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Early mammalian development
Involvement of DNA replication cycles in the timing of early mouse
development
57
H. Alexandre*, Laboratoire de Cytologie et Embryologie moliculaires, University libre de Brwcelles, B-1640
Rhode-St-Genese, Belgium
Early morphogenesis responds to a biological clock wound up at fertilization. It has been shown, in
different biological systems, that cell cleavage per se is not a prerequisite for scheduled tissue-specific
enzymes expression (Satoh, 1982) or even for some early morphogenetic events (Lillie, 1902; Kimber and
Surani, 1981). On the contrary, a critical DNA replication cycle has been proposed to be responsible for cell
commitment during embryogenesis. This hypothesis has been verified for early mouse embryogenesis, using
antiproliferative drugs affecting essentially DNA replication. A transient polyamine depletion in early
cleaving embryos was shown to induce an early cavitation with regard to the elapsed cell cycles. More
recently, we used a more direct way, namely the specific inhibition of DNA polymerase a by aphidicolin: the
4th DNA replication cycle appeared to be the one responsible for the trophectoderm determination.
Moreover, trophectodermal cells are able to undergo their typical morphological differentiation in vitro
(spreading, outgrowth and nucleus enlargement) in total absence of any additional DNA synthesis from the
32 to 64 cell-blastocyst stage onwards. This total inhibition of DNA replication was achieved by aphidicolin
but not by either dideoxythymidine or dideoxythymidine-triphosphate, which is in disagreement with the
recent findings of Siegel and Kalf (1982) concerning the involvement of DNA polymerase ft instead of DNA
polymerase a in the DNA endoreduplication in giant trophoblast cells.
Finally, preliminary experiments also indicate that aphidicolin induces, in F9 teratocarcinoma cells,
morphological modifications similar to those recorded at the onset of their retinoic acid-induced differentiation (B. Capone & H. Alexandre).
KIMBER, S. J. & SURANI, M. A. H. (1981). Morphogenetic analysis of changing cell associations following
release of 2-cell and 4-cell mouse embryos from cleavage arrest. /. Embryol. exp. Morph. 61, 331-345.
LILLIE, F. R. (1902). Differentiation without cleavage in the egg of the annelid, Chaetopterus
pergamentaceus. Arch. Entwicklungsmech. Org. 14, 477.
S*TOH, N.
SIEGEL, R.
(1982). Timing mechanisms in early embryonic development. Differentiation 22, 156^163.
L. & KALF, G. F. (1982). DNA polymerase fi Involvement in DNA Endoreduplication in Rat
Giant Trophoblast Cells. J. Biol. Chem. 257, 1785-1790.
Isolation of human X-coded sequences expressed in human/mouse
teratocarcinoma hybrids
F. J. Benham 1, K. E. Davies 2, M. V. Miles 3 and P. N. Goodfellow 3. lPaediatric Research Unit, Guy's
Hospital Medical School, London SE19RT.2Biochemistry Department, St. Mary's Hospital Medical School,
London W2. 3Human Molecular Genetics, Imperial Cancer Research Fund, London WC2
We have introduced single human chromosomes into the mouse embryonal carcinoma (E.C.) cell line
PCC4 by (1) microcell transfer of a human X or X/autosome translocation into an HPRT-denvation of
PCC4 or by (2) introduction of the dominant selectable marker Ecogpt into a human genome followed by
microcell transfer of the human chromosome which contains the gpt gene into PCC4. These lines retain the
capacity to differentiate in vitro. The lines are being used to investigate regulation of human genes by the
embryonic environment of the E.C. cell, for example, expression of HLA genes where results indicate that
the E.C. cell does not regulate Class I genes, but does regulate Class II genes. The hybrids also provide a
system for isolating X-coded human gene sequences which are expressed in the E.C. hybrids, some of which
may be developmentally regulated. Three X-coded sequences which recognise single mRNAs in human and
the E.C. hybrid material have been isolated from an X chromosome library. One of these maps to Xp21 to
Xp22 and identifies a highly conserved multigene family. The X sequence, however, represents an inserted,
processed pseudogene. Additional X-coded sequences are being isolated and investigated.
58
Early mammalian development
Neural differentiation in teratocarcinomas and developing brain
H. Bliithmann, University of Geneva, Dipartement de Biochimie, 1211 Geneve 4, Switzerland
Stem cells of teratocarcinomas can differentiate into various cell types and immature tissues and are
comparable to pluripotent embryonic cells in the postimplantation embryo. From such a multidifferentiating
teratocarcinoma, two neuroblastoma-like tumors, TDN 2151 and TDN 2283, were obtained by repeated in
vivo passaging, which homogeneously show immature neural differentiation.
In the present study, overall synthesis of cellular proteins - resolved on two-dimensional gels - served as a
very rigorous and general criterion for comparing in vivo differentiation of nerve cells in teratocarcinomas
with brain development in the mouse foetus. Proteins synthesized during muscle differentiation in
developing limbs were used as a control for a developmentally unrelated tissue.
Comparison of the very complex protein patterns revealed the following differentiation-related new
protein syntheses: a) aberrantly expressed proteins, which appeared in the neurogenic tumors but not in
foetal brain, b) general proteins, which appeared in at least one of the neurogenic tumors and were also
present in foetal brain and limbs, c) nerve-specific proteins, which appeared in the neurogenic tumors and
were detected in brain but not in muscle tissue of developing limbs. Four nerve-specific proteins with MT of
29 kd, 26 kd, 20 kd and 16 kd are the most promising ones for further studies of the molecular basis of
differentiation.
Development of the thymus in dogs
B. Bodey, W. Calvo, O. Priimmer and T. M. Fliedner, Department of Clinical Physiology and Occupational
Medicine, University Ulm, D-7900, Ulm, West Germany
The development of the thymus was studied with histological, electron microscopical (TEM) and
histochemical methods in 100 fetuses (beagle) from 47 litters, between day 19 of gestation and day 21 after
birth. The three earliest samples investigated were 19,20 and 21 day old embryos in which no organogenesis
was present. The thymus development could be divided in three stages: 1) Formation of epithelial palisades
(31 to 35 days of gestation; 26 fetuses); 2) Begin of lymphopoiesis (36 to 38 days; 13 fetuses); 3)
Development of medulla and Hassall bodies (HB) (39 days of gestation to perinatal period; 58 fetuses and
newborns). The epithelial anlagen were seen at the 31st day of gestation showing the characteristic palisade
structure of the endoderm derived epithelium. They originated from the ventral part of the 3rd and 4th
pharyngeal pouches. Four days later the lymphopoiesis and the reticularisation of the epithelial cells begins.
Between days 36 and 38 of gestation the rapid proliferation of lymphoid and reticulo-epithelial (RE) cells
resulted in the differentiation of the cortical and medullary areas. The first HB could be found at the 38th
day, when both areas could be well recognized. The HB grow out of the cells of RE network, undergoing
special morphological changes. The palisade structure of the epithelial cells remains at this stage at the
surface of the cortex, the cytoplasmic processes develop deeper in the cortex and especially in the medulla.
There was a rich content of PAS positive granules in the cytoplasma of RE cells. This material was glycogen
as demonstrated by TEM. The PAS reaction was much more abundant at the very surface of the cortex in
contact with the interlobular connective tissue (ICT), than in deeper areas and more in the early stages of
the development than after birth. The HB showed a weak diffuse reaction in most of the cytoplasm of the
RE cells and a strong, coarse, granular reaction under the membrane. Some lymphocytes were also PAS
positive but did not show glycogen. The ultrastructural observations demonstrated the presence of
desmosomes connecting RE cells to one another. Desmosomes were not found between RE and
lymphocytes. The HB showed their main development between days 45 to 54 of gestation beginning with
hypertrophy of the RE cells. The growth of the thymus into the mesenchymal matrix resulted in the
tabulation of the organ by connective tissue cells and fibers. Reticulin was demonstrated by the Gomori's
method and was abundant in the ICT but few fibers penetrated into the cortex. The mast cells were first seen
at the 35th day of gestation, being abundant in the ICT; few were present in cortex and more in medulla
near the HB. Small groups of neutrophil cell precursors appeared in the ICT from the 40th day on, but no
eosinophils were found. Cysts were not present up to the 21st day after birth.
Early mammalian developments
Oocyte coded glucose phosphate isomerase is gradually replaced by
embryo coded enzyme during preimplantation development of the
mouse: a fine analysis on single embryos
59
Denis Duboule 1 and KurtBurki 2, Laboratory for Cell Differentiation, University of Geneva, Switzerland.
l
CNRS du LGME, Unite" de Biologie moliculaire et de Ginie ginitique, Faculti de Midecine, 11, rue
Humann, F-67085 Strasbourg Cedex, France. 2Dept. Biologie Animale, University of Geneva, 154 route de
Malagnou, CH-1224 Geneva, Switzerland
Mouse embryos were derived from eggs heterozygous for alleles of the dimeric enzyme glucose phosphate
isomerase (Gpi-la/Gpi-lb) fertilized with sperm carrying a third allele (Gpi-lc). This particular combination
makes it possible to study the cfollowing phenomena during early development:
1) The appearance of Gpi-l coded isozyme-subunits as an indication of activity of the paternally derived
gene.
2) A shift of the initial oocyte type hybrid pattern to the final embryo type pattern as an indication of
activity of the maternally derived gene.
3) The presence of GPI-1AB dimers to estimate the persistence of oocyte coded enzyme throughout early
development.
4) The temporary presence of GPI-1BC dimer in Gpi-la/Gpi-lc embryos as a possible indication of the
simultaneous expression of the paternally derived allele and the maternal message. The different isozymes
present in single embryos were separated by electrophoresis on cellulose acetate gels. The results show that
the oocyte coded glucose phosphate isomerase is gradually replaced by embryo coded enzyme. Expression
of the paternally derived allele is first detected at the morula stage. At this stage the translation of
maternally derived message seems to be either exhausted or below the detection limit of our systems. Some
oocyte coded enzyme nevertheless persists until after implantation.
Transcription of two mitochondrial genes in perinatal and postnatal
developing rat liver
P. Cantatore, P. Loguercio Polosa, F. Fracasso, Z. Flagella, A. de Montalvo andM. N. Gadaleta, Institute
of Biological Chemistry, University of Bari, Bari, Italy
Studies on mitochondrial (mt) DNA transcription in HeLa cells have revealed the existence of two
transcriptional units on the H strand of mt DNA coding the first one for the two rRNAs and for two tRNAs
and the second one for all the mRNAs and for the remaining tRNA. The measurement of the steady-state
levels of mitochondrial rRNAs and of some mRNAs in HeLa cells, in adult rat liver and in sea urchin
embryos at gastrula stage has shown that rRNA concentration is quite similar in these three systems whereas
the concentration of mRNAs is higher in rat liver. These results suggest the possibility of a different
regulation of mitochondrial rRNA and mRNA synthesis. We have approached this problem by studying the
steady-state levels of two mt RNA species namely the 16S rRNA and the mRNA for the subunit I of the
cytochrome oxidase (Col) during rat liver development. By using a technique based on the hybridization
between total mt RNA and radioactive mt DNA probes, we have found that, in correspondence of the
perinatal period of life, there is an increase in the number of molecules per mitochondrion of the Col
mRNA, while the concentration of 16S rRNA species remains substantially unchanged. This differential
expression of mt genome during development is in agreement with the increase in oxidative phosphorilation
and with the upsurge of the cytochrome oxidase activity observed at birth in rat liver.
60
Early mammalian development
Cell heterogeneity in the mouse inner cell mass
Julia C. Chisholm, Department of Anatomy, University of Cambridge, Downing Street,
Cambridge CB2 3DY
Until the expanded blastocyst stage of development, the inside cells of the mouse embryo remain
developmentally labile and will generate trophectoderm (TE) when placed experimentally in an atypical
outside position. At later times, extraembryonic endoderm (ExEnd) is generated. The time course of this
restriction on the developmental potency of inner cell mass (ICM) cells has been investigated and it is found
that cells within one ICM do not all become committed at the same time.
ICMs were isolated at time points from 1 to 12 h after the earliest signs of cavitation. Among ICMs
isolated from blastocysts of each age post cavitation there was wide heterogeneity in cell number, as
estimated by nuclear counts, and in developmental potential, as judged by both light and electron
microscopic observation of the morphological changes occurring during in vitro culture of the ICMs. Thus
the initiation of cavitation does not appear to be related either to the developmental cell cycle of inside cells
or to their state of commitment.
Some single ICMs developed both TE and ExEnd cells when isolated and placed in culture, suggesting
that separate populations of committed and uncommitted cells can coexist in the same ICM and that
differential expression of their potency is possible in some instances. This mixed potential for development
was also seen in aggregates made by pairing ICMs from early (2 h) and expanded (12 h) blastocysts.
These results indicate that commitment of ICM cells may take place as an independent event in each cell,
at a time unrelated to the absolute age of the embryo or to manifestations of cell cycle events (blastocoel
formation) in outside cells. Since cell cycles between and within embryos are asynchronous, it is suggested
that the time of commitment of ICM cells may be related to the number of developmental cell cycles that
each has undergone.
Histogenetic factors in the development of the EPU
P. V. Crespo, C. Santisteban and A. Campos, Departamento de Histologiay Embriologia General, Facultad
de Medicina, Granada, Spain
The epidermal proliferative unit can be described as consisting of two large areas located above and below
the horny basal keratinocyte, and termed columnar and subcolumnar respectively.
The purpose of the present communication is to analyse the processes of differentiation and evolution of
the EPU. A total of 24 Wistar rats were studied, which were sub-divided into four chronological groups:
fetal, newborn, four days and adult specimens. The material was also classified into three topographical
groups: back, ear and abdomen.
Using semithin sections we carried out a quantitative study of the cellular elements that make up the EPU
in the aforementioned chronological periods and topographical zones. All the data were studied and
subjected to multivariate statical analysis in order to elucidate possible correlations. A total of 13 factors
were defined which influence the histogenetic process in the EPU. Only three appeared to be significantly
implicated in the epidermal unit.
Factor I is related to perinatal specific proliferation. Factor II is related to differentiation of the horny
cells of the EPU. It partipates to a minor degree in the perinatal stages, but its importance gradually
increases to an important degree in adult stages. Factor III which regulates subcolumnar differentiation does
not vary according to chronological stages.
Early mammalian development
The effects of lectins on blastocyst and embryonal carcinoma cell
development
61
G. B. Dealtry and M. H. Sellens, Department of Biology, Essex University, Colchester, C043SQ Essex
Cellular interactions via surface glycoconjugates are thought to play an important role in development.
The blockage of these surface receptors by lectins and specific antibodies has been shown to alter or prevent
further development of pre-implantation mouse embryos (Reeve, 1982) and embryonal carcinoma cells
(Martin, Grabel and Rosen, 1980). In the present study a panel of 7 lectins (BSL, ConA, DBA, Lotus
Lectin, PNA, UEA and WGA) previously shown by us to bind to both the inner cell
mass and
trophectoderm of the mouse blastocyst were added at concentrations of 25, 50 and 100 /Agml"1 to blastocyst
cultures for varying time intervals, and their effects on cell growth and differentiation were studied. A
similar protocol was used to study the effects of these lectins on embryoid body formation and endoderm
development by embryonal carcinoma cells fPSMB G27 line). WGA was cytotoxic. The remaining lectins,
whilst allowing some cell division, induced characteristic changes in cell growth and/or disrupted cell
contacts, which ultimately led to the failure of blastocyst outgrowth and embryoid body development.
MARTIN, G. R., GRABEL, L. B. & ROWEN, S. D. (1980). Use of teratocarcinoma cells as a model system for
studying the cell surface during early mammalian development. In The cell surface: mediator of
developmental processes, (ed. S. Subtelny and N. K. Wessels), pp. 325-348. Academic Press, London.
REEVE, W. J. D. (1982). Effect of concanavalin A on the formation of the mouse blastocyst. /. Reprod.
Immunol. 4, 53-64.
DNA polymerase activity in 7*5 day mouse trophoblast
W. L. Dean and J. Rossant, Department of Biological Sciences, Brock University, St. Catharines, Ontario,
Canada L2S3A1
In the postimplantation mouse embryo, the diploid ectoplacental cone (EPC) is known to undergo a
diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by
replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the
formation of giant nuclei. Studies of 13-5 day rat trophoblast have indicated a relatively low rate of DNA
polymerase a, the normal eukaryotic replicase, in comparison to that of DNA polymerase a. These results
have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase
enzyme. In order to determine whether a switch from DNA polymerase a to DNA polymerase (3 is a
necessary concomitant of the diploid to giant cell transformation, the embryonic ectoderm, extraembryonic
ectoderm and EPC from 7-5 day mouse embryos were treated with aphidicolin, a specific reversible inhibitor
of eukaryotic DNA polymerase a, on various days after explantation.
DNA synthesis was measured in
control and treated embryos following a 2 h pulse with methyl 3H-thymidine. Scintillation counts of the
embryonic ectoderm revealed that DNA synthesis was consistently inhibited by greater than 90 % in the
presence of aphidicolin. The extraembryonic ectoderm and the EPC were inhibited to a lesser extent. The
extent of the inhibition of DNA synthesis in the extraembryonic ectoderm and EPC varied between
81-95 % and 82-98 % respectively. A qualitative measure of the effects of aphidicolin on these tissues was
provided by autoradiography after various days in culture. DNA synthesis in the embryonic tissues was
completely inhibited at all stages in explant culture. In contrast, both the extraembryonic ectoderm and the
EPC were observed to possess some heavily labelled cells in the presence of 10 /xg/ml aphidicolon after two
days in culture. These results suggest that DNA polymerase a is the primary replicating enzyme responsible
for endoreduplication in mouse trophoblast giant cells. There appears to be a small but significant
population of trophoblast giant cells which incorporate label in the presence of aphidicolin and hence
employ a non alpha polymerase for replication.
62
Early mammalian development
Coloboma mutants: isoenzyme patterns of LDH in blastocysts and
chromosome anomalies in adult mice
O. A. Dryanovska, N. G. Kolevska and S. G. Nonchev, Institute of Genetics, Sofia 1113, Bulgaria
Early embryos from the crosses between mice, heterozygous for the gene Coloboma, were studied on the
4th, 5th and 6th days of pregnancy. In the progeny 16 % of the blastocysts and 21 % of the embryos at the
beginning of implantation show morphological and functional anomalies. The blastulation is delayed and
the primary differentiation in trophoblast and embryoblast is altered in the first of these two groups. After
the flushing of the uterine horns on the days 5 and 6 p . c , unattached late blastocysts were found, with
degenerative trophoblast and/or embryoblast outgrowths. The abnormal early and late blastocysts were
used for the LDH-spectra investigation, together with apparently normal embryos from the same matings.
Microdiscelectrophoresis revealed only a single band of LDH-1 in the two groups of embryos at the early
blastocysts stage. The day 5 embryos, attached to the uterine wall contain LDH-5 fraction, while the
heteromeric isozyme LDH-4 appeared in day 6 attached embryos. However, in the unattached blastocysts
flushed from the same uterine horns, a clear amount of LDH-5 is observed as well as a faint band of LDH-1.
The presence of LDH-5 indicates that the embryo genome is activated at least in respect of the genes,
responsible for the subunit-ar synthesis. The LDH-1 band in those embryos could be determined either by
ovocyte storage products, or is an expression of newly activated 0-subunit genes. It is tempting to speculate
that these flushed blastocysts are more probably homozygous for the gene Coloboma. The effect of the
mutant gene double dose could interfere with implantation, but not with the process of early differentiation,
expressed by the shift from LDH-1 to LDH-5 synthesis. In addition, cytogenetical analysis of adult mice
Cm/+ was performed on spleen and bone-marrow cells. The G-banding preparations indicate a difference in
the length and segmentation between the two chromosome 12 homologous. In all the metaphase plates the
subterminal E and the terminal F fragments of chromosome 12 are absent. This deletion, if related to
Coloboma condition, should be investigated in the early embryos from Cm/+xCm/+ crosses.
Proteolytic activity in the placenta, decidua and embryos of rats
Amos Fein, Varda Eyal and Laslo Nebel, Department of Embryology & Teratology, Tel Aviv Univ. School
of Medicine, SHEBA Med. Cent., TelHashomer, Israel
There is accumulating evidence that proteolytic enzymes play an important role in implantation in
rodents. Rising proteolytic activity was found during development of mouse blastocyst and during
postimplantation stages in rats. The aim of the present investigation was to demonstrate the activity of two
proteinases, an exopeptidase activity against leucine 6-naphtyl-amide (L-NA) and an endopeptidase activity
against benzoyl-arganine j8-naphtyl-amide (BA-NA). These activities were measured in the postimplantation rat embryo as well as in placenta and decidual tissues. Rats were killed on days 11, 12 and 13 of
gestation. The embryos were separated from the decidual and placental tissues. For.the assay of proteolytic
activity, tissue homogenates were prepared. After 90 min of incubation with L-NA or BA-NA, the
concentration of the end products was determined colorimetrically. L-NA activity increased with time in the
placenta and was unchanging in the embryo and decidua. This enzyme was under the inhibitory effect of
bivalent cations. This inhibition can be removed by treatment with EDTA, after which the enzyme's activity
increases. The enzyme activity seem to have a Cathepsin-B function. BA-NA activity was increasing with
time in the embryo and in the decidua, decreasing in the placenta. In view of the changes in the activity of
the enzymes manifest in the placenta and decidua during these specific days of placentation, it is tempting to
presume that they may have a regulatory role in this process.
Early mammalian development
Decidual cells are not derived from the bone marrow
63
D. J. FowlisandJ. D. Ansell, Department of Zoology, University of Edinburgh, West Mains Road,
Edinburgh
Recently published data have suggested that artificially induced deciduomata, produced in mice after
irradiation and transfusion of semi-ajlogeneic bone marrow, are derived from the donor marrow. Donor and
host cells were distinguished serologically with anti-H2 antisera (Kearns, M. and Lala, P. K. (1982). /. Exp.
Med. 155, 1537-1554). Our evidence, obtained from chimaeras formed with alloenzyme-marked, but
histocompatible, bone marrow, contradicts this view.
Female CBA/Ca mice carrying the phosphoglycerate kinase alloenzyme marker, PGK-1B, were given
varying doses of radiation and repopulatea with congenic CBA-PGK-1A bone marrow cells. These mice
were then either naturally mated or their uteri were traumatised to induce deciduomata artificially. The
PGK phenotypes of the resultant decidual masses were analysed after electrophoresis. Whilst the donor
bone marrow cells contributed to haematopoiesis to a greater or lesser extent, dependent on the dose of
radiation and the number of injected cells, no evidence was found for the differentiation of decidual cells
from transfused bone marrow precursors.
The use of allo-enzyme markers in this case provides a more reliable indication of the possible
descendants of transfused bone marrow. In the previously published observations, non-specific binding of
the serological reagents, possibly to Fc receptors on decidual cells, may have been misleading.
Histone gene expression in early mouse embryos
D. H. Giebelhaus*, R. A. Graves, W. F. MarzluffandG. A. Schultz, Department of Medical Biochemistry,
The University of Calgary, Calgary, Alberta, Canada and Department of Chemistry, Florida State University,
Tallahassee, Florida, U.S.A.
Through use of Northern blots and hybridization with a homologous histone H3 recombinant DNA
probe, it has previously been shown that there is a pool of histone H3 mRNA in the unfertilized egg which is
largely eliminated by the mid two-cell stage. Histone H3 mRNA content then increases progressively
through the eight-cell stage to the blastocyst due to transcription from the zygote genome (Giebelhaus, D.
H., Heikkila, J. J. and Schultz, G. A., 1983. Changes in the quantity of histone and actin messenger RNA
during the development of pre-implantation mouse embryos. Devi. Biol. 98: 148-154). In the current study
we have quantitated the number of histone H3 mRNA molecules at several stages of development and
comapred these values to absolute rates of synthesis of histone H3 proteins. Assuming all mRNAs are
translatable, it is possible to calculate theoretical translational efficiencies. Translational efficiency was
predicted to increase from 0-5 histone H3 polypeptides/mRNA/minute at the two-cell stage to about 2
polypeptides/mRNA/minute in the blastocyst. Finally, we have applied SI nuclease mapping techniques to
begin to identify which of four different H3 genes isolated from the mouse genome are utilized during this
early developmental period. In the unfertilized egg, about 45 % of the H3 transcripts are derived fromgene
H3.614 on chromosome 3 while about 25 % come from gene H3.2 on chromosome 13. Less than 5 % are
derived from gene H3.1 which is adjacent to H3.2 (Sittman, D. B., Chiu, I., Pan, C , Cohn, R. H., Kedes,
L. H. and Marzluff, W. F., 1981. Isolation of two clusters of mouse histone genes. Proc. natn. Acad. Sci.
U.S.A. 78: 4078-4082). In the blastocyst, only a small percentage of the histone H3 mRNA transcripts are
derived from the same gene set. The results indicate that while histone mRNA content is similar in the egg
and blastocyst, the genes which are expressed have changed.
This work was supported by the MRC (Canada) and the NIH (U.S.A.).
64
Early mammalian development
Age determination by somite counting: a simple and accurate method
/ . F. Goedbloed and A. E. Smits-Van Prooije, Department of Anatomy, University of Leiden,
Wassenaarseweg 62, 2333 AL Leiden, The Netherlands
The relation between 'developmental age' and the number of somites was studied in the mouse and rat
embryo, in a period of development ranging from 0 to 43 somites.
The 'developmental age' is based on the logarithm of the volume (Goedbloed, 1976). The developmental
age shows a linear relation to the number of somites. With help of the straight line thus produced, the
'developmental age' can be estimated from the number of somites with an accuracy of less than 0-1 day, and
therefore it can be considered a better age estimate than those based on the usual methods.
In the mouse, the first somite is formed at 7-75 days after fertilization; the formation of each next somite
takes 1-68 h ( ± 100 min). In the rat, the first somite is formed at 8-95 days after fertilization; the formation
of the next somites takes 2-24 h (2 h 15 min) each. This relation holds for the formation of somite 1 to 43.
If a mouse and a rat embryo with the same number of somites are compared, their volumes turn out to be
the same. It would be interesting to know whether this is a general phenomenon in mammalian embryos.
GOEDBLOED, J. F. (1976). The embryonic and postnatal growth of rat and mouse IV. Acta Anat. 95: 8-33.
Cell division in the inner cell mass of the mouse blastocyst is restricted
before implantation
Alan Handyside and Susan Hunter, MRC Experimental Embryology and Teratology Unit, Woodmansterne
Road, Carshalton, Surrey SM5 4EF
The mouse blastocyst consists of an outer layer of trophectoderm (TE) cells surrounding a fluid-filled
cavity and specialised for implantation, and an inner cell mass (ICM) from which the embryo is formed. The
numbers of these cells in individual blastocysts has been studied by differential labelling of the nuclei in situ
with two polynucleotide-specific fluorochromes (Handyside and Hunter, 1984). This allows them to be
distinguished by fluorescence microscopy on the basis of their colour: TE nuclei appear pink or red, and
ICM nuclei blue or unlabelled depending on the filter combination used. Blastocysts were sampled at
intervals either in vivo or in vitro under various conditions in which implantation was allowed to proceed
normally or was suppressed. Under normal conditions in vivo in non-implanted blastocysts (84-114 h post
coitum), the number of TE cells increased steadily over two doublings and then rapidly levelled off. In
contrast, after an initial increase, the number of ICM cells first declined and then increased abruptly before
reaching a plateau in excess of twice the number present at cavitation. During the period of decline, many
ICMs contained disintegrating nuclei indicating extensive cell death. In the absence of implantation in vivo
or in vitro, no further increase in the number of ICM cells was observed unless the TE collapsed. We
propose that the mouse blastocyst functions to regulate the development of the ICM before implantation by
(1) eliminating late-dividing cells and synchronising cell division, and (2) restricting further cell division to
maintain the ICM at the appropriate stage. In this way, the initiation of embryonic growth may be
synchronised with implantation.
A. H. & HUNTER, S. (1984). A rapid procedure for the visualisation of the trophectoderm and
inner cell mass nuclei of the mouse blastocyst using polynucleotide-specific fluorochromes. /. exp. Zool.
(submitted).
HANDYSIDE,
Early mammalian development
65
Sequence of morphological and molecular events during the first cell
cycle of mouse embryogenesis
Sarah K. Howlettand Virginia N. Bolton, Department ofAnatomy, Downing Street, Cambridge CB23DY
The precise timing and sequence of various morphological and molecular events during the first cell cycle
of mouse development following fertilisation in vitro were investigated. The timing of development through
the first cell cycle was found to be initiated by an event associated with sperm penetration rather than with
germinal vesicle breakdown. Initiation of DNA replication occurred randomly in either pronucleus of a
given egg, beginning approximately 11 h after fertilisation and lasting 6-7 h. Careful study of polypeptide
synthetic profiles revealed three sets of polypeptides exhibiting changes during the first few hours of
development (45KD, 35KD complex and 30KD). Pulse chase experiments showed that some, but not all, of
the changes result from post-translational modifications. The remaining changes are presumed to result
from differential mRNA utilisation. Most of the polypeptide synthetic changes observed also occur in the
ageing unfertilised egg, but proceed more slowly. It is suggested that an endogenous programme of
developmental change (the oocyte programme) is set in train by the terminal events of oocyte maturation
and accelerated by the events of fertilisation, which activate the endogenous fertilisation programme.
Histological examination of tissues in a series of chimaeric rats between
congenic strains varying at the RT1 locus
P. M. Iannaccone 1, J. C. Howard 2 and W. C. Weinberg 1. Northwestern University Medical School,
Chicago, IL 60611, U.S.A. Agricultural Research Council, Babraham, Cambridge CB2 4AT, England
A series of chimaeric rats were produced by aggregating morulae of FYG-RTF strain animals with
morulae of PVG-/?77C strain animals. The tissues of these two strains are histologically distinguishable by
virtue of a difference in a class I major histocompatibility complex (MHC) molecule. Monoclonal antibodies
directed against various epitopes of these molecules were iodinated and applied to fresh cryostat sections of
frozen tissues from these animals. Autoradiograms were prepared by dipping the sections in photographic
emulsion. The strain of origin of the tissue could be unequivocally established by the uniform accumulation
or uniform absence of grains over the tissues. The exception was brain tissue, which was unlabeled due to
low determinant density in the substance of the brain. Blood vessels within the brain were strongly labeled.
This observation was confirmed by incubating monoclonal antibody which had been preabsorbed with either
spleen or brain cell suspensions, with frozen sections of either spleen or brain. Preabsorption with spleen
removed all specific labeling, preabsorption with brain did not detectably affect reactivity of the antibody.
Red blood cells (RBC) from eight animals born in two litters following amalgamation of morulae were
analyzed with a cell sorter. Chimaerism of the RBC's was established for four of these animals which ranged
from 7-5 % to 70-4 % WG-RTP cells. Frozen sections of the livers
of these chimaeras confirmed the
presence of visceral chimaerism with random patchiness of PVG-/?77a or PVG-/?77C cells depending on the
proportion of the two cell types present in the tissue. Digital quantitation of the histological sections
demonstrated a range of RTl* cells in the liver from 34 % to 86 %. This analysis revealed a linear
relationship between the proportion of the major cell type and the number of patches of the minor
cell type/unit area examined ( r = 0-99). The average patch size also depended on the proportion of the two
cell types present (for example, with 53 % RT1* liver cells the average patch size was -020 ± -004 mm2).
The large variation in patch size was the result of geometric complexity of the patches even in unbalanced
chimaeras. Distinctive patterns of mosaicism were present in other tissues with possible implications for
organogenesis. The adrenal cortex appeared striped indicating that the cortex may have been developed by
clonal proliferation of primordial cells randomly assorted along the medulla. The thymus demonstrated
cortical patchiness indicative of stem cell proliferation. Kidney, major salivary glands, uterus, ovaries,
heart, lung and spleen were also examined in one animal sacrificed for this purpose. This work was
supported by DHHS grant CA29078 and CA34913.
66
Early mammalian development
The toxicity of tritiated amino acids to the mouse embryo
H. M. Killen and J. Carroll, Department of Biochemistry, Trinity College, Dublin 2, Ireland
The addition of tritiated amino acids inhibited growth in vitro of the mouse embryo. The toxicity of the
H-amino acid was greatest at the 2-cell stage3 of development and 50 % of these survived to the blastula
stage in the presence of the following doses of H-amino
acid: glutamic acid 10 /xCi/ml, leucine 1 /i,Ci/ml and
tryptophan 003 /LtCi/ml. The high toxicity of 3H-tryptophan (greater than that of 3H-thymidine) was not
relieved by the presence
of a large excess of non-radioactive tryptophan derivatives. The 2-cell embryo
rapidly incorporated 3H-tryptophan into proteins and autoradiographic examination of thin sections showed
a net concentration of this radioactivity in the nuclear region. Comparative analysis by 2-dimensional
electrophoresis in polyacrylamide gels of nuclear proteins labelled with either 35S-methionine or
3
H-tryptophan showed that the latter was selectively incorporated into a limited sub-set of the chromosomal
proteins. The significance of these observations are also examined in the context of the patterns of
developmental stage-specific peptide synthesis.
3
The functioning embryonic heart: early onset of normal development or
deviation into malformation
H. W. Klein and P. Krediet, Anatomy, Erasmus University Rotterdam (NL) and Veterinary Anatomy,
R.U.C.A., Antwerp, Belgium
In the 4 mm mammalian, including human, embryo out of the cardiac loop two separate ventricular
pouches develop, a left (proximal) and a right (distal) one. A linear zone in between these pouches, roughly
the curvatura maior of the loop, connecting the mid-caudo-dorsal side of the AV canal and the left
cranio-ventral side of the outflow tract or bulbus, does not elongate. This zone becomes a ridge, thus
forming the luminar border of the interventricular septum. Further outgrowth of the pouches in ventral and
caudal direction simulates infolding of the caudo-dorsal part of the AV-canal, forming the caudo-dorsal
AV-cushion. This 'infolding' also affects the caudal atrial wall.
The pumping action of both ventricles now creates two blood streams out of the atrium through the
dividing AV-canal. Both ventricles pump blood into the bulbus, the left one over the interventricular
septum, along the caudo-dorsal side of the bulbus in a right-dorsal direction, and the right ventricle along
the cranio-ventral side in a left dorsal direction. Both flows turn in a right-hand way around each other.
Bulbus contractions press the cardiac jelly in between these blood streams, thus forming two spiralling
bulbus ridges.
Any disturbance of formation of two blood streams of equal size influences the morphology of the outflow
tracts and of the ventricles.
Early mammalian development
In situ localization of collagen oc\ (I) mRNA in developing mouse
embryos
67
Michael Kuehn, Jiirgen Lohler and Rudolf Jaenisch, Heinrich-Pette-Institutfur Experimentelle Virologie und
Immunologie an der Universita't Hamburg, 2000 Hamburg 20, West Germany
The importance of type I collagen in the embryonic development of the mouse has recently been
illustrated by the recessive lethal mutation in the collagen al(I) gene found in Movl3 mice (Schnieke et al.
1983). We have analyzed the developmental expression of this gene in normal mice using the method of in
situ hybridization of 3 H labeled DNA probes to mRNA in frozen tissue sections. To determine both the
point at which expression can first be detected as well as the cell types responsible for collagen arl(I)
expression during embryogenesis, we have analyzed embryos at days 8, 9-5, 11,13 and 16 of gestation (the
day of the vaginal plug is day 1). A low level of collagen al(l) expression has been detected in day 8 embryos
in mesenchymal cells in the head region of the embryo proper as well as mesodermal cells in the visceral yolk
sac (VYS) and amnion. At 9-5 days these same cell types are more heavily labeled suggesting a significant
increase in mRNA expression for orl(I) collagen between day 8 and day 9. Testing for the presence of
protein, Adamson and Ayers (1979) reported that both the mesodermal and endodermal portions of the
VYS produce type I collagen in embryos as early as day 10. Using in situ hybridization, however, we have
found that in embryos from day 8 through day 13 only the mesodermal cells located on the inner face of the
VYS are labeled. By day 11, embryos begin to show very heavy labeling in the umbilical cord, VYS
mesoderm and surrounding the vascular system. This is consistent with the need for increased amounts of
collagen to be laid down in these areas affected by an increase in mechanical forces imparted by the growing
embryo. The need for collagen I in the vascular system is evidenced by the fact that Movl3 homozygous
animals die at day 12 due to vascular rupture (Lohler et al. submitted]). Embryos in the last third of
development show heavy labeling in all mesenchymal cells and their derivatives. This pattern of mRNA
expression correlates with the pattern of protein distribution in later stage embryos as determined by
antibody staining (Lohler et al. submitted), which is not markedly different from the adult protein
distribution pattern.
ADAMSON, E. D. & AYERS, S. E. (1979). The localization and synthesis of some collagen types in developing
mouse embryos. Cell 16, 953-965.
SCHNIEKE, A., HARBERS, K. & JAENISCH, R. (1983). Embryonic lethal mutation in mice induced by
retrovirus insertion into the arl(I) collagen gene. Nature 304, 315-320.
Fate mapping of the endoderm in pre-somite mouse embryos by
intracellular microinjection of horseradish peroxidase
K. A. Lawson x, J. J. Meneses 2andR. A. Pedersen 2>3. ^Hubrecht Laboratory, Utrecht, The Netherlands.
2
Laboratory ofRadiobiology and Environmental Health and ^Department of Anatomy, University of
California, San Francisco, U.S.A.
Studies on early mouse development show that primary ectoderm-derived cells can contribute
descendants to the definitive gut of the foetus; the fate of the primary endoderm is still uncertain. We have
used intracellular microinjection and embryo culture to trace endoderm cells and their descendants from the
early primitive streak stage to early somite stages. Horseradish peroxidase was injected iontophoretically
into one axial endoderm cell and the embryos cultured in DMEM+50 % rat serum for 1 or 2 days. Labelled
cells were visualised in whole mounts and their exact localisation and number determined histologically. 1)
Descendants of axial endoderm cells overlying and immediately anterior to the early primitive streak spread
over the entire axis of the embryo during the following 24 h (early neural plate stage) and can contribute to
endoderm covering the heart, the longitudinal axis, and to the foregut of the 3-6 somite embryo. 2)
Descendants of mid-line embryonic endoderm cells anterior to the distal tip of the early primitive streak
stage embryo are localised anterior to the prospective foregut imagination at the neural plate stage and
contribute to the anterior visceral yolk sac of the early somite embryo. 3) Axial embryonic endoderm at the
early streak stage has a median cell cycle time of circ. 8 h and no detectable dying or non-proliferating cells.
By the late streak stage at least 42 % will die without dividing within the next 24 h. (Supported by the Royal
Dutch Academy of Sciences and the U.S. Department of Energy.)
68
Early mammalian development
Presence of an electron dense layer on polar trophoblastic cells of
cultured embryos
T. LeclipteuxandJ. Remade, Uniti de Biochimie CelMaire, F.N.D.P. Ruede Bruxelles61, B-5000, Namur,
Belgium
Products of the major histocompatibility complex play an important role in allograph rejection. Cells of
the trophoblast and derivative tissues seem to lack these antigens (Searle, 1976). This observation could
explain the maintenance of the close relationship existing between the foetus and the mother during
pregnancy.
In order to study these H-2 antigens at the time of implantation, we developed histochemical approaches
including the immunogold staining method. Embryos were assayed for the presence of H-2 antigens before
implantation and after 5 days culture in BMOC-3+10 % FCS. H-2 antigens were detected on the
trophoblastic cells but they disappeared after implantation in vitro (LecUpteux & Remade, 1983).
Nevertheless besides the disappearance of the H-2 antigens, we show here that a strong non specific staining
of gold particles can be observed on a dense layer overlying the apical part of the embryo. Polar
trophoblastic cells are likely those which are covered by this layer while others bordering it are free of any
dense material. Particles are also present within the layer. This can be explained if the layer is slightly
porous. It is difficult to explain why this layer is present on this type of trophoblastic cell but following the
report of Searle (Searle, 1982), one hypothesis could be that it is masking H-2 antigens in a manner similar
to a layer present on the labyrinthine trophoblast, which is located at the maternal-foetal circulatory
interface.
LECLIPTEUX, T. & REMACLE, J. (1983). Disappearance of paternal histocompatibility antigens from hybrid
mouse blastocyst at the time of implantation. FEBS Letters 157, (2), 277.
SEARLE, R. F., SELLENS, M. H., ELSON, J., JENKINSON, E. J. & BILLINGTON, W. D. (1976). Detection of
alloantigens during preimplantation development and early trophoblast differentiation in the mouse by
immunoperoxidase labeling. / . Exp. Med. 143, 348.
SEARLE, R. F. (1982). Trophoblast and MHC antigens. Immunology today 3, 63.
Tooth-forming potential of mammalian neural crest
A. G. S. Lumsden, Department of Anatomy, Guy's Hospital Medical School, London SE1 9RT
It is widely supposed that the mesenchyme contributing to mammalian tooth development (through
interaction with oral ectoderm) originates, in whole or in part, from the cranial neural crest. This
supposition has lacked the support of direct experimental evidence; techniques of ablation and
3
H-thymidine labelling, by means of which the cranial neural crest origin of odontogenic mesenchyme has
been demonstrated in amphibians (Chibon, 1967), have not been successfully applied to mammalian
embryos.
In the present study, the developmental potential of mammalian premigratory neural crest has been
investigated in intraocular grafted recombinations of explanted crest and homologous competent epithelia.
Slivers of crest tissue were excised from the free margins of the posterior mesencephalic-anterior
rhombencephalic (CNC) and trunk (TNC) regions of the neural plate of E8 (6-8 somite) CD1 mouse
embryo; mandibular arch epithelium (ME) and limb bud epithelium (LE) were isolated from E10 (30-35
somite) embryos by cold trypsinisation. The tissue fragments were suspended in saline and injected via fine
glass cannula into anterior chambers. When lodged in the iridiocorneal angle, heterotypic tissues adhered to
each other and became vascularised by ciliary vessels. After 12 days the grafts were recovered, fixed,
sectioned in wax, and stained with alcian blue-chlorantine fast red (Bee and Thorogood, 1980). Cartilage,
perichondral bone, membrane bone and neural tissue developed extensively in combinations of CNC with
epithelium. ME grafted alone developed into small epithelial whorls. Teeth and large hair follicles were
formed by both CNC/ME and TNC/ME but not by CNC/LE grafts.
The results indicate that mammalian neural crest has an odontogenic potential, that this is expressed when
these cells are associated with a regionally appropriate epithelium, that normal crest cell migration is not a
prerequisite for differentiation, that the neural crest is odontogenically uncommitted prior to migration, and
that tooth determination is likely to be a function of the oral ectoderm.
CHIBON, P. (1967). Etude experientale par ablations greffes et autoradiographie, de l'origine des dents chez
l'amphibien urodele. Archs oral Biol. 12, 745-753.
BEE, J. & THOROGOOD, P. (1980). The role of tissue interactions in the skeletogenic differentiation of avian
neural crest cells. Devi. Biol. 78, 47-62.
Early mammalian development
Stage specific fucosylated glycoproteins in pre-implantation mouse
embryos
69
Hilary A. MacQueen \ Susan J. Kimber 2 and Peter R. Bagley 2 . l Department ofBiology, Open University,
Walton Hall, Milton Keynes, MK76AA.2Experimental Embryology and Teratology Unit, MRC
Laboratories, Woodmansterne Road, Carshalton, Surrey SM5 4EF
Fucosylated glycoconjugates are important constituents of the cell surface in the pre-implantation embryo
(Solter & Knowles, 1978; Gooi et al. 1981) and there is now evidence for the involvement of fucosylated
molecules in adhesion of mouse embryonal carcinoma cells (Grabel et al. 1981, 1983) and compaction of
8-cell mouse embryos (Kimber & Bird, 1983; Bird & Kimber, in press). We have incubated embryos with
medium containing 14C-fucose and harvested them at various stages in development from 2-cell to
blastocyst. Solubilized embryos were subjected to SDS-PAGE electrophoresis on 10 % acrylamide gels.
Various bonds were detected after development of the resulting fluorographs; in particular several
glycopeptides appeared in a stage specific manner. A glycopeptide appearing at the 8-cell stage might be
involved in compaction. The nature of these bands is being investigated.
BIRD, J. M. & KIMBER, S. J. Dev. Biol. (in press).
Gooi, H. C ,
156-158.
FEIZI,
T., KAPADIA, A., KNOWLES, B. B., SOLTER, D. & EVANS, M. J. (1981). Nature 292,
GRABEL, L. B., GLABE, C. G., SINGER, M. S., MARTIN, G. R. & ROSEN, S. D. (1981). Biochem. Biophys.
Res. Commun. 102, 1165-1171.
GRABEL, L. B., SINGER, M. S., MARTIN, G. R. & ROSEN, J. D. (1983). /. Cell Biol. 96,
KIMBER, S. J. & BIRD, J. M. (1983). Mouse News Letter 69, 14-15.
SOLTER, D. & KNOWLES, B. B. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 5565-5569.
1532-1537.
Oligosyndactyly: A lethal mutation in the mouse that results in a mitotic
arrest very early during development
Terry Magnusoh* and Charles J. Epstein, Department of Pediatrics, University of California, San Francisco,
California 94143, U.S.A.
Oligosyndactyly (Os) is a radiation-induced mutation located on mouse chromosome 8. In heterozygotes,
the mutation results in syndactyly, abnormal muscle development, and diabetes insipidus. When present in
the homozygous state, the mutation is lethal very early during development. Large numbers of cells
accumulate in metaphase and the embryo dies during implantation. To date, no evidence has been reported
which links the effects found in heterozygous mice with the embryonic lethality that occurs in homozygous
embryos. We began work on the Os mutation by studying homozygous embryos since they remain free of
the complicating effects caused by the presence of the wild-type gene product(s). Our primary working
hypothesis has been that the Os mutation results in abnormal spindle formation or blocked chromosome
movement. We have found that Os homozygous embryos begin to accumulate cells in metaphase at the
mid-blastocyst stage. Nevertheless, the embryos are able to hatch from the zona pellucida, attach to the
culture dish and form blastocyst outgrowths. At this time as many as 70 % of the cells in an embryo are
arrested in metaphase, with mitotic spindles being present. The appearance of the mutant phenotype is not
temperature sensitive. Since tubulin is the major component of mitotic spindles, 2D gel protein patterns
were examined, and no qualitative differences were detected in tubulins synthesized by homozygous,
normal littermate or wild-type embryos. In addition, it was found that the spindle microtubules of
homozygous embryos are not hyperstable. They can be depolymerized by cold-treatment or by incubation in
colcemid. However, after a suitable recovery time, the spindles do reform. This treatment does not result in
rescue of the mutant phenotype. When homozygous embryos were examined for the pericentriolar material
of centrosomes, immunologically reactive material was found located at opposite spindle poles. In addition,
normal appearing centrioles were found in homozygous embryos examined by thin-section electron
microscopy.
70
Early mammalian development
An interaction between the chromosomes, the plasma membrane and the
cytoskeleton in the mouse 1-cell oocyte
B. Maro*, M. H. Johnson, G. Flach and S. Pickering, Department of Anatomy, Downing Street,
Cambridge CB2 3 DY
Unfertilized mouse oocytes and eggs 1-8 h after fertilization in vitro were examined at light microscope
level for structural changes, distribution of actin, tubulin, and lamins, surface distribution of microvilli
(assessed from Concanavalin A binding pattern), chromosomal distribution and condensation, and
pronuclear formation and migration. The influence of the drugs cytochalasin D and nocodazole (alone or in
combination and applied continuously or in pulses of varying duration) on these parameters was
investigated. Characteristic changes in the distribution of surface microvilli, subcortical actin and tubulin
were observed in association with the formation of the fertilization cone, rotation of the second meiotic
spindle, extrusion of the second polar body, and formation and migration of pronuclei. Particularly striking
was the clear association under a number of conditions between the presence of chromosomes subcortically
and both a focal concentration of actin plus a localized loss of microvilli. This association was only observed
when the chromosomes were not enveloped in a pronuclear membrane and did not obviously depend upon
the presence of microtubules associated with the chromosomes. The possibility that the chromosomes
themselves might cause local organisational changes in the overlying cytocortex, and the significance of such
an effect, is considered.
Presence of nucleolar vesicles and vacuoles in the rat oocyte
C. Merveille, A. M. Renard and E. Baeckeland, Department of Embryology, University of Li^ge, Ruede
Pitteurs20, B-4020 Liege, Belgium
The present investigations were undertaken to study the modifications of the nucleolus under the
influence of gonadotropins in the follicular oocytes of immature rats. Nucleolar vacuoles and vesicles in the
follicular oocytes of mature and immature rats were investigated accordingly.
First, 28-day-old rats were injected with 40 UI of FSH and the oocytes were observed after 24 h, 48 h,
72 h and 96 h. Second, other 28-day-old rats were injected once, twice or three times with 40 UI of FSH
with an interval between the injections of 24 h; in this last case, the oocytes were observed 24 h after the last
injection. Oocytes of mature rats were also examined at prooestrus, oestrus, meteoestrus and dioestrus
stages.
In the three experiments, some nucleoli exhibited vesicles or vacuoles but their frequency was very
different for each condition. In mature rats nearly all nucleoli showed closed cavities. In immature rats their
presence seemed to be dependent upon stimulation with FSH.
LH does not seem to influence the presence of nucleolar vesicles: one injection of 25 UI of LH, 24 h after
an injection of 40 UI of FSH and the observation of the nucleolus 1 h after this injection did not show a
change in the percentage of nucleoli with vacuoles or vesicles. The nature and the significance of these
vacuoles and vesicles are discussed.
Early mammalian development
71
Regulation of heat-shock protein synthesis in mouse preimplantation
embryo
M. Morange*1, O. Bensaude * and C. Babinet 2. 1Uniti de Ginitique cellulaire du College de France et de
I'Institut Pasteur. 2Uniti de Ginitique des Mammiferes, Institut Pasteur, 25 rue du Docteur Roux,
75724 Paris Cedex 15
Heat-shock proteins (HSP) are very conserved polypeptides, the synthesis of which is stimulated after a
stress (heat, exposure to some chemicals, viral infection) in cells of all organisms from bacteria to man.
However, this stimulation is not observed in cells of very early Drosophila or sea urchin embryos.
In the mouse, at the two-cell stage, the first proteins detected, the synthesis of which requires activation of
the embryonic genome, have been shown to be HSPs; later, at the eight-cell stage, two HSP are the proteins
synthesized spontaneously at the highest level, while the other HSP cannot be stimulated by stress.
Interestingly, cell lines in mouse embryonal carcinoma also express high constitutive levels of the same two
HSP. Moreover, in cells of some lines, the synthesis of the other HSP cannot be induced by any stress.
However, in vitro differentiation of these cells brings the usual inducible phenotype.
Effect on mouse development of high-speed centrifugation at the
pronuclei stage
Jacques Mulnard and Frangoise Puissant, Department of Anatomy and Embryology and Human
Reproduction Research Unit, The Free University of Brussels Medical School, Rue Evers2, B-1000 Brussels,
Belgium
Fertilized one-cell eggs obtained from superovulated Fl (C57BLxCBA) hybrid females mated with OF1
males were submitted to accelerations up to 90-000 g during 30 min at 37 °C in a Sorvall ultracentrifuge.
After dispersion of the cumulus by hyaluronidase, they were either prepared for light and electron
microscopy or placed in culture for 96 h together with non-centrifuged control eggs from the same female.
At 90000 g, practically all lipidic inclusions are expelled in the centripetal part of the perivitelline space.
The stratification of the other organelles is moderate, indicating a high degree of cytoplasmic viscosity.
Three zones can be recognized along the centrifugation axis: 1) a centripetal hyaline cap in which a few
elements of granular endoplasmic reticulum are sedimented, 2) a large mitochondrial layer containing also
Golgi complexes and 'jigsaw' vesicular aggregates, 3) a small centrifugal hyaline cap in which a sperm tail is
occasionally seen. All three layers contain filamentous material, isolated ribosomes and small polyribosomes, secondary lysosomes, vesicular and cisternal agranular endoplasmic reticulum associated with
multivesicular bodies. The pronuclei remain in the central area: they are elongated in the centrifugation axis
by migration of their fused nucleoli towards the centrifugal pole. After 96 h in vitro a variable proportion of
the centrifuged embryos form morphologically normal early blastocysts, some of which were transferred to
the uterine horns of 3-day pseudopregnant foster-mothers. Out of 28 transfers, three 13-day normal
embryos and two healthy youngs have hitherto developed from eggs centrifuged at 90-000 g at the pronuclei
stage. There were no significant differences of developmental timing or survival ratio between the
centrifuged and control embryos.
72
Early mammalian development
An investigation into the levels of cytochrome P450 present in the rat
visceral yolk sac between 9*5 and 19*5 days of gestation
M. K. Pratten, K. M. Holness and F. Beck, Department of Anatomy, The Medical School, University of
Leicester, Leicester, LEI 7RH
Cytochrome P450 is an important component of the microsomal drug metabolising system in adult liver.
However, it has been shown that drug metabolising enzymes are absent from the liver of the foetus or
neonate of many animal species (Neims, Warner, Loughnan & Aranda, 1976). It has been suggested that
the visceral yolk sac functions, for at least part of gestation, as the foetal liver (Padykula, 1958). We have
therefore investigated the levels of cytochrome P450 present in the rat visceral yolk sac at different stages of
gestation between 9-5 and 19-5 days.
Yolk sacs were homogenised and the levels of cytochrome P450 measured using the method of O'Brien
and Rahimtula (1978) and protein by the Lowry method (1951) so that it was possible to compare the
specific activity throughout the later stages of pregnancy.
No evidence for the presence of the enzyme was found prior to 14-5 days of gestation. The levels of
cytochrome P450 were found to increase at later stages and then remain constant until 18-5 days when a loss
of enzyme activity was observed. These results suggest that the rat visceral yolk sac may be instrumental in
drug metabolism between 14-5 and 18-5 days of gestation. Further studies on the levels of other enzymes
involved and their modulations are now in hand.
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the
Folin Phenol reagent. /. Biol. Chem. 193, 265-275.
NEIMS, A. H., WARNER, M., LOUGHNAN, P. M. & ARANDA, J. V. (1976). Developmental aspects of the
hepatic cytochrome P450 monoxygenase system. Anna. Rev. Pharmacol. Toxicol. 16, 427-445.
O'BRIEN, P. S. & RAHIMTULA, A. D. (1978). A peroxidase assay for cytochrome P450. In Methods in
Enzymology, (Editors in Chief S. P. Colowick & N. O. Kaplan), Volume LII, Part C 'Biological
oxidations: microsomal, cytochrome P450 and other hemoprotein systems', (eds S. Heischer & L.
Packer), pp. 407-412. Academic Press, New York.
PADYKULA, H. A. (1958). A histochemical and quantitative study of enzymes of the rat's placenta. /. Anat.
92, 118-129.
Aggregation between early mouse embryos and embryonal carcinoma
cells: A model for blastocyst transformation
/. Reima
l>2
, J. Wartiovaara 2>3 and E. Lehtonen l. Departments of1 Pathology, 2Electron Microscopy and
^Medical Biology, University of Helsinki, Helsinki, Finland
The mouse blastocyst contains two distinct tissues, viz. the inner cell mass (ICM) and the trophectoderm.
The blastocyst differentiation is initiated at 'compaction' of the 8-cell stage blastomeres, involving
redistribution of cell surface projections, reorganization of cytoskeletal structures and formation of
membrane junctions. We have investigated the preimplantation development by studying aggregation
between embryo and F9 embryonal carcinoma (EC) cells. In this model system, the EC cells (similarly as
isolated ICM cells) tend to segregate into an inside position in the chimaeric embryo.
In aggregation experiments (Lehtonen et al., J. Embryol. exp. Morph. 81, 1984), the 8-cell-stage
blastomeres acted as in normal preimplantation development. The aggregates compacted and formed
chimaeric morulae with EC cells surrounded by the embryo cells. Establishment of cell contacts by
microvilli and larger cell processes, and eventually spreading of the blastomeres over the EC cells took
place. In the chimaeric embryos, the embryo and EC cells had cell projections similar to those between ICM
and trophectoderm cells in vivo. Cytoskeletal organization involved microtubules parallel to contact areas
between embryo and EC cells. The cell projections contained orientated microtubules and bundles of
microfilaments. Similarly as in compacting morulae in vivo, membrane specializations developed. Typically,
adherent junctions and close contacts, later also gap junctions, were formed between embryo and EC cells.
Cellular features connected with blastocyst transformation in vivo seem to be repeated in the
embryo-EC-aggregation model in vitro. The aggregation model allows a study of the precise time sequence
of the process connected with blastocyst transformation.
Early mammalian development
12>
Anti-progesterone monoclonal antibody affects early embryonic
development in the mouse by an indirect rather than direct action
V. Rider, AFRC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT
Previous studies showed that pregnancy in two inbred strains of mice (BALB/c, CBA) was prevented by
passive immunization with anti-progesterone monoclonal antibody (Wang, Rider, Heap and Feinstein,
1984). Preliminary investigation indicated that the rate of embryo development was retarded in
antibody-treated mice and we have investigated this aspect in more detail.
Nulliparous females were injected intra-peritoneally 32 h post coition (p.c.) with ascites fluid containing
4-5 nmol immunoglobulin (IgG). At autopsy on days 3 and 4 p.c. the reproductive tract was flushed with
phosphate-buffered saline, embryos were collected and their developmental stages assessed. At day 3 p.c.
embryos were at the 4 cell stage regardless of treatment. By day 4, however, there was a marked effect of
antibody treatment since most embryos from treated-females had not begun cavitation (BALB/c 16 %
blastocysts; CBA 4 %) while embryos from control animals were at the blastocyst stage (BALB/c, 93 %;
CBA 62 %).
To determine whether altered cleavage rates were due to a direct action of IgG, BALB/c females were
superovulated with 5 i.u. pregnant mare serum gonadotrophin followed 48 h later by 5 i.u. human chorionic
gonadotrophin (hCG). Two-cell stage embryos were collected from oviducts 44-46 h post hCG in
BMOC-culture medium. Purified anti-progesterone IgG (range 0-1-0 mg/ml) was added to the medium on
the day of culture. At 98 h post hCG greater than 80 % of the embryos cultured were blastocysts at each
dose tested.
The results indicate that progesterone is important in early stages of cleavage in vivo, and that antibody
treatment probably alters the tubal environment producing a deleterious effect on embryo development
rather than exerting a direct influence on the early embryo.
WANG, M.-Y., RIDER, V., HEAP, R. B. & FEINSTEIN, A. (1984). / . Endocrin. 101, 95-100.
Cell lineage analysis in the mouse embryo using molecular probes
J. Rossant*1, J. Sanford 2, V. M. Chapman 2andG. Andrews 3 .'Department of Biological Sciences, Brock
University, St. Catharines, Ontario, Canada. 2Department of Molecular Biology, Roswell Park Memorial
Institute, Buffalo, N.Y., U.S.A. 3NIEHS, Research Triangle Park, N.C., U.S.A.
Experimental manipulation of the early mouse embryo, combined with the use of sensitive genetic
markers, has established clearly the later derivatives of the first cell lineages to be formed in the embryo. For
example, embryos derived from blastocysts reconstituted with M. caroli inner cell masses and M. musculus
trophectoderm have been analyzed using in situ DNA-DNA hybridization with a probe to M. musculus
satellite DNA. These experiments have shown that both the ectoplacental cone and the extra-embryonic
ectoderm of the 7-5 day embryo are pure derivatives of the trophectoderm, whereas the trophectoderm
derivatives of the later chorioallantoic placenta are intimately interspersed with maternal and inner cell mass
derivatives. Other experiments have shown that pure populations of primitive endoderm derivatives can be
isolated from the visceral and parietal yolk sacs between 12 and 15 days of pregnancy.
We have used the information obtained from these studies to ask whether there are any common features
of the molecular biology of the derivatives of the early cell lineages which might shed light on the
mechanisms of their establishment and maintenance. Examination of the extent of methylation of several
repetitive DNA elements revealed consistent undermethylation in all derivatives of the extraembryonic
lineages, trophectoderm and primitive endoderm. Recent studies have shown that this undermethylation
extends to transcribed sequences, including major urinary proteins, albumin and alpha-fetoprotein genes.
The latter example is interesting since alpha-fetoprotein has been previously shown to be expressed and
undermethylated in the visceral endoderm, but the present study has shown that it is also undermethylated
in extra-embryonic tissues in which it is not expressed. These studies do not eliminate the possibility that
specific sequences in the 5' region of genes may remain methylated and act in a controlling manner, but they
show that global methylation is not required for control of gene activity in these mammalian cells. Studies
are currently in progress to determine at what stage in development these cell-lineage-specific patterns of
methylation are established and whether undermethylation always accompanies differentiation of
extraembryonic cell types. The possible significance of undermethylation for the regulatory capacity of the
extraembryonic cell lineages will be discussed.
74
Early mammalian development
The shaping of the mesometrial proamniotic cavity in the mouse: Are
there various mechanisms?
S. Sandor, Maria Checiu, Laboratory of Embryology, Center of Hygiene and Public Health, Timisoara,
Romania
1) In a part (about 20 %) of several hundred mouse embryos (RAP strain) examined, the shaping of the
extraembryonic part of the proamniotic cavity differs from the normally described variant:
- Early imagination of the mesometrial pole of the embryoblast (together with the covering trophoblast)
gives rise to an early cavity communicating with the uterine lumen. Subsequent proliferation of the
surrounding trophoblast separates this 'channel' from the uterine lumen.
- Another possible, unusual, mechanism (which was not supported by our microscopic findings) may be a
secondary communication with the uterine lumen - by the rupture of its roof- of a very early formed cavity.
2) The observations reported attest to the relatively frequent appearance of a phylogenetically older
morphogenetic mechanism, with respect to an essential step in the embryogenesis of Muridae: the shaping
of the proamniotic cavity.
Cytoskeleton and nuclear lam in organization during mammalian
fertilization and early development
Gerald Schatten*1, Calvin Simerly \ Heide Schatten 1, ChristiCline l and Gerd Maul2, department of
Biological Science, Florida State University, Tallahassee, FL 32306, U.S.A. 2The Wistar Institute,
Philadelphia, PA 19104, U.S.A.
The organization of the cytoskeleton and the nuclear lamina were studied in oocytes during fertilization
and preimplantation embryos from mice and hamsters after natural mating. Microtubules, detected with
antitubulin immunofluorescence and electron microscopy, comprise the meiotic apparatus of the
unfertilized oocyte. After sperm incorporation a new class of microtubules forming into about a dozen
cytoplasmic asters assemble in and around the developing pronuclei. As the pronuclei are positioned at the
oocyte center, the microtubules form into a dense cytoplasmic matrix. This matrix disassembles at prophase
when microtubular sheaths appear to envelope the adjacent, but separate, pronuclei. The first mitotic
apparatus is quite unusual, barrel-shaped and devoid of asters, reminiscent of plant spindles. During
cleavage a dense bundle of interzonal microtubules appear and radial monasters position the blastomere
nuclei at the cell centers. By third division typical animal-type mitotic apparatus with fusiform spindles are
observed. Interestingly the incorporated sperm centriole does noj seem to serve as a micro tubule organizing
center (MTOC) during mouse fertilization; contrary to dogma, the active MTOCs during mammalian
fertilization may be of maternal origin. Microfilaments, localized with rhodamine-phalloidin, are detected at
the oocyte surface during polar body formation and in the formed incorporation cone. In pronucleate
oocytes, microfilaments seem to colocalize with the microtubules in the cytoskeletal matrix. Nonerythrocyte spectrin or fodrin also is localized subjacent to the oocyte surface. Immunofluorescence
localization with monoclonal antibodies specific to nuclear lamins A, B and C indicate that the sperm
nucleus, which does not have lamins, acquires these nuclear lamins as the male pronucleus develops. The
meiotic chromosomes similarly do not have any associated lamins, though the female pronucleus acquires
these nuclear proteins immediately after the completion of meiosis. Though all nuclei within the fertilized
oocyte cytoplasm have the nuclear lamins, remarkably the second polar body nucleus remains devoid of
lamins, suggesting a pathway for nuclear degeneration. These studies support the hypothesis that specific
cytoskeletal and nuclear lamina alterations are required for the motility events necessary for the successful
completion of mammalian fertilization. Supported by grants from the NIH and NSF.
Early mammalian development
Prenatal repair of clefts in the secondary palate of rats
75
P. M. Schtipbach and H. E. Schroeder, Department of Oral Structural Biology, Dental Institute, University
of Zurich, CH-8028 Zurich, Switzerland
In a previous study we have shown that partial clefts of the hard palate with soft palate integrity, can be
produced through amniocentesis performed in Sprague-Dawley rats at day 16 of gestation. Such clefts result
from fusion and complete union of the posterior shelf portions, and fusion in the posterior region occurs
independently from fusion in the anterior region. The purpose of the present study was to answer the
questions: (1) whether or not such clefts, once being experimentally induced, can be repaired prenatally by
further late closure of the shelves in the anterior direction, and (2) how long does the medial shelf edge
epithelium retain its competence to fuse? A refined technique of amniotic sac puncturing was employed at
day 16-2 of gestation in order to produce a series of total clefts and rare forms of partial clefts in
Sprague-Dawley rat fetuses. In a first study generating a total of 396 fetuses of a precise, individually
determined age, 301 fetuses were examined microscopically and 95 upper jaws were examined in the
scanning electron microscope (SEM) and in serial epon sections. Total clefts were found in 48-9 % of
fetuses examined at day 17-8 and in 21*8 % of those examined at day 19-3. Partial clefts were observed in
141 % and 18-5 % of fetuses examined at days 17-8 and 19-3, respectively. At day 19-3, 16-1 % of the
fetuses showed a very inconspicuous, small abnormality (with residual clefting and incomplete fusion with
the nasal septum) in the region of the palatine foraminae. Morphological observations suggested that under
conditions of detained palatal closure (1) fusion of the soft palatal shelves commences independently from
and prior to fusion of the hard palate, (2) delayed palatal shelf fusion proceeding in the anterior may occur
with or without sickle-shaped clefts remaining in the anterior hard palate, and (3) in fetuses with
small sickle-shaped clefts, fusion of the palatal shelves with the nasal septum does not occur, implying
incomplete prenatal repair. In a second study, total clefts were produced by amniotic sac puncturing at day
15-5 of gestation. About 28 % of 178 fetuses showed a total cleftpalate. In 12 selected upper jaws (age
ranging from 16-2 to 21-5 days), the medial shelf edge epithelia (MEE) were examined in the SEM and in
serial epon sections. The density of superficial cells in various states of differentiation was counted
morphometrically. In the soft palate, critical density of ciliated cells among MEE-cells was reached at day
170-17-5, and in the hard palate at day 17-5-18-0. At the dorsal aspect of the nasal septum, ciliated cells
appeared at day 17-5-17-7. These data imply, that epithelial competence to fuse is retained longer in the
anterior than in the posterior region.
Extraneous signals affect the growth and differentiation of cultured rat
embryonic shields
N. Skreb, V. Crnek-Kunstelj and F. Bulit, Institute of Biology, Faculty of Medicine, Zagreb, Yugoslavia
A modified organ culture of rat embryonic shields provides favourable conditions for a period of two
weeks for the differentiation of main tissue types. Since the normal process of cell differentiation requires
various extraneous signals during the critical period of embryonic development we tried to analyse the
impact of various factors on the growth and differentiation of cultured rat embryonic shields.
In the first part of our study either N602 dibutyryl 3'5' cyclic adenosine monophosphate (dbcAMP,
Sigma, lmM) or retinoic acid (RA, Sigma, 10, /LIM) was added to Eagle's minimal essential medium
supplemented with about 40 % of rat serum. If dbcAMP was added dunng the first week of culture the
growth was severely retarded. If added during the second week the skeletal muscle appeared more
frequently. If RA was added in the second week the growth was unaffected, but the differentiation of the
skeletal muscle seemed to be stimulated, whereas that of the cartilage was impeded. In the second part of
the study the embryonic rat shields were cultured only in Eagle's minimal essential medium. The growth was
practically inexistent, and the survival was significantly better in the medium supplemented with serum. The
differentiation proceeded with relatively few consequences. Only the incidence of neuroblasts and skeletal
muscle were significantly decreased in the serum-free medium.
76
Early mammalian development
Mesoderm formation in the rat embryo, cultured in vitro, analysed with
a lectin-coated colloidal gold marker
A. E. Smits-Van Prooije*, R. E. Poelmann and Chr. Vermeij-Keers, Department of Anatomy, University of
Leiden, 2333 AL Leiden, The Netherlands
Rat embryos are cultured in vitro in a rotating-bottle system. Because transplantation techniques as
achieved by e.g. Le Douarin (1982) are not possible in these embryos, a marker was developed that, after
injection into the embryos, would be taken up by the neural crest cells. Gesink et al. (1983) showed that the
plasma membrane contains many receptors for wheat germ agglutinin (WGA). To visualize this lectin,
colloidal gold (Au, 12 nm) was coupled to WGA. Injected into the amniotic cavity of the in vitro cultured
rat embryos, the WGA-Au probe is endocytosed by all cells bordering that cavity and stored in
membrane-bound vesicles; the content of these vesicles is not exocytosed during subsequent development
and can therefore serve as a marker for ectoderm-derived cells, such as primitive streak-, ectodermal
placode- and neural crest-derived cells. In order to discern mesoderm cells produced by these sources, it is
necessary to investigate a series of embryos with varying survival times (2 to 30 h). Computer-aided 3-D
reconstructions are made from the serially sectioned embryos. Some results are:
- In the trunk, the neural crest not only produces the spinal ganglia, but some cells are also added to the
sclerotome.
- The segmented mesoderm caudal to the last pair of somites, contains enough cells to produce another 5
pairs of somites. After injection, the 6th newly formed pair of somites is the first to be labeled.
- The mesoderm of the fore-limb is heavily labeled; the somites located at the same level are not. The
origin of these cells remains to be elucidated.
GESINK, A. F., POELMANN, R. E., SMITS-VAN PROOIJE, A. E. & VERMEU-KEERS, CHR. (1983). The cell
surface coat during closure of the neural tube, as revealed by concanavalin A and wheat germ agglutinin.
/. Anat. 137, 418-419.
LE DOUARIN, N. (1982). The neural crest. Cambridge University Press.
Appearance of trophoblastic giant cells in normal and transformed
(YSCA) visceral yolk sac
H. Sobis, Y. L. Lu, L. Van Hove and M. Vandeputte,
University of Leuven, Rega Institute, Department of Immunopathology, B-3000 Leuven, Belgium
Twelve day-old rat visceral yolk sac was cultured in tubes on a roller system during 48 h. This method
favours the proliferation of yolk sac cells probably by formation of three-dimensional structures. The
spherical pieces of the membrane were placed in organ culture or implanted into the omentum of adult
syngeneic rats. After 2-3 weeks the appearance of giant cells was observed in vitro as well as in vivo. In both
cases it was preceded by the appearance of poorly differentiated cells from which the giant cells differentiate
as documented by autoradiography using tritium-labelled thymidine. The trophoblastic nature of the giant
cells is illustrated by the presence of A5-3j3-hydroxysteroid dehydrogenase using pregnolone as substrate and
by the presence of progesterone in the organ culture medium.
Yolk sac derived yolk sac carcinoma cells were cloned and subclones, prepared by single cell cloning,
were injected into syngeneic rats. In growing tumors A5-3/Miydroxysteroid dehydrogenase-positive giant
cells were found among yolk sac endodermal cells. The level of progesterone in the serum of tumor-bearing
animals was elevated when the giant cells were present in the tumor.
Early mammalian development
Sister chromatid exchange in preimplantation mouse embryos: a
sensitive assay for genotoxic effects in early embryos
77
H. Spielmann, R. Vogel, Ch. Krugerand C. Engeholm, M. v. Pettenkoferlnst., Federal Health Office
(BGA), 1 Berlin 33, W. Germany
A sensitive technique was developed for the differential staining of sister chromatids in preimplantation
mouse embryos during in vitro culture for two cell
cycles. 4-cell and 8-cell embryos and also morulae and
blastocyst were cultured in media containing 10~6 M 5-bromodesoxyuridine (BrdU) for one cell cycle and
without BrdU for the next cell cycle (total culture period 48 h). 0-1 /ig/ml Colcemid was added 3 h before
chromosome preparation. Sister chromatid exchanges (SCEs) can be visualized after staining with the
Fluorescence-Plus-Giemsa technique. SCE frequencies per chromosome were 0,6 for 4-cell and 8-cell
embryos and also for morulae and blastocysts compared to 0,3 per chromosome in bone marrow of adult
mice that were exposed to BrdU in vivo. A positive SCE-staining could never be obtained in
preimplantation mouse embryos in vivo after BrdU treatment of the mother animals. Increased SCE-rates
were also obtained in preimplantation mouse embryos during culture in media containing rat or human sera
which are supporting embryonic differentiation in vitro. A significantly higher SCE rate was seen in embryos
cultured in media supplemented with either 10 % rat sera from animals treated with cyclophosphamide
(CPA) or 10 % human sera from cancer patients treated with cytostatic drugs. Culture media containing
sera from these two sources were also inducing diplopchromosomes in mouse morulae and blastocysts,
however, diplochromosomes could never be detected in media containing either BSA or rat or human sera
from untreated animals or patients. SCE frequencies were increased dose related in 4-cell and 8-cell mouse
embryos flushed from oviducts 1 h after CPA treatment of the mother using 5-20 mg/kg. Other sensitive
methods (e.g. cell number, growth and differentiation in culture or in vivo after transfer to foster mothers)
could so far not detect embryotoxic effects in preimplantation mouse embryos treated with 20 mg/kg CPA
or less in vivo. SCE-determinations in early mouse embryos may be useful to study embryotoxic effects and
repair mechanisms.
Regulation of cell number following aggregation of morulae and
formation of chimaeric blastocysts in the mouse
Joan Sutherland and Alan Handyside, MRC Experimental Embryology and Teratology Unit,
Woodmansterne Road, Carshalton, Surrey SM5 4EF
The outer and inner cells of the 16-cell morula normally give rise to the trophectoderm (TE) and inner cell
mass (ICM) of the blastocyst, respectively. Under different experimental conditions, these cells either retain
their normal fate, or express developmental lability. Using a double labelling technique which allows the TE
and ICM nuclei of individual blastocysts to be distinguished (see abstract page 64), we have examined how
the aggregation of morulae affects the allocation of cells to the two cell types in the resulting chimaeric
blastocyst. If in this situation, the cells are not labile, the numbers of TE and ICM cells in double
blastocysts, for example, should both be twice the numbers in single blastocysts. Alternatively, if the cells
are labile, the proportion of TE and ICM cells should be altered according to the reduced surface
area/volume ratio i.e. the numbers of these cells should be less, and greater than twice those in single
blastocysts, respectively. The ratio of cell numbers in double chimaeric blastocysts and single blastocysts
were examined soon after cavitation and again after transfer to the oviducts of pseudopregnant females and
recovery from the uterus three days later. The ratio of TE cells was significantly less than double, both
initially and after transfer. However, there was no corresponding increase in the ratio of ICM numbers in
the same blastocysts, and total cell number was also significantly less than twice in double blastocysts. We
suggest that during aggregation the outer cells which form the initial contact are reallocated as inner cells.
But the resulting increase in the proportion of inner cells, which divide at a slower rate than the outer cells,
reduces ICM and total cell number in early double blastocysts. After transfer, the numbers of ICM cells in
chimaeric blastocysts probably fail to catch up because of the increased vulnerability of late dividing cells to
cell death. It, therefore, appears that in addition to early postimplantation regulation, the number of ICM
cells in chimaeric blastocysts is partially regulated before implantation.
78
Early mammalian development
Studies on the behaviour of nuclei in thymocyte-oocyte homospecific
mouse cell hybrids
D. Szollosi*, Renata Czo/owska, A. K. Tarkowski, J. Modlinski, I.N.R.A. Station centrale de Physiologie
animate, Jouy-en-Josas, France and Department of Embryology, University of Warszawa, Warszawa,
Poland
Cell hybrids were formed between mouse thymocytes and ovulated oocytes with polyethylene glycole
(PEG) and Sendi virus under two different conditions (Czolbwska et al. J. Cell Science, in press): 1. hybrids
were formed followed by parthenogenetic activation of the oocytes, 2. parthenogenetic activation of oocytes
followed by hybridisation with thymocytes.
Under the first condition usually a small number of thymocytes (2-4) hybridize with the oocytes and
following 6-22 h of culture the thymocyte nuclei are structurally identical to the female pronucleus formed
as a result of activation by alcohol. In each 'pronucleus' a single, giant nucleolus develops and remains
throughout the culture period. Under the second condition up to ten thymocytes were observed to enter the
oocyte cytoplasm. Thymocyte nuclei showed a number of different images after 6-22 h of culture. They
ranged from compact chromatin masses lying freely within the oocyte cytoplasm to nuclei surrounded by
forming nuclear envelope elements. They are usually multilaminar in nature (quadri- to hexalaminar), at
least in regions, but only the innermost appears to constitute the usual, continuous nuclear envelope.
Nucleoli form along the nuclear envelope. The observations permit us to say that as soon as thymocytes
enter the oocyte cytoplasm their respective nuclear envelope is removed, while a new envelope is
reconstituted along the surface of the chromatin mass from membrane components recruited from the
toplasm. Other details of nuclear behaviour shall also be presented
(Research supported in part by a Research Exchange programme between France and Poland.)
Germ cell differentiation and gonad formation in the mouse embryos
treated with cadmium during the early organogenesis stages
P. P. L. Tarn and W. K. Liu, Department of Anatomy, Faculty of Medicine, The Chinese University of Hong
Kong, Shatin, N. T., Hong Kong
Cadmium has been shown to interfere with spermatogenesis and ovulation in adult mouse, rat and golden
hamster, but its effects on the prenatal development of primordial germ cells and fetal gonads were not fully
known. In the present study, cadmium chloride (5-6 mg/kg) was given intraperitoneally to the pregnant
mice at 7-5-8-5 days p.c. At this age, the mouse embryos were at the primitive-streak to early-somite stages
of development and the primordial germ cells were for the first time detectable by their strong alkaline
phosphatase activity. The growth of the cadmium-treated embryos was initially retarded but an accelerated
restorative growth ensued so that at birth both the body size and the body weight were within normal
ranges. Histological examination of the genital ridges of 13-5-day embryos revealed that in both sexes the
size of the ridges was reduced and there were fewer primordial germ cells in the ridges. In the testes of
16-5-day embryos, seminiferous tubules and interstitial tissues were formed normally but the testes were
small in size and the number of spermatogonia found inside the tubules was about 65 % of the normal. In
the ovaries, primordial follicles were formed but the ovaries contained much fewer meiotic oocytes. About
18-50 % of the cadmium-treated embryos showed exencephaly at the diencephalic and mesencephalic
region. There were however no consistent variations in the extent of germ cell reduction and gonadal
deficiency between the exencephalic embryos and those having a normal head morphology. A defective
hypothalmo-pituitary axis which may be associated with exencephaly was unlikely to be the cause of poor
gonadal development because both structures seemed to have developed normally in the exencephalic and
the non-execephalic treated embryos. The growth pattern of cadmium-treated embryos was reminiscent of
that of mitomycin C-treated mouse embryos which showed a depletion of germ cells, poor gonadal
development and a high incidence of postnatal infertility (Tam & Snow, 1981). A. mismatch in the growth
and differentiation of tissues during rapid restorative growth may be the causative factor for most errors of
morphogenesis.
P. P. L. & SNOW, M. H. L. (1981). Proliferation and migration of primordial germ cells during
compensatory growth in the mouse embryos. /. Embryol. exp. Morph. 64, 133-149.
TAM,
Early mammalian development
Ovary development in the bandicoot (Peramelidae, Marsupialia)
79
Suzanne L. Ullmann, Department of Zoology, University of Glasgow, Glasgow, G12 8QQ, Scotland
Primordial germ cell (PGC) morphology and migration appear to conform to the eutherian pattern (1),
but the origins of the somatic elements of the gonad are controversial (2).
Early stages of bandicoot gonadal development are similar in both sexes: blastema cells differentiate
directly into two cell types which give rise to (i) Sertoli cells (cfcf) or medullary cord cells ( $ 9 ) and (ii)
stroma cells. Sex cord formation during testis development in the native cat (3) and bandicoot (2) does not
involve mesothelial proliferations, as described for most eutherians.
In the prospective ovary fibroblasts separate medulla from a narrow cortex in which, by day 5 post
partum, most PGCs are located. By day 10 oogonia largely replace PGCs and the cortex has thickened: a
mesothelial contribution to it at this stage is probable. The medulla shrinks progressively.
Meiosis begins on day 21 and primordial follicles appear a week later. Follicle cells seem to arise from the
stroma and not the rete, as in the mouse (4). By day 34 the first primary follicles are present with a
paranuclear complex within the oocyte (5).
1) ULLMANN, S. L. (1981). Observations on the primordial germ cells of bandicoots (Peramelidae,
Marsupialia). /. Anat. 132, 581-595.
2) ULLMANN, S. L. (1981a). Sexual differentiation of the gonads in bandicoots (Peramelidae, Marsupialia).
In Development and Function of Reproductive Organs. Byskov & Peters (Eds). Elsevier, North-Holland.
3) ULLMANN, S. L. (1984). Early differentiation of the testis in the native cat Dasyurus viverinus
(Marsupialia). /. Anat. (in press).
4) BYSKOV, A. G. (1978). The anatomy and ultrastructure of the rete system in the fetal mouse ovary. Biol.
Reprod. 19, 720-735.
5) ULLMANN, S. L. (1978). Observations on the primordial oocyte of the bandicoot Isoodon macrourus
(Peramelidae, Marsupialia). /. Anat. 128, 619-631.
Scanning electron microscopic studies of the visceral yolk sac entoderm
in early somite rat embryos from streptozotocin diabetic rats and from
rats fed a high sucrose diet, in vivo studies
/. Zusman and A. Ornoy, Laboratory of Teratology, Department of Anatomy and Embryology, Hebrew
University - Hadassah Medical School, Jerusalem, Israel
Offspring of diabetic rats exhibit congenital malformations, growth retardations and reduced litter
average. Similar findings were found in rats fed a 50 % sucrose diet. In order to find whether there is a
correlation between embryonic malformations and placental changes, we studied by SEM the morphological characteristics of visceral yolk sac entodermal cells in H i and 13£ day old embryos from streptozotocin
(STZ) induced diabetic rats and from rats fed a 50 % sucrose diet before and during pregnancy. Normal rats
fed a regular diet served as controls. In both control groups, SEM studies showedhomogeneous epithelial
cells covered with uniform rod shaped microvillii. In H i day old embryos from rats fed a high sucrose diet,
many cells were devoid of microvilli and instead had apical crater formation and numerous membranes
folds. By 13i days, changes were more severe consisting of vesicular blebs of various sizes attached to the
cell membranes. Sometimes the microvillii were broad and short, resembling blebs. Some cells were
connected among themselves by membranes or cytoplasmic bridges. Similar changes were observed in
embryos from STZ-diabetic rats (40 or 65 mg/kg of body weight, on day 0 or 5 of gestation respectively). In
this group, however, both the craters and blebs were smaller and more numerous than in embryos from the
high sucrose diet fed animals. It is possible that part of the embryonic effects observed in diabetic animals or
in rats fed a high sucrose diet are due to the above described visceral yolk sac lesions. Supported in part by
the US - Israel Binational Grant No. 2774/82, by the Grant No. 015-4846 from the Israel Ministry of Health
and by the Richard Meyer Grant for Teratological research.