Meccanismi molecolari di infertilità femminile nel bovino ... · Meccanismi molecolari di...

I Seminari del Dipartimento 30 November 2012

Meccanismi molecolari di infertilità

femminile nel bovino e nel suino

Fulvio Gandolfi Dipartimento di Scienze Veterinarie per la Salute, la Produzione

Animale e la Sicurezza Alimentare

OOCYTE QUALITY

Why is it so important?

Department of Health, Animal Science and Food Safety (VESPA)

Department of Health, Animal Science and Food Safety (VESPA)

Development is the result of a continous series

of steps each depending on the previous one

Oocyte quality is the first and fundamental step

Department of Health, Animal Science and Food Safety (VESPA)

Follicle development last AS

2 thirds of pregnancy

Folliculogenesis is controlled both within and

outside the ovary

Department of Health, Animal Science and Food Safety (VESPA)

Department of Health, Animal Science and Food Safety (VESPA)

L’animale pre-pubere

I contaminanti ambientali L’uso dell’AMPc

L’mRNA materno

La prima clonazione di un gene di pluriptenza

negli animali domestici

Department of Health, Animal Science and Food Safety (VESPA)

2012: dal laboratorio al campo

Il ruolo delle infezioni croniche

Department of Health, Animal Science and Food Safety (VESPA)

Perché si elimina una bovina

Department of Health, Animal Science and Food Safety (VESPA)

Mastite e fertilità in letteratura

Mastite prima di FA

Mastite dopo 1a FA, dopo gravidanza o nessuna mastite

Mastite prima di FA

Mastite dopo 1a FA

Mastite dopo gravidanza o nessuna mastite

Department of Health, Animal Science and Food Safety (VESPA)

Barker AR, Schrick FN, Lewis MJ, Dowlen HH, Oliver SP. Influence of

clinical mastitis during early lactation on reproductive performance

of Jersey cows. Journal of Dairy Science 1998;81: 1285-1290.

Schrick FN, Hockett ME, Saxton AM, Lewis MJ, Dowlen HH, Oliver SP.

Influence of subclinical mastitis during early lactation on

reproductive parameters. J Dairy Sci 2001;84: 1407-1412.

Santos JEP, Cerri RLA, Ballou MA, Higginbotham GE, Kirk JH. Effect of

timing of first clinical mastitis occurrence on lactational and

reproductive performance of Holstein dairy cows. Animal

Reproduction Science 2004;80: 31-45.

Hansen PJ, Soto P, Natzke RP. Mastitis and fertility in cattle - possible

involvement of inflammation or immune activation in embryonic

mortality. Am J Reprod Immunol 2004;51: 294-301.

Department of Health, Animal Science and Food Safety (VESPA)

Mastite e fertilità in letteratura:

pochi i lavori disponibili

1. Reproductive efficiency was decreased by the

presence of clinical mastitis alone because a greater

proportion of cows with mastitis remained non-

pregnant over time.

2. Moreover, a greater proportion of cows with mastitis

or diseases remained non-pregnant by 224

postpartum.

3. Furthermore, the negative effects on reproduction

were exacerbated when cows experienced both

clinical mastitis and other diseases.

1. Clinical mastitis before first service is a negative factor

that increases the days to first service and the days to

conception in dual-purpose cows under tropical

conditions.

2. Therefore, measures for mastitis prevention should be

implemented to reduce the effect of this additional

factor over the postpartum anestrus incidence in dual-

purpose cows.

3. Further research to understand the mechanism of the

association between mastitis and reproduction should

be granted.

Mastite e fertilità: le conferme

Department of Health, Animal Science and Food Safety (VESPA)

2009

2010

QUESTI LAVORI SONO DESCRITTIVI E

STABILISCONO UNA CONNESSIONE TRA

MASTITE E FERTILITÀ

Ma quali meccanismi collegano la mastite con la fertilità?

Department of Health, Animal Science and Food Safety (VESPA)

I possibili bersagli della mastite

Department of Health, Animal Science and Food Safety (VESPA)

La mastite ha un effetto

sull’attività ovarica?

Department of Health, Animal Science and Food Safety (VESPA)

Correlation between chronic mastitis and

ovarian activity in dairy cattle:

Grading of animals

based on mastitis

intensity

Somatic cell count

Bacteriological score

Morphological and

molecular analysis of

the ovaries

Macroscopic analysis

Stereology

Image analysis

Immuno-localization

Experimental design

Department of Health, Animal Science and Food Safety (VESPA)

Aseptic collection from

udder quarters

Immediately stored at

+4°C

Post-slaughter pairs

of ovaries of 74 animals

Stored with saline

solution set at 32-34°C

Milk

Ovaries

Sample collection

Department of Health, Animal Science and Food Safety (VESPA)

Grading of mastitic infection

by NMC procedures

Department of Health, Animal Science and Food Safety (VESPA)

Animals were distributed in 4 classes

based on pathological degree

Department of Health, Animal Science and Food Safety (VESPA)

Follicle count

Department of Health, Animal Science and Food Safety (VESPA)

Whole slide

images

MacBiophotonics

Image J

Standardization of

surface area

Results expressed

as Follicle/cm2

Stereological procedure

Department of Health, Animal Science and Food Safety (VESPA)

Mastitis causes an

altered transition

from primary to

secondary follicles

Department of Health, Animal Science and Food Safety (VESPA)

Mastitis causes a

significant

decrease in the

density of blood

vessels in the

cortical area

Department of Health, Animal Science and Food Safety (VESPA)

Mastitis

causes a

significant

increase of

fibrous stroma

Department of Health, Animal Science and Food Safety (VESPA)

Localization and expression of GDF-9

Gilchrist RFD 2011

Department of Health, Animal Science and Food Safety (VESPA)

Mastitis

inhibits

GDF-9

expression

Department of Health, Animal Science and Food Safety (VESPA)

Conclusioni

Department of Health, Animal Science and Food Safety (VESPA)

La mastite ha un effetto sull’attività

ovarica?

SI Department of Health, Animal Science and Food Safety (VESPA)

Ipotesi di lavoro

Questi effetti sono mediati da sostanze liberate dal processo

infiammatorio

Hansen 2004 AJRI

Department of Health, Animal Science and Food Safety (VESPA)

Effetto di sostanze liberate durante

un’infiammazione sulla qualità dell’ovocita

Soto P ey al. (2003) Am J Reprod Immunol, 50: 263-272.

Lipopolisaccaride PGF2a

Ossido Nitrico

Lipopolisaccaride PGF2a

Ossido Nitrico

Lipopolisaccaride PGF2a

TNF-a

Department of Health, Animal Science and Food Safety (VESPA)

Il ruolo dello stress ambientale

Department of Health, Animal Science and Food Safety (VESPA)

Enne, G., P. V. Beccaro, and C. Tarocco. 1977. Fertility in swine.

Summer infertility in a herd in the Po valley. Clin. Vet 100(11):794-

803.

Enne, G., P. V. Beccaro, and C. Tarocco. 1979. A note on the effect

of climate on fertility in pigs in the Padana (Po) valley of Italy. Anim

Prod 28(1):115-117.

Department of Health, Animal Science and Food Safety (VESPA)

L’infertilità stagionale nella

scrofa

Nucleous

HSF-1 monomeric form (inert)

HSF-1 trimeric form

(active)

HSE

Cytosol

HSP

mRNA HSP70

HSP40

NEF

Thermal stress

Misfolded protein

Folded protein

HSP40

HSP70

HSP40

HSP70 NEF

Misfolded protein

Misfolded protein

The HSP chaperone machinery

What happens in

porcine ovary?

Is HSP chaperone machinery

expressed?

Department of Health, Animal Science and Food Safety (VESPA)

The HSP machinery is expressed in pig ovary

Nucleotide Exchange Factors

Heath Shock Transcription Factors

Department of Health, Animal Science and Food Safety (VESPA)

Immunoistochemical analisys showed an

ubiquitary localization

Department of Health, Animal Science and Food Safety (VESPA)

Functional response studies

Ovaries exposed to 42°C for 1 h

In vitro maturation

(IVM)

Semiquantivative

analysis

COCs isolation from follicles of 3

to 5 mm

Ovaries maintained at 37°C (CTR)

COCs isolation from follicles of 3

to 5 mm

In vitro maturation

(IVM)

Semiquantivative

analysis

Ovaries collected at the slaughterhouse

Statistical analysis was performed by t-student test

Department of Health, Animal Science and Food Safety (VESPA)

Functional response studies: IVM

p < 0,05 0

10

20

30

40

50

60

70

80

90

MII MI GV Degenerated

CTR

Treated

*

*

* *

Control group Treated group

Department of Health, Animal Science and Food Safety (VESPA)

Functional response studies:

Semi-quantivative PCR

Oocytes

Granulosa Cells

Department of Health, Animal Science and Food Safety (VESPA)

Functional response studies:

western blots

Oocytes

Granulosa Cells

Department of Health, Animal Science and Food Safety (VESPA)

Seasonal effect on in vivo Expression

of HS genes

Oocytes

Granulosa Cells

Department of Health, Animal Science and Food Safety (VESPA)

Department of Health, Animal Science and Food Safety (VESPA)

Seasonal effect on in vivo Expression

of HS proteins

Oocytes

Granulosa Cells

Conclusions

An HSP complex is present in the oocyte and in the follicle

Specific localization in oocytes, granulosa and theca cells.

Thermal stress induces a specific mRNA synthesis up-regulation of Hsp

chaperone machinery in the oocyte but not in the cumulus cells.

Comparison of ovarian tissues collected at different times of the year,

revealed that thermal stress is directly involved in infertility during

the hot season.

Can HSP be stimulated?

Department of Health, Animal Science and Food Safety (VESPA)

Department of Health, Animal Science and Food Safety (VESPA)

Department of Health, Animal Science and Food Safety (VESPA)

RADIOTERAPIA E FERTILITA’

SALVAGUARDIA DELLA FERTILITA’

FEMMINILE

Congelamento ovarico

Innesti corticali VS ovaio intero

CONGELAMENTO

CONVEZIONALE

CONGELAMENTO

DIREZIONALE

DISEGNO SPERIMENTALE

CONGELAMENTO CONVEZIONALE (Planer)

Planer, The Kryo 560M -1

(Planer PLC, United Kingdom)

Il metodo di congelamento convenzionale prevede l'abbassamento graduale della

temperatura in una camera di congelamento.

PROBLEMA CALORE LATENTE

+ 2°C

+ 0 °C - 5 °C - 5 °C

(A.Arav, US Patent 5,873,254)

CONGELAMENTO DIREZIONALE

(Multithermal gradient ™ freezing)

Multithermal gradient ™ (MTG) freezing

•Permette di generare un gradiente di temperatura lineare in un organo/tessuto da

congelare;

• Il trasferimento di calore ha una direzione opposta a quella del campione durante il

congelamento;

• Il congelamento avviene attraverso un gradiente lineare di temperatura in modo che

la velocità di raffreddamento e la propagazione del ghiaccio possone essere

precisamente controllati.

Cooling Rate 1 = V(T1-T0)/d1

T1

T2

T3

T4

- 70°C - 40°C + 4°C

DISEGNO SPERIMENTALE

PLANER

AZOTO

MTG

AZOTO

OVAIO

INTERO

CONCLUSIONI

• I risultati mostrano come dopo congelamento dell’ovaio intero con PLANER il tessuto

ovarico risulta danneggiato. Anche con una corretta perfusione del crioprotettore, dopo il

congelamento convezionale, si osservano danni vascolari già a livello dei vasi di grosso

calibro e una generale diminuzione del numero di follicoli e un’ incapacità da parte di questi

di riprendere lo sviluppo.

• Al contrario, il congelamento dell’ovaio intero mediante il metodo direzionale, sembra

preservare in modo ottimale il tessuto ovarico. A livello di vascolarizzazione non si sono

osservati grossi danni e la maggior parte dei follicoli mostra una morfologia normale e una crescita

follicolare molto simile al tessuto fresco.

Il congelamento dell’ovaio intero mediante MTG preserva meglio il

tessuto ovarico rispetto al congelamento con PLANER

Lo strumento è a disposizione di chiunque sia interessato

La squadra

Tiziana Brevini Georgia Pennarossa

Sara Maffei

Araianna Vanelli

Mahabub Rahaman

GRAZIE PER L’ATTENZIONE

Con quali meccanismi?

Blocca lo sviluppo follicolare sia nella fase sotto il

controllo intraovarico che nella fase a regolazione

endocrina

Department of Health, Animal Science and Food Safety (VESPA)

Con quali meccanismi?

Porta ad una regressione del letto vascolare

Induce una trasformazione fibrotica

Inibisce l’espressione dei fattori che stimolano lo sviluppo follicolare

Altri meccanismi sono sicuramente attivi e richiedono ulteriori studi

D

B

C

Department of Health, Animal Science and Food Safety (VESPA)

VELOCITA’ DI CONGELAMENTO: 0.1 mm/s

CONGELAMENTO

-80

-70

-60

-50

-40

-30

-20

-10

0

10

0 2 4 10 20 30 40 50 60 70 78

TEM

PER

AT

UR

A (

°C

)

MINUTI

MTG

-120

-100

-80

-60

-40

-20

0

20

40

0 1 5 6 14 24 34 44 54 64 74 84 94 98 102 106

TEM

PER

AT

UR

A (°

C)

MINUTI

PLANER

-0,5 ºC/min da +4 ºC a -40ºC;

-5 ºC/min da -40ºC a -100ºC.

1. Analisi Morfologica

2. Studi di Vascolarizzazione

3. Sviluppo Follicolare

PLANER VS MTG

1. ANALISI MORFOLOGICA: midollare MTG

MTG

PLANER

PLANER

1. ANALISI MORFOLOGICA: corticale

MTG PLA

NER

2. STUDI DI

VASCOLARIZZAZIONE

VASCOLARIZZAZIONE: perfusione

ovaio con indian ink

1 2

3 4

FRESH

Small vessels

(3µm-20 µm)

Medium

vessels

(21µm-75 µm)

Large vessels

(<75µm)

MTG PLANER

Scale bars 50µm

P VALUE < 0,05

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

SMALL VESSELS MEDIUM VESSELS LARGE VESSELS

% P

ERFU

SED

VES

SELS

CORTEX

FRESH

PLANER

MTG

a a

b

a

b

a

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

SMALL VESSELS MEDIUM VESSELS LARGE VESSELS

% P

ERFU

SED

VES

SELS

MEDULLA

FRESH

PLANER

MTG

a a a

a a a

b b b

3. SVILUPPO FOLLICOLARE IN VITRO

Coltura di frammenti di corticale ovarica (2mmx2mm)

Durata coltura: 7 giorni

2. SVILUPPO FOLLICOLARE

P VALUE < 0,05

0

10

20

30

40

50

60

70

80

90

100

FRESH PLANER MTG

%

MORPHOLOGY NORMAL FOLLICLE

0 DAY

7 DAY

a

a

b

c

a

a

2. SVILUPPO FOLLICOLARE

0

10

20

30

40

50

60

70

80

90

100

0 DAY 7 DAY

%

MTG

Primordial

Intermediate

Primary

a

c

e

b

d f

0

10

20

30

40

50

60

70

80

90

100

0 DAY 7 DAY

%

PLANER

Primordial

Intermediate

Primary

a

c e

b d

f

0

10

20

30

40

50

60

70

80

90

100

0 DAY FRESH 7 DAY FRESH

%

FRESH

Primordial

Intermediate

Primary

c

e

b

d f

a

P VALUE < 0,05

STUDI FUTURI

MTG VS PLANER

• Validare i risultati ottenuti in vitro mediante studi in vivo.

Corticale ovarica VS ovaio intero

• Analisi morfologia dei tessuti trapiantati nei topi;

• Valutazione crescita follicolare;

• Analisi vascolarizzazione del tessuto trapiantato.

Mastite e fertilità, i primi dati

Mastite prima di FA

Mastite dopo 1a FA, dopo gravidanza o nessuna mastite

Mastite prima di FA

Mastite dopo 1a FA

Mastite dopo gravidanza o nessuna mastite

Department of Health, Animal Science and Food Safety (VESPA)