(Received 19th September 1969)

TRANSFER

TO

THE MOUSE

OVIDUCT

OF EGGS

WITH AND WITHOUT THE

ZONA PELLUCIDA

R. A. BRONSON and ANNE McLAREN

Institute

_of

Animal

_Genetics,

_{Edinburgh University}

(Received

19th

_September

₁₉₆₉₎

Summary.

When

_{pre-implantation embryos}

were transferred to the

oviducts of

_{pseudopregnant recipient}

_females,

on the 1st

day

post

coitum,

the

_percentage

of females

_becoming

_pregnant

was

_greater

when a_very small inoculum volumewasused. The number of_eggs

injected

_per

ovi-duct,

in the _rangefrom two to ten, did not

_{significantly}

affect the

per-centage

developing

into foetuses. The pregnancyratewaslower forone\x=req-\ cell than for two-cell and

_eight-cell

_{eggs, morulae and}

_blastocysts,

but the

_percentage

_{of eggs}

_developing

into live foetuses in those females which became

_pregnant

was similar for all

stages.

Blastocysts

transferredtothe oviducts of

recipients

onthe first

day

p.c.

passed

down the oviduct ata rate similarto that of the

_recipients'

own unfertilized eggs.

Removal of the zona

pellucida

with pronase

prevented

almost

entirely

the

_development

of

_eight-cell

_eggs to foetuses after transfer to oviducts of

_recipients

on the 1st

day

p.c.,

although development

in vitro was not affected. Removal of thezona did _notreduce the

proportion

of

blastocysts

developing successfully,

after transfer to either oviduct or uterus. Itwasconcluded that thezona

pellucida

is necessary for the tran-sit of

_cleavage

_stages

_through

_{the oviduct in normal pregnancy.}

The rate of

_{transmigration}

between uterine horns

_following

transfer to the oviduct was _verylow

(0\m=.\4%).

INTRODUCTION

In

_1959,

_{Tarkowski demonstrated that two-cell eggs could}

_{develop successfully}

after transfer to the oviducts of

_{pseudopregnant recipient}

mice. Of the trans¬ ferred

_embryos,

_70%

survivedto

birth,

orto

_autopsy

between the 15th and 19th

day

of pregnancy. Thissuccessratewas_greaterthan that of_anyother method of

egg transfer in mice. Whether the method would prove

satisfactory

for the

transfer of_eggs atother_stages of

development

was not

fully

answered. Alower

proportion

of

_blastocysts

_{than of two-cell eggs}

_developed

into live foetuses after transfer to the

oviduct,

and Tarkowski therefore

_speculated

that the shock

*_{Present address:}

DepartmentofSurgery,New YorkUniversityMedicalCenter,550 FirstAvenue,

New_York,N.Y. 10016.

t AgriculturalResearch Council Unit of Animal Genetics. 129

causedto the

blastocysts

by

the actof transfer

might

weaken their

capacity

for

implantation

in the uterus. A similar

_finding

was

reported by Noyes

& Dick¬ mann

(1961)

forthe rat.

The _present

_experiments

were

designed

to

explore

the effect of the

_stage

of

eggs

transferred,

andto determine whetheran intactzona

pellucida

is

required

fornormal

_development

in vivo.

MATERIALS AND METHODS

The mice used were from the

randomly

bred

0_

strain. One-cell eggs were recovered in

_phosphate

buffered saline

_(PBS)

from donors on the 1st

day

post

coitum

_{(p.c.), by incising}

the

_ampulla

of the

_oviduct,

and then treated with

hyaluronidase

(Sigma, Type

1,

350 NF

_units/mg)

to remove the cumulus

oophorus.

The

_{hyaluronidase}

solutionwas made up ata concentration of

0-5%

in PBS

_containing

10

_mg/ml

of

_{polyvinyl pyrrolidone,}

added to reduce the stickiness of the eggs. The time

required

to

_disperse

the

cumulus,

at room

temperature, was 3 to 5 _{min. The treated eggs} were then washed in three

changes

of 1 ml PBS.

Two-cell _eggswere obtainedon the

morning

of the2nd

day

p.c.

by

flushing

the excised oviduct with PBS.

_Eight-cell

_eggswere

expressed by

blunt dissection from the uterine end of the oviductonthe 3rd

day

_p.c.,

and morulae and blasto¬

cysts were flushed from the uterine horns on the 4th

day

_p.c.

(vaginal plug

found on 1st

day p.c.).

The zona

pellucida

was removed from the eggs with _pronase

(Calbiochem,

grade,

45,000

PUK

_units/g),

at a concentration of

0-5%

(Mintz, 1962).

This solution was made _{up in} PBS

containing

10

mg/ml

of

polyvinyl pyrrolidone,

incubated at 37° C for 2

_hr,

and

_{dialysed overnight}

in the cold

_{(4° C) against}

PBS

_{(Mintz, 1967). Eggs}

were treated with _pronasesolution atroom _tempera¬ ture and observed under the

_{dissecting microscope. They}

were removed from the pronase either when the zonahad

just

vanished

(8

to 12

_min)

(Treatment

1)

or when a faint

ring

ofzona still remained

(7

to 11

min) (Treatment 2).

This

remnant ofzonahad dissolved

by

the time of transfer. After removal from the

pronase, the eggs were carried

through

two

_washings

with 1 ml of PBS

_each,

and

_placed

in transfer medium

_(10%

calfserum in

PBS,

Calf Serum No.

1,

natural

_clot,

_heated,

Wellcome

_Labs.).

Transfers were carried out

according

to a modification of the method of Tarkowski

_{(1959, 1970).}

On the 1st

_day

_p.c.,

_recipient

mice mated to vasecto¬ mized males were anaesthetized with

intraperitoneal

Nembutal

(0-0125 ml/g

body weight

of6

_mg/ml

_solution).

A_{transverse, dorsal skin incision}wasmadeto the left of the midline. The left_ovarycould

_usually

beseen

through

the

posterior

abdominal

_wall,

whichwas thenincised. The ovarian fat

pad

was

grasped

with smooth

_forceps

and

_{pulled through}

the

_wound,

_carrying

the_ovary,oviductand

uterine horn with it. The distal end of the _mesentery of the uterus was also incised to aid in ease of retraction. A small arterial

bull-dog clamp, grasping

the ovarian fat

_pad

and

_lying

across the dorsum of the mouse,

provided

the

correct amount of traction. The remainder of the

_operation

was carried out under the

_{dissecting microscope,}

at 28

_{magnification.}

A_strong

_point-source

of

Transfer of

eggs

illuminationwas used.

By

carefulobservation of thetubo-ovarian

junction,

the infundibulum of the oviduct could be located within the ovarian bursa as the

ovarywasrotated.Atransverseincisionwas thenmadeinthe bursa with

iridec-tomy scissors. The

edges

of the bursawere retracted with fine

pointed forceps,

and then the

_{funnel-shaped opening}

of the infundibulum could

_usually

be observed

_directly.

A fine

_tungsten

wire needlewas

helpful

attimes to locate the ostium oftheinfundibulum.

Before the

_operation,

the _eggs to be transferred were taken up into a

finely

drawn Pasteur

_{pipette by capillary}

action. The

_tip

of the

_pipette

was

placed

into the infundibulum and

_passed

into the oviduct asfaras

possible, usually

to the first bend. The fluid

_containing

the_eggswasthen

injected

withahandbulb. An

_approximate

estimate

_{by weight}

ofthe minimum volume of fluid used for transfer was 0-1 to 0-4

µ .

In contrast to transfers carried out to the uterus

(McLaren

&

_Michie,

_1956),

the introduction ofasmall amount of air into the

oviductwas notdetrimentaltothesuccessof the transfer

operation.

In

fact,

the air served as a useful marker that the entireinoculum had been

injected.

In all cases, unless otherwise

noted,

the transfers were carried out into the leftoviduct. The

_right

side servedas acontrol that the vasectomized maleswere indeed

_consistently

sterile.

_Autopsies

were

performed

after the 10th

day

p.c.

Pregnant

mice are defined as those

recipients possessing

atleast onelive foetus at the timeof

_autopsy.

A small number of decidua

(i.e. moles)

were

observed,

but these have notbeen includedin any ofthe

_analyses

since

they

donotneces¬

sarily

indicate the _presence ofan

embryo. Only

four

recipient

uteriwerefound at

_autopsy

to contain decidua in the absence of live foetuses.

Blastocysts

weretransferred totheuterusof

recipients

on the 3rd

day

accord¬

ing

to the method of McLaren & Michie

_(1956).

_Eggs

were cultivated in vitro

according

to the method ofBrinster

_(1963).

RESULTS

The success of the transfer

operations depended critically

_upon the volume

injected

into the oviduct

_{(Table 1).}

A six-fold reduction in the volume ofthe

inoculum increased the_pregnancyratefrom

25%

to

63%

( 2( )

=

5-7,

<0·02).

The increase in the number of live foetuses in those

_recipients

which became

pregnant was not

statistically significant.

In

subsequent experiments,

the smaller volume was used.

The

_stage

of

_development

of_{the transferred eggs}

_{(Table 2)}

also had a

sig¬

nificant _{effect upon} the _pregnancy rate

(heterogeneity

2(3)

=

8·3,

<0·05).

This is accounted for

_by

the lower

_proportion

of

_pregnancies

_{among females}

receiving

one-cell _eggs

_{( 2( )}

=

7-61, P<0-01,

for the

comparison

between

one-cell_eggs and the

_rest).

In those females which became

_pregnant,

_however,

thesurvival of the transferred _eggswasunrelated totheir_stage of

_development.

Because _{of the lower pregnancy} rate after transfer of one-cell eggs, it was

necessary to examine the effectofthe number of eggs

injected

for one-cell and later_stages

_separately.

When small numbers _{of eggs} are

injected,

somefemales

willfailto become_pregnant not

_{through reproductive}

failure orfactors

causing

loss of the inoculumas a

whole,

but becausenoneofthe_eggssurvives the various

hazards to which _eggs are

subject

as individuals. Somewhat lower _pregnancy rates

will, therefore,

be

_expected

afterthe

_injection

ofsmallernumbers_{of eggs,} but thesuccessrateinthose femaleswhichbecome_pregnantwill be

correspond¬

ingly higher.

However,

with survival rates of the order found in the

_present

study

this effect will be small.Infact

_{(Table 3),}

the numberof_eggs

_injected

had no

significant

effect either on the _pregnancy rate or on the

_percentage

of eggs transferred to

_pregnant

females and

subsequently

recovered as live foetuses.

Table 1

the effect of volume_injected on success rateof

transfers of one- or two-cell eggs to the

oviducts of recipients on the 1st day postcoitum

Sizeof inoculum* Large Small No._of recipients 12 66 pregnant 25 63 Pregnant recipients No._ofeggs transferred 12 139 °/„Uve foetuses 58 71

*_{The relative size of the inoculum in thetwo}

groupswas

determined_{by weight} tobe6 : 1.

Table 2

theeffect of stage of development of eggs trans¬ ferredto the oviducts of recipients on the 1stday

p.c. Developmental stage No._of recipients pregnant Pregnantrecipients No._ofeggs transferred % Uve foetuses One-cell Two-cell Eight-cell Morulae and blastocysts 60 19 10 15 53-3 73-7 90-0 73-3 110 68 36 63 75-5 73-5 69-4 66-7

The _pregnancy rate with one-cell _eggs was

consistently

lower however _many

eggs were

injected.

To determine the effectof

_removing

the zona

pellucida,

eight-cell

_eggswere treated with_pronasebefore transfertotheoviduct. As describedinthe Methods

section,

two _exposure times for _pronase treatment were used.

Very

few _eggs

developed successfully

ontransfer to theoviduct after either treatment

_(Table

4).

The

_viability

of the_{eggs removed from the pronase before}

_lysis

was

complete

(Treatment

2)

was

independently

checked

by

invitro

culture,

andwas foundnot

to differ from that of control eggs, with thezona intact

(Table 4).

When

_blastocysts

weretreated with pronase _to removethe _zona, a different

picture

was seen

(Table

5).

If the zona was allowed to

lyse

completely

in the

pronase solution

(Treatment 1),

no

development

occurred;

but after the less

Transfer of

eggs

was not

significantly

below that of

_blastocysts

with the zonaintact.

Evidently

the

_degree

_{of exposure} to _pronase was critical.

To confirm that normal

_development

after removal of the zona and subse¬

quent

transfertotheoviductwas

possible

atthe

blastocyst

but not atthe

eight-cell

_stage,

afurther small

experiment

was carriedout

(Table 6).

The difference was in the

expected

direction and

statistically highly significant.

Table 3

the effect of number of eggs transferred to the oviducts of recipients on the 1st day post coitum

No. ofeggs transferred 2 3 4 5 6 7 10 One-cell_eggs No._of recipients 16 19 18 6 0 0 1 pregnant 56-3 47-4 55-6 50-0 100-0 ratein pregnant females 83-3 70-4 72-5 80-0 80-0 Other_stages No._of recipients 4 7 14 5 6 6 2 pregnant 75-0 71-4 64-3 100-0 83-3 83-3 100-0 y0success ratein pregnant females 66-7 73-3 61-1 72-0 80-0 62-9 80-0 Table 4

the effect of removing the zona with pronase on the suc¬ CESSFUL DEVELOPMENT OF EIGHT-CELL EGGSin VWO ANDin Vitro

Category Pronasetreatment1, transferredtooviducts Pronasetreatment_2, transferredtooviducts* Pronasetreatment_2, cultured in vitro*

Eggs placed directlyin culture withzonaintact*

No.of recipients 23 6 No.of eggs 92 21 27 23

°/o ofeggsdevelopingtolive

foetuses(in vivo) orblasto¬

cysts(in vitro)

3 0 84 88

*

Eight-cell eggs from each donor mouse were divided into three groups.

Onegroupwas_{treated withpronase}toremovethezonaand then transferredto

recipients on the 1st _dayp.c.; the other two _groups were cultured, with or

without thezona.

Some

_histological

observationswere

made,

after fixation of oviducts in formol

saline,

sectioning

at8µand

staining

with

haematoxylin

and eosin. Five oviducts were examined within an hour of transfer oftwo-cell eggs

(pseudopregnant

recipients,

\

day p.c.).

Transferred _eggs were observed in

all,

near or even adherenttothe

clump

ofcumuluscells

surrounding

the native one-celleggs, or

occasionally

nearer the infundibular end ofthe

ampulla.

No transferred eggs werefound

beyond

the cumulus. At

1^

days

p.c.,

sixoviducts of

pregnant

females showed two-cell _{eggs in} the _upper

_part

of the

_isthmus,

_{grouped fairly}

close

together.

At the same _stage of

pseudopregnancy,

after the introduction of

blastocysts

with or without the zona

pellucida

24 hr

previously,

one-cell unfertilized_eggswereobservedin

approximately

thesame

region

ofthe

isthmus,

with

_{blastocysts nearby. Blastocysts}

were seenin five out of six and five out of seven

oviducts,

respectively,

after transfer of control and zona-free eggs; in the

Table 5

the effect of removing the zona with pronase on the successful development of blastocysts aftertransfer to the oviduct or uterus

Category Pronasetreatment1, transferredtooviducts Pronasetreatment_2, transferredtooviducts* Pronasetreatment_2, transferredtoone horn ofuterus*

Blastocystswithzona_intact,

transferredto_oppositehornof uterus*

No.of

recipients pregnantNo.

0 3 3 _<! Totalno.of blastocysts transferred 25 17 24 19 No.of foetuses

*_{Blastocysts from each donormouse were divided into three}

groups. One group was

treated with pronase to remove the zona pellucida and transferred to the oviducts of recipientson_the1stday_p.c.One_groupwastreatedwith pronasetoremovethezonaand

transferredtothe leftuterinehorn of_recipientsonthe 3rd_dayp.c. The_{remaining group}

wasnottreatedwith pronase,but transferred_directlytotherightuterine horn ofthesame

recipients (3rd day p.e.). One recipient received zona-free _blastocysts into both uterine

horns,inerror.

Table 6

theeffect of stageof development of zona-free eggs on success rate of transfers to the oviduct

Stageof developmentof zona-freeeggs No.ofeggs transferred (total) No. of live foetuses Eight-cell Blastocyst 29 28 1 14

successful

transfers,

the_recoveryrateswere twelveoutof

twenty-four

andfour¬ teen out of

twenty-six

_{blastocysts, respectively.}

Of the twelve

blastocysts

ob¬ served in the control group, one was still surrounded

by

a zona and one was

half-way

out; the

remaining

tenhad shed thezona.Five

empty

zonae werealso

seen,

although

the stain usedwasnot

_optimal

foridentificationof thezona.

DISCUSSION

In the

_present

_work,

the _pregnancy rate _among females

receiving

transfers of one-cell eggs to the oviductwas

significantly

lower than when two-cell orlater

Transfer of

eggs

stagesweretransferred. The

_non-pregnant

femaleswere

evenly

distributed with

respect to clutches of one-cell _eggs from individual donors

_{(heterogeneity}

Z2(8)

=

5-7);

hence the lower

pregnancy rate is more

likely

to be due to a

higher

rateof transfer failure thantofailure of

fertilization,

or

greater

sensitivity

to adverse

conditions,

on the

_part

of whole clutches. In those females which became

_pregnant,

the

_percentage

_{of eggs}

_developing

into live foetuses was as

high

forone-cell asfor later _stages.

Apart

from the lower _pregnancy rate with one-cell _eggs, the

_percentage

of

eggs

developing

into live foetuses was similar for all

_stages

tested,

even for

blastocysts

removed from theuterusnearthetime of

implantation.

This

finding

is incontrast to that of Tarkowski

(1959).

Examination of the excised oviducts 24 hr

_following

transferrevealedthatthe

_mobility

ofthetransferred

_blastocysts

did notdiffer from that of the

one-cell,

unfertilized eggs of the

pseudopregnant

host. The

_{blastocysts appeared morphologically}

normal. The

_shedding

ofthe

zona

pellucida by blastocysts

retained in the

oviduct,

with

persistence

of the

empty zonae, has been

reported previously by

Orsini & McLaren

(1967).

Of 263 live foetuses found at _autopsy, all but one were in the uterine horn

corresponding

tothe

injected

oviduct. This low rate of

transmigration

between

horns is consistent with the

_finding

ofRunner

_(1951),

that one out of

_eighty

embryos

examined had

_migrated

to the

_opposite

horn

_following

transfer of one-cell _eggs to the ovarian

_capsule.

It

_suggests

that the

_{significantly higher}

rate

(four

outof

_forty-six)

reported

_by

McLaren & Michie

_{(1954) following}

_egg transfertotheuterus was, as

they suspected,

associated with the pressure of the fluid medium in _{which the eggs} were

injected,

and is not

typical

of normal

pregnancy.

Thecorrelation betweenthevolumeof inoculum andthesuccessof_eggtrans¬

fers to the oviduct _may be due to the back-flow of_eggs into the

periovarian

space whenthe inoculum is

large,

orit_maybe related tothe distance

_along

the oviduct that the_eggswereflushedatthe time of transfer. The

larger

the volume

injected,

the further

_along

the oviduct would one

expect

the eggs to be im¬

mediately

after

_transfer,

_{and the earlier the eggs}

_might

reach the uterus. In

rabbits,

themuscular

_activity

oftheuterus

_just

after ovulation

_expels

mostof the

eggstransferred toiton the 1st

day

of

pseudopregnancy.

Evenon the2nd and 3rd

_day

of

_{pseudopregnancy,}

the uterine environment is toxic tothe eggs, and

only

a small

proportion

of eggs

_develop

into young iftransferredto the uterus

(Chang, 1950).

Within _{the range} tested

_(two

to ten _{eggs per}

oviduct),

the

_percentage

of transferred eggs

_developing

tofoetuseswas

independent

of the number

injected.

Most of the

_autopsies

were carried out on the 10th

day

p.c.,

before foetal

mortality

due to

crowding

would be

expected

to occur

(McLaren

&

Michie,

1959).

However,

with the

_larger

numbers of_eggs

_transferred,

all the

_implanted

foetuses would be

_unlikely

to survive to term.

The _exposure time of _eggs to _pronase

proved

critical. When the zona was

completely

dissolved

_by

the_pronase

_solution,

_exposing

_{the eggs} tothe fullcon¬ centration of the _enzyme, none of the zona-free

blastocysts developed

into live foetuses after transfertooviducts of

_recipients

onthe 1st

day

p.c.

A_very

slightly

decreased _exposure time

_{permitted development}

of most of the

blastocysts

(Tables

5 and

_6).

The deleterious effects of_pronase on mouse _eggs have been noted

_previously.

McLaren

_{(1969), using}

untreated _pronase, found that there was a

large

and

highly significant

reduction inthe totalnumber of

implantation

sites,

when

_blastocysts

were transferred to the uteri of

_{recipients following}

removal of the zona with _pronase. Mintz

(1967) suggested

incubation and

dialysis

of pronase solution to diminish its

_toxicity,

a recommendation which wasfollowed in the

present

experiments.

Confirmatory

evidence that

_blastocysts

do not

_require

a zona

pellucida

for successful transit

_through

the oviduct is

_{provided by}

the

_histological

observa¬ tions. Most of the untreated

_blastocysts

_{spontaneously}

lost the zona

pellucida

within a

day

after transfer to the

_oviduct,

when

_they

were

midway

from the infundibulum to the uterus, _yet

they

were no less

capable

of

completing

the transit

_through

the oviduct and

_developing

into foetuses thanwereearlier_stages which retained the zona

throughout

their

sojourn

in the oviduct. Tarkowski

(1961)

also observed that zona-free

_{blastocysts (single}

or

fused)

were able to traverse the oviduct and

_{implant successfully}

in theuterus.

In

_striking

contrast is the fate of zona-free

_eight-cell

_{eggs. Even when the} reduced time of_exposureto_pronasewas

used,

such_eggstransferredtooviducts of

_recipients

on the 1st

day

p.c.

_veryseldom

developed

into foetuses. This could notbe accounted foron the basis of_pronase

toxicity,

because

80%

of

eight-cell

eggs from the same

donor,

treated with _pronase for the same

length

of

time,

reached the

_blastocyst

_stagewhen

_placed

into culture. Such

_blastocysts,

cultured zona-free from the

_eight-cell

_stage,

_may be transferred to the uteri of

_pseudo¬

pregnant

females,

with

_subsequent

normal

_{development (Tarkowski,}

_1961;

Mintz,

1965).

The same situation

probably prevails

in the rabbit

since,

in this

_species,

_Moore,

Adams & Rowson

₍₁₉₆₈₎

were unable to recover zona-free blastomeres from the two- and four-cell _stage after transfer to the oviduct.

Thus,

the zona

pellucida

_appears to be _necessary for the successful transit of

_{cleavage-stage}

_eggs

_through

the

_oviduct,

while not _{necessary for the transit}

of

_blastocysts.

Electron

_micrographs

ofrat _eggs reveal intercellular connections between the

_trophoblast

cells of

_{blastocysts (Schlaf}

ke &

_Enders,

_1967).

These

junctional complexes,

absent in

_eight-cell

_{eggs, increase the}

_stability

of the bond betweenthecells and_may

_prevent

their

_{disaggregation}

ontransit

through

the oviduct.

_Eggs

in culture remain

_{relatively still,}

_diminishing

the chances of

disaggregation

of blastomeresoncethezonais removed. On the other

hand,

_eggs within the oviduct are

transported

by

peristalsis

to the uterus. The zona

pel¬

lucida and the fluid in the

_{perivitelline}

_{space may} therefore act as a shock

absorber,

preventing

the

_separation

of blastomeres from the

_cleaving

_egg.

NOTEADDED IN PROOF

The paper of Modfinski

(1970)

came to our attention after the

present

paper had been

prepared.

Modlinski's

findings

are similar to our own,

with the

_exception

that

_blastocysts

transferred to the oviduct showed a

somewhat lower

_percentage

ofsuccessful

_development

than did earlier

_stages.

Transfer

of

eggs

eggs adhered to the

_epithelium

of the oviduct within a few hours of

transfer,

and did not

_undergo

_{any further}

cleavage.

Modlinski,J. (1970) Therole of thezonapellucidain thedevelopmentofmouse_eggsin vivo.J.

Em-bryol.exp. Morph. (in press).

ACKNOWLEDGMENT

Weare

grateful

forfinancial_supportfrom the FordFoundation

(A.M.)

and the National Institutes of Health

_(R.A.B.;

Grant No. 5T01 GMO

_1777).

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