Developmental competence of goat oocytes from follicles of different

Developmental

competence

of

_goat

_oocytes

from follicles of different

size

_categories

_following

_maturation,

fertilization and culture

in vitro

N.

Crozet,

M.

Ahmed-Ali and

M. P.

Dubos

INRA, Unit\l=e'\de

Biologie

de laF\l=e'\condation, Station de

Physiologie

Animale, 78352

_{Jouy-en-Josas}

c\l=e'\dex,

France

Theaim of the

_present

_study

was to

investigate

the effect ofsizeof

follicle

from which

goat

oocytes

originate

on their

subsequent

ability

to be fertilized and to

undergo

early

embryonic

development

in vitro. Nonatretic follicles

_larger

than 2 mm in diameter were

dissected and distributed into three _groups

_according

to size

_(small:

2\p=n-\3mm; medium:

3.1\p=n-\5mm;

large:

>5

_mm).

Cumulus\p=n-\oocyte

_complexes

wereisolated from the follicles and

only

those witha

_compact

multilayered

cumuluswereselected forin vitro maturation.After

maturation,

70%,

83% and 97% of

oocytes

from

small,

medium and

_large

follicles,

respectively,

were at

metaphase

II. After in vitro

fertilization,

no

significant

difference was

observedinthe

_cleavage

rate 40h afterinseminationbetween

_oocytes

from small

_(46%)

and medium

_{(55%) follicles,}

and between

_oocytes

from

_large

follicles

_(69%)

and ovulated

oocytes (75%).

Afterin vitro

_culture,

_{significantly}

more

embryos

from small follicles arrested

beforeorat

the

8-16 cell

_stage

(84%

compared

with

53%,

45% and 39% of

embryos

from

medium and

_large

follicles and ovulated

_oocytes,

_{respectively).}

The

_proportion

of morulae and

_blastocysts

obtained was 10% and6% from small

follicles,

35%

and

12%from medium

follicles,

29% and 26% from

_large

follicles and 20% and 41% from ovulated

_oocytes.

Oocytes

from small and medium follicles

_yielded

a

significantly

lower

proportion

of hatched

blastocysts

(0%

and

_3%,

_{respectively)}

than did those from

_large

follicles and from ovulated

oocytes (15%

and 34%,

respectively).

These results indicate that

_{developmental}

_competence

of

_goat

_oocytes

is

_acquired

_{progressively}

_during

follicular

_growth

and

that

_only

a small

proportion

of

oocytes,

those isolated from

_large

antral

follicles,

have the

_capacity

to

progress to the

blastocyst

stage

following

in vitro maturation, fertilization and culture.

Introduction

Oocyte

maturation, characterized

by

germinal

vesicle break¬ down, formation of the first meiotic

_{spindle (metaphase}

I),

expulsion

ofthefirst

_{polar body}

andarrest in

_metaphase

ofthe

second meioticdivision

_(metaphase

_II), occursin

preovulatory

follicles inresponse to the surge of

_{gonadotrophins}

and leads

toanovulatedfertilizableoocyte.Mammalianoocytesreleased

from their follicular environment are able to

_undergo

_spon¬ taneous nuclear maturation in vitro (Pincus and Enzmann, 1935). However, oocytes that reach

_metaphase

II in vitro are

not

necessarily

_competent

to

_support

normal fertilization and

further

_embryonic

_development

_(Thibault

and Gérard, 1970;

Moor and Trounson, 1977).

_Although

culture conditions for

in vitro_oocytematuration

_(IVM)

in

_large

domestic

_species

have been

_improved,

the

_{developmental}

_competence

of in vitro

matured

_oocytes,

_following

in vitro fertilization (IVF), remains

limited.In

_goats,

the

_percentage

ofoocytes

reaching

metaphase

II in vitro is

_{approximately}

90% and

_high

fertilizationrateshave been achieved forin vitromatured

_oocytes

(Youniset

al,

1991;

De Smedtet

al,

1992).In addition,IVM andIVF have resulted

in the birth of live

_offspring

(Crozet et al, 1993;

_Keskintepe

et

_al,

1994). However,

_only

aboutathird ofthe _eggs

resulting

from IVM andIVF did

_develop

to

morula—blastocysts

in vivo

orin vitro (Crozetetal, 1993;

Keskintepe

etal, 1993) and,

by

co-culture with oviduct

_epithelial

cells, fewer than 10% devel¬

oped

into

blastocysts

(Keskintepe

et

al,

1993).Fromthesedata,

itisclearthat

_only

a small

proportion

of

goat

oocytesselected

for IVM can

complete

the full

cytoplasmic

maturation which confersonthemthe

ability

to

_support

embryonic

development.

Mammalian _oocytes

_acquire

meiotic

_competence

towards

the end of their

_{growth phase.}

As

_reported

in mice and

_pigs

(Sorensen and Wassarman, 1976; Motlik et

al,

1984) meiotic

competence

is

_acquired

_{progressively during}

follicular

_growth.

Goat _oocytes

_acquired

the

ability

to initiate

_resumption

of

meiosis in

early

antral follicles 0.5-0.8mm indiameterand to

reach

_metaphase

I in follicles 1—1.8mm indiameter(De Smedt

et

al,

1994).

_Although

86%ofoocytesfrom follicles

_larger

than

2mm_progress to

metaphase

II(De Smedtet

_al,

1992),

only

a

small

_proportion

of themcan

support

embryonic

development

(Crozetet

al,

1993),

_suggesting

that the

_capacity

to

_complete

cytoplasmic

maturation

_develops

beyond

the

_acquisition

of

meiotic

_competence.

Incattle, oocytes

originating

fromfollicles

larger

than 6mm in diameter

yield

a

significantly higher

Received 7November1994.

percentage

of

blastocysts

than do those from smaller follicles (Tan and Lu, 1990;

McCaffrey

et

al,

1992;

_Lonergan

et

al,

1994).Whetheroocyte

quality

isrelated tofolliclesizein

_goats

has never been determined. This

point

deserves attention

owing

to the increased interest in

_producing

_{goat zygotes}

in vitro, for gene transfer

technology.

Furthermore,

obtaining

populations

of_oocytes able orunable to

_support

embryonic

development, according

to follicle size,may

represent

a useful

tool withwhich to

_investigate

the mechanisms

_controlling

the

acquisition

of

_{developmental}

competence.

Theaimof the

_present

study

wasto

investigate

the effect of

folliclesizeonthe

ability

of

goat

oocytestobefertilizedandto

undergo early

embryonic

development

in vitro.

_{Developmental}

competence

of the

embryos

resulting

from IVM and IVF was

evaluated

by

co-culture with oviduct

_epithelial

cells. Co-culture

of one-cell

_goat

_embryos

_produced

in vivo with oviduct

epithelial

cells allows a

high

percentage

of them to

develop

beyond

the

_stage

of

_cleavage

arrest (8—16

cells) (Sakkas

etal, 1989) and to progress to the

_blastocyst

_{stage (Prichard}

et

al,

1992).

Preimplantation development

invitro,which has

_already

been

_proposed

as a check

point

for

studying

bovine oocyte

maturation (Rose and Bavister, 1992), can therefore also be

used in

_goats.

The

_present

resultsleadtotheconclusionthatin

_goats,

_only

oocytesfrom follicles about5 mmin diametercanachieve full

developmental

competence

through

astandardIVM

procedure.

Oocytes

originating

from small antral follicles that

_complete

nuclearmaturation in vitrocan

support

normalfertilization,but

most of them cannot

_{develop beyond}

the

_stage

of

_cleavage

arrest.

Materials and Methods

Oocyte

collection

Goats fromFrench

_Alpine

andSaanenbreedswere used.To

obtain ovulated oocytes, does were

synchronized

for oestrus

by

treatment for 10

days

with

_intravaginal

sponges

impreg¬

nated with 45 _mg

_{fluorogestone}

acetate(Intervet,

Angers)

and

by

injectionof 100_µg ofa

prostaglandin

analogue

(Estrumate:

Pitman-Moore, Meaux) 34h before sponge removal.

Super-ovulation was obtained

by

injecting

16_mg

pig

FSH

(pFSH,

Rhòne-Mérieux,

Lyon)

in

_decreasing

doses twice a

day,

for 3

days.

Inthe lastfour_injectionstheFSHLHratiowasdecreased

from 8 to 1.5 and 0.5

by

addition of

_purified

_pig

LH

(Rhône-Mérieux).

The

_goats

werekilled30hafteri.m.injection of 100µg LHRH (UCB,

Bioproducts,

Nanterre) administered

41h aftersponge removal.

Ovarian _oocytes were obtained

by

synchronizing

does for

oestrusasdescribedabove.Behaviouraloestruswasmonitored,

and follicular

_growth

was stimulated

by

three injections of

pFSH

(3, 2and2

_mg)

_starting

on

day

14or 15 oftheoestrous

cycle

at48, 40 and24h before

_slaughter.

The

_genital

tracts and ovaries were

transported

from the

abattoir to the

_laboratory

in a warm chamber

(30°C)

within

5 min.

Ovulated _oocytes were

immediately

recovered

by

retro¬

grade flushing

of the oviducts withadefinedmedium buffered

with

_Hepes

(DM- ,

_pH

7.8) (Crozet et

al,

1987), at 30-35°C.

Oocyte

maturation

The ovaries were dissected and nonatretic follicles were

selected and

_separated

intothreegroups

according

todiameter:

small, 2—3mm; medium, 3.1—5mm; and

large,

>5 mm. The

procedure

forIVMwasasdescribed

by

DeSmedtetal (1992).

Cumulus-oocyte

complexes

and

_granulosa

cells were isolated

from the follicles and were washed in warm TO99 medium

(Gibco

BRL,

_Eragny)

buffered with 20mmol

_Hepes

1

~

(pH

7.35) and

_containing

4mmol

NaHC03

1_1.

The same

medium enriched with 10%

(v/v)

heat-inactived calf serum

(Dutscher,

Paris), 1_µg oestradiol

ml-1

and 10_µg FSH ml~:

and 10_{µg LH} ml~ l

was used for the culture. The

cumulus-oocyte

complexes

(5—20

_complexes)

were co-cultured with

granulosa

cells (1 10 ml_

₎

from follicles

_larger

than 3mm

indiameter,in 2ml ofmedium,at

38.5°C,

with

gentle

_agitation

for27h. Beforein vitro

fertilization,

the_oocytesweremechani¬

cally

denuded from the cumulus cells in TC199 medium

containing

150IU

_{hyaluronidase}

ml-1

(Serva,

_Heidelberg)

and

were

extensively

washed in TO99 medium. Extrusion of the first

_polar

_body

was assessed

by

examination undera dissect¬

ing

microscope,

and

only

matured oocytes were selected for

fertilization.

Sperm

capacitation

and

_{fertilization}

The

_procedure

for sperm

capacitation

and IVF was as

reported

by

De Smedt et al (1992). Fresh

_ejaculates

obtained froma

particular

male of_proven

fertility

werediluted with8ml

DM- medium. After

_{centrifugation}

for 10 min at 200g, the

supernatants

were discarded and 50

µ

of the

_pellet

was

overlaid with 2ml of DM- enriched with 20%

(v/v)

of heat-inactived oestrous

_sheep

serum. The

spermatozoa

were

allowed to

_swim-up

for 1.5h at

38.5°C,

under air. The

supernatants containing

highly

motile

_spermatozoa

were

recovered.

_Sperm

motility

wasevaluated

by light

microscopy,

and _sperm concentration was measured with a Thoma cell.

Spermatozoa

were diluted to 1 x107 ml~1 with DM-H

containing

20% oestrous

_sheep

serum

(pH

7.3) and incubated

for 5—6h at 38.5°C in

_stoppered

tubes. After assessment of

motility,

the

_spermatozoa

werediluted forfertilizationtoafinal concentration of 1x

106

cells

ml"I

in DM-H+20%

(v/v)

oestrous

_sheep

serum. The

pH

ofthe medium was

adjusted

to 7.7

_by

addition of NaOH. The fertilization medium was

supplemented

with 7.75 mmol calcium Iactate 1

~

*

(i(+)-lactic

acid, hemicalcium salt;

Sigma,

St Louis, MO).

Five to ten ovulated or

mechanically

denuded in vitro

matured oocyteswereadded toeach testtube

_containing

1ml of the sperm

suspension

and incubated for 17h at 38.5°C in

stoppered

tubes.

Source

and

culture

_of

oviduct

_epithelial

cells

Oviduct cellswereobtained from does

serving

as sourcesof

ovaries. The oviducts were dissected, washed twice with PBS

and flushed with TO99

_Hepes

medium. Oviduct mucosal

tissue was extracted and transferred to a conical tube with

10ml TC199

Hepes

medium. The cells were dissociated

by

repeated

aspirations

with a

syringe.

After

decanting

the _super¬

natant, the

_pellet

(1

ml)

was

resuspended

in 20ml TO99 Downloaded from Bioscientifica.com at 03/13/2021 07:06:57PM

bufferedwith

_NaHC03,

_containing

2%

_(v/v)

ultroserG

_(Gibco

BRL,

Eragny),

1%

_(v/v)

200mmol

_glutamine

1" 1

(Gibco BRL),

1mg, 5

hydroxytryptamine

ml-1

_(Sigma),

100 iu

_penicillin

ml" 1

(Gibco

BRL), 100_µg

_{streptomycine}

ml":

(Gibco BRL),

250_µg

_fungizone

ml~1

(Gibco

BRL) and8%

_(v/v)

heat-treated

fetal calfserum

(Dutscher).

The cells weretransferredto tissue

culturedishes (Nunc,

Poly

Labo,

Paris)

_previously

coated with

0.5%

_(v/v)

_gelatin

_(swine skin

_type

_I,

_Sigma).

Part of the cell

suspension

was transferred to a 25cm tissue culture

flask,

as

control. The cells were incubated at 39°C with 5%

_C02

in

humidified air.

Thirty

hours after

_{explantation,}

non-attached cells and debris were

partially

removed. The medium was

replaced

after 48h and

_culturing

lasted 96h. The

monolayers

were used for co-culture with _eggs after

they

had reached

confluence

_(within

5

_days)

and_{up to} 2 _{weeks thereafter.}

Co-culture

_of

_embryo

and oviduct cells

Seventeen hours after _oocyte insemination, the _eggs were

washed with fertilization medium and _with

_B2

_{medium (INRA}

Ménézo)

_containing

10%

_(v/v)

fetal calf serum.

They

were

transferred to the

_monolayers

in 0.5ml

_B2

_supplemented

with

10%

_(v/v)

fetal calf serum and were cultured for 8-9

days

at

39°C with 5%

_C02

inhumidified air.

At intervals of 24

h,

half of the medium was

replaced

and

embryos

wereexamined for

cleavage

underaninvertedmicro¬

scope. Gametes and

embryos

were

manipulated

in a room at

30-35°C

Cytological procedures

After24h ofco-culture, one-cell _eggswereremoved, fixed inaceticacid (90%

v/v)-ethanol (l/3, v/v),

for24hat4°C and

stained with lacmoid before nuclear examination

by

_light

microscopy.

Those eggs

containing

more than two

pronuclei

with a_{sperm tail close} to eachmale

pronucleus

wereclassified

as

polyspermic.

Eight

days

after

fertilization,

two

_blastocysts

from thesmall, four from the

medium,

three from the

_large

sizefolliclegroups and four derived from ovulated _oocytes were fixed in 0.5%

(v/v)

acetic acid in ethanol (70%

v/v),

for 1h at room

temperature,

rehydrated

in 50%

_(v/v)

and 30%

_(v/v)

ethanol

and washed in PBS for 30 min.

_They

were embedded in

gelatin

10%

_(v/v)

inPBS and cooled in

_isopentane

(

—

130°C).

Cryostat

sections (10 µ ) were

exposed

for 15 s to Hoechst

dye

33258

_(Sigma)

for chromatin

_staining,

rinsed in PBS and mounted in Citifluor

(City University, London)

before

examination and

_counting

the number of cells under an

epifluorescence

microscope.

Statistical

_analyses

Statistical

_analyses

wereconducted

by

meansof

Chi-squared

analyses

or the Fisher exact test, as

appropriate.

Each group

was

compared

withits

neighbour

andtothegroupofovulated

oocytes.

Results

A total of 543

healthy

antral follicles were dissected from23

does,ofwhich173 wereclassifiedas small

follicles,

2—3mmin

Table 1.

Recovery

andselection of

_{cumulus-oocyte}

_complexes

from follicles of different size

Number ofintact

Number of COCs selected for

Source ofoocytes isolated COCs IVM(%)

Small follicles (2-3 mm) 173 107_(62)a

Medium follicles (3.1-5 mm) 157 132

(84)b

Largefollicles (>5mm) 213 185

(87)b

Datafrom23_goatsin nineexperiments.COC:_{cumulus-oocyte}complex.

a

Values in the same column with different _superscripts are significantly

different(P<_0.001).

Table 2. In vitro maturation of

_goat

oocytes from follicles of different size

Number of Number ofoocytes

Sourceof _oocytes oocytes cultured in

_metaphase

II(%)

Small follicles(2-3 mm) 105 74 (70)a

Medium follicles(3.1-5 mm) 127 105

(83)b

Large

follicles (>5mm) 159 154_(97)c Resultsarefromnineexperiments.

Valuesinthesamecolumn withdifferentsuperscriptsaresignificantlydifferent

ab(P<_0.05),

bc(P

<_0.01).

diameter; 157 as medium

follicles,

3.1-5 mm in diameter; and 213 as

large

follicles,

>5 mm in diameter.

Only

cumulus-oocyte

complexes

witha

compact,

multilayered

cumulus were

selected for IVM. The

_proportion

of

_{cumulus-oocyte}

com¬

plexes

selected in each

_category

is shown in Table 1.

_Signifi¬

cantly

more

high-quality

cumulus-oocyte

complexes

were

obtained from the medium and

_large

follicle size _groups than from the small size _group (84% and 87%

_compared

with 62%;

<0.001).

Thenumber ofoocytes

reaching

metaphase

IIwithin27hof

culture increased

_{progressively}

with follicular size: _70%, 83% and 97% ofoocytes from the small,medium and

_large

_groups

completed

nuclear maturation,

_respectively

(Table

2).

_Signifi¬

cant differences were found between the small and medium

groups (P<_0.05) and between the medium and

large

_groups

(P<0.01).

Only

mature oocytes that exhibited a

polar

body

were

subsequently

used for IVF. In each

_experiment,

inaddition to

oocytesfrom the differentgroups,ovulatedoocyteswereused as controls. Semen from a

single

male was used

throughout

these

_experiments

to avoiddifferencesin

_fertilizing

_ability

and

embryonic

development

capacity

among males. The

cleavage

rate 40h after inseminationincreased

_{progressively}

from46%

in the small group to69% inthe

_large

group andwas 75%in

ovulated oocytes

(Table

3). No

_significant

difference was

observed in the

_cleavage

rate between the small and medium

groups and between the

large

and ovulated oocyte groups.

However, oocytes from the

_large

_group

_yielded

a

significantly

higher

proportion

of

_cleaving

embryos

than did oocytes from the medium group (P<0.05). Uncleaved eggs were either

Table 3. In vitro fertilization of

_goat

oocytesfrom follicles ofdifferent size

Number of Number of cleaved eggs Number of Sourceofoocytes inseminatedoocytes 40h afterinsemination (%)

_polyspermic

eggs(%)

Smallfollicles (2-3 mm) 68 31 (46)a 9 (13)

Medium follicles (3.1-5 mm) 93 51 (55f 10 (11)

Largefollicles (>5mm) 143 98

(69)b

12 ₍₈₎

Ovulated 128 96

(75)b

12 (9)

Resultsarefromnineexperiments. a_Valuesinthe

samecolumn withdifferentsuperscriptsaresignificantlydifferent(P<_0.05).

Fig. 1.

_{Developmental}

_competenceofgoat oocytes from follicles of differentsizecultivated for8daysafter fertilizationin vitro. small

(2-3 mm); (0): medium (3.1-5 mm); (D):

_large

(>5mm); (S): ovu¬

lated _oocytes. Resultsare fromnine_experiments. Values inthesame group with different numbers of asterisks are

significantly

different

(embryo

<8-16 cells and morulaeP<0.01;

_blastocysts

P<0.05).

Fig. 2.

_{Developmental}

_competence of oocytes from follicles of different sizecultivated for9daysafter fertilizationin vitro.( small

(2-3mm); (0): medium (3.1-5 mm); ( ):

_large

(>5mm); (US): ovu¬

lated_oocytes.Resultsarefrom six_experiments.Values withdifferent

numbers of asterisks are

significantly

different: *'**(P<_0.05);

unfertilized or

penetrated by

more than one

spermatozoon.

Polyspermy, primarily dispermy,

which affected 13%, 11%, 8% and 9% of the inseminated oocytes from the small, medium,

large

and ovulated oocyte groups,

respectively,

was the

only

abnormality

detected.

Developmental

competence

was evaluated

by

examining

cleaved

_embryos

fromthe different_groupseach

_day

_during

the

culture

_{period. Significantly}

more

embryos

from the small

group arrested before or at the 8-to-16 cell

_stage

(84%

compared

with 53%, 45% and 39% in the

medium,

large

and ovulatedoocyte groups,

respectively

(P<_0.01)

_(Fig.

_1).

The

_proportions

of morulae and

_blastocysts

found 8

_days

after fertilization were 10% and 6%,

respectively,

in the small

group,35% and12%inthemediumgroup, 29%and 26%inthe

large

group and 20% and 41% in the ovulated oocyte group

(Fig.

1).

Oocytes

from medium-sized antral follicles

yielded

significantly

more morulae than did those from small antral

follicles (P< 0.01), but

_they

yielded

_{significantly}

fewer blasto¬ cysts than did oocytes derived from

_large

follicles (P<0.05). However, it should be noted that

_embryos

_developing

to the

morula

_stage

8

_days

after fertilizationwere

probably

notviable.

Significantly

more ovulated oocytes

developed

to the blasto¬

cyst

stage (41%

compared

with 26% in the

_large

_group)

(P<0.05)

(Fig.

1). The

_proportions

of hatched

_blastocysts

in

the

_large

and ovulated

_oocyte

_groups, 15% and 34%,

respec-tively,

9

days

after fertilization

(Fig.

2), were

approximately

double those found on the

_previous

day

(data

not

shown).

Oocytes

derived from

_large

follicles

_yielded

_{significantly}

fewer hatched

_blastocysts

than did ovulated _{oocytes (P<0.01)}

_(Fig.

2).Inthe small and medium_groups, the

_percentage

ofhatched

blastocysts

was _very low (zero and3%,

respectively)

(Fig.

2).

Six

_blastocysts

of the sevenobtained 8

days

after fertiliza¬

tion from the small and medium groups were examined on

frozen sections, after Hoechst

_dye

_staining.

They

contained fewer than50

cells,

and their innercellmass wasundetectable.

In _contrast,

_blastocysts

from the

_large

and ovulated

_oocyte

groupsatthis timecontainedover 150cells and theirinnercell

mass had over 20 cells.

Discussion

Results fromthe

_{present study}

demonstrateaclear

relationship

between folliclesizeand thein vitro

_{developmental}

_competence

of

_goat

oocytesmatured and fertilizedin vitro

_by

the

_procedure

reported

by

DeSmedtetal(1992).Whereas

_oocytes

_originat¬

ing

from follicles >5 mm in diameter

yielded

a

proportion

of

blastocysts

comparable

tothat of ovulatedoocytes,those from

small antral follicles lacked

_{developmental}

_competence.

In

cattle,

the size of follicles from which oocytes are derived

affects their

_{developmental}

_competence

(Tan and Lu, 1990;

Galli and Moor, 1991;Pavloketal 1992;

_Lonergan

et

al,

1994) but, to our

knowledge,

this

report

is the first to document a

relationship

between folliclesizeandoocyte

_quality

in

_goats.

In

both ruminant

_species,

oocytes from

_large

follicles

yielded

a

significantly

higher

percentage

of

_blastocysts

than did oocytes from small antral follicles. Bovineoocytes from medium antral follicles

_yielded

fewer

blastocysts

than did oocytes from

_large

follicles. However, identical

_hatching

rates

_suggest

that

embryos

derived from medium and

_large

follicles are of

equal

biological quality

(Tan and Lu, 1990; Pavlok et

al,

1992;

Lonergan

et

al,

1994). In contrast,

_goat

oocytes derived from mediumantral folliclesexhibited_poor

_{developmental}

_capacity,

as shown

by

the low rates of

blastocyst

formation and

hatching.

The small number of cells in the whole blasto¬

cysts and in the inner cell masses further

suggested

that

embryos

from this group were unable to

_support

subsequent

development.

Fertilization

abnormalities,

such as failure of _sperm

de-condensation and male

_pronuclear

formation,

thatare

likely

to

be due to insufficiences in

_cytoplasmic

maturation, were not

observed in fertilized

_goat

oocytes from follicles >2mm in

diameter.

_Polyspermy,

which affected

_{approximately}

10% of bothin vitroand in vivo matured_oocytes,most

likely

resulted

from IVFconditions. Thesedata indicatedthat a

large

_propor¬

tion of

_goat

oocytes from follicles >2mm in diameterwere

competent

to

_support

normal fertilization under the

_present

IVM/IVF conditions. In contrast, bovineoocytes from follicles >2mm in diameter

display

a

high

incidence of fertilization

abnormalities after IVM/IVF

(Pavlok

et

al,

1992).

De Smedt et al. (1994)

_reported

that the

_acquisition

of

meiotic

_competence

by

_{goat oocytes}

occurs

progressively

during

follicular

_growth.

The oocytes become

_capable

of

resuming

meiosis in antral follicles 0.5-0.8mm in diameter.

They

canreach

metaphase

Iinfollicles1.0—1.8mmindiameter

and_progress to

_metaphase

IIin

_larger

follicles.Datafrom the

present

study

further indicated that the

proportion

ofoocytes

completing

nuclear maturation

_{significantly}

increased as fol¬

licles

_enlarged

from 2 to 5 mm. Full maturationinvolves both

nuclear and

_cytoplasmic

events that confer on the _oocyte the

capacity

for

_supporting

normal fertilization and

_{early embry¬}

onic

_development

(Thibault

and Gérard, 1970; Moor and

Trounson, 1977).Theresults

_reported

here

_clearly

indicatethat

competence

to

_{undergo cytoplasmic}

maturation was also

acquired progressively

by

theoocyte,

_during

follicular

_growth.

In

_agreement

with the

_present

_{findings, Eppig}

et al (1994)

reported

that mouse oocytes

acquire

their

developmental

competence

ina

stepwise

manner.

Approximately

halfof

goat

oocytes fromfollicles 2-3mmin diameterwere

_competent

to

support

normalfertilization,butmostof themfailedto

develop

beyond

the 8—16cell

_stage.

The lack of some

_type

ofmRNA

could be one reason for

cleavage

arrest in

embryos

from this group. De Smedtetal (1994)

reported

that

goat

oocytesfrom

follicles 2mm in diameter are still

actively

engaged

in RNA

synthesis,

and entera

period

of low

transcriptional

activity

in

3mm follicles. Half of the oocytes from follicles 3-5mm in

diametercould

_develop

in vitro

_beyond

the8-16cell

_stage,

but

mostof them arrested at the morula

_stage,

beforeblastomere

compaction.

One

_{possible explanation}

could be that

_specific

maternal _mRNAs, critical for

_compaction

and for

_blastocyst

differentiation, accumulate intheoocyte

during

thefinal

_phase

offollicular

_growth.

Our

_findings

indicated that_oocytes from

large

follicleswere

superior

tooocytesfrom smalland medium

antral follicles in terms of

_{developmental}

_capacity

to the

blastocyst

stage.

This

finding

suggests

that in follicles about 5mm indiameter

_goat

oocyteshave

_synthesized

thematernal

factors

_required

to

_support

normal maturation, fertilization and

early embryonic

development.

Furtherstudiesare_necessaryto

provide

information on these maternal factors

responsible

for

the

_acquisition

of

_{developmental}

_competence.

The

availability

of oocytes from follicles of different size

_progressing

to

different

_stages

of

early embryonic

development

represents

an

important

tool for such studies.

The

_present

_findings

also

_emphasize

the necessity, for studies or

applications

that

require

full

developmental

poten¬

tial,toselectoocytes

according

to folliclesize.

_Keskintepe

elal (1994)

_reported

that 40%of

_goat

embryos

resulting

fromIVM

andIVF_progressin vitrotothe morula

_stage

but fail to

_develop

into

_blastocysts.

In another series of

_experiments,

_using

co-culture with oviduct

_epithelial

cells,

only

a few

blastocysts

wereobtained

(Keskintepe

et

al,

1993).The authors concluded

that methods for

_sustaining

in vitro

_development

up to the

blastocyst

stage

ofin vitromatured and fertilized

_goat

oocytes have not yet been

_developed

_(Keskintepe

et

_al,

1994). How¬ ever, oocytes from follicles 2-5mm in diameter wereused in

these

_experiments,

and as shown in the

_{present study,}

most

oocytesfrommediumantralfolliclesfailedtoprogress

beyond

the morula

_{stage. By}

_selecting

oocytesfrom

_large

follicles,

30%

of

blastocysts

were obtained under the IVM/IVF and

embryo

culture conditions used in the

_{present study.}

This

blastocyst

ratefell withintherangeof values

reported

forbovine

oocytes

using

standard IVM/IVF and

embryo

culture

_procedures

(Tan and Lu, 1990; Pavlok et

al,

1992;

_Lonergan

et al, 1994).

Administration of

_{pFSH beginning}

3

_days

before

_slaughter

has been shown to increase the number of

_large

follicles in the

ovariesof heifers and hence toincreasethenumber ofoocytes

competent

to

_{support embryonic}

development

following

IVM/ IVF and

_embryo

culture

_(Lonergan

et al, 1994). We can

speculate

that

_FSH-priming,

asusedinthe

present experiments,

mayalsoincreasethe number of

large

follicles in

goat

ovaries.

It would be

_interesting

for further

_applications

to compare

the

_{embryo yield}

_{per ovary} of

_goats

with or without

pFSH

treatment.

Although

the

_{developmental}

_competence

of

_goat

_oocytes

from follicles >5 mm in diameter and of those of ovulated

oocytes were

comparable,

ovulated oocytes were

_superior

especially

in termsof

blastocyst

hatching.

Thismay be dueto

the conditions for oocyte maturation that are critical for

subsequent

embryonic

development,

conditionsin vivo

_offering

abetterenvironmentthanconditionsin vitro. It isevident,both

from ourdata and from those ofothers, that

preimplantation

development

is a valuable criterion for

evaluating

the con¬

ditions for

_oocyte

maturation in vitro. The introduction of ovulated oocytes, as a control in the

experiments,

is also

important

for

_interpreting

the results obtained.

In the

_present

_study,

the

_viability

ofIVM/IVF

_embryos

was

not confirmed

_by

transfer to

_recipient

_goats,

the

only

conclu¬

sive test of

_{developmental}

_competence.

However,

previous

experiments

indicated that in vitro matured and fertilized oocytes

_produced

_through

the IVM/IVF

_procedure

described

are viable and can

produce

term

_offspring

(Crozet et

al,

1993).

In

conclusion,

the

_present

results indicate that the _compe¬

tence to

_undergo

_cytoplasmic

maturation is

_acquired

_progres¬

sively by

the

_goat

oocyte

during

final follicular

_growth.

The

capacity

tobe

normally

fertilized andto

_support

early

_cleavage

is

_acquired

_{concomitantly}

to meiotic

_competence.

Further

differentiation ofthe _oocytes enables them _to

_develop

_at

_first,

through

the

_period

of

_cleavage

arrest and thereafter, to

progressto the

blastocyst

_stage.

Itis

_important

tostress,forfurther

_{applications,}

that

_only

a

small

_proportion

of

_goat

oocytes isolated from antral follicles

have the

_capacity

to

_support

_embryonic

_development

in vitro. Purified FSH was a

gift

provided

by

the _InstitutRhône-Mérieux,

Lyon, France. The authors thank A. Solari for statistical analysis, C.

Sévellec forassistance inIVMandY.Lavergnefor technicalassistance.

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