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Introduction fertilized and developing eggs for a time in the mother's body, so
that a degree of viviparity was attained (ovoviviparity), but still it was
Nature has evolved many stratagems by which organisms can necessary to invest the egg with all the nutrients required to
reproduce themselves. The earliest animals lived in the sea, and develop to a stage where free living was possible.
when multicellular animals developed, their internal body fluids With the evolution of mammals two great advances were made:
reflected the composition of their environment - salt water. Colo- firstly, with the exception of the egg-laying monotremes, the
nisation of the land, and indeed of fresh water, required that embryo now derived protection and nutrition from the mother via a
animals were able to regulate their internal environment. However, placental connection Secondly, after birth, further nutrition was
this posed further complications in that reproduction required a provided by the mother in the form of milk, thus freeing the new-
degree of replication of the ancestral marine environment. Different born from the demands of food-seeking, and allowing it to benefit
animal groups have devised various ingenious ways of coping with from a prolonged period of learning under sheltered conditions. In
these problems. Amphibians are for the most part tied to water for marsupials, of which more later, attachment to a nipple in the
their reproduction, although some species are able to take advan- mother's pouch occurs at such an early stage that the development
tage of minute quantities of water, or its infrequent availability, even that is completed there is more akin to the fetal stages in eutherians,
to the extent of laying their eggs in an aqueous jelly high amongst It is interesting to note that evolution of maternal nourishment of the
the leaves of trees, or in burrows in desert regions, Furthermore, developing embryo is not unique to mammals; some reptiles and
some amphibians have astonishingly rapid development, to take snakes have yolk sac placentas, or more rarely allantoic placentas.
advantage of what h"le rainfall there is. In other amphibian species, or in some cases both. There are also some elasmobranchs in
one orotherof the parents carries the developing embryos, thereby which Ihe embryonic yolk sac attaches to Ihe uterine wall and
protecting them from desiccation and predation (Young, 1962), derives nourishment from it (Amoroso, 1952). However, only in
Development of a more or less self-contained (i.e. cleidoic-lilerally mammals is placentation almost universal.
'closed box') egg in reptiles and birds meant that the liquid milieu Convenient though viviparity is for the organism concerned, it
and nutrients needed for early development could be provided poses considerable problems for the scientist who wishes to
independent of the surroundings, and by the time of hatching Ihe
organism would be able to regulate its own internal environment . \b!m,';l/tiolH I/lt'f/;II th;'/JtI/N't:c!t', dtTidual t"lrMI: ('t', ('1I1hnot1it" ("Iran: IC,
and resist the hostile dehydrallng eltects of dry land. Some groups iIlIlTlt'rlialdYl("l1l1iJII~t'.I: \IE\I, \Jilli.llal r."I'llli,11 \It-diul1I (":;'.~]t"'I: U\IE\I,
further improved the protection of the young by retaining their Dulht't"("o', \1~,dilic;lIioll Hr [;I~I(", \h-diul1I.
-Address for reprints: Imperial Cancer Research Fund, Department ofZoo!ogy, University ofOltford, South Parks Road, Oxford OX, 3PS, United Kingdom. FAX:
+44,1865,281310. e-mail: cockroft@icrf.icnet.uk
01l4-6~R~/97/S05.00
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Pnn.,-" In Spain
128 D.L. Cockr,,(r
Watchglass Embryos Serum ble to explantation with their yolk, and indeed such preparations
can be continuedto hatching (Perry, 1988).
Preimplantation mammalian cultures
Placentation
When implantation takes place a more or less intimate attach- Fig. 4. Pressure chamber apparatus. Pressure chamber containing
ment forms between embryo and mother. In marsupials in utero, Clrcularors rhar can operate ar two or three atmospheres of pressure.
UO DL Cocknift
Culture medium
Along with the introduclion of lhe rolating bottle systems_
improvements were made in the culture medium. Table 1 gives
some idea of the variety of media that have been applied to
postimplanlation embryos in culture. Early tests had shown that,
for rat embryos, homologous serum was best (New, 1966). When
this was prepared by the standard technique, wilh lhe blood
allowed to clot at 4'C overnight followed by centrifugation, it was
satisfactory for somite-stage embryos, but head-fold and earlier
stages were frequenlly abnormal. Chris Steele, whilst a PhD
student in Denis New's laboratory, discovered that preparing the
serum from blood centrifuged immediately after withdrawal from
Fig. 7. Culture of 9.S-day rat embryos. fAI Rat embryos e;o,planredar 9.5
days. before (a,b) and after (c,d) a 48 h culture. Embryos are shown
dissected free of their e)o;traembryomc membranes (a,C), and with the
membranes Intact (b. d), as during culture. IB) With renewal of the medium
after 48 h (bJ, culture of rat embryos from rhe head-fold stage (al can be
contmued to 72 h (el (shown 'ree of the membranes wlrh which they were
cultured) Bars, I mm.
TA8LE 2
Rar
8.5lpml a (pnmltlve streak) 60-65 -25
951am) a Ihead.fo!dl 48-72 26-35
10.5 lam) 10-15 45 35-40
115tpml 26-28 18-24 -40 Fig. 8. Dissection procedure for 9.S-day rat embryo. Sequence of
12.5 (pm) 40-44 (open yolk sac! 42 50-55 operations In removalofthedecldua (stippled) from the 95-.day(earlyhead4
13.5 (pm) 51-55 (open yolk sac) 42 60-63 fOld) rat conceptus, using watchmaker's forceps. ExplantatlOn is com-
-- - -- - - - - - - - -- -- -- -- -- -- -- -- - - -- -- -- pleted bv reflection of the parietal volk sac. (From Cockroft, 1990)
Vt'r/t'hrll/l' clii/Un' 1I1l'/hods 133
Oxygen
Maintenance of the optimum oxygen level is
also critical to the success of cultures. Early
somite stage embryos develop well in culture
medium equilibrated with air (with 5% carbon
dioxide). Older embryos require higher oxygen
concentrations, largely to compensate for the
lack of an adequate placental transport system.
But a more surprising discovery was that pre-
somite embryos require lower oxygen levels
than that obtained by equilibration with air (Le.
less than 20% oxygen), and forthem 5% oxygen
was found to be beneficial (New et al., 1976a).
The culture procedure that has been most widely
adopted and adapted is to incubate head-fold or
early somite stage rat (or mouse) embryos for
two days in rotating bottles, in immediately cen-
trifuged rat serum equilibrated initially with 5%
oxygen, raised to 20% oxygen around the time
that a heart-beat and blood circulation develop
(Fig. 7). It has been demonstrated that this
regime gives development barely distinguish-
able from in vivo (New et a/., 1976b) and since
this procedure was established, there has been
little substantive improvement in method, but a
burgeoning of applications of the technique in
diverse developmental fields. Cultures of the
head-fold rat embryo can reliably be continued
for 72 h (Fig. 7) If the culture serum is renewed,
Fig. 9. Dissection of 9.5-,10.5- and 1 1.5-day rat embryos. Srages In rhe d'ssecr,on oflA) 9.5-
but often the shorter period is sufficient to show
day,IBI lOS-day, and ICI , 7.S-day rar embryos In each case: (a' On removal from rl:e urerus.
the effect under study, and therefore more eco-
rhe conceprus is embedded in marernal decidua; (b} half rhe deCIdua has been removed. showmg
nomical. rhe position of rhe conceptus withm; (e) rhe decidua has been complerely removed: (d) wIth the
Although the culture methods were largely parietal yol~ sac opened. the conceptus ISready for culrure Bars, 1 mm
developed using rat embryos, which are some.
what easier to work with, particularly at early stages, similar Dissection techniques
results are also attainable with the mouse (Sadler, 1979; Sadler For most of the stages that have been successfully cultured,
and New, 1981; Lawson ela/., 1986, 1987; Martin and Cockroft, the dissection procedures are fundamentally similar (Figs. 8 and
1997). Some popular culture protocols for the rat and mouse are 9): the conceptus is separated from the uterus, the decidua in
shown in Table 2. which it is embedded is peeled off, and the parietal yolk sac
13.\ D.L C(lckn~rl
Marsupials
Unfortunately there is no true equivalent with
mammalian postimplantation culture of the em-
bryo-transfer technique that has proved so valu-
able in the study of the preimplantation mamma-
lian embryo. A notable attempt was made by
injection of 6th and 7th day mouse embryos into
the decidua of 5th or 6th day pregnant mice, using
pigmentation and enzyme markers to distinguish
host and donor conceptuses (Beddington, 1985).
Whilst a small proportion of such embryos
reimplanted and developed into 12th-14th day
fetuses, none went to term, possibly due to re-
tarded development of the allantoic placenta,
Denis New had previously brought a different
approach to this problem, reasoning that if the
Fig. 10. Culture of 13.5.day rat embryos. (AI Rar embryo e.,,-planredat 13.5 days with open present techniques could carry development only
(visceral) yolk sac is scud: to a fabnc--covered. collagen--coared. glass coverslip, for anchorage
to the limb-bud stage, it would be interesting to
withm a Circulator (see Figs. 2 and 3). (81 Rat embryos e),planted at 13.5 days before (a) and
(b) 42 h of culture with open yolk sacs. Bars, 10 mm examine the possibility of culturing embryos of a
after
species that is actually born at about this stage,
(Reichert's membrane with Its adherent trophoblast and parietal Le. a marsupial. To this end some trials were made with the
endoderm cells) is opened, exposing the visceral yolk sac endo- Virginian opossum in collaboration with Merle Mizell (New and
derm layer (see New, 1971; Cockroft, 1990). However, there are Mizeil, 1971; New ef al., 1977), and some deveiopmentin cuiture
some variations for older and younger embryos that are worthy of of opossums at various stages was achieved, including with
mention. One of the most difficult stages to culture successfully is embryos close to birth (Fig. 11). The practical difficulties of
the primitive-streak (or pre-streak) stage, (8.5 days in the rat, 7.5 working in quarantine with such a large and reproductively intran-
days inthe mouse); so far, even the most successful cultureshave sigent animal meant that the goai of transferring cultured embryos
shown a 20% malformation rate (e.g. Buckley et al.. 1978). One to the pouch for continued development was not attained. Never-
study appeared to improve on this, modifying the explantation theless, marsupials remain an attractive subject for culture stud-
procedure by transecting the extraembryonic visceral yolk sac ies - see, for example, the contribution by Seiwood ef al. to this
and thus removing the ectoplacental cone (Fujinaga and Baden, volume.
1991). Embryos explanted by this procedure did indeed develop
well, with 100% (41/41) of 8.5-day rat embryos developing nor- Future challenges
mally to the 26-somite stage. But equaily noteworthy was that in The articles in this volume represent only a fraction of the
their study, the control embryos explanted by the 'old' technique applications which have benefited from vertebrate culture tech.
also developed very well to the same stage with only 7.3% (3/41) niques. Whilst acknowledging ail that has been achieved, many
developing abnormally. Thus it appears that some factor other challenges remain. Those that come most immediately to my
than removal of the ectoplacental cone may have been responsi- mind are:
ble for the good development of primitive streak rat embryos in a) Improving the culture of early postimplantation stages (primi-
this study. Notwithstanding, the authors had fewer embryos tive and pre-primitive streak).
damaged (and therefore discarded) during dissection by their b) Obtaining culture over the implantation period; some remark-
novel technique. and found the method easier, which would able success in this area has been reportedby Hsu and his
Ver!c!Jra{c clI/flIrt' me/lwd, 135
Summary
--
136 D.L. Cockro/i
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