Development 102. 793-803 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

793

The preimplantation pig embryo: cell number and allocation to

trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

V. E. PAPAIOANNOU1 and K. M. EBERT2

^Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA2Department of Anatomy and Cellular Biology, 200 Westboro Road, North Grafton, MA 01536, USA

Summary

Total cell number as well as differential cell numbersrepresenting the inner cell mass (ICM) and trophecto-derm were determined by a differential staining tech-nique for preimplantation pig embryos recoveredbetween 5 and 8 days after the onset of oestrus. Totalcell number increased rapidly over this time span andsignificant effects were found between embryos of thesame chronological age from different females. Innercells could be detected in some but not all embryos of12-16 cells. The proportion of inner cells was low inmorulae but increased during differentiation of ICMand trophectoderm in early blastocysts. The pro-portion of ICM cells then decreased as blastocystsexpanded and hatched.

Some embryos were cultured in vitro and otherswere transferred to the oviducts of immature mice as asurrogate in vivo environment and assessed for mor-phology and cell number after several days. Althoughtotal cell number did not reach in vivo levels, morpho-logical development and cell number increase wassustained better in the immature mice than in vitro.The proportion of ICM cells in blastocysts formed invitro was in the normal range.

Key words: pig, blastocyst, ICM, trophectoderm, cellnumber, in vivo, in vitro.

Introduction

In spite of the diversity of placentation types ineutherian mammals, early preimplantation develop-ment is remarkably similar in different species. Em-bryos reach the morula stage and then develop intothe blastocyst with two tissues, the inner cell mass andthe surrounding trophectoderm. From that point,however, development of the embryo, in particularthe trophectoderm, diverges considerably in differentspecies. Although investigations into the mechanismsof control of this early morphogenesis have beendone mainly in the mouse, other mammalian species,including those of economically important agricul-tural animals, are increasingly the subject of exper-imental work (see Papaioannou & Ebert, 1986a forreview). In this study of the early development of thedomestic pig, we set out to lay the groundwork forcomparative experimental studies that will determineto what extent mechanisms discovered in the mousecan be generalized to a species with a very differenttype of placentation. In addition, these basic studies

on blastocyst formation may help elucidate the causesof the high embryonic mortality of 22-41 % seen inpigs (Perry & Rowlands, 1962).

Unlike the situation in the mouse, the trophecto-derm of the pig blastocyst continues dividing and theblastocyst expands into a tubular structure up to ametre in length before implantation occurs (Geisert etal. 1982; Perry & Rowlands, 1962). We have exam-ined the allocation of cells to this tissue in the pigembryo during the morula-to-blastocyst transition inorder to compare this stage of pig embryogenesis withthe mouse. To accomplish this, data on normalembryo cell number were collected and the allocationof cells to the two primary cell lineages of theblastocyst was determined. These data were thenused in the evaluation of both in vitro culture and anin vivo surrogate environment, the immature mouseoviduct. The cell-counting method of Ebert et al.(1985) and the method of Handyside & Hunter (1984)for the visualization of inner cell mass (ICM) and

794 V. E. Papaioannou and K. M. Ebert

trophectoderm nuclei have been substantially modi-fied to provide a simple, rapid method of differentialcell counts of pig blastocysts.

Materials and methods

Embryo collection31 gilts between 9 and 16 months of age were used toprovide pig embryos. 20 were pure-bred Yorkshire, 3 pure-bred Duroc and 8 were Yorkshire-Duroc hybrids. Pigswere bred either naturally or by artificial insemination toproven fertile Yorkshire boars. Females were checked twicedaily at 9 a.m. and 4p.m. for signs of oestrus and then bredtwo or three times at 6 to 12 h intervals. Breeding wasusually done at the first sign of oestrus or between 6 and12 h later. Timing of embryos is from the onset of detectableoestrus. Embryo collection was begun between 8 and9 a.m., thus day-5 embryos, for example, were collected4-75-5 days after onset of oestrus.

Pig embryos were collected by retrograde flushing of theuterus with 30 ml of sterile PBS after cannulating theovarian end of the uterus. Embryos were transferred toHam's F12 medium (Flow Laboratories) with 10% fetalcalf serum, maintained at 37°C for 1-2 h and then scoredfor gross morphology using a Wild dissecting microscopewith transmitted illumination and phase microscopy. Someembryos were collected during surgery from animals main-tained under halothane (2-4 %) and nitrous oxide (66 %)while others were flushed from reproductive tracts excisedwithin lOmin of death from an overdose of barbiturate.Counts of corpora lutea were made on fresh ovaries.

Development of a rabbit anti-pig antiserumA rabbit anti-pig antiserum was developed in order to applythe technique of immunosurgery (Solter & Knowles, 1975)to pig embryos for the purpose of making differential cellcounts of the ICM and trophectoderm of the blastocyst(Handyside & Hunter, 1984). Adult female pig spleen washomogenized in PBS (2-4 g per 25 ml) and 3 ml was injectedsubcutaneously into two New Zealand White male rabbits.Booster injections were given 14 and 21 days later and theanimals were then killed and exsanguinated. Blood samplesfor antibody testing were taken before each booster injec-tion. Serum was collected, heat inactivated and tested forantibody titres and specificity using primary monolayercultures of pig oviduct fibroblasts, an established line ofSTO mouse embryonic fibroblasts (Martin & Evans, 1975;Ware & Axelrad, 1972), and mouse blastocysts. Mouseblastocysts were recovered in modified PB1 medium(Whittingham & Wales, 1969; Papaioannou & West, 1981)3-5 days post coitum from naturally mated, random-bredCD-I mice (Charles River).

Cultures of fibroblasts in multiwell plates were exposedto different dilutions of rabbit serum in culture medium for30min, washed three times and then incubated with 10%guinea-pig complement (Pelfreez Biologicals, Roger, AR)for 30min. Exposure to culture medium with either anti-body or complement alone provided controls. Cell viabilitywas determined by trypan-blue-dye exclusion. Duplicatecultures were scored and between 200 and 300 cells were

scored per culture. Mouse blastocysts were treated asdescribed for pig blastocysts below, using propidium ipdideto identify dead cells, but leaving the zona pellucida intact.

Immunosurgery and double dye technique fordifferential ICM/trophectoderm cell counts of pigblastocystsThe procedure for immunosurgery and differential nuclearstaining of ICM and trophectoderm depends on the comp-lement-mediated killing of trophectoderm cells that havebeen exposed to antiserum made against cell surfaceantigens and then washed before complement. Inner cellmass cells are protected by their position within thetrophectoderm since antibodies will not go through thetight junctions associated with the single-cell-thick troph-ectoderm layer (Solter & Knowles, 1975). Differentialstaining is accomplished by the use of two chromatin-specific fluorochromes with different fluorescent spectra;one, propidium iodide (PI; Sigma), which is excluded fromvital cells, and the other, bisbenzimide (Hoechst 33258,Sigma), which enters vital or nonvital cells.

To determine optimal immunosurgery and staining con-ditions for pig blastocysts, several parameters of the tech-niques used on mouse embryos (Handyside & Hunter,1984; Solter & Knowles, 1975; V. E. Papaioannou & K. M.Ebert, unpublished data) were varied. The zona pellucida(ZP) was either left intact, dissected open with a microma-nipulator or treated with acidic Tyrode solution (AT;pH2-5; Nicolson etal. 1975) for up to 3min to effect partialor complete removal. Two embryos were treated with ATand complement but no antiserum to test for nonspecificAT or complement toxicity. Since bisbenzimide is a vitalnuclear dye easily taken up by intact or lysed cells, it wasadded to complement along with the nonvital dye, PI. PIwas tested at concentrations between 10 and 100 ̂ g ml"1

and final wash time after staining was varied from a fewseconds to 1 h. The final protocol we developed for 6- to 8-day pig embryos is a rapid procedure that, unlike that ofHandyside & Hunter (1984), does not involve fixation ofembryos. Details are as follows:

ZP-contained embryos are treated with AT with constantvisual monitoring for up to 3 min to dissolve the ZP. Theyare then incubated for 30 min at 37 °C in 5 % CO2 in air in a-MEM (Flow Laboratories) plus 10 % rabbit anti-pig anti-serum, washed three times for 5 min each wash in a-MEM + 5 % fetal calf serum and 5 % newborn calf serum,and then incubated for 30 min in a-MEM with 10 % guinea-pig serum as a source of complement. PI and bisbenzimideare added at a final concentration of lO^gml"1 to thecomplement solution. Finally, the embryos are washed verybriefly in 0--MEM with fetal and newborn calf serum andsquashed under a coverslip as described by Ebert et al.(1985). Dyed embryos are examined using a Zeiss StandardRA fluorescent microscope with an HBO 200W/4 lampunder transmittance illumination. Excitation filter UG 1(365 nm) is used in combination with barrier filter 41(410 nm) resulting in bisbenzimide-stained nuclei fluor-escing blue and nuclei stained with both fluorochromesfluorescing pink (Fig. 1A,B). Classification of nuclei can beconfirmed by viewing with interference green excitationfilters (546 nm) and barrier filter 59 (590 nm; Fig. 1C) which

Pig blastocysts in vivo and in vitro 795

excludes blue fluorescence. The entire area under thecoverslip is scanned to detect scattered nuclei. Whennuclear numbers are low, counts can be made directly. Withhigh numbers of nuclei, colour photographs are taken forcounting at a later time. The number of nuclei counted isconsidered to represent the number of cells in the embryo.Occasionally, nuclear fragments were detected in culturedembryos which presumably represent nuclei from cells thathad begun to degenerate (Handyside & Hunter, 1986).

Total cell countsIn most cases, total cell number was determined afterimmunosurgery and differential staining but in some em-bryos total cell number was determined using bisbenzimidealone (Fig. IE; Ebert et al. 1985). In these cases, the ZPwas partially dissolved with AT or slit with glass needles ona micromanipulator to allow the embryo to escape duringsquashing without undue distortion of the nuclei. In ad-dition, during the development of the differential cell-counting technique, some preparations were unsuitable fordetermining ICM and trophectoderm counts due to the lackof adequate dye distinction or other technical reasons, butwere adequate for the determination of total nuclearnumbers.

Embryo dissection and cultureOne day-7 blastocyst was mechanically dissected using aLeitz micromanipulator (Gardner, 1972; Papaioannou,1981) and the separated ICM was subjected to immunosur-gery using the differential dye protocol above. Some day-5and day-6 embryos were cultured at 37°C or 39°C in 0-25 mldrops of Ham's F12 medium under oil in 5 % CO2 in air andscored daily under phase microscopy for morphologicaldevelopment. Others were transferred to oviducts of imma-ture, random-bred CD-I female mice, between 17 and 22days of age (Papaioannou & Ebert, 1986b). After 2 or 3days, these embryos were recovered from culture or theimmature mice, and scored for morphology and processedfor total or differential cell counts as described above.

StatisticsOne-way analysis of variance was used after log transform-ation to compare cell number of embryos within andbetween females at each age, cell number between different

Fig. 1. Fluorescence micrographs of nuclei dyed withbisbenzimide or differentially dyed with bisbenzimide andpropidium iodide (PI). (A) Whole mount of differentiallydyed pig blastocyst. Blue ICM nuclei are visibleinternally. x512. (B) Squash of differentially dyedblastocyst. Blue ICM nuclei and pink trophectodermnuclei are easily distinguished. X512. (C) Same as Bviewed with propidium iodide optics. Only Pi-stainednuclei fluoresce. (D) Day-7 inner cell mass mechanicallydissected and double dyed following treatment withantiserum and complement. Blue nuclei from inner cellsare evident and could be counted after squashing. X512.(E) Squash of day-5 bisbenzimide-dyed morula withsperm heads in zona pellucida. x200. (F) Anomalouscystic structure (probably nonembryonic) dyed withbisbenzimide. X512.

ages, and also to compare the proportion of ICM cellswithin different morphological groupings. Student's f-testwas used to compare the proportion of ICM to total cells incultured and noncultured expanded blastocysts.

Results

Anti-pig antiserum

At the first bleeding, both rabbits appeared to beproducing anti-pig antibodies effective in comp-lement-mediated cell lysis at dilutions of 1:10. At thesecond bleeding, more extensive tests showed thatnearly all cells in pig fibroblast cultures were lysed at1:20 dilution whereas antiserum alone or comp-lement alone left all cells viable. The final serumsample from one of the rabbits was tested in ad-ditional experiments and found to lyse >99 % of pigfibroblasts at 1:10 (Fig. 2) with approximately 50%viability resulting at a dilution of 1:40. It was obviousthat many lysed cells lifted off the culture dish andwere thus not counted. Consequently, cell lysis atlower concentrations of antiserum is probably muchgreater than indicated by the curve in Fig. 2.

To determine that this antiserum is specific to pigcell surface antigens, it was also tested on mousefibroblasts and mouse blastocysts. At a dilution of1:10, it was found ineffective at complement-mediated cell lysis of mouse fibroblasts (331/332 cellsviable) and trophectoderm (no dead cells in 11treated blastocysts). This anti-pig antiserum wasfrozen as aliquots for use in immunosurgery of pigembryos.

Development of immunosurgery and double dyetechnique for differential ICM/trophectoderm cellcounts of pig blastocystsThe antigens recognized by the anti-pig antiserumwere found to be present, as judged by complement-mediated cell lysis following exposure to antiserum,

100

.2 50-

1:10 1:40 1:160Anti serum dilution

1:640

Fig. 2. Complement-mediated cell lysis of pig primaryfibroblasts following exposure to different dilutions ofrabbit anti-pig antiserum. Results are combined from twoseparate dilution series. Antiserum alone or complementalone left all cells viable.

796 V. E. Papaioannou and K. M. Ebert

on all cells of the 6- to 8-cell uncompacted pig embryo(data not shown), on outside cells of compactedmorulae and on the trophectoderm of the blastocyst.When the ICM from a day-7 blastocyst was dissectedaway from the trophectoderm (Fig. 3A) and sub-jected to immunosurgery, the outside cells of theICM clump were also lysed (24 ICM cells lysed out ofa total of 65) indicating that antigens on these cells arerecognized as well (Figs 3B, ID). Two blastocyststreated with AT for ZP removal and subjected to theimmunosurgery procedure but with antiserum omit-ted showed no evidence of dead cells, indicating thatneither AT nor GPC is toxic to pig trophectodermcells and that cell lysis is dependent on the presence ofantibody to pig cell surface antigens.

In the pig, unlike the mouse, it was found that ZPremoval was necessary to allow access of the antibodyto the embryonic cells. In 28 5- to 7-day embryossubjected to immunosurgery with an intact ZP, nocells or only a few cells lysed as judged by theirappearance under phase and fluorescence mi-croscopy. In one group of day-6 embryos from asingle gilt, three blastocysts were treated with intactZP and three after ZP removal. The percentage ofdead cells in these embryos was 21, 0 and 0 with theZP intact, and 77, 64 and 71 with the ZP removed,indicating that the intact ZP effectively reduced oreliminated lysis. Removal of the ZP after antibodytreatment but before treatment with complement didnot improve lysis. Partial dissolution of the ZP withAT resulted in better lysis of trophectoderm butresults from 23 embryos were mixed; some had verylittle lysis while others were similar to ZP-free em-bryos of the same age. Tearing the ZP with needles orremoving it completely gave consistently good immu-nosurgery resulting in lysis of the outside layer ofcells, leaving an intact inner core. Two day-7 blasto-cysts were pipetted after immunosurgery and thelysed trophectoderm layer separated easily from the

Fig. 3. (A) Day-7 blastocyst after dissection intotrophectoderm and ICM. (B) ICM after treatment withrabbit anti-pig serum and complement. Bar, 100fim.

ICM which appeared round and smooth and similarto the ICM dissected mechanically (Fig. 3A). Thus,the differential cell counts reported were obtainedonly from embryos that were already ZP-free or hadthe ZP removed entirely prior to exposure to anti-body.

With ZP removal, the immunosurgery and dyeprotocol outlined in Materials and methods gives areliable, obvious distinction between lysed cells inwhich the nucleus appears bright pink, and intact cellsin which the nucleus appears blue (Fig. 1A,B). Twofactors, dye diffusion and quenching of fluorescencecould alter the picture. PI present in the medium ordiffusing out of other nuclei slowly diffuses into ICMcell nuclei once these cells are squashed. In addition,constant u.v. exposure over several minutes appearsto quench mainly the Hoechst dye so that blue nucleibecome paler and pink nuclei become more red.However, cell counts or photographs can be madebefore either of these factors obliterates the distinc-tion between lysed and intact cells.

Partial dissolution of the ZP of pig blastocysts hasseveral advantages for total cell counts not involvingimmunosurgery. The ZP of the pig is thicker andtougher than that of the mouse and when an intact ZPis broken by squashing, the resulting pressure fre-quently causes distortion and smearing of the nuclei,making cell counts unreliable. In addition, the squashtechnique used for making cell counts (Ebert et al.1985) does not always completely free the pig embryofrom the ZP, resulting in the possibility of inter-ference with nuclear counts by the numerous brightlyfluorescing sperm heads contained within the ZP,particularly following artificial insemination(Fig. IE). Softening the ZP helps prevent both ofthese problems.

Morphology and cell number of day-5 to -8 pigembryosOf the thirty-one pigs used in this study, nine (29 %)comprising five Yorkshire, one Duroc and threehybrids, produced only unfertilized oocytes or greatlyretarded embryos which are not included in theanalysis. The retarded embryos were twenty-seven 1-to 4-cell embryos collected from three pigs. Twenty-two pigs produced one or more normally developingembryos. Recovery of embryos from these femaleswas 86 % efficient as judged by comparison of cor-pora lutea and embryo counts. The Yorkshire femalesovulated 14-6 ±2-6 (S.D.) oocytes on average, theDuroc females 12-0 ± 3-5, while the hybrids averaged11-5 ±2-1. Table 1 summarizes the numbers andmorphology of embryos obtained from each breed ateach age.

87 % of embryos appeared morphologically normalat the time of recovery. There was a clear progression

Pig blastocysts in vivo and in vitro 797

Table 1. Breed, number and morphology of pig embryos collected between day 5 and 8

Day ofgestation

5

6

7

8

Breed

Yorkshire

YorkshireDuroc

York, x Duroc

YorkshireDuroc

York, x Duroc

Yorkshire

No. offemales

4

813

212

1

Total no. ofembryos

55

951026

191228

10

Degenerating/unfertilized

0

6*5t6i00

16§

0

Morula

37

1952

100

0

Number of

Early

18

2804

000

0

Blastocysts

Expanded

0

420

14

718

0

Hatched

0

000

11114

10

* Consisting of two uncleaved embryos, one 4-cell, and three fragmenting embryos (17 and 18 nuclei) from four females.t Consisting of five uncleaved embryos.$ Consisting of six uncleaved embryos from a single female.§ Consisting of sixteen uncleaved embryos from a single female that also had one blastocyst.

in morphological development with time after onsetof oestrus (Fig. 4A-C). At day 5, most embryos weremorulae or early blastocysts and most had expandedby day 6. By day 7 and 8, most embryos had hatchedfrom the ZP and were large expanded blastocysts.

Total cell number for all embryos collected be-tween 5 and 8 days after onset of oestrus and themean cell number at each day are presented in Fig. 5.Analyses of variance indicated that the mean cellnumber of embryos was significantly different be-tween females at 5, 6 and 7 days (P< 0-001) and thatoverall means were significantly different at differentages (P< 0-001).

The relationship between cell number and morpho-logical development is indicated in Fig. 6. On thewhole, there was a good correlation between cellnumber and morphological development, but cellnumber within each morphological category variedwidely and there was considerable overlap betweencategories. Compaction of blastomeres was seen in allembryos in this study including one with as few as 8cells and these were morphologically classified asmorulae. Blastocoel formation usually occurred afterthe 4th cleavage division (16 cells or more) althoughthree embryos with 9, 12 and 15 total cells containedcavities. About half the embryos with 17-32 cellscontained a cavity as did nearly all embryos with agreater total cell number. Only four embryos contain-ing more than 32 cells (five cleavage divisions) wereclassified as morulae. These were from two femalesthat also contained expanded blastocysts. Hatchingfrom the ZP occurred only in day-7 or -8 embryos andthe lowest cell number of a hatched blastocyst was 80,although ZP-contained embryos with as many as 135cells were also found.

Three anomalous cystic structures were found onday 6 (Figs 7, IF). These were recovered from aYorkshire female with 13 CL from which 10 ZP-contained expanded blastocysts were also recovered.Although they superficially resembled hatched,expanded blastocysts, the cystic structures wereclearly different from any of the embryos seen in thisstudy. There was no evidence of any cytoplasmiclipid, they were larger and more elongated than 6- to8-day blastocysts and the knob of cells correspondingto ICM was not clearly internal but could have beenon the surface. They were dyed with bisbenzimidealone and found to contain a total of 274, 283 and 365nuclei with 15, 26 and 37 nuclei contained in the innerclump, while the average cell number of eight blasto-cysts recovered from the same gilt was 58. Althoughthese structures were not considered to be embryonicand may be pieces of cystic endometrium, a positiveidentification could not be made.

Allocation of cells to inner cell mass andtrophectodermDifferential cell counts of inner and outer cells weresuccessfully made on a total of 62 6- to 8-dayembryos. One or two inner cells were detected inthree morulae with as few as 12-15 cells whereas twomorulae with 15 and 16 cells contained no inner cells.In addition, two embryos classified morphologicallyas early blastocysts with 24 and 49 total cells con-tained no inner cells. These were from a day-6 femalewith embryos ranging from a 16-cell morula to an 80-cell expanded blastocyst. Fig. 8 presents the meanproportion of ICM cells in embryos of differentmorphological classifications. In the morula, themean proportion of ICM cells is 0-14. This figure risesat the initiation of blastocoel formation and continues

798 V. E. Papaioannou and K. M. Ebert

Fig. 4. Phase micrograph of pig embryos. (A-C) Embryos recovered at day 5, 6 and 7 after the onset of oestrus,respectively. (D-F) Embryos recovered at day 5 and cultured for 1 day in vitro (D), 2 days in vitro (E) or 2 days in animmature mouse oviduct (F). Bar, 100 ̂ m.

Pig blastocysts in vivo and in vitro 799

to rise with expansion of the blastocyst but thendeclines in more advanced blastocysts. Analysis ofvariance shows a significant difference in proportionof ICM cells to total cells between these groups.

Culture of day-5 and -6 embryos in vitro and inimmature mouse oviductsSome randomly chosen embryos were cultured for1-3 days either in vitro at 37 °C or 39 °C or inimmature mouse oviducts. Table 2 and Fig. 4 indicatethe morphology of these embryos on the day ofrecovery, during in vitro culture and after recoveryfrom immature mice. Day-5 embryos progressedfrom morulae to blastocysts after two days either invitro or in vivo, but did not hatch from the ZP andappeared to be deteriorating by 3 days in vitro. Thethree embryos scored at this time were dyed with PIand bisbenzimide without immunosurgery in order todetect dead cells, and a high proportion of pink nucleiindicated that these embryos contained many dead

10001

bo «•oO*

IT ioo

£3

10

5 6 7Days after onset of oestrus

Fig. 5. Total cell number of embryos recovered between5 and 8 days after the onset of oestrus. For each day,different symbols represent the embryos from anindividual gilt. Star = mean cell number of each agegroup.

cells. Fragmented nuclei were also seen. Day-6 em-bryos progressed morphologically during 2 days invitro or in vivo and some of these in each culturecondition hatched from the ZP. Two embryos fromeach type of culture had several fragmented nuclei,indicating cell death and nuclear degeneration.

Cell numbers were determined for embryos fromfive females. From each, some embryos were countedat the time of recovery and others were cultured.Because significant differences between female ef-fects on cell number were found (Fig. 5), results arepresented as the factor increase in mean cell numberfor embryos from each female. These data are pre-sented in Fig. 9 and Table 3. It is clear that embryoscollected at day 5 show a greater increase in cellnumber than embryos collected at day 6 and thatembryos cultured in vivo in an immature mouseoviduct fare better than in vitro cultured embryos, a

IX!E

10'

20

15

10

15

10

5'

15

10

5

r~i

n

i—i

—

t

1He

lasttchedocysts

In•

Expandedslastocysts

Earlyblastocysts

Morulae

9- 1 7 - 3 3 - 6 5 - 121- 241-16 32 64 120 240 480

Total cell number

481-960

Fig. 6. The frequency of embryos with different total cellnumbers at various morphological stages of development.The arrow indicates the median cell number at each stageand each bar of the histogram represents a cell doubling.

800 V. E. Papaioannou and K. M. Ebert

result suggested by the morphology. However, aftereither type of culture, embryos were considerablyretarded in cell number compared with embryos ofthe same chronological age that developed entirely invivo. Differential cell counts were made on sevenembryos recovered at day 6 and cultured in vitro for 1day at 37°C (n = 3, average proportion of ICM =0-24) or 2 days at 39°C (n = 4, average proportion ofICM = 0-34). These were all expanded blastocysts atthe time of counting and the proportion of ICM cellswas not different in the two groups and was also notsignificantly different from morphologically similar,but chronologically younger, expanded blastocystsdeveloping entirely in vivo (F>0-l) .

Fig. 7. Phase micrograph of three anomalous cysticstructures recovered from a day-6 pig. Bar, 100^m.

0-3

0-2

0 1

o(5)

(24)

"(21)

Morula Early Expanded Hatchedblastocyst blastocyst blastocyst

Morphology

Fig. 8. The proportion of ICM cells in embryos ofdifferent morphological classifications (mean ± standarddeviation). Numbers in parentheses are the number ofembryos counted in each group. Embryos with no innercells have been omitted.

Discussion

By adapting the differential ICM/trophectodermcell-counting technique of Handyside & Hunter(1984) to the pig blastocyst, we have been able for thefirst time to detail the allocation of cells to the twoprimary cell lineages in a domestic mammal. Rabbit

Table 2. Morphology of embryos collected on day 5or 6 after the onset of estrus, scored and thencultured either in vitro or in the oviducts of

immature mice for 1-3 days

Day

55 + 1 in vitro5+2 in vitro5 + 3 in vitro5+2 in mouse oviducts

66+1 in vitro6+2 in vitro6+2 in mouse oviducts

* One embryo was not

Morula

14

1

822

recovered

Morphology

Early

342

621

Blastocyst

Expanded Hatched

3436*

54 22 3

from the mouse oviduct.

U

x4-

x3

x2-

X l '

/

/

/ /

16)o

/

/

/

/ (,_./ .o

^ /

/ ^ (5) _ ^ -

(3)

-°(5)

(4)

5 6 7 8Days after onset of oestrus

Fig. 9. Increase in cell number in embryos collected atday 5 or 6 and cultured for 1-3 days either in vitro (solidlines) or in the oviducts of immature mice (dotted lines).The X-axis indicates the chronological age of the embryosin days after the onset of oestrus and the Y-axis is thefactor by which the mean cell number of groups ofembryos from each female increased. Points are averagedfrom several females. Number in parenthesis is numberof embryos at each point. Three embryos collected at day5 and cultured in vitro for 3 days (to day 8) were culturedat 39 °C.

Pig blastocysts in vivo and in vitro 801

Table 3. Mean cell number (±standard error) in embryos collected on day 5 or 6 from five females duringculture in vitro or in immature mouse oviducts for 1-3 days

Day

5

5

5

6

6

* Culture

Recovery

Cell no. (n)

13-7 ±1-6 (9)

17-3 ±2-0 (6)

31-1 ±4-8 (10)

37-0 ± 9-5 (4)

104-0 ±31-1(2)

at 39 °C.Number of embryos counted is in

1

99-6 ±9-0 (5)

parenthesis.

Day of culture

2

35-3 ± 10-7 (3)67-0 ±3-8 (3)

49-0 ± 13-5 (4)62-7 ±2-3 (3)

47-5 ±11-6 (4)91-2 ±9-4 (5)

3

89-3 ±25-3 (3)'

Culture condition

in vitroin mouse oviducts

in vitroin mouse oviducts

in vitro

in vitroin mouse oviducts

in vitro

antibodies to cell surface antigens from pig spleencells were effective in complement-mediated lysis ofoutside cells of compact morulae and trophectodermcells of the pig blastocyst in line with ultrastructuralobservations of junctional complexes in these cells at120 h postinsemination (140 h after first oestrus; Nor-berg, 1973). In addition, the presence of cell surfaceantigens on ICM cells was indicated by their suscepti-bility to complement-mediated lysis when ICM cellswere separated from trophectoderm by mechanicaldissection and then subjected to immunosurgery. Thepresence of nonlysed internal cells of the ICMsuggests that tight junctions might also be presentbetween ICM cells although we cannot rule out thepossibility that some ICM cells do not possess cellsurface antigens recognized by our antiserum.

The presence of an intact ZP interferes withimmunosurgery of pig blastocysts. This can be at-tributed to a low permeability of the ZP to antibodiesrather than complement since removal of the ZP afterantiserum but before complement exposure resultedin little or no lysis. This suggests that the pig ZP is lesspermeable to immunoglobulins than the mouse ZPsince removal of the ZP from mouse embryos is notnecessary to achieve complete immunosurgery(unpublished observations).

The morphology and developmental stages of therecovered embryos fits well with other studies of pigembryos (see Davis, 1985 for review). Davis & Day(1978) found compacted morulae on day 5 or 6 with asfew as eight nuclei. Perry & Rowlands (1962) re-covered 89 % of ovulated eggs from the uterus(compare with our 86 %) and found that 78 % werenormal between the 6th and 9th day (compare with87 % normal between day 5 and 8 in the presentstudy).

Total cell number increases rapidly between days 5and 8, representing between four and five cell doub-lings. There was a significant difference in mean cellnumber between groups of embryos collected fromdifferent females on the same day of pregnancy.Assuming cleavage time is similar between embryos,this probably reflects inaccuracy in assessing the timeof ovulation by standard oestrus detection methods aswell as variability in the relationship between ovu-lation and the degree of behavioural oestrus. Cellnumber of embryos within females was fairly close,suggesting that fertilization of all embryos in a givenfemale occurred at about the same time and thatthere is some degree of synchrony of the cleavagedivisions within the group.

There is a clear association between cell numberand morphological development. The overlap in cellnumber between different morphological categoriesrepresents the range at morphological transitionpoints. Cells are first located in an internal position asjudged by their protection from complement-mediated lysis between the 3rd and 4th cleavagedivisions (between 8 and 16 cells). In the mouse,inner cells are first seen at about this time histologi-cally (Barlow et al. 1972; Herbert & Graham, 1974)and by immunofluorescence techniques (Handyside,1981), although the time of protection of inner cellsfrom complement-mediated lysis has been variouslyreported as 32 cells or more (Handyside, 1978;McLaren & Smith, 1977).

Formation of a blastocoel was apparent in threeembryos with fewer than 16 cells but occurred mostlyafter the 4th cleavage division. A few embryos withmore than 32 cells did not appear to have a blasto-coel. However, because of the large amount of lipidpresent in pig embryos, the blastocoelic cavity inearly blastocysts is sometimes difficult to detect withphase microscopy so it is possible that these embryos

802 V. E. Papaioannou and K. M. Ebert

were early blastocysts misclassified as morulae. Onthe whole, our data support the notion that blastocystformation in the pig usually begins after the 4thcleavage (Hunter, 1974; King et al. 1982; Perry &Rowlands, 1962), one division earlier than the mouse(Smith & McLaren, 1977). Hatching from the ZPoccurred on day 7, for the most part after the 7thcleavage division (121-240 cells).

The two cavitating embryos lacking ICM cells werean unusual finding. There was no reason to suspectthat the ICMs were 'lost' during the counting pro-cedure, leading to the conclusion that these embryoswere vesicles of pure trophectoderm. Althoughnaturally occurring trophectoderm vesicles have notbeen reported in the mouse, a number of experimen-tal procedures that decrease total cell number willresult in the formation of trophectoderm vesicles(e.g. Ansell & Snow, 1975; Snow, 1976; Tarkowski,1971) presumably due to a decrease in the number ofinternally located cells at the time of compaction. Noretardation of cell division was indicated for these pigvesicles since their total cell number was within thenormal range and higher than several morulae (con-taining inner cells) from the same female. However,it is possible that the vesicles resulted from a raresituation at the 8- to 16-cell stage in which no cellswere internalized.

Both ICM and trophectoderm averaged just overthree doublings between the early blastocyst andhatched blastocyst stage. However, if the proportionof ICM cells is examined in different morphologicalcategories, it is clear that this proportion dropssignificantly in the most advanced embryos from ahigh point in the early blastocyst, suggesting that thegrowth of the ICM component slows down comparedto the trophectoderm. In the mouse, the blastocystimplants soon after its formation with some cells ofthe trophectoderm ceasing division to form gianttrophoblast cells. In contrast, the pig blastocystcontinues to increase in size, rapidly elongatingbefore implantation. This size increase and change inmorphology occurs between 10 and 12 days of ges-tation and is accompanied by active mitosis in thetrophectoderm and overall increase in DNA content(Geisert et al. 1982). Thus, it is not surprising that thekinetics of cell number increase and distribution ofcells within the blastocyst differs in these two species.In the mouse blastocyst, the rate of increase oftrophectoderm cell number is much greater than theICM in spite of similar mitotic indices and there isevidence for cells of the ICM contributing to thetrophectoderm (Copp, 1978) as well as considerableICM cell death (Handyside & Hunter, 1986). In thepig, we found no evidence of cell death in the ICM. Inthe absence of data relating to mitotic index, ourfigures indicate that the trophectoderm continues

growing at a rapid pace while the ICM slows downbetween the expanded and hatched blastocyst stages,but we are unable to determine whether this rep-resents a slowing in cell division rate or a contributionof ICM cells to the trophectoderm.

It is well known that in vitro culture retardsdevelopment of mouse embryos (Smith & McLaren,1977; Papaioannou & Ebert, 19866) and that ad-equate culture conditions have not yet been deter-mined for the preimplantation pig embryo (Davis,1985; Davis & Day, 1978). In this study, morphologi-cal development of day-5 and day-6 embryos culturedeither in vitro or in immature mice for 24-48 h wassimilar to other reports of in vitro culture (Davis &Day, 1978) and comparable to in vivo embryos.However, cell counts indicated that cultured embryoswere retarded compared to in vivo embryos of thesame chronological age. The few embryos cultured at39°C, the normal body temperature of pigs, did notshow any dramatic difference from embryos culturedat 37°C. Embryos that were transferred to immaturemouse oviducts fared better in terms of cell numberincrease than those cultured in Ham's F12 for com-parable periods of time suggesting that the immaturemouse oviduct may be a better surrogate environ-ment than the in vitro culture conditions tested. Asimilar situation has been reported for mouse em-bryos (Papaioannou & Ebert, 19866).

The lower cell number we observed both in imma-ture mice and in vitro culture is likely to result from acombination of retardation and cell death since nu-clear fragments were seen in several embryos fromeach culture condition. It has yet to be determined towhat extent this lower cell number has compromisedthe potential of these cultured embryos for furtherdevelopment. The observation that the proportion ofICM cells is appropriate for the total cell number andmorphological stage indicates that at least this par-ameter of blastocyst development has not been dis-turbed.

We would like to thank Ann Schmidt, Tom Smith, SusanCamper and Doug Harper for expert technical assistance.This material is based upon work supported by the USDepartment of Agriculture Competitive Research GrantsOffice under agreement No. 85-CRCR-1-1890 and NIHgTant SSS-2(A) 5 RO1 HD21616. We also thank CindyWelch for help with preparation of the manuscript.

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(Accepted 13 January 1988)