/ . Embryol. exp. Morph. Vol. 36, l,pp. 133-144, 1976 133

Printed in Great Britain

Comparison of growth in vitro and in vivo ofpost-implantation rat embryos

By D. A. T. NEW1, P. T. COPPOLA1 AND D. L. COCKROFT1

From the Physiological Laboratory, Cambridge

SUMMARYRat embryos explanted at early head-fold stage and grown in vitro by improved culture

methods were compared with littermates in vivo. Very similar rates of growth and differ-entiation were obtained over a period of 48 h, while the embryos developed to around the25-somite stage.

INTRODUCTION

Methods for studying the development of mammalian embryos in vitro are ofvalue only to the extent that the growth and differentiation of the embryosresemble that in vivo. In recent years, techniques have been devised for growingpost-implantation rat and mouse embryos in vitro throughout the period oforganogenesis (reviews by New, 1973; Kochhar, 1975; Steele, 1975). Theembryos are explanted with their membranes and grown in serum, or in mixturesof serum and synthetic culture media, with carefully regulated levels of oxygena-tion. Under these conditions the explanted embryos differentiate well but it hasusually been found that growth, as determined by rate of protein synthesis, isslower than in vivo (Berry, 1968; Robkin, Shepard & Tanimura, 1972; New,1973; Cockroft, 1973, 1976).

A likely explanation for the retarded growth of the older embryos is that theylack a functional allantoic placenta. But the allantoic blood circulation does notbecome established in normal development until about the 17-somite stage (11days of gestation in the rat). It seems possible that at the earlier stages oforganogenesis, normal rates of growth might be supported simply by an im-proved nutrient medium combined with appropriate concentrations of O2 andCO2 in the gas phase. It has recently been found that the nutrient serum can beimproved by preparing it from blood centrifuged immediately after withdrawalfrom the donor rat and by heat-inactivating it before use (Steele, 1972; Steele &New, 1974; New, Coppola & Cockroft, 1976), and that young embryos benefitfrom a reduction of oxygen in the gas phase to 5 % (M. H. Jacobs, unpublished;New et ah 1976). In this paper, rat embryos explanted at early head-fold stage(9| days of gestation) and grown under the improved culture conditions arecompared with similar embryos grown in vivo.

1 Authors' address: Physiological Laboratory, Downing Street, Cambridge CB2 3EG, U.K.

134 D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT

4 embryosgrown

Fig. 1. Selection of embryos from the two uterine horns for comparison of growthin vivo and in vitro.

MATERIALS AND METHODS

Embryos were obtained from rats of CFHB strain during the morning of the10th day of gestation (9£ days post coiturri). In the embryos at this stage, neuralfolds were just beginning to appear but no other organs had yet formed (Fig.3 A). An allantoic rudiment was present as a small bud at the hind end of theembryo. The length of the whole conceptus (embryo + membranes), excludingthe ectoplacental cone, was about 1-5 mm.

Comparisons of growth in vitro and in vivo were made between embryos fromthe two uterine horns of each rat. The rat was anaesthetized with ether and onehorn of the uterus drawn out through a small incision in the abdominal wall.This horn and its blood vessels were ligated at the cervical end between thefirst and second implantation sites (Fig. 1). The blood vessels were also ligatedat the ovarian end. The ovary and uterine horn were then cut free except for theimplantation site next to the vagina, which was left in place. The whole opera-tion took about 20 min and within half an hour the animal had recovered fromthe anaesthetic and was actively moving round the cage and licking itself. Carewas taken at all stages of the operation to avoid disturbing the uterine horn andblood vessels of the opposite side. To check whether the operation had anyeffect on the growth of the embryos in vivo, we (i) compared the embryos at 32and 48 h after operating with those of similar age in unoperated animals, and(ii) allowed development in a few operated rats to proceed to term and com-pared the newborn young with those of unoperated animals.

It is known that in mice, foetal weight can be affected by both number andposition of the foetuses in the uterine horn (Healy, McLaren & Michie, 1961;McLaren, 1965). We therefore used in this study only rats that had 5-10embryos in each horn with a difference of not more than three embryos betweenthe two horns, and the embryos to be compared were taken from similar posi-tions in the two horns (Fig. 1). The mean and standard error of the number of

Rat embryos in vitro and in vivo 135

Table 1. Embryo growth in operated and unoperated rats

Mean somites Mean protein (jig)per embryo per embryo

Embryos at 9£ days+ 32 h from:Operated ratsUnoperated rats (1973)

Embryos at 9\ days+ 48 h from:Operated ratsUnoperated ratsUnoperated rats (1973)

15-714-5

24-624-723-5

5551

219223190

implantation sites in the horns used to provide embryos for growth in vitro was7-7 ±0-3, in those for growth in vivo 7-3 ±0-3.

The embryos for culturing were explanted with the visceral yolk-sac andectoplacental cone intact but with the Reichert membrane torn open (Fig. 3 A).They were incubated at 38 °C in small cylindrical stoppered bottles rotated con-tinuously at 40-50 rev/min for periods of 32 or 48 h (New, Coppola & Terry,1973). The bottles were of 30 ml capacity containing 4 ml of culture serum, butsome of the embryos were transferred after 24 h of incubation to bottles of60 ml capacity containing 8 ml of serum. Usually, four embryos were placed ineach bottle. The culture serum was obtained from CFHB rats, without regardto the sex or age of the animals, and pooled before use. The serum was alwaysprepared from blood centrifuged immediately after extraction from the rat (I.C.serum of Steele & New, 1974) and was heat-inactivated (56 °C for 30 min)before being added to the culture bottles. The gas phase was 5 % O2/5 % CO2/90 % N2 for the first 22-24 h, followed by 20 % O2/5 % CO2/75 % N2. Some ofthe cultures received a further gassing at 32 h with 20 % O2/5 %CO2/75 % N2 or40 % O2/5 % CO2/55 % N2.

Development of the embryos, in vitro and in vivo, was assessed by the numberof somites formed, the condition of the blood circulation, the closure of theneural tube, the adoption by the embryo of the foetal (ventrally concave)position, and the fusion of allantois and chorion. Growth was assessed bymeasuring the crown-rump length and the yolk-sac diameter, and by determin-ing the protein content of the embryo (without its membranes) by the colori-metric method of Lowry, Rosebrough, Farr & Randall (1951).

RESULTS

Growth of embryos in operated and unoperated rats

(i) Table 1 shows the mean values for somite number and protein content ofall the embryos grown in vivo in one horn of the uterus for 32 h and 48 h afterremoval of the opposite horn (i.e. all the in vivo embryos listed in Tables 2 and3). These values are compared with those of embryos of the same age in

136 D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT

Table 2. Comparison of embryos grown for 32 h in vivo and in vitro

(All values are means per embryo)

Ratno.

1

2

3

4

5

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

Numberof

embryos

44

44

44

33

44

Yolk sacdiam (mm)

3-42-8

3-42-9

3-42-9

2-22-5

3-72-9

Crown-rump

length (mm)

2-22-3

2-22-2

202-3

Notdetermined

2-42-5

Somitenumber

17016-2

14-815-8

15-515-5

12-7150

170180

Protein (/*g)(mean ± S.E.)

59±751 ±346±452 ±5Not

determined38 + 643 ±176±274 ±6

unoperated rats. Data for the latter were obtained from four unoperated pregnantrats examined as part of the present series of experiments, and from a curve ofnormal embryo growth prepared previously (New, 1973). The comparison givesno indication that growth of the embryos in the operated rats is retarded.

(ii) Embryonic development in four operated rats was allowed to continue toterm. Birth of normal young occurred at 22, 22, 24 and 24 days of gestationrespectively, with a mean litter number of 8-2 and a mean weight for eachnewborn of 6-1 g. Four unoperated animals all gave birth at 21 to 22 days ofgestation, with a mean litter number of 12-0 and a mean weight per newborn of6-0 g. The slight difference in the time of birth of the two groups makes ituncertain how far the weights of the newborn can be used for exact comparisonof rates of embryo growth. However, the results do not suggest that eithergrowth or development of the embryos in the operated rats was affectedsignificantly.

Comparison of embryos in vitro and in vivo after 32 h

Thirty-two hours after the time of explantation, all the embryos in culture hada good yolk-sac blood circulation. Those embryos with 17 or more somites, andan occasional embryo with 16 somites also showed a rudimentary blood circu-lation in the allantois. In all the 19 embryos grown in vivo, and in 15 out of the19 grown in vitro, the allantois was now tightly fused with the chorion; in theremaining four it had enlarged but remained 'free' in the extra-embryoniccoelom. Apart from this, all the embryos appeared to be normal and withoutmalformations (Fig. 3B).

Table 2 gives further details. In four out of the five rats used for the experi-ment, the mean yolk-sac diameter of the embryos in vitro was less than in vivo.

Rat embryos in vitro and in vivo 137Scries

250

200

oc. 150

100

50

65'

-h

82"

D

89 ", 112

Treatment/'// vitro

At 24 h

At 32 h

-0",'. O, 20",; o2

60 ml bottle8 ml new serum

20",; O,

20% O,

20",;O2

40 •;,; o .

20 ", ;o,

60 ml bottle

8 ml new serum

40';,, O2

Fig. 2. Comparison of final embryo protein in vivo and in vitro, after five differentculture treatments. Each rectangle in the histogram shows the mean and standarderror of 12 embryos. Further details of the five series of embryos (A-E) are given inTable 3.

But there was no significant difference in crown-rump length, somite number, orprotein content.

It appears that, for the first 32 h after explantation, the rate of growth anddevelopment of the embryo itself is the same in vitro as in vivo. But in some ofthe explants there may be slight abnormalities of the embryonic membranes.

Comparison of embryos in vitro and in vivo after 48 h

Fifteen rats were used in this experiment, providing a comparison between 60embryos grown for 48 h in vitro with 60 left to continue growth for the sameduration in vivo. Nearly all the explanted embryos still had a good yolk-sacblood circulation at the end of the culture period and many also had a circu-lation in the allantois. In about 20 % of the cultured embryos, the allantois hadfailed to fuse with the chorion. All the embryos had turned into the ventrally-concave 'foetal' position, but in a few (< 10 %) the posterior tip of the trunkwas bent sideways as though the final stages of turning had not been completed.These two abnormalities appeared to be unrelated. In view of the sensitivity ofneural tube development to variations in the culture conditions (New et al.1976), all the cultured embryos were examined particularly carefully forabnormalities in this region, but no failures of closure of the tube or othermalformation of brain or spinal cord were observed.

138 D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT

Table 3. Comparison of embryos grown for 48 h in vivo and in vitro

(All values are means per embryo)

RatSeries no.

Number Crown-of Yolk sac rump Somite Protein (/*g)

embryos diam (mm) length (mm) number (mean ± S.E.)

B

D 10

11

12

13

14

15

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

In vivoIn vitro

3-73-4

4-441

4-54-3

4-33-8

4-43-6

4 13-4

403-9

4-34-2

4-23-6

4-641

4-44-3

4-24-2

4-34-3

3-940

4-23-9

3-32-7

3-93-6

403-83-83-6

403-33-530

3-43-3

3-63-7

3-73-4

3-83-9

3-83-8

3-73-8

2-53-8

3-43-7

3-43-4

23022-526-726-7

26-525-5

24-8240

24-323-5

23-3220

23-823-5

24-824-5

24-822-3

25-025-3

25-825-3

24-825-3

25026-8

23-325-3

23-323-3

178 ±7101 ±12292+13190±3

255 ±12179±3

226+19173 ±7

252 ±12163 ±13

195 ±21136 ±6

177 ±6144±9

182± 13184± 13

191 ±3122 ±9

233 ± 14201 ±6

274 ±8209 ±13

223 ±40240 ±11

224 ±15268 ±17

201 ± 17233 ±10

182±5177 ±5

Five variants of the culture procedure were tried, each using three of thefifteen rats, as follows:

Series A. The culture bottles were regassed with 20 % O2 (i.e. 20%O2/5%CO2/75%N2) a t24h but not subsequently; the serum was not renewed.

Series B. Regassed with 20 % O2 at 24 h and transferred to 8 ml fresh serumin a larger (60 ml) bottle.

Series C. Regassed with 20 % O2 at 24 h and at 32 h; serum not renewed.Series D. Regassed with 20 % O2 at 24 h, and with 40 % O2 at 32 h; serum not

renewed.

Rat embryos in vitro and in vivo

Table 4. Comparison of different culture treatments on embryosgrown for 48 h in vitro

139

Treatment atA

24 h

20%O2

20%O2

60 ml bottle8 ml new serum

20 % O2

20%O2

60 ml bottle8 ml new serum

32 h

20%O2

[20 %o,J

40%O2

40%O2J

No. ofembryos

12

12

12

12

Yolk sacdiam(mm)

3-8

3-8

3-7

3-7

Crown-rump

length (mm)

3-6

3-6

3-4

3-5

Somitenumber

23-9

23-6

23-9

23-9

Protein (/*g)(mean ± S.E)

162 ± 11

180± 11

171 ±12

181 ±11

Series E. Regassed with 20 % O2 at 24 h, and transferred to 8 ml fresh serumin a larger (60 ml) bottle. Gassed with 40 % O2 at 32 h.

Figure 2 and Table 3 give further details. In Series A, the average number ofsomites in the cultured embryos was similar to that of the comparable embryosin vivo but the relative protein content was only 65 %. In Series B to E theprotein of the cultured embryos progressively increased until in Series E it was112 % of that of the controls. In each of Series A, B and C the protein contentsof the embryos in vitro were significantly different from those in vivo (P < 0-01);in Series D and E the differences were insignificant (P > 0-1).

Comparison of different treatments for embryos cultured for 48h

To obtain further information about the variants of culture method used inthe previous experiment, 48 head-fold embryos were explanted and grownunder four different culture treatments, care being taken that embryos from thesame litter were always divided equally between the four treatments.

The culture details and the results are summarized in Table 4. All the cultureswere regassed at 24 h and at 32 h. The results show slightly increased embryoprotein when the O2 level was raised at 32 h to 40 %. Embryo protein was alsohigher in those cultures that were transferred at 24 h to 60 ml bottles with 8 mlnew serum. This may have been an effect of the fresh serum, or an effect of theincreased gas space providing more oxygen, or a lower CO2 concentrationresulting in a serum pH closer to normal values; at the end of the culture, thepH of the serum in the 60 ml bottles was 7-4-7-5, in the 30ml bottles 7-1-7-3.

But in general the results of the four treatments were similar, and the differ-ences of embryo protein content are not statistically significant (P > 0-1).

It should be noted that three of these treatments resemble those given to thecultured embryos of Series C, D and E of Fig. 2. None is comparable withSeries A or B.

140 D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT

Fig. 3. (A) Head-fold stage embryos as explanted at 9\ days of gestation. (B)Embryos grown for 32 h in vivo (upper row) and in vitro (lower row). The embryosare all from rat 1 of Table 2. Photographed after removal of the embryonic mem-branes. (C) Embryos grown for 48 h in vivo (upper row) and in vitro (lower row).The embryos are all from rat 14 of Table 3. Photographed after removal of theembryonic membranes. All the photographs are at the same magnification.

Rat embryos in vitro and in vivo 141

* 4 ** -

Fig. 4. (A) Optic vesicle of embryo after 48 h in culture. The retinal area and lensepidermis are thickening, and invagination of the optic cup is beginning. (B) Opticvesicle of a littermate of the embryo shown in (A), which was allowed to develop forthe corresponding period in vivo. The vesicle is rounded and no retinal or lensthickening has taken place. (C) Auditory vesicle of the embryo shown in (A).Closure is just being completed. (D) Auditory vesicle of the embryo shown in (B),which is at a similar stage of development to that shown at (C). All the photographsare at the same magnification.

142 D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT

Comparative histology of embryos grown in vitro and in vivo

A histological examination was made of 12 embryos (obtained from threerats) that had been grown in vitro for 48 h, and of a further 12 embryos, litter-mates of the cultured embryos, that had been allowed to develop in vivo for thecorresponding period. The culture schedule was the same as for the embryos ofSeries E, Fig. 2. No abnormal tissue degeneration was found in any of theembryos examined, though small differences in degree of development of corre-sponding in vitro and in vivo embryos were noted.

In the cultured embryos from one rat, one embryo was retarded, but the otherthree were more advanced than the corresponding in vivo embryos with respectto eye development (Fig. 4 A, B) and closure of the posterior neuropore. Inother respects, such as closure of the auditory vesicles (Fig. 4C, D), develop-ment in vitro was similar to that in vivo. The cultured embryos from the secondrat were slightly less well developed in all respects than the in vivo embryos,though the differences were within the range of variation often found in embryosfrom the same rat. For the third rat, optic vesicle development and closure ofthe posterior neuropore were again slightly more advanced in vitro than in vivo,though in other respects the embryos were equivalent.

It appears that histological differentiation of the embryos grown in culture isvery similar to that of the littermates in vivo.

DISCUSSION

The results show that the rates of growth and differentiation of all theembryos grown for 32 h in culture were similar to those of littermates in vivo(Table 2 and Fig. 3 B).

The embryos grown in culture for 48 h were more variable. This is perhapsnot surprising in view of the rapidly increasing demands of the embryo on theculture system. At 32 h after explanation, the embryos are at about the 16-somite stage. During the period 32-48 h, littermates in vivo synthesize aboutthree times as much protein as during the previous 32 h. A culture system that issufficient to support normal rates of growth up to 32 h may be inadequate forlonger periods. The inadequacies may include diminished O2, excessively highCO2 and low pH, and insufficient nutrients or accumulating waste products.

Another reason why it becomes more difficult to maintain normal rates ofgrowth with increasing age of the embryo, is the failure of development of theallantoic placenta. At 32 h, blood is just beginning to circulate in the allantois,and from then on the embryos in vivo can be presumed to have the support of afunctional allantoic placenta. In embryos in culture, there may be an embryonicblood circulation in the allantois but there is no maternal blood, nor any systemof spaces allowing passage of the culture medium through the placenta, and it isvery unlikely that the allantois provides any support for growth of the embryo.

Rat embryos in vitro and in vivo 143Our results show that if incubation of the 32-h cultures is continued to 48 h,

without any other treatment, the embryo has only about 65 % of the proteincontent of littermates in vivo (Fig. 2, Series A). But if the culture bottles are re-gassed at 32 h with 20 % O2/5 % CO2/75 % N2, final embryo protein in vitrorises to over 80 % of that in vivo (Series C). Further small gains can be made byraising the O2 level at 32 h to 40 % and by transferring the embryos during theculture period to a larger bottle with fresh serum; the protein content of theembryos in vitro then becomes very similar to that of the controls in vivo(Series D, E).

The regassing at 32 h is clearly important. The fact that a gas mixture con-taining 40 % O2 gave slightly better results than 20 % O2 (Fig. 2, Series C, D;and Table 4) suggests that at least part of the effect on embryonic growth resultsfrom a raised oxygen level. But these concentrations must give oxygen tensionsin the culture serum of around 320mmHg and 160mmHg respectively, farhigher than those found anywhere in the uterus. It seems likely that theirbeneficial effect on embryos in vitro is to cause increased transport of oxygen tothe embryo by the yolk-sac, which compensates for the lack of a functionalallantoic placenta.

Regassing also removes the excess CO2 resulting from embryonic respiration,and by restoring the CO2 level to 5 %, helps to maintain the serum pH at around7-4. Both these effects are potentially beneficial but our data are insufficient toestablish whether they were significant. Cultures in which the serum was renewed(and increased) at 24 h showed improved embryonic growth (Fig. 2, Series Bcompared with A, and E compared with D; also Table 4) but these were alwaystransferred to 60 ml bottles with a larger gas volume; it is uncertain thereforewhether the improvement was primarily the result of the renewed serum or offactors - respiration, pH, etc. - related to the gas volume.

Is embryonic differentiation at 48 h the same in vitro as in vivo ? Table 3 showsthat under optimum culture conditions (Series D and E) the number of newsomites formed by the embryos in culture is the same as in the littermates in vivo.In overall appearance, the two groups of embryos are usually indistinguishable(Fig. 3C). Histologically, the resemblance is also very close (Fig. 4). The onlyabnormalities that we have observed were in a few embryos (< 10 %) where theposterior tip of the trunk was bent sideways - possibly a result of incompleteturning of the embryo (Deuchar, 1975)-and in about 20% of the embryoswhere the allantois failed to fuse with the chorion.

We conclude that the available culture methods enable rat embryos explantedat head-fold stage to be grown in vitro for 48 h at the same rate of growth anddifferentiation as in vivo. We have described in another paper (New et ah 1976)the further growth of these embryos for 72-95 h.

We would like to thank Mrs S. M. Jackson for valuable technical assistance and theMedical Research Council for financial support.

144 D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT

REFERENCES

BERRY, C. L. (1968). Comparison of in vivo and in vitro growth of the rat foetus. Nature,Lond. 219, 92-93.

COCKROFT, D. L. (1973). Development in culture of rat foetuses explanted at 12-5 and 13-5days of gestation. J. Embryol. exp. Morph. 29, 473-483.

COCKROFT, D. L. (1976). Comparison of in vitro and in vivo development of rat foetuses.DevlBiol.48, 163-172.

DEUCHAR, E. M. (1975). Reconstitutive ability of axial tissue in early rat embryos afteroperations and culture in vitro. J. Embryol. exp. Morph. 33, 217-226.

HEALY, M. J. R., MCLAREN, A. & MICHIE, D. (1961). Foetal growth in the mouse. Proc. R.Soc. B 153, 367-379.

KOCHHAR, D. M. (1975). The use of in vitro procedures in teratology. Teratology 11, 273-287.LOWRY, O. J., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measure-

ment with the folin phenol reagent. / . biol. Chem. 193, 265-275.MCLAREN, A. (1965). Genetic and environmental effects on foetal and placental growth in

mice. / . Reprod. Fertil. 9, 79-98.NEW, D. A. T. (1973). Studies on mammalian fetuses in vitro during the period of organo-

genesis. In The Mammalian Fetus in vitro (ed. C. R. Austin), pp. 15-65. London: Chapmanand Hall.

NEW, D. A. T., COPPOLA, P. T. & COCKROFT, D. L. (1976). Improved development of head-fold rat embryos in culture resulting from low oxygen and modifications of the cultureserum. / . Reprod. Fertil. 48 (In the Press).

NEW, D. A. T., COPPOLA, P. T. & TERRY, S. (1973). Culture of explanted rat embryos inrotating tubes. J. Reprod. Fertil. 35, 135-138.

ROBKIN, M. A., SHEPARD, T. H. & TANIMURA, T. (1972). A new in vitro culture technique forrat embryos. Teratology 5, 367-376.

STEELE, C. E. (1972). Improved development of rat 'egg-cylinders' in vitro as a result offusion of the heart primordia. Nature New Biology lil, 150-151.

STEELE, C. E. (1975). The culture of post-implantation mammalian embryos. In The EarlyDevelopment of Mammals (ed. M. Balls & A. E. Wild), pp. 61-79. Cambridge UniversityPress.

STEELE, C. E. & NEW, D. A. T. (1974). Serum variants causing the formation of doublehearts and other abnormalities in explanted rat embryos. / . Embryol. exp. Morph. 31,707-719.

{Received 13 January 1976; revised 12 March 1976)