Follicular size affects the meiotic competence of in vitro matured prepubertal and adult oocytes in sheep

Original

article

Follicular size affects

the

meiotic

_competence

of in vitro

matured

_prepubertal

and

adult

_oocytes

in

_sheep

Sergio

Ledda

Luisa

_Bogliolo

Giovanni

_Leoni,

Salvatore Naitana

Department

of Animal

Biology, Veterinary Faculty,

via Vienna 2, 07100 Sassari,

Italy

(Received 28 December 1998;

accepted

24 March ₁₉₉₉₎

Abstract - Non-atretic follicles dissected from

prepubertal

and adult ovaries were allocated in

three groups: a) < 1 mm; b) 1-2 mm; c) > 2 mm. After 24 h of _maturation, a lower _percentage of adult oocytes from _group a _(P <

_0.01)

reached

_metaphase

II than those from _groups b and c _(70.4 versus 89.5 and

_{95.5). Prepubertal}

_oocytes showed similar results _(P < 0.01; 27.2 versus 79.8 and 81.8). There

was a

_significant

difference (P < _0.01) in _meiotic

_progression

between

_prepubertal

and adult _oocytes

of < 1 mm follicles. The diameter of

_prepubertal

_{oocytes derived from group} a was

_{significantly}

lower _(P < _0.01)

_compared

to _groups b and c _{( 138.1} versus 142.1 and _145.6); adult _oocytes showed similar results _(P < _0.01; 142.2 versus 157.2 and _158.1

_Oocytes

with the same diameter derived from different follicles of

_prepubertal

and adult ovaries showed similar meiotic

_progression

rates.

@ Inra/Elsevier, Paris

ovary / oocyte / follicle size / meiotic competence / adult and

prepubertal sheep

Résumé &horbar; Lien entre la taille et la

_{compétence méiotique d’ovocytes d’agnelles}

et de brebis

matu-rés in vitro. Des follicules

non-atrétiques

sélectionnés à

_partir

d’ovaires d’ovins

prépubères

et adultes

ont été

_répartis

en _{trois groupes selon leur diamètre : a)} < 1 mm ; b) 1-2 mm ; c) > 2 mm.

_Après

24 h de maturation les ovocytes dérivant des ovaires d’animaux adultes appartenant au _groupe a ont

atteint la MII dans des _pourcentages nettement inférieurs

_(p

< _0,01) à ceux observés pour _{les groupes} b et c _(70,4 versus 89,5 et 95,5). Des résultats

_identiques

ont été obtenus avec les _{ovocytes provenant}

d’animaux

_{prépubères (p}

< 0,01 ; 27,2 versus _79,8 et

_81,8).

Une

_grande

différence

_(p

< _0,01) dans le

potentiel

à

_reprendre

la méiose existe entre les _ovocytes de

_prépubères

et des adultes _provenant de

fol-licules < 1 mm. Le _diamètre des _ovocytes

_d’agneaux

_provenant de follicules du _groupe a était nettement

inférieur

_(p

_{< 0,01 )}

_par rapport à ceux _provenant de follicules _{des groupes b ou} c ( 138,1 versus _142,1 et 145,6). Des résultats

_identiques

ont _{été observés pour les ovocytes provenant} d’animaux adultes

(p

< 0,01, 142,2 versus 157,2 et 158,1). Des _ovocytes de même diamètre provenant d’ovaires de

prépubères

et d’adultes ne diffèrent _{pas dans} leur

_aptitude

à

_reprendre

la méiose. &copy; _{Inra/Elsevier,} Paris follicule / _ovocyte /

_{compétence méiotique}

/ ovins

_prépubères

et adultes

*

Correspondence

and

_reprints

E-mail: vetfis@ssmain.uniss.it

1. INTRODUCTION

It is known that mammalian

_oocytes

released from the

_inhibitory

influences of their follicles and cultured in vitro under efficient conditions are able to

_{spontaneously}

resume meiosis and

_complete

maturation

[10].

Several data

_{indicate, however,}

that many of these

oocytes

fail to be

_fertilised,

develop

or

_produce

a

_high

_percentage

of viable

_embryos

_{[12, 18, 29].}

_During

_oocyte

growth,

physiological

events occur in both the nucleus and the

_{cytoplasm allowing}

the

oocyte

to achieve final

_{developmental}

com-petence.

Nuclear

_progression

from the ger-minal vesicle

_(GV)

to the

_metaphase

II

_(Mll)

stage can occur in

_oocytes

_{that may} not have

undergone complete cytoplasmic

matura-tion

_[2].

_Many

intracellular

_changes

are

required

in the

_cytoplasm,

such as

_organelle

distribution

_{[28, 29]}

and

_protein

_synthesis

at different

_stages

of meiosis

_progression

[15, 22].

Moreover,

the meiotic

_competence

in bovine

_species

is

_closely

related to the follicular

_dimension;

_oocytes derived from small follicles showed a lower

_capacity

to resume meiosis and to

_develop

_up to

_early

embryonic

stages [4].

Previous works

_[8,

19, 23]

have

_clearly

demonstrated that the

acquisition

of meiotic competence after in vitro maturation is achieved

_{progressively}

as the follicular size increases in several domes-tic

_{species. Oocytes}

derived from follicles less than 2 mm in diameter did not resume or

complete

meiosis in vitro.

In

_sheep,

Moor and

_{Trounson [21] also}

indicated that meiotic and

_{developmental}

competence

is

_higher

in

_oocytes

recovered from

_large

follicles

_(atretic

and

_non-atretic)

than in those derived from small follicles.

They

also showed that

_oocytes

cultured in intact follicles

_(<

2

_mm)

are

_incapable

of

resuming

meiosis. A

_positive

correlation between follicular size and meiotic and

developmental

competence

has also been

reported

by

Cogni6

et al.

_[5].

_They

showed that a different

_genotype

can affect the

abil-ity

of

_oocytes

recovered from follicles of different sizes to resume meiosis in vitro.

A lower

_{developmental capacity}

has also been observed after in vitro maturation of oocytes obtained from

_prepubertal

animals

[9, 26, 27].

This can be related to defective conditions of

_oocyte

maturation and can also be due to the reduced size of

_oocytes

and follicular

_population

_[17]

and a small size of volume fractions of mitochondria and cortical

_granules

_[25].

The aim of this work was to examine the

relationship

between the follicular size and meiotic _competence of

_oocytes

derived from

prepubertal

and adult

_sheep

in different

cat-egories

of follicles after in vitro maturation.

’

2. MATERIALS AND METHODS

2.1.

_Recovery

of the

_oocytes

Ovaries from

_prepubertal

_(30!0

_{days, body}

mass 6-12

_kg)

and adult Sarda

_sheep

were col-lected from a local abattoir and

_transported

in Dulbecco’s

_{phosphate-buffered}

saline (PBS;

Sigma,

Saint-Louis, MO)

containing

50 U

peni-cillin mL-’ and 50 _pg

_streptomycin

mL-1 (SIGMA) at 20-25 °C,

arriving

at the

_laboratory

within 1 h. Ovaries were washed three times in

fresh PBS with

_penicillin

and

_{streptomycin.}

Non-atretic follicles were obtained from the ovaries

_using

microblades and fine

_forceps

under

a

_{dissecting microscope}

and

_single

selected fol-licles were obtained from both

_prepubertal

and adult ovaries. After dissection, the follicles were

measured with a micrometer and

_placed

accord-ing

to diameter in the three

_following

_groups:

a) < 1 mm; b) 1.0-2.0 mm; c) > 2 mm. The walls

of the follicles of the _{different groups} were

opened

and the oocytes were released in the TCM 199 medium (with Earle’s salts and bicarbonate;

Sigma) supplemented

with 25 mmol

_Hepes

1-1

(Sigma), penicillin

and

_{streptomycin,}

and 0.1 %

(w/v)

polyvinyl

alcohol _(PVA;

_Sigma).

The

cumulus-oocyte complexes

(COCs), selected

according

to

_{morphological}

criteria (intact COCs

with several dense cumulus cell

_layers

and

homogeneous cytoplasm),

were released in the medium and washed three times in the same

medium before in vitro maturation.

After denudation of the cumulus cells

_by

mechanical

_pipetting,

several _oocytes obtained

from each follicle-sized _{group of}

_prepubertal

and adult

_{sheep, including}

the zona

_pellucida,

were

measured

using

an inverted

microscope equipped

with a calibrated

eyepiece.

Their outside

diame-ters were recorded.

2.2. In vitro maturation

The COCs obtained from

_juveniles

were allo-cated to the

_following

maturation _system:

pre-pubertal

and adult oocytes were cultured in

par-allel for 24 h in TCM 199 medium

_supplemented

with 10 % (v/v) heat-treated foetal calf serum

(FCS;

Sigma)

in the presence of 10 pg FSH mL-’

and 10 _pg LH mL-1

_(Pergonal,

Serono, Rome).

Only

25-30 of the _oocytes were _put in a

_single

Petri dish _{(35 mm)}

_containing

2 mL of the culture medium which was maintained on a _rotary shaker

at low

_{speed during}

the entire incubation

_period

to avoid the attachment

_{of granulosa}

cells to the bottom of the Petri dish. The culture conditions

were the

_following:

a humidified

_atmosphere

with 5 %

_C0

₂

in the air and a _temperature of 39 °C. At the end of the maturation

_period,

the

oocytes from each group were fixed for 24 h in

acetic acid ethanol solution (1:3, v/v), stained

with 1 % (w/v) Lacmoid

(Sigma)

and examined under a

_{phase-contrast}

_microscope

at a

magnifi-cation of x 200-400. The _stages of nuclear

mat-uration were assessed after evaluation, as the

germinal

vesicle (GV),

metaphase

I (M I),

metaphase

II _{(M II)} and

_degenerated.

Some _oocytes from

_prepubertal

ovary < 1 mm follicles were cultured for 48 h in the same

cul-ture conditions as those described above,

exam-ined after 24 h of fixation and stained with 1 %

Lacmoid.

2.3. Statistical

_analysis

Differences in meiotic

_{progression according}

to the follicular size of

prepubertal

and adult

oocytes were

_subjected

to

_{chi-squared analysis}

or

the Fisher exact test. Mean _oocyte diameters in the follicle size

_categories

were

_compared

sta-tistically by

ANOVA. Differences were consid-ered

_significant

at P < 0.05.

3. RESULTS

After 24 h of incubation the

_percentage

of

metaphase

II

_oocytes

derived from ovaries of adult and

_prepubertal

animals increased

progressively according

to follicle sizes

(from

< 1 to > 2 mm

_(table

_!.

There was a

significant

difference

(P

<

_0.01)

in the

per-centage

of M II between oocytes derived from < 1 mm follicles

_compared

to

_oocytes

of > 1 and > 2 mm of both

_prepubertal

(27.2

2 versus 79.8 and 81.8

_%)

and adult

_(70.6

ver-sus 89.7 and 95.5

_%)

_{sheep. Oocytes}

col-lected from < 1 mm follicles of

_prepubertal

animals also showed a

_{significantly}

_(P

<

_0.01)

marked decrease in the

_ability

to resume

complete

meiosis

_compared

to adult

_oocytes.

This difference can be related to a

signifi-cantly

lower

_(P

<

_{0.01) oocyte}

diameter in the three

_categories

of follicles

(< 1,

1-2 and > 2

_mm)

of

_prepubertal

_(138.1,

142.1 I and 145.6

_l

_tm)

_compared

to adult

_sheep

Oocytes

derived from < 1 mm follicle size cultured in vitro for 48 h showed a sim-ilar

_percentage

of M II as those cultured for 24 h

_(29.7

versus

_27.2).

4. DISCUSSION

This work shows that the

_ability

to

progress in the meiotic

cycle

is

achieved

progressively

as follicle and

_oocyte

diam-eter increases in both adult and

_prepubertal

sheep.

However,

in contrast to the observa-tions of De Smedt et al.

[8]

in _goats,

_sheep

oocytes derived from very small follicles

(<

1

_mm)

of adult ovaries can _{progress in} meiosis in a

_high

_percentage

and 70 % of total

_oocytes

reach

_metaphase

II.

Several studies in other domestic

_species

[6,

11, 23],

indicate that

_{significantly}

more

oocytes derived from > 2 mm follicles matured in _{vitro up} to M

II,

while

_oocytes

collected from follicles of less that 2 mm are

_incapable

of

_resuming

or

_completing

meiosis. In

_prepubertal

_goats it has been

reported

[20]

that

_complete

meiotic

com-petence was achieved in

_oocytes

derived from 3 mm in diameter follicles and similar results have been obtained in adult

_goats

by

De Smedt et al.

_[8].

_They

observed that the

oocyte

was able to resume meiosis in < 1 mm antral follicles but

only

oocytes

derived from follicles

_larger

than 3 mm were

_fully

com-petent and able to reach

_metaphase

II. These authors also showed that

_oocytes

from 2 mm follicles were still

_{actively engaged}

in RNA

_synthesis,

whereas RNA

_synthesis

was

reduced in

_oocytes

from 3 mm follicles. Motlik et al.

_[24]

_reported

that

_porcine

oocytes

derived from < 0.7 mm follicles remained at the

_germinal

vesicle _stage

main-taining activity

in RNA

_synthesis.

The

capacity

to resume meiosis also seems to

be related to the location of the follicles in the ovaries. Arlotto et al.

_[1]

_reported

that

oocytes

of all diameters collected from the

cortical

_region

_{of the ovary} are

_{significantly}

more _competent than

_oocytes

located in a sub-cortical area. More

_recently

_Hyttel

et

al.

_[14]

showed a direct correlation

_only

between bovine

_oocyte

dimension and mei-otic

_{developmental}

_competence

after the maturation of

_oocytes

derived from folli-cles of different sizes.

In

_sheep,

after the culture of intact folli-cles of different

sizes,

Moor and Trounson

[21]

showed that _oocytes derived from < 2 mm follicles were

meiotically

incom-petent

while on the

_{contrary oocytes}

from

bigger

follicles were

_meiotically

and

devel-opmentally

fully

competent.

This is

appar-ently

in contrast with the results

_reported

in this _paper but our culture conditions were different and

_oocytes

were cultured outside of the follicles and not in intact follicles as Moor and Trounson

_reported.

These condi-tions _may have allowed the

_oocyte

to

progress to meiosis because it cannot be influenced

by

the

_inhibitory

control of meio-sis exerted

_by

the follicular environment.

Cogniè

et al.

_[5]

also found that the

_ability

of ovine

_oocytes

to resume meiosis and to

_gain

the

_ability

to

_develop

_up to the

_blastocyst

enlarges.

They

also showed that this

abil-ity

is related to the

_genotype

of the ewe. In

fact, oocytes

from < 1 mm follicles of FecB gene carriers were able to resume meiosis in

higher

percentages

than those from non-car-riers.

_They

also found that the _pattern of oocyte

growth

in

_preantral

follicles was related to the

_genotype

and that FecB carri-ers had

_larger

_oocytes

than those from non-carriers. Our data showed that in Sarda ewes the

_ability

to resume meiosis is

_high

from oocytes derived from < 1 mm follicles and

may be related to the

_genotype

of this breed. In fact the

_oocytes

derived from these

folli-cles,

which are even smaller than those from

larger

follicles,

are

_capable

of

_being

meiot-ically

competent.

In contrast, the

_experiment

of maturation

of prepubertal

oocytes indicated that

_oocytes

derived from < 1 mm follicles were

inca-pable

of

_resuming

and

_completing

meiosis

compared

to adult _oocytes

₍₂₇

versus 70 of

M

_II)

and a

_higher

_percentage

of the

_oocytes

remained arrested at the GV and M I

_stage.

This difference in meiotic

_progression

_may be due to the smaller _oocyte diameter _in

pre-pubertal

oocytes

(138

versus 142

₁₁

_m)

prob-ably indicating

an

incomplete

_growth

_phase.

The

_acquisition

of meiotic

_competence

may be

_closely

related to

_oocyte

diameter because

at _{142 11m} in diameter both

_prepubertal

and adult

sheep

oocytes

show similar nuclear

progression

(79.8

versus

_70.6).

In

_fact,

bovine _oocytes derived from small follicles showed a low diameter of < _{100 11m and} are unable to resume meiosis or are arrested at

M

_I;

moreover these

_oocytes

show an intense

_{transcriptional activity}

_[11].

] .

In

_prepubertal

_oocytes

the lower

capa-bility

to resume and

_complete

meiosis can be due to either inefficient

_coupling

between the somatic

_compartment

and the

_oocyte

that can decrease the

_signals

involved in meiotic

_resumption

or the lack of

_key

molecules

_regulating

the meiosis

_cycle.

In

fact,

it has been observed that the gap

junc-tions increase with the follicular dimensions

[13].

Prepubertal

oocytes

have also shown a

less functional

_coupling

between the corona cell and

_oocytes

[16].

The increase in gap

junctions

between somatic cells and the

oocyte

is also related to the follicular dimen-sion

_during

the

_{folliculogenesis.}

Further-more, it has been observed that

_growing

oocytes

are still

_engaged

in

_high

RNA syn-thesis _{that may} be crucial for the

produc-tion of molecules

_directly

involved in cell

cycle

progression.

In

_fact,

it has been observed

_[3]

that

_prepubertal

ovine

_oocytes

showed a lower HI

_activity

and a lower MPF

_biological

_activity

after fusion of M II

prepubertal

oocytes

with GV

_{stage oocytes.}

Similarly

it has been

_reported

that calf

oocytes

had lower H and MAPK activities

compared

to adult

_{oocytes [7].}

This lower

competence

in

_prepubertal

_oocytes

has also been observed after fertilisation and culture in vivo and in vitro _up to the

_blastocyst

_stage

[17].

In

_conclusion,

these

_experiments

showed

that, contrary

to observations in

_prepubertal

sheep,

a

_high

_percentage

of adult

_oocytes

derived from very small follicles are able

to reach full meiotic

_progression.

Meiotic

competence

may,

however,

be

closely

related to _oocyte diameter in

_sheep.

ACKNOWLEDGEMENT

This work was

_{supported by}

MURST (cofi-nanziamento

1998)

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