Early mammalian development 57Involvement of DNA replication cycles in the timing of early mouse

developmentH. 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, indifferent biological systems, that cell cleavage per se is not a prerequisite for scheduled tissue-specificenzymes expression (Satoh, 1982) or even for some early morphogenetic events (Lillie, 1902; Kimber andSurani, 1981). On the contrary, a critical DNA replication cycle has been proposed to be responsible for cellcommitment during embryogenesis. This hypothesis has been verified for early mouse embryogenesis, usingantiproliferative drugs affecting essentially DNA replication. A transient polyamine depletion in earlycleaving embryos was shown to induce an early cavitation with regard to the elapsed cell cycles. Morerecently, we used a more direct way, namely the specific inhibition of DNA polymerase a by aphidicolin: the4th 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 the32 to 64 cell-blastocyst stage onwards. This total inhibition of DNA replication was achieved by aphidicolinbut not by either dideoxythymidine or dideoxythymidine-triphosphate, which is in disagreement with therecent findings of Siegel and Kalf (1982) concerning the involvement of DNA polymerase ft instead of DNApolymerase 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 differentia-tion (B. Capone & H. Alexandre).

KIMBER, S. J. & SURANI, M. A. H. (1981). Morphogenetic analysis of changing cell associations followingrelease 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, Chaetopteruspergamentaceus. Arch. Entwicklungsmech. Org. 14, 477.

S*TOH, N. (1982). Timing mechanisms in early embryonic development. Differentiation 22, 156^163.SIEGEL, R. 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/mouseteratocarcinoma hybrids

F. J. Benham 1, K. E. Davies 2, M. V. Miles 3 and P. N. Goodfellow 3. lPaediatric Research Unit, Guy'sHospital 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 linePCC4 by (1) microcell transfer of a human X or X/autosome translocation into an HPRT-denvation ofPCC4 or by (2) introduction of the dominant selectable marker Ecogpt into a human genome followed bymicrocell transfer of the human chromosome which contains the gpt gene into PCC4. These lines retain thecapacity to differentiate in vitro. The lines are being used to investigate regulation of human genes by theembryonic environment of the E.C. cell, for example, expression of HLA genes where results indicate thatthe E.C. cell does not regulate Class I genes, but does regulate Class II genes. The hybrids also provide asystem for isolating X-coded human gene sequences which are expressed in the E.C. hybrids, some of whichmay be developmentally regulated. Three X-coded sequences which recognise single mRNAs in human andthe E.C. hybrid material have been isolated from an X chromosome library. One of these maps to Xp21 toXp22 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 developmentNeural differentiation in teratocarcinomas and developing brainH. 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 arecomparable to pluripotent embryonic cells in the postimplantation embryo. From such a multidifferentiatingteratocarcinoma, two neuroblastoma-like tumors, TDN 2151 and TDN 2283, were obtained by repeated invivo passaging, which homogeneously show immature neural differentiation.

In the present study, overall synthesis of cellular proteins - resolved on two-dimensional gels - served as avery rigorous and general criterion for comparing in vivo differentiation of nerve cells in teratocarcinomaswith brain development in the mouse foetus. Proteins synthesized during muscle differentiation indeveloping limbs were used as a control for a developmentally unrelated tissue.

Comparison of the very complex protein patterns revealed the following differentiation-related newprotein syntheses: a) aberrantly expressed proteins, which appeared in the neurogenic tumors but not infoetal brain, b) general proteins, which appeared in at least one of the neurogenic tumors and were alsopresent in foetal brain and limbs, c) nerve-specific proteins, which appeared in the neurogenic tumors andwere detected in brain but not in muscle tissue of developing limbs. Four nerve-specific proteins with MT of29 kd, 26 kd, 20 kd and 16 kd are the most promising ones for further studies of the molecular basis ofdifferentiation.

Development of the thymus in dogsB. 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) andhistochemical methods in 100 fetuses (beagle) from 47 litters, between day 19 of gestation and day 21 afterbirth. The three earliest samples investigated were 19,20 and 21 day old embryos in which no organogenesiswas 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 andnewborns). The epithelial anlagen were seen at the 31st day of gestation showing the characteristic palisadestructure of the endoderm derived epithelium. They originated from the ventral part of the 3rd and 4thpharyngeal 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) cellsresulted in the differentiation of the cortical and medullary areas. The first HB could be found at the 38thday, when both areas could be well recognized. The HB grow out of the cells of RE network, undergoingspecial morphological changes. The palisade structure of the epithelial cells remains at this stage at thesurface 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 glycogenas demonstrated by TEM. The PAS reaction was much more abundant at the very surface of the cortex incontact with the interlobular connective tissue (ICT), than in deeper areas and more in the early stages ofthe development than after birth. The HB showed a weak diffuse reaction in most of the cytoplasm of theRE cells and a strong, coarse, granular reaction under the membrane. Some lymphocytes were also PASpositive but did not show glycogen. The ultrastructural observations demonstrated the presence ofdesmosomes connecting RE cells to one another. Desmosomes were not found between RE andlymphocytes. The HB showed their main development between days 45 to 54 of gestation beginning withhypertrophy of the RE cells. The growth of the thymus into the mesenchymal matrix resulted in thetabulation of the organ by connective tissue cells and fibers. Reticulin was demonstrated by the Gomori'smethod and was abundant in the ICT but few fibers penetrated into the cortex. The mast cells were first seenat the 35th day of gestation, being abundant in the ICT; few were present in cortex and more in medullanear the HB. Small groups of neutrophil cell precursors appeared in the ICT from the 40th day on, but noeosinophils were found. Cysts were not present up to the 21st day after birth.

Early mammalian developments 59Oocyte coded glucose phosphate isomerase is gradually replaced byembryo coded enzyme during preimplantation development of the

mouse: a fine analysis on single embryosDenis Duboule 1 and KurtBurki 2, Laboratory for Cell Differentiation, University of Geneva, Switzerland.

lCNRS du LGME, Unite" de Biologie moliculaire et de Ginie ginitique, Faculti de Midecine, 11, rueHumann, 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 phosphateisomerase (Gpi-la/Gpi-lb) fertilized with sperm carrying a third allele (Gpi-lc). This particular combinationmakes it possible to study the following phenomena during early development:

1) The appearance of Gpi-lc coded isozyme-subunits as an indication of activity of the paternally derivedgene.

2) A shift of the initial oocyte type hybrid pattern to the final embryo type pattern as an indication ofactivity of the maternally derived gene.

3) The presence of GPI-1AB dimers to estimate the persistence of oocyte coded enzyme throughout earlydevelopment.

4) The temporary presence of GPI-1BC dimer in Gpi-la/Gpi-lc embryos as a possible indication of thesimultaneous expression of the paternally derived allele and the maternal message. The different isozymespresent in single embryos were separated by electrophoresis on cellulose acetate gels. The results show thatthe oocyte coded glucose phosphate isomerase is gradually replaced by embryo coded enzyme. Expressionof the paternally derived allele is first detected at the morula stage. At this stage the translation ofmaternally derived message seems to be either exhausted or below the detection limit of our systems. Someoocyte coded enzyme nevertheless persists until after implantation.

Transcription of two mitochondrial genes in perinatal and postnataldeveloping rat liver

P. Cantatore, P. Loguercio Polosa, F. Fracasso, Z. Flagella, A. de Montalvo andM. N. Gadaleta, Instituteof Biological Chemistry, University of Bari, Bari, Italy

Studies on mitochondrial (mt) DNA transcription in HeLa cells have revealed the existence of twotranscriptional units on the H strand of mt DNA coding the first one for the two rRNAs and for two tRNAsand the second one for all the mRNAs and for the remaining tRNA. The measurement of the steady-statelevels of mitochondrial rRNAs and of some mRNAs in HeLa cells, in adult rat liver and in sea urchinembryos at gastrula stage has shown that rRNA concentration is quite similar in these three systems whereasthe concentration of mRNAs is higher in rat liver. These results suggest the possibility of a differentregulation of mitochondrial rRNA and mRNA synthesis. We have approached this problem by studying thesteady-state levels of two mt RNA species namely the 16S rRNA and the mRNA for the subunit I of thecytochrome oxidase (Col) during rat liver development. By using a technique based on the hybridizationbetween total mt RNA and radioactive mt DNA probes, we have found that, in correspondence of theperinatal period of life, there is an increase in the number of molecules per mitochondrion of the ColmRNA, while the concentration of 16S rRNA species remains substantially unchanged. This differentialexpression of mt genome during development is in agreement with the increase in oxidative phosphorilationand with the upsurge of the cytochrome oxidase activity observed at birth in rat liver.

60 Early mammalian developmentCell 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 remaindevelopmentally labile and will generate trophectoderm (TE) when placed experimentally in an atypicaloutside position. At later times, extraembryonic endoderm (ExEnd) is generated. The time course of thisrestriction on the developmental potency of inner cell mass (ICM) cells has been investigated and it is foundthat 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 ICMsisolated from blastocysts of each age post cavitation there was wide heterogeneity in cell number, asestimated by nuclear counts, and in developmental potential, as judged by both light and electronmicroscopic observation of the morphological changes occurring during in vitro culture of the ICMs. Thusthe initiation of cavitation does not appear to be related either to the developmental cell cycle of inside cellsor to their state of commitment.

Some single ICMs developed both TE and ExEnd cells when isolated and placed in culture, suggestingthat separate populations of committed and uncommitted cells can coexist in the same ICM and thatdifferential expression of their potency is possible in some instances. This mixed potential for developmentwas 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 (blastocoelformation) in outside cells. Since cell cycles between and within embryos are asynchronous, it is suggestedthat the time of commitment of ICM cells may be related to the number of developmental cell cycles thateach has undergone.

Histogenetic factors in the development of the EPUP. 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 belowthe horny basal keratinocyte, and termed columnar and subcolumnar respectively.

The purpose of the present communication is to analyse the processes of differentiation and evolution ofthe 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 topographicalgroups: back, ear and abdomen.

Using semithin sections we carried out a quantitative study of the cellular elements that make up the EPUin the aforementioned chronological periods and topographical zones. All the data were studied andsubjected to multivariate statical analysis in order to elucidate possible correlations. A total of 13 factorswere defined which influence the histogenetic process in the EPU. Only three appeared to be significantlyimplicated in the epidermal unit.

Factor I is related to perinatal specific proliferation. Factor II is related to differentiation of the hornycells of the EPU. It partipates to a minor degree in the perinatal stages, but its importance graduallyincreases to an important degree in adult stages. Factor III which regulates subcolumnar differentiation doesnot vary according to chronological stages.

Early mammalian development 61The effects of lectins on blastocyst and embryonal carcinoma cell

developmentG. 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 preventfurther 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, LotusLectin, PNA, UEA and WGA) previously shown by us to bind to both the inner cell mass andtrophectoderm of the mouse blastocyst were added at concentrations of 25, 50 and 100 /Agml"1 to blastocystcultures for varying time intervals, and their effects on cell growth and differentiation were studied. Asimilar protocol was used to study the effects of these lectins on embryoid body formation and endodermdevelopment 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 cellcontacts, 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 forstudying the cell surface during early mammalian development. In The cell surface: mediator ofdevelopmental 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 trophoblastW. 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 adiploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized byreplication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and theformation of giant nuclei. Studies of 13-5 day rat trophoblast have indicated a relatively low rate of DNApolymerase a, the normal eukaryotic replicase, in comparison to that of DNA polymerase a. These resultshave suggested that endoreduplication in trophoblast giant cells may not employ the normal replicaseenzyme. In order to determine whether a switch from DNA polymerase a to DNA polymerase (3 is anecessary concomitant of the diploid to giant cell transformation, the embryonic ectoderm, extraembryonicectoderm and EPC from 7-5 day mouse embryos were treated with aphidicolin, a specific reversible inhibitorof eukaryotic DNA polymerase a, on various days after explantation. DNA synthesis was measured incontrol and treated embryos following a 2 h pulse with methyl 3H-thymidine. Scintillation counts of theembryonic ectoderm revealed that DNA synthesis was consistently inhibited by greater than 90 % in thepresence of aphidicolin. The extraembryonic ectoderm and the EPC were inhibited to a lesser extent. Theextent of the inhibition of DNA synthesis in the extraembryonic ectoderm and EPC varied between81-95 % and 82-98 % respectively. A qualitative measure of the effects of aphidicolin on these tissues wasprovided by autoradiography after various days in culture. DNA synthesis in the embryonic tissues wascompletely inhibited at all stages in explant culture. In contrast, both the extraembryonic ectoderm and theEPC were observed to possess some heavily labelled cells in the presence of 10 /xg/ml aphidicolon after twodays in culture. These results suggest that DNA polymerase a is the primary replicating enzyme responsiblefor endoreduplication in mouse trophoblast giant cells. There appears to be a small but significantpopulation of trophoblast giant cells which incorporate label in the presence of aphidicolin and henceemploy a non alpha polymerase for replication.

62 Early mammalian developmentColoboma mutants: isoenzyme patterns of LDH in blastocysts and

chromosome anomalies in adult miceO. 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 the4th, 5th and 6th days of pregnancy. In the progeny 16 % of the blastocysts and 21 % of the embryos at thebeginning of implantation show morphological and functional anomalies. The blastulation is delayed andthe primary differentiation in trophoblast and embryoblast is altered in the first of these two groups. Afterthe flushing of the uterine horns on the days 5 and 6 p .c , unattached late blastocysts were found, withdegenerative trophoblast and/or embryoblast outgrowths. The abnormal early and late blastocysts wereused 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 earlyblastocysts stage. The day 5 embryos, attached to the uterine wall contain LDH-5 fraction, while theheteromeric isozyme LDH-4 appeared in day 6 attached embryos. However, in the unattached blastocystsflushed 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 byovocyte storage products, or is an expression of newly activated 0-subunit genes. It is tempting to speculatethat these flushed blastocysts are more probably homozygous for the gene Coloboma. The effect of themutant 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 miceCm/+ was performed on spleen and bone-marrow cells. The G-banding preparations indicate a difference inthe length and segmentation between the two chromosome 12 homologous. In all the metaphase plates thesubterminal E and the terminal F fragments of chromosome 12 are absent. This deletion, if related toColoboma condition, should be investigated in the early embryos from Cm/+xCm/+ crosses.

Proteolytic activity in the placenta, decidua and embryos of ratsAmos 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 inrodents. Rising proteolytic activity was found during development of mouse blastocyst and duringpostimplantation stages in rats. The aim of the present investigation was to demonstrate the activity of twoproteinases, an exopeptidase activity against leucine 6-naphtyl-amide (L-NA) and an endopeptidase activityagainst benzoyl-arganine j8-naphtyl-amide (BA-NA). These activities were measured in the postimplan-tation rat embryo as well as in placenta and decidual tissues. Rats were killed on days 11, 12 and 13 ofgestation. The embryos were separated from the decidual and placental tissues. For.the assay of proteolyticactivity, tissue homogenates were prepared. After 90 min of incubation with L-NA or BA-NA, theconcentration of the end products was determined colorimetrically. L-NA activity increased with time in theplacenta and was unchanging in the embryo and decidua. This enzyme was under the inhibitory effect ofbivalent cations. This inhibition can be removed by treatment with EDTA, after which the enzyme's activityincreases. The enzyme activity seem to have a Cathepsin-B function. BA-NA activity was increasing withtime in the embryo and in the decidua, decreasing in the placenta. In view of the changes in the activity ofthe enzymes manifest in the placenta and decidua during these specific days of placentation, it is tempting topresume that they may have a regulatory role in this process.

Early mammalian development 63Decidual cells are not derived from the bone marrow

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 afterirradiation and transfusion of semi-ajlogeneic bone marrow, are derived from the donor marrow. Donor andhost 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, buthistocompatible, bone marrow, contradicts this view.

Female CBA/Ca mice carrying the phosphoglycerate kinase alloenzyme marker, PGK-1B, were givenvarying doses of radiation and repopulatea with congenic CBA-PGK-1A bone marrow cells. These micewere then either naturally mated or their uteri were traumatised to induce deciduomata artificially. ThePGK phenotypes of the resultant decidual masses were analysed after electrophoresis. Whilst the donorbone marrow cells contributed to haematopoiesis to a greater or lesser extent, dependent on the dose ofradiation and the number of injected cells, no evidence was found for the differentiation of decidual cellsfrom transfused bone marrow precursors.

The use of allo-enzyme markers in this case provides a more reliable indication of the possibledescendants of transfused bone marrow. In the previously published observations, non-specific binding ofthe serological reagents, possibly to Fc receptors on decidual cells, may have been misleading.

Histone gene expression in early mouse embryosD. 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 DNAprobe, it has previously been shown that there is a pool of histone H3 mRNA in the unfertilized egg which islargely eliminated by the mid two-cell stage. Histone H3 mRNA content then increases progressivelythrough 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 RNAduring the development of pre-implantation mouse embryos. Devi. Biol. 98: 148-154). In the current studywe have quantitated the number of histone H3 mRNA molecules at several stages of development andcomapred these values to absolute rates of synthesis of histone H3 proteins. Assuming all mRNAs aretranslatable, it is possible to calculate theoretical translational efficiencies. Translational efficiency waspredicted to increase from 0-5 histone H3 polypeptides/mRNA/minute at the two-cell stage to about 2polypeptides/mRNA/minute in the blastocyst. Finally, we have applied SI nuclease mapping techniques tobegin to identify which of four different H3 genes isolated from the mouse genome are utilized during thisearly developmental period. In the unfertilized egg, about 45 % of the H3 transcripts are derived fromgeneH3.614 on chromosome 3 while about 25 % come from gene H3.2 on chromosome 13. Less than 5 % arederived 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 arederived from the same gene set. The results indicate that while histone mRNA content is similar in the eggand 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 developmentAge 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 ratembryo, 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 developmentalage 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, andtherefore 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 somitetakes 1-68 h ( ± 100 min). In the rat, the first somite is formed at 8-95 days after fertilization; the formationof 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 bethe 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 restrictedbefore implantation

Alan Handyside and Susan Hunter, MRC Experimental Embryology and Teratology Unit, WoodmansterneRoad, Carshalton, Surrey SM5 4EF

The mouse blastocyst consists of an outer layer of trophectoderm (TE) cells surrounding a fluid-filledcavity and specialised for implantation, and an inner cell mass (ICM) from which the embryo is formed. Thenumbers of these cells in individual blastocysts has been studied by differential labelling of the nuclei in situwith two polynucleotide-specific fluorochromes (Handyside and Hunter, 1984). This allows them to bedistinguished by fluorescence microscopy on the basis of their colour: TE nuclei appear pink or red, andICM nuclei blue or unlabelled depending on the filter combination used. Blastocysts were sampled atintervals either in vivo or in vitro under various conditions in which implantation was allowed to proceednormally or was suppressed. Under normal conditions in vivo in non-implanted blastocysts (84-114 h postcoitum), the number of TE cells increased steadily over two doublings and then rapidly levelled off. Incontrast, after an initial increase, the number of ICM cells first declined and then increased abruptly beforereaching a plateau in excess of twice the number present at cavitation. During the period of decline, manyICMs contained disintegrating nuclei indicating extensive cell death. In the absence of implantation in vivoor in vitro, no further increase in the number of ICM cells was observed unless the TE collapsed. Wepropose 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 tomaintain the ICM at the appropriate stage. In this way, the initiation of embryonic growth may besynchronised with implantation.

HANDYSIDE, A. H. & HUNTER, S. (1984). A rapid procedure for the visualisation of the trophectoderm andinner cell mass nuclei of the mouse blastocyst using polynucleotide-specific fluorochromes. / . exp. Zool.(submitted).

Early mammalian development 65Sequence of morphological and molecular events during the first cell

cycle of mouse embryogenesisSarah K. Howlettand Virginia N. Bolton, Department of Anatomy, Downing Street, Cambridge CB23DY

The precise timing and sequence of various morphological and molecular events during the first cell cycleof mouse development following fertilisation in vitro were investigated. The timing of development throughthe first cell cycle was found to be initiated by an event associated with sperm penetration rather than withgerminal vesicle breakdown. Initiation of DNA replication occurred randomly in either pronucleus of agiven egg, beginning approximately 11 h after fertilisation and lasting 6-7 h. Careful study of polypeptidesynthetic profiles revealed three sets of polypeptides exhibiting changes during the first few hours ofdevelopment (45KD, 35KD complex and 30KD). Pulse chase experiments showed that some, but not all, ofthe changes result from post-translational modifications. The remaining changes are presumed to resultfrom differential mRNA utilisation. Most of the polypeptide synthetic changes observed also occur in theageing unfertilised egg, but proceed more slowly. It is suggested that an endogenous programme ofdevelopmental change (the oocyte programme) is set in train by the terminal events of oocyte maturationand accelerated by the events of fertilisation, which activate the endogenous fertilisation programme.

Histological examination of tissues in a series of chimaeric rats betweencongenic 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 withmorulae of PVG-/?77C strain animals. The tissues of these two strains are histologically distinguishable byvirtue of a difference in a class I major histocompatibility complex (MHC) molecule. Monoclonal antibodiesdirected against various epitopes of these molecules were iodinated and applied to fresh cryostat sections offrozen tissues from these animals. Autoradiograms were prepared by dipping the sections in photographicemulsion. The strain of origin of the tissue could be unequivocally established by the uniform accumulationor uniform absence of grains over the tissues. The exception was brain tissue, which was unlabeled due tolow 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 eitherspleen or brain cell suspensions, with frozen sections of either spleen or brain. Preabsorption with spleenremoved 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 wereanalyzed with a cell sorter. Chimaerism of the RBC's was established for four of these animals which rangedfrom 7-5 % to 70-4 % WG-RTP cells. Frozen sections of the livers of these chimaeras confirmed thepresence of visceral chimaerism with random patchiness of PVG-/?77a or PVG-/?77C cells depending on theproportion of the two cell types present in the tissue. Digital quantitation of the histological sectionsdemonstrated a range of RTl* cells in the liver from 34 % to 86 %. This analysis revealed a linearrelationship between the proportion of the major cell type and the number of patches of the minorcell type/unit area examined ( r = 0-99). The average patch size also depended on the proportion of the twocell 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 unbalancedchimaeras. Distinctive patterns of mosaicism were present in other tissues with possible implications fororganogenesis. The adrenal cortex appeared striped indicating that the cortex may have been developed byclonal proliferation of primordial cells randomly assorted along the medulla. The thymus demonstratedcortical 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 wassupported by DHHS grant CA29078 and CA34913.

66 Early mammalian developmentThe 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 the3H-amino acid was greatest at the 2-cell stage of development and 50 % of these survived to the blastulastage in the presence of the following doses of 3H-amino acid: glutamic acid 10 /xCi/ml, leucine 1 /i,Ci/ml andtryptophan 003 /LtCi/ml. The high toxicity of 3H-tryptophan (greater than that of 3H-thymidine) was notrelieved by the presence of a large excess of non-radioactive tryptophan derivatives. The 2-cell embryorapidly incorporated 3H-tryptophan into proteins and autoradiographic examination of thin sections showeda net concentration of this radioactivity in the nuclear region. Comparative analysis by 2-dimensionalelectrophoresis in polyacrylamide gels of nuclear proteins labelled with either 35S-methionine or3H-tryptophan showed that the latter was selectively incorporated into a limited sub-set of the chromosomalproteins. The significance of these observations are also examined in the context of the patterns ofdevelopmental stage-specific peptide synthesis.

The functioning embryonic heart: early onset of normal development ordeviation 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 ventricularpouches develop, a left (proximal) and a right (distal) one. A linear zone in between these pouches, roughlythe curvatura maior of the loop, connecting the mid-caudo-dorsal side of the AV canal and the leftcranio-ventral side of the outflow tract or bulbus, does not elongate. This zone becomes a ridge, thusforming the luminar border of the interventricular septum. Further outgrowth of the pouches in ventral andcaudal direction simulates infolding of the caudo-dorsal part of the AV-canal, forming the caudo-dorsalAV-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 thedividing AV-canal. Both ventricles pump blood into the bulbus, the left one over the interventricularseptum, along the caudo-dorsal side of the bulbus in a right-dorsal direction, and the right ventricle alongthe 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 spirallingbulbus ridges.

Any disturbance of formation of two blood streams of equal size influences the morphology of the outflowtracts and of the ventricles.

Early mammalian development 67In situ localization of collagen oc\ (I) mRNA in developing mouse

embryosMichael Kuehn, Jiirgen Lohler and Rudolf Jaenisch, Heinrich-Pette-Institutfur Experimentelle Virologie und

Immunologie an der Universita't Hamburg, 2000 Hamburg 20, West GermanyThe 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 insitu hybridization of 3H labeled DNA probes to mRNA in frozen tissue sections. To determine both thepoint 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 (theday of the vaginal plug is day 1). A low level of collagen al(l) expression has been detected in day 8 embryosin mesenchymal cells in the head region of the embryo proper as well as mesodermal cells in the visceral yolksac (VYS) and amnion. At 9-5 days these same cell types are more heavily labeled suggesting a significantincrease in mRNA expression for orl(I) collagen between day 8 and day 9. Testing for the presence ofprotein, Adamson and Ayers (1979) reported that both the mesodermal and endodermal portions of theVYS produce type I collagen in embryos as early as day 10. Using in situ hybridization, however, we havefound that in embryos from day 8 through day 13 only the mesodermal cells located on the inner face of theVYS are labeled. By day 11, embryos begin to show very heavy labeling in the umbilical cord, VYSmesoderm and surrounding the vascular system. This is consistent with the need for increased amounts ofcollagen to be laid down in these areas affected by an increase in mechanical forces imparted by the growingembryo. The need for collagen I in the vascular system is evidenced by the fact that Movl3 homozygousanimals die at day 12 due to vascular rupture (Lohler et al. submitted]). Embryos in the last third ofdevelopment show heavy labeling in all mesenchymal cells and their derivatives. This pattern of mRNAexpression correlates with the pattern of protein distribution in later stage embryos as determined byantibody staining (Lohler et al. submitted), which is not markedly different from the adult proteindistribution 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 byintracellular microinjection of horseradish peroxidase

K. A. Lawson x, J. J. Meneses 2andR. A. Pedersen 2>3. ^Hubrecht Laboratory, Utrecht, The Netherlands.2Laboratory 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 contributedescendants to the definitive gut of the foetus; the fate of the primary endoderm is still uncertain. We haveused intracellular microinjection and embryo culture to trace endoderm cells and their descendants from theearly primitive streak stage to early somite stages. Horseradish peroxidase was injected iontophoreticallyinto one axial endoderm cell and the embryos cultured in DMEM+50 % rat serum for 1 or 2 days. Labelledcells 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 spreadover the entire axis of the embryo during the following 24 h (early neural plate stage) and can contribute toendoderm 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 streakstage embryo are localised anterior to the prospective foregut imagination at the neural plate stage andcontribute to the anterior visceral yolk sac of the early somite embryo. 3) Axial embryonic endoderm at theearly 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 RoyalDutch Academy of Sciences and the U.S. Department of Energy.)

68 Early mammalian developmentPresence of an electron dense layer on polar trophoblastic cells of

cultured embryosT. LeclipteuxandJ. Remade, Uniti de Biochimie CelMaire, F.N.D.P. Ruede Bruxelles61, B-5000, Namur,

BelgiumProducts 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 couldexplain the maintenance of the close relationship existing between the foetus and the mother duringpregnancy.

In order to study these H-2 antigens at the time of implantation, we developed histochemical approachesincluding the immunogold staining method. Embryos were assayed for the presence of H-2 antigens beforeimplantation and after 5 days culture in BMOC-3+10 % FCS. H-2 antigens were detected on thetrophoblastic 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 stainingof gold particles can be observed on a dense layer overlying the apical part of the embryo. Polartrophoblastic cells are likely those which are covered by this layer while others bordering it are free of anydense material. Particles are also present within the layer. This can be explained if the layer is slightlyporous. It is difficult to explain why this layer is present on this type of trophoblastic cell but following thereport of Searle (Searle, 1982), one hypothesis could be that it is masking H-2 antigens in a manner similarto a layer present on the labyrinthine trophoblast, which is located at the maternal-foetal circulatoryinterface.

LECLIPTEUX, T. & REMACLE, J. (1983). Disappearance of paternal histocompatibility antigens from hybridmouse 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 ofalloantigens during preimplantation development and early trophoblast differentiation in the mouse byimmunoperoxidase 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 (throughinteraction with oral ectoderm) originates, in whole or in part, from the cranial neural crest. Thissupposition has lacked the support of direct experimental evidence; techniques of ablation and3H-thymidine labelling, by means of which the cranial neural crest origin of odontogenic mesenchyme hasbeen demonstrated in amphibians (Chibon, 1967), have not been successfully applied to mammalianembryos.

In the present study, the developmental potential of mammalian premigratory neural crest has beeninvestigated 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-anteriorrhombencephalic (CNC) and trunk (TNC) regions of the neural plate of E8 (6-8 somite) CD1 mouseembryo; mandibular arch epithelium (ME) and limb bud epithelium (LE) were isolated from E10 (30-35somite) embryos by cold trypsinisation. The tissue fragments were suspended in saline and injected via fineglass cannula into anterior chambers. When lodged in the iridiocorneal angle, heterotypic tissues adhered toeach 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 withepithelium. ME grafted alone developed into small epithelial whorls. Teeth and large hair follicles wereformed 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 whenthese cells are associated with a regionally appropriate epithelium, that normal crest cell migration is not aprerequisite for differentiation, that the neural crest is odontogenically uncommitted prior to migration, andthat 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 69Stage specific fucosylated glycoproteins in pre-implantation mouse

embryosHilary 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, MRCLaboratories, 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 fucosylatedmolecules in adhesion of mouse embryonal carcinoma cells (Grabel et al. 1981, 1983) and compaction of8-cell mouse embryos (Kimber & Bird, 1983; Bird & Kimber, in press). We have incubated embryos withmedium containing 14C-fucose and harvested them at various stages in development from 2-cell toblastocyst. Solubilized embryos were subjected to SDS-PAGE electrophoresis on 10 % acrylamide gels.Various bonds were detected after development of the resulting fluorographs; in particular severalglycopeptides appeared in a stage specific manner. A glycopeptide appearing at the 8-cell stage might beinvolved in compaction. The nature of these bands is being investigated.

BIRD, J. M. & KIMBER, S. J. Dev. Biol. (in press).Gooi, H. C , FEIZI, T., KAPADIA, A., KNOWLES, B. B., SOLTER, D. & EVANS, M. J. (1981). Nature 292,

156-158.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, 1532-1537.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.

Oligosyndactyly: A lethal mutation in the mouse that results in a mitoticarrest 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 inthe homozygous state, the mutation is lethal very early during development. Large numbers of cellsaccumulate in metaphase and the embryo dies during implantation. To date, no evidence has been reportedwhich links the effects found in heterozygous mice with the embryonic lethality that occurs in homozygousembryos. We began work on the Os mutation by studying homozygous embryos since they remain free ofthe complicating effects caused by the presence of the wild-type gene product(s). Our primary workinghypothesis has been that the Os mutation results in abnormal spindle formation or blocked chromosomemovement. We have found that Os homozygous embryos begin to accumulate cells in metaphase at themid-blastocyst stage. Nevertheless, the embryos are able to hatch from the zona pellucida, attach to theculture dish and form blastocyst outgrowths. At this time as many as 70 % of the cells in an embryo arearrested in metaphase, with mitotic spindles being present. The appearance of the mutant phenotype is nottemperature sensitive. Since tubulin is the major component of mitotic spindles, 2D gel protein patternswere 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 ofhomozygous embryos are not hyperstable. They can be depolymerized by cold-treatment or by incubation incolcemid. However, after a suitable recovery time, the spindles do reform. This treatment does not result inrescue of the mutant phenotype. When homozygous embryos were examined for the pericentriolar materialof 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 electronmicroscopy.

70 Early mammalian developmentAn interaction between the chromosomes, the plasma membrane and the

cytoskeleton in the mouse 1-cell oocyteB. 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 microscopelevel for structural changes, distribution of actin, tubulin, and lamins, surface distribution of microvilli(assessed from Concanavalin A binding pattern), chromosomal distribution and condensation, andpronuclear formation and migration. The influence of the drugs cytochalasin D and nocodazole (alone or incombination and applied continuously or in pulses of varying duration) on these parameters wasinvestigated. Characteristic changes in the distribution of surface microvilli, subcortical actin and tubulinwere observed in association with the formation of the fertilization cone, rotation of the second meioticspindle, extrusion of the second polar body, and formation and migration of pronuclei. Particularly strikingwas the clear association under a number of conditions between the presence of chromosomes subcorticallyand both a focal concentration of actin plus a localized loss of microvilli. This association was only observedwhen the chromosomes were not enveloped in a pronuclear membrane and did not obviously depend uponthe presence of microtubules associated with the chromosomes. The possibility that the chromosomesthemselves might cause local organisational changes in the overlying cytocortex, and the significance of suchan effect, is considered.

Presence of nucleolar vesicles and vacuoles in the rat oocyteC. 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 theinfluence of gonadotropins in the follicular oocytes of immature rats. Nucleolar vacuoles and vesicles in thefollicular 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 FSHwith an interval between the injections of 24 h; in this last case, the oocytes were observed 24 h after the lastinjection. Oocytes of mature rats were also examined at prooestrus, oestrus, meteoestrus and dioestrusstages.

In the three experiments, some nucleoli exhibited vesicles or vacuoles but their frequency was verydifferent for each condition. In mature rats nearly all nucleoli showed closed cavities. In immature rats theirpresence 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 afteran injection of 40 UI of FSH and the observation of the nucleolus 1 h after this injection did not show achange in the percentage of nucleoli with vacuoles or vesicles. The nature and the significance of thesevacuoles and vesicles are discussed.

Early mammalian development 71Regulation of heat-shock protein synthesis in mouse preimplantation

embryoM. 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 astress (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 ofthe embryonic genome, have been shown to be HSPs; later, at the eight-cell stage, two HSP are the proteinssynthesized 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 twoHSP. 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 thepronuclei stage

Jacques Mulnard and Frangoise Puissant, Department of Anatomy and Embryology and HumanReproduction 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 OF1males 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 electronmicroscopy 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 fewelements of granular endoplasmic reticulum are sedimented, 2) a large mitochondrial layer containing alsoGolgi complexes and 'jigsaw' vesicular aggregates, 3) a small centrifugal hyaline cap in which a sperm tail isoccasionally seen. All three layers contain filamentous material, isolated ribosomes and small polyribo-somes, secondary lysosomes, vesicular and cisternal agranular endoplasmic reticulum associated withmultivesicular bodies. The pronuclei remain in the central area: they are elongated in the centrifugation axisby migration of their fused nucleoli towards the centrifugal pole. After 96 h in vitro a variable proportion ofthe centrifuged embryos form morphologically normal early blastocysts, some of which were transferred tothe uterine horns of 3-day pseudopregnant foster-mothers. Out of 28 transfers, three 13-day normalembryos and two healthy youngs have hitherto developed from eggs centrifuged at 90-000 g at the pronucleistage. There were no significant differences of developmental timing or survival ratio between thecentrifuged and control embryos.

72 Early mammalian developmentAn investigation into the levels of cytochrome P450 present in the rat

visceral yolk sac between 9*5 and 19*5 days of gestationM. 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 orneonate of many animal species (Neims, Warner, Loughnan & Aranda, 1976). It has been suggested thatthe visceral yolk sac functions, for at least part of gestation, as the foetal liver (Padykula, 1958). We havetherefore investigated the levels of cytochrome P450 present in the rat visceral yolk sac at different stages ofgestation between 9-5 and 19-5 days.

Yolk sacs were homogenised and the levels of cytochrome P450 measured using the method of O'Brienand Rahimtula (1978) and protein by the Lowry method (1951) so that it was possible to compare thespecific 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 ofcytochrome P450 were found to increase at later stages and then remain constant until 18-5 days when a lossof enzyme activity was observed. These results suggest that the rat visceral yolk sac may be instrumental indrug metabolism between 14-5 and 18-5 days of gestation. Further studies on the levels of other enzymesinvolved 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 'Biologicaloxidations: 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 carcinomacells: 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, involvingredistribution of cell surface projections, reorganization of cytoskeletal structures and formation ofmembrane junctions. We have investigated the preimplantation development by studying aggregationbetween embryo and F9 embryonal carcinoma (EC) cells. In this model system, the EC cells (similarly asisolated 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-stageblastomeres acted as in normal preimplantation development. The aggregates compacted and formedchimaeric morulae with EC cells surrounded by the embryo cells. Establishment of cell contacts bymicrovilli and larger cell processes, and eventually spreading of the blastomeres over the EC cells tookplace. In the chimaeric embryos, the embryo and EC cells had cell projections similar to those between ICMand trophectoderm cells in vivo. Cytoskeletal organization involved microtubules parallel to contact areasbetween embryo and EC cells. The cell projections contained orientated microtubules and bundles ofmicrofilaments. 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 theembryo-EC-aggregation model in vitro. The aggregation model allows a study of the precise time sequenceof 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 actionV. 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 bypassive immunization with anti-progesterone monoclonal antibody (Wang, Rider, Heap and Feinstein,1984). Preliminary investigation indicated that the rate of embryo development was retarded inantibody-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 containing4-5 nmol immunoglobulin (IgG). At autopsy on days 3 and 4 p.c. the reproductive tract was flushed withphosphate-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 ofantibody 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 weresuperovulated with 5 i.u. pregnant mare serum gonadotrophin followed 48 h later by 5 i.u. human chorionicgonadotrophin (hCG). Two-cell stage embryos were collected from oviducts 44-46 h post hCG inBMOC-culture medium. Purified anti-progesterone IgG (range 0-1-0 mg/ml) was added to the medium onthe day of culture. At 98 h post hCG greater than 80 % of the embryos cultured were blastocysts at eachdose tested.

The results indicate that progesterone is important in early stages of cleavage in vivo, and that antibodytreatment probably alters the tubal environment producing a deleterious effect on embryo developmentrather 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 probesJ. Rossant*1, J. Sanford 2, V. M. Chapman 2andG. Andrews 3.'Department of Biological Sciences, Brock

University, St. Catharines, Ontario, Canada. 2 Department of Molecular Biology, Roswell Park MemorialInstitute, 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 geneticmarkers, has established clearly the later derivatives of the first cell lineages to be formed in the embryo. Forexample, embryos derived from blastocysts reconstituted with M. caroli inner cell masses and M. musculustrophectoderm have been analyzed using in situ DNA-DNA hybridization with a probe to M. musculussatellite DNA. These experiments have shown that both the ectoplacental cone and the extra-embryonicectoderm of the 7-5 day embryo are pure derivatives of the trophectoderm, whereas the trophectodermderivatives of the later chorioallantoic placenta are intimately interspersed with maternal and inner cell massderivatives. Other experiments have shown that pure populations of primitive endoderm derivatives can beisolated 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 featuresof the molecular biology of the derivatives of the early cell lineages which might shed light on themechanisms of their establishment and maintenance. Examination of the extent of methylation of severalrepetitive DNA elements revealed consistent undermethylation in all derivatives of the extraembryoniclineages, trophectoderm and primitive endoderm. Recent studies have shown that this undermethylationextends 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 andundermethylated in the visceral endoderm, but the present study has shown that it is also undermethylatedin extra-embryonic tissues in which it is not expressed. These studies do not eliminate the possibility thatspecific sequences in the 5' region of genes may remain methylated and act in a controlling manner, but theyshow that global methylation is not required for control of gene activity in these mammalian cells. Studiesare currently in progress to determine at what stage in development these cell-lineage-specific patterns ofmethylation are established and whether undermethylation always accompanies differentiation ofextraembryonic cell types. The possible significance of undermethylation for the regulatory capacity of theextraembryonic cell lineages will be discussed.

74 Early mammalian developmentThe 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,

Romania1) 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 thesurrounding trophoblast separates this 'channel' from the uterine lumen.

- Another possible, unusual, mechanism (which was not supported by our microscopic findings) may be asecondary 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 oldermorphogenetic mechanism, with respect to an essential step in the embryogenesis of Muridae: the shapingof the proamniotic cavity.

Cytoskeleton and nuclear lam in organization during mammalianfertilization and early development

Gerald Schatten*1, Calvin Simerly \ Heide Schatten 1, ChristiCline l and Gerd Maul2, department ofBiological 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 fertilizationand preimplantation embryos from mice and hamsters after natural mating. Microtubules, detected withantitubulin immunofluorescence and electron microscopy, comprise the meiotic apparatus of theunfertilized oocyte. After sperm incorporation a new class of microtubules forming into about a dozencytoplasmic asters assemble in and around the developing pronuclei. As the pronuclei are positioned at theoocyte center, the microtubules form into a dense cytoplasmic matrix. This matrix disassembles at prophasewhen microtubular sheaths appear to envelope the adjacent, but separate, pronuclei. The first mitoticapparatus is quite unusual, barrel-shaped and devoid of asters, reminiscent of plant spindles. Duringcleavage a dense bundle of interzonal microtubules appear and radial monasters position the blastomerenuclei at the cell centers. By third division typical animal-type mitotic apparatus with fusiform spindles areobserved. Interestingly the incorporated sperm centriole does noj seem to serve as a micro tubule organizingcenter (MTOC) during mouse fertilization; contrary to dogma, the active MTOCs during mammalianfertilization may be of maternal origin. Microfilaments, localized with rhodamine-phalloidin, are detected atthe oocyte surface during polar body formation and in the formed incorporation cone. In pronucleateoocytes, microfilaments seem to colocalize with the microtubules in the cytoskeletal matrix. Non-erythrocyte spectrin or fodrin also is localized subjacent to the oocyte surface. Immunofluorescencelocalization with monoclonal antibodies specific to nuclear lamins A, B and C indicate that the spermnucleus, which does not have lamins, acquires these nuclear lamins as the male pronucleus develops. Themeiotic chromosomes similarly do not have any associated lamins, though the female pronucleus acquiresthese nuclear proteins immediately after the completion of meiosis. Though all nuclei within the fertilizedoocyte cytoplasm have the nuclear lamins, remarkably the second polar body nucleus remains devoid oflamins, suggesting a pathway for nuclear degeneration. These studies support the hypothesis that specificcytoskeletal and nuclear lamina alterations are required for the motility events necessary for the successfulcompletion of mammalian fertilization. Supported by grants from the NIH and NSF.

Early mammalian development 75Prenatal repair of clefts in the secondary palate of rats

P. M. Schtipbach and H. E. Schroeder, Department of Oral Structural Biology, Dental Institute, Universityof Zurich, CH-8028 Zurich, Switzerland

In a previous study we have shown that partial clefts of the hard palate with soft palate integrity, can beproduced through amniocentesis performed in Sprague-Dawley rats at day 16 of gestation. Such clefts resultfrom fusion and complete union of the posterior shelf portions, and fusion in the posterior region occursindependently from fusion in the anterior region. The purpose of the present study was to answer thequestions: (1) whether or not such clefts, once being experimentally induced, can be repaired prenatally byfurther late closure of the shelves in the anterior direction, and (2) how long does the medial shelf edgeepithelium retain its competence to fuse? A refined technique of amniotic sac puncturing was employed atday 16-2 of gestation in order to produce a series of total clefts and rare forms of partial clefts inSprague-Dawley rat fetuses. In a first study generating a total of 396 fetuses of a precise, individuallydetermined age, 301 fetuses were examined microscopically and 95 upper jaws were examined in thescanning electron microscope (SEM) and in serial epon sections. Total clefts were found in 48-9 % offetuses examined at day 17-8 and in 21*8 % of those examined at day 19-3. Partial clefts were observed in141 % and 18-5 % of fetuses examined at days 17-8 and 19-3, respectively. At day 19-3, 16-1 % of thefetuses showed a very inconspicuous, small abnormality (with residual clefting and incomplete fusion withthe nasal septum) in the region of the palatine foraminae. Morphological observations suggested that underconditions of detained palatal closure (1) fusion of the soft palatal shelves commences independently fromand prior to fusion of the hard palate, (2) delayed palatal shelf fusion proceeding in the anterior may occurwith or without sickle-shaped clefts remaining in the anterior hard palate, and (3) in fetuses withsmall sickle-shaped clefts, fusion of the palatal shelves with the nasal septum does not occur, implyingincomplete prenatal repair. In a second study, total clefts were produced by amniotic sac puncturing at day15-5 of gestation. About 28 % of 178 fetuses showed a total cleftpalate. In 12 selected upper jaws (ageranging from 16-2 to 21-5 days), the medial shelf edge epithelia (MEE) were examined in the SEM and inserial epon sections. The density of superficial cells in various states of differentiation was countedmorphometrically. In the soft palate, critical density of ciliated cells among MEE-cells was reached at day170-17-5, and in the hard palate at day 17-5-18-0. At the dorsal aspect of the nasal septum, ciliated cellsappeared at day 17-5-17-7. These data imply, that epithelial competence to fuse is retained longer in theanterior than in the posterior region.

Extraneous signals affect the growth and differentiation of cultured ratembryonic 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 twoweeks for the differentiation of main tissue types. Since the normal process of cell differentiation requiresvarious extraneous signals during the critical period of embryonic development we tried to analyse theimpact 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 mediumsupplemented with about 40 % of rat serum. If dbcAMP was added dunng the first week of culture thegrowth was severely retarded. If added during the second week the skeletal muscle appeared morefrequently. If RA was added in the second week the growth was unaffected, but the differentiation of theskeletal muscle seemed to be stimulated, whereas that of the cartilage was impeded. In the second part ofthe study the embryonic rat shields were cultured only in Eagle's minimal essential medium. The growth waspractically inexistent, and the survival was significantly better in the medium supplemented with serum. Thedifferentiation proceeded with relatively few consequences. Only the incidence of neuroblasts and skeletalmuscle were significantly decreased in the serum-free medium.

76 Early mammalian developmentMesoderm formation in the rat embryo, cultured in vitro, analysed with

a lectin-coated colloidal gold markerA. 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 asachieved by e.g. Le Douarin (1982) are not possible in these embryos, a marker was developed that, afterinjection into the embryos, would be taken up by the neural crest cells. Gesink et al. (1983) showed that theplasma 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 culturedrat embryos, the WGA-Au probe is endocytosed by all cells bordering that cavity and stored inmembrane-bound vesicles; the content of these vesicles is not exocytosed during subsequent developmentand can therefore serve as a marker for ectoderm-derived cells, such as primitive streak-, ectodermalplacode- and neural crest-derived cells. In order to discern mesoderm cells produced by these sources, it isnecessary to investigate a series of embryos with varying survival times (2 to 30 h). Computer-aided 3-Dreconstructions 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 thesclerotome.

- The segmented mesoderm caudal to the last pair of somites, contains enough cells to produce another 5pairs 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. Theorigin of these cells remains to be elucidated.

GESINK, A. F., POELMANN, R. E., SMITS-VAN PROOIJE, A. E. & VERMEU-KEERS, CHR. (1983). The cellsurface 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 methodfavours the proliferation of yolk sac cells probably by formation of three-dimensional structures. Thespherical pieces of the membrane were placed in organ culture or implanted into the omentum of adultsyngeneic rats. After 2-3 weeks the appearance of giant cells was observed in vitro as well as in vivo. In bothcases it was preceded by the appearance of poorly differentiated cells from which the giant cells differentiateas documented by autoradiography using tritium-labelled thymidine. The trophoblastic nature of the giantcells is illustrated by the presence of A5-3j3-hydroxysteroid dehydrogenase using pregnolone as substrate andby 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 giantcells were found among yolk sac endodermal cells. The level of progesterone in the serum of tumor-bearinganimals was elevated when the giant cells were present in the tumor.

Early mammalian development 77Sister chromatid exchange in preimplantation mouse embryos: a

sensitive assay for genotoxic effects in early embryosH. 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 preimplantationmouse embryos during in vitro culture for two cell cycles. 4-cell and 8-cell embryos and also morulae andblastocyst were cultured in media containing 10~6 M 5-bromodesoxyuridine (BrdU) for one cell cycle andwithout BrdU for the next cell cycle (total culture period 48 h). 0-1 /ig/ml Colcemid was added 3 h beforechromosome preparation. Sister chromatid exchanges (SCEs) can be visualized after staining with theFluorescence-Plus-Giemsa technique. SCE frequencies per chromosome were 0,6 for 4-cell and 8-cellembryos and also for morulae and blastocysts compared to 0,3 per chromosome in bone marrow of adultmice that were exposed to BrdU in vivo. A positive SCE-staining could never be obtained inpreimplantation mouse embryos in vivo after BrdU treatment of the mother animals. Increased SCE-rateswere also obtained in preimplantation mouse embryos during culture in media containing rat or human serawhich are supporting embryonic differentiation in vitro. A significantly higher SCE rate was seen in embryoscultured 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 containingsera 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 serafrom untreated animals or patients. SCE frequencies were increased dose related in 4-cell and 8-cell mouseembryos flushed from oviducts 1 h after CPA treatment of the mother using 5-20 mg/kg. Other sensitivemethods (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 CPAor less in vivo. SCE-determinations in early mouse embryos may be useful to study embryotoxic effects andrepair mechanisms.

Regulation of cell number following aggregation of morulae andformation 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 cellmass (ICM) of the blastocyst, respectively. Under different experimental conditions, these cells either retaintheir normal fate, or express developmental lability. Using a double labelling technique which allows the TEand ICM nuclei of individual blastocysts to be distinguished (see abstract page 64), we have examined howthe aggregation of morulae affects the allocation of cells to the two cell types in the resulting chimaericblastocyst. If in this situation, the cells are not labile, the numbers of TE and ICM cells in doubleblastocysts, for example, should both be twice the numbers in single blastocysts. Alternatively, if the cellsare labile, the proportion of TE and ICM cells should be altered according to the reduced surfacearea/volume ratio i.e. the numbers of these cells should be less, and greater than twice those in singleblastocysts, respectively. The ratio of cell numbers in double chimaeric blastocysts and single blastocystswere examined soon after cavitation and again after transfer to the oviducts of pseudopregnant females andrecovery from the uterus three days later. The ratio of TE cells was significantly less than double, bothinitially and after transfer. However, there was no corresponding increase in the ratio of ICM numbers inthe same blastocysts, and total cell number was also significantly less than twice in double blastocysts. Wesuggest 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 inchimaeric blastocysts probably fail to catch up because of the increased vulnerability of late dividing cells tocell death. It, therefore, appears that in addition to early postimplantation regulation, the number of ICMcells in chimaeric blastocysts is partially regulated before implantation.

78 Early mammalian developmentStudies on the behaviour of nuclei in thymocyte-oocyte homospecific

mouse cell hybridsD. 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. hybridswere formed followed by parthenogenetic activation of the oocytes, 2. parthenogenetic activation of oocytesfollowed by hybridisation with thymocytes.

Under the first condition usually a small number of thymocytes (2-4) hybridize with the oocytes andfollowing 6-22 h of culture the thymocyte nuclei are structurally identical to the female pronucleus formedas a result of activation by alcohol. In each 'pronucleus' a single, giant nucleolus develops and remainsthroughout the culture period. Under the second condition up to ten thymocytes were observed to enter theoocyte cytoplasm. Thymocyte nuclei showed a number of different images after 6-22 h of culture. Theyranged from compact chromatin masses lying freely within the oocyte cytoplasm to nuclei surrounded byforming nuclear envelope elements. They are usually multilaminar in nature (quadri- to hexalaminar), atleast 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 thymocytesenter the oocyte cytoplasm their respective nuclear envelope is removed, while a new envelope isreconstituted 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 embryostreated 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 HongKong, Shatin, N. T., Hong Kong

Cadmium has been shown to interfere with spermatogenesis and ovulation in adult mouse, rat and goldenhamster, but its effects on the prenatal development of primordial germ cells and fetal gonads were not fullyknown. In the present study, cadmium chloride (5-6 mg/kg) was given intraperitoneally to the pregnantmice at 7-5-8-5 days p.c. At this age, the mouse embryos were at the primitive-streak to early-somite stagesof development and the primordial germ cells were for the first time detectable by their strong alkalinephosphatase activity. The growth of the cadmium-treated embryos was initially retarded but an acceleratedrestorative growth ensued so that at birth both the body size and the body weight were within normalranges. Histological examination of the genital ridges of 13-5-day embryos revealed that in both sexes thesize of the ridges was reduced and there were fewer primordial germ cells in the ridges. In the testes of16-5-day embryos, seminiferous tubules and interstitial tissues were formed normally but the testes weresmall in size and the number of spermatogonia found inside the tubules was about 65 % of the normal. Inthe ovaries, primordial follicles were formed but the ovaries contained much fewer meiotic oocytes. About18-50 % of the cadmium-treated embryos showed exencephaly at the diencephalic and mesencephalicregion. There were however no consistent variations in the extent of germ cell reduction and gonadaldeficiency between the exencephalic embryos and those having a normal head morphology. A defectivehypothalmo-pituitary axis which may be associated with exencephaly was unlikely to be the cause of poorgonadal development because both structures seemed to have developed normally in the exencephalic andthe non-execephalic treated embryos. The growth pattern of cadmium-treated embryos was reminiscent ofthat of mitomycin C-treated mouse embryos which showed a depletion of germ cells, poor gonadaldevelopment and a high incidence of postnatal infertility (Tam & Snow, 1981). A. mismatch in the growthand differentiation of tissues during rapid restorative growth may be the causative factor for most errors ofmorphogenesis.

TAM, P. P. L. & SNOW, M. H. L. (1981). Proliferation and migration of primordial germ cells duringcompensatory growth in the mouse embryos. /. Embryol. exp. Morph. 64, 133-149.

Early mammalian development 79Ovary development in the bandicoot (Peramelidae, Marsupialia)

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 differentiatedirectly 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 notinvolve mesothelial proliferations, as described for most eutherians.

In the prospective ovary fibroblasts separate medulla from a narrow cortex in which, by day 5 postpartum, most PGCs are located. By day 10 oogonia largely replace PGCs and the cortex has thickened: amesothelial 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 thestroma and not the rete, as in the mouse (4). By day 34 the first primary follicles are present with aparanuclear 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 entodermin 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 litteraverage. Similar findings were found in rats fed a 50 % sucrose diet. In order to find whether there is acorrelation between embryonic malformations and placental changes, we studied by SEM the morphologic-al characteristics of visceral yolk sac entodermal cells in Hi and 13£ day old embryos from streptozotocin(STZ) induced diabetic rats and from rats fed a 50 % sucrose diet before and during pregnancy. Normal ratsfed a regular diet served as controls. In both control groups, SEM studies showedhomogeneous epithelialcells covered with uniform rod shaped microvillii. In Hi day old embryos from rats fed a high sucrose diet,many cells were devoid of microvilli and instead had apical crater formation and numerous membranesfolds. By 13i days, changes were more severe consisting of vesicular blebs of various sizes attached to thecell membranes. Sometimes the microvillii were broad and short, resembling blebs. Some cells wereconnected among themselves by membranes or cytoplasmic bridges. Similar changes were observed inembryos from STZ-diabetic rats (40 or 65 mg/kg of body weight, on day 0 or 5 of gestation respectively). Inthis group, however, both the craters and blebs were smaller and more numerous than in embryos from thehigh sucrose diet fed animals. It is possible that part of the embryonic effects observed in diabetic animals orin rats fed a high sucrose diet are due to the above described visceral yolk sac lesions. Supported in part bythe US - Israel Binational Grant No. 2774/82, by the Grant No. 015-4846 from the Israel Ministry of Healthand by the Richard Meyer Grant for Teratological research.