Upload
tranngoc
View
216
Download
0
Embed Size (px)
Citation preview
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)