Gdf9 and bmp15

Role of Growth Differentiation Factor

9 &

Bone Morphogenetic Protein 15

IN

ASSISTED REPRODUCTIVE TECHNOLOGIES

Muzamil AhmadM.V.Sc. Student

Animal Biotechnology

Recent reproductive technologies have opened up

possibilities in accelerating genetic gain and

enhancing production potential in livestock .

Introduction :

These technologies include Artificial insemination (AI). Multiple ovulation and embryo transfer

(MOET). In vitro production of embryos. Animal Cloning. Estrus synchronization. Marker assisted selection ,and Transfer of genes between animals.

(transgenesis).

In vitro production of embryos (IVEP)has emerged as a

reliable alternative method to conventional ovarian

super stimulation methods

Important research tool for animal embryology.

Previous studies have clearly demonstrated that, while the intrinsic quality of the oocyte determines the proportion of oocytes developing to blastocysts.

The post-fertilization culture environment has the

biggest influence on blastocyst quality. [Russell et al.,

2006] However, the exact influence of culture conditions

during critical events is still unknown.

Invitro maturation: platform for invitro embryo production

IVM

Animal cloning

IVF

In vitro embryo

production

Transgenic animal

production

Oocyte in vitro maturation (IVM) is a reproductive technology that enables mature oocytes to be generated ex vivo.

IVM involves removal of cumulus-oocyte-complexs (COCs) and culturing them into standard culturing medium for 24h upto meiosis II.

The efficiency of IVM technologies or assisted reproductive technologies (ARTs) is limited by

The oocyte developmental competence ,and

Subsequent embryo development as compared to in vivo.

a) Meiotic Maturation b) Fertilization c) Preimplantation development d) Development to term /successful pregnancy

Oocyte Developmental Competence

Oocytes gradually and sequentially acquire

developmental competence during the course of

folliculogenesis as the oocyte grows and its

companion somatic cells differentiate. (Eppig et al., 1994).

(1) The origin of the oocyte. (Lonergan et al., 1994)

(2) Follicle health. (Blondin et al.,1995)

(3) Hormonal stimulation. (Sirard et al., 2006)

(4) Communication between the oocyte and its surrounding

cumulus cells (CCs) (Krisher, 2004)

Factors affecting oocyte competence

Previously it was thought that oocyte is a passive recipient of Cumulus Cell function.

But now it is evidenced oocyte is a key regulator of its developmental competence by regulating its micro environment via the secretion of paracrine molecules like GDF9 , BMP15 etc.

Role of GDF 9 and BMP 15

Oocyte plays active role throughout folliculogenesis

via secretion of paracrine factors ( like GDF9 &

BMP15) that maintain an appropriate

microenvironment for the acquisition of its

developmental competence. (Dong et al., 1996; Eppig et al., 1997;Gilchrist et al., 2004).

Falck and colleagues demonstrated in 1959 that intact rabbit preovulatory follicles do not luteinize when transplanted into the anterior chamber of the eye.

In contrast to oocyte-free explants of either the follicle wall or granulosa cells that do undergo morphological luteinization.

(Falck 1959).

Subsequently, Nalbandov and colleagues confirmed this work using rabbit dominant follicles in situ, showing that removal of the oocyte caused spontaneous luteinization of granulosa and theca cells and elevated secretion of progesterone to levels produced by corpora lutea.

( Nalbandov 1970).

It is known that bidirectional interactions occurs between oocyte and cumulus cells.

Bidirectional interaction occurs via; gap junction and paracrine signaling.

Helps in growth and development of both oocyte and cumulus cells.

( Eppig et al.,1997,2002;Matzuk et al., 2002)

Cumulus cell and oocyte interaction

Bidirectional interaction between cumulus cells and oocyte

Growth differentiation factor 9 (GDF9)and Bone morphogenetic

protein 15 (BMP15) are two closely related members of the

Transforming Growth Factor-β (TGF-β ) superfamily.

TGF-β superfamily ; large family of structurally related

proteins.

Controls proliferation, differentiation, development etc in

variety of cells/tissues.

GDF9 & BMP15

GDF9 & BMP15 are expressed and translated in oocytes .

Synthesized as preproproteins, consisting of a signal peptide, a large proregion and a mature region.

( Shimasaki et al., 2004)

Contd…..

Properties : Lack of 4th cysteine residue (otherwise conserved in

other members of TGF-β superfamily)

Function mostly as homodimer/and or sometimes as heteriodimers.

Required for female fertility: as mutations (homozygus) leads to infertility due to block in follicullogenisis at early stage.

GDF9 and BMP 15 have recently been shown to

signal through known TGF-β superfamily receptors

to activate the SMAD intracellular cascade

GDF9 and BMP15 signaling

Two pathways BMP pathway and TGFβ/activin pathway which respectively activate SMAD1/5/8 and SMAD2/3 messengers

BMP15 utilizes BMP pathway to activate SMAD1/5/8

GDF9 utilizes SMAD2/3

Proliferation of Granulosa cells

Differentiation of Granulosa cells

Prevention of apoptosis

Functions of GDF9 & Bmp15

Inhibition of Luteinization

Cumulus cell expansion

Metabolism

GDF 9 and BMP15 stimulates granulosa cell proliferation and DNA synthesis.

Expression of Ccnd2 mRNA cell cycle transcript >>>>>cyclin d 2 protein of cell cycle

Assessed by uptake of [H3]thymidine (Glichrist et al.,2006)

Proliferation

Oocyte secreted factors i-e,GDF9 & BMP15 differentiates the granulosa cells into mural cells

(MGCs) and cumulus cells (CCs) at antral stage of follicle.

MGCs under influence of FSH CCs under influence of OSFs (Hussein et.al.,2005;Diaz et al.,2007)

Differentiation

BMP15 prevent Cumulus Cell apoptosis by maintaining a localized gradient of anti-apoptotic factors within the COC

(Hussein et al., 2005)

Expression of anti-apoptotic gene Bcl2

Suppression of pro-apoptotic protein Bax

Apoptosis

Suppression of LH receptor (LHCGR) ( Eppig et al.,1997)

Inhibition of FSH induced progestrone production

Inhibition of luteinization

Two signaling pathways:- (i) Gonadotrophin or epidermal growth factor (EGF)

stimulation and (ii) Paracrine signals of oocyte (GDF9 &BMP15)

Synthesise extracellular matrix (ECM)

Hyaluronan (HA) major component

tumor necrosis factor alpha induced protein 6 (TNFAIP6)

Pentraxin 3(PTX3) (Russell et al., 2006)

Cumulus expansion

Prior to the LH surge, cumulus cells require GDF-9 to support the metabolic cascades such as glycolysis and sterol biosynthesis

(Sugiura et al. 2005).

Evidence for this comes from findings of reduced

cholesterol synthesis from acetate in cumulus-oocyte complexes of Gdf9 − /−mutant mice.

(Sugiura et al. 2005).

Metabolism

Cumulus cells and oocyte regulatory loop

OSFs(GDF9 &BMP15) enhance oocyte devlopmental competence

Hussein et al.,2005..

Blastocyst

Inner cell

mass

BlastocystICM

IVF

IVF

Treatment No.of oocytes used

% Cleavage Rate

% Blastocycst from cleaved

oocytesCONTROL 205 86.5 ±3.2 41.0 ±0.9

GDF9 191 88.1 ±2.0 49.5 ±3.9

BMP15 189 88.7 ±4.2 57.5 ±2.4

GDF9+BMP15 184 88.9 ±2.9 55.1 ±4.5

293H 187 80.4 ±2.2 27.1 ±3.1

Table:effect of GDF9 and/or BMP15 during IVM on oocyte developmental competence

(Hussein et al.,2005)

It is now clear that oocyte regulates the cumulus cell functions like differentiation, apoptosis, expansion, luteinization

And cumulus cells provide microenvironment to oocytes for development

In order to increase the efficiency of ARTs the OSFs (GDF9 & BMP15) may be utilized in culture media

Conclusion

Thank you