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UNCLASSIFIED AD NUMBER AD848641 NEW LIMITATION CHANGE TO Approved for public release, distribution unlimited FROM Distribution authorized to U.S. Gov't. agencies and their contractors; Critical Technology; DEC 1968. Other requests shall be referred to Department of the Army, Fort Detrick, Attn: Techical Release Branch/TID. Frederick, MD 21701. AUTHORITY BDL, D/A ltr, 29 Sep 1971 THIS PAGE IS UNCLASSIFIED

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Page 1: UNCLASSIFIED AD NUMBER - DTICmammalian embryo. Seidel"'2 and Tarkovski, 3 utilizing rabbit and mouse ova, respectively, demonstrated the equipotency of the blastomeres ... of blastulation

UNCLASSIFIED

AD NUMBER

AD848641

NEW LIMITATION CHANGE

TOApproved for public release, distributionunlimited

FROMDistribution authorized to U.S. Gov't.agencies and their contractors; CriticalTechnology; DEC 1968. Other requests shallbe referred to Department of the Army,Fort Detrick, Attn: Techical ReleaseBranch/TID. Frederick, MD 21701.

AUTHORITY

BDL, D/A ltr, 29 Sep 1971

THIS PAGE IS UNCLASSIFIED

Page 2: UNCLASSIFIED AD NUMBER - DTICmammalian embryo. Seidel"'2 and Tarkovski, 3 utilizing rabbit and mouse ova, respectively, demonstrated the equipotency of the blastomeres ... of blastulation

IAD 4~

TECHNICAL, MANUSCRIPT 480

D)EVELOPMENT IN VITRO OF "HALF EGGS"AND SINGLE BLASTOMERES ISOLATED FROM

TWO., IFOUR-, AND EIGHT-CELL MOUSE OVA

jam L SpearsE3.E

JwqyS. W.I&.r Xu<LU 1 4-E fn E

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MENUME 1968 '0

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0 DEPARTMENT OF THE ARMY

Frederick, Maryland

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Copy

Page 4: UNCLASSIFIED AD NUMBER - DTICmammalian embryo. Seidel"'2 and Tarkovski, 3 utilizing rabbit and mouse ova, respectively, demonstrated the equipotency of the blastomeres ... of blastulation

DEPARTMENT OF THE ARMYFort: Detrick

Frederick, Maryland 21701

TECHNICAL MANUSCRIPT 480

DEVELOPMENT IN VITRO OF "RhAi EGGS" ANDSINGLE BIASTOMERES ISOLATED FROM

NWO-, FOUR-, AND EIGHT-CELL MOUSE OVA

James R. Spears

Jerry S. Walker

I

Process Development DivisionAGENT DEVELOPMENT AND ENGINEERING IAM(ATORIES

Project 1B533001ID426 December 1968

14

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2

In conducting the research described in this report, theinvestigators adhered to the "Guide for Laboratory AninalFacilities and Care," as promulgated by the Comittee onthe Guide for laboratory Animal Facilities and Care of theInstitute of Laboratory Animal Resources, National Academyof Sciences-National Research Council.

t _______________________

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3

ABSTRACT

The extent of mouse blastomere lability was studied by isolatingand growing in vitro blastomeres as various components of two-,four-, and eight-cell ova. The results were in accord with theview that blastomeres of the eight-cell ovum and possibly evenlatexr stages have the capability of becoming either trophoblastor inner cell mass, depending on location in the cell aggregateat the time of blastulation. The formation of "trophoblasticvesicles" from single blastomeres of four- and eight-cellova is a result of an inadequate number of cells at time ofcavitation.

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Abstract .* 3

I. INT3ODUC'nom .* 7

*II.* MATERIALS AND METHODS . 8A. Animals . 8B. Techniques .................. 8

III. RESULTS AND DISCUSS19 ....... . 10

Literature Cited .. .. .................... .............. 19Distribution List. .... .................... ................... 21DD Form 1473 .. .... ................ . ........................23

FIGURES

1. Twin Blastomeres from a Two-Cell Ovum with Intact Control . 132. TWin Component. from a Four-Cell Ovum with Control at the Time

of Explantation .. .. .o. .. .. .o. .. .. .. . ... o13

3o Twin Components from an Eight-Cell Ovum with Control at theTime of Explantation.......... ........................................ 13

4. Twin Morula Typical of Those Developed from One-Half of Two-,Four-, or Zight-Cell Ova.oo........................14

5. Twin Blastocysts Developed from Halves of an Eigoht-C~ell Ov~umoafter 30fHours in vitro............................... .. 14

6. Twin Blastocysus Developed from Srtures (Morula) Shown inFigure 4 .. .... .................... ............ ........ 1

7. Trophoblastic Vesicles Developed from 'OneDias tomere of anEight-Cell Ovum. ........................... 16

8. The Inherent Ability of Bias'tomeres'to Cvttafer FvMitotic Divisions, Regardless of the Total Number of CellsPresent or the Cell Stage from which Single Blastomere wasIsolated.o............................ ..... . . . . 17

1. Proportions of Two-, Four-, and Eight-Cell Ova Recovered atVarious Time Intervals after Linteinising Hormone Injection .. * 8

2. Explantation Protocol . . . . . . ... . . .i * it .'. 93. Percentages of Morula- and Blastocyst-Like Sructures Developing

from "One Half" of Two-, Four-, and Eight-Cell Mouse Ova o . . . 10

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1. INTRODUCTION

Regulation of development in the embryo has been observed in severalinvertebrate species, but this notion has not yet been resolved in themammalian embryo. Seidel"' 2 and Tarkovski, 3 utilizing rabbit and mouseova, respectively, demonstrated the equipotency of the blastomeresof the two-cell ovum. These experiments consistP of destroying oneblastomere of the two-cell ovum by puncturing through the zona pellucidawith a glass needle and allowing the continued development of theremaining blastomere. When the latter were transferred to recipientfemales, the birth of several living young was observed. Attemptsto sustain development from four-cell ova with three destroyed blastomeresfailed. *Tarkowski3 concluded that the rodent egg was characterizedby polarity and bilateral symetry and that progressive segregation ofthe cytoplasm into dorsal and ventral sections gave rise to innercell mass and trophoblastic cells, respectively.

•intz4 presented results from studies involving the experimentalproduction of genetically mosaic embryos indicating that the mouseovum is still developmentally labile at the eight-cell stage andpossibly even beyond. Her conclusions were based on the fact thatselective sorting out or migration of cells did not occur in chimericaggregations from mouse ova fused at the cleavage stage just precedingblastulation. There was complete integration with the formation of asingle blastocyst after the usual in vitro culture period. This doessuggest that early determination of blastomeres has not occurred.Tarkowski and Wroblewska 5 have since spoken against Tarkowski's earlierconclusions and demonstrated that all blastomeres from four- and eight-cell ova have the inherent ability to develop into trophoblastic cells.

Dalcq6 and INilnard, 7,8 utilizing cytochemical and cytological

techniques, concluded that there did exist a "bilateral symetrical"organization in the uncleaved ovum. The findings of Dalcq! gave wayfor the interpretations that Tarkowski 3 and Seidel 1" proposed from theresults of experiments mentioned previously.

Kulnard's conclusions7,s were based on experimental results ofcytochemical studies specifically designed for the detection of acidphosphatase activity associated with cells of the inner cell mass.

Our investigation was conducted in an effort to determine the extentof blastomere lability when isolated as various components of two-, four..,and eight-cell ova. The interpretations drawn from these experimentsare in accord with views of Tarkowski and Wroblewskas and ,iintz'; thatis, the blastomeres of the eight-cell ovum and possibly even laterstages have the capability of becoming either trophoblast or inner cellmass, depending on their location in the cell aggregate at the timeof blastulation. The formation of "trophoblastic vesicles" from singleblastomeres of four- and eight-cell ova is a result of an inadequatenumber of cells at time of cavitation.

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11. PATERIALS AND MNKTWS

A. ANIMBIS

This work was performed with C5 7 BL/6CUM and random-bred Swiss albinomice. All animals were at least 6 weeks old when used.

B. TECIQUES

Ovulation was induced by intraperitoneal (IP) injection of 5 internationalunits (IU) of pregnant wares' serum (IMS) followed 40 to 48 hours laterby an IP injection of human chorionic gonadotropin (HM). The techniquesof recovery, manipulation, and culture of intact ova are as described in aprevious comunication° vlt'2 the exception that 0.6 ml of 4% methylcellulosewas added to each 10-al portion of culture medium. The ova were recoveredat appropriate time intervals after BG injection so as to assure thepresence of the desired cell stages (Table 1).

TAMLZ 1. 0OPC3TIMOMS (V TWO-, FOUR-, AMN EIHT-CMLL OVA RCOEED ATVARIOUS TIEITUVALS AF LUZIEINIZID BORNf INJECTION

Hours from Injection Percentage of Each Cell Stage Recovere&e/of Luteinizing Hormone to lecovery 2-Cell 4-Cell 8-Cell

48 99.549 76 2450 58 4251 70 3052 28 7253 7 9354 8 9255 15 8556 1.5 98.557 1 9958 1 9959 96 460 68 3261 36 6462 5 95

a. Data taken from 1,497 ova recovered from 272 mice.

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After recovery, the ova were deposited in a 0.5% solution of StreptD-myces griseus protease (Pronase®, CalBiochem Co.) at room temperatureto remove the zona pellucidae.' 0 The Pronase was prepared in Hanksbalanced salt solution without bicarbonate. Dissolution of pellucidmembranes was complete in 3 to 5 minutes. After removal of the zonapellucidae, the ova were washed several times in culture medium to removeany trace of Pronase. After sufficient washing, the ava were repeatedlypipetted through a micropipette (inside diameter approx. 50ý±), whichfacilitated blastomere separation. Blastomeres of two-cell ova separatedoulte readily; in most cases, four-cell ova separated into two two-cellcomponents and, with additional pipetting, separated into iour singleblastomeres. The eight-cell ova rarely separated into two four-cellcomponents; generally, they separated irregularly, one or two cells ata time. Pipetting was continued until the desired number of ceilsremaired (four), and the cells that were "shelled off" were reassembledin aggregates containing four blastomeres or explanted as four separatecultures, each containing a single blastomere. Versene (EDTA) wasinitially employed to facilitate blastomere separation; however, thispractice was discontinued when toxic effects were observed.

The component parts of two-, four-, and eight-cell ova that werecultured and the number of blastomeres in these parts are given inTable 2.

TABLE 2. EXPIA.ITATIOW PROTOCOL

No. of Cellsin Intact No. of Cells in Fraction Explanted

ovum 1/2 1/4 1/8

2 1 - -

4 2 1 -

8 4 - 1

Ir. these experiments, no attempt was made to identify explantedblastomeres with their original intact ovum. Instead, blastomereswere randomly selected from a common pool in a microdrop that containedcells of the sam cleavage stage. However, in one group of experiments,the two halves from the same intact ovum were explanted together inthe same microdrop, along with a control; care was taken to preventcontact which might result in recombination or fusion into theoriginal intact form. In all experiments, one or more intact ova, ofthe same developmental stage as that from which the blastomeres ca'.,were deposited in each microdrop to serve as controls and for photographicdemonstration.

4u

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III. RESULTS AND DISCUSSION

A total of 717 halves from two-, four-, and eight-cell ova (Fig. 1-3)were cultured; their development efficiency is recorded in Table 3.Overall, 73.2% of these component parts developed into distinct morpho-logical entities as morulae or blastocysts (Fig. 4-6). The percentageof components developing was noticeably higher for those isolated from thelater cleavage stages. The percentages of components developing toblantocyst when isolated from two-, four-, and eight-cell ova were41, 58, and 66%, respectively (Table 3).

TALEZ 3. IERCEUTAGE CF MMUIA- AND BLASTOCYST-LIKE STRUCTURES DEVEOPIMimE "Oing WAX?" or TWO-, FOUR1-, AMD EIGHT-CELL MVUSE OVA

Cell State Total No.at Time of Slastomeres TotalIsolation Explanted Morula, Blastocyst %.

2 427 102 (24%) 176 (417,) 65.1

4 199 51 (261) 117 (581) 84.4

B 91 19 (201) 60 (661) 85.7

Total 717 172 353 73.2

Some diff iculty was encountered In consistently de~montratingdevelopmnt of single blastomeres from both four- and eight-cell ova.In one series of =!mimernets, seven of eight blastomeres from an eight-cellovas developed Into the ebaracteristic "tropboblastic vesicles" asdescribed by Tarkewski and Uroblevska .5 These structures contained novisible inner cell MOBs (Fig. 7).

in the intact owu, four cleavage divisions can be observed quitereadily. Immediately after the third division, there seem to be a fusionof blas tomeres that prevents f urther separation and also mkes thenumer of subsequent intermitotic divisions difficult to ascertain.Nomever, by caref ul saminatlon, one can estimate with reasonable accuracythe total - 'or of cells and, In turn, determine the numer of mitoticdivisions prior to blastulation. We gave particular attention to the numberof cleavage divisions each component ("one half" of two-, f our-, or eight-cell ova; 1/4 of four-cell ova; and 1/S of S-cell ova) undewent. Thisphenomenon is Illustrated in Figure 8.

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When one blastomere is isolated from a two-cell ovum and grown in vitrofor 72 hours, it undergoes five mitotic divisions. Blastomeres fromfour-cell ova and four from eight-cell ova undergo four and three mitoticdivisions, respectively. The total number of cells in each of thecomponents is exactly equal at the time of cavitation, which occursbetween the fifth and sixth mitotic division.

Those structures developing from single blastomeres of four- and aigbt-:ell ova underwent four and three mitotic divisions, respectively. Irthis case, the single blastomere fxim the four-cell ovum contained no morethan 16 cells at the end of the in vitro culture period; those blastomeresfrom eight-cell ova contained a maximum of eight cells. Irrespectiveof the number of cells, after the fifth mitotic division (counting fromthe intact ovum) cavitation proceeded. Blastomeres developing as anaggregate in the Intact ovum or separated into component parts appearto have an inherent ability to cavitate after a predetermined numberof intermitotic divisions.

Structures that developed from a single blastomeres from eight-cellova never contained embryonic inner cell mass. These findings seem tosubstantiate the interpretations proposed by Tarkowski and Wroblewska.sAny particular cell can become inner cell mass or trophoblast, dependingor -hether it lies inside the cell mass or on the periphery of the mass.In the case of the "trophoblastic vesicles" formed from single blastomeresof eight-cell ova, a maximum of eight cells are formed from three mitoticdivisions after isolation from the intact eight-cell ovum. When cavitationoccurs, this presents a difficult situation from the standpoint of geometryalone. Upon secretion of the blastocoelic fluid, resulting in the formationof the blastocoele, the number of cells is insufficient to form a cell-lined cavity and still "cut off" or "trap" inner cells that wouldconstitute the embryonic inner cell mass.

In separating eight-cell ova into two four-cell halves, blastomereswere particularly difficult to remove other than by "shelling off"one, two, or three blastomeres at a time until only four blastomeresremained. Those blastomeres "shelled off" frequently showed obviousdamage upon reaggregation, and, in most cases, deteriorated or becamenecrotic shortly after explanation. This gave rise to the possibilitythat only ventral components were being explanted, which would accountfor the preponderance and increased incidence of "blastocyst-like"structures developing from the "half eggs" of four- and eight-cellova. However, experiments consisting of explanting pairs of blastomeresfrom four-cell ova, in which blastocysts developed from each halfwith visible inner cell mass, indicated this was not the case. Thisalso seems to indicate that no regional determination has occurred at

the four-cell stage. Attempts to achieve comparable results from eachfour-cell component of eight-cell ova were not as successful. However,a limited number of four-blastomre pairs from eight-cell ova diddevelop into smaller, but quite normal-appearing, twin blastocysts(Fig. 5).

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The mechanism of monozygotic twinning is poorly understood in the rodentand other mamals. Transfer experiments are being conducted to determineif identical twins can arise from the fission of two-, four-, and eight-cellova after development to blaotocyst in vitro.

The importance of the ovum c..lture technique for experimental twinningstudies has been indicated.5 Tarkowski and Wroblewska6 mentioned thatnaked blastumeres transferred to the oviducts of mice did not surviveand could not be recovered for some unknown reason. One of us12 transferred336 separated blastomeres from 168 two-cell rat ova into the oviductsof 51 recivient animals and Gbserved no implantation sites on femalesautopsied or living young from females allowed to go to term. Two-cellova deprived of the zona pellucidae likewise failed to develop, whereas75% of intact two-cell controls resulted in the birth of living young.In this case either the importance of zona pellucidae or developm.entalfailure attributable to Pronase damage is indicated.

Living young produced from each half of an eight-cell ovum would proveconclusively that the blastomeres of the eight-cell ovum are completelyequipotent and that regional determination occurs at later stages ofdevelopment. The fact that Tarkowski 3 and Seidel31 2 demonstrated thatsingle blastomeres of the two-cell ovum could give rise to living young,and interpretations drawn from the results of this study, do not ruleout the possible existence of another mechanism of twinning. On severaloccasions, Spears has ncted a subsequent separation into two quite normal-appearing structures after the fusion of early preimplantation ova (two-,four-cell). The work of Mintr 4 and Tarkowskil 3 with chimeric embryossuggests the possibility that complete integration of genotypes couldresult, with the subsequent fission into two embryos which would beindistinguishable because of this complete genetic integration priorto differentation. Experiments are being planned to test this hypothesis.

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FIGURE 1. Twin Blastomeres from a Two-Cell Ovum with Intact Control.A, at the time of explantation; B, after 28 hours in vitro. 200X

FIGURE 2. Twin Components from a FIGURE 3. Twin Components from anFour-Cell Ovum with Control at the Eight-Cell Ovum with Control at theTime of Explantation. 20OX Ti.. of Explantation. 200X

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FIGURE 4. Twin Morula Typical of ThoseDeveloped from One-Half of Two-, Four-, orEight-Cell Ova. This partic~slar pairdeveloped from halves of a four-cell ovumafter 30 hours in vitro. Control is in thecenter. 20OX

FIGURE 5. Twin Blestocyats Developed fromHalves of an tight-Cell Ovu, after 30 Hoursin vitro. Two control ova are present. 200X

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FIGURE 6. Twin Blastocysts Developed from Structures (lorula)Shown in Figure 4. Photographed the fo~llowing day after 45 hoursin vitro. Control embryo is beginning to hatch. 200X

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LITERATURE CITED

1. Seidel, F. 1952. Die Entwicklungspotenzen einer isoliertenBlastomere des Zveizellenstadiums in Saugetierei. Naturwissenschaften39:355-356.

2. Seidel, F. 1960. Die Entwicklungsfahigkeiten isolierterFurchungszellen aus dem Ei des Kaninchens Oryctolazus cuniculus.Arch. Entvickl. Organ. 152:43-130.

3. Tarkowski, A.K. 1959. Experiments on the development of isolatedblastomeres of mouse eggs. Nature 184:1286-1287.

4. Mintz, B. 1964. Formation of genetically mosaic mouse embryos andearly development of "lethal (t1 2 /tl 2 ) normal" mosaics. J. Exp. Zool.157:273-292.

5. Tarkowski, A.K.; Wroblewska, J. 1967. Development of blastomeresof mouse eggs isolated at the 4- and 8-cell stage. J. Embryol.Exp. Horphol. 18:155-180.

6. Dalcq, A.M. 1957. Introduction to general embryology, p. 103-128.Oxford University Press, London.

7. Mulnard, J.G. 1965. Studies on the regulation of mouse ova in vitro,p. 123-138. In A Ciba Foundation Symposium: Preimplantation stagesof pregnancy. J. & A. Churchill Ltd., London.

8. HMlnard, J.G. 1965. Aspects cytochemiques de la regulation in vitrode l'oeuf de souris apres destruction d'un des blastomeres dustade II: 1. La phosphomonoesterase acide. Hem. Bull. Acad. Roy.Med. Belg. 5:31-67.

9. Spears, J.R.; Walker, J.S. August 1968. A comparison of the in vitroviability of ova from hormone-treated mice with ova from spontaneousovulations, (Technical Manuscript 473). Process Development Division,Fort Detrick, Frederick, Maryland.

10. Mintz, B. 1962. Experimental study of the developing mmlian egg:

Removal of the zona pellucida. Science 138:594-595.

11. Spears, J.R. 1966. A study of the developmental capacities of isolatedblastomeres of rat ova. Ph.D. Thesis, University of Kentucky,Lexington, Ky.

12. Tarkovski, A.K. 1963. Studies on mouse chimeras developed from eggsfused in vitro. Rat. Cancer Inst. Monogr. 11:51-71.

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23Unclassified

DOCCWENT CONTROL DATA. - R D(s.emffv ""m.is.es of ade .0 "" d.ia m mtl awm 1 OW 60 ==1 NOW as.g Oumo t

1. Oweav'4A~i AcYWv;ý:yyw 1119"4T "CWfIYV CLASINISCATION

Department of the Army eMIL 6Fort Detrick, FrederickI Marylandx 21701

9. ma,.mi TITLE

DEVELOPMENT IN VITRO OF "HALF EGGS" AND SINGLE BIASITMUES ISOLATED FROM TWO-,FOUR-, AND EIGHT-CELL MOUSE OVA

a- -uS-mom ammS AN. Miem. NiN18 saw "0)

James R. SpearsJerry S. Walker

A- ftSPOT DATE p.. TOTAL. no. or OASES a, No. or aIe,December 1968 23 12

G& CON TO&C T 00 GRANT NO. OL emt"UN*TOW6 IMPORT WSUNIOR

&5P'w~cTN- 1353300 lD426 Technical Manuscript 480

go. m5O1TN1WTION m

Qualified requesters my obtain copies of this publication from DDC.Foreign announcement and dissemination of this publication by DOC is not authorized.Release or announcement to the public Is not authorized.

It. 0110416411T4Aft NOTES. is. mOeNam MUTANT ACTIVITV

Department of the ArmyFort Detrick, Frederick, Maryland, 21701

28. AORAACT

\LThe extent of mouse blastomere lability was studied by isolating and growing

in vitro blastomeres as various components of two-, four-, and eight-cell ova.The results were in accord with the view that blastomeres of the eight-cell ovumand possibly even later stages have the capability of becoming either troipboblastblastulation. The formation of "trophoblastic vesicles" from single blastomresof four- and eight-cell ova is a result of an inadequate number of calls at timeof Cavitation.()

14. Key WordsBlastomeresovaEmryonic developmentHouse

.rnrea473 ~ ~ ~Ucasfe

mas commnest

N. *,