Trends

in

Folic

Acid

Awareness

and

Behavior

in

the

United

States

The

Gallup

Organization

for

the

March

of

Dimes

Foundation

Surveys

Objective

To

summarize

changes

in

folic

acid

awareness

knowledge

and

behavior

among

women

of

childbearing

age

in

the

United

States

since

the

U.S.

Public

Health

Service

-LRB-

USPHS

-RRB-

folic

acid

recommendation

and

later

fortification

Methods

Random-digit

dialed

telephone

surveys

were

conducted

of

approximately

women

-LRB-

per

survey

year

-RRB-

aged

years

from

in

the

United

States

Results

The

percentage

of

women

reporting

having

heard

or

read

about

folic

acid

steadily

increased

from

in

to

in

Of

all

women

surveyed

in

knew

folic

acid

prevented

birth

defects

an

increase

from

in

The

proportion

of

women

who

reported

learning

about

folic

acid

from

health

care

providers

increased

from

in

to

in

The

proportion

of

all

women

who

reported

taking

a

vitamin

supplement

containing

folic

acid

increased

slightly

from

in

to

in

Among

women

who

were

not

pregnant

at

the

time

of

the

survey

in

reported

taking

a

vitamin

containing

folic

acid

daily

compared

with

in

Conclusions

The

percentage

of

women

taking

folic

acid

daily

has

increased

modestly

since

Despite

this

increase

the

data

show

that

the

majority

of

women

of

childbearing

age

still

do

not

take

a

vitamin

containing

folic

acid

daily

Health

care

providers

and

maternal

child

health

professionals

must

continue

to

promote

preconceptional

health

among

all

women

of

childbearing

age

and

encourage

them

to

take

a

vitamin

containing

folic

acid

daily

Introduction

Spina

bifida

and

anencephaly

the

most

common

neural

tube

defects

-LRB-

NTDs

-RRB-

are

serious

birth

defects

that

occur

early

in

pregnancy

often

before

a

woman

realizes

she

is

pregnant

Important

studies

show

that

intake

of

the

B

vitamin

folic

acid

reduces

the

incidence

of

NTDs

by

when

taken

before

conception

and

during

the

first

trimester

of

pregnancy

-LSB-

-RSB-

This

compelling

research

prompted

the

U.S.

Public

Health

Service

-LRB-

USPHS

-RRB-

recommendation

that

all

women

of

childbearing

age

who

are

capable

of

becoming

pregnant

consume

micrograms

-LRB-

mcg

-RRB-

of

folic

acid

daily

-LSB-

-RSB-

For

women

who

previously

had

an

infant

with

an

NTD

and

were

planning

a

pregnancy

it

was

recommended

that

they

consume

a

much

larger

dose

mcg

of

folic

acid

per

day

at

least

one

month

before

becoming

pregnant

and

during

the

first

three

months

of

pregnancy

-LSB-

-RSB-

In

the

USPHS

recommendation

was

reaffirmed

by

the

Institute

of

Medicine

which

stated

that

women

capable

of

becoming

pregnant

should

take

micrograms

of

synthetic

folic

acid

daily

from

fortified

foods

or

supplements

or

a

combination

of

the

two

in

addition

to

consuming

natural

folate

from

a

varied

diet

-LSB-

-RSB-

Additionally

in

the

Food

and

Drug

Administration

-LRB-

FDA

-RRB-

began

requiring

folic

acid

fortification

of

enriched

breads

cereals

flours

pastas

rice

and

other

grain

products

-LSB-

-RSB-

to

aid

in

increasing

folic

acid

consumption

by

an

estimated

average

of

mcg

daily

in

women

of

childbearing

age

Other

organizations

such

as

the

American

Academy

of

Pediatrics

and

the

American

College

of

Obstetricians

and

Gynecologists

have

supported

the

USPHS

recommendation

-LSB-

-RSB-

National

and

state

organizations

implemented

educational

and

media

campaigns

during

the

to

promote

the

awareness

and

consumption

of

folic

acid

among

women

of

childbearing

age

-LSB-

-RSB-

Since

the

implementation

of

national

policies

the

number

of

pregnancies

affected

by

NTDs

in

the

U.S

has

decreased

approximately

from

affected

pregnancies

prior

to

fortification

to

affected

pregnancies

after

fortification

-LSB-

-RSB-

Despite

these

efforts

the

percent

reduction

anticipated

by

the

USPHS

in

has

not

been

forthcoming

This

report

summarizes

results

from

nine

surveys

conducted

between

after

the

USPHS

recommendation

and

folic

acid

fortification

It

documents

the

steady

increase

in

awareness

and

knowledge

of

folic

acid

and

the

as

yet

modest

change

in

consumption

of

a

vitamin

containing

folic

acid

among

U.S.

women

of

childbearing

age

-LSB-

-RSB-

Table

knowledge

behavior

and

source

of

knowledge

regarding

folic

acid

among

women

of

childbearing

age

United

States

Heard

or

read

about

folic

Knew

folic

acid

can

help

prevent

birth

Knew

folic

acid

should

be

taken

before

Take

folic

acid

daily

-LRB-

all

women

-RRB-

Take

folic

acid

daily

-LRB-

non-pregnant

women

-RRB-

of

knowledgea

Health-care

sources

of

folic

acida

Green

leafy

vegetables

Fortified

cereals

March

of

Dimes

Birth

Defects

Foundation

From

the

margin

of

error

for

estimates

is

In

the

margin

of

error

for

estimates

was

Note

Not

available.aOf

women

who

report

being

aware

of

folic

acidbStatistical

estimates

were

weighted

to

reflect

the

total

population

of

women

aged

years

in

the

contiguous

United

States

who

resided

in

households

with

telephones

Methods

The

data

for

this

study

include

a

national

sample

of

approximately

women

-LRB-

per

survey

year

-RRB-

aged

years

from

The

March

of

Dimes

Birth

Defects

Foundation

contracted

the

Gallup

Organization

to

conduct

random-digit-dialed

telephone

surveys

Since

response

rates

have

ranged

from

to

The

response

rate

for

the

survey

was

-LRB-

women

-RRB-

Statistical

estimates

were

weighted

based

on

Current

Population

Survey

-LRB-

CPS

-RRB-

data

to

reflect

the

total

population

of

women

aged

years

in

the

contiguous

United

States

who

resided

in

households

with

telephones

In

the

sampling

design

included

an

oversampling

of

women

ages

In

the

margin

of

error

for

estimates

based

on

the

total

sample

size

was

and

in

it

was

The

questionnaire

and

methods

used

were

described

previously

and

were

the

same

in

all

survey

years

-LSB-

-RSB-

Results

Table

displays

the

percentage

of

women

reporting

awareness

of

and

knowledge

about

folic

acid

and

their

daily

use

of

vitamins

containing

folic

acid

Between

and

the

percentage

of

women

reporting

having

heard

or

read

about

folic

acid

increased

from

in

to

in

Since

awareness

of

folic

acid

has

remained

relatively

constant

at

about

Of

all

women

surveyed

in

knew

that

folic

acid

prevented

birth

defects

compared

to

in

Also

in

of

women

knew

that

folic

acid

should

be

taken

before

pregnancy

compared

to

in

The

proportion

of

all

surveyed

women

who

reported

taking

a

vitamin

supplement

containing

folic

acid

daily

has

remained

almost

unchanged

but

increased

gradually

from

in

to

in

but

decreased

to

in

Of

women

surveyed

in

who

were

aware

of

folic

acid

identified

green

leafy

vegetables

as

good

sources

of

folic

acid

However

in

fortified

cereals

were

added

to

the

list

of

possible

sources

and

only

of

women

identified

fortified

cereals

as

a

good

source

of

folic

acid

Table

also

displays

women

's

reported

sources

of

information

about

folic

acid

From

to

the

proportion

of

women

who

reported

learning

about

folic

acid

from

health

care

providers

showed

a

two-fold

increase

from

in

to

in

Of

women

surveyed

in

reported

that

their

health

care

provider

discussed

the

benefits

of

folic

acid

with

them

compared

to

in

Of

the

same

group

of

women

in

reported

being

told

by

their

health

care

provider

that

folic

acid

prevented

birth

defects

However

only

of

women

in

reported

that

their

health

care

provider

said

folic

acid

needs

to

be

taken

before

pregnancy

This

statistic

has

ranged

from

in

to

in

since

its

inclusion

in

the

survey

in

Table

of

women

of

childbearing

age

who

reported

awareness

of

folic

acid

by

selected

sociodemographic

characteristics

United

States

group

-LRB-

yrs

-RRB-

Less

than

high

High

school/Trade/Technical

Vocational

College

-LRB-

any

-RRB-

household

income

Source

March

of

Dimes

Birth

Defects

Foundation

From

the

margin

of

error

for

estimates

is

In

the

margin

of

error

for

estimates

was

aStatistical

estimates

were

weighted

to

reflect

the

total

population

of

women

aged

years

in

the

contiguous

United

States

who

resided

in

households

with

telephones

For

the

last

ten

years

the

percentage

of

women

learning

about

folic

acid

from

media

sources

such

as

radio

and

television

increased

from

in

to

in

whereas

information

about

folic

acid

obtained

from

magazines

or

newspapers

decreased

from

in

to

in

These

results

are

shown

in

Table

Table

displays

the

percentage

of

women

of

childbearing

age

who

reported

awareness

of

folic

acid

by

selected

sociodemographic

characteristics

In

the

women

least

likely

to

be

aware

of

folic

acid

were

non-white

women

-LRB-

-RRB-

women

aged

years

-LRB-

-RRB-

those

women

with

less

than

a

high

school

education

-LRB-

-RRB-

and

those

with

a

household

income

of

less

than

-LRB-

-RRB-

These

results

are

consistent

with

years

prior

to

Table

displays

the

percentage

of

women

of

childbearing

age

who

reported

taking

folic

acid

daily

by

selected

sociodemographic

characteristics

Of

all

women

surveyed

in

those

least

likely

to

consume

a

vitamin

containing

folic

acid

daily

include

non-white

women

-LRB-

-RRB-

those

women

aged

-LRB-

-RRB-

those

with

less

than

a

high

school

education

-LRB-

-RRB-

and

those

with

household

incomes

of

or

less

-LRB-

-RRB-

Among

women

who

were

not

pregnant

at

the

time

of

the

survey

in

reported

taking

a

vitamin

containing

folic

acid

daily

compared

with

in

Among

women

who

were

surveyed

in

who

were

currently

pregnant

reported

taking

a

vitamin

containing

folic

acid

daily

compared

to

in

Table

of

women

of

childbearing

age

who

reported

taking

folic

acid

daily

by

selected

sociodemographic

characteristics

United

States

White

Non-White

Hispanic

Non-Hispanic

group

-LRB-

yrs

-RRB-

Less

than

high

High

school/Trade/Technical

Vocational

College

-LRB-

any

-RRB-

household

income

Pregnancy

status

Currently

pregnant

Not

currently

March

of

Dimes

Birth

Defects

Foundation

From

the

margin

of

error

for

estimates

is

In

the

margin

of

error

for

estimates

was

Note

Not

availableaStatistical

estimates

were

weighted

to

reflect

the

total

population

of

women

aged

years

in

the

contiguous

United

States

who

resided

in

households

with

telephones

In

of

childbearing

age

women

reported

they

did

not

take

daily

supplements

The

most

common

reasons

for

not

taking

any

daily

vitamin

or

mineral

supplements

are

that

they

forget

to

take

them

stated

by

of

women

or

do

n't

feel

they

need

them

as

reported

by

of

women

However

in

of

women

who

were

not

currently

taking

daily

vitamins

or

mineral

supplements

reported

they

would

be

likely

to

start

taking

a

multivitamin

daily

if

advised

by

their

health

care

provider

or

physician

Discussion

The

findings

in

this

study

show

that

whereas

a

large

percentage

of

women

have

heard

of

folic

acid

only

a

small

proportion

are

aware

that

folic

acid

prevents

birth

defects

and

should

be

taken

before

pregnancy

Furthermore

despite

the

USPHS

recommendation

in

-LSB-

-RSB-

the

percentage

of

women

taking

a

vitamin

supplement

containing

folic

acid

daily

has

only

modestly

increased

between

and

There

was

a

substantive

increase

in

the

percentage

of

women

taking

folic

acid

daily

in

but

this

increase

was

not

sustained

in

Clearly

the

observed

increases

in

awareness

and

knowledge

about

folic

acid

are

not

translating

into

increased

multivitamin

use

Increased

awareness

of

the

importance

of

folic

acid

is

due

in

part

to

educational

outreach

activities

undertaken

over

the

years

by

CDC

the

March

of

Dimes

Birth

Defects

Foundation

and

its

chapters

the

Spina

Bifida

Association

of

America

and

members

of

the

National

Council

on

Folic

Acid

These

activities

culminated

in

a

national

meeting

in

January

to

coordinate

a

folic

acid

promotional

campaign

-LRB-

May

through

September

-RRB-

with

continuing

independent

educational

outreach

efforts

-LSB-

-RSB-

Although

there

were

no

substantial

changes

in

folic

acid

use

recent

dietary

habits

might

provide

insight

into

the

sudden

increase

in

folic

acid

use

in

A

recent

study

found

that

women

on

low-carbohydrate

diets

were

more

likely

to

take

folic

acid

daily

than

women

on

other

diets

however

in

folic

acid

use

was

similar

across

dieting

types

-LSB-

-RSB-

The

results

from

the

survey

may

be

an

anomalous

finding

given

that

many

of

the

findings

returned

to

levels

-LRB-

e.g.

multivitamin

use

-RRB-

Our

findings

may

suggest

the

need

for

more

continued

and

increased

media

messages

to

encourage

women

to

consume

adequate

amounts

of

folic

acid

every

day

through

fortified

foods

dietary

supplements

and

a

folate-rich

diet

Furthermore

communities

should

identify

food

products

that

provide

recommended

levels

of

folic

acid

available

in

local

grocery

stores

and

encourage

women

to

select

diets

with

sufficient

folic

acid

Recent

educational

efforts

alone

do

not

appear

to

be

adequate

to

demonstrate

an

impact

on

changing

behavior

with

regard

to

folic

acid

consumption

The

findings

from

this

report

can

be

used

to

develop

multi-pronged

targeted

public

health

programs

to

increase

the

number

of

women

of

childbearing

age

consuming

a

vitamin

containing

folic

acid

daily

Demographic

sociocultural

and

environmental

factors

impact

behavior

and

more

attention

must

be

given

to

these

factors

Results

from

these

surveys

indicate

that

variations

exist

in

folic

acid

awareness

knowledge

and

use

among

women

of

different

race/ethnicity

age

groups

education

levels

and

socioeconomic

status

This

suggests

that

the

linkage

between

folic

acid

awareness

knowledge

and

use

is

more

complicated

than

simply

increasing

awareness

and

knowledge

Non-white

women

those

between

the

ages

of

those

less

educated

and

those

of

a

lower

socioeconomic

status

are

associated

with

both

lower

folic

acid

knowledge

and

lower

use

of

a

vitamin

containing

folic

acid

Targeted

messages

and

teachable

opportunities

of

communication

for

younger

women

women

of

racial

and

ethnic

groups

and

women

of

low

socioeconomic

status

need

to

be

identified

and

mobilized

to

increase

the

number

of

childbearing

age

women

taking

folic

acid

daily

regardless

of

pregnancy

plans

Our

results

indicated

that

despite

the

fact

that

some

women

learn

about

folic

acid

from

their

health

care

provider

most

women

are

not

currently

following

their

health

care

provider

's

advice

even

though

they

might

feel

the

recommendation

is

important

This

is

apparent

when

looking

at

the

proportion

of

women

taking

a

multivitamin

daily

In

addition

women

report

they

do

not

take

a

vitamin

containing

folic

acid

because

they

feel

they

do

n't

need

to

or

are

not

receiving

the

folic

acid

message

from

their

health

provider

More

research

is

needed

to

understand

what

the

motivators

and

barriers

are

for

consuming

a

multivitamin

containing

folic

acid

daily

to

design

appropriate

interventions

-LRB-

whether

service

delivery

policy

media

messages

or

educational

in

nature

-RRB-

for

women

of

childbearing

age

It

is

important

to

continue

to

target

educational

efforts

at

health

care

providers

with

emphasis

on

the

importance

of

women

of

childbearing

age

taking

folic

acid

daily

to

prevent

NTDs

But

education

of

health

care

providers

may

not

be

enough

by

itself

Changing

health

care

provider

practices

is

not

merely

a

matter

of

knowledge

acquisition

Public

health

programs

need

to

forge

stronger

alliances

with

the

health

care

delivery

systems

at

the

local

and

national

level

to

develop

and

incorporate

meaningful

folic

acid

messages

into

routine

counseling

and

existing

health

programs

and

services

since

preconceptional

counseling

is

not

available

to

all

women

of

childbearing

age

Strategies

are

needed

to

understand

the

context

of

patient

and

provider

interaction

to

promote

consumption

of

a

daily

vitamin

containing

folic

acid

among

all

women

of

childbearing

age

regardless

of

whether

or

not

they

are

planning

a

pregnancy

The

findings

from

this

survey

are

subject

to

several

important

limitations

First

because

women

under

the

age

of

were

not

included

in

the

survey

the

full

extent

of

folic

acid

knowledge

and

consumption

among

the

entire

population

of

women

of

childbearing

age

is

unknown

Second

women

without

telephones

were

excluded

therefore

data

may

underestimate

the

number

of

women

of

childbearing

age

from

low

socioeconomic

groups

Third

folic

acid

knowledge

and

consumption

of

nonparticipants

might

have

been

different

from

those

of

participants

participating

women

were

more

highly

educated

than

the

total

U.S.

population

The

prevalence

of

consuming

vitamin

supplements

might

have

been

higher

among

these

women

than

among

U.S.

women

in

general

because

vitamin

consumption

is

positively

correlated

with

education

-LSB-

-RSB-

Fourthly

folic

acid

use

may

have

been

under

ascertained

since

we

did

not

have

information

on

women

's

dietary

folate

consumption

or

their

folate

levels

Further

self-report

of

dietary

intake

such

as

taking

multivitamins

could

be

biased

by

social

desirability

the

tendency

of

respondents

to

respond

in

a

way

so

as

to

avoid

disapproval

rather

than

reporting

actual

beliefs

or

actions

Finally

surveys

of

a

more

representative

sample

of

women

of

childbearing

age

in

the

United

States

are

needed

to

obtain

more

precise

estimates

of

folic

acid

use

among

this

population

Policy

and

practice

implications

Fortification

efforts

are

an

effective

method

of

reducing

the

number

of

NTD-affected

pregnancies

and

have

led

to

a

decline

in

the

number

of

NTD-affected

pregnancies

since

the

mandate

that

enriched

cereal

grain

flours

be

fortified

with

mcg

per

gram

-LRB-

g

-RRB-

-LSB-

-RSB-

Although

this

is

a

substantial

decline

it

does

not

reach

the

reduction

estimated

by

the

USPHS

in

-LSB-

-RSB-

While

the

U.S.

is

seeing

a

decline

in

the

number

of

NTDs

other

countries

have

seen

similar

or

greater

declines

In

Chile

for

example

wheat

flour

was

fortified

in

with

mcg

folic

acid

per

gram

of

the

pre-mix

already

used

in

wheat

flour

Nine

public

hospitals

in

Santiago

Chile

which

account

for

approximately

of

the

births

in

that

country

reported

a

decline

in

the

number

of

NTDs

-LSB-

-RSB-

Canada

's

flour

was

fortified

with

folic

acid

in

with

mcg

of

folic

acid

per

g

of

grain

and

certain

provinces

reported

almost

a

decline

of

NTDs

-LSB-

-RSB-

Some

countries

have

chosen

to

recommend

the

use

of

folic

acid

supplementation

for

women

of

childbearing

age

instead

of

fortification

One

study

examined

data

from

thirteen

birth

defect

registries

from

areas

in

Europe

and

Israel

with

over

cases

of

spina

bifida

and

anencephaly

to

determine

the

impact

of

recommendations

on

the

rate

of

NTDs

-LSB-

-RSB-

Although

some

countries

saw

a

slight

decline

in

rates

the

majority

of

countries

saw

no

detectable

decrease

in

the

rates

of

NTDs

using

recommendations

for

supplementation

alone

-LSB-

-RSB-

This

small

decline

implies

that

although

recommendations

might

be

an

important

aspect

of

reducing

the

rate

of

NTDs

programs

such

as

fortification

could

have

a

broader

and

more

effective

impact

on

the

overall

rates

of

NTDs

Countries

who

try

to

use

a

combination

of

methods

such

as

fortification

and

supplementation

along

with

recommendations

and

education

programs

might

see

a

greater

decline

in

the

rates

of

NTDs

Conclusions

The

Healthy

People

objectives

call

for

an

increase

to

in

the

number

of

nonpregnant

women

aged

years

consuming

daily

amounts

of

mcg

of

folic

acid

through

fortified

foods

or

dietary

supplements

-LRB-

Objective

-RRB-

and

to

increase

the

median

red

blood

cell

-LRB-

RBC

-RRB-

folate

level

to

ng/ml

among

nonpregnant

women

aged

to

years

by

-LRB-

Objective

-RRB-

-LSB-

-RSB-

Although

substantial

increases

in

RBC

folate

levels

among

women

of

childbearing

age

has

been

achieved

-LSB-

-RSB-

more

aggressive

efforts

are

needed

to

increase

the

number

of

women

consuming

mcg

of

folic

acid

daily

through

fortified

food

or

dietary

supplement

to

reduce

the

prevalence

of

NTDs

To

succeed

in

eliminating

all

folic

acid

preventable

birth

defects

beyond

what

fortification

has

already

achieved

more

must

be

done

besides

educating

women

and

their

health

care

providers

Pregnancies

and

births

affected

by

spina

bifida

or

anencephaly

have

profound

physical

emotional

and

financial

effects

on

families

and

communities

This

year

there

will

be

an

estimated

NTD-affected

pregnancies

This

study

reinforces

the

need

for

public

health

practitioners

to

mobilize

multiple

sectors

including

health

care

professionals

government

organizations

state

and

local

organizations

advocacy

groups

and

concerned

citizens

to

coordinate

efforts

and

activities

to

help

alleviate

a

devastating

public

health

issue

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

multivitamin use
folic acid consumption
childbearing age women


Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases Examination of factors regulating oocyte chromatin remodeling is crucial to circumvent embryonic aneuploidy and resulting defects . Aurora kinases -LRB- AURK -RRB- are involved in regulation of chromatin remodeling , however , little attention has been paid to AURKs in regard to oocyte maturation . Meiotically incompetent mouse oocytes contain transcripts for all three Aurk isoforms : A , B and C. Upon achieving meiotic competence , oocytes showed significant increases in transcript levels of all three Aurk isoforms and transcript levels remained unchanged as oocytes progressed through meiosis , with AurkA being the predominant isoform . Inhibition of oocyte AURKs during the prophase -- metaphase I -LRB- MI -RRB- transition via inhibitor ZM447439 -LRB- ZM -RRB- had no effect on germinal vesicle breakdown . However , meiotic spindles were malformed , and microtubule organizing centers and chromatin were scattered . Chromosomal spreads of MI oocytes indicated AURK inhibition resulted in abnormal chromosome condensation . Furthermore , inhibition of AURK during prophase I -- MII prevented completion of MII and extrusion of the polar body . Inhibition of AURKs during the MI -- MII transition resulted in significantly fewer cells progressing to MII and induced aberrant chromatin remodeling . Further analysis indicated that inhibition of AURKs resulted in absence of histone-H3 phosphorylation at serine 10 and 28 . These data suggest a ZM-sensitive AURK may be an oocyte histone-H3 kinase capable of regulating chromatin remodeling throughout oocyte meiosis , both pre - and post-MI . Introduction In vitro oocyte maturation offers immense potential for treatment of infertility , however , current systems are relatively inefficient -LRB- Tan et al. , 2007 -RRB- . Additionally , mammalian oocytes are notorious for high rates of chromosomal abnormalities -LRB- Hassold and Hunt , 2001 -RRB- , resulting in subsequent embryonic aneuploidy , infertility and congenital defects . Unfortunately , components of successful oocyte maturation and regulation of these events remains enigmatic . Therefore , understanding regulatory mechanisms involved in oocyte meiotic maturation , especially those controlling chromatin remodeling , is imperative to establish therapies to improve current assisted reproductive technologies and circumvent oocyte-derived infertility and aneuploidy-induced congenital defects . Remodeling of chromosomes during oocyte meiosis begins when homologues initially pair and condense via actions of the synaptonemal complex during initiation of prophase I , accompanied by homologous recombination and crossing-over events -LRB- Vallente et al. , 2006 -RRB- . Chromatin subsequently decondenses as oocytes enter a phase of quiescence prior to completing prophase I . In response to the pre-ovulatory gonadotropin surge , follicle-enclosed oocytes resume meiosis and homologues condense in preparation for a reductional division -LRB- Mehlmann , 2005 -RRB- . A bipolar meiotic spindle forms , consisting of polymerized microtubules , and attaches to homologues at their centromeres . Subsequently , physical contact between homologous pairs at chiasmata counteract forces pulling apart homologues , resulting in alignment of chromosomes along the metaphase plate , signaling completion of metaphase I -LRB- MI -RRB- . The meiotic spindle then facilitates separation and segregation as homologues are pulled toward opposite spindle poles at the beginning of anaphase . Oocytes progress through telophase , resulting in disproportionate cytokinesis and extrusion of the first polar body signaling completion of meiosis I -LRB- Wang and Sun , 2006 -RRB- . Subsequently , oocytes forego DNA replication and re-arrest at MII . Reversible phosphorylation , controlled via actions of protein kinases and phosphatases , is an extremely important regulator of oocyte meiosis , and well-suited to the rapid changes required during the cell cycle -LRB- see reviews , Swain , 2007 ; Swain and Smith , 2007 -RRB- . Extensive research has been conducted on the role of kinases such as cyclin-dependant kinase -LRB- CDK1 -RRB- and mitogen-activated protein kinase -LRB- MAPK -RRB- in mammalian oocytes -LRB- Motlik et al. , 1998 ; Abrieu et al. , 2001 -RRB- . Additionally , roles for protein phosphatases -LRB- PPP -RRB- in control of oocyte germinal vesicle breakdown -LRB- GVBD -RRB- -LRB- Alexandre et al. , 1991 ; Gavin et al. , 1991 , 1994 ; Swain et al. , 2003 -RRB- , spindle dynamics -LRB- Alexandre et al. , 1991 ; Lu et al. , 2002 -RRB- and chromatin remodeling have been identified -LRB- Mailhes et al. , 2003 ; Swain et al. , 2007 -RRB- . However , relatively little attention has been paid to aurora kinases -LRB- AURK -RRB- in regard to mammalian oocyte maturation . AURKs are a family of serine/threonine protein kinases responsible for regulating several mitotic cell cycle events important to maintaining proper cellular ploidy , including chromosome condensation , spindle dynamics and cytokinesis -LRB- see review , Carmena and Earnshaw , 2003 -RRB- . Thus , considering high rates of aneuploidy associated with oocyte meiosis , AURKs may play an instrumental role in the female gamete was well . Based primarily on research from lower eukaryotes and mitotically dividing mammalian cells , three AURKs exist categorized by sequence differences and subcellular localization ; AURKA , AURKB and AURKC -LRB- Nigg , 2001 -RRB- . Aurora-A has been identified in mammalian oocytes localized to nuclei in GV-intact oocytes ; to the spindle , spindle poles and condensing chromatin during MI ; and to spindle poles at MII -LRB- Yao and Sun , 2005 -RRB- . Neutralization of oocyte AURKA activity delays GVBD and distorts MI spindle organization -LRB- Yao et al. , 2004 -RRB- . Aurora-B kinase is known as the equatorial kinase and localizes to condensed chromatin during mitosis and meiosis in lower eukaryotes , where it is thought to regulate chromosome condensation -LRB- Hsu et al. , 2000 ; Murnion et al. , 2001 -RRB- and homologue separation -LRB- Kaitna et al. , 2002 ; Rogers et al. , 2002 -RRB- . Aurora-C kinase is the least studied AURK isoform , previously reported to be testes-specific with localization to centrosomes -LRB- Tseng et al. , 1998 ; Kimura et al. , 1999 ; Hu et al. , 2000 -RRB- ; though transcript for AurkC has now been identified in human cumulus -- oocyte complexes -LRB- Assou et al. , 2006 -RRB- and reports indicate that it binds to chromosomal passenger proteins during mitosis -LRB- Li et al. , 2004 ; Sasai et al. , 2004 ; Yan et al. , 2005 -RRB- . Objectives of this study were to determine which Aurk isoform transcripts are present in oocytes ; examine functional roles of AURK activity during various meiotic stages of mouse oocyte maturation , and identify AURK phosphoprotein substrates and targets of actions , focusing primarily on regulation of chromatin remodeling during the first meiosis . Materials and Methods All procedures described within were reviewed and approved by The University Committee on Use and Care of Animals at the University of Michigan and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals . Mouse stimulation and oocyte collection Meiotically incompetent GV-intact oocytes were collected from 11-day-old female CF1 mice -LRB- Harlan , Indianapolis , IN -RRB- . Meiotically competent GV-intact oocytes were collected from 20 -- 23-day-old CF1 female mice , 42 -- 44 h following injection with 10 IU eCG -LRB- Sigma , St Louis , MO -RRB- . Oocytes were isolated by manual rupturing of antral ovarian follicles in Hepes-buffered human tubal fluid medium -LRB- HTFH ; Irvine Scientific , Santa Ana , CA -RRB- supplemented with 0.3 % w/v polyvinylpyrrolidone -LRB- Sigma -RRB- . RNA isolation , reverse transcription and real-time PCR Oocyte total RNA was extracted from 50 oocytes at each development stage using Picopure RNA isolation kit -LRB- Arcturus Bioscience , Mountain View , CA -RRB- following manufacturer 's instructions . Oocyte cDNA was synthesized using 125 pmol random hexamer , 500 µM dNTP , 20 U RNase inhibitor and 62.5 U MultiScribeTM reverse transcriptase -LRB- ABI systems -RRB- in a final volume 50 µl . Primers for mouse AurkA , AurkB and AurkC were designed with no sequence overlap between isoforms -LRB- AurkA -- forward primer : 5 ′ cactagcaaagagccaacca 3 ′ , reverse primer : 5 ′ ggtggcttcaatagggtgtt 3 ′ ; AurkB -- forward primer : 5 ′ cctgacctactgccacaaga 3 ′ , reverse primer : 5 ′ gccaaagtctgcaatcttca 3 ′ ; AurkC -- forward primer : 5 ′ ctgccatgagaagaaggtga 3 ′ , reverse primer : 5 ′ gtccagagtcccacacattg 3 ′ -RRB- . Real-time PCR was performed on Applied Biosystems 7300 Real-Time PCR system . Each PCR was performed with 1.5 oocyte equivalents of cDNA added to SYBR Green PCR Master Mix -LRB- Applied BioSystems , Foster City , CA -RRB- . In addition , control reactions were conducted consisting of no template with primers and master mix . Real-time PCR reactions were carried out for 40 cycles -LRB- 95 °C for 15 s , 60 °C for 1 min -RRB- after initial 10 min incubation at 95 °C . Following PCR , products were isolated and run on a 2 % agarose gel for 60 min at 100 V to verify size of the amplified product . Additionally , DNA was isolated from gels using QIAquick Gel Extraction Kit -LRB- Qiagen , Chatsworth , CA -RRB- and subjected to DNA sequencing to verify identity of the product . Standard curve method was used to compare relative abundance of a single Aurk isoform between oocyte meiotic stages using normalization of β-actin levels . Data were collected over three replicates , with triplicate samples for each isoform and fold increases were based on meiotically incompetent GV-intact oocytes levels , which were normalized to 1 . Statistical significance was determined using unpaired Student 's t-test , P < 0.05 -RRB- . To examine relative abundance of all three isoforms within a single time point , we ensured primer efficiency of all samples were within a 5 % range of an internal β-actin control . We then analyzed data using comparative Ct method . Oocyte culture and AURK inhibition Aurora kinase inhibitor ZM447439 -LRB- ZM , Astra Zeneca , Wilmington , DE -RRB- was dissolved in dimethylsulphoxide -LRB- DMSO -RRB- to obtain a 10 mM stock . Stock solution was dissolved in HTF to obtain final concentrations of 0.625 , 1.25 , 2.5 , 5 , 10 and 20 µM . Control treatments contained DMSO . To assess effects of AURK inhibition on oocyte maturation , meiotically competent GV-intact oocytes -LRB- prophase I -RRB- were placed into culture in presence or absence of varying doses of ZM . Oocytes were assessed for GVBD and MII development at 2 and 16 h , respectively . Experiments were performed in triplicate and statistical differences in development were assessed using chi-square analysis with differences considered significant if P < 0.05 . To determine effects of AURK on chromosome condensation and spindle formation during the prophase I to MI transition , prophase I oocytes were matured in vitro to MI -LRB- 7 h -RRB- in the presence or absence of ZM -LRB- 10 µM -RRB- then subjected to immunocytochemistry -LRB- ICC -RRB- or processed for chromosome spreading . Prophase-I oocytes were also cultured to a time point consistent with MII to assess spindle and chromatin characteristics following extended AURK inhibition . To assess effects of AURK inhibition on oocyte meiosis during the MI -- MII transition , oocytes were matured for 7 or 9 h in the absence of any chemical manipulation to allow normal spindle formation and chromatin remodeling . Oocytes were then cultured to MII -LRB- an additional 9 or 7 h -RRB- in presence or absence of 10 µM ZM , followed by assessment of chromatin positioning and spindle configuration . All experiments were performed in triplicate and nonparametric parameters were analyzed for significant differences by Chi-square . Finally , to begin to determine substrates for oocyte AURK , histone-H3 phosphorylation at ser10 and ser28 was assessed following AURK inhibition at various time points utilizing ICC and western blot analysis . Immunocytochemistry To examine effects of AURK inhibition on spindle formation and metaphase chromosome positioning , MI and MII oocytes were attached to poly-lysine coated coverslips , and fixed in 2 % -LRB- w/v -RRB- paraformaldehyde with 0.05 % -LRB- v/v -RRB- Triton X-100 in phosphate-buffered saline -LRB- PBS -RRB- -LRB- pH = 7.3 -RRB- for 30 min . Oocytes were then blocked overnight with 2 % -LRB- w/v -RRB- bovine serum albumin , 0.1 M glycine and 5 % -LRB- w/v -RRB- dry milk in PBS at 4 °C . Oocytes were incubated with β-tubulin antibody -LRB- Sigma , 1:200 -RRB- and pericentrin antibody -LRB- Abcam , 1:1000 -RRB- . Negative controls included non-immune mouse serum in place of primary antibody . After three 5 min washes with blocking solution , samples were reacted with the appropriate Alexa 568 and 488 conjugated secondary antibodies -LRB- Molecular Probes -RRB- at a 1:750 dilution for 1 h at 37 °C . Following washing , slides were incubated with Hoescht 33 342 -LRB- 1 µg/ml -RRB- in PBS for 20 min at 37 °C . Coverslips were then mounted on glass slides with 90 % glycerol in PBS for fluorescence microscopic visualization under × 1000 on a confocal microscope . Chromosomal spreading and analysis Following culture to MI in ZM -LRB- 10 µM -RRB- , oocytes were collected and prepared for chromosomal spreading -LRB- Hodges and Hunt , 2002 -RRB- . Briefly , zona pellucida were removed by exposure to 1 % pronase in HTFH . Zona-free oocytes were then washed and fixed by carefully placing them onto a microscope slide dipped in a solution of 1 % paraformaldehyde in distilled water -LRB- pH 9.2 -RRB- containing 0.15 % Triton X and 3 mM dithiothreitol . Slides were then placed into a humidified chamber overnight before being subjected to triplicate 5 min washes in PBS and air-dried at room temperature . To analyze chromosomal condensation , slides were placed into a 1 % solution of Hoescht 33 342 in PBS for 10 min and subjected to three more washes in PBS . Glycerol mounting solution and a coverslip were added and slides were sealed . Chromosomal spreads were analyzed blind to treatment at × 1000 on a Leica DMR microscope . Statistical differences between treatment groups were analyzed using chi-square analysis . Electrophoresis and western blot analysis To assess effects of AURK inhibition on oocyte histone-H3 phosphorylation , groups of oocytes -LRB- n = 100 -RRB- were prepared for western blot analysis . Oocytes were placed in 2 × sodium dodecyl sulphate -LRB- SDS -RRB- -- polyacrylamide gel electrophoresis -LRB- PAGE -RRB- sample loading buffer -LSB- 80 mM Tris -- HCl -LRB- pH = 6.8 -RRB- , 20 % glycerol , 4 % SDS , 4 % β-mercaptoethanol , 0.04 % bromophenol blue -RSB- , vortexed and placed on ice for 15 min . Following sonication on ice for 10 s , samples were denatured at 90 °C for 10 min and loaded for electrophoresis . Total protein from equal numbers of mouse oocytes was loaded in each lane and separated by one-dimensional SDS -- PAGE . Resolving gels were cast using 12 % acrylamide ; stacking gels contained 5 % acrylamide . HeLa cell histone lysate was used as a positive control for recognizing phospho-ser10 . Gels were equilibrated and transferred to Hybond-P PVDF transfer membrane -LRB- Amersham Life Science , Little Chalfont Buckinghamshire , UK -RRB- by Semi-Dry Electrophoretic Transfer Cell -LRB- Bio-Rad Laboratories , Hercules , CA -RRB- according to the manufacturer 's instructions . Blots were blocked in 5 % nonfat milk in Tris-buffered saline -LRB- TBS -RRB- +0.5 % Tween -LRB- TBST -RRB- at room temperature for 1 h and incubated with the appropriate primary antibody diluted in TBST + 5 % nonfat milk overnight at 4 °C with agitation . Antibodies included anti-phospho-ser10-histone H3 -LRB- 1:1000 , Upstate -RRB- and anti-phospho-ser28-histone-H3 antibody -LRB- 1:500 , Upstate -RRB- . After complete washing in TBST , blots were incubated with the appropriate horse-radish peroxidase-conjugated IgG secondary antibody -LRB- diluted 1:2000 -RRB- at room temperature for 1 h , washed in TBST and developed with ECL Plus reagents -LRB- Amersham Life Sciences -RRB- according to the manufacturer 's instructions . To verify equal protein loading of lanes to allow densitometric analysis blots were stripped for 30 min in a 50 °C water bath with agitation in a stripping buffer -LRB- 62.5 mM Tris -- HCl , pH 6.7 , 100 mM β-mercaptoethanol and 2 % SDS -RRB- . Completely stripped blots were blocked in 5 % nonfat milk in TBST for 1 h at room temperature , then incubated with histone-H3 antibody -LRB- diluted 1:1000 , Chemicon -RRB- overnight at 4 °C with agitation and processed further as described above . Band densities were assessed using NIH imaging software , Image J. Results AURK isoforms in mouse oocytes To determine specific AURK isoforms present in mouse oocytes , isoform-specific primers were designed for AurkA , AurkB and AurkC . Transcripts were amplified utilizing real-time PCR for all three isoforms in meiotically incompetent GV-intact oocytes . Significant increases in transcript levels for all three Aurk isoforms were identified in meiotically competent GV-intact and meiotically maturing oocytes , compared with incompetent oocytes -LRB- Fig. 1 -RRB- . No differences in transcript levels were identified for any isoform between meiotically competent GV-intact , GVBD , MI or MII oocytes . Additionally , AurkA appeared to be the predominant isoform transcript , displaying an approximate 12-fold increase compared with AurkB and AurkC , which displayed comparable levels , in meiotically competent GV-intact oocytes -LRB- data not shown -RRB- . Single bands were present on agarose gels for each isoform , corresponding to predicted sizes -LRB- AurkA -- 90 bp , AurkB -- 100 bp , AurkC -- 145 bp -RRB- . Sequencing verified that amplified gene products shared 100 % homology with mouse Aurk isoforms -LRB- data not shown -RRB- . Figure 1 : Graphical representation demonstrating fold-increases of aurora kinase -LRB- Aurk -RRB- isoform levels between various oocyte types obtained from real-time PCR . Day 11 germinal vesicle intact -LRB- GVI -RRB- oocytes were used at controls and normalized to 1 . -LRB- A -RRB- AurkA , -LRB- B -RRB- AurkB and -LRB- C -RRB- AurkC . Presence of an asterick represents statistical significance compared with groups without an asterick , P < 0.05 . AURKs and oocyte meiotic progression To determine the influence of AURK on oocyte meiotic progression , meiotically competent GV-intact oocytes -LRB- prophase I -RRB- were matured in varying doses of the AURK inhibitor ZM and development was checked at 2 and 16 h. Though a significant reduction was observed with 2.5 μM , Inhibition of AURK had no effect on oocyte GVBD at 2 h at any other dose examined compared with control treatments -LRB- Table I -RRB- . Furthermore , no differences were visually apparent at the light microscope level between ZM-treated and control oocytes at a time point consistent with MI development -LRB- 7 h ; Fig. 2 , inset -RRB- . However , concentrations of ZM at 2.5 , 5 , 10 and 20 µM prevented all oocytes from progressing to MII at 16 h -LRB- Fig. 3 -RRB- . Concentrations of 1.25 and 0.675 µM allowed a small portion of oocytes to extrude the first polar body at 16 h , which was significantly less than the percentage of MII oocytes obtained from control treatments , P < 0.01 -LRB- Table I -RRB- . Additionally , culturing GV-intact oocytes for 7 h to MI in presence of 5 or 10 µM ZM , followed by thorough washing and 9 h of culture in the absence of the AURK inhibitor , indicated that , although ZM may be washed out from blocking the ATP binding pockets , defects caused by ZM treatment were not reversible , as oocytes were unable to complete meiosis and extrude the first polar body -LRB- data not shown -RRB- . Based on these data , a dose of 10 µM ZM was selected for future experiments to ensure all AURK isoforms were inhibited . This dose has been used in other studies on mammalian oocytes -LRB- Jelinkova and Kulbeka , 2006 -RRB- , and is lower than the 20 µM used in other studies -LRB- Gadea and Ruderman , 2005 -RRB- . Figure 2 : Representative micrographs demonstrating effect of aurora kinase -LRB- AURK -RRB- inhibition on oocyte meiotic spindle formation . Inhibition of mouse oocyte AURKs with 10 µM ZM447439 -LRB- ZM -RRB- for 7 h had no observable effect on oocyte development at the light microscope level -LRB- inset images -RRB- . However , immunocytochemical examination demonstrated that morphology of the meiotic spindle of ZM-treated oocytes appeared abnormal , with polymerized microtubules -LRB- β-tubulin : green -RRB- , microtubule organizing centers MTOCS -LRB- pericentrin : red/orange -RRB- and chromatin -LRB- blue -RRB- was scattered around the metaphase plate , compared with vehicle treated controls . Furthermore , 16 h treatment of oocytes with ZM resulted in arrest of oocytes at a MI-like stage -LRB- inset -RRB- . Immunocytochemical examination demonstrated aberrant polymerized microtubules , MTOCs and condensed chromatin -LRB- blue -RRB- and MTOCs -LRB- red/orange -RRB- . Polar body of control MII oocytes is indicated by PB . Figure 3 : Representative micrographs of oocyte chromosome spreads demonstrating negative impact of AURK inhibition on oocyte chromatin remodeling . Treatment of oocytes with ZM447439 -LRB- ZM-10 µM -RRB- for 7 h resulted in metaphase I oocytes -LRB- MI -RRB- with significantly greater defects in bivalent formation , compared with controls -LRB- P < 0.0001 -RRB- . Table I. Development of mouse oocytes following treatment in varying doses of Aurora kinase inhibitor ZM447439 -LRB- ZM -RRB- . ZM concentration -LRB- µM -RRB- 2 h GVBD 16 h MII 0 115/121 -LRB- 95 % -RRB- a 38/52 -LRB- 73 % -RRB- a 0.625 29/30 -LRB- 97 % -RRB- 8/30 -LRB- 27 % -RRB- b 1.25 30/35 -LRB- 86 % -RRB- 8/26 -LRB- 31 % -RRB- b 2.5 84/99 -LRB- 85 % -RRB- b 0/54 -LRB- 0 % -RRB- c 5 59/66 -LRB- 89 % -RRB- 0/28 -LRB- 0 % -RRB- c 10 55/64 -LRB- 86 % -RRB- 0/28 -LRB- 0 % -RRB- c 20 55/64 -LRB- 85 % -RRB- 0/28 -LRB- 0 % -RRB- c Significant differences in development between treatments within a time point are indicated by different superscripts , P < 0.01 . AURKs and oocyte spindle morphology and chromatin remodeling Prophase -- MI transition To begin to determine a temporal window when AURK inhibition may be conveying observed phenotypes , experiments examined the effects of AURK inhibition during the prophase I -- MI transition . Aberrant spindle morphology and improper positioning of chromatin were observed in oocytes cultured for 7 h in presence of 10 µM ZM -LRB- 19 % normal , n = 32 -RRB- compared with controls -LRB- 91 % normal , n = 35 ; P < 0.0001 ; Fig. 2 -RRB- . Similar patterns were also obtained from treatments containing 2.5 , 5 and 20 µM ZM -LRB- data not shown -RRB- . To examine effects of AURK inhibition on oocyte chromosome condensation in greater detail , chromosomal spreads of MI oocytes were examined following 7 h of ZM -LRB- 10 µM -RRB- treatment from prophase I. Inhibition of AURKs resulted in oocytes with a significant reduction in normal chromosome condensation -LRB- 0 % , n = 21 -RRB- compared with control treatments -LRB- 95 % , n = 22 -RRB- , as evidenced by the inability to resolve bivalents , P < 0.001 -LRB- Fig. 3 -RRB- . Prophase I -- MII transition Culture of oocytes for 16 h in 10 µM ZM during the prophase I -- MII transition indicated that arrest of oocytes was not due to inability of microtubule polymerization as β-tubulin staining indicated microtubules polymerized around chromatin -LRB- Fig. 2 -RRB- . Additionally , pericentrin staining indicated apparent microtubule organizing centers -LRB- MTOC -RRB- assembly . However , spindle formation and MTOC localization were disrupted compared with untreated controls . Furthermore , chromatin was scattered throughout the meiotic spindle , with the pattern of normal chromatin remodeling significantly reduced following ZM treatment -LRB- 0 % , n = 44 -RRB- compared with controls -LRB- 81 % , n = 32 -RRB- , P < 0.001 . MI -- MII transition To determine effect of AURK inhibition following normal chromatin remodeling and spindle formation , oocytes were cultured for 7 or 9 h in the absence of ZM -LRB- cells typically at MI or AI , respectively -RRB- . Subsequently , these oocytes were cultured to a time point consistent with MII -LRB- an additional 9 or 7 h -RRB- in presence or absence of ZM . Treatment of oocytes for 9 h with ZM resulted in significantly less cells progressing to MII -LRB- 53 % , n = 117 -RRB- compared with 80 % of control oocytes -LRB- n = 121 -RRB- , P < 0.05 . Treatments of oocytes for 7 h with ZM also resulted in significantly fewer cells progressing to MII -LRB- 58 % , n = 88 -RRB- , compared with controls -LRB- 73 % , n = 78 ; P < 0.05 -RRB- . All MII oocytes obtained following ZM treatment displayed scattered chromatin around the metaphase plate and slightly irregular shaped spindles -LRB- Fig. 4 -RRB- . Figure 4 : Representative micrographs of oocytes cultured to a time point consistent with MII in presence or absence of 10 µM AURK inhibitor ZM447439 during the MI-MII transition . Control MI oocytes developed normally to MII , extruding the first polar body and displaying condensed chromatin on the metaphase plate -LRB- blue -RRB- within the meiotic spindle with normally condensed β-tubulin -LRB- red -RRB- -LRB- A -RRB- . However , following AURK inhibition , a portion of oocytes arrested prior to MII , displaying scattered chromatin within a malformed meiotic spindle -LRB- B -RRB- . Those oocytes that did complete MII under AURK inhibition displayed disorganized chromatin within the meiotic spindle -LRB- C -RRB- . Similar patterns of chromatin disorganization and inability to complete cytokinesis and progress to MII were also observed following culture of oocytes to MII -LRB- 7 h -RRB- following AURK inhibition from a time point where cells are beginning to enter anaphase -LRB- E and F -RRB- compared with controls -LRB- D -RRB- . AURKs and oocyte histone phosphorylation Because of reported roles for histone-H3 phosphorylation in chromatin remodeling , phosphorylation of histone-H3 following AURK inhibition was examined as a possible cause of aberrant condensation during the prophase I to MI transition . Western blot analysis and ICC utilizing phospho-ser10 histone-H3 antibody was performed . Inhibition of AURK for 2 or 7 h with 10 µM ZM resulted in a total lack of histone-H3 ser10 phosphorylation -LRB- Fig. 5A and B -RRB- . Treatment of oocytes with ZM also completely inhibited phosphorylation of histone-H3 at ser28 , as evidenced by ICC and western blot -LRB- Fig. 6A and B -RRB- . These data raise the possibility that a ZM-sensitive AURK is an oocyte histone-H3 kinase and that histone-H3 phosphorylation may be influencing normality of oocyte metaphase chromatin condensation and subsequent separation and segregation of homologues . Figure 5 : Representative micrographs and western blot demonstrating inhibition of oocyte aurora kinases -LRB- AURK -RRB- results in ser10-histone-H3 hypophosphorylation . -LRB- A -RRB- Culture of GV-intact oocytes for 2 h to allow germinal vesicle breakdown -LRB- GVBD -RRB- in presence of 10 µM ZM447439 resulted in a total lack of ser10-Histone-H3 phosphorylation -LRB- d -RRB- , compared with controls -LRB- a -RRB- . A similar reduction in ser10 phosphorylation was observed following 7 h -LRB- MI -RRB- of culture in the presence of ZM -LRB- j -RRB- compared with controls -LRB- g -RRB- . Chromatin was stained with Hoescht and is pictured in blue -LRB- b , e , h , k -RRB- . Overlays are also indicated -LRB- c , f , i , l -RRB- . -LRB- B -RRB- Western blot analysis confirmed that ZM treatment inhibits ser10 phosphorylation -LRB- n = 100 oocytes/lane -RRB- . Figure 6 : Representative micrographs and western blot demonstrating inhibition of oocyte aurora kinases -LRB- AURK -RRB- results in ser28-histone-H3 hypophosphorylation . Chromatin is stained in blue , while phospho-ser28-histone-H3 is stained in red . -LRB- A -RRB- Culture of GV-intact oocytes for 2 h to allow germinal vesicle breakdown -LRB- GVBD -RRB- in presence of 10 µM ZM447439 -LRB- ZM -RRB- resulted in a total lack of ser28 phosphorylation -LRB- b -RRB- , compared with controls -LRB- a -RRB- . A similar reduction in ser28 phosphorylation was observed following 7 h -LRB- MI -RRB- of culture in the presence of ZM -LRB- d -RRB- compared with controls -LRB- c -RRB- . -LRB- B -RRB- Western blot analysis confirmed that ZM treatment inhibits ser28 phosphorylation -LRB- n = 100 oocytes/lane -RRB- . Discussion Maintaining integrity of chromatin remodeling is especially important in the oocyte considering its extreme susceptibility to aneuploidy , primarily during the first meiosis -LRB- Hassold and Hunt , 2001 -RRB- . AURKs are a family of serine/threonine kinases that regulate various structural elements and mechanistic events associated with the dynamic process of chromatin remodeling . Therefore , examination of AURKs during oocyte maturation is of interest when attempting to discern causative factors and molecular signaling pathways involved in aberrant oocyte chromosome modifications . We have determined that mouse oocytes contain transcripts for all three Aurk isoforms : AurkA , AurkB and AurkC and that levels of these transcripts increase significantly as oocytes gain meiotic competence , but do not change as oocytes progress through meiosis to MII . The predominant Aurk isoform transcript in meitocally competent and maturing oocytes appears to be AurkA . In agreement with these findings , during preparation of this manuscript , transcripts for AurkA , AurkB and AurkC were also reported in fully-grown immature bovine oocytes , with AurkA as the predominant isoform -LRB- Uzbekova et al. , 2007 -RRB- . To determine effects of AURK on oocyte maturation , we utilized the highly selective pharmacological AURK inhibitor , ZM . Inhibition of AURKs via ZM occurs through blockage of the ATP binding site at an adjacent cleft not present in other kinases -LRB- Ditchfield et al. , 2003 -RRB- . Although ZM is a selective inhibitor of AURKs , it does inhibit other kinases , including CDK1 , MAPK and CDC25 . However , concentrations of ZM much higher than those utilized in the majority of our studies -LRB- 20 µM -RRB- had no effect on CDK1 , CDC25 or MAPK activities in Xenopus egg extracts , indicating ZM did not directly affect these kinases , or affect any upstream regulatory kinases involved in their activation -LRB- Gadea and Ruderman , 2005 -RRB- . Furthermore , cellular characteristics and phosphorylation patterns following inhibition of CDK1 -LRB- Marchal et al. , 2001 ; Kubelka et al. , 2002 ; Swain et al. , 2003 ; Bui et al. , 2004 -RRB- and MAPK -LRB- Tong et al. , 2003 ; Yu et al. , 2007 -RRB- in mammalian oocytes are dramatically different than those observed following ZM treatment in this study ; suggesting observed effects are indeed the result of AURK inhibition . It should be mentioned that ZM displays differential inhibitory action toward different AURK isoforms , demonstrating inhibition of AURKB ∼ 20 times more potently than AURKA -LRB- Girdler et al. , 2006 -RRB- . Thus , differential phenotypes observed in our study using lower concentrations of ZM may indicate AURKB-specific functions . Future experiments will attempt to determine if lower doses of ZM , as well as utilization of other AURK inhibitors with differential inhibitory actions , can verify this and determine isoform-specific functions within the mammalian oocyte . AURK do not appear to play a role in oocyte meiotic resumption or regulation of oocyte nuclear envelope -LRB- NE -RRB- integrity during GVBD . Although ZM appeared to cause a slight delay in NE disassembly , no significant differences were apparent at any dose of ZM examined other than 2.5 μM . This is in agreement with findings that inhibition of bovine oocyte AURKs with VX680 has no effect on GVBD -LRB- Uzbekova et al. , 2007 -RRB- . However , these findings are in contradiction to a study utilizing pig oocytes , which demonstrated lack of GVBD following exposure to elevated doses of ZM -LRB- 7 -- 10 µM ; Jelinkova and Kubelka , 2006 -RRB- . Difference may be the result of varying experimental conditions or species-specific differences , as Jelinkova and Kulbelka -LRB- 2006 -RRB- utilized porcine cumulus -- oocyte complexes from abattoir ovaries , whereas the current study utilized denuded oocytes obtained from gonadotropin stimulated mice . In our study , inhibition of AURKs during 16 h of culture inhibited progression to MII and polar body extrusion , similar to developmental observations in clam oocytes following treatment with another AURK inhibitor , Hesperadin -LRB- George et al. , 2006 -RRB- , and bovine oocytes treated with VX680 -LRB- Uzbekova et al. , 2007 -RRB- . Failure to complete cytokinesis has also been observed following AURK inhibition during mitosis -LRB- Ditchfield et al. , 2003 -RRB- . Evidence exists suggesting AURKB may be the specific isoform responsible for failure of oocytes to extrude the first polar body , as specific inhibition of AURKB in Drosophila cells prevented cytokinesis -LRB- Adams et al. , 2001 ; Giet and Glover , 2001 -RRB- . Interestingly , AURKB was localized to the region of the contractile ring in bovine MII oocytes ; -LRB- Uzbekova et al. , 2007 -RRB- . However , AURKA and AURKC were also found in the vicinity of the contractile ring in bovine oocytes , confounding interpretation . Whether failure to complete cytokinesis and extrude the polar body in our study is a primary effect of AURK inhibition on cytokinesis , or a secondary effect due to chromosome remodeling or spindle defects remains to be elucidated . To begin to determine possible causes for oocyte arrest prior to MII , we examined effects of AURK inhibition on chromatin remodeling and spindle formation during the prophase I -- MI transition . Inhibition of AURKs during this time point resulted in improper positioning of chromatin , as evidenced by scattering throughout the meiotic spindle . This is in agreement with recent studies of AURK inhibition in mouse -LRB- Wang et al. , 2006 -RRB- , pig -LRB- Jelinkova and Kubelka , 2006 -RRB- and bovine oocytes -LRB- Uzbekova et al. , 2007 -RRB- reporting abnormal chromosome positioning at MI . Additionally , chromosomal spreading in our study indicates AURK inhibition negatively affects ability of MI oocyte chromosomes to condense properly and resolve bivalents . This may be due to premature decondensation , similar to that observed following AURK inhibition in Xenopus egg extracts -LRB- Gadea and Ruderman , 2005 -RRB- . It has been reported that AURKB inhibition via RNAi is responsible for chromosome misalignment in Drosophila cultured cells and results in amorphous chromatin -LRB- Adams et al. , 2001 -RRB- and only partial condensation -LRB- Giet and Glover , 2001 -RRB- . Thus , AURKB may be the AURK isoform responsible for chromatin defects observed in mammalian oocytes in this study . Indeed , a recent report demonstrates AURKB localized to condensed chromatin in MI and MII bovine oocytes -LRB- Uzbekova et al. , 2007 -RRB- . Failure of ZM-treated oocytes to progress to MII does not appear to be the result of inability to polymerize microtubules or form MTOCs , as indicated by β-tubulin and pericentrin staining . This is in contrast to AURK inhibition studies in Xenopus egg extracts , where ZM treatment resulted in failure to form the mitotic spindle -LRB- Gadea and Ruderman , 2005 -RRB- . However , fidelity of spindle function in our study remains in question , as spindle morphology and MTOC localization was disrupted following AURK inhibition . AURKs , such as AURKA , regulate several components of the spindle apparatus and spindle poles -LRB- see review , Ducat and Zheng , 2004 -RRB- . Indeed , AURKA localized to spindle poles in mouse -LRB- Yao et al. , 2004 -RRB- and pig oocytes -LRB- Yao and Sun , 2005 -RRB- , but not bovine oocytes -LRB- Uzbekova et al. , 2007 -RRB- and specific neutralization of the kinase resulted in disorganization of the meiotic spindle . This disorganization is in agreement with initial reports of ZM influences on intact somatic cells -LRB- Ditchfield et al. , 2003 -RRB- . Thus ZM inhibition of oocyte AURKA may account for observed defective spindle phenotypes . Alternatively , condensed chromosomes direct formation of the spindle apparatus via nucleation/stabilization of microtubules -LRB- Merdes and Cleveland , 1997 ; Khodjakov et al. , 2000 -RRB- . Therefore , defects in meiotic spindle morphology and scattering of chromatin observed in these experiments may be the result of aberrant chromatin condensation , possibly controlled by AURKB -LRB- see review , Shannon and Salmon , 2002 -RRB- . Reports in Xenopus mitotic cell-free extracts indicate AURK inhibition does interfere with chromatin driven microtubules assembly -LRB- Gadea and Ruderman , 2005 -RRB- . To begin to determine if the inability of ZM-treated oocytes to reach MII following AURK inhibition was due only to defects incurred during the prophase -- MI transition , or if AURKs had roles at other meiotic transition time points , we matured oocytes in vitro to time points consistent with MI and AI oocytes , thus allowing normal spindle formation and chromatin remodeling to occur . Subsequently , we then cultured oocytes in presence of ZM to a time point consistent with MII . Inhibition of AURKs during the MI-MII transition resulted in a portion of oocytes failing to segregate chromosomes and extrude the first polar body , while those that did complete MII displayed severely scattered chromatin . Thus , it appears as if AURKs may not only control spindle formation and chromatin condensation during early meiotic events , but also regulate separation and/or segregation of oocyte chromosomes during later meiosis . Effects may be directly on meiotic spindle components , possibly regulated by AURKA . Alternatively , defective chromosome remodeling could also be due to ZM inhibition of AURKB . Aurora B regulates kinetochores and their interactions with microtubules -LRB- Kaitna et al. , 2002 ; Cimini et al. , 2006 -RRB- . Another possible explanation for aberrant separation/segregation following AURK inhibition during late oocyte meiosis may be interferences with regulation of cohesion . Aurora B regulates release of chromosome cohesion during meiosis in Caenorhabditis elegans , apparently via phosphorylation of REC-8 -LRB- Rogers et al. , 2002 -RRB- . Future studies will attempt to determine if defects include premature separation of sister chromatids due to premature release of cohesion , or if aberrant phenotypes are the result of failure to separate homologous chromosomes . To begin to determine possible targets of oocyte AURKs responsible for observed defects in chromosomal remodeling , we examined the phoshorylation state of histone-H3 at ser10 and ser28 . It is thought that phosphorylation of histone-H3 may cause the histone to act as a receptor or recruitment factor for condensation factors -LRB- Hirano , 2000 -RRB- , or possibly reduce the affinity of histone-H3 for DNA and make the relatively compact chromatin fiber more readily accessible to remodeling factors -LRB- Hirano , 2000 -RRB- , such as the condensin complex . Condensin is a multi-subunit protein complex that play a central role in chromosome compaction and condensation and is reported to co-localize and bind with phosphorylated histone-H3 -LRB- Schmiesing et al. , 2000 ; Ball et al. , 2002 -RRB- . Interestingly , AURKB controls association of condensin with chromosomes during mitosis -LRB- Lipp et al. , 2007 ; Takemoto et al. , 2007 -RRB- , and may be functioning in a similar manner during mammalian oocyte meiosis , though this remains unknown . It should be mentioned that reports for the requirements of histone-H3 phosphorylation in chromatin condensation varies greatly -LRB- Van Hooser et al. , 1998 ; Wei et al. , 1998 , 1999 ; Goto et al. , 1999 ; Kaszas and Cande , 2000 ; Schmitt et al. , 2002 -RRB- . These contradictions also appear to hold true in regard to oocyte meiosis , with differing reports on temporal and spatial localization and correlation with condensation -LRB- Jelinkova and Kubelka , 2006 ; Wang et al. , 2006 ; Swain et al. , 2007 -RRB- . However , contradictions may be explained by differences in experimental conditions or perhaps species-specific differences . Regardless , contradictions in the literature make it increasingly evident that differences in the role of histone phosphorylation exist depending on the organism and/or type of cellular division examined -LRB- Fuchs et al. , 2006 -RRB- , and that the specific histone-H3 AURK within mammalian oocytes remains unknown . In summary , these studies indicate AURK plays a significant role in mouse oocyte maturation involving progression to MII , acting in both early -LRB- prophase -- MI transition -RRB- and late meiotic events -LRB- MI -- MII transition -RRB- . When AURK is inhibited in early meiosis , microtubules polymerize and MTOCs form , but spindle morphology and MTOC localization is disrupted . Furthermore , metaphase chromatin does not condense or position normally . Inhibition of oocyte AURKs during late meiosis -LRB- MI -- MII transition -RRB- , following chromatin condensation and spindle formation , negatively affects separation and segregation of chromosomes . This aberrant chromatin remodeling following AURK inhibition in oocytes appears to be due , in part , to hypophosphorylation of histone-H3 at both ser10 and ser28 . We report amplification and sequencing of all three Aurk isoforms transcripts : AurkA , AurkB and AurkC . Levels of these transcripts increase as oocytes achieve meiotic competence , with AurkA being the predominant isoform . Future studies will focus on determining protein expression levels of AURK isoforms , as well as specific functional roles and intracellular targets . Funding Funding for research by Gary D. Smith provided by NIH RO1 grant #HD046768 -01 A2 . Support for Jason E. Swain provided by an NIH T32 grant . Partial support for Jingwen Wu provided from the Lyle C. Roll Research Fund to Professor James O. Woolliscroft , Dean of Medical School , University of Michigan .

O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

chromosomal disorders
signal transduction