/ . Embryol. exp. Morph. Vol. 37, pp. 217-226, 1977 2 1 7

Printed in Great Britain

Evaluation of the technique of immunosurgeryfor the isolation of inner

cell masses from mouse blastocysts

By A. H. HANDYS1DE1 AND S. C. BARTON1

From the Department of Anatomy, University of Cambridge

SUMMARYInner cell masses (ICMs) immunosurgically-isolated from 3^-day mouse blastocysts were

examined for trophoblast cell contamination and developmental capacity. Blastocysts werepreincubated in rabbit anti-mouse antiserum, washed thoroughly and then incubated incomplement. The ICMs were then easily dissected by drawing through a fine pipette.

Various experiments confirmed that the trophectoderm had been completely removed bythis treatment. Firstly, the ICMs did not bind a fiuorescein-conjugated antibody directedagainst rabbit IgG, indicating the absence of cells exposed to the rabbit antiserum during theimmunosurgical procedure. Secondly, ICMs dissected from blastocysts preincubated in asuspension of melanin granules did not include any of the trophoblast cells that had phago-cytosed the granules. And, thirdly, the protein synthetic profile of these ICMs was similar tomicrosurgically dissected ICMs, and in particular, trophoblast specific spots were absent.

The developmental capacity of immunosurgically-isolated ICMs was tested by injectingthem into blastocysts and transferring to the uterus of 2^-day pseudopregnant recipients.Extensive chimaerism was detected in the majority of implants, 5-6 days after transfer, butonly in ICM-derived tissues. This demonstrates both the lack of trophoblast cell contamina-tion and functional viability of these ICMs.

INTRODUCTION

The 3^-day mouse blastocyst consists of two distinct groups of cells: theinner cell mass (ICM) and the trophectoderm. The properties of these tissueshave been extensively studied after their microsurgical separation (Gardner,Papaioannou & Barton, 1973; Rossant, 1975a, b; Van Blerkom, Barton &Johnson, 1976). However, the difficulty of the microsurgical procedure limits therate at which blastocysts can be dissected. Furthermore, some ICM cells maybecome damaged due to the use of needles to pull the ICM away from theoverlying trophectoderm. An alternative technique for the isolation of ICMs,termed immunosurgery, has recently been described (Solter & Knowles, 1975).Blastocysts were preincubated in anti-species antiserum, washed thoroughlyand then incubated in complement. Since the antiserum was prevented fromgaining access to the ICM by the tight junctions between the surrounding

1 Authors' address: Department of Anatomy, Downing Street, Cambridge CB2 3DY.

218 A. H. HANDYSIDE AND S. C. BARTON

trophectoderm cells, only trophoblast tissue lysed on the addition of complement,leaving the ICMs intact. This technique has the potential advantage of allowingmany ICMs to be recovered without the risk of mechanical damage to any of thecells. In the experiments of Solter & Knowles, the purity of recovered ICMs wasassessed by the failure to detect trophoblastic-type outgrowths in vitro. Wereport here a more systematic assessment of the purity of immunosurgically-derived ICMs, as well as a test of their developmental capacity.

The first three experiments reported here were carried out to determinewhether immunosurgically isolated ICMs were contaminated with any tropho-blast cells resistant to complement lysis. First, a fluorescent-conjugated anti-Igantiserum was used to detect antibody bound to the surface of any remainingtrophoblast cells after immunosurgery. Secondly, blastocysts were incubated insuspensions of melanin granules prior to immunosurgery, so that the tropho-blast cells, which selectively phagocytose the granules (Gardner, 1975), couldbe identified by phase contrast and electron microscopy at various stages in theimmunosurgical procedure. Thirdly, the protein synthetic profile of immuno-surgically-isolated ICMs was compared with the profiles of microsurgicallyisolated ICMs and trophoblast vesicles (Van Blerkom et ah 1976).

In a fourth experiment, the viability and developmental potential of immuno-surgically-isolated ICMs was studied to determine whether exposure to comple-ment or any other factors had affected them adversely. The ICMs were injectedinto genetically distinct host blastocysts and then transferred to pseudopregnantrecipients. The contribution of the donor ICMs to post-implantation tissues wasassessed by use of glucose-phosphate isomerase (GPI) isoenzyme markersseveral days later (Gardner et al. 1973).

MATERIALS AND METHODSImmunosurgery

Blastocysts were flushed from the uteri of superovulated females early on thefourth day after mating. The zonae were removed by incubating in Pronase(Calbiochem 0-5 % Tris-citrate buffer pH 7-0) for 5-10 min at 37 °C, or byminimal (15-30 sec) exposure to acidic Tyrode's solution, pH 2-5, adjustedwith N/5 HC1, containing 0-4 % polyvinylpyrrolidone followed by transfer tomedium PBl+10% Foetal Calf Serum (FCS) (Whittingham & Wales, 1969),which was used thereafter for handling the embryos. Blastocysts treated withacidic Tyrode to remove their zonae implanted and developed normally aftertransfer to pseudopregnant recipients.

Blastocysts were then preincubated in a rabbit antiserum to mouse embryohomogenate (Gardner & Johnson, 1973) diluted 1 in 10 with PB1 for 15 mins at37 °C followed by three 5-min washes in PBl+10% FCS. They were thentransferred to guinea-pig complement (Flow Labs.) made up to the specifiedvolume with the diluent supplied and then diluted 1 in 10 with PB1, and in-

Immunosurgical isolation of ICMs from mouse blastocysts 219cubated for up to 45 mins at 37 °C. These conditions had been shown inpreliminary experiments to give optimal lysis with this antiserum and comple-ment. The ICMs were dissected free of lysed trophoblast cells by drawing theembryos through a fine pipette and were then transferred to fresh medium.

Immunofluorescence

Control blastocysts, blastocysts after incubation with the rabbit antiserumand immunosurgically-isolated ICMs were incubated under oil in separatewells of a cytotoxicity slide for 15 min at 4 °C in fluorescein-conjugated goatanti- rabbit-IgG (Miles Labs.) diluted 1 in 10 with PB1 (Muggleton-Harris& Johnson, 1975). The embryos were then transferred with minimal amounts ofmedium through three more wells containing drops of PB1+10 % FCS to washoff excess conjugate before removing the slide from the oil, applying a coverslipand viewing with a Zeiss epifluorescent microscope (incident fluorescent lightsource HBO 200 with excitation filter system 427902 and barrier filter system427903).

Incorporation of melanin granules

Blastocysts were incubated for 2 h in a suspension of melanin granules inPB1 + 10 % at 37 °C before immunosurgery. The suspension was prepared byscraping fragments of melanin from the pigmented layer of the eye of C57B1 micein a little medium, and decanting to eliminate large fragments (Gardner, 1975).

Protein synthetic analysis

Groups of 8-24 control blastocysts and of immunosurgically-isolated ICMswere incubated for 2̂ —4 h in protein-free medium 16 (Whittingham, 1971) in thepresence of [35S]methionine. The embryos were then lysed and the labelled pro-tein analysed by two-dimensional electrophoresis and fluorography as describedelsewhere (O'Farrell, 1975; Van Blerkom et al 1976).

Injection of ICMs into blastocysts

ICMs were immunosurgically dissected from blastocysts derived frommatings homozygous for GPI-lb and injected into host blastocysts homozygousfor GPI-la, using of a Leitz micromanipulator. The injection chimaeras werethen transferred to 2^-day pseudopregnant CFLP recipients homozygous forGPI-la. Six or seven days after transfer the conceptuses were explanted and dis-sected into ectoplacental cone, mural trophoblast (including Reichert's mem-brane), yolk-sac, amnion and allantois (analysed together) and conceptus.Eachof these five samples of tissue was then washed separately in drops of medium,and lysed by freezing and thawing in 5 /A. of distilled water. The lysates werethen run on starch slab gels at 4 °C, 170 V for 45-60 min to determine the GPIisoenzyme variants present. Samples of diluted blood from GPI-typed animalswere run as controls (Gardner et ah 1973).

220 A. H. HANDYSIDE AND S. C. BARTON

FIGURE 1

Phase contrast micrographs of 3^-day blastocyst after incubation in a suspension ofmelanin granules (A) prior to lysis, granules present in the trophoblast cells (x 400);(B) undergoing complement lysis after antibody treatment (x 400) and (C) isolatedICMs post-lysis aggregating in culture and showing no melanin granules ( x 400).

Immunosurgical isolation of ICMs from mouse blastocysts 221

Electron microscopy

Control blastocysts, blastocysts after incubation with melanin granules, andICMs isolated immunosurgically from blastocysts that had been incubated withgranules were prefixed for 30 min at 4 °C in Karnovsky's fixative (Karnovsky,1965), washed twice in 0-1 M sodium cacodylate and then postfixed in 1 %osmium tetroxide for 45 min at 4 °C. They were then dehydrated in gradedconcentrations of acetone before embedding in araldite. Sections, 5 nm thick,were stained with lead citrate and uranyl acetate. Unstained sections wereviewed at 60 kV. Sections were viewed on the Philips 300 electron microscope.

RESULTSImmunofluorescence

After incubation in rabbit antiserum both polar and mural trophoblastshowed an intense and specific uniform surface staining. However immuno-surgically-isolated ICMs did not show any fluorescence, thus indicating thatnone of the outer trophoblast cells remained present on the ICM after treatmentwith complement.

Melanin granules

Blastocysts before dissection showed presence of granules both in mural andpolar trophoblast under phase contrast and by electron microscopy (Figs. 1 A,2A). After immunosurgery cells bearing granules were absent (Figs. 1 C, 2B).Furthermore, isolated ICMs aggregated spontaneously in culture (Fig. 1C), aproperty not manifested by trophoblast cells or intact blastocysts.

Protein synthetic patterns

The patterns obtained with immunosurgically-isolated ICMs were similarto those obtained with microsurgically-isolated ICMs of equivalent age. Inparticular all trophoblast specific spots were absent but those characteristicof the ICM were present (Fig. 3). The absolute incorporation of label wasgreater than that obtained with microsurgically-isolated ICMs.

ICM injection into blastocysts

Of 18 injection chimaeras, 16 transferred blastocysts subsequently implanted.The ICM-derived tissues (amnion, allantois, yolk-sac, embryo) of 14 out ofthese 16 showed contributions of donor ICM GPI-type. Two conceptuses alsoshowed some contribution of the donor ICM tissue to trophoblast-derivedtissues (Fig. 4). This was probably due to contamination of mural trophoblastwith distal endoderm and of ectoplacental cone by adherent allantois duringdissection (Gardner et al. 1973).

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222 A. H. HANDYSIDE AND S. C. BARTON

FIGURE 2

Electron micrograph of (A) mural trophoblast showing granules both on the surfaceand within the cytoplasm, and a tight junction between two trophoblast cells(x 15000); (B) ICM immunosurgically-isolated from blastocyst incubated inmelanin granules, with no adherent or contained granules (x 3900).

Immuno surgical isolation of ICMs from mouse blastocysts 223

y -' / /

_ . 4

y / -

FIGURE 3

Two-dimensional electrophoresis patterns of radioactive proteins synthesized by(A) microsurgically-isolated trophoblast; (B) immunosurgically-isolated ICMs;(C) microsurgically-isolated ICMs. (Arrowheads indicate trophoblast characteristicspots, and arrows those characteristic of ICM.) Separation horizontally by iso-electric focusing and vertically by SDS gradient electrophoresis.

15-2

224 A. H. HANDYSIDE AND S. C. BARTON

Individualparts analysed

Explanted at 9\ days

Recipient 1

I 2 3Ectoplacental I 11 11 I

mnp I I ' I I—I

Recipient 2

1 2 3 4 5

Mural trophoblast • • • • • • • !

Yolk sac

Amnion

Embryo

Explanted at 8^ days

Recipient 1 Recipient 2

1 2 3 4 1 2 3 4

• • • • • • • •• • O - • • • •• • • • ammm

N.A. N.A.

N.A. N.A.

Fig. 4. GPI-type analysis of conceptuses obtained after transfer of blastocysts, injectedwith immunosurgically-isolated ICMs, to pseudopregnant recipients. • , Chimaeric;D, host type; N.A., not analysed.

DISCUSSION

Trophoblast cells could not be detected after immunosurgical isolation ofICMs from 3^-day mouse blastocysts, as judged by several independent criteria.

Firstly, no cells could be detected by immunofluorescent staining of boundantibody or by presence of melanin granules despite the fact that both thesemarkers were present on or in all trophoblast cells before immunosurgery.Secondly, the patterns of protein synthesis of the ICMs showed none of thespots specific for microsurgically-isolated trophectoderm but did show ICMspecific spots (Van Blerkom et al. 1976). And thirdly, chimaeras formed byinjection of immunosurgically-isolated ICMs into host blastocysts showeddonor ICM contribution to trophoblast-derived tissues in only two cases. Acontaminant allantois was almost certainly responsible for the contributionto the ectoplacental tissue of the 9-j-day embryo since this organ was missingfrom the embryonic dissection. The trace of activity found in mural trophoblastand associated Reichert's membrane of these older embryos was probably dueto distal endoderm.

Additional evidence on the purity of immunosurgically-isolated ICMs hascome from the analysis of three interspecific chimaeras made by injectingimmunosurgically-isolated rat ICMs into mouse blastocysts (M. H. Johnson andR. L. Gardner, personal communication). The rat ICMs contributed only toembryonic ectoderm and to extra-embryonic and embryonic endoderm, but notto trophoblast, ectoplacenta and extra-embryonic ectoderm, indicating posses-sion of the same developmental potential as microsurgically-isolated ICMs(Gardner & Johnson, 1975).

These experiments have also reduced doubts that the ICMs are deleteriously

Immunosurgical isolation of ICMs from mouse blastocysts 225affected by the process of immunosurgery. The high proportion of chimaericembryos recovered and the generally high contribution to embryonic tissuesindicate that the functional viability of these ICMs is at least as good as theirmicrosurgically-dissected counterparts. Indeed the increased incorporation of[35S]methionine into protein by immunosurgically-isolated ICMs as comparedwith microsurgically-isolated ICMs may be a reflexion of the better conditionof the cells.

Immunosurgery allows only the isolation of ICMs and although an alternativetechnique has been described for the isolation of trophoblast vesicles, reserva-tions have recently been expressed about the use of [3H]thymidine in thisapproach (Rossant & Kelly, 1976). Microsurgical methods may still be the onlyway of recovering this component of the blastocyst. The real advantages ofimmunosurgery are the ease with which larger numbers of blastocysts can bedissected and the possibility of obtaining ICMs from strains which are difficultto handle microsurgically.

We would like to thank Dr Martin H. Johnson for help and advice, Debbie Eager, MitziGooding, and John Ward for their technical assistance, and Debby Hickman for preparingthe manuscript. The work was supported by an M.R.C. project grant to M.H.J., a grant fromthe Wellcome Trust, and a grant from the Ford Foundation to Professor C. R. Austin.

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GARDNER, R. L., PAPAIOANNOU, V. E. & BARTON, S. C. (1973). Origin of the ectoplacentalcone and secondary giant cells in mouse blastocysts reconstituted from isolated trophoblastand inner cell mass. / . Embryol. exp. Morph. 30, 561-572.

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O'FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. / .biol. Chem. 250, 4007-4020.

ROSSANT, J. (1975 a). Investigation of the determinative state of the mouse inner cell mass. I.Aggregation of isolated inner cell masses with morulae. J. Embryol. exp. Morph. 33, 979-990.

ROSSANT, J. (19756). Investigation of the determinative state of the mouse inner cell mass.II. The fate of isolated inner cell masses transferred to the oviduct. /. Embryol. exp. Morph.33, 991-1001.

ROSSANT, J. & KELLY, S. J. (1976). The effect of short-term labelling in [3H]thymidine on theviability of mouse blastomeres: alone and in combination with unlabelled blastomeres./. Embryol. exp. Morph. 35, 95-106.

226 A. H. HANDYSIDE AND S. C. BARTON

SOLTER, D. & KNOWLES, B. B. (J975). Immunosurgery of mouse blastocyst. Proc. natn.Acad. Sci. U.S.A. 72, 5099-5102.

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Aust. J. biol. Sci. 22, 1065-1068.

{Received 11 August 1976, revised 27 September 1976)