3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

Growth Factors (BMP-15 AND GDF-9) Gene Expression

Of Mice Oocytes In Vitro
S. Rahayu
Departement of Biology, Faculty of Mathematics and Natural Sciences
Brawijaya University, Malang, Indonesia
e-mail: fatma.author1@unair.com srahayu@ub.ac.id
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Abstract
The oocyte-secreted polypeptide growth factors,
growth differentiation factor 9 (GDF-9) and bone
morphogenetic protein 15 (BMP-15, also known as
GDF-9B) have both been shown to be essential for
ovarian follicular growth and function. Bone
morphogenetic protein-15, an oocyte growth factor
belonging to the transforming growth factor-
superfamily, has recently been shown to be
necessary for normal female fertility in mammals.
In mice, the use of knock-out models has shown
that GDF-9 is essential for normal follicular
development with BMP-15 regulating the
fertilization potential of oocytes. The aim of this
research was to find out the influenced volume of
IVM oocyte media to BMP-15 and GDF-9 gene
expression. Oocytes were collected by puncturing
the surface of the mice ovaries with sterile needles
27 G . Furthermore oocytes were matured within
Hx medium during 22 hours at 38,5 C temperature
in the incubator with 5% CO2 to achive metaphase
II (M II) oocytes. Oocytes were cultured in 3
variations media droplet volume of 50 ul / droplet,
100 ul / droplet and 200 ul / droplet. After RT-PCR
and agarose gel electrophoresis, relative mRNA
abundance of BMP-15 and GDF-9 were analyze in
each group of oocytes. The BMP-15 gene
expression of oocytes were obtained when oocytes
were cultured in 50, 100 and 200 ul / droplet media.
The Highest expression of BMP-15 gene obtained
when oocytes were culture in 200 ul / droplet
media. The GDF-9 gene expression of oocytes were
obtained when oocytes were cultured in 200 ul /
droplet media.

Keywords: BMP-15 gene, GDF-9 gene, Metaphase

II (M II), mice oocytes
1

Introduction

The development of ovarian follicles is a complex

process dependent upon endocrine regulation
involving
hypophyseal
gonadotropins,
and
regulated locally by ovarian factors. This process is
characterized by differentiation and proliferation of
granulose cells and by enlargement of the oocyte
(Hirshfield 1991, Braw-Tel & Yossefi 1997), and is
regulated by several growth factors (Gougeon 1996,
McGee & Hsueh 2000, Picton & Gosden 2000). Of
intra ovarian growth factors, attention has been
focused on members of the transforming growth
factor- (TGF- ) super family (Erickson &
Shimasaki 2000). Bone morphogenetic protein-15
(BMP-15), an oocyte growth factor belonging to the
transforming growth factor- superfamily, has
recently been shown to be necessary for normal
female fertility in mammals (Moore et al., 2003).
The Bone morphogenetic protein 15 (BMP-15) are
key regulators of follicular development (Shimasaki
et al. 2004). Silva et al. (2004) reported that the
mRNAs and proteins of GDF-9 and BMP-15 are
expressed in goat ovarian follicles at all stages of
their development.

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BMP-15 is oocyte-specific proteins secreted by

growing oocytes in mouse and humans (Dube et al.
1998, Otsuka et al. 2000). The BMP-15 gene is
located on the X chromosome and in rodents its
mRNA and protein are expressed in oocytes from
the primary stage through ovulation (Dube et al.,
1998). They are known to control folliculogenesis
by acting on GC in developing follicles. Studies in
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A. Rahayu, Growth Factors (BMP-15 AND GDF-9) Gene Expression Of Mice Oocytes In Vitro

covering of compacted cumulus cells were used in

this study. Furthermore oocytes were matured
within Hx medium during 22 hours at 38,5 C
temperature in the incubator with 5% CO2 to
achive metaphase II (M II) oocytes. Oocytes were
cultured in 3 variations media droplet volume of 50
ul/droplet, 100 ul/ droplet and 200 ul/droplet, under
paraffin oil.

genetic mutations have elucidated the role of these

proteins in regulating the primary to secondary
follicle
transition.
BMP-15-deficient
ewes
(Galloway et al., 2000) is infertile due to a
complete block in folliculogenesis at the primary
follicle stage. Mutations in BMP15 gene result in
growth arrest at the primary stage. (Galloway et al.
2000, Hanrahan et al. 2004).
GDF-9 mRNA and GDF-9 protein are not only
expressed at the primary follicle stage but are also
present in oocytes throughout growth to the
ovulatory phase (Elvin et al. 1999, 2000, McGrath
et al. 1995). In GDF-9 knockout mice (Dong et al.
1996), the absence of GDF-9 resulted in a block in
folliculogenesis at the type 3b stage (late, onelayer
primary follicle stage), indicating that GDF-9 is
associated with early folliculogenesis. The
expression of GDF-9 mRNA and protein was
confined to oocytes of primary and large follicles in
rats (Hayashi et al. 1999, Jaatinen et al. 1999), mice
(McGrath et al. 1995, Dong et al. 1996) and
humans
(Aaltonen
et
al.1999).
Growth
differentiation factor-9 (GDF9) and bone
morphogenetic protein 15 (BMP15) are oocytespecific proteins secreted by growing oocytes in
rodents, sheep, and humans (McGrath et al. 1995,
Dube et al. 1998, Fitzpatrick et al. 1998, Aaltonen
et al. 1999, Bodensteiner et al. 1999, Otsuka et al.
2000). They are known to control folliculogenesis
by acting on GC in developing follicles. Studies in
genetic mutations have elucidated the role of these
proteins in regulating the primary to secondary
follicle transition. Mutations in GDF9 (Dong et al.
1996) and BMP15 (Galloway et al. 2000, Hanrahan
et al. 2004) result in growth arrest at the primary
stage.

Reverse Transcription PCR

To confirm the expression of BMP-15 and GDF-9
mRNA, RT-PCR analysis was performed on the
isolated oocytes. Total RNA from the three oocyte
groups was isolated in parallel following the
manufacturer's instructions. To avoid contamination
with genomic DNA, total RNA preparations were
treated with DNase. Total RNA extraction and
DNA synthesis was performed using Cell-to-cDNA
MII. PCR was performed using Ex Taq polymerase.
The sequence for primer pair was BMP-15 sense, 5AGCAACCAGGTGACAGGA-3, and antisense
primer,
5-CCTCCTTTACCAGGTCA-3.
The
sequence for primer pair was GDF-9 sense, 5AGCAACCAGGTGACAGGA-3, and antisense
primer, 5-CCTCCTTTACCAGGTCA-3.
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3

Result

Morphological change were observed before and

after in vitro culturing of oocytes (Figure 1). We
also obtained M II oocytes after 22 h in vitro
oocytes culturing (Figure 1). Morphological of
cumulus oocyte complexes will be change during
oocytes maturation (Yakoo M and Eimei S, 2000).

2 Methodology
Animals
Mice used in this study were maintained at the
Tohoku University animal house. Twenty-one-dold mice were injected with 5 IU PMSG, and
ovaries were collected 46 h later. All animals were
housed under controlled humidity, with a 12-hr
light and a2-hr dark phase, temperature, and fed ad
libitum.

After RT-PCR and agarose gel electrophoresis,

relative mRNA abundance of BMP-15 and GDF-9
were analyze in each group of oocytes. The BMP15 gene expression of oocytes were obtained when
oocytes were cultured in 50, 100 and 200 ul/droplet
media. The highest expression of BMP-15 gene
obtained when oocytes were culture in 200
ul/droplet media (Figure 2). The GDF-9 gene
expression of oocytes were obtained when oocytes
were cultured in 200 ul / droplet media (Figure 3).

Oocytes collecting
Mice were killed by the cervical dislocation.
Ovaries were cleaned free of adherent adipose and
connective tissues and placed in HEPES-buffered
tissue cultured medium-199 (H-TCM-199; ICN
Biomedicals Inc., Costa Mesa, CA) supplemented
with 0.1% (wt/vol) BSA (H-TCM-199/BSA).
COCs were isolated by puncturing antral follicles
with 27 G needles and collected in L-15Meiumd/BSA.
Only COCs with a uniform
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3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

BMP15 production in cultured primary follicles are

not different from in vivo.
M

(a)

9 10 11 12

(b)

Figure 3: RT-PCR analysis of GDF-9 expression

of oocytes grown in vitro. M : Marker DNA.:
oocytes were cultured in 50 ul / droplet media
(Lane 1-2); 100 ul / droplet media (lane 3-4);
200 ul / droplet media (Lane 5-6). Control of
PCR (mG3PDH) (Lane 7-12).

(c)
Figure 1: Follicle culture in vitro before (a) and
after (b) culturing oocytes. (c) MII Oocytes
obtained from in vitro culture

1 2

The paracrine factor GDF-9 secreted by oocytes

plays important roles in the intercommunication
between oocytes and follicle cell. Althought, Kim
et al (2004) had worked on oocytes maturated in
vitro and in vivo and foun no difference of the
GDF-9 gene expression between them. Shimizu et
al (2004), found that The injection of porcine
GDF-9 gene fragments resulted in an increase in the
number of primary, secondary and tertiary follicles,
concomitant with a decrease in the number of
primordial follicles. These results indicated that
exogenous
GDF-9
can
promote
early
folliculogenesis in the porcine ovary, and that a
technique for direct ovarian injection of GFD-9
gene fragments may contribute to a novel therapy
for prevention and treatment of infertility associated
with ovarian dysfunction. GDF-9 and BMP15
appear to be key regulators of normal follicular
development and ovulation rate in cattle (Juengel
et al , 2009).

Figure 2: RT-PCR analysis of BMP-15 gene

expression of oocytes grown in vitro.
M : Marker DNA.: oocytes were cultured in 50
ul / droplet media (Lane 1-2); 100 ul / droplet
media (lane 3-4); 200 ul / droplet media (Lane 56).
As members of the TGF- superfamily, BMP and
GDF9, and their receptors, have been shown to play
important roles during mammalian folliculogenesis
(Shimasaki et al. 2004, Juengel & McNatty 2005).
From mRNA analysis, length of BMP-15, was 170
bp (Figure 2). Our result showed that BMP-15 gene
expressed in the oocytes in vitro maturation.

Conclusions

The BMP-15 gene expression of oocytes in vitro

maturation depend on volume of media culture
droplet. The highest expression of BMP-15 gene
obtained when oocytes were culture in 200
ul/droplet media. The GDF-9 gene expression of
oocytes in vitro maturation depend on volume of
media culture droplet. The GDF-9 gene expression
of oocytes were obtained when oocytes were
cultured in 200 ul / droplet media.

The paracrine factor BMP-15 secreted by oocytes

plays important roles in the intercommunication
between oocytes and follicle cell. Although, Kim et
al (2004) had worked on oocytes maturated in vitro
and in vivo and found no difference of the BMP-15
gene expression between them. Shimizu et al
(2004), found that the injection of porcine BMP-15
gene fragments resulted in an increase in the
number of primary, secondary and tertiary follicles,
concomitant with a decrease in the number of
primordial follicles. These results indicated that
exogenous
BMP-15
can
promote
early
folliculogenesis. Sadeu J.C. et al (2008), found that

References
[1] Braw-Tel R & Yossefi S. 1997. Studies in
vivo and in vitro on the initiation of follicle

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A. Rahayu, Growth Factors (BMP-15 AND GDF-9) Gene Expression Of Mice Oocytes In Vitro

of
cultured
early
ovarian
Endocrinology 140 12361244.

growth in the bovine ovary. Journal of

Reproduction and Fertility. 109: 165171.

follicles.

[12] Jaatinen R, Laitinen MP, Vuojolainen K,

Aaltonen J, Louhio H, Heikinheimo K,
Lehtonen E & Ritvos O 1999 Localization of
growth differentiation factor-9 (GDF-9)
mRNA and protein in rat ovaries and cDNA
cloning of rat GDF-9 and its novel homolog
GDF-9B.
Molecular
and
Cellular
Endocrinology 156 189193.

[2] Dube JL, Wang P, Elvin J, Lyons KM, Celeste

AJ & Matzuk MM. 1998. The bone
morphogenetic protein 15 gene is X-linked
and expressed in oocytes. Molecular
Endocrinology .12: 18091817.
[3] Dong J, Albertini DF, Nishimori K, Kumar
TR, Lu N & Matzuk MM 1996 Growth
differentiation factor-9 is required during early
ovarian folliculogenesis. Nature 383 531535.

[13] Juengel JL & McNatty KP. 2005. The role of

proteins of the transforming growth factorbeta superfamily in the intraovarian regulation
of
follicular
development.
Human
Reproduction Update. 11: 143160.

[4] Erickson GF & Shimasaki S. 2000. The role

of the oocyte in folliculogenesis. Trends in
Endocrinology and Metabolism.11:193198.
[5] Galloway SM, McNatty KP, Cambridge LM,
Laitinen MPE, Juengel JL, Jokiranta TS,
McLaren RJ, Luiro K, Dodds KG,
Montgomery GW. 2000. Mutations in an
oocyte-derived growth factor gene (BMP15)
cause increased ovulation rate and infertility
in a dosage-sensitive manner. Nature
Genetics. 25: 279283.

[14] Kim DH., Ko DS., Lee HC., Lee HJ., Park

WI., Kim SS. 2004. Comparassion of
maturation, fertilization, development and
gene expression of mouse oocytes grown in
vitro and in vivo. J. Assist Reprod Genet. 21
(7) : 233-240
[15] McGee EA & Hsueh AJ .2000. Initial and
cyclic recruitment of ovarian follicles.
Endocrine Review 21 200214. McLaughlin
EA., Mclver SC. 2009. Awakening the
oocytes : Controling primordial follicle
development. Reproduction 137 : 1-11

[6] Fitzpatrick SL, Sindoni DM, Shughrue PJ,

Lane MV, Merchenthaler IJ & Frail DE 1998
Expression of growth differentiation factor-9
messenger ribonucleic acid in ovarian and
nonovarian rodent and human tissues.
Endocrinology 139 25712578

[16] McGrath SA, Esquela AF & Lee SJ 1995

Oocyte-specific
expression
of
growth/differentiation factor-9. Molecular
Endocrinology 9 131136

[7] Gougeon A. 1996. Regulation of ovarian

follicular development in primate: facts and
hypothesis. Endocrine Reviews.17:121155.

[17] Moore R. K. , Otsuka F. and Shimasaki, S.

2003.
Molecular
Basis
of
Bone
Morphogenetic Protein-15 Signaling in
Granulosa Cells. The Journal Of Biological
Chemistry. 278 (1) : 304310

[8] Hirshfield AN.1991. Development of follicles

in the mammalian ovary. International Review
of Cytology. 124:43101
[9] Hanrahan JP, Gregan SM, Mulsant P, Mullen
M, Davis GH, Powell R & Galloway SM 2004
Mutations in the genes for oocyte-derived
growth factors gdf9 and bmp15 are associated
with both increased ovulation rate and sterility
in cambridge and belclare sheep (ovis aries).
Biology of Reproduction 70 900909.

[18] Otsuka F, Yao Z, Lee T, Yamamoto S,

Erickson GF & Shimasaki S. 2000. Bone
morphogenetic protein-15. Identification of
target cells and biological functions. Journal
of Biological Chemistry. 275 : 39523 -39528.
[19] Picton HM & Gosden RG. 2000. In vitro
growth of human primordial follicles from
frozen-banked ovarian tissues. Molecular and
Cellular Endocrinology. 166:2735.

[10] Hanrahan JP, Gregan SM, Mulsant P, Mullen

M, Davis GH, Powell R & Galloway SM.
2004. Mutations in the genes for oocytederived growth factors gdf9 and bmp15 are
associated with both increased ovulation rate
and sterility in cambridge and belclare sheep
(ovis aries). Biology of Reproduction. 70:
900909.

[20] Sadeu, J.C., Adriaenssens, T. and Smitz, J.

2008. Expression of growth differentiation
factor 9, bone morphogenetic protein 15, and
anti-Mu llerian hormone in cultured mouse
primary follicles. Reproduction . 136: 195
203

[11] Hayashi M, McGee EA, Min G, Klein C, Rose

UM, van Duin M & Hsueh AJ 1999
Recombinant growth differentiation factor-9
(GDF-9) enhances growth and differentiation

[21] Shimasaki S, Moore RK, Otsuka F &

Erickson GF. 2004. The bone morphogenetic
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3rd International Conferences and Workshops on Basic and Applied Sciences 2010

protein system in mammalian reproduction.

Endocrine Reviews. 25:72101.
[22] Silva, JRV, van den Hurk R, van Tol HTA,
Roelen BAJ, Figueiredo JR. 2004. Experssion
of growth differentiation factor 9 (GDF-9) and
bone morphogenetic protein 15 (BMP-15) and
BMP receptors in the ovaries of goats. Mol.
Reprod. Dev. 70: 11-19.
[23] Shimizu T., Yasunori M., Masaki Y., Yumi
H., Hiroshi S. and Eimei S. 2004. Molecular
cloning of porcine growth differentiation
factor 9 (GDF-9) cDNA and its role in early
folliculogenesis: direct ovarian injection of
GDF-9 gene fragments promotes early
folliculogenesis. Reproduction. 128: 537543
[24] Yokoo Masaki and Eimei Sato. 2000.
Cumulus-Oocyte Complex Interaction During
Oocyte Maturation. International Review of
Cytology. 235 : 2.

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ISBN: 978-979-19096-1-7