(Received 1st July 1966)

STUDY OF

STAGES OF

IMPLANTATION IN MICE

C. A. FINN and ANNE McLAREN

Department of

Biological

Sciences,

Wye

College

( University of London), Ashford,

Kent,

andA.R.C. Unit

_of

Animal

_Genetics,

Institute

_of

Animal

_Genetics,

West Mains

_Road,

_Edinburgh

(Received

1st

_{July 1966)}

Summary.

In order to

study

the

changes

_{taking place}

in the

_blastocyst

and

_locally

in the uterus at thesite of the

_blastocyst

attachment,

preg-nantmicewere killedatintervals between 3

days

21hrand 4

days

16 hr

post

coitum. The uteri were tested for the presence of Pontamine Blue areas

(indicating

increased

permeability

of

capillaries)

and

histochemic-ally

foralkaline

_phosphatase

inthe stroma, andalso examined

histologic-ally. By

classifying

each female for the various features

_studied,

the

follow-ing

order of appearancewas established: Pontamine Blue

reactivity,

`W\x=req-\

bodies'

_emerging

from

_blastocyst,

local oedema of the uterine stroma, alkaline

_phosphatase

in_{the uterine stroma,}

_histological

decidualization. Giant cell transformation ofthe

_trophoblast

occurredatabout the same

time as the _emergence of W-bodies from the

blastocyst.

Blastocyst

elongation began

after the_{appearance of Pontamine}Blue

_reactivity

and

before

_histological

_{decidualization,}

but was not

_sequentially

related to

the other features studied.

INTRODUCTION

Implantation

ofova in rodents involvesa

complex

series of

morphological

and

biochemical interactions between the

_blastocyst

_{and the uterus,}

_culminating

in the attachment of the

_embryo

to the mother

by

means of the

placenta.

Recently

three

_separate

local reactions have been demonstrated to occur soon

after the

_blastocyst

makes contact with the endometrium.

Psychoyos

(1961)

showed,

originally

in_{rats, that}the blood vesselsnear the site ofan

implanting

blastocyst

become more

permeable,

so that afterthe

injection

ofa

large

mole¬

cular

_{weight dye,}

such as Pontamine

Sky

Blue,

a coloured band _appears across theuterus where a

blastocyst

is

present.

Thisreactionoccurs before _any

other

_macroscopic

evidenceof

_implantation

canbe seenintheuterus.

_Secondly

Wilson

₍₁₉₆₃₎

_{demonstrated the passage of}

_cells,

which he called

_'primary

invasive

_cells',

from the

_blastocyst

into the uterine

_epithelium,

and

_thirdly

Finn & Hinchliffe

₍₁₉₆₄₎

_{showed that the enzyme alkaline}

_phosphatase

appeared

in the endometrial stroma around an

implanting

blastocyst.

Histo-*_{Present address: Department ofPhysiology, Royal Veterinary College, Royal College Street,}

London,N.W.I.

260

logically,

the uterine stroma around an

implanting

blastocyst

shows marked oedema at an

early

_stage, with the formation ofthe

'primary

decidual

zone',

consisting

of

_{closely packed}

cells around the uterine

_lumen,

_following

later

(Krehbiel, 1937).

The

_object

of the

_present

_experiments

was to

study

these reactions in the same animals in order to obtain information about the order in which

they

appeared.

This isa_necessary

preliminary

to _anycausal

analysis

of the_processes

involved. In

_particular,

the

_question

of howtheuterusis stimulatedto

respond

locally

to the_{presence of}a

blastocyst

isstill_{open. Blandau}

(1949) reported

that

placing glass

and

_paraffin

beadsintheuteruswould induce decidual

_changes

in rats and

_guinea-pigs

; thishasoften been

interpreted

as

demonstrating

that itis the pressureof the

_blastocyst

_against

the uterine

_epithelium

whichis theeffective stimulus in normal pregnancy.

However,

there are almost

certainly

other

factors

_involved,

since unfertilizedmouseova

(Orsini

&

McLaren,

unpublished),

deadrat

blastocysts

(Segal

&

Nelson,

1958)

andfixed sea-urchinova

(Alden

&

Smith,

1959)

allfailtoinduceadecidual response.

The

_{basically staining}

RNA-rich bodies contained in Wilson's

_'primary

invasive cells' are

easily

seen under the

microscope.

The cells

themselves,

however,

are not so _{easy to} see. We shall therefore refer

throughout

not to

'primary

invasive

_cells',

butto

'W-bodies',

atermwhich makesno

assumptions

about theirfunction.

MATERIAL AND METHODS

Females

_belonged

to the

_randomly

bred

_{0_strain,}

and were

nulliparous.

They

were

paired

with males of their own strain in the

afternoon,

examined for

copulation plugs

the

_following

_morning,

and killed between 3

_days

21 hr and 4

_days

16 hr

_post

coitum

_(assuming

coitustook

_place

at01.00hours

_during

the

_night

of

_mating).

The

_experiment

was carried out in two

_parts,

the first in

August

andthesecond in

_November;

thetwobatchesof micedidnotdiffer

significantly

fromone

another,

andhavetherefore been combined.

Fifteen minutes before each animal was

killed,

0-25 ml ofa 1

%

solution of

Pontamine

_Sky

Blue 5BXwas

injected

intravenously.

The

position, intensity

and

number of blue areas in the two uterine horns was recorded after death. One

uterine horn ofeach animal was fixed in cold 80

%

alcohol; after

remaining

overnight

in a

refrigerator

these horns were

posted

in ice from

Edinburgh

to

Wye,

where

_they

were sectioned and examined for the presence of alkaline

phosphatase

by

the Gomori

_technique.

The other horn of each female was

fixed in either AFA

₍₃₀

ml

_{95% alcohol,}

10 ml commercial

_formalin,

10 ml

glacial

acetic

_acid,

50 ml

_water)

orin

Carnoy's

fluid;

these hornswere cleared

according

to the method described

by

Orsini

(1962a,

b)

sothat the Pontamine

Sky

Blueareascould be examinedmore

closely

;

they

werethen

serially

sectioned,

stained with Iron

_Haematoxylin

orwith

Methyl

GreenandToluidine

Blue,

and

examined

_{histologically.}

All

_blastocysts

inthese sectionswere

photographed,

so that

_they

could be more

readily

compared.

Left and

right

horns were allotted

alternately

to the two treatments.

Pontamine Blue reactions wererecorded as :

—

(no

Early

of implantation

areas, too faint to count

accurately),

+

(faint

blue

_areas),

+ 4-

(strong

blue

areas, + 4- +

(strong

blue areas, with central

opacity

visible after

clearing).

Alkaline

_phosphatase

reactions of the uterine stroma were recorded as:

—

(negative),

+

_(faintly

positive),

+ 4-

_{(strongly positive) (PI.}

_1,

_Figs.

1 to

4).

Localized oedema of the stroma was recorded as: —

(absent)

or +

(present)

(PI.

2,

Figs.

2 and

_4).

The decidual _response of the uterine endometriumwas classifiedas :

—

(absent)

or +

_{(present) (PI.}

_2,

Fig.

_{6). Blastocysts}

wereclassified as

round,

oval or

elongate (PI.

2,

Figs.

1,

3 and

5).

Where the

blastocyst

itself was

distorted,

its

original shape

could often be deduced from the form of the uterine chamber.

_Emerging

W-bodies

_(PL

_3,

_Figs.

1 to

₇₎

were recorded as:

—

(absent),

+?

_(sparse

and

_dubious),

+

_(sparse

and

_definite),

+ +

_(numerous

and

_definite).

Both authors

_{independently}

examined the sectioned uteri for

W-bodies;

their classifications werein

good

_agreement.

RESULTS

Of the

_twenty-nine

_females,

three were not

_pregnant

(one

of these had un¬ fertilized eggs in the

_uterus).

Data on the

remaining

twenty-six

are listed in

Table

_1,

in order oftimeof

_killing.

Six showedno _responseto the testfor alka¬

line

_phosphatase,

even in theserosal

region

which is

normally

strongly

positive.

All alkaline

_phosphatase

must therefore have been

inactivated,

_probably

as a result of

overheating

in transit. Three other uteri were lost in transit before

they

couldbe examined

_{histologically}

_{for the presence of W-bodies}anddecidual

response. The uterine horns tested forthe presenceof alkaline

phosphatase

and those examined

_{histologically}

had shown an _{average of}4-2 and 4-5 Pontamine

Blue areas

respectively.

Onehundred

blastocysts

were examined forW-bodies. There was some

variability

within uterine

horns,

particularly

with

_respect

to

blastocyst

_shape.

This is indicated in Table 1. Infemale

_12,

one

implantation

site showed alkaline

_phosphatase

in the _{stroma, while the other three} sites in the horn were

negative.

In female

5,

one

blastocyst

was at an earlier _stage of

development

than the other

_four,

and still showed remnants of the zona

pellucida.

Itwas not associated witha Pontamine Blue _area. In female

_11,

_one

of the two

_blastocysts

was _{very advanced in}

development,

and showed no

W-bodies;

the other was less

advanced,

and showed W-bodies.

In females 19 and

_20,

the

_blastocysts

in the uterine horn examined histo¬

logically

wereclustered nearthe middle of the horn. Inthe other

females,

_they

were

spread

along

the

length

of the horn. On three occasions

(females

8,

10 and

_19),

two

_blastocysts

were found

sharing

the same

implantation

chamber

(PI.

2,

Fig.

7).

The Pontamine Blue reaction

As may be seen from Table 1 the Pontamine Blue reaction was

already

evident at22.15

hours,

whenthefirstmouse waskilled. Ofthethirteenfemales

killed_up to

midnight,

two were

negative,

seven were

faintly

positive

and four

strongly

positive.

Of the thirteen killed after

_midnight,

one was

negative,

three

faintly

positive

andnine

_{strongly positive.}

In the three

_negative

_females,

all the

262 C. A. Finn

Table 1

OBSERVATIONS ON FEMALE MICE KILLED DURING THE 4 NIGHT AND 5 DAY AFTER A COPULATION PLUG HAD BEEN FOUND

Female Timekillingof (GMT)

Pontamine

Blue Blastocysts W-bodies_emerging

Alkaline phosphatase Oedema Decidual response 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 22.15 22.45 22.45 23.00 23.00 23.15 23.15 23.30 23.30 23.45 23.45 24.00 24.00 00.15 00.15 00.30 00.45 00.45 01.00 01.15 08.30 09.30 11.00 14.00 17.00 17.15 + + + + +? + + + 4-4-? + + + + + + +- + + + + 4-4-+ ? + + + 4-+ 4-+ + ++ + + 4-4-Oval Round,oval Round,oval Round Round,oval Round Round,oval Round Round Round,oval Round,oval Oval Oval Oval Round,oval Round,oval Oval Round Round Round Elongate Round Oval Oval Elongate Round + + + + + + ++ + + 4-4-4-? ++ 4- + + + + + + 4-4-+ 4-4-+ 4- + 4-+ + + 4-+ 4-+ 4-+ +

*_{InTables 2 and3,female 23 isomitted,}asthe absence of W-bodies isprobably secondary.

Table 2

thenumbers of uteri found withnegative,faintly positive or strongly positive reactions with pontamine blue, classified with respectto the other changes

studied

_(data

extracted from table

₁₎

W-bodies Oedema Blastocyst shape Alkaline-phosphatase Decidual response +, +4-+ Elongate Oval

Roundand oval Round 4-PontamineBlue 4-4-,+++ No.offemales classifiedfor both 21 23 26 20 23

Early

oedemaor

positive

alkaline

phosphatase

reactions. The time of_appearance of

the other

_changes,

relative to the Pontamine Blue

_reaction,

is summarized in

Table 2.

The alkaline

_phosphatase

reaction

The

_{alkaline-phosphatase}

results correlated rather well with the time of

killing.

The first

_positive

reaction was seen in the mouse killed at 23.45 hours.

By

the

_{following morning,}

all the mice

_successfully

testedwere

positive (5/5).

The

_relationship

between _{the time of appearance} of alkaline

_phosphatase

and

the Pontamine Blue reactionisshown in Table 2. Thetwoare

closely

correlated,

butthe Pontamine Blue reaction

_clearly

occurs first.

Alkaline

_{phosphatase activity}

in theuterineendometrium first

_appeared

as a

positive

area inthestromain the

_{neighbourhood}

ofthe

_blastocyst.

Inthe

_early

responses the

activity

was

present,

not in the area of stroma

_immediately

adjacent

to the

_epithelium,

but as a

crescent-shaped

area in the middle of the subendometrialstroma

_(PI.

_1,

Figs.

2 and

_3).

This hadnotbeenobservedin the earlier

_{study (Finn}

&

_{Hinchliffe, 1964)}

;

there,

however,

the first responses

were not examined until the

_morning

of the 5th

_day

_post

_coitum,

so that the

initiation of the_response, which from the

_present

data_appears to start

during

the

_previous

_night,

had beenoverlooked.

Alkaline

_phosphatase

is

_present

in the

_blastocysts

beforeit canbe detected in

the uterine stroma, andin

_{early specimens}

is found around the entire circum¬

ference, indicating

perhaps

the continued _presence of the zona

pellucida.

In thelater_stages,atabout the timeofits appearanceinthe uterinestroma, alkaline

phosphatase disappears

from the

_abembryonic

_regions

ofthe

_blastocyst,

_except

foranoccasional

cell,

butthe innercellmassremains

strongly

positive.

The

_migration

_of

W-bodies

W-bodies weremost

_clearly

seen in the

epithelium

after

staining

with

Tolui-dine

_Blue,

but

_they

were also

fairly readily

identified after

staining

with

iron-haematoxylin.

With the

_{light microscope they appeared}

as

specks

of

basically

staining

material in which it was not

_possible

to differentiate _any internal structure.

They

varied

_considerably

in

_shape

,some

being

circular whilst others were

elongated

in the _more

typical

form described

by

Wilson

(1963).

It_was usual to find more than one in each

blastocyst,

and sometimes as _many as a dozen were found. Cells

containing

W-bodies were sometimes observedin the wall ofthe

_blastocysts,

but

_only

in those

_specimens

from which other W-bodies

were

already emerging.

Wilson

(1963) reported

the _presenceof

cells,

which he believed to be identical with the

_primary

invasive cells later to be seen in the

trophoblast,

in the inner cell mass _up to 12 hr earlier than the earliest _stages

studied here.

Emerging

W-bodies were

always

accompanied by

a

positive

Pontamine Blue

reaction,

but in at least six cases

(Nos.

2, 5, 7, 10,

13 and

16)

their emergence

preceded

the

_development

of a

positive alkaline-phosphatase

reaction. The

apparent absence of W-bodies in the later

_stages

of the

_implantation

_reaction,

e.g. female

23,

is consistentwiththe

findings

of Wilson

(1963),

and is

probably

due tothe

_difficulty

of

_detecting

themoncethedecidualreaction has started.

Blastocyst

morphology

The

_changes

in _{the appearance of the}

_blastocysts

were

fairly

well correlated with the other reactions. Thus

_3/7

uteri in which

_only

round

_blastocysts

were

found showed an

alkaline-phosphatase

reaction,

and

3/8

showed

W-bodies,

whereasinuteri

_{containing only}

ovalor

elongated blastocysts,

the

corresponding

proportions

were

5/6

and

5/6,

respectively.

On the other

hand,

blastocyst

morphology

was _veryvariable with

_respect

to time; thefirstfemale killed con¬

tainedoval

_blastocysts,

whereas intwo out ofthe six animals killed on the 5th

day

the

_blastocysts

were round.

The

_{trophoblastic giant}

cell reaction

_{(Dickson, 1963)}

was

closely

correlated

with the_emergenceof the W-bodies. The timeoflossof thezona

pellucida

could not be ascertained with _any

certainty

as the zona is removed

completely

by

AFA and

_{partially by Carnoy's}

fluid. Traces ofzonae were seen around the

blastocysts

of females

_8,

19 and20. Oedemaanddecidual reactions

Like the

_{alkaline-phosphatase}

_reaction,

the _appearance of oedema in the uterine stroma showed

_good

correlation with the timeof

killing.

Thus,

of the

twenty-three

females

_studied,

_only

one killed before 23.30 hours

(Table 1)

showed

_oedema,

whereas

_only

two of those killed after that time failed to showit.

Ofthe nine femalesnot

showing

oedema,

sixwere

positive

for Pontamine

Blue,

andthree

_already

showed the _presence of W-bodies in the uterine

_epithelium.

None of the females without oedema showed a

positive

alkaline

phosphatase

reaction,

but four with oedema had not _yet

developed

alkaline

phosphatase.

On the other

_hand,

the

_massing

of stroma cells around the anti-mesometrial side of the uterine

_lumen,

which was taken as indicative ofthe

beginning

of the _{decidual response, occurred after the appearance}of alkaline

_phosphatase.

The four mice

_showing

a decidual response

(Table

1)

were all

positive

for

alkaline

_phosphatase,

but one female

positive

for alkaline

phosphatase (No.

18)

hadnot

_yet

developed

_any

histological

signs

of decidualization.

DISCUSSION

Ourresults

_present

a consistent

picture

of the

chronology

of the

early

_stages of

implantation

in the mouse. In the strain of mice

used,

the Pontamine Blue

reaction,

anindex of

locally

increased

capillary permeability,

was

positive

in the

majority

of

_pregnant

females before

_midnight

on the4th

day

_post

coitum. Inthe

present

study,

this was the earliest indication that the uterus was

reacting

locally

tothe presenceofa

blastocyst.

The timeof appearance of the Pontamine

Blue reaction is consistent with that

_{reported by}

Orsini & McLaren

_(1967).

The relative

_ordering

of the

_phenomena

studiedcanbest be

appreciated

if the

dataare

displayed

asinTable3. Allthefemalesexaminedfall intoone orother of

the six

_{'stages' (though}

one or two females have been omitted from theTable

on account of

_{incomplete classification).}

The fact that the females can be so

arranged

implies

that,

asfarasthe

present

dataare

concerned,

thefivecharac¬

of implantation

any one will also show all those earlier in the list.

(This

neednot

necessarily

meanthat

they

are also

causally

dependent.) Change

of

shape

of the

blastocyst,

on the other

hand,

is

independent

ofmost of the characters listed:

blastocyst

elongation

was not seen in

stage-1

females,

and was _present in all

stage-6

females,

but

_stages

_2,

4and 5 all include some females

containing only

round

blastocysts,

andothersin which

_elongation

had

_begun.

The appearance of alkaline

phosphatase

in the uterine stroma,

_indicating

that this has become a

region

of increased cellular

activity,

followed the

Pontamine Bluereaction

_by

notmore thana fewhours. The localization of the

early

alkaline

_phosphatase

reaction is

_puzzling:

the initial

_signal

for

_activity

Table 3

the _sequential dependence of the observed changes

Stage 1 2 3 4 5 6 Pontamine Blue + + + + + W-bodies + + + + Stromal oedema + + Alkaline phosphatase + + Decidual response Females (6),8, 19 3,4,20 (0,2,7 5, 10, 13, 16 14,18,22 11, 12,21

Numbersin_parenthesesindicate females whichwerenotclassifiedfor alkaline_phosphatase activity. Females_9, 15and _17,also unclassified for alkaline_{phosphatase activity,}have been omitted since_theycould have_belongedeitherto_stage4orto_stage5.

to

begin

must come from the

blastocyst

in the uterine

lumen,

_yet it was not

those

_regions

of the stroma

adjacent

to the lumen which showed the reaction

first.

_Possibly

the initial distribution of alkaline

_phosphatase

is related to the

pattern ofvascularization of thestroma.

The emergenceof W-bodiesfromthe

_blastocyst

andtheir

_penetration

into the uterine

_{epithelium appeared}

to take

place

before the appearanceof the alkaline

phosphatase

but after the Pontamine Blue reaction. If this is confirmed it

wouldmeanthatthe W-bodiesarenot

responsible

for

informing

theuterus that a

blastocyst

is

_present.

We therefore remain in

ignorance

both of_the function

of these remarkable

_objects,

and of the natureof theinitial

signal

from blasto¬

cystto uterus.

ACKNOWLEDGMENTS

We

_{gratefully acknowledge}

financial

_support

from the Ford Foundation

(A.McL.)

and the Medical Research Council

_(C.A.F.),

and would liketothank Mr K. Barber for histechnical assistance.

REFERENCES

Alden,R.H. &_Smith,M._{J. (1959) Implantation}of therat_{egg.IV. Some effects of artificial}ovaon

therat uterus._{J. exp. Z°°l·} 142,215.

Blandau,R._{J.(1949) Embryo-endometrialinterrelationship}in theratandguinea pig.Anat. Ree.104,

Dickson, . D. _{(1963)Trophoblastic giant}cell transformation ofmouseblastocysts.J. Reprod.Fert. 6,

465.

Finn, C. A. &Hinchliffe,J.R. ₍₁₉₆₄₎ Reaction of themouseuterus_{during implantation}and deci-duoma formationasdemonstrated _{by changes} inthe distribution of alkaline _{phosphatase. J.}

Reprod.Fert._8,331.

Krehbiel,R. H. _{(1937) Cytological}studies of the decidual reaction in therat_{duringearlypregnancy}

andin theproductionof deciduomata._{Physiol. <W.}10,212.

Orsini, M.W. _{(1962a) Technique}of_{preparation, study}and_photographyof_{benzyl-benzoate}cleared material for_{embryological}studies._{J. Reprod.}Fert.3,283.

Orsini,M.W._{(1962b) Study}of_{ova-implantation}inthe_{hamster,rat,mouse,guinea-pig}andrabbit in cleareduterinetracts._{J. Reprod.}Fert.3,278.

Orsini, M. W. & McLaren, A. (1967) Loss ofthe zonapellucida in _mice,and the effect of tubai ligation andovariectomy.J. Reprod.Fert. _(Inpress).

Psychoyos, A. (1961) Perméabilitécapillaire et decidualization utérine. C. r. _{hebd. Séanc. Acad. Sci.,}

Paris, 252, 1515.

Segal, S.J. & _Nelson, W. O. ₍₁₉₅₈₎ An _orally active compound with _{antifertility}effects inrats. Proc. Soc.exp.Biol.Med.98,431.

Wilson, I.B. ₍₁₉₆₃₎ Anewfactorassociatedwith theimplantationof themouse_egg.J. Reprod.Fert. 5,281.

EXPLANATION OF PLATES

Plate 1

Fig. 1.Transverse section ofuterusof female_{3, negative}for alkaline_{phosphataseactivity}

inthe uterinestroma. Note the presence of alkaline_{phosphataseactivity}in theserosa,

around the uterineglands,and inthe_blastocyst. 70.

Fig.2.Transversesection ofuterusof female _{14,faintlypositive}for alkalinephosphatase activityinthe uterinestroma. Notetheoedema intheouterareas ofthe mucosa, con¬

trastingwith the_{closelypacked} cells around the uterine lumen ₍ = primary decidual

zone).Thealkaline_{phosphatase activity begins} neartheouter_marginof the_expanding

primarydecidual zone, 70.

Fig. 3. _Highpowerview ofFig. 2,to show_earlydistribution of alkalinephosphatase activity, 200.

Fig.4. Transversesection ofuterusof female24, stronglypositivefor alkalinephosphatase activityintheuterinestroma. X60.

Plate 2

Fig.1. 'Round'_blastocyst,from female15._Althoughthe_blastocystis_crumpled,its_original

shape canbe deducedfrom the shapeof the anti-mesometrial chamber inwhich it is situated.Hereandinalltheother_figures,the space in the uterine lumen andbetweenthe

blastocystand the uterine epithelium is a _shrinkage artefact. In vivo, the walls of the uterine lumenarepressed tightly togetheratthis_stageof_pregnancy, 220.

Fig.2.Transversesection ofuterusof female5,toshow oedema oftheuterinestroma, 70.

Fig.3. 'Oval'blastocyst,from female3. 300.

Fig.4._Highpowerview of uterinestromaof female_13,toshow oedema, 300. Fig.5. _{'Elongate' blastocyst,}from female 21. 220.

Fig. 6. Transversesection ofuterusof female_{21, showingenlarging}areaof cellular_hyper¬

plasiaaroundtheuterine lumen ₍= primarydecidualzone). This femalewasclassified

as_showingadecidual_{response. The}decidualcellreaction isnot_yetevident. X70.

Fig. 7.Transversesection ofuterusof female19, showingtwo_blastocystsinasingle

anti-mesometrial chamber. Thecorrugatedbordertothe uterine lumen wherethe wallsare

pressed togetheris characteristic ofthis_stageof_pregnancyor_{pseudopregnancy,} and is

Early

stages

of implantation

Plate 3

Figs. 1 to_{7. Transverse sections ofblastocysts,showingW-bodies(indicated by arrows).} Figs.2 to5are_{of uteri stained withMethyl Green}and Toluidine_Blue,Figs. 1,6 and7 with iron haematoxylin. The focal _{plane has,} in each_case, been selected toshow the W-bodiesasclearlyaspossible,_withthe result thattherestof the sectionisoftensome¬

whatoutof focus.

Fig. 1. A _blastocyst from female _16, attached _{subterminally.} One and _perhaps two

W-bodiesare_faintlyseen in the uterineepithelium adjacenttothe_blastocyst;afurther

twoare seen enteringthe uterineepithelium ontheoppositeside of thelumen, _against

which the_blastocystwaspressedbefore fixationshrinkagetookplace. One ofthese was

stillattachedtothe_blastocystbyafilament,whichhasbroken;thebroken ends ofthe

filamentcan_beseen_{protrudingfrom theblastocyst}and from the_W-body. 300.

Fig. 2. The W-bodiesin_{embryonic region}of_blastocyst.Female5. 300.

Fig. 3. OneW-body crossingfrom the blastocystinto the uterine _epithelium,another possible W-body alreadyinthe _epithelium, 300.

Fig. 4.Sameblastocystasin_{Fig. 2.}TwoW-bodies_crossingfromblastocystinto uterine epithelium,oneofthemstretchedout toform afilament, 300.

Fig. 5. Adifferent _{blastocyst, also from female 5. Two W-bodiesentering} the uterine epithelium, 300.

Fig.6. Two W-bodies_{enteringthe uterineepithelium}andathirdaboutto enter.Female 22. x400.

Fig.7. Two W-bodies_{enteringthe uterineepitheliumfrom}a_blastocystatasubterminal

attachmentsite.The antimesometrial end of theuterine lumenisatthebottom left-hand