the prostaglandin transporter (pgt): a novel …cme-utilities.com/mailshotcme/material for...

1

Prof. Ariel Hourvitz

Reproduction Lab and IVF Unit-Sheba Medical Center

Sackler school of medicine, Tel Aviv University

The Prostaglandin Transporter (PGT): A novel indispensable mediator of

ovulation

From Basic Research to Clinical Implications

• Consultant to EarlySense®

Disclosure

Clinical Reports – First IVM Pregnancy

ED Program

270 Oocytes were aspirated from 23 ovaries

ET of 5 embryos to a woman with POF

Triplets pregnancy

1: Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon

Pregnancy after in vitro fertilization of human immature oocytes collected from non-stimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril. 1991 Jan;55(1):109-13.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1986950&query_hl=2&itool=pubmed_DocSum




IVM Protocols First US-day 2-3 (AFC)

Second US- day 6

hCG administration when leading follicle is 12-14 mm and ES>6 mm

OPU day 10-14

Estrogen supplementation

4

1 2 3 4 5 6 7 8 9 Day of Cycle

AFC

Hormonal

Profile

4 mm 10 mm 12-14 mm

OPU

Leading Follicle: 12-14 mm

ES>6mm

hCG

6 mm

FSH

In vitro maturation (IVM) of oocytes

Advantages:

• Does not require gonadotropin stimulation

• Shorter treatment

• Less expensive

• Simpler

• Safer, avoiding OHSS

Unfortunately …

• Maturation rate ~50-70%

• reduced developmental potential

• Lower fertilization rate

• Lower pregnancy and live birth rates 5

IVM / IVF

Not only a treatment tool……

Hypothesis:

• Using the IVM / IVF model we generated a cDNA library of genes significantly up or down-regulated during the final stages of in vivo maturation and ovulation.

• We hypothesize that these regulated ovulatory genes might be critical determinants of ovulation and oocyte developmental potential.

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Materials and Methods

Ovu

latio

n

Primordial Primary Secondary Pre- Antral Pre- Antral Ovulatory

3-5 Months

FSH Independent

14 days

FSH Dependent

IVM IVF

GV-IVM

sample

M2-IVF

sample

Flow chart of

RNAseq • Total RNA extraction

• cDNA library

preparation

• WTA

• Raw read whole

library

• Transcripts

assembly

• Align reads to

quantify expression

• Bioinformatics

analysis

Different signature for GV-IVM and M2-IVF samples

• Cluster analysis of relative expression of RNAseq samples from GV-IVM and M2-IVF

• 1100 genes were significantly upregulated in the expanded CC cells and 646 were downregulated

GVCOC1 GVCOC2 MIICOC MIICOC MIICOC

Yerushalmi et al., MHR, 2014

Major processes during cumulus maturation/expansion

Ingenuity IPA analysis

Yerushalmi et al., MHR, 2014

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Prostaglandin transporter (PGT)

regulation of PGE2 signaling is

essential for ovulation

Why did we choose to

explore Prostaglandin

Transporter (PGT)?

1. PGT is among the highly up-regulated genes in Expanded Cumulus

Cells Obtained from MII Oocytes (CCMII) Compared With Compact

Cumulus Cells Obtained from GV Oocytes (CCGV)

Base Mean Sample

83.6 CCGV

32825.2 CCMII

Fold change:392.3

RNAseq

0

5

10

15

20

25

30

35

*

CCGV CCMII

qRT-PCR

0.3

26.9

• Prostaglandins (PGs) have been recognized as key mediator of ovulation for more than 30 years.

• Ovarian PGs production is increased in response to the LH surge.

• PGs biosynthesis is under the control of the Cyclooxygenase (COX) enzyme.

• PGE2 is the major COX-2 product that mediates the ovulation process.

• Disruption of PGE2 production or PGE2 receptor

expression prevent ovulation.

• PGE2 is essential for follicle rupture, oocyte release, cumulus expansion.

2.PGT involved in the transport of

Prostaglandins

• PGs are organic anions and diffuse poorly

through plasma membranes.

• Their diffusion rate is too low to initiate biological

response.

• Carrier-mediated transport mechanism is needed

for PGs to cross the biological membranes

3. There is no report on PGT expression,

regulation and function in the ovary.

In vivo Expression Pattern of

PGT mRNA in Human

Granulosa Cells

Expression of PGT mRNA in Cumulus (CGCs)

and Mural Granulosa Cells (MGCs)

NS

Yerushalmi et al., Science Trans Med, 2016

PGT mRNA Expression Pattern During the

Late Antral Follicle Development in mGCS

Preovulatory

(>17 mm)

0

5

10

15

20

25

PG

T/β

-actin m

RN

A (

*10^3

)

Small

(<10 mm)

Large

(10–14 mm)

a

b

c

PGT mRNA Expression in CGCs According to

Maturation Stage of the Corresponding Oocyte.

0

5

10

15

20

25

GV MI MII

PG

T/β

-actin m

RN

A (

*10

^3)

a a

b

In vitro PGT Regulation in

Human Granulosa Cells

hCG Effect on PGT Expression in Cultured

MGCs Cells

0

10

20

30

40

50

60

Control 1U 10U

b

b

a PG

T m

RN

A (

fold

change)

hCG

Control 1U 10U

34 46

a b

Control 1U/ml hCG Yerushalmi et al., Science Trans Med, 2016

Early Antral Post ovulatory Corpus Luteum

CL

CL

CL

GC

GC

GC

A

A

A

A

A

A

GC T

T

T GC

A-Antrum GC-granulosa cells T-Theca cells V-Luteal blood Vessels

A D H

B E I

C F J

V

V

T

T

T T

G

PGT expression in the human ovary during folliculogenesis

Intracellular Signaling Pathways Regulating

PGT Gene Expression: cAMP,PKC Pathways

0

20

40

60

80

100

120

140

FSK Control hCG PMA FSK

+

PMA

PG

T m

RN

A (

fold

change)

b

b

a

b

ab

Intracellular Signaling Pathways Regulating

PGT Gene Expression: cAMP-PKA pathway

0

5

10

15

20

25

30

FSK Control hCG

+

H89

hCG FSK

+

H89

PG

T m

RN

A (

fold

change)

b

b

a

a a

Intracellular Signaling Pathways Regulating PGT

Gene Expression: PI3K/Akt, MAPK pathways P

GT

mR

NA

(fo

ld c

hange)

0

5

10

15

20

25

30

35

U0126

+

hCG

Control LY294002

+

hCG

hCG

a

b

b

a

Intracellular Signaling Pathways Regulating PGT

Gene Expression: PKA, PKC and MAPK pathways

0

2

4

6

8

10

12

14

16

Cont FSK FSK+U0126 PMA PMA+U0126

PG

T m

RN

A (

fold

change)

a

b

a a

c

• In the preovulatory follicles PGT is induced in

response to LH surge.

• LH-induced expression

of PGT is mediated by

PKA and PKC pathways

• Both pathways signal through

the activation of the

MEK-ERK pathway.

Summary & Conclusions


In vitro PGT Function in

Human Granulosa Cells

• PGT has been identified as major PGE2

transporter.

• Three possible roles were postulated for PGT:

1. PGT mediate the efflux of newly-

synthesized PGs from cells.

2. PGT mediate the influx of PGs for

their inactivation.

3. PGT mediate vectorial PG transport .

Prostaglandin Transporter (PGT)

1.

2.

3.

PGT Mediated Uptake of PGE2

0

50

100

150

200

250

300

Control hCG hCG

+

BCG

BCG

PG

E2 p

g/m

l

a a

b

c


Time- Course of PGT Mediated PGE2 Uptake

0

20

40

60

80

100

120

140

160

180

200

0 10 20 30 40 50 60 70

Cont

hCG

[3H

]PG

E2 U

pta

ke (

cpm

)

Time (min)


33 33 33

hCG

PGE2

COX2

Cytoplasm

PGT

PGE2

Uptake

In Vivo Mouse Model

The Role of PGT in the

Ovulatory Process

Superovulation Protocol

10U PMSG

48h

10U hCG

23-25 day old

48h

10U hCG+ DIDS 10U PMSG

16h

Collection of

oocytes from

oviducts

PGT mRNA Expression Pattern in the Mouse

Ovary Throughout the Pre-ovulatory Period

0

5

10

15

20

25

30

Saline 0h 3h 6h 9h 12h

PG

T/β

-actin m

RN

A (

*10^3

)

hCG

a

b b

c

c

b

Dose- dependent Inhibition of hCG-induced

Ovulation by DIDS in PMSG-primed mice

Range of

oocytes in

ovulating

mice

Mean no.

ova/ovulating mice

No. ovulating

mice/no. treated

(%)

10-111 56.2±6.6 16/16 (100%) hCG

55-74 60.6±3.5 5/5(100%) hCG+Vehicle

- 9 1/25 (4%) hCG+DIDS 50

mg/kg

1-51 22±6.8 7/12 (58.3%) hCG+DIDS 20

mg/kg

10-60 27.4±6.8 9/13 (69.2%) hCG+DIDS 10

mg/kg

7-77 53.5 ±5.1 12/12 (100%) hCG+DIDS 5

mg/kg


Inhibition of Ovulation with BCG

Range of

oocytes in

ovulating mice

Mean no. ova/ovulating

mice

No. ovulating

mice/no. treated

(%)

10-111 56.2±6.6 16/16 (100%) hCG

- 0 0/4 (0%) hCG+BCG 500mg/kg

- 0 0/4 (0%) hCG+BCG 350mg/ml

- 1 1/4(25%) hCG+BCG 300mg/ml

12-61 58.4±7.4 15/16(93.8%) hCG+BCG 250mg/ml

Ovulation cascade

antral follicles

preantral follicle

Ovary cortex

cortical primordial follicles

• Oocyte maturation

• Luteinization

• Cumulus

expansion

• Follicular rupture

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Induction of ovulation in mice-Ovarian Histology

48hr PMSG

X40

X100

Thick wall X200

Compact cumulus

X100

X1000

Intact granulosa and Theca Layers

Cumulus

Expansion

X40

GC invasion and membrane

thinning

X40

Follicle rupture

X40

Induction of ovulation in mice-Ovarian Histology

post

hCG

(12-16h)

PGT inhibition by DIDS block hCG effects (12hr)

hCG+DIDS

Intact GC and theca layers Compact CC + GV oocyte

hCG

GC invasion and membrane thinning Cumulus expansion


PGT inhibition by DIDS block hCG effects (16hr) hCG

Early corpus luteum

hCG+DIDS

Compact CC + GV oocyte

PGT inhibition by DIDS block hCG effects (40hr) hCG

Corpus luteum

hCG+DIDS

Compact CC + GV oocyte


Progesterone levels


• LH targets

Molecular effect of inhibition of PGT by DIDS on Ovulation

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0

500

1000

1500

48h PMSG 3h hCG 3hhCG+DIDS

AREG

/β-a

ctin

mR

NA

p<0.001 p<0.001

0

200

400

600

800


EREG

/β-a

ctin

mR

NA

p<0.001 p<0.001

• LH targets

• Cumulus expansion and ovulation


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0

0.5

1

1.5

2

2.5

PMSG 3h hCG 3hhCG+DIDS

AD

AM

TS1

/β-a

ctin

mR

NA

p=0.002 p=0.002

0

0.5

1

1.5

2

2.5

3


CTS

L/β

-act

in m

RN

A

p=0.016 p=0.018

0

100

200

300

400


TNFA

IP6/β

-act

in m

RN

A

p<0.001 p<0.001

• LH targets

• Cumulus expansion and ovulation

• Steroidogenesis


48

0

0.2

0.4

0.6

0.8

1

1.2

1.4


p4

50

SCC

/β-a

ctin

m

RN

A NS

0

2

4

6

8

10

12

14


StA

R /β

-act

in m

RN

A p=0.046 p=0.042

• PGT blockage results in – Complete blocking of all ovulatory processes – Inhibition in the molecular level

• PGT is responsible for PGE2 influx in granulosa cells • PGT blocking leads to accumulation of extracellular PGE2.

Summary

49

Mechanism?

• Elevated extracellular PGE2 levels leads to EP receptor desensitization.

• This effect may be mitigated by PGT expression.

• PGT is a modulator of EP receptor signaling

Chi et al., 2014

Surface EP4

PGE2↑↑

PGT

PGT

PGE2

Surface EP4

Sensitized cell

Surface EP4

Internalized EP4

PGT

Desensitized cell

PGT

PGE2

Surface EP4

Internalized EP4

PGE2

PGE2 desensitization in Granulosa cells culture

• Primary granulosa cells were treated with PGE2. • cAMP levels were measured after 10 min to estimate

EP receptor activity (short term desensitization). • EP4 receptor levels following incubation with PGE2

for 24h (long term desensitization).

hCG+PGT inhibitor

COX-2

PGE2 PGE2

PGE2 receptor desensitization

PGT

Blocked PGE2 signaling

No ovulation

hCG

COX-2

PGE2 PGE2

Ovulation

Normal PGE2 Signaling

PGT

Modeling the role of PGT in LH/hCG-induced ovulation

PG receptor desensitization

LH-PG signaling in ovulation

Heng-Yu Fan, et al., Science 2009

Ben-ami et al. MHR 2006

Shrestha K et al. Reproduction 2015

LH

EGF like (AREG, EREG)

ERK 1/2

FSH action Ovulation

Oocyte maturation

COC expansion

Follicle rupture

Luteinization

EP2 signaling

PGT

EGF like (AREG, EREG)

TNFAIP6 StAR

PGE2↓ PGE2↑

COX-2

Sensitized

cell

PR ADAMTS1

CTSL

Translational therapeutic significance

Non hormonal contraception – the PG cascade

The combined approach

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Rubens Fadini

Giovanni Cotticio

Mariabeatrice Dal Canto

Fausta Brambillasca

Biogenesi Reproductive Medicine

Centre, Istituti Clinici Zucchi

Monza, Italy

Collaborators:

IVF Unit

Sheba Medical

Center

http://www.ramat-gan.muni.il/NR/rdonlyres/66E61DC3-BDCC-4897-B9A0-4DFEA178AD2A/0/ביתחוליםש2יבא.jpg

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Reproduction Research Lab

Svetlana Merkman

Yuval Yung

Gil Yerushalmi

Ettie Maman

the prostaglandin transporter (pgt): a novel …cme-utilities.com/mailshotcme/material for...

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