. E. (93). Serum and plasma preparation for potassium analysis: Effects of anticoagulants, storage time and temperature before separation, and haemoloysis. S. Afr. J. Med. Sci. 8, 99-. DEHAAN, R. L. (98 C?). Modification of cell-migration patterns in the early chick embryo. Proc. natn. Acad. Sci. U.S.A., 3-3. DEHAAN, R. L. (98). Cell migration and morphogenetic movements. In A Symposium on the Chemical Basis of Development (ed. W. D. McElroy and B. Glass), pp Baltimore: Johns Hopkins Press. DEMANT, P., CAPKOVA, J., HINZOVA, E. & VORACOVA, B. (93). The role of histocompatability--linked Ss-Slp region in the control of mouse complement. Proc. natn. Acad. Sci. U.S.A., LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (9). Protein measurement with the folin phenol reagent. /. biol. Chem. 93, -. NEW, D. A. T. (9). Methods for the culture of post-implantation embryos of rodents. In Methods in Mammalian Embryology (ed. J.C.Daniel), pp San Francisco: Freeman. NEW, D. A. T., COPPOLA, P. T. & TERRY, S. (93). Culture of explanted rat embryos in rotating tubes. /. Reprod. Fert. 3, 3-38.

13 Serum variants and double hearts 9 NEW, D. A. T. & DANIEL, J. C. (99). Cultivation of rat embryos explanted at - and 8- days of gestation. Nature, Loud. 3, -. SEVERN, C. B. & HOLYOKE, E. A. (93). Human acardiac anomalies. Am. J. Obstet. Gynec., STEELE, C. E. (9). Improved development of'rat egg-cylinders' in vitro as a result of fusion of the heart primordia. Nature New Biology, Lond. Ill, -. (Received January 9) E M B 3

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