Abstract

BACKGROUND: The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes and cumulus cells. METHODS: Using oligonucleotide microarrays, genome-wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS: In addition to known genes, such as DAZL, BMP15 or GDF9, oocytes up-regulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14 and IL4 and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-to-cell signalling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A and SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, including CDC25A and SOCS7. CONCLUSION: The identification of genes that were up- and down-regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumours.

Introduction

The quality of oocytes obtained following controlled ovarian stimulation (COS) for assisted reproductive technology (ART) varies considerably. Although most oocytes are amenable to fertilization, only half of those fertilized complete preimplantation development and even fewer implant. During follicle growth, the oocyte obtains the complement of cytoplasmic organelles and accumulates mRNAs and proteins that will enable it to be fertilized and to progress through the first cleavage divisions until embryonic genes start to be expressed. Transcriptional activity decreases as the oocyte reaches maximal size (Fair et al., 1995), and later on, the oocyte depends on stored RNAs for normal function during maturation, fertilization and early embryonic development (Moor et al., 1998). After oocyte retrieval, the mature oocyte [metaphase II (MII)] and even some immature oocytes [germinal vesicle (GV) and metaphase I (MI)] are surrounded by the cumulus oophorus. Several layers of cumulus cells surround the oocyte in the antral follicle and play an important support and regulation role in oocyte maturation (Dekel and Beers, 1980; Larsen et al., 1986).

Analysis of oocyte maturation using microarray analysis techniques could detail the genes involved in this process and the specific checkpoints regulating acquisition of full competence for ovulation and fertilization. The understanding of the molecular processes involved in the development of a competent oocyte under COS conditions could guide the choice of ovarian stimulation protocols and lead to improvements in oocyte quality, oocyte culture and manipulation. Some studies demonstrate that changes in gene expression during COS, such as GDF9 or bone morphogenic protein 15 (BMP15) in oocytes, or pentraxin 3 (PTX3) in cumulus cells, can be monitored for selecting appropriate oocytes for fertilization and embryos for replacement (Elvin et al., 1999; Yan et al., 2001; Zhang et al., 2005). Therefore, transcriptome studies in human oocytes and cumulus cells could contribute to not only elucidate the mechanisms of oocyte maturation but could also provide valuable molecular markers of abnormal gene expression in oocytes with reduced competence. The aims of the present study were to establish (i) whole-genome transcriptome of human immature and mature oocytes and cumulus cells, (ii) specific gene-expression signatures of immature and mature oocytes and cumulus cells and (iii) genes whose expression progressively increase during oocyte maturation.

Materials and methods

Oocytes and cumulus cells

Oocytes and cumulus cells were collected from patients consulting in our centre for conventional IVF (cIVF) or for ICSI (male infertility). This study has received institutional review board’s approval. Patients were stimulated with a combination of GnRH agonist (Decapeptyl PL 3; Ipsen, Paris, France) and recombinant FSH (Puregon, Organon, Saint Denis, France and Gonal F, Serono, Randolph, MA, USA) or Menopur (Ferring). Ovarian response was evaluated by serum estradiol level and daily ultrasound examination to observe follicle development. Retrieval of oocytes occurred 36 h after HCG administration and was performed under ultrasound guidance. Cumulus cells were removed from a mature oocyte (MII) 21 h after insemination. Immature oocytes (GV and MI) and unfertilized MII oocytes were collected 21 or 44 h after insemination or after microinjection by ICSI. Cumulus cells and oocytes were frozen at –80°C in RLT buffer (RNeasy kit, Qiagen, Valencia, CA, USA) before RNA extraction. Pools of 20 GV (seven patients, age 30 years ± 4.6), 20 MI (six patients, age 30.1 years ± 6.7) and 16 MII oocytes (six patients, age 34 years ±4.5) were analysed by DNA microarrays. All these oocytes were from couples referred to our centre for cIVF (tubal infertility) or for ICSI.

Complementary RNA preparation and microarray hybridization

RNA was extracted using the micro RNeasy Kit (Qiagen), and the RNA integrity was assessed by using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). RNA quantity was also assessed for some samples using the Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Complementary RNA (cRNA) was prepared according to the manufacturer’s protocol ‘small sample protocol II’, starting from total RNA (ranging from ∼4 ng pooled oocytes to 100 ng cumulus cells) and hybridized to HG-U133 plus 2.0 GeneChip pan-genomic oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA). HG-U133 plus 2.0 arrays contain 54 675 sets of oligonucleotide probes (probeset) which correspond to ≈39 000 unique human genes or predicted genes. The GeneChip system is a robust microarray system with more than 3000 publications using this technology (http://www.affymetrix.com/community/publications/index.affx), little lab-to-lab variability and a good accuracy and precision (Irizarry et al., 2005). Primary image analysis of the arrays was performed by using GeneChip Operating Software 1.2 (GCOS) (Affymetrix), resulting in a single value for each probeset (signal). Data from each different array experiment were scaled to a target value of 100 by GCOS using the ‘global scaling’ method. The dataset was floored to 2, that is, each signal value under 2 was given the value 2.

Statistical analysis

Samples were analysed using a pairwise comparison using the GCOS 1.2 software. Interestingly, this algorithm provides the information of whether a gene is expressed with a defined confidence level or not (detection call). This ‘call’ can be either ‘present’ when the perfect match probes are significantly more hybridized than the mismatch probes, ‘absent’ when both perfect match and mismatch probes display a similar fluorescent signal or ‘marginal’ when the probeset complies neither to the ‘present’ nor to the ‘absent’ call criteria. A gene was denoted as exclusively expressed in one category when this gene displayed a detection call ‘present’ in this given category and ‘absent’ or ‘marginal’ in all the other three categories. A gene was considered as over- or underexpressed in a category when all the three possible pairwise comparisons showed a significant change in P-value (P ≤ 0.01) according to the GCOS 1.2 software and a ratio ≥ 3 or ≤ 0.333 for the genes increased or decreased, respectively. We also determined a list of genes whose expression progressively increased during oocyte maturation by selecting the probesets with a significant increase according to the GCOS 1.2 algorithm and matching the following ratio constraints: cumulus < GV (with GV/cumulus ≥ 3), GV < MI (MI/GV ≥ 1.73) and MI < MII (MII/MI ≥ 1.73), where cumulus, GV, MI and MII stand for signal values in these samples. Note that 1.73 × 1.73 = 3. Gene annotation was based on Unigene Build 176.

For hierarchical clustering, data were filtered [15 000 genes with a significant expression (‘present’ detection call) in at least one sample and with the highest variation coefficient], log transformed, median centred and processed with the CLUSTER and TREEVIEW software packages with the average linkage method and an uncentred correlation (Eisen et al., 1998). Gene ontology (GO) annotations (http://www.geneontology.org/) were obtained and analysed via the Fatigo website tool (http://www.fatigo.org/) using level-three annotations. In some cases, we used the GO annotations downloaded from the Affymetrix NetAffx database. Genes with a role in cell-to-cell communication function were obtained by filtering the genes based on the following criteria: cellular component comprising the terms ‘membrane’ or ‘extracellular’. Bibliographical search was carried out in Pubmed using boolean logic. For each gene G present in Tables I and II, using its Hugo-approved abbreviation or any of its aliases, we looked for publication matching the query ‘gene G AND (gamete or ‘germ cell’ or ‘germ cells’ or egg or eggs or oocytes or oocyte or meiosis)’ for genes found preferentially expressed in oocytes, and the query ‘gene G AND (gamete or ‘germ cell’ or ‘germ cells’ or egg or eggs or oocytes or oocyte or cumulus or granulosa)’ for genes overexpressed in cumulus cells.

The expression, including signal values, of all genes cited in Tables I and II can be examined on our website (http://amazonia.montp.inserm.fr/the_human_oocyte_transcriptome.html) as online supplemental data. The expression of these genes in various normal tissue transcriptome datasets, including ovarian and testis samples, is provided through the Amazonia! database Web page (unpublished data).

Table I.

Genes significantly overexpressed in oocytes

Gene symbol Gene title Fold ratio Probeset Chromosomal location Species References 
Gamete markers       
DAZL Deleted in azoospermia like 976.3 206588_at chr3p24.3 Homo sapiens Nishi et al. (1999), Cauffman et al. (2005) 
DDX4/VASA DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 1181.5 221630_s_at chr5p15.2-p13.1 Homo sapiens Castrillon et al. (2000) 
DPPA3/STELLA Developmental pluripotency-associated 3 10389.1 231385_at chr12p13.31 Homo sapiens Saitou et al. (2002) 
Maturation-promoting factor and related factors       
CCNB1 Cyclin B1 157.0 228729_at chr5q12 Homo sapiens Heikinheimo et al. (1995) 
CCNB2 Cyclin B2 308.8 202705_at chr15q22.2 Bos taurus (cow) Wu et al. (1997) 
CDC2 Cell division cycle 2, G1 to S and G2 to M 18.4 210559_s_at chr10q21.1 Mus musculus (mouse) Kalous et al. (2005) 
CDC25A Cell division cycle 25A 90.8 1555772_a_at chr3p21 Mus musculus (mouse) Wickramasinghe et al. (1995) 
CDC25B Cell division cycle 25B 9.7 201853_s_at chr20p13 Mus musculus (mouse) Lincoln et al. (2002) 
CDC25C Cell division cycle 25C 75.2 205167_s_at chr5q31 Capra hircus (goat) Gall et al. (2002) 
Spindle checkpoint       
BUB1 BUB1 budding uninhibited by benzimidazoles 1 homologue 20.6 209642_at chr2q14 Homo sapiens Steuerwald et al. (2001) 
BUB1B / BUBR1 BUB1 budding uninhibited by benzimidazoles 1 homologue beta 117.8 203755_at chr15q15 Xenopus laevis (frog) Abrieu et al. (2000) 
CENPA Centromere protein A 88.1 204962_s_at chr2p24-p21 Mus musculus (mouse) Schatten et al. (1988) 
CENPE Centromere protein E 113.9 205046_at chr4q24-q25 Mus musculus (mouse) Duesbery et al. (1997) 
CENPH Centromere protein H 14.3 231772_x_at chr5p15.2 NR  
MAD2L1/MAD2 MAD2 mitotic arrest deficient-like 1 53.5 203362_s_at chr4q27 Mus musculus (mouse) Wassmann et al. (2003) 
APC/C complex, securins and cohesions       
ANAPC1/APC1 Anaphase-promoting complex subunit 1 6.8 218575_at chr2q12.1 NR  
ANAPC10/APC10 Anaphase-promoting complex subunit 10 7.0 207845_s_at chr4q31 NR  
CDC20 CDC20 cell division cycle 20 273.9 202870_s_at chr1p34.1 Mus musculus (mouse) Chang et al. (2004) 
PTTG1 Pituitary tumour-transforming 1 58.2 203554_x_at chr5q35.1 Mus musculus (mouse) Yao et al. (2003) 
PTTG3 Pituitary tumour-transforming 3 50.4 208511_at chr8q13.1 NR  
STAG3 Stromal antigen 3 76.9 219753_at Hs.323634 Homo sapiens Prieto et al.(2004) 
Epigenetic remodelling       
DNMT1 DNA (cytosine-5-)-methyltransferase 1 49.8 201697_s_at chr19p13.2 Homo sapiens Huntriss et al. (2004) 
DNMT3B DNA (cytosine-5-)-methyltransferase 3 beta 24.2 220668_s_at chr20q11.2 Homo sapiens Huntriss et al. (2004) 
HDAC9 Histone deacetylase 9 342.6 1552760_at chr7p21.1 Mus musculus (mouse) De La Fuente et al. (2004) 
H1FOO H1 histone family, member O, oocyte specific 414.5 1553064_at chr3q21.3 Homo sapiens Tanaka et al. (2003) 
HCAP-G Chromosome condensation protein G 260.1 218663_at chr4p15.33 NR  
Meiosis, miscellaneous       
AKAP1 A kinase (PRKA) anchor protein 1 72.1 210625_s_at Hs.78921 Rattus norvegicus (rat) Carr et al. (1999) 
MCM3 MCM3 minichromosome maintenance deficient 3 21.2 201555_at chr6p12 Xenopus laevis (frog) Kubota et al. (1995) 
MOS v-mos Moloney murine sarcoma viral oncogene homologue 72.5 221367_at chr8q11 Homo sapiens Pal et al. (1994) 
SPAG16 Sperm-associated antigen 16 393.4 240898_at chr2q34 NR  
TUBB4Q Tubulin, beta polypeptide 4, member Q 866.5 211915_s_at chr4q35 NR  
FBXO5/EMI1 F-box protein 5 414.2 234863_x_at chr6q25-q26 Mus musculus (mouse) Paronetto et al. (2004) 
AURKC Aurora kinase C 49.1 211107_s_at chr19q13.43 NR  
Extracellular matrix, growth factors, cell surface, signalling       
BMP15 Bone morphogenetic protein 15 31.0 221332_at chrxp11.2 Homo sapiens Aaltonen et al. (1999) 
BMP6 Bone morphogenetic protein 6 38.3 206176_at chr6p24-p23 Mus musculus (mouse) Lyons et al. (1989) 
GDF9 Growth differentiation factor 9 83.0 221314_at chr5q31.1 Homo sapiens Aaltonen et al. (1999) 
FGFR2 Fibroblast growth factor receptor 2 8.4 208228_s_at chr10q26 Mus musculus (mouse) Haffner-Krausz et al. (1999) 
FGF9 Fibroblast growth factor 9 (glia-activating factor) 43.8 206404_at chr13q11-q12 NR  
FGF14 Fibroblast growth factor 14 28.1 221310_at chr13q34 NR  
KIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologue  205051_s_at chr4q11-q12 Homo sapiens Liu (2006) 
IL4 Interleukin 4 52.5 207538_at chr5q31.1 NR  
TNFSF13 / APRIL Tumour necrosis factor superfamily, member 13 131.6 210314_x_at chr17p13.1 NR  
ERBB4 v-erb-a erythroblastic leukemia viral oncogene homologue 4 32.3 206794_at chr2q33.3-q34 NR  
FZD3 Frizzled homologue 3 17.9 219683_at chr8p21 NR  
GPR37 G protein-coupled receptor 37 (endothelin receptor type B like) 64.4 209631_s_at chr7q31 NR  
GPR39 G protein-coupled receptor 39 1234.0 229105_at chr2q21-q22 NR  
GPR51 G protein-coupled receptor 51 5.7 209990_s_at chr9q22.1 NR  
GPR126 G protein-coupled receptor 126 11.7 213094_at chr6q24.1 NR  
GPR143 G protein-coupled receptor 143 94.1 206696_at chrxp22.3 NR  
GPR160 G protein-coupled receptor 160 11.3 223423_at chr3q26.2 NR  
ZP1 Zona pellucida glycoprotein 1 (sperm receptor) 86.6 237335_at 11q12.2 Homo sapiens Lefievre et al. (2004) 
ZP2 Zona pellucida glycoprotein 2 (sperm receptor) 1558.8 207933_at chr16p12 Homo sapiens Hinsch et al. (1998) 
ZP3 Zona pellucida glycoprotein 3 (sperm receptor) 87.6 204148_s_at chr7q11.23 Homo sapiens Grootenhuis et al. (1996) 
ZP4 Zona pellucida glycoprotein 4 52.2 231756_at chr1q43 Homo sapiens Eberspaecher et al. (2001) 
SLC5A11 Solute carrier family 5 (sodium/glucose cotransporter), member 11 144.7 237254_at chr16p-p11 NR  
SOCS7 Suppressor of cytokine signalling 7 26.3 2265772_at chr17q12 NR  
Transcription factors       
SOX15 SRY (sex-determining region Y)-box 15 127.2 206122_at chr17p13 NR  
SOX30 SRY (sex determining region Y)-box 30 618.5 207678_s_at chr5q33 NR  
OTX2 Orthodenticle homologue 2 (Drosophila) 5022.8 242128_at chr14q21-q22 NR  
FOXR1 Forkhead box R1 344.1 237613_at chr11q23.3 NR  
Imprinted gene       
MEST Mesoderm-specific transcript homologue 39.2 202016_at chr7q32 Homo sapiens Salpekar et al. (2001) 
Apoptosis       
BNIP1 BCL2/adenovirus E1B 19kDa interacting protein 1 20.8 207829_s_at chr5q33-q34 NR  
BIRC5 Baculoviral IAP repeat-containing 5 (survivin) 23.2 202094_at chr17q25 NR  
BCL2L10 BCL2-like 10 (apoptosis facilitator) 623.3 236491_at chr15q21 Mus musculus (mouse) Burns et al. (2003) 
Gene symbol Gene title Fold ratio Probeset Chromosomal location Species References 
Gamete markers       
DAZL Deleted in azoospermia like 976.3 206588_at chr3p24.3 Homo sapiens Nishi et al. (1999), Cauffman et al. (2005) 
DDX4/VASA DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 1181.5 221630_s_at chr5p15.2-p13.1 Homo sapiens Castrillon et al. (2000) 
DPPA3/STELLA Developmental pluripotency-associated 3 10389.1 231385_at chr12p13.31 Homo sapiens Saitou et al. (2002) 
Maturation-promoting factor and related factors       
CCNB1 Cyclin B1 157.0 228729_at chr5q12 Homo sapiens Heikinheimo et al. (1995) 
CCNB2 Cyclin B2 308.8 202705_at chr15q22.2 Bos taurus (cow) Wu et al. (1997) 
CDC2 Cell division cycle 2, G1 to S and G2 to M 18.4 210559_s_at chr10q21.1 Mus musculus (mouse) Kalous et al. (2005) 
CDC25A Cell division cycle 25A 90.8 1555772_a_at chr3p21 Mus musculus (mouse) Wickramasinghe et al. (1995) 
CDC25B Cell division cycle 25B 9.7 201853_s_at chr20p13 Mus musculus (mouse) Lincoln et al. (2002) 
CDC25C Cell division cycle 25C 75.2 205167_s_at chr5q31 Capra hircus (goat) Gall et al. (2002) 
Spindle checkpoint       
BUB1 BUB1 budding uninhibited by benzimidazoles 1 homologue 20.6 209642_at chr2q14 Homo sapiens Steuerwald et al. (2001) 
BUB1B / BUBR1 BUB1 budding uninhibited by benzimidazoles 1 homologue beta 117.8 203755_at chr15q15 Xenopus laevis (frog) Abrieu et al. (2000) 
CENPA Centromere protein A 88.1 204962_s_at chr2p24-p21 Mus musculus (mouse) Schatten et al. (1988) 
CENPE Centromere protein E 113.9 205046_at chr4q24-q25 Mus musculus (mouse) Duesbery et al. (1997) 
CENPH Centromere protein H 14.3 231772_x_at chr5p15.2 NR  
MAD2L1/MAD2 MAD2 mitotic arrest deficient-like 1 53.5 203362_s_at chr4q27 Mus musculus (mouse) Wassmann et al. (2003) 
APC/C complex, securins and cohesions       
ANAPC1/APC1 Anaphase-promoting complex subunit 1 6.8 218575_at chr2q12.1 NR  
ANAPC10/APC10 Anaphase-promoting complex subunit 10 7.0 207845_s_at chr4q31 NR  
CDC20 CDC20 cell division cycle 20 273.9 202870_s_at chr1p34.1 Mus musculus (mouse) Chang et al. (2004) 
PTTG1 Pituitary tumour-transforming 1 58.2 203554_x_at chr5q35.1 Mus musculus (mouse) Yao et al. (2003) 
PTTG3 Pituitary tumour-transforming 3 50.4 208511_at chr8q13.1 NR  
STAG3 Stromal antigen 3 76.9 219753_at Hs.323634 Homo sapiens Prieto et al.(2004) 
Epigenetic remodelling       
DNMT1 DNA (cytosine-5-)-methyltransferase 1 49.8 201697_s_at chr19p13.2 Homo sapiens Huntriss et al. (2004) 
DNMT3B DNA (cytosine-5-)-methyltransferase 3 beta 24.2 220668_s_at chr20q11.2 Homo sapiens Huntriss et al. (2004) 
HDAC9 Histone deacetylase 9 342.6 1552760_at chr7p21.1 Mus musculus (mouse) De La Fuente et al. (2004) 
H1FOO H1 histone family, member O, oocyte specific 414.5 1553064_at chr3q21.3 Homo sapiens Tanaka et al. (2003) 
HCAP-G Chromosome condensation protein G 260.1 218663_at chr4p15.33 NR  
Meiosis, miscellaneous       
AKAP1 A kinase (PRKA) anchor protein 1 72.1 210625_s_at Hs.78921 Rattus norvegicus (rat) Carr et al. (1999) 
MCM3 MCM3 minichromosome maintenance deficient 3 21.2 201555_at chr6p12 Xenopus laevis (frog) Kubota et al. (1995) 
MOS v-mos Moloney murine sarcoma viral oncogene homologue 72.5 221367_at chr8q11 Homo sapiens Pal et al. (1994) 
SPAG16 Sperm-associated antigen 16 393.4 240898_at chr2q34 NR  
TUBB4Q Tubulin, beta polypeptide 4, member Q 866.5 211915_s_at chr4q35 NR  
FBXO5/EMI1 F-box protein 5 414.2 234863_x_at chr6q25-q26 Mus musculus (mouse) Paronetto et al. (2004) 
AURKC Aurora kinase C 49.1 211107_s_at chr19q13.43 NR  
Extracellular matrix, growth factors, cell surface, signalling       
BMP15 Bone morphogenetic protein 15 31.0 221332_at chrxp11.2 Homo sapiens Aaltonen et al. (1999) 
BMP6 Bone morphogenetic protein 6 38.3 206176_at chr6p24-p23 Mus musculus (mouse) Lyons et al. (1989) 
GDF9 Growth differentiation factor 9 83.0 221314_at chr5q31.1 Homo sapiens Aaltonen et al. (1999) 
FGFR2 Fibroblast growth factor receptor 2 8.4 208228_s_at chr10q26 Mus musculus (mouse) Haffner-Krausz et al. (1999) 
FGF9 Fibroblast growth factor 9 (glia-activating factor) 43.8 206404_at chr13q11-q12 NR  
FGF14 Fibroblast growth factor 14 28.1 221310_at chr13q34 NR  
KIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologue  205051_s_at chr4q11-q12 Homo sapiens Liu (2006) 
IL4 Interleukin 4 52.5 207538_at chr5q31.1 NR  
TNFSF13 / APRIL Tumour necrosis factor superfamily, member 13 131.6 210314_x_at chr17p13.1 NR  
ERBB4 v-erb-a erythroblastic leukemia viral oncogene homologue 4 32.3 206794_at chr2q33.3-q34 NR  
FZD3 Frizzled homologue 3 17.9 219683_at chr8p21 NR  
GPR37 G protein-coupled receptor 37 (endothelin receptor type B like) 64.4 209631_s_at chr7q31 NR  
GPR39 G protein-coupled receptor 39 1234.0 229105_at chr2q21-q22 NR  
GPR51 G protein-coupled receptor 51 5.7 209990_s_at chr9q22.1 NR  
GPR126 G protein-coupled receptor 126 11.7 213094_at chr6q24.1 NR  
GPR143 G protein-coupled receptor 143 94.1 206696_at chrxp22.3 NR  
GPR160 G protein-coupled receptor 160 11.3 223423_at chr3q26.2 NR  
ZP1 Zona pellucida glycoprotein 1 (sperm receptor) 86.6 237335_at 11q12.2 Homo sapiens Lefievre et al. (2004) 
ZP2 Zona pellucida glycoprotein 2 (sperm receptor) 1558.8 207933_at chr16p12 Homo sapiens Hinsch et al. (1998) 
ZP3 Zona pellucida glycoprotein 3 (sperm receptor) 87.6 204148_s_at chr7q11.23 Homo sapiens Grootenhuis et al. (1996) 
ZP4 Zona pellucida glycoprotein 4 52.2 231756_at chr1q43 Homo sapiens Eberspaecher et al. (2001) 
SLC5A11 Solute carrier family 5 (sodium/glucose cotransporter), member 11 144.7 237254_at chr16p-p11 NR  
SOCS7 Suppressor of cytokine signalling 7 26.3 2265772_at chr17q12 NR  
Transcription factors       
SOX15 SRY (sex-determining region Y)-box 15 127.2 206122_at chr17p13 NR  
SOX30 SRY (sex determining region Y)-box 30 618.5 207678_s_at chr5q33 NR  
OTX2 Orthodenticle homologue 2 (Drosophila) 5022.8 242128_at chr14q21-q22 NR  
FOXR1 Forkhead box R1 344.1 237613_at chr11q23.3 NR  
Imprinted gene       
MEST Mesoderm-specific transcript homologue 39.2 202016_at chr7q32 Homo sapiens Salpekar et al. (2001) 
Apoptosis       
BNIP1 BCL2/adenovirus E1B 19kDa interacting protein 1 20.8 207829_s_at chr5q33-q34 NR  
BIRC5 Baculoviral IAP repeat-containing 5 (survivin) 23.2 202094_at chr17q25 NR  
BCL2L10 BCL2-like 10 (apoptosis facilitator) 623.3 236491_at chr15q21 Mus musculus (mouse) Burns et al. (2003) 

NR, not reported.

A Pubmed search using each synonym for this gene (as listed by LocusLink) and one of the following keywords, oocyte, germ cell, gamete, egg, meiosis, did not retrieve any significant result. Expression values of all the genes described in this table can be accessed on our website (see Materials and methods) or can be downloaded as supplemental data.

Table II.

Genes significantly overexpressed in cumulus oophorus cells

Gene symbol Gene title Fold ratio Probeset Chromosomal location Species References 
Hormone and hormone receptors       
LHCGR Luteinizing hormone/ choriogonadotrophin receptor 47.4 207240_s_at Chr2p21 NR  
PGRMC1 Progesterone receptor membrane component 1 13.4 201121_s_at chrxq22-q24 Rattus norvegicus (rat) Park and Mayo (1991) 
PGRMC2 Progesterone receptor membrane component 2 7.4 213227_at Chr4q26 Homo sapiens Tokuyama et al. (2001) 
STAR Steroidogenic acute regulator 33.0 204548_at Chr8p11.2 Homo sapiens Devoto et al. (2001) 
GNRH1 Gonadotrophin-releasing hormone 1 (luteinizing-releasing hormone) 26.7 207987_s_at 8p21-p11.2 Homo sapiens Leung et al. (2003) 
Prostaglandins biosynthesis       
PTGS1 Prostaglandin–endoperoxide synthase 1 5.0 215813_s_at Chr9q32-q33.3 NR  
PTGS2/COX-2 Prostaglandin–endoperoxide synthase 2 18.7 204748_at Chr1q25.2-q25.3 Rattus norvegicus (rat) Sirois et al. (1993), Davis et al. (1999) 
PTGIS Prostaglandin I2 (prostacyclin) synthase 10.8 208131_s_at Chr20q13.11–13 NR  
PTGER2 Prostaglandin E receptor 2 5.4 206631_at Chr14q22 Homo sapiens Narko et al. (2001) 
BMP and BMPR superfamily       
BAMBI BMP and activin membrane-bound inhibitor homologue 9.3 203304_at Chr10p12.3–11.2 NR  
BMP1 Bone morphogenetic protein 1 10.6 202701_at Chr8p21 NR  
BMP8B Bone morphogenetic protein 8b 19.4 235275_at Chr1p35-p32 NR  
BMPR2 Bone morphogenetic protein receptor, type II 7.2 225144_at Chr2q33-q34 Rattus norvegicus (rat) Vitt et al. (2002) 
INHA Inhibin, alpha 5.2 210141_s_at Chr2q33-q36 Homo sapiens Jaatinen et al. (1994) 
INHBA Inhibin, beta A activin A, activin AB alpha polypeptide 34.8 210511_s_at Chr7p15-p13 Homo sapiens Rabinovici et al. (1992) 
TNF and TNFR superfamily       
TNFSF11/OPGL/RANKL Tumour necrosis factor (ligand) superfamily, member 11 79.9 210643_at Chr13q14 NR  
TNFRSF1A/TNF-R Tumour necrosis factor receptor superfamily, member 1A 14.9 207643_s_at chr12p13.2 NR  
TNFRSF10B/DR5 Tumour necrosis factor receptor superfamily, member 10b 5.9 209295_at chr8p22-p21 NR  
TNFRSF12A Tumour necrosis factor receptor superfamily, member 12A 4.6 218368_s_at chr16p13.3 NR  
Complement       
CFHL1 Complement factor H-related 1 105.8 215388_s_at chr1q32 NR  
C7 Complement component 7 135.8 202992_at chr5p13 NR  
IF I factor (complement) 34.7 203854_at chr4q25 NR  
CFH Complement factor H 40.0 213800_at chr1q32 NR  
C1S Complement component 1, s subcomponent 28.0 208747_s_at chr12p13 NR  
C1R Complement component 1, r subcomponent 9.1 212067_s_at chr12p13 NR  
CLU Clusterin (complement lysis inhibitor) 140.9 208791_at chr8p21-p12 Rattus norvegicus (rat) Hurwitz et al. (1996) 
Secreted (other) 
CXCL1 Chemokine (C-X-C motif) ligand 1 8.0 204470_at chr4q21 Homo sapiens Karstrom-Encrantz et al. (1998) 
IL1B Interleukin 1, beta 11.7 39402_at chr2q14 Homo sapiens de los Santos et al. (1998) 
IL8 Interleukin 8 12.9 202859_x_at chr4q13-q21 Homo sapiens Runesson et al. (2000) 
TIMP1 Tissue inhibitor of metalloproteinase 1 (erythroid-potentiating activity, collagenase inhibitor) 25.3 201666_at chrxp11.3–23 Homo sapiens O’Sullivan et al. (1997) 
TIMP3 Tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory) 19.9 201150_s_at chr22q12.3 NR  
PAPPA Pregnancy-associated plasma protein A, pappalysin 1 54.3 224942_at chr9q33.2 Homo sapiens Stanger et al.(1985) 
PTX3 Pentaxin-related gene, rapidly induced by IL-1 beta 25.4 206157_at chr3q25 Homo sapiens Zhang et al.(2005) 
CD molecules       
CD24 CD24 antigen 126.9 208650_s_at chr6q21 Homo sapiens Hourvitz et al. (2000) 
CD44 CD44 antigen 17.0 212063_at chr11p13 NR  
CD47 CD47 antigen 4.6 213857_s_at chr3q13.1 NR  
CD58 CD58 antigen/LFA3 18.6 216942_s_at chr1p13 Homo sapiens Hattori et al. (1998) 
CD59 CD59 antigen p18–20 16.4 228748_at chr11p13 NR  
CD63 CD63 antigen 7.7 200663_at chr12q12-q13 Rattus norvegicus (rat) Espey and Richards (2002) 
CD74 CD74 antigen 5.9 209619_at chr5q32 NR  
CD81 CD81 antigen 6.1 200675_at chr11p15.5 NR  
CDW92 CDW92 antigen 4.7 224596_at chr9q31.2 NR  
CD99 CD99 antigen 4.1 201029_s_at chrxp22.32 Homo sapiens Gordon et al. (1998) 
CD151 CD151 antigen 8.4 204306_s_at chr11p15.5 NR  
CD200 CD200 antigen 22.9 209583_s_at chr3q12-q13 NR  
Membrane bound (other)       
ADAMTS1 A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 58.2 222162_s_at chr21q21.2 Mus musculus (mouse) Russell et al. (2003) 
CSPG2 Chondroitin sulfate proteoglycan 2 (versican) 5.5 211571_s_at chr5q14.3 Mus musculus (mouse) Russell et al. (2003) 
SEMA3A Semaphorin 3A 43.2 206805_at chr7p12.1 NR  
SEMA6A Semaphorin 6A 9.8 225660_at chr5q23.1 NR  
SEMA6D Semaphorin 6D 38.5 226492_at chr15q21.1 NR  
TM4SF1 Transmembrane 4 superfamily member 1 83.8 215034_s_at chr3q21-q25 NR  
TM4SF6 Transmembrane 4 superfamily member 6 13.2 209108_at chrxq22 NR  
TM4SF8 Transmembrane 4 superfamily member 8 5.4 200973_s_at chr15q24.3 NR  
TM4SF10 Transmembrane 4 superfamily member 10 7.5 209656_s_at chrxp11.4 NR  
Transcription factors       
CEBPB CCAAT/enhancer binding protein (C/EBP), beta 7.4 212501_at chr20q13.1 Sus scrofa (pig) Gillio-Meina et al. (2005) 
GATA6 GATA-binding protein 6 56.8 210002_at chr18q11.1–11.2 Homo sapiens Suzuki et al. (1996) 
Other       
PRDX2 Peroxiredoxin 2 6.2 39729_at chr19p13.2 Bos taurus (cow) Leyens et al. (2004) 
PRDX4 Peroxiredoxin 4 31.2 201923_at chrxp22.11 Bos taurus (cow) Leyens et al. (2004) 
PRDX5 Peroxiredoxin 5 5.3 1560587_s_at chr11q13 Bos taurus (cow) Leyens et al. (2004) 
PRDX6 Peroxiredoxin 6 23.2 200844_s_at chr1q25.1 Bos taurus (cow) Leyens et al. (2004) 
CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) 17.0 202284_s_at chr6p21.2 Mus musculus (mouse) Jirawatnotai et al. (2003) 
CDKN1B Cyclin-dependent kinase inhibitor 1B (p27, Kip1) 17.0 209112_at chr12p13.1-p12 Mus musculus (mouse) Robker and Richards (1998) 
CTSK Cathepsin K (pycnodysostosis) 11.8 202450_s_at chr1q21 Rattus norvegicus (rat) Oksjoki et al. (2001) 
Gene symbol Gene title Fold ratio Probeset Chromosomal location Species References 
Hormone and hormone receptors       
LHCGR Luteinizing hormone/ choriogonadotrophin receptor 47.4 207240_s_at Chr2p21 NR  
PGRMC1 Progesterone receptor membrane component 1 13.4 201121_s_at chrxq22-q24 Rattus norvegicus (rat) Park and Mayo (1991) 
PGRMC2 Progesterone receptor membrane component 2 7.4 213227_at Chr4q26 Homo sapiens Tokuyama et al. (2001) 
STAR Steroidogenic acute regulator 33.0 204548_at Chr8p11.2 Homo sapiens Devoto et al. (2001) 
GNRH1 Gonadotrophin-releasing hormone 1 (luteinizing-releasing hormone) 26.7 207987_s_at 8p21-p11.2 Homo sapiens Leung et al. (2003) 
Prostaglandins biosynthesis       
PTGS1 Prostaglandin–endoperoxide synthase 1 5.0 215813_s_at Chr9q32-q33.3 NR  
PTGS2/COX-2 Prostaglandin–endoperoxide synthase 2 18.7 204748_at Chr1q25.2-q25.3 Rattus norvegicus (rat) Sirois et al. (1993), Davis et al. (1999) 
PTGIS Prostaglandin I2 (prostacyclin) synthase 10.8 208131_s_at Chr20q13.11–13 NR  
PTGER2 Prostaglandin E receptor 2 5.4 206631_at Chr14q22 Homo sapiens Narko et al. (2001) 
BMP and BMPR superfamily       
BAMBI BMP and activin membrane-bound inhibitor homologue 9.3 203304_at Chr10p12.3–11.2 NR  
BMP1 Bone morphogenetic protein 1 10.6 202701_at Chr8p21 NR  
BMP8B Bone morphogenetic protein 8b 19.4 235275_at Chr1p35-p32 NR  
BMPR2 Bone morphogenetic protein receptor, type II 7.2 225144_at Chr2q33-q34 Rattus norvegicus (rat) Vitt et al. (2002) 
INHA Inhibin, alpha 5.2 210141_s_at Chr2q33-q36 Homo sapiens Jaatinen et al. (1994) 
INHBA Inhibin, beta A activin A, activin AB alpha polypeptide 34.8 210511_s_at Chr7p15-p13 Homo sapiens Rabinovici et al. (1992) 
TNF and TNFR superfamily       
TNFSF11/OPGL/RANKL Tumour necrosis factor (ligand) superfamily, member 11 79.9 210643_at Chr13q14 NR  
TNFRSF1A/TNF-R Tumour necrosis factor receptor superfamily, member 1A 14.9 207643_s_at chr12p13.2 NR  
TNFRSF10B/DR5 Tumour necrosis factor receptor superfamily, member 10b 5.9 209295_at chr8p22-p21 NR  
TNFRSF12A Tumour necrosis factor receptor superfamily, member 12A 4.6 218368_s_at chr16p13.3 NR  
Complement       
CFHL1 Complement factor H-related 1 105.8 215388_s_at chr1q32 NR  
C7 Complement component 7 135.8 202992_at chr5p13 NR  
IF I factor (complement) 34.7 203854_at chr4q25 NR  
CFH Complement factor H 40.0 213800_at chr1q32 NR  
C1S Complement component 1, s subcomponent 28.0 208747_s_at chr12p13 NR  
C1R Complement component 1, r subcomponent 9.1 212067_s_at chr12p13 NR  
CLU Clusterin (complement lysis inhibitor) 140.9 208791_at chr8p21-p12 Rattus norvegicus (rat) Hurwitz et al. (1996) 
Secreted (other) 
CXCL1 Chemokine (C-X-C motif) ligand 1 8.0 204470_at chr4q21 Homo sapiens Karstrom-Encrantz et al. (1998) 
IL1B Interleukin 1, beta 11.7 39402_at chr2q14 Homo sapiens de los Santos et al. (1998) 
IL8 Interleukin 8 12.9 202859_x_at chr4q13-q21 Homo sapiens Runesson et al. (2000) 
TIMP1 Tissue inhibitor of metalloproteinase 1 (erythroid-potentiating activity, collagenase inhibitor) 25.3 201666_at chrxp11.3–23 Homo sapiens O’Sullivan et al. (1997) 
TIMP3 Tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory) 19.9 201150_s_at chr22q12.3 NR  
PAPPA Pregnancy-associated plasma protein A, pappalysin 1 54.3 224942_at chr9q33.2 Homo sapiens Stanger et al.(1985) 
PTX3 Pentaxin-related gene, rapidly induced by IL-1 beta 25.4 206157_at chr3q25 Homo sapiens Zhang et al.(2005) 
CD molecules       
CD24 CD24 antigen 126.9 208650_s_at chr6q21 Homo sapiens Hourvitz et al. (2000) 
CD44 CD44 antigen 17.0 212063_at chr11p13 NR  
CD47 CD47 antigen 4.6 213857_s_at chr3q13.1 NR  
CD58 CD58 antigen/LFA3 18.6 216942_s_at chr1p13 Homo sapiens Hattori et al. (1998) 
CD59 CD59 antigen p18–20 16.4 228748_at chr11p13 NR  
CD63 CD63 antigen 7.7 200663_at chr12q12-q13 Rattus norvegicus (rat) Espey and Richards (2002) 
CD74 CD74 antigen 5.9 209619_at chr5q32 NR  
CD81 CD81 antigen 6.1 200675_at chr11p15.5 NR  
CDW92 CDW92 antigen 4.7 224596_at chr9q31.2 NR  
CD99 CD99 antigen 4.1 201029_s_at chrxp22.32 Homo sapiens Gordon et al. (1998) 
CD151 CD151 antigen 8.4 204306_s_at chr11p15.5 NR  
CD200 CD200 antigen 22.9 209583_s_at chr3q12-q13 NR  
Membrane bound (other)       
ADAMTS1 A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 58.2 222162_s_at chr21q21.2 Mus musculus (mouse) Russell et al. (2003) 
CSPG2 Chondroitin sulfate proteoglycan 2 (versican) 5.5 211571_s_at chr5q14.3 Mus musculus (mouse) Russell et al. (2003) 
SEMA3A Semaphorin 3A 43.2 206805_at chr7p12.1 NR  
SEMA6A Semaphorin 6A 9.8 225660_at chr5q23.1 NR  
SEMA6D Semaphorin 6D 38.5 226492_at chr15q21.1 NR  
TM4SF1 Transmembrane 4 superfamily member 1 83.8 215034_s_at chr3q21-q25 NR  
TM4SF6 Transmembrane 4 superfamily member 6 13.2 209108_at chrxq22 NR  
TM4SF8 Transmembrane 4 superfamily member 8 5.4 200973_s_at chr15q24.3 NR  
TM4SF10 Transmembrane 4 superfamily member 10 7.5 209656_s_at chrxp11.4 NR  
Transcription factors       
CEBPB CCAAT/enhancer binding protein (C/EBP), beta 7.4 212501_at chr20q13.1 Sus scrofa (pig) Gillio-Meina et al. (2005) 
GATA6 GATA-binding protein 6 56.8 210002_at chr18q11.1–11.2 Homo sapiens Suzuki et al. (1996) 
Other       
PRDX2 Peroxiredoxin 2 6.2 39729_at chr19p13.2 Bos taurus (cow) Leyens et al. (2004) 
PRDX4 Peroxiredoxin 4 31.2 201923_at chrxp22.11 Bos taurus (cow) Leyens et al. (2004) 
PRDX5 Peroxiredoxin 5 5.3 1560587_s_at chr11q13 Bos taurus (cow) Leyens et al. (2004) 
PRDX6 Peroxiredoxin 6 23.2 200844_s_at chr1q25.1 Bos taurus (cow) Leyens et al. (2004) 
CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) 17.0 202284_s_at chr6p21.2 Mus musculus (mouse) Jirawatnotai et al. (2003) 
CDKN1B Cyclin-dependent kinase inhibitor 1B (p27, Kip1) 17.0 209112_at chr12p13.1-p12 Mus musculus (mouse) Robker and Richards (1998) 
CTSK Cathepsin K (pycnodysostosis) 11.8 202450_s_at chr1q21 Rattus norvegicus (rat) Oksjoki et al. (2001) 

NR, not reported.

A Pubmed search using each synonym for this gene (as listed by LocusLink) and one of the following keywords, oocyte, germ cell, gamete, egg, cumulus, granulosa, did not retrieve any significant result. Expression values of all the genes described in this table can be accessed on our website (see Materials and methods) or can be downloaded as supplemental data.

Results

Identification of genes expressed in human oocytes and cumulus cells

Total cRNA was synthesized from pools of GV-, MI- or MII-stage oocytes, or cumulus cells, then labelled and hybridized to pan-genomic oligonucleotide microarrays. We analysed the detection call (GCOS 1.2 software) of all 54 675 probes in oocyte and cumulus samples. Oocytes express in average 8728 genes. The lowest number of genes expressed was found in MII oocytes (n = 5633) and highest in GV oocytes (n = 10 869) (Table III). We found that expression variations between MI, MII and GV samples were low as illustrated by tight scatter plots and high correlation coefficients (0.63–0.92), as opposed to a marked difference of expression between the cumulus sample and the oocyte samples as illustrated by dispersed scatter plots and low correlation coefficients (0.39–0.50) (Figure 1A).

Table III.

Genes expressed in oocytes and cumulus cells

 Germinal vesicle Metaphase I Metaphase II Cumulus 
Expressed genesa 10 869 9682 5633 10 610 
Exclusive genesb 739 326 234 1829 
Genes overexpressedc 104 444 2600 
Genes underexpressedd 803 1514 
 Germinal vesicle Metaphase I Metaphase II Cumulus 
Expressed genesa 10 869 9682 5633 10 610 
Exclusive genesb 739 326 234 1829 
Genes overexpressedc 104 444 2600 
Genes underexpressedd 803 1514 
a

Genes (based on Unigene Build 176) that had at least one probe with a detection call ‘present’.

b

Genes (based on Unigene Build 176) that had a detection call ‘present’ only in one sample category.

c

Genes significantly overexpressed in one sample compared with all other samples, with a fold ratio of at least 3.

d

Genes significantly underexpressed in one sample compared with all other samples, with a fold ratio of at least 0.333.

Figure 1.

Global gene-expression variation. (A) Scatter plots. Each sample was plotted against all other samples to visualize the expression variation. Only the 26 662 probes with at least one sample with a ‘present’ detection call were included. All signal values were floored to 2. Red circles, probes overexpressed in the sample specified on the left side; green circles, probes overexpressed in the sample specified at the bottom of each plot; grey circles, probes whose expression does not vary significantly between the two samples. For each couple of sample, the Pearson’s correlation coefficient was computed (r), based on the signal of probes with at least one sample with a ‘present’ detection call. GV, germinal vesicle; MI, metaphase I; MII, metaphase II. (B) Hierarchical clustering. The expression signatures of oocytes and cumulus cells were visualized by hierarchical clustering on the 15 000 probesets with the highest variation coefficient. The colours indicate the relative expression levels of each gene, with red indicating an expression above median, green indicating expression under median and black representing median expression. Cluster (a) was a group of genes overexpressed in oocyte (GV–MI–MII), including genes such as DAZL, GDF9, BMP15, ZP1,2,3,4. Cluster (b) was group of genes overexpressed in cumulus cells, including genes such as CD24, Activin A, PAPPA, TNTSF11, LHCGR and INHA.

Figure 1.

Global gene-expression variation. (A) Scatter plots. Each sample was plotted against all other samples to visualize the expression variation. Only the 26 662 probes with at least one sample with a ‘present’ detection call were included. All signal values were floored to 2. Red circles, probes overexpressed in the sample specified on the left side; green circles, probes overexpressed in the sample specified at the bottom of each plot; grey circles, probes whose expression does not vary significantly between the two samples. For each couple of sample, the Pearson’s correlation coefficient was computed (r), based on the signal of probes with at least one sample with a ‘present’ detection call. GV, germinal vesicle; MI, metaphase I; MII, metaphase II. (B) Hierarchical clustering. The expression signatures of oocytes and cumulus cells were visualized by hierarchical clustering on the 15 000 probesets with the highest variation coefficient. The colours indicate the relative expression levels of each gene, with red indicating an expression above median, green indicating expression under median and black representing median expression. Cluster (a) was a group of genes overexpressed in oocyte (GV–MI–MII), including genes such as DAZL, GDF9, BMP15, ZP1,2,3,4. Cluster (b) was group of genes overexpressed in cumulus cells, including genes such as CD24, Activin A, PAPPA, TNTSF11, LHCGR and INHA.

We visualized the respective gene expressions across all samples using hierarchical clustering. Average linkage hierarchical clustering on 15 000 genes showed that oocytes cluster together, demonstrating a common gene expression, but are only distantly related to cumulus cells (Figure 1B). These results highlight that feminine germ cells and their nourishing neighbour cumulus cells display very different expression profiles, in agreement with a very different but complementary biological function and with cell lineage disparity.

Specific transcription program in each sample type

We next examined which genes were specific to each sample category, using two different approaches. First, we determined the genes that were only detected in one sample and not in the three other samples. These genes were called ‘exclusively expressed’ (Table III). As expected, cumulus cells have the largest number of exclusively expressed genes (n = 1829), likely because they display a very different transcriptome as compared with oocytes (n = 234–739). Second, we considered the probes that were over- or underexpressed in one sample compared with all three other samples, with a fold ratio of at least 3. Again, cumulus cells show the largest lists of genes, overexpressing 2600 and underexpressing 1514 genes as compared with oocytes. Using this rather stringent criteria (fold change of at least 3 between a given sample and the three other samples), we found very few genes over- or underexpressed in GV and MI oocytes. This shows that very few genes modify their expression between GV and MI oocytes, as opposed to MII oocytes that overexpress more than 400 genes and underexpress more than 800.

We compared functional GO annotations of overexpressed genes versus underexpressed genes in oocytes and cumulus cells. We observed that certain functional annotations were more represented in either oocytes or cumulus cells (Figure 2). There were significantly more genes involved in ‘response to stimulus’, ‘secretion’ and ‘extracellular matrix’ in cumulus cells, suggesting that cumulus cells are more active in cell-to-cell communication processes. Conversely, genes annotated ‘reproduction’, ‘ubiquitin ligase complex’, ‘microtubule-associated complex’, ‘microtubule motor activity’, ‘nucleic acid binding’ and ‘ligase activity’ were significantly more frequently associated with genes overexpressed in oocytes, in agreement with the major processes involved in meiosis and implying microtubules’ attachment to chromosomes and the ubiquitin ligase complex APC/C regulation.

Figure 2.

Differential gene ontology (GO) annotations between oocytes and cumulus cells. We compared the frequency of level-three GO annotations of genes overexpressed in oocytes to those of genes overexpressed in cumulus cells. The statistical analysis was made using the Fatigo website (http://www.fatigo.org/) using Unigene cluster ID. Histograms show the percentage of genes with the specified GO annotation in the group of genes overexpressed in oocytes (purple) or in cumulus (green).

Figure 2.

Differential gene ontology (GO) annotations between oocytes and cumulus cells. We compared the frequency of level-three GO annotations of genes overexpressed in oocytes to those of genes overexpressed in cumulus cells. The statistical analysis was made using the Fatigo website (http://www.fatigo.org/) using Unigene cluster ID. Histograms show the percentage of genes with the specified GO annotation in the group of genes overexpressed in oocytes (purple) or in cumulus (green).

Whole-genome transcriptome of oocytes

We observed that 1514 genes were expressed with at least a threefold increase in oocytes, that is, underexpressed in cumulus cells when compared with oocytes. Selected genes are highlighted in Table I, which is also available as Web supplemental data, including the expression histogram for each gene (http://amazonia.montp.inserm.fr/the_human_oocyte_transcriptome.html). This list includes genes already recognized as specifically expressed in male and female germinal cells in mammals, such as DAZL, the RNA helicase DDX4/VASA or DPPA3/STELLA (full names are listed in Table I). Numerous well-recognized actors of meiosis were highly expressed in oocytes: the components of the maturation-promoting factor (MPF) (CDC2/CDK1, CCNB1 and CCNB2), CDC25 phosphatases (CDC25A, CDC25B and CDC25C), components of the spindle checkpoint (BUB1, BUBR1, MAD2L1/MAD2, CENP-A and CENP-E), CDC20, which is a component of the anaphase-promoting complex (APC/C) and a downstream target, the meiosis-specific sister chromatid arm cohesin STAG3 (Figure 3A). As expected, we observed the overexpression of genes known to be specific to oocytes such as the Zona Pellucida genes (ZP1, ZP2, ZP3 and ZP4), members of the transforming growth factor (TGF)-β superfamily such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 6 and 15 (BMP6 and BMP15), FGFR2, the chromatin remodelling molecules histone deacetylase HDAC9 and the oocyte-specific H1 histone H1FOO (Figure 3B). Thus, the data are in complete agreement with the published studies. Interestingly, we show here that many genes, previously found expressed in oocytes in various animal models, are indeed highly expressed in human oocytes. Hence, our microarray data are of sufficient scope and accuracy to pave the way to a systematic gene-expression exploration of oocyte and cumulus transcriptome.

Figure 3.

Schematic representation of selected genes involved in meiosis and cumulus–oocyte complex (A) Meiosis. Actors of meiosis in oocytes: components of the maturation-promoting factor (MPF), components of the spindle checkpoint, components of the anaphase-promoting complex (APC/C), the downstream targets such as the securin PTTG3 and regulators. Genes in pink are up-regulated in oocytes. Genes that are specific to meiosis are highlighted by an orange hexagon. Genes in white did not display a significant modification in gene expression between oocytes and somatic cells (cumulus cells). See Table I for full name and references. (B) Cumulus–oocyte complex. Genes overexpressed in oocytes (pink) or overexpressed in cumulus (green) that are involved in the cumulus–oocyte complex interactions. Oocyte genes included members of the TGF-β superfamily such as growth differentiation factor 9 (GDF9), fibroblast growth factor 9 and 15 (FGF9, 15), bone morphogenetic protein 6 and 15 (BMP6, 15). Conversely, in cumulus cells, the genes overexpressed included hormonal receptors such as luteinizing hormone/choriogonadotrophin receptor (LHCGR), progesterone receptor membrane component 1 and 2 (PGRMC1, 2), interleukin IL1beta, chemokines (IL8) and CD24 antigen, inhibin alpha (INHA), activin A (INHBA). Genes in red are up-regulated in the oocytes compared with cumulus cells. Genes in green are up-regulated in the cumulus cells compared with oocytes. Genes shown in blue are expressed in oocytes and cumulus cells such as gap junction protein alpha (GJA1). See Table II for complete list of full names and references.

Figure 3.

Schematic representation of selected genes involved in meiosis and cumulus–oocyte complex (A) Meiosis. Actors of meiosis in oocytes: components of the maturation-promoting factor (MPF), components of the spindle checkpoint, components of the anaphase-promoting complex (APC/C), the downstream targets such as the securin PTTG3 and regulators. Genes in pink are up-regulated in oocytes. Genes that are specific to meiosis are highlighted by an orange hexagon. Genes in white did not display a significant modification in gene expression between oocytes and somatic cells (cumulus cells). See Table I for full name and references. (B) Cumulus–oocyte complex. Genes overexpressed in oocytes (pink) or overexpressed in cumulus (green) that are involved in the cumulus–oocyte complex interactions. Oocyte genes included members of the TGF-β superfamily such as growth differentiation factor 9 (GDF9), fibroblast growth factor 9 and 15 (FGF9, 15), bone morphogenetic protein 6 and 15 (BMP6, 15). Conversely, in cumulus cells, the genes overexpressed included hormonal receptors such as luteinizing hormone/choriogonadotrophin receptor (LHCGR), progesterone receptor membrane component 1 and 2 (PGRMC1, 2), interleukin IL1beta, chemokines (IL8) and CD24 antigen, inhibin alpha (INHA), activin A (INHBA). Genes in red are up-regulated in the oocytes compared with cumulus cells. Genes in green are up-regulated in the cumulus cells compared with oocytes. Genes shown in blue are expressed in oocytes and cumulus cells such as gap junction protein alpha (GJA1). See Table II for complete list of full names and references.

We observed that several genes previously reported to be expressed in male germ cells are also highly expressed in human oocytes, in all maturation stages, such as aurora kinase C (AURKC), SOX30 or sperm associated antigen 16 (SPAG16/PF20). Still, the majority of the genes we found overexpressed in oocytes were not yet reported to be associated with gamete biology. Some of these previously unrecognized ‘oocyte genes’ are listed in Table I and comprise several functional categories. After fertilization, the spindle checkpoint inhibition is released and the APC/C complex degrades the securins, resulting in an entry into anaphase. We found that genes of the centromere protein CENPH that interacts with the spindle checkpoint, and the anaphase-promoting complex subunits ANAPC1 and ANPC10 are highly expressed in oocytes. Moreover, the securing genes PTTG1 and PTTG3 are 58 and 50 times more expressed in oocytes than in cumulus cells, respectively. We found several growth factors and growth factor receptors significantly overexpressed in oocytes (IL-4, FGF9, FGF14 and TNFSF13/APRIL), transcription factors (SOX15, OTX2 and FOXR1), three anti-apoptosis molecules (BCL2L10, BNIP1 and BIRC5/Survivin) and the glucose transporter (SLC5A11).

Whole-genome transcriptome of cumulus cells

Conversely, we observed that 2600 genes are overexpressed in cumulus cells compared with all three oocyte samples. The cumulus sample we studied was obtained from an MII oocyte during ovulation. First, we observed a marked expression of the LH receptor LHCGR in cumulus cells, which primes these cells to respond to the LH surge. Second, we observed that genes overexpressed in MII cumulus cells comprise the main genes that are induced by the LH surge during ovulation (Table II). We observed a very high expression of the progesterone receptors PGRMC1 and PGRMC2, and the steroidogenic acute regulator (STAR) that are induced by LH. Similarly, we found that eicosanoids biosynthesis enzymes, such as the two prostaglandin endoperoxide synthetase (PTGS1) and PTGS2/COX2 and the prostaglandin I2 (prostacyclin) synthase (PTGIS), the prostaglandin receptor (PTGER2) and two downstream effectors of this signalling pathway, interleukin IL1beta and pentaxin-related 3 (PTX3), are also overexpressed in cumulus cells. These genes were mostly described in animal models, and we show here for the first time that the RNA expression of these genes is also highly induced in human cumulus cells obtained after ovulation. Two chemokines are highly produced by cumulus cells, CXCL1/GRO-alpha and IL8, in agreement with the invasion of the granulosa by leucocytes during ovulation. Interestingly, the metalloprotease ADAMTS1, as well as its target versican whose cleavage has been shown to contribute to the proteolytic disintegration of the cumulus matrix, was also highly induced. The transcription factor CEBPB, induced after the gonadotrophin surge and after mediating the up-regulation of inhibin alpha (INHA), is found overexpressed in our post-stimulation cumulus cells. Accordingly, INHA and INHBA/activin A are 5 and 34 times more expressed in cumulus cells than in oocytes, respectively. Another transcription factor characteristic of granulosa cells, GATA6, is also highly overexpressed in comparison with oocytes. We observed the up-regulation of peroxiredoxins (PRDX2, PRDX4, PRDX5 and PRDX6), which are part of a family of peroxidases involved in antioxidant protection and cell signalling and recently reported in bovine ovaries (Leyens et al., 2004), as well as a lysosomal cysteine proteinase, cathepsin K (CTSK). Gene codings for protein found in follicular fluid such as PAPPA are also found overexpressed in cumulus cells. Thus, genes found overexpressed in cumulus cells by our whole-genome transcriptome analysis recapitulate previous expression studies on post-LH surge granulosa cells carried out in various species.

Considering that cell-to-cell communication genes are a functional category that plays an essential role in the maturation of the cumulus–oocyte complex, we focused on genes filtered on the GO cellular localization annotations ‘membrane’ or ‘extracellular’ (see Materials and methods); 615 genes passed this filter. The most noticeable genes from this list were ligands (BMP1 and BMP8B) or receptors (BAMBI and BMPR2) from the TGF-β superfamily, ligands (TNFSF11/OPGL/RANKL) or receptors from the tumour necrosis factor receptor (TNFR) superfamily (TNFRSF1A/TNF-R, TNFRSF10B/DR5 and TNFRSF12A), components of the complement (CFHL1, C7, IF, CFH, C1S and C1R) and one inhibitor of the complement system (CLU), semaphorins (SEMA3A, SEMA6A and SEMA6D), tetraspanins (TM4SF1, TM4SF6, TM4SF8 and TM4SF10) and various CD members (CD24, CD44, CD47, CD58, CD59, CD63, CD74, CD81, CDW92, CD99, CD151 and CD200). Table II lists these genes, and references key publications relevant to female reproductive biology. Furthermore, components, such as connexin 43, of the cumulus–oocyte complex signalling pathways were retrieved. We found that this connexin was expressed at a high signal in both cumulus cells and in all oocyte categories, in line with its extracellular domains that provide strong and specific homophilic adhesion properties. Most interestingly, many of these genes were never before highlighted as expressed in granulosa cells.

Differences in gene expression variation during oocyte maturation

An important feature of our work is that we established a transcriptome for each of the three stages of oocyte maturation: GV, MI and MII. We were thus able to identify genes whose expression gradually increased during oocyte maturation (see Materials and methods). Fifty-two probesets were retrieved, including the phosphatase CDC25A, PCNA and SOCS7. However, most of the resulting genes are poorly characterized or only predicted coding sequences. All these genes are candidate markers for oocyte cytoplasmic and/or nuclear maturation (Figure 4).

Figure 4.

Expression histograms of selected genes in oocytes and cumulus cells. Histograms show signal values of 20 genes that are differentially expressed between oocyte (purple) and cumulus cells (green). Gene expression is measured by pan-genomic HG-U133 Plus 2.0 Affymetrix oligonucleotides microarrays, and the signal intensity for each gene is shown on the y-axis as arbitrary units determined by the GCOS 1.2 software (Affymetrix). GV, germinal vesicle; MI, metaphase I; MII, metaphase II; C, cumulus.

Figure 4.

Expression histograms of selected genes in oocytes and cumulus cells. Histograms show signal values of 20 genes that are differentially expressed between oocyte (purple) and cumulus cells (green). Gene expression is measured by pan-genomic HG-U133 Plus 2.0 Affymetrix oligonucleotides microarrays, and the signal intensity for each gene is shown on the y-axis as arbitrary units determined by the GCOS 1.2 software (Affymetrix). GV, germinal vesicle; MI, metaphase I; MII, metaphase II; C, cumulus.

Discussion

We undertook to establish the molecular transcriptome phenotype of the human oocyte and its surrounding cumulus cells by using oligonucleotide microarrays covering most of the genes identified in humans. Relying on a recently developed technique of double in vitro transcription, which amplifies more than 100 000 times the initial RNA input, we were able to establish the expression profile of pooled oocytes from distinct maturation stages and from cumulus cells of MII oocytes. Thus, for the first time, we report in human samples the variation of gene expression during oocyte nuclear maturation, and that of its neighbouring cumulus cells, at whole-genome scale. A global analysis of the number of genes detected in each sample category showed a progressive decrease of the number of genes expressed during oocyte nuclear maturation, with the lowest number of genes expressed found in MII oocytes compared with GV or MI oocytes. This is in agreement with the significant decrease, both in quantity and in diversity, of maternal RNAs observed in mouse oocytes (Bachvarova et al., 1982; Wang et al., 2004). Indeed, GV and MI oocytes over- or underexpressed few genes compared with the other samples (Table III), reflecting a very similar expression profile. By contrast, MII oocytes differed markedly, underexpressing specifically many genes (n = 803), which may be explained by the RNA content decrease. In addition, MII oocytes overexpress 444 genes, which may be because of a specific expression pattern related to the near completion of meiosis or to the longer in vitro incubation time secondary to the IVF procedure (21 or 44 h after insemination).

Hierarchical clustering demonstrated that oocyte expression profiles were markedly different from those of cumulus cells (Figure 1). We compared the oocyte samples with the cumulus cells and we found that 1514 genes were up-regulated in oocytes, whereas 2600 genes were up-regulated in cumulus cells. Analysing these lists of genes, we observed that oocytes markedly overexpressed genes involved in meiosis process such as MPF, APC/C and spindle checkpoint complexes. Full completion of meiosis is only accomplished after fecundation because metaphase exit is prevented by the activity of cytostatic factor (CSF) that will only be relieved by gamete fusion. As expected, EMI1, which was recently found to be part of CSF, is highly expressed in all oocyte samples, as well as MOS. We also found that the two major cyclin-dependent kinase inhibitors CDKN1A/p21 and CDKN1B/p27, acting at the G1-S transition, were found markedly down-regulated in oocytes as compared with cumulus cells (Table II). The separation of sister chromatids at the metaphase-to-anaphase transition is activated by proteases called separases, which are activated by the destruction of the inhibitory chaperone securins. Interestingly, we found two securins highly expressed in all oocyte pools: PTTG1 and PTTG3. These securins are expressed at least 15 times more in oocytes than in cumulus cells, CD34+ sorted bone marrow cells, B lymphocytes or mesenchymal stem cells (data not shown). PTTG1 expression was reported in mice oocytes, but not human oocytes, whereas PTTG3-marked expression in oocytes was not previously noted. Considering that post-ovulation oocytes are germinal cells that have just escaped the very long meiosis I arrest and are due to the second meiosis arrest, securins, that are crucial to these processes, must be expressed at a high level. We propose that PTTG1 and PTTG3 play this role in oocytes (Figure 3). The metaphase-to-anaphase transition is associated with a rapid drop of securin protein level mediated by the proteases of the separase family. Degradation of securins leads to the destruction of cohesins, a ring structure formed by a multisubunit complex that holds sister chromatids together. We confirm the specific up-regulation of the meiosis-specific cohesin subunit STAG3 in human oocytes, whereas the mitotic cohesin STAG2 is markedly down-regulated in oocytes compared with cumulus cells or other somatic cells (data not shown). Thus, as for the securins, two homologues of an essential component of the cell division machinery are differentially expressed between human oocytes and somatic cells, implying that one homologue (the cohesin STAG2) is operating during mitosis, whereas the other homologue (the cohesin STAG3) is replacing the first one during the very specialized cell division process of meiosis.

The high conservation of many of the molecular determinants of gametogenesis in the animal kingdom, sometimes from yeast to mammals, suggests that genes found in mammals’ oocytes should be expressed in human oocytes. We provide here the unambiguous demonstration for many genes that they are indeed strongly overexpressed in the three pools of oocytes (Table I). These genes include CENPA, CENPE, PTTG1, FBXO5/EMI1 and BMP6. These results underscore the consistency of our approach. Furthermore, the inventory of human genes essential for nuclear and cytoplasmic oocyte maturation is an important step towards the comprehensive understanding of oocyte biology.

Although female and male gametes differ in many aspects, they share a common meiosis machinery. Indeed, we see here that genes reported to be expressed specifically in spermatozoa are also highly overexpressed in oocytes in comparison with somatic cumulus cells. This is the case for aurora kinase C (AURKC), sperm associated antigen 16 (SPAG16/PF20) and SOX30 (Osaki et al., 1999; Horowitz et al., 2005; Yan et al., 2005). Three aurora kinases have been identified (AURA/STK6, AURKB and AURKC) that share a conserved catalytic domain and play a role in centrosome separation and maturation, spindle assembly and segregation, and cytokinesis (Giet et al., 2005). Whereas AURA and AURKB are involved in mitosis in somatic cells, AURKC was only found highly expressed in testis, suggesting a tissue-specific role in meiosis. It is therefore of special interest to observe that AURKC is also 49 times more expressed in pure oocyte samples than in somatic cells. Because AURKB and AURKC have a similar cellular localization and a similar biological activity such as SURVIVIN/BIRC5 binding, we propose that AURKC is replacing AURKB during meiosis in both male and female gametes. In line with this proposition, our data show that in oocyte samples, AURKB expression is close to background, whereas SURVIVIN/BIRC5, a known partner of the AURKC complex (Yan et al., 2005), is also strongly overexpressed.

We found the specific up-regulation in oocytes of two methyltransferase enzymes (DNMT1 and DNMT3B), one histone deacetylase (HDAC9) and an oocyte-specific histone (H1FOO). Interestingly, the chromosome condensation protein G (HCAP-G), which is a component of the condensin complex that mediates genome-wide chromosome condensation at the onset of mitosis and directly interacts with DNMT3B (Geiman et al., 2004), is also found preferentially expressed in oocytes, suggesting that this condensin is essential to the nuclear maturation of oocytes. Keeping in line with epigenetic modifications of the genome, we screened our list of oocyte genes for imprinted genes. Of note, one paternally imprinted gene, MEST, was highly overexpressed in all three oocyte samples as compared with cumulus cells, whereas other paternally imprinted genes such as IGF2 and NNAT were not.

We noted the overexpression of two pro-apoptotic genes in oocytes (BNIP1 and BCL2L10). These findings strongly argue in favour of a model where the survival of oocytes is mediated by external signals provided by surrounding cumulus cells rather than by intrinsic cues such as overexpression of anti-apoptotic factors. Accordingly, we found many receptors for growth factors overexpressed on oocytes, including a BMP receptor (BMPR2), the receptor for the stem cell factor (KIT), a member of the EGF receptor family (ERBB4), and a frizzled receptor (FZD3) member of the WNT pathway. In addition, we observed 6 poorly characterized G protein-coupled receptors in oocytes (GPR37, GPR39, GPR51, GPR126, GPR143 and GPR160). The fact that oocytes overexpress these growth factor receptors strongly suggests that the ligands of these receptors are involved in conveying surviving and maturation cues from the oophorus cumulus to the oocytes. Conversely, oocytes express many growth factors. Among the genes, we noted the remarkable overexpression of a ligand from the TNF superfamily, TNFSF13/APRIL, which we found to be expressed 131 times more in oocytes than in cumulus cells. We did not see a significant expression of the two TNF receptors for APRIL, TNFRSF13B/TACI and TNFRSF17/BCMA (data not shown). But it was recently described that APRIL’s binding to proteoglycan was necessary for the survival signal conveyed by this cytokine to targets cells (Ingold et al., 2005). Because cumulus cells overexpress several proteoglycans such as CSPG2/versican (Table II) and SYNDECAN4 (data not shown), APRIL could mediate a comparable trophic signal from the oocyte to the surrounding cumulus cells.

We also focused our analysis on genes for which the expression increased progressively during oocyte meiosis. We postulate that they could be interesting candidate genes for oocyte maturation. Indeed, if these genes fail to be up-regulated in MII-stage oocytes, it is likely that the maturation process was defective. Genes increasing progressively during oocyte maturation include SOCS7. This gene is part of a family of proteins negatively regulating intracellular signal transduction cascades (Krebs and Hilton, 2000). Its overexpression in MII-stage oocytes may indicate the shutting down of specific cytokine signalling. For this category, it must also be noted that many genes are still not characterized and remain without any hint about their function (20 of 48 genes, i.e. 42%). It is not a surprise if so many genes from this list have escaped bioinformatics or biological functional investigations to date, because (i) MII-stage oocytes are a very rare cell type, (ii) it is a very specialized cell type expressing numerous genes that may not be found in any other tissue type, including genes devoid of any molecular motif found in other tissues and (iii) we used pan-genomic microarray to study for the first time gene expression of this cell type without any selection bias. It will be essential to describe in detail the function of these genes to obtain further insights into oocyte biology.

In order to decipher the tight relationship weaved between the oocyte and its surrounding follicle cells, we also analysed the transcriptome profile of cumulus cells. Indeed, 24% of the 2600 genes overexpressed in cumulus cells are annotated either ‘membrane’ or ‘extracellular’, demonstrating a strong bias towards genes involved in cell-to-cell communication processes. The signalling pathways involved comprise progesterone and its receptors, eicosanoids and several enzymes involved in their biosynthesis and chemokines. We showed in this study that cumulus cells up-regulated hormonal receptors and hormones such as LHCGR, Inhibin alpha, Inhibin beta A, GNRH1 and progesterone receptor membrane component 1 and 2.

Interestingly, cumulus cells overexpress BMPR2, which is the receptor for GDF9 that is overexpressed by oocytes, demonstrating a typical intercellular communication process (Figure 4). In addition to the inhibins INHA and INHBA, we also observed the overexpression of BMP1 and BMP8B, as well as the pseudoreceptor BAMBI, lacking an intracellular serine/threonine kinase domain and thus negatively regulating TGF-β signalling. Another important growth factor superfamily found to be overexpressed in cumulus cells is the TNF superfamily. The marked overexpression of TNFSF11/OPGL/RANKL (80 times more expressed in cumulus cells than in oocytes) is intriguing and awaits further investigations. Magier et al. (1990) suggested a positive effect of cumulus cells on fertilization, and a protective effect and a possible beneficial effect on further embryo development. In addition, Platteau et al. (2004) suggested that the exogenous luteinizing hormone activity may influence treatment outcome in IVF but not in ICSI. We provide here molecular evidence for cumulus cell expression of hormones and growth factors that could mediate these functions.

Another puzzling observation is the increased expression of seven complement factors or closely related genes. Whether this overexpression is involved in the cellular destruction process taking place in the antrum during ovulation needs to be considered. Finally, cumulus cells express several other cell-surface gene families such as semaphorins, first identified for their role in neuron guidance, tetraspanins, with one member, CD9, directly involved in fertilization (Le Naour et al., 2000), and many other CD molecules with various functions (Table II). Very interestingly, some genes overexpressed in granulosa cells are also found expressed in ovarian tumours. We found, for example, a high expression in cumulus cells of CD24 and CD99, which are expressed in ovarian tumours and have been proposed as either diagnostic tools (Choi et al., 2000) or as prognostic tools (Kristiansen et al., 2002). These findings suggest that many of the genes overexpressed in cumulus samples, including the cell-surface markers of cumulus cells listed in Table II, could provide ovarian cancer markers.

We pooled oocytes according to their maturation stage for this first, exploratory, whole-genome transcriptome analysis. This strategy levelled down differences that would be associated with different IVF settings such as maternal age, sperm exposure or in vitro incubation time. In order to describe the expression modifications that may be related to specific conditions, we are currently analysing the transcriptome of oocytes pooled according to the hormonal profile at day 3, maternal age or ovarian stimulation protocol. Nevertheless, to appreciate variations in gene expression according to each patient idiosyncrasies, we will need to achieve reliable transcriptome analysis from single oocytes.

In conclusion, DNA microarray provided us with the opportunity to analyse human oocytes and cumulus cell-expression profiles on a genome scale and permitted significant progress in the understanding of the molecular events involved in the processes governing oocyte maturation. Many of the genes described here may well provide markers to monitor health, viability and competence of oocytes. In addition, underpinning oocyte growth factor receptors should help to design optimal in vitro culture conditions for oocyte and early embryo development.

Acknowledgements

We are grateful to Stephan Gasca, Irène Fries, Benoit Richard, Benoit Latucca and Benoît Crassou for helpful discussions. We thank all members of our ART team for their assistance during this study. This study was supported by grants from Ferring and Organon Pharmaceuticals, France.

References

Aaltonen
J
Laitinen
MP
Vuojolainen
K
Jaatinen
R
Horelli-Kuitunen
N
Seppa
L
Louhio
H
Tuuri
T
Sjoberg
J
Butzow
R
, et al.  . 
Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis
J Clin Endocrinol Metab
 , 
1999
, vol. 
84
 (pg. 
2744
-
2750
)
Abrieu
A
Kahana
JA
Wood
KW
Cleveland
DW
CENP-E as an essential component of the mitotic checkpoint in vitro
Cell
 , 
2000
, vol. 
102
 (pg. 
817
-
826
)
Bachvarova
R
Burns
JP
Spiegelman
I
Choy
J
Chaganti
RS
Morphology and transcriptional activity of mouse oocyte chromosomes
Chromosoma
 , 
1982
, vol. 
86
 (pg. 
181
-
196
)
Burns
KH
Owens
GE
Ogbonna
SC
Nilson
JH
Matzuk
MM
Expression profiling analyses of gonadotropin responses and tumor development in the absence of inhibins
Endocrinology
 , 
2003
, vol. 
144
 (pg. 
4492
-
4507
)
Carr
DW
Cutler
RE
Jr
Cottom
JE
Salvador
LM
Fraser
ID
Scott
JD
Hunzicker-Dunn
M
Identification of cAMP-dependent protein kinase holoenzymes in preantral- and preovulatory-follicle-enriched ovaries, and their association with A-kinase-anchoring proteins
Biochem J
 , 
1999
, vol. 
344
 
Pt 2
(pg. 
613
-
623
)
Castrillon
DH
Quade
BJ
Wang
TY
Quigley
C
Crum
CP
The human VASA gene is specifically expressed in the germ cell lineage
Proc Natl Acad Sci USA
 , 
2000
, vol. 
97
 (pg. 
9585
-
9590
)
Cauffman
G
Van de Velde
H
Liebaers
I
Van Steirteghem
A
DAZL expression in human oocytes, preimplantation embryos and embryonic stem cells
Mol Hum Reprod
 , 
2005
, vol. 
11
 (pg. 
405
-
411
)
Chang
HY
Levasseur
M
Jones
KT
Degradation of APCcdc20 and APCcdh1 substrates during the second meiotic division in mouse eggs
J Cell Sci
 , 
2004
, vol. 
117
 (pg. 
6289
-
6296
)
Choi
YL
Kim
HS
Ahn
G
Immunoexpression of inhibin alpha subunit, inhibin/activin betaA subunit and CD99 in ovarian tumors
Arch Pathol Lab Med
 , 
2000
, vol. 
124
 (pg. 
563
-
569
)
Davis
BJ
Lennard
DE
Lee
CA
Tiano
HF
Morham
SG
Wetsel
WC
Langenbach
R
Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1beta
Endocrinology
 , 
1999
, vol. 
140
 (pg. 
2685
-
2695
)
De La Fuente
R
Viveiros
MM
Burns
KH
Adashi
EY
Matzuk
MM
Eppig
JJ
Major chromatin remodeling in the germinal vesicle (GV) of mammalian oocytes is dispensable for global transcriptional silencing but required for centromeric heterochromatin function
Dev Biol
 , 
2004
, vol. 
275
 (pg. 
447
-
458
)
de los Santos
MJ
Anderson
DJ
Racowsky
C
Simon
C
Hill
JA
Expression of interleukin-1 system genes in human gametes
Biol Reprod
 , 
1998
, vol. 
59
 (pg. 
1419
-
1424
)
Dekel
N
Beers
WH
Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus
Dev Biol
 , 
1980
, vol. 
75
 (pg. 
247
-
254
)
Devoto
L
Kohen
P
Gonzalez
RR
Castro
O
Retamales
I
Vega
M
Carvallo
P
Christenson
LK
Strauss
JF
III
Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase
J Clin Endocrinol Metab
 , 
2001
, vol. 
86
 (pg. 
5633
-
5639
)
Duesbery
NS
Choi
T
Brown
KD
Wood
KW
Resau
J
Fukasawa
K
Cleveland
DW
Vande Woude
GF
CENP-E is an essential kinetochore motor in maturing oocytes and is masked during mos-dependent, cell cycle arrest at metaphase II
Proc Natl Acad Sci USA
 , 
1997
, vol. 
94
 (pg. 
9165
-
9170
)
Eberspaecher
U
Becker
A
Bringmann
P
van der Merwe
L
Donner
P
Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries
Cell Tissue Res
 , 
2001
, vol. 
303
 (pg. 
277
-
287
)
Eisen
MB
Spellman
PT
Brown
PO
Botstein
D
Cluster analysis and display of genome-wide expression patterns
Proc Natl Acad Sci USA
 , 
1998
, vol. 
95
 (pg. 
14863
-
14868
)
Elvin
JA
Clark
AT
Wang
P
Wolfman
NM
Matzuk
MM
Paracrine actions of growth differentiation factor-9 in the mammalian ovary
Mol Endocrinol
 , 
1999
, vol. 
13
 (pg. 
1035
-
1048
)
Espey
LL
Richards
JS
Temporal and spatial patterns of ovarian gene transcription following an ovulatory dose of gonadotropin in the rat
Biol Reprod
 , 
2002
, vol. 
67
 (pg. 
1662
-
1670
)
Fair
T
Hyttel
P
Greve
T
Bovine oocyte diameter in relation to maturational competence and transcriptional activity
Mol Reprod Dev
 , 
1995
, vol. 
42
 (pg. 
437
-
442
)
Gall
L
Ruffini
S
Le Bourhis
D
Boulesteix
C
Cdc25C expression in meiotically competent and incompetent goat oocytes
Mol Reprod Dev
 , 
2002
, vol. 
62
 (pg. 
4
-
12
)
Geiman
TM
Sankpal
UT
Robertson
AK
Chen
Y
Mazumdar
M
Heale
JT
Schmiesing
JA
Kim
W
Yokomori
K
Zhao
Y
, et al.  . 
Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery
Nucleic Acids Res
 , 
2004
, vol. 
32
 (pg. 
2716
-
2729
)
Giet
R
Petretti
C
Prigent
C
Aurora kinases, aneuploidy and cancer, a coincidence or a real link?
Trends Cell Biol
 , 
2005
, vol. 
15
 (pg. 
241
-
250
)
Gillio-Meina
C
Hui
YY
LaVoie
HA
Expression of CCAAT/enhancer binding proteins alpha and beta in the porcine ovary and regulation in primary cultures of granulosa cells
Biol Reprod
 , 
2005
, vol. 
72
 (pg. 
1194
-
1204
)
Gordon
MD
Corless
C
Renshaw
AA
Beckstead
J
CD99, keratin, and vimentin staining of sex cord-stromal tumors, normal ovary, and testis
Mod Pathol
 , 
1998
, vol. 
11
 (pg. 
769
-
773
)
Grootenhuis
AJ
Philipsen
HL
de Breet-Grijsbach
JT
van Duin
M
Immunocytochemical localization of ZP3 in primordial follicles of rabbit, marmoset, rhesus monkey and human ovaries using antibodies against human ZP3
J Reprod Fertil Suppl
 , 
1996
, vol. 
50
 (pg. 
43
-
54
)
Haffner-Krausz
R
Gorivodsky
M
Chen
Y
Lonai
P
Expression of Fgfr2 in the early mouse embryo indicates its involvement in preimplantation development
Mech Dev
 , 
1999
, vol. 
85
 (pg. 
167
-
172
)
Hattori
N
Fujiwara
H
Maeda
M
Yoshioka
S
Higuchi
T
Mori
T
Ohishi
N
Minami
M
Fujii
S
Ueda
M
Human large luteal cells in the menstrual cycle and early pregnancy express leukotriene A4 hydrolase
Mol Hum Reprod
 , 
1998
, vol. 
4
 (pg. 
803
-
810
)
Heikinheimo
O
Lanzendorf
SE
Baka
SG
Gibbons
WE
Cell cycle genes c-mos and cyclin-B1 are expressed in a specific pattern in human oocytes and preimplantation embryos
Hum Reprod
 , 
1995
, vol. 
10
 (pg. 
699
-
707
)
Hinsch
E
Hagele
W
van der Ven
H
Oehninger
S
Schill
WB
Hinsch
KD
Immunological identification of zona pellucida 2 (ZP2) protein in human oocytes
Andrologia
 , 
1998
, vol. 
30
 (pg. 
281
-
287
)
Horowitz
E
Zhang
Z
Jones
BH
Moss
SB
Ho
C
Wood
JR
Wang
X
Sammel
MD
Strauss
JF
III
Patterns of expression of sperm flagellar genes: early expression of genes encoding axonemal proteins during the spermatogenic cycle and shared features of promoters of genes encoding central apparatus proteins
Mol Hum Reprod
 , 
2005
, vol. 
11
 (pg. 
307
-
317
)
Hourvitz
A
Widger
AE
Filho
FL
Chang
RJ
Adashi
EY
Erickson
GF
Pregnancy-associated plasma protein-A gene expression in human ovaries is restricted to healthy follicles and corpora lutea
J Clin Endocrinol Metab
 , 
2000
, vol. 
85
 (pg. 
4916
-
4920
)
Huntriss
J
Hinkins
M
Oliver
B
Harris
SE
Beazley
JC
Rutherford
AJ
Gosden
RG
Lanzendorf
SE
Picton
HM
Expression of mRNAs for DNA methyltransferases and methyl-CpG-binding proteins in the human female germ line, preimplantation embryos, and embryonic stem cells
Mol Reprod Dev
 , 
2004
, vol. 
67
 (pg. 
323
-
336
)
Hurwitz
A
Ruutiainen-Altman
K
Marzella
L
Botero
L
Dushnik
M
Adashi
EY
Follicular atresia as an apoptotic process: atresia-associated increase in the ovarian expression of the putative apoptotic marker sulfated glycoprotein-2
J Soc Gynecol Investig
 , 
1996
, vol. 
3
 (pg. 
199
-
208
)
Ingold
K
Zumsteg
A
Tardivel
A
Huard
B
Steiner
QG
Cachero
TG
Qiang
F
Gorelik
L
Kalled
SL
Acha-Orbea
H
, et al.  . 
Identification of proteoglycans as the APRIL-specific binding partners
J Exp Med
 , 
2005
, vol. 
201
 (pg. 
1375
-
1383
)
Irizarry
RA
Warren
D
Spencer
F
Kim
IF
Biswal
S
Frank
BC
Gabrielson
E
Garcia
JG
Geoghegan
J
Germino
G
, et al.  . 
Multiple-laboratory comparison of microarray platforms
Nat Methods
 , 
2005
, vol. 
2
 (pg. 
345
-
350
)
Jaatinen
TA
Penttila
TL
Kaipia
A
Ekfors
T
Parvinen
M
Toppari
J
Expression of inhibin alpha, beta A and beta B messenger ribonucleic acids in the normal human ovary and in polycystic ovarian syndrome
J Endocrinol
 , 
1994
, vol. 
143
 (pg. 
127
-
137
)
Jirawatnotai
S
Moons
DS
Stocco
CO
Franks
R
Hales
DB
Gibori
G
Kiyokawa
H
The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 cooperate to restrict proliferative life span in differentiating ovarian cells
J Biol Chem
 , 
2003
, vol. 
278
 (pg. 
17021
-
17027
)
Kalous
J
Solc
P
Baran
V
Kubelka
M
Schultz
RM
Motlik
J
PKB/AKT is involved in resumption of meiosis in mouse oocytes
Biol Cell
 , 
2005
, vol. 
98
 (pg. 
111
-
123
)
Karstrom-Encrantz
L
Runesson
E
Bostrom
EK
Brannstrom
M
Selective presence of the chemokine growth-regulated oncogene alpha (GROalpha) in the human follicle and secretion from cultured granulosa-lutein cells at ovulation
Mol Hum Reprod
 , 
1998
, vol. 
4
 (pg. 
1077
-
1083
)
Krebs
DL
Hilton
DJ
SOCS: physiological suppressors of cytokine signaling
J Cell Sci
 , 
2000
, vol. 
113
 
Pt 16
(pg. 
2813
-
2819
)
Kristiansen
G
Denkert
C
Schluns
K
Dahl
E
Pilarsky
C
Hauptmann
S
CD24 is expressed in ovarian cancer and is a new independent prognostic marker of patient survival
Am J Pathol
 , 
2002
, vol. 
161
 (pg. 
1215
-
1221
)
Kubota
Y
Mimura
S
Nishimoto
S
Takisawa
H
Nojima
H
Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor
Cell
 , 
1995
, vol. 
81
 (pg. 
601
-
609
)
Larsen
WJ
Wert
SE
Brunner
GD
A dramatic loss of cumulus cell gap junctions is correlated with germinal vesicle breakdown in rat oocytes
Dev Biol
 , 
1986
, vol. 
113
 (pg. 
517
-
521
)
Le
Naour F
Rubinstein
E
Jasmin
C
Prenant
M
Boucheix
C
Severely reduced female fertility in CD9-deficient mice
Science
 , 
2000
, vol. 
287
 (pg. 
319
-
321
)
Lefievre
L
Conner
SJ
Salpekar
A
Olufowobi
O
Ashton
P
Pavlovic
B
Lenton
W
Afnan
M
Brewis
IA
Monk
M
, et al.  . 
Four zona pellucida glycoproteins are expressed in the human
Hum Reprod
 , 
2004
, vol. 
19
 (pg. 
1580
-
1586
)
Leung
PC
Cheng
CK
Zhu
XM
Multi-factorial role of GnRH-I and GnRH-II in the human ovary
Mol Cell Endocrinol
 , 
2003
, vol. 
202
 (pg. 
145
-
153
)
Leyens
G
Knoops
B
Donnay
I
Expression of peroxiredoxins in bovine oocytes and embryos produced in vitro
Mol Reprod Dev
 , 
2004
, vol. 
69
 (pg. 
243
-
251
)
Lincoln
AJ
Wickramasinghe
D
Stein
P
Schultz
RM
Palko
ME
De Miguel
MP
Tessarollo
L
Donovan
PJ
Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation
Nat Genet
 , 
2002
, vol. 
30
 (pg. 
446
-
449
)
Liu
K
Stem cell factor (SCF)-kit mediated phosphatidylinositol 3 (PI3) kinase signaling during mammalian oocyte growth and early follicular development
Front Biosci
 , 
2006
, vol. 
11
 (pg. 
126
-
135
)
Lyons
KM
Pelton
RW
Hogan
BL
Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development
Genes Dev
 , 
1989
, vol. 
3
 (pg. 
1657
-
1668
)
Magier
S
van der Ven
HH
Diedrich
K
Krebs
D
Significance of cumulus oophorus in in-vitro fertilization and oocyte viability and fertility
Hum Reprod
 , 
1990
, vol. 
5
 (pg. 
847
-
852
)
Moor
RM
Dai
Y
Lee
C
Fulka
J
Jr
Oocyte maturation and embryonic failure
Hum Reprod Update
 , 
1998
, vol. 
4
 (pg. 
223
-
236
)
Narko
K
Saukkonen
K
Ketola
I
Butzow
R
Heikinheimo
M
Ristimaki
A
Regulated expression of prostaglandin E(2) receptors EP2 and EP4 in human ovarian granulosa-luteal cells
J Clin Endocrinol Metab
 , 
2001
, vol. 
86
 (pg. 
1765
-
1768
)
Nishi
S
Hoshi
N
Kasahara
M
Ishibashi
T
Fujimoto
S
Existence of human DAZLA protein in the cytoplasm of human oocytes
Mol Hum Reprod
 , 
1999
, vol. 
5
 (pg. 
495
-
497
)
O’Sullivan
MJ
Stamouli
A
Thomas
EJ
Richardson
MC
Gonadotrophin regulation of production of tissue inhibitor of metalloproteinases-1 by luteinized human granulosa cells: a potential mechanism for luteal rescue
Mol Hum Reprod
 , 
1997
, vol. 
3
 (pg. 
405
-
410
)
Oksjoki
S
Soderstrom
M
Vuorio
E
Anttila
L
Differential expression patterns of cathepsins B, H, K, L and S in the mouse ovary
Mol Hum Reprod
 , 
2001
, vol. 
7
 (pg. 
27
-
34
)
Osaki
E
Nishina
Y
Inazawa
J
Copeland
NG
Gilbert
DJ
Jenkins
NA
Ohsugi
M
Tezuka
T
Yoshida
M
Semba
K
Identification of a novel Sry-related gene and its germ cell-specific expression
Nucleic Acids Res
 , 
1999
, vol. 
27
 (pg. 
2503
-
2510
)
Pal
SK
Torry
D
Serta
R
Crowell
RC
Seibel
MM
Cooper
GM
Kiessling
AA
Expression and potential function of the c-mos proto-oncogene in human eggs
Fertil Steril
 , 
1994
, vol. 
61
 (pg. 
496
-
503
)
Park
OK
Mayo
KE
Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge
Mol Endocrinol
 , 
1991
, vol. 
5
 (pg. 
967
-
978
)
Paronetto
MP
Giorda
E
Carsetti
R
Rossi
P
Geremia
R
Sette
C
Functional interaction between p90Rsk2 and Emi1 contributes to the metaphase arrest of mouse oocytes
EMBO J
 , 
2004
, vol. 
23
 (pg. 
4649
-
4659
)
Platteau
P
Smitz
J
Albano
C
Sorensen
P
Arce
JC
Devroey
P
Exogenous luteinizing hormone activity may influence the treatment outcome in in vitro fertilization but not in intracytoplasmic sperm injection cycles
Fertil Steril
 , 
2004
, vol. 
81
 (pg. 
1401
-
1404
)
Prieto
I
Tease
C
Pezzi
N
Buesa
JM
Ortega
S
Kremer
L
Martinez
A
Martinez
AC
Hulten
MA
Barbero
JL
Cohesin component dynamics during meiotic prophase I in mammalian oocytes
Chromosome Res
 , 
2004
, vol. 
12
 (pg. 
197
-
213
)
Rabinovici
J
Spencer
SJ
Doldi
N
Goldsmith
PC
Schwall
R
Jaffe
RB
Activin-A as an intraovarian modulator: actions, localization, and regulation of the intact dimer in human ovarian cells
J Clin Invest
 , 
1992
, vol. 
89
 (pg. 
1528
-
1536
)
Robker
RL
Richards
JS
Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1
Mol Endocrinol
 , 
1998
, vol. 
12
 (pg. 
924
-
940
)
Runesson
E
Ivarsson
K
Janson
PO
Brannstrom
M
Gonadotropin- and cytokine-regulated expression of the chemokine interleukin 8 in the human preovulatory follicle of the menstrual cycle
J Clin Endocrinol Metab
 , 
2000
, vol. 
85
 (pg. 
4387
-
4395
)
Russell
DL
Doyle
KM
Ochsner
SA
Sandy
JD
Richards
JS
Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation
J Biol Chem
 , 
2003
, vol. 
278
 (pg. 
42330
-
42339
)
Saitou
M
Barton
SC
Surani
MA
A molecular programme for the specification of germ cell fate in mice
Nature
 , 
2002
, vol. 
418
 (pg. 
293
-
300
)
Salpekar
A
Huntriss
J
Bolton
V
Monk
M
The use of amplified cDNA to investigate the expression of seven imprinted genes in human oocytes and preimplantation embryos
Mol Hum Reprod
 , 
2001
, vol. 
7
 (pg. 
839
-
844
)
Schatten
G
Simerly
C
Palmer
DK
Margolis
RL
Maul
G
Andrews
BS
Schatten
H
Kinetochore appearance during meiosis, fertilization and mitosis in mouse oocytes and zygotes
Chromosoma
 , 
1988
, vol. 
96
 (pg. 
341
-
352
)
Sirois
J
Levy
LO
Simmons
DL
Richards
JS
Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Identification of functional and protein-binding regions
J Biol Chem
 , 
1993
, vol. 
268
 (pg. 
12199
-
12206
)
Stanger
JD
Yovich
JL
Grudzinskas
JG
Bolton
AE
Relation between pregnancy-associated plasma protein A (PAPP-A) in human peri-ovulatory follicle fluid and the collection and fertilization of human ova in vitro
Br J Obstet Gynaecol
 , 
1985
, vol. 
92
 (pg. 
786
-
792
)
Steuerwald
N
Cohen
J
Herrera
RJ
Sandalinas
M
Brenner
CA
Association between spindle assembly checkpoint expression and maternal age in human oocytes
Mol Hum Reprod
 , 
2001
, vol. 
7
 (pg. 
49
-
55
)
Suzuki
E
Evans
T
Lowry
J
Truong
L
Bell
DW
Testa
JR
Walsh
K
The human GATA-6 gene: structure, chromosomal location, and regulation of expression by tissue-specific and mitogen-responsive signals
Genomics
 , 
1996
, vol. 
38
 (pg. 
283
-
290
)
Tanaka
Y
Kato
S
Tanaka
M
Kuji
N
Yoshimura
Y
Structure and expression of the human oocyte-specific histone H1 gene elucidated by direct RT-nested PCR of a single oocyte
Biochem Biophys Res Commun
 , 
2003
, vol. 
304
 (pg. 
351
-
357
)
Tokuyama
O
Nakamura
Y
Muso
A
Honda
K
Ishiko
O
Ogita
S
Expression and distribution of cyclooxygenase-2 in human periovulatory ovary
Int J Mol Med
 , 
2001
, vol. 
8
 (pg. 
603
-
606
)
Vitt
UA
Mazerbourg
S
Klein
C
Hsueh
AJ
Bone morphogenetic protein receptor type II is a receptor for growth differentiation factor-9
Biol Reprod
 , 
2002
, vol. 
67
 (pg. 
473
-
480
)
Wang
QT
Piotrowska
K
Ciemerych
MA
Milenkovic
L
Scott
MP
Davis
RW
Zernicka-Goetz
M
A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo
Dev Cell
 , 
2004
, vol. 
6
 (pg. 
133
-
144
)
Wassmann
K
Niault
T
Maro
B
Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes
Curr Biol
 , 
2003
, vol. 
13
 (pg. 
1596
-
1608
)
Wickramasinghe
D
Becker
S
Ernst
MK
Resnick
JL
Centanni
JM
Tessarollo
L
Grabel
LB
Donovan
PJ
Two CDC25 homologues are differentially expressed during mouse development
Development
 , 
1995
, vol. 
121
 (pg. 
2047
-
2056
)
Wu
B
Ignotz
G
Currie
WB
Yang
X
Dynamics of maturation-promoting factor and its constituent proteins during in vitro maturation of bovine oocytes
Biol Reprod
 , 
1997
, vol. 
56
 (pg. 
253
-
259
)
Yan
C
Wang
P
DeMayo
J
DeMayo
FJ
Elvin
JA
Carino
C
Prasad
SV
Skinner
SS
Dunbar
BS
Dube
JL
, et al.  . 
Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function
Mol Endocrinol
 , 
2001
, vol. 
15
 (pg. 
854
-
866
)
Yan
X
Cao
L
Li
Q
Wu
Y
Zhang
H
Saiyin
H
Liu
X
Zhang
X
Shi
Q
Yu
L
Aurora C is directly associated with Survivin and required for cytokinesis
Genes Cells
 , 
2005
, vol. 
10
 (pg. 
617
-
626
)
Yao
YQ
Xu
JS
Lee
WM
Yeung
WS
Lee
KF
Identification of mRNAs that are up-regulated after fertilization in the murine zygote by suppression subtractive hybridization
Biochem Biophys Res Commun
 , 
2003
, vol. 
304
 (pg. 
60
-
66
)
Zhang
X
Jafari
N
Barnes
RB
Confino
E
Milad
M
Kazer
RR
Studies of gene expression in human cumulus cells indicate pentraxin 3 as a possible marker for oocyte quality
Fertil Steril
 , 
2005
, vol. 
83
 
Suppl. 1
(pg. 
1169
-
1179
)
© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org