Effect of age on the transcriptome

of the human MII oocyte

Marie Louise Grøndahl, DVM, PhD

Fertility Clinic, Hvidovre Hospital,

Copenhagen University Hospital

Denmark

Contents

• Cytoplasmic maturation

• The transcriptome of the human MII oocyte• The transcriptome of the human MII oocyte

• Effect of age on the MII transcriptome

Maturity of the oocyte

• ‘Nuclear maturation’ is used to describe the oocyte maturation stages of the chromosome compartment: GV, MI and MII

• ‘Cytoplasmic maturation’ covers the rest: the organelles, the transcriptome and the proteins stored in the cytoplasma during oogenesis.

Developmental competence

• timely translation of stored maternal transcripts

provide the ooplasma with new proteins

• post translational modification of stored or newly

synthesized protein sets the exact timing for

cellular eventscellular events

• processes involved in degradation of proteins and

mRNAs remove no longer needed molecules

The microarray technique has made it possible to approach the transcriptome part of the ’cytoplasmic maturation’.

The transcriptome of

the human MII oocytethe human MII oocyte

Materials and Methods

• 15 Women undergoing IVF/ICSI treatment

• Short antagonist protocol, Puregon and Orgalutran (Organon, Denmark)

• One COC and follicular fluid with mural granulosa cells was donated, if > 6 COCs granulosa cells was donated, if > 6 COCs available for the treatment

• Zona were removed with Tyrodes solution (MediCult, Denmark)

• The oocyte was flash frozen within one hour after ovum pick up

extraction and

amplification and

labelling of

mRNA (cRNA)(PicoPureTM Isoloation Kit (Arcturus, USA))

hybridization

to the gene chip

(R, dChip, Partek,

Ingenuity)

(Human Genome U133 2.0 Plus array fromAffymetrix (Affymetrix Inc., USA))

Results

Patient and treatment data

’Old’

Number of donated follicles 15 5

Age (years) 33.1± 2.7(27-39)

37,8±1,3(37-39)

FSH CD2-3 (IU/l) 5.9±2.0(2.0-13.0)

8,9±3,3 *(6,3-13,0)

BMI 22.6± 2.7 21,0 ±1,4BMI 22.6± 2.7 21,0 ±1,4

Reason for treatment Male factor: 10

Ovulationdefect: 3

Unexplained: 2

Male factor: 2

Ovulation defect: 1

Cycle number 1.2± 0.4 1,2± 0,4

FSH total (IU) 1785±383(1200-4050)

2345±1003*(1400-4050)

FSH days 8.9±1.6 8.8±0.8

No of oocytes 11.6±4.6 11,2± 4,6

“Follicle size” (ml) 2.5±0.9 2,5±1,0

Results / Discussion

• 10,428 transcripts representing 7,470 genes were present in the human MII oocyte

____________________________________

Sample Number of Sample Number of

transscriptswhole genome arrays

HumanIndividual

MII oocytes

8,125 (Woods et al. 2007)

Pooled

(3-10)

MII oocytes

4,801 - 12,031 (Wells and Patrizio, 2008), (Jones et al.,

2008),(Assou et al.,2006)

(Jaroudi et al., 2009). (Kocabas et al.,

2006).

Murine Pooled

(25-500)

MII oocytes

10,977- 13,892 (Hamatani et al., 2004)

(Pan et al., 2008)

ResultsEnrichment analysis

• In this list of expressed genes in the MII

oocytes, 5,213 of the expressed genes have

a biological and a molecular process term a biological and a molecular process term

assigned

• 2,257 genes have no functional term

assigned, yet.

Results

Biological functionCellular process

Biological regulation

Metabolic process

Developmental process

Establishment of localization

Response to stimulus

Multicellular organismal

process

e.g.

establishment of protein localization (20.0)

establishment of RNA localization (7.1) ‘reproductive process’

‘gamete generation’ (100 genes)

‘fertilization’ (14 genes)

process

Biological adhesion

Muti-organism process

Reproductive process

Immune system process

Localization

Growth

Locomotion

Rythmic process

Pigmentation

cellular metabolic process (65.7)

cellular organization (27.6)

cell cycle (24.4)

cell division (19.4)

microtubule based processes (6.5)

chromosome segregation (5.3)e.g.

macromolecular metabolic process (29.3)

electron transport chain (10.3)

Figures in ( ): enrichment score > 1 is significant; p < 0.001

Molecular function

Binding

Catalytic activity

Transcription regulator activity

Molecular transducer activity

Transporter activity

protein binding (45.5)

antigen binding (44.8)

nucleotide binding (21.8)

nucleic acid binding (20.7)

carbohydrate binding (10.4)

chromosome binding (6.8)

e.g

translation initiation factor activity (9.3)

translation elongation factor activity (2.8)

Transporter activity

Enzyme regulator activity

Structural molecular activity

Translational regulator activity

Electron carrier activity

Auxillar transport protein

activityAntioxidant activity

Proteasome regulator activity

e.g.

ligase activty (15.7)

isomerase activity (7.4)

transferase activity (6.5)

RNA splicing activity (5.5)

Figures in ( ): enrichment score > 1 is significant; p < 0.001

MII oocyte transcriptome

• List of expressed genes

• Biological function

• Molecular function

• Signalling pathways• Signalling pathways

• …….

Competent or incompetent oocyte?

• What characterize the developmental

machinery in a competent oocyte?

• Does the transcriptome part of the

‘cytoplasmic maturation’ differ?

• Age is a significant quality parameter!

20

25

30

35

40

45

%

Pos hCG

Ongoing

Implantationsrate

/ OPU

/ OPU

Hvidovre Hospital, 2003-2007

1st cycle

535 cycles

Transfer: 88%

DET:40%

1007 cycles

Transfer:86%

DET:65%

365 cycles

Transfer:75%

DET:60%

0

5

10

15

<30 years 30-36 years 37-39 years

Birth/ OPU

The effect of age on

the transcriptome of

the human MII oocytethe human MII oocyte

<36 years x 37-39 years

Results Patient and treatment data

Younger Older

Number of donated

oocytes

10 5

Age (years) 31.1± 2.7 (27-35) 37.8±1.3 (37-39)

FSH CD2-3 (IU/l) 4.5±1.7 8.9±3.3 *

Length of infertility

(years)

2.1±1.5 1.6±0.9

BMI 23.4± 2.5 21.0 ±1.4

Data are mean ± standard deviation. * P< 0.05

BMI 23.4± 2.5 21.0 ±1.4

Reason for treatment Male factor: 8

Ovulation defect: 2

Male factor: 2

Un-explained: 2

Ovulation defect: 1

Cycle number 1.2± 0.4 1.2± 0.4

FSH total (IU) 1,505±383 2,345±1,003*

FSH days 8.9±1.6 8.8±0.8

No of oocytes 11.9±3.8 11.2± 4.6

“Follicle size” (ml) 2.5±0.8 2.5±1.0

• 351 transcripts, 342

unique genes, (4,5%)

showed significant

oocyt-1

oocyt-2

oocyt-3

oocyt-4

oocyt-5

oocyt-6

oocyt-7

oocyt-8

oocyt-9

oocyt-10

oocyt-11

oocyt-12

oocyt-13

oocyt-14

oocyt-15

203765_at225342_at241443_at223282_at215048_at206448_at202794_at212989_at1568780_at

208859_s_at202275_at201844_s_at226079_at205809_s_at222667_s_at230552_at226614_s_at223195_s_at233076_at238353_at200607_s_at227267_at228368_at230027_s_at201702_s_at227016_at210778_s_at234339_s_at214358_at223849_s_at201783_s_at206114_at225650_at203319_s_at232116_at223101_s_at91952_at226833_at221871_s_at216693_x_at203094_at

219099_at219175_s_at228972_at201861_s_at226843_s_at216088_s_at209072_at204524_at200679_x_at201040_at207213_s_at204928_s_at1563321_s_at215732_s_at204318_s_at215942_s_at45653_at221503_s_at208620_at217795_s_at219239_s_at208779_x_at218719_s_at213198_at234725_s_at204003_s_at232204_at212152_x_at215264_at209407_s_at219924_s_at

212085_at203252_at38158_at202140_s_at210276_s_at218215_s_at213698_at213669_at206499_s_at223425_at210775_x_at227818_at212669_at223541_at219229_at234985_at1552540_s_at225091_at219437_s_at203491_s_at223382_s_at200993_at210649_s_at242959_at213286_at33148_at219146_at227152_at235728_at242647_at1557388_at

age type

28 28 29 29 30 30 32 34 35 35 37 37 38 39 39yo yo yo yo yo yo yo yo yo yo ol ol ol ol ol

Results / Discussion

134

showed significant

expression change with

age, fold change >1.5,

p <0.05

• 103 genes > 2 fold

• 239 genes >1.5 fold

1557388_at226815_at

218241_at239612_at217791_s_at236076_at1555882_at208286_x_at220072_at235645_at204507_s_at202296_s_at226767_s_at218676_s_at219682_s_at204222_s_at205591_at221765_at218533_s_at208627_s_at214451_at226176_s_at210950_s_at214838_at215535_s_at1558173_a_at218317_x_at222548_s_at223232_s_at200964_at217959_s_at242918_at236243_at

209094_at213353_at229573_at213122_at222273_at227636_at227641_at201503_at225811_at213206_at212183_at224955_at226430_at202903_at222243_s_at206245_s_at219119_at218683_at226660_at225264_at221825_at228562_at209272_at205347_s_at222209_s_at203860_at219147_s_at212071_s_at226052_at230102_at214097_at235141_at

230263_s_at201225_s_at224592_x_at223090_x_at210102_at227442_at201589_at209206_at224788_at222572_at225363_at202606_s_at217954_s_at201223_s_at221493_at238021_s_at200733_s_at212526_at200969_at227075_at206656_s_at224413_s_at215338_s_at231867_at203075_at203801_at228318_s_at234675_x_at213085_s_at224315_at202317_s_at212628_at

229835_s_at201826_s_at226085_at1554095_at204796_at223210_at203494_s_at1562921_at212774_at204824_at232183_at218342_s_at209409_at220030_at230785_at1562275_at203513_at221963_x_at213452_at225711_at216095_x_at230718_at226312_at225684_at218396_at223077_at225878_at225517_at235299_at1554043_a_at1557057_a_at

1553928_at239012_at218209_s_at244599_at228181_at223883_s_at203020_at221008_s_at235048_at218772_x_at244640_at225623_at202691_at226974_at206272_at206364_at223022_s_at229970_at219532_at1560990_a_at220945_x_at238890_at218422_s_at208828_at224990_at222390_at202515_at218259_at203983_at200749_at222606_at201535_at

208924_at242458_at209555_s_at229053_at201595_s_at218133_s_at201630_s_at224964_s_at206451_at225679_at212714_at218640_s_at203208_s_at222990_at218446_s_at1558905_at212335_at225023_at225337_at226425_at219312_s_at233899_x_at221918_at241342_at209847_at205583_s_at216944_s_at218877_s_at219276_x_at225611_at228396_at235158_at

204127_at220147_s_at219673_at219797_at228328_at235165_at219580_s_at229615_at212719_at228185_at205198_s_at212465_at201344_at225051_at242366_at228776_at1555274_a_at224810_s_at226917_s_at201887_at238719_at213348_at226032_at225810_at238992_at226807_at208838_at230352_at209130_at217894_at228920_at

206286_s_at232112_at1568791_s_at212747_at1568763_s_at1569110_x_at209707_at224209_s_at234464_s_at226550_at243624_at208999_at225546_at229131_at227425_at213405_at227547_at212448_at218754_at227731_at209295_at241344_at218465_at219702_at207538_at207539_s_at

-2.0 -1.6 -1.1 -0.7 -0.2 0.2 0.7 1.1 1.6 2.0

218

Results / Discussion

Age groups

(number of

biological

replicates)

Number of

Differentially

expressed genes

Array platform

Human <36 years (10)

>36 years (5)

351 transcripts / 4.5%

103 genes > 2 fold,

239 genes >1.5 fold

48,000 transcripts

Affymetrix, Human

Genome U133 2.0 Plus

Present study

Human

Oocytes failed to

fertilize after

<32 years (2)

32-40 years (1)

> 40 years (2)

608 transcripts / 28%

> 1.5 fold change

8,500 transcripts

Affymetrix ‘Human

Genome Focus Array’

Steuerwald et al.,

2007

fertilize after

IVF/ICSI

> 40 years (2) > 1.5 fold change Genome Focus Array’

Murine

MII oocytes

harvested 18 hrs

after hCG

5-6w (3)

42-45w (3)

530 transcripts /5%

99 transcripts > 2.0

431 transcripts > 1.5

22,000 transcripts

Agilent technolo-

gies NIA 22K array

Hamatani et al.,

2004

Murine

MII oocytes

harvested 16 hrs

after hCG

6 weeks (4) MII

66 weeks (4)

GV

2129 transcripts/ 13%

2001 transcripts >1.4

128 transcripts>2.0

335 transcripts / 2%

298 transcripts >1.4

47 transcripts > 2.0 fold

39,000 transscripts

Affymetrix ‘murine

genome array’

MOE430 v2

Pan et al., 2008

ResultsqRT-PCR confirmation of microarray data

Gene Symbol Ratio by microarray

5 older oocytes (37-39 years)

Ratio by real time PCR

2 older oocytes (39 years)5 older oocytes (37-39 years)

10 younger oocytes (27-35 years)

2 older oocytes (39 years)

4 younger oocytes (26-35 years)

SMAD2SMAD family member

2

0.42 0.38

MRPL43mitochondrial

ribosomal protein L43

2.50 1.40

ANAPC4anaphase promoting

complex subunit 4

0.65 0.08

ResultsEnrichment analysis of the differentially expressed genes

Cellular process

Biological regulation

Metabolic process

Establishment of

local ization

Developmental process

Response to stimulus

Multicellular organismal

process

Multi-organismal process

Biological adhesion

Reproductive process

Localization

Annotational term p-value

Cell compartment Intracellular 188 0.000074

Nucleus 75 0.004391

Cell cycle Cell cycle 27 0.002681

Mitotic cell cycle 13 0.00148

Regulation of mitosis 5 0.005004

Spindle / microtubule Cytoskeleton organization and biogenesis 19 0.00078

Spindle organization and biogenesis 4 0.000733

Cellular organization Cellular component organization and biogenesis 61 0.004947

Regulation of cellular component organization 6 0.002685

Golgi vesicle transport 9 0.001402

DNA DNA metabolic process 28 0.000248

DNA replication 10 0.003983

Nucleobase, nucleoside, nucleotide metabolic process 87 0.000507

Hydrolase activity, acting on ester bonds 21 0.004135Hydrolase activity, acting on ester bonds 21 0.004135

DNA repair DNA repair 10 0.016809

Response to DNA damage stimulus 13 0.010501

RNA Regulation of transcription from RNA polymerase II promoter 15 0.008099

Negative reg of transcript from RNA polymerase II promoter 7 0.008154

Metabolism Metabolic process 171 0.000035

Biopolymer metabolic process 122 0.000000

Catalytic activity 107 0.009242

Post translational

protein modification

Protein modification process 46 0.000565

Post-translational protein modification 43 0.000088

Protein catabolic process 12 0.000749

Ubiquination Protein ubiquitination 6 0.00222

Ubiquitin ligase complex 5 0.00569

Energy ATP binding 36 0.007735

Purine ribonucleotide binding 43 0.007701

Apoptosis Programmed Cell death 20 0.035156

Signalling and Protein kinase activity 28 0.009097

Developmental competence

• timely translation of stored maternal transcripts

provide the ooplasma with new proteins

• post translational modification of stored or newly

synthesized protein sets the exact timing for synthesized protein sets the exact timing for

cellular events

• processes involved in degradation of proteins and

mRNAs remove no longer needed molecules

Results ubiquination

• The most prominent function of ubiquitination is labelling of proteins

for protesomal degradation by the ubiquitin ligases. The proteasomal

degradation pathway is essential for many cellular processes including

the cell cycle regulation and progression, where cyclins are

ubiquitinated, i.e. is cyklin A degradated by the ubiquitin ligase,

anaphase promoting complex

• Ubiquilin 1 (1.6 fold), an anaphase promoting complex subunit,

ANAPC4 (1.6 fold) and two ubiquitin-conjugating enzyme genes

UBE2D (1.9 fold) and UBE4B (1.8 fold) were down regulated while

three genes for ubiquitin specific peptidases USP2, USP34 and USP42

were up-regulated (2.9, 1.6 and 1.6 fold, respectively).

• These findings showing mainly reduction in the ubiquitination by age

is in accordance with previous findings on murine (Hamatani et al.,

2004) and human oocytes (Steuerwald, et al., 2007).

Results cell cycleNumber one signalling network affected by age

‘organism development and cell cycle’

Activin A receptor 1B was up-regulated (1.5 fold) in

the older oocytes compared to the younger oocytes

Down-regulated

Up-regulated

SMAD2 was down-regulated (2.8 fold) in the

older oocytes compared to the younger oocytes

SMAD2

Table II. mRNA expression pattern in human oocytes and preimplantation embryos. The figures show the number of oocytes and preimplantation embryos that were positive, and the figure in parentheses shows the number of patients that donated the material

Gene Oocyte (3) 4-cell (3) 8-cell (4) Morula (6) Blastocyst (10)

TGFßR-I 3/5 0/4 0/5 0/6 7/1015/15TGFßR-I 3/5 0/4 0/5 0/6 7/10

TGFßR-II 4/5 0/4 0/5 0/6 0/10

Smad2a 5/5 4/4 5/5 6/6 8/10

TGFßR-I = transforming growth factor (TGF)ß receptor type I;

TGFßR-II = TGFß receptor type II.

aRT–PCR product obtained using primer for Smad 2/3 was by

sequencing shown to be Smad2.

Österlund and Fried, 2000

15/15

TGFßR-III 15/15

15/15

Results/Discussion

SMAD2

• SMAD2 was significantly down regulated (1.5 fold) in MII

oocytes from aged compared to young mice. Pan et al 2008

• Presence and dosage of SMAD2 have been shown to have • Presence and dosage of SMAD2 have been shown to have

an essential role in early embryonic development in studies

with mutant mice and in vitro culture of mice oocytes with

inhibition of SMAD2 signalling. Weinstein, Yang et al., 1998,

Nomura and Li, 1998.

• Separase was up-regulated (1.6 fold) in the older oocytes compared to the younger oocytesOver-expression of separase induces premature separation of chromatids, lagging chromosomes, and anaphase bridges (Zhang, Kuznetsov et al., 2008)

Results meiosis, mitosisseparase

EME1

Kuznetsov et al., 2008)

• EME1 (essential meiotic endonuclease 1) gene expression

was down-regulated (1.6 fold) in older oocytes compared to

younger Small decreases in gene dosage of EME1 promote re-replication and

polyploid cells in addition to chromosome aberrations as DNA gaps and

breaks (Hiyama, Katsura et al., 2006).

Results meiosis, mitosis/spindle

CSPP

• CSPP (centrosome/spindle pole associated protein) was up-

regulated 1.7 fold) in the aged oocytesOver-expression of CSPP has been shown to impair mitosis and promote multi-

polar spindles (Patzke, Stokke et al., 2006)

• Ran (member RAS oncogene family/GTP-binding nuclear protein Ran)

was down regulated by 1.8 fold, (p = 0.0041)

-The Ran GTPase mediates chromatin signaling to control cortical

polarity during polar body extrusion in mouse oocytes. Deng et al. 2008

-Involved in the acentriolar spindle assembly during meiosis i mouse

oocytes.

-Ran was 1.4 fold up regulated in MII oocytes from aged mice as

compared to younger. Pan et al 2008.

Conclusion

• Significant difference in the transcriptome of the MII oocyte between younger and older oocyte

• 342 of 7470 expressed genes were differentially expressed between the two age groups

• Analysis of the these 342 genes indicates alterations in• Analysis of the these 342 genes indicates alterations in

-ubiquination, generally reduced

-cell cycle, top signalling network affected

-mitosis/spindle, suggests molecular basis for the age-associated increase in aneuploidy in human oocytes and pre-embryos

• Gene expression profiles in cumulus and granulosa cells are also affected by age

When and how are the changes

induced in the oocyte?

• ‘limited oocyte pool’ hypothesis, where the limited number

of antral follicles available in older women could lead to suboptimal

oocyte for ovulation

• ‘over time’ hypothesis, while resting in the prophase, changes • ‘over time’ hypothesis, while resting in the prophase, changes

happen to the oocytes or its milieu inducing damage to the

meiotic/developmental machinery

Thanks

Rehannah BorupMicroarray Centeret

Rigshospitalet

Jeanette Bogstad, Helle Meinertz, VibekeBoujida Hartvig, Thue Bryndorf Anita Elverdal, Pernille AndersenKirsten Langbøl,Marianne Pedersen,Mette Villads Nielsen, Tanja Schrøder

Claus Yding AndersenRigshospitalet

Mette Villads Nielsen, Tanja SchrøderFertility ClinicHvidovre Hospital

Organon, Denmark‘a division of Schering-Plough’