Growth of opossum embryos in vitro duringor gano genesis
By D. A. T. NEW,1 M. MIZELL2 AND D. L. COCKROFT1
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 grownin culture for periods of 20-30 h. Many of the explants had a good heartbeat and bloodcirculation in embryo and yolk sac after 12 h, and a few after 24 h. Growth of the embryosincluded 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. Embryosexplanted during the last day of gestation showed persistent and vigorous body movementsin culture, particularly of the forelimbs, head and tongue.
INTRODUCTION
In recent years, methods have been developed for growing rat and mouseembryos in culture during the period of organogenesis, i.e. throughout thesecond week of gestation (reviews by New, 1973; Kochhar, 1975; Steele, 1975).Although these methods already have important applications for the study ofdevelopmental 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 besupported for longer periods, to late fetal stages or beyond. At present thelimiting factor is the failure of the complex allantoic placenta to develop inculture. The simpler yolk sac of the explanted rat or mouse embryo grows welland, with its network of blood capillaries, acts as a respiratory and nutritiveorgan supporting growth of the embryo until the stage of early limb develop-ment. But further growth requires the extra support of the allantoic placentaand until a culture system has been devised that includes, or substitutes for, thisplacenta, it seems unlikely that more advanced development of rodent embryoscan be obtained in vitro.
An alternative approach is to develop culture methods for a mammal thatrelies more on the yolk sac and less on the allantois. This suggests one of themarsupials, of which the most readily available in the Northern hemisphere is
1 Authors' addresses: Physiological Laboratory, Cambridge CB2 3EG, U.K.2 Author's address: Laboratory of Tumor Cell Biology, Tulane University, New Orleans,
La. 70118, U.S.A.
112 D. A. T. NEW, M. MIZELL AND D. L. COCKROFT
Didelphys marsupialis virginicma, the common American opossum. The gesta-tion period of this animal is only 12f days and as many as 15-20 or more em-bryos can often be obtained from each pregnant female. The allantois is presentonly as a simple sac that enlarges with urine during the last 3 days of gestationand probably has no placental function. But the yolk-sac, which in rats is about7 mm in diameter on the 13th day of gestation, develops in the opossum toover 20 mm in diameter, with folds closely apposed to those of the uterineendometrial surface.
The opossum young are born at a stage of development corresponding aboutto 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 toteats 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 developmentwhich are usually hidden in the uterus of eutherian mammals. If culture methodscould be devised for opossum embryos as successful as those now available forrodents, it might be possible to conduct long-term experiments on embryossubjected to a test treatment at an early stage in culture and then reared aspouch young. In a preliminary trial, New & Mizell (1972) obtained limitedgrowth of opossum embryos explanted at 10 and 11 days of gestation. Thepresent 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 Marmosaopossums (Barnes & Wolf, 1971) in captivity, they are still much more difficultto maintain in self-perpetuating colonies than the common laboratory animals.We therefore used only Didelphys opossums recently caught in the wild. Theanimals, trapped in Florida, were purchased from a dealer during the breedingseason in the early part of each year from 1974 to 1976 and housed in theLaboratory of Tumour Cell Biology, Tulane University, New Orleans. All thefemales were found to have pouch young on arrival. At different times duringthe period February to April, the young were removed and the females placedwith males to mate during the first post-lactational oestrus, following the proce-dure of Mizell, Ramsey, Warriner & Spencer (1970). The females were thensent by overnight plane to London and immediately taken, under quarantine,to Cambridge.
Fifteen to eighteen days after removal of the pouch young, the animalswere opened under penthrane anaesthetic, blood was withdrawn from the dorsalaorta to provide culture serum, and the ovaries and uterus were excised. Eachuterine horn was opened under Hanks saline (with 70 mg/1 bicarbonate to givepH 6-5-7-0) by cutting with fine scissors through the muscle wall along the
Opossum embryos in vitro 113antimesometrial side where the tissue is free of large blood vessels. The endo-metrium was then gently torn open with forceps to expose the embryos whichvaried 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, theembryos, with their yolk-sacs, are surrounded by a keratinous shell membrane(Tyndale-Biscoe, 1973) and lie free in the uterus. These embryos were transferredwith a wide pipette directly to the culture vessels.
Embryos of 10 days and older have lost the shell membrane and adhere to theendometrium by the vascular area of the yolk-sac, which comprises about athird to a half of the yolk-sac surface. The strength of adhesion increases withage but with care, yolk-sacs of even 11^-day embryos could be freed withoutdamage to the vascular area. A satisfactory procedure was first to pull off theentire endometrium (with embryos) from the uterine muscle and then, underlow magnification and transmitted light, gently to pull each yolk-sac free.Besides their adhesion to the endometrium, the yolk-sacs of embryos older than10 days are fused to each other in the avascular region and these junctionscannot be pulled apart. To separate these embryos, most of the yolk-sacs werecut in the avascular region, so that each explant consisted of the embryo/fetuswith the entire vascular area of the yolk-sac but only part of the avascular regiontogether with the amnion and the small allantoic sac.
During the last day of gestation (i.e. after 11^ days) the yolk sacs are sofirmly attached to the endometrium that we were unable to remove them with-out damage. But some embryos at this stage were successfully explanted withthe 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 theallantois, which was explanted intact, had enlarged to a sac 8-15 mm in dia-meter and was completely covered with a network of blood capillaries whichprobably assisted respiration in culture.
The time between removal of the uterus from the opossum and the beginningof incubation of the explanted embryos in culture was 1-3 h. For most of thisperiod the embryos were maintained at 5-10 °C but were allowed to warm toroom temperature during the later stages of explantation.
Glass-stoppered reagent bottles (of two sizes, 60 ml and 250 ml) were usedas culture chambers. About one-fifth of the volume of the bottle was filled withculture medium and embryos, and the remainder gassed with the requiredO2/CO2/N2 mixture. Embryos younger than 9 days were cultured in 2-3 ml ofmedium per embryo with 10-40 % O2 and 5 % CO2 in the gas phase. Embryosof 9 days or older were cultured in 8-12 ml of medium per embryo with 95 %O2/5 % CO2 in the gas phase (New & Mizell, 1972). The bottles were laidhorizontally on rollers and rotated continuously at 30-40 rev./min duringculture. A few of the older embryos were cultured in a 'circulator' system asdescribed 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 (Table1) 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/1bicarbonate. The plasma expander Ficoll 70 (Pharmacia Fine Chemicals) wasadded at 60 mg/ml to some of the media with' 199' to raise the osmolarity of themacromolecular content to that of whole serum. A few cultures were als.o madein Hanks or Tyrode saline with 20 % serum. Aseptic precautions were takenduring explantation and culture, and all culture media contained 50/tg/mlstreptomycin.
The cultures were usually examined at intervals of 2-3 h during the first 12 hof incubation and then at intervals of 6 h. Survival and development in culturewere assessed by the condition of the heart-beat, blood circulation, yolk-sac andallantois, and by the changes in the external features of the embryo/fetus.Growth in many of the older embryos was determined by measurements ofcrown-rump length and by assays of total fetal protein (excluding the mem-branes) by the colorimetric method of Lowry, Rosebrough, Farr & Randall(1951).
The embryonic development of the opossum has been described in detail inthe valuable monograph of McCrady (1938). However, as this monograph* isnot widely available, the following brief indication of some of the relevantMcCrady stages is given for reference:
Stage 21 (7^ days). Primitive streak, notochord and medullary plate. Yolksac 4 mm diam., surrounded by shell membrane.
neuropore and otocysts closed. Mesonephric tubules and liver diverti-culum. Allantois rudiment.
Stage 30 (10 days). Forelimbs paddle-shaped. Hind limb-buds. Secondary(convex) lumbar flexure. Tail forming. Amnion complete. Shell membranelost and area vasculosa of yolk-sac now adheres to endometrium.
Stage 31 (10^ days). Forelimb digital ridge. Allantois 2 mm diam. Yolk-sac20 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. Epitri-
chium 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 embryosin each animal varied from 6 to 23 with an average of 14. The gestation agevaried between 1\ and \2\ days, with 8/14 animals at 10^-11^- days, and indi-cated that oestrus and mating had occurred 4-8 days after removal of the pouchyoung 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 opos-sums and grown in culture for 20-30 h. The table shows that many of the em-bryos maintained a blood circulation for 6-12 h and some for over 24 h, andthat there were substantial increases in embryo protein content during theculture period. Further details are as follows:
Embryos explanted at stages 21-22. Head process embryos, yolk-sacs4-0-4-5 mm diam {opossum 1). 16 embryos
Most of the yolk-sacs remained fully expanded within the shell membranefor over 8 h. Those in whole serum looked normal while those in the 199/serumformed several small ' blisters' on the surface. But when the culture was endedat 24 h, all the yolk sacs in whole serum had collapsed away from the shellmembrane while 5/8 of those in 199/serum were still expanded. The embryosin 199/serum had attained Stage 23, with 3-5 somites, a well-marked medullaryplate and head folds. The better development in 199/serum may have resultedfrom the lower pH (final pH 6-0) as compared with that in whole serum (finalpH 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 manyof the yolk-sacs had collapsed after \ h (in the 199 medium), or after 2 h (in theTyrode medium). The shell membrane remained distended. Reasons for thispoor result as compared with litter 1 may have included (i) a delay of about \ hduring explantation, when the embryos were examined in Hanks saline and wereseen 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 beatingheart was formed and a blood circulation which persisted in culture for over18 h (in whole serum) or for 12-18 h (in the 199 media). Prominent anterior
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 coversnearly 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 119
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 theyolk-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.
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 theyolk-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 membranehad disappeared) and were attached1 to each other; they were separated atexplantation and opened in the avascular area. Half the embryos maintained ablood circulation for over 12 h in culture. The embryos in whole serum andTyrode media developed better than those in the Hanks or 199 media (Table 1).Digital buds formed on the forelimb. Somites behind the hind limb-budincreased from 5-6 to 8-13 (Fig. 3). The allantois increased in diameter to2-0-4-5 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 inculture, and in the best it was maintained for over 24 h. The hind limbs de-veloped a digital ridge and the allantois increased in diameter to 13-15 mm (Fig4). Development of the anterior part of the embryos was retarded and no oralshield or epitrichium was formed. The embryos enlarged considerably in culture(Fig. 4) and the protein content of some almost doubled, but part of this in-crease may have been the result of oedema.
Oedema developed quickly in culture (it could be observed after two hours insome embryos) and was particularly conspicuous round the neck and shoulders.The oedema appeared to be reduced in the media with the higher macromole-cular content (whole serum, or 199/serum with Ficoll) but was not eliminated.Haemorrhage was also common and occurred within the embryo as well asfrom the membranes.
Several of the better-developed embryos became very active and showedmovements of the tongue, trunk, limbs and tail which persisted to the end of theculture 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 theyolk-sac cut away and the vitelline vessels ligated. No significant difference wasnoted between the embryos in bottles and those in circulators.
Many of the explanted embryos maintained a blood circulation for several
Opossum embryos in vitro 121hours, and the protein content increased over the culture period. No signs ofoedema were observed.
Several of the embryos showed persistent and vigorous body movements inculture. One embryo removed from culture after 11 h (Fig. 5) and placed incold saline continued to make active movements of the forelimbs, head andtongue; when the mouth was touched with a pair of forceps, the tongue wasdepressed and there were apparent attempts to suck the forceps.
DISCUSSION
The results show that opossum embryos can be grown in culture for periodsup to 30 h at all stages of development from the primitive streak to the latefetus. (The possibility of growing younger embryos has not been excludedbut this study was concerned only with the period of organogenesis and youngerembryos were not examined.) Many of the explanted embryos maintained ablood circulation for several hours, some for over 24 h. Development of theembryos in culture was often equivalent to 12-24 h in vivo. Particularly interest-ing was the appearance of active movements in the older foetuses, including atleast one instance of an apparent sucking reflex, suggesting that it may even-tually be possible to rear opossums from fetuses grown in culture and trans-ferred to the pouch.
Padykula & Taylor (1971) have described changes of the opossum endo-metrium during pregnancy which indicate secretory and transport activity bythe glands and surface epithelium increasing after the 10th day. Renfree (1975)found the protein content of opossum endometrium exudate to be similar bothin concentration and in electrophoretic distribution to that of serum, exceptthat the exudate contained a few prealbumins absent from the serum. Too littleexudate can be extracted from the uterus to provide a culture medium for anybut 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 varyingamounts of bicarbonate. In general, development of the embryos in dilutedserum was as good as in whole serum, but in one culture which contained noserum (two embryos of opossum 9) growth was very poor. Most of the othervariations in the media had no discernible effect on development, but low pHappeared to benefit the youngest embryos and a high macromolecular contentin the medium reduced oedema in the stage-33 embryos.
The oxygen tension in the uterine artery of most mammals is about 90-100mmHg (Comline & Silver, 1975), equivalent to a culture gas phase containingabout 12 % O2. The oxygen levels used for most of these cultures were thereforemuch higher than the embryos would experience in the uterus. They were basedon our previous results (New & Mizell, 1972) which showed better developmentof 11-day opossum embryos in 95 % O2 than in 20 % O2. Such high oxygenlevels 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 ultimatelybe a factor limiting embryonic growth.
The present study was designed to examine the growth of opossum embryosexplanted at all stages of organogenesis and incubated under several differentculture conditions. Although most of the embryos showed some developmentin culture, the results suggest that embryos explanted at about 8-̂ -9 days ofgestation (stages 26-27) are particularly promising for future work. These liefree in the uterine cavity and, unlike the older embryos, can be explanted rapidlyand with the entire yolk sac intact. They have shown consistently good growthin culture and it would be interesting now to study their development in awider 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 LauraCharbonnet and Ms Christi Mortensen (Tulane) for valuable technical assistance. Financialsupport was provided by the Medical Reseacrh Council, London and by United States PublicHealth Service Grant No. CA-011901 from the National Cancer Institute, and a grant fromthe Cancer Association of Greater New Orleans.
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{Received 22 February 1977, revised 29 April 1977)
