Cumulin: a new oocyte‐secreted factor of potential use as an IVM media additive

Cumulin: a new oocyte‐secreted factor of potential use as an IVM media additive

David G. Mottershead

Mottasis Oy Ltd, Helsinki, Finland Robinson Research Institute,

School of Medicine, University of Adelaide, Australia.

Oocyte-Secreted Factors GDF9 & BMP15 Regulate Granulosa Cells and Oocyte Developmental Competence

Gilchrist RB (2011) Reprod. Fert. Dev. 23:23-31

GDF9 and BMP15 as TGF-β family members

pro/mature forms

GDF9 BMP15

•

TGF- β

Pro-region

Mature region

(Shi et al., 2011; Harrison et al., 2011)

•  TGF-β family mature regions normally form covalent dimers

•  GDF9 & BMP15 lack 4^th Cys of the 7 conserved Cys usually found in the mature region, hence they form non-covalent dimers

Mature Pro-region

SS

RXXR

Pro-BMP15

Mature Pro-region

SS

RXXR

Pro-GDF9

GDF9/BMP15 Synergism

purified mature regions of GDF9 and BMP15 (R&D Systems)

(Mottershead et al., 2012)

GDF9

BMP15

• 

(Mottershead et al., 2012)

•  proteins tested at 12.5 ng/ml

GDF9/BMP15 Synergism on Cumulus Cells

GDF9/BMP15 synergism is mediated via the SMAD3 pathway

SMAD3 (CAGA) pathway

SMAD1/5/8 (BRE) pathway

(Mottershead et al., 2012)

GDF9 BMP15

GDF9 BMP15 GDF9/BMP15

What is the mechanistic basis of GDF9 / BMP15 synergism?

Mature Pro-region

SS His₈

RRRR

Pro-BMP15

•

Mature Pro-region

SS

RRRR

Pro-GDF9

•

His₈

Ser-Cys mutations

Can GDF9 and BMP15 form a covalent heterodimer?

•  Co-expressed in HEK-293T cells

•  Serum-free production media mixed with IMAC resin

•  Final step rpHPLC

GDF9/BMP15 covalent heterodimer is bioactive

(Mottershead et al., 2015)

•

GDF9:BMP15 heterodimer bioactivity comparison

•

+

• 

3 H-ThymidineIncorporation

0 10 20 30 40 50

GDF9/BMP15 variants (ng/mL) Pro-GDF9 Pro-BMP15

Pro-GDF9 + Pro-BMP15 Pro-Mature proteins

Mature proteins

Pro-cumulin

(non-covalent heterodimer) Cumulin

(Mottershead et al., 2015)

Cumulin activates both SMAD signaling pathways

•  in human COV434 granulosa tumour cells

SMAD2/3 activation

proGDF9

proBMP15 proGDF9

+proBMP15

+

proCumulin:

Cumulin

•

(covalent)

SMAD1/5/8 activation

(Mottershead et al., 2015)

GDF9/BMP15 variants (ng/ml)

BMP15 covalent homodimer does not synergize with GDF9

• 

•

BMP15_Ser-Cys

+

•

BMP15_Ser-Cys

GDF9 BMP15

+

BMP15

GDF9 SMAD1/5/8 activation

GDF9/BMP15 variants (ng/ml)

(Mottershead et al., 2015)

Cumulin activates gene expression associated with cumulus differentiation

(Mottershead et al., 2015)

• 

•

0   10   20   30   40   50   60   70   80  

Cont   B15   G9   B+G_100   Co3_20   He_20  

D7BL

a

a a

b

ab

a

80 70 60 50 40 30 20 10 Blastocyst at D7/cleaved (%) 0

Cont.

+

proCumulin stimulates developmental competence in the porcine IVM model

proBMP15 proGDF9

proGDF9 + proBMP15

Cumulin

(covalent)

proCumulin

(Mottershead et al., 2015)

•

Key Findings 1

1). It is possible to produce a GDF9/BMP15 mature region covalent heterodimer,

which is very bioactive.

2). For DNA synthesis in granulosa cells cumulin [1] is the most active form, followed by pro-cumulin [2], and finally the GDF9 + BMP15 [3] combination.

•

Cumulin

+

• [3]

[1]

Cumulin

[2]

Pro-cumulin

Key Findings 2

3). The BMP15 covalent homodimer does not synergize with wild type GDF9, a further indication that the basis of GDF9/BMP15 synergism is

heterodimerization.

4). Pro-cumulin [2] stimulates oocyte developmental competence, whereas cumulin [1] (mature region only) does not!!

• ⁺ - ^{+ +++}

[1] •

Cumulin

[2]

Pro-cumulin

Model of human GDF9/BMP15 action in vivo

Oocyte

Granulosa cell (cumulus cell)

Potential Uses of Cumulin in Reproductive Medicine

1). IVM media development?

2). Ovarian cryopreservation?

3). Measurement of Cumulin levels for oocyte selection?

Acknowledgements

Funding

-  NHMRC Australia - COOK Medical

- Faculty of Health Sciences, University of Adelaide

Robinson Research Institute,

University of Adelaide. Hudson Institute of Medical Research, Melbourne

Craig Harrison Sara Al-Musawi David Mottershead

Robert Gilchrist

*

Melissa White Jing-Jie Li

Satoshi Sugimura Lesley Ritter

Georgia Martin Jaqueline Sudiman Andrew Trotta Jun-Yan Shi

Julius-von-Sachs Institute University Wuerzburg

Thomas Mueller

*