/ . Embryol. exp. Morph. Vol. 41,pp. 111-123. 1977
\\\
Printed in Great Britain © Company of Biologists Limited 1977
Growth of opossum embryos in vitro during
or gano genesis
By D. A. T. NEW,1 M. MIZELL 2 AND D. L. COCKROFT 1
From the Physiological Laboratory, Cambridge,
and the Laboratory of Tumor Cell Biology, Tulane University, New Orleans
SUMMARY
Opossum embryos, explanted between primitive streak and late fetal stages, were grown
in culture for periods of 20-30 h. Many of the explants had a good heartbeat and blood
circulation in embryo and yolk sac after 12 h, and a few after 24 h. Growth of the embryos
included formation of the neural tube and body flexures, increase in the number of somites,
differentiation of the limbs and digits, and development of the amnion and allantois. Embryos
explanted during the last day of gestation showed persistent and vigorous body movements
in culture, particularly of the forelimbs, head and tongue.
INTRODUCTION
In recent years, methods have been developed for growing rat and mouse
embryos in culture during the period of organogenesis, i.e. throughout the
second week of gestation (reviews by New, 1973; Kochhar, 1975; Steele, 1975).
Although these methods already have important applications for the study of
developmental mechanisms in mammals (e.g. Berry, 1971; Morriss & Steele,
1974; Deuchar, 1975, 1976; Cockroft & New, 1975; Robkin, Shepard & Dyer,
1976), their value would be much enhanced if growth of the embryos could be
supported for longer periods, to late fetal stages or beyond. At present the
limiting factor is the failure of the complex allantoic placenta to develop in
culture. The simpler yolk sac of the explanted rat or mouse embryo grows well
and, with its network of blood capillaries, acts as a respiratory and nutritive
organ supporting growth of the embryo until the stage of early limb development. But further growth requires the extra support of the allantoic placenta
and until a culture system has been devised that includes, or substitutes for, this
placenta, it seems unlikely that more advanced development of rodent embryos
can be obtained in vitro.
An alternative approach is to develop culture methods for a mammal that
relies more on the yolk sac and less on the allantois. This suggests one of the
marsupials, of which the most readily available in the Northern hemisphere is
1
Authors' addresses: Physiological Laboratory, Cambridge CB2 3EG, U.K.
Author's address: Laboratory of Tumor Cell Biology, Tulane University, New Orleans,
La. 70118, U.S.A.
2
112
D. A. T. NEW, M. MIZELL AND D. L. COCKROFT
Didelphys marsupialis virginicma, the common American opossum. The gestation period of this animal is only 12f days and as many as 15-20 or more embryos can often be obtained from each pregnant female. The allantois is present
only as a simple sac that enlarges with urine during the last 3 days of gestation
and probably has no placental function. But the yolk-sac, which in rats is about
7 mm in diameter on the 13th day of gestation, develops in the opossum to
over 20 mm in diameter, with folds closely apposed to those of the uterine
endometrial surface.
The opossum young are born at a stage of development corresponding about
to that of the mouse foetus of the same age, except that the forelimbs, pancreas,
and a few other organs are precociously developed. The newborn attach to
teats in the maternal pouch and are accessible for studies (e.g. Burns, 1945;
Miller, Block, Rowlands & Kind, 1965; Mizell & Isaacs, 1970; Jurgelski,
Hudson, Falk & Kotin, 1976; Hindes & Mizell, 1976) on stages of development
which are usually hidden in the uterus of eutherian mammals. If culture methods
could be devised for opossum embryos as successful as those now available for
rodents, it might be possible to conduct long-term experiments on embryos
subjected to a test treatment at an early stage in culture and then reared as
pouch young. In a preliminary trial, New & Mizell (1972) obtained limited
growth of opossum embryos explanted at 10 and 11 days of gestation. The
present paper reports the results of a more extensive study.
MATERIALS AND METHODS
Despite some remarkable successes in breeding Didelphys (e.g. McCrady,
1938; Feldman & Self, 1973; Jurgelski & Porter, 1975) and the smaller Marmosa
opossums (Barnes & Wolf, 1971) in captivity, they are still much more difficult
to maintain in self-perpetuating colonies than the common laboratory animals.
We therefore used only Didelphys opossums recently caught in the wild. The
animals, trapped in Florida, were purchased from a dealer during the breeding
season in the early part of each year from 1974 to 1976 and housed in the
Laboratory of Tumour Cell Biology, Tulane University, New Orleans. All the
females were found to have pouch young on arrival. At different times during
the period February to April, the young were removed and the females placed
with males to mate during the first post-lactational oestrus, following the procedure of Mizell, Ramsey, Warriner & Spencer (1970). The females were then
sent by overnight plane to London and immediately taken, under quarantine,
to Cambridge.
Fifteen to eighteen days after removal of the pouch young, the animals
were opened under penthrane anaesthetic, blood was withdrawn from the dorsal
aorta to provide culture serum, and the ovaries and uterus were excised. Each
uterine horn was opened under Hanks saline (with 70 mg/1 bicarbonate to give
pH 6-5-7-0) by cutting with fine scissors through the muscle wall along the
Opossum embryos in vitro
113
antimesometrial side where the tissue is free of large blood vessels. The endometrium was then gently torn open with forceps to expose the embryos which
varied in age, in different animals, from 1\ to Yl\ days of gestation.
Up to the time of appearance of the limb-buds at 9% days of gestation, the
embryos, with their yolk-sacs, are surrounded by a keratinous shell membrane
(Tyndale-Biscoe, 1973) and lie free in the uterus. These embryos were transferred
with a wide pipette directly to the culture vessels.
Embryos of 10 days and older have lost the shell membrane and adhere to the
endometrium by the vascular area of the yolk-sac, which comprises about a
third to a half of the yolk-sac surface. The strength of adhesion increases with
age but with care, yolk-sacs of even 11^-day embryos could be freed without
damage to the vascular area. A satisfactory procedure was first to pull off the
entire endometrium (with embryos) from the uterine muscle and then, under
low magnification and transmitted light, gently to pull each yolk-sac free.
Besides their adhesion to the endometrium, the yolk-sacs of embryos older than
10 days are fused to each other in the avascular region and these junctions
cannot be pulled apart. To separate these embryos, most of the yolk-sacs were
cut in the avascular region, so that each explant consisted of the embryo/fetus
with the entire vascular area of the yolk-sac but only part of the avascular region
together with the amnion and the small allantoic sac.
During the last day of gestation (i.e. after 11^ days) the yolk sacs are so
firmly attached to the endometrium that we were unable to remove them without damage. But some embryos at this stage were successfully explanted with
the vitelline vessels ligated to minimize loss of blood from the area vasculosa.
Such explanted embryos lacked the support of a yolk-sac but by this stage the
allantois, which was explanted intact, had enlarged to a sac 8-15 mm in diameter and was completely covered with a network of blood capillaries which
probably assisted respiration in culture.
The time between removal of the uterus from the opossum and the beginning
of incubation of the explanted embryos in culture was 1-3 h. For most of this
period the embryos were maintained at 5-10 °C but were allowed to warm to
room temperature during the later stages of explantation.
Glass-stoppered reagent bottles (of two sizes, 60 ml and 250 ml) were used
as culture chambers. About one-fifth of the volume of the bottle was filled with
culture medium and embryos, and the remainder gassed with the required
O 2 /CO 2 /N 2 mixture. Embryos younger than 9 days were cultured in 2-3 ml of
medium per embryo with 10-40 % O 2 and 5 % CO 2 in the gas phase. Embryos
of 9 days or older were cultured in 8-12 ml of medium per embryo with 95 %
O2/5 % CO 2 in the gas phase (New & Mizell, 1972). The bottles were laid
horizontally on rollers and rotated continuously at 30-40 rev./min during
culture. A few of the older embryos were cultured in a 'circulator' system as
described for rat embryos by Cockroft (1973).
The incubation temperature was 35 °C, to conform with normal opossum
114
D. A. T. NEW, M. MIZELL AND D. L. COCKROFT
body temperature (Tyndale-Biscoe, 1973). The various culture media used (Table
1) included untreated or heat-inactivated (56 °C for 30 mins) opossum serum,
and mixtures of '199' tissue-culture medium with 20 % serum and 0-5-2-0 g/1
bicarbonate. The plasma expander Ficoll 70 (Pharmacia Fine Chemicals) was
added at 60 mg/ml to some of the media with' 199' to raise the osmolarity of the
macromolecular content to that of whole serum. A few cultures were als.o made
in Hanks or Tyrode saline with 20 % serum. Aseptic precautions were taken
during explantation and culture, and all culture media contained 50/tg/ml
streptomycin.
The cultures were usually examined at intervals of 2-3 h during the first 12 h
of incubation and then at intervals of 6 h. Survival and development in culture
were assessed by the condition of the heart-beat, blood circulation, yolk-sac and
allantois, and by the changes in the external features of the embryo/fetus.
Growth in many of the older embryos was determined by measurements of
crown-rump length and by assays of total fetal protein (excluding the membranes) by the colorimetric method of Lowry, Rosebrough, Farr & Randall
(1951).
The embryonic development of the opossum has been described in detail in
the valuable monograph of McCrady (1938). However, as this monograph* is
not widely available, the following brief indication of some of the relevant
McCrady stages is given for reference:
Stage 21 (7^ days). Primitive streak, notochord and medullary plate. Yolk
sac 4 mm diam., surrounded by shell membrane.
Stage 23 (8 days). Head-fold. 4 somites.
Stage 25 (8^ days). Neural tube forming. Fusion of lateral heart tubes. 12-13
somites. Yolk sac 8 mm diam.
Stage 27 (9 days). Heart begins beating and blood circulates. Cervical flexure.
Lung buds. Amnion forming.
Stage 28 (9^ days). Forelimb-buds. Primary (concave) lumbar flexure. Anterior
neuropore and otocysts closed. Mesonephric tubules and liver diverticulum. Allantois rudiment.
Stage 30 (10 days). Forelimbs paddle-shaped. Hind limb-buds. Secondary
(convex) lumbar flexure. Tail forming. Amnion complete. Shell membrane
lost and area vasculosa of yolk-sac now adheres to endometrium.
Stage 31 (10^ days). Forelimb digital ridge. Allantois 2 mm diam. Yolk-sac
20 mm diam.
Stage 32 (11 days). Forelimb digital buds. Hind limb club-shaped.
Stage 33 (11J days). Forelimb digits. Hind limb paddle-shaped. Crown-rump
length 8-0 mm. Allantois 10 mm diam. Yolk sac 25 mm diam.
Stage 34 (12 days). Forelimb digits with claws. Hind limb digital ridge. Epitrichium covers eyes, ears and sides of mouth. Oral shield formed.
Stage 35 (12^ days). Oral shield lost. Crown-rump length 10-11 mm. Birth.
Opossum embryos in vitro
115
RESULTS
Fourteen pregnant opossums were used in this study. The number of embryos
in each animal varied from 6 to 23 with an average of 14. The gestation age
varied between 1\ and \2\ days, with 8/14 animals at 10^-11^- days, and indicated that oestrus and mating had occurred 4-8 days after removal of the pouch
young with a peak at 5-6 days, in close agreement with the findings of Hartman
(1923), Renfree (1974) and Feldman & Ross (1975).
Table 1 summarizes the results from 98 embryos taken from 11 of the opossums and grown in culture for 20-30 h. The table shows that many of the embryos maintained a blood circulation for 6-12 h and some for over 24 h, and
that there were substantial increases in embryo protein content during the
culture period. Further details are as follows:
Embryos explanted at stages 21-22. Head process embryos, yolk-sacs
4-0-4-5 mm diam {opossum 1). 16 embryos
Most of the yolk-sacs remained fully expanded within the shell membrane
for over 8 h. Those in whole serum looked normal while those in the 199/serum
formed several small ' blisters' on the surface. But when the culture was ended
at 24 h, all the yolk sacs in whole serum had collapsed away from the shell
membrane while 5/8 of those in 199/serum were still expanded. The embryos
in 199/serum had attained Stage 23, with 3-5 somites, a well-marked medullary
plate and head folds. The better development in 199/serum may have resulted
from the lower pH (final pH 6-0) as compared with that in whole serum (final
pH 7-2).
Embryos explanted at stages 22-23. Embryos with 2-4 somites,
yolk-sacs 5-5-6-5 mm diam. {opossum 2) 16 embryos
None of the embryos developed beyond the stage at explantation and many
of the yolk-sacs had collapsed after \ h (in the 199 medium), or after 2 h (in the
Tyrode medium). The shell membrane remained distended. Reasons for this
poor result as compared with litter 1 may have included (i) a delay of about \ h
during explantation, when the embryos were examined in Hanks saline and were
seen to form rapidly enlarging 'blisters' in the yolk-sac, (ii) the higher O2
concentration (20-40 %) in the gas phase of the culture, and (iii) the higher pH
(final pH 7-2-7-3) of the culture media.
Embryos explanted at stages 26-27. Embryos with 12-20 somites,
yolk-sacs 9-13 diam. {opossums 3 and 4) 17 embryos
Most of these embryos developed in culture and attained stage 28. A beating
heart was formed and a blood circulation which persisted in culture for over
18 h (in whole serum) or for 12-18 h (in the 199 media). Prominent anterior
27
(9d)
30
(10 d)
4
32-33
33
(11* d)
33
(11* d)
7
8
9
30-31
(10-10* d)
6
5
3
2
21-22
(7*d)
22-23
(8d)
26
(8*-9 d)
1
no.
Opossum
199+0-5 g/l+s
Serum
199+2 g/l+s
Tyrode+s
199+1 g/l + s+f
Serum
Serum (H.I.)
199+2 g/1
199+2 g/l+s
199+1 g/l + s
199+1 g/l+s + f
Serum (H.I.)
199 + 2 g/l+s
199+2 g/l+s
Hanks+2 g/l+s
Tyrode+s
Tyrode+s
199+1 g/l+s+f
Serum
Serum (H.I.)
199 + 0-5 g/l + s
Tyrode+s
199+0-5 g/l+s
199+0-5 g/l+s
4
4
4
2
3
2
2
2
2
1
2
2
1
2
2
2
3
1
2
1
95
95
95
95
95
95
20-40
20-40
10
(%)
embryos
Culture medium
Oxygen
of
Number
OO OO OO OO
Initial
stage
(McCrady)
o
o
o
o
o
L
o
o
c
c
o
o
o
o
c
c
o
c
o
o
o
Amnion
c
c
c
c
c
c
c
c
c
c
o
o
o
Yolk-sac
0-2
6-12
12-18
12-18
6-12
6-12
6-12
>24
6-12
0-2
6-12
12-18
18-24
18-24
12-18
12-18
6-12
12-18
18-24
0-2
(h)
Blood
circ.
3305
3340
2745
1005
815
Mean
initial
protein
Og)
4940
1240
1315
1340
1370
5140
4320
3930
6395
5060
1290
1160
1340
Mean
final
protein
Og)
31
31-32
31-32
32
33
33
32-33
33-34
33
33
33-34
23
22
22-23
22-23
28
28
28
28
28
31-32
31-32
32
30-31
Final
stage
(McCrady)
Table 1. Growth and development of opossum embryos explanted at different ages and grown in different culture media
o
o o o o
H
o
r
o
o
o
d
d
r
N
W
f
m
H
p
>
2
1
1
1
4
3
7
3
199+0-5 g/l+s
199+0-5 g/1
199 + 0-5 g/1
Tyrode+s
199 + 2 g/l+s
199+1 g/l+s
199+2 g/l+s
199+2 g/l+s
95
95
(%>
embryos
Culture medium
Oxygen
of
Number
o
o
L
o
0
c
c
0
0
0
0
0
o
c
Amnion
o
o
Yolk-sac
>24
3-6
6-12
>24
3-6
3-6
6-12
6-12
Blood
circ.
(h)
8000
4790
Mean
initial
protein
(/*g)
Blood circ. (h) = maximum number of hours embryonic circulation maintained in culture.
(.) = not determined. Protein = protein content of embryo without membranes.
Abbreviations:
Culture Medium
g/1 = g/litre bicarbonate
s = 20 % opossum serum
f = 6 % Ficoll plasma expander
H.I. = heat-inactivated.
Yolk-sac and amnion
c = left closed and intact
o = torn open
L = vitelline vessels ligated
—= not yet formed (amnion).
34
11
(12 d)
33-34
(Hi—12 d)
10
9 (cont.)
Opossun l
no.
Initial
stage
(McCrady)
Table 1 {cont.)
8825
5340
4020
4000
5680
6450
6590
Mean
final
protein
(/*g)
33-34
33
33
33-34
33-34
33-34
34
34
Final
stage
(McCrady)
I
118
D. A. T. NEW, M. MIZELL AND D. L. C O C K R O F T
Fig. 1. Embryos from opossum no. 3 as explanted (left) and after 26 h in culture
(right). Photographed after removal of embryonic membranes.
Fig. 2. Embryo from opossum no. 4 after 22 h in culture. Area vasculosa covers
nearly half the yolk-sac. Transparent membrane is the shell membrane, torn open.
Fig. 3. Embryos from opossum no. 5 as explanted (left) and after 21 h in culture
(right). Photographed after removal of embryonic membranes.
Opossum embryos in vitro
Fig. 4. Embryos from opossum no. 8, as explanted (left) and after 26 h in culture
(right). Closed sac is the allantois; crumpled membrane is the area vasculosa of the
yolk-sac.
Fig. 5. Embryos from opossum no. 1.1, as explanted (left) and after 11 h in culture
(right). The cultured embryo showed spontaneous movements of headandforelimbs.
Photographed after removal of membranes.
119
120
D. A. T. NEW, M. MIZELL AND D. L. COCKROFT
limb-buds appeared and the embryonic axis developed the lumbar flexure
(Fig. 1). There was continued extension of the amnion and expansion of the
yolk-sac and shell membrane (Fig. 2).
Embryos explanted at stages 30-31. Forelimb paddle-shaped.
Allantois 1-2-1-5 mm diam {opossums 5 and 6) 14 embryos
The yolk-sacs were now adhering to the endometrium (the shell membrane
had disappeared) and were attached1 to each other; they were separated at
explantation and opened in the avascular area. Half the embryos maintained a
blood circulation for over 12 h in culture. The embryos in whole serum and
Tyrode media developed better than those in the Hanks or 199 media (Table 1).
Digital buds formed on the forelimb. Somites behind the hind limb-bud
increased from 5-6 to 8-13 (Fig. 3). The allantois increased in diameter to
2-0-4-5 mm. The protein content of the foetus increased by 30-60 %.
Embryos explanted at stages 32-33. Hind limb paddle-shaped.
Allantois 8-10 mm diam {opossums 7, 8, 9) 18 embryos
Most of these embryos showed a blood circulation for several hours in
culture, and in the best it was maintained for over 24 h. The hind limbs developed a digital ridge and the allantois increased in diameter to 13-15 mm (Fig
4). Development of the anterior part of the embryos was retarded and no oral
shield or epitrichium was formed. The embryos enlarged considerably in culture
(Fig. 4) and the protein content of some almost doubled, but part of this increase may have been the result of oedema.
Oedema developed quickly in culture (it could be observed after two hours in
some embryos) and was particularly conspicuous round the neck and shoulders.
The oedema appeared to be reduced in the media with the higher macromolecular content (whole serum, or 199/serum with Ficoll) but was not eliminated.
Haemorrhage was also common and occurred within the embryo as well as
from the membranes.
Several of the better-developed embryos became very active and showed
movements of the tongue, trunk, limbs and tail which persisted to the end of the
culture period (24-30 h).
Embryos explanted at stages 33-34. Allantois 12-15 mm diam
{opossums 10 and 11) 17 embryos
The yolk-sacs could not be separated from the endometrium without damage.
Most of the embryos were cultured in bottles with the (damaged) yolk-sacs.
Three embryos from opossum 11 were cultured in circulators with most of the
yolk-sac cut away and the vitelline vessels ligated. No significant difference was
noted between the embryos in bottles and those in circulators.
Many of the explanted embryos maintained a blood circulation for several
Opossum embryos in vitro
121
hours, and the protein content increased over the culture period. No signs of
oedema were observed.
Several of the embryos showed persistent and vigorous body movements in
culture. One embryo removed from culture after 11 h (Fig. 5) and placed in
cold saline continued to make active movements of the forelimbs, head and
tongue; when the mouth was touched with a pair of forceps, the tongue was
depressed and there were apparent attempts to suck the forceps.
DISCUSSION
The results show that opossum embryos can be grown in culture for periods
up to 30 h at all stages of development from the primitive streak to the late
fetus. (The possibility of growing younger embryos has not been excluded
but this study was concerned only with the period of organogenesis and younger
embryos were not examined.) Many of the explanted embryos maintained a
blood circulation for several hours, some for over 24 h. Development of the
embryos in culture was often equivalent to 12-24 h in vivo. Particularly interesting was the appearance of active movements in the older foetuses, including at
least one instance of an apparent sucking reflex, suggesting that it may eventually be possible to rear opossums from fetuses grown in culture and transferred to the pouch.
Padykula & Taylor (1971) have described changes of the opossum endometrium during pregnancy which indicate secretory and transport activity by
the glands and surface epithelium increasing after the 10th day. Renfree (1975)
found the protein content of opossum endometrium exudate to be similar both
in concentration and in electrophoretic distribution to that of serum, except
that the exudate contained a few prealbumins absent from the serum. Too little
exudate can be extracted from the uterus to provide a culture medium for any
but the youngest embryos and we therefore used serum in most of our cultures,
usually combined 1:4 with medium 199, Tyrode or Hanks saline with varying
amounts of bicarbonate. In general, development of the embryos in diluted
serum was as good as in whole serum, but in one culture which contained no
serum (two embryos of opossum 9) growth was very poor. Most of the other
variations in the media had no discernible effect on development, but low pH
appeared to benefit the youngest embryos and a high macromolecular content
in the medium reduced oedema in the stage-33 embryos.
The oxygen tension in the uterine artery of most mammals is about 90-100
mmHg (Comline & Silver, 1975), equivalent to a culture gas phase containing
about 12 % O 2 . The oxygen levels used for most of these cultures were therefore
much higher than the embryos would experience in the uterus. They were based
on our previous results (New & Mizell, 1972) which showed better development
of 11-day opossum embryos in 95 % O 2 than in 20 % O 2 . Such high oxygen
levels may temporarily compensate for a reduced respiratory surface, e.g. a
122
D. A. T. NEW, M. MIZELL AND D. L. COCKROFT
damaged yolk-sac, but after a time are themselves harmful to embryonic tissues
(New & Coppola, 1970; New, Coppola & Cockroft, 1976) and may ultimately
be a factor limiting embryonic growth.
The present study was designed to examine the growth of opossum embryos
explanted at all stages of organogenesis and incubated under several different
culture conditions. Although most of the embryos showed some development
in culture, the results suggest that embryos explanted at about 8-^-9 days of
gestation (stages 26-27) are particularly promising for future work. These lie
free in the uterine cavity and, unlike the older embryos, can be explanted rapidly
and with the entire yolk sac intact. They have shown consistently good growth
in culture and it would be interesting now to study their development in a
wider range of culture conditions and under lower oxygen concentrations.
We would like to thank Mrs S. M. Jackson and Mr B. S. Riar (Cambridge) and Mrs Laura
Charbonnet and Ms Christi Mortensen (Tulane) for valuable technical assistance. Financial
support was provided by the Medical Reseacrh Council, London and by United States Public
Health Service Grant No. CA-011901 from the National Cancer Institute, and a grant from
the Cancer Association of Greater New Orleans.
REFERENCES
R. D. & WOLF, H. G. (1971). The husbandry of Marmosa mitis as a laboratory
animal. Int. Zoo Yearbook 11, 50-54.
BERRY, C. L. (1971). The effects of an antiserum to the contractile proteins of the heart on the
developing rat embryo. /. Embryol. exp. Morph. 25, 203-212.
BURNS, R. K. (1945). Bisexual differentiation of the sex ducts in opossums as a result of
treatment with androgen. /. exp. Zool. 100, 119-140.
COCKROFT, D. L. (1973). Development in culture of rat foetuses explanted at 12-5 and 13-5
days of gestation. /. Embryol. exp. Morph. 29, 473-483.
COCKROFT, D. L. & NEW, D. A. T. (1975). Effects of hyperthermia on rat embryos in culture.
Nature, Lond. 258, 604-606.
COMLINE, R. S. & SILVER, M. (1975). Placental transfer of blood gases. Br. med. Bull. 31,
25-31.
DEUCHAR, E. M. (1975). Reconstitutive ability of axial tissues in early rat embryos after
operations and culture in vitro. J. Embryol. exp. Morph. 33, 217-226.
DEUCHAR, E. M. (1976). Regeneration of amputated limb-buds in early rat embryos. /.
Embryol. exp. Morph. 35, 345-354.
FELDMAN, D. B. & Ross, P. W. (1975). Methods for obtaining neonates of known age from
the Virginia opossum (Didelphis marsupialis virginiana). Lab. Anim. Sci. 25, 437-439.
FELDMAN, D. B. & SELF, J. L. (1973). Establishment of a helminth-free opossum colony.
Lab. Anim. Sci. 23, 855-857.
HARTMAN, C. G. (1923). The oestrous cycle in the opossum. Am. J. Anat. 32, 353-421.
HINDES, R. D. & MIZELL, M. (1976). The origin of immunoglobulins in opossum embryos.
Devi Biol. 53,49-61.
BARNES,
JURGELSKI, W., HUDSON, P. M., FALK, H. L. & KOTIN, P. (1976). Embryonal neoplasms in
the opossum: A new model for solid tumours of infancy and childhood. Science, N. Y. 193,
328-331.
W. & PORTER, M. E. (1975). The opossum (Didelphys virginiana Kerr) as a biomedical model. III. Breeding the opossum in captivity: Methods. Lab. Anim. Sci. 24,
412-425.
KOCHHAR, D. M. (1975). The use of in vitro procedures in teratology. Teratology 11, 273-287.
JURGELSKI,
Opossum embryos in vitro
123
O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, A. J. (1951). Protein measurement with the Folin phenol reagent. /. biol. Chem. 193, 265-275.
MCCRADY, E. (1938). The Embryology of the Opossum. American Anatomical Memoirs No.
16. Philadelphia: Wistar Institute of Anatomy and Biology.
MILLER, J. F. A. P., BLOCK, M., ROWLANDS, D. T. & KIND, P. (1965). Effect of thymectomy
on hematopoietic organs of the opossum 'embryo'. Proc. Soc. exp. Biol. Med. 118, 916—
921.
MIZELL, M. & ISAACS, J. J. (1970). Induced regeneration of hindlimbs in the newborn opossum. Am. Zool. 10, 141-155.
MIZELL, M., RAMSEY, D. E., WARRINER, R. A. & SPENCER, R. (1970). Laboratory development and use of the American opossum Didelphys virginiana. Am. Zool. 10, 539.
MORRISS, G. M. & STEELE, C. E. (1974). The effect of excess vitamin A on the development
of rat embryos in culture. /. Embryol. exp. Morph. 32, 505-514.
NEW, D. A. T. (1973). Studies on mammalian fetuses in vitro during the period of organogenesis. In The Mammalian Fetus in vitro (ed. C. R. Austin), pp. 15-65. London: Chapman
& Hall.
NEW, D. A. T. & COPPOLA, P. T. (1970). Effects of different oxygen concentrations on the
development of rat embryos in culture. J. Reprod. Fert. 21, 109-118.
NEW, D. A. T. & MIZELL, M. (1972). Opossum fetuses grown in culture. Science, N. Y. 175,
533-536.
NEW, D. A. T., COPPOLA, P. T. & COCKROFT, D. L. (1976). Comparison of growth in vitro
and in vivo of post-implantation rat embryos. /. Embryol. exp. Morph. 36, 133-144.
PADYKULA, H. A. & TAYLOR, J. M. (1971). Ultrastructural differentiation of the endometrium
of the opossum (Didelphis marsupialis virginiana) during pregnancy. Anat. Rec. 169,394-395.
RENFREE, M. B. (1974). Ovariectomy during gestation in the American opossum, Didelphis
marsupialis virginiana. /. Reprod. Fert. 39, 127-130.
RENFREE, M. B. (1975). Uterine proteins in the marsupial Didelphis marsupialis virginiana
during gestation. /. Reprod. Fert. 42, 163-166.
ROBKIN, M. A., SHEPARD, T. H. & DYER, D. C. (1976). Autonomic receptors of the early rat
embryo heart. Proc. Soc. exp. Biol. Med. 151, 799-803.
STEELE, C. E. (1975). The culture of post-implantation mammalian embryos. In The Early
Development of Mammals (ed. M. Balls & A. E. Wild), pp. 61-79. Cambridge University
Press.
TYNDALE-BISCOE, H. (1973). Life of Marsupials. London: Edward Arnold.
LOWRY,
{Received 22 February 1977, revised 29 April 1977)