Hum. Reprod. Advance Access originally published online on March 29, 2006
Human Reproduction 2006 21(7):1705-1719; doi:10.1093/humrep/del065
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The human cumulus–oocyte complex gene-expression profile
1 CHU Montpellier, Institut de Recherche en BiothÉrapie, Hôpital Saint-Eloi, Montpellier, France; 2 INSERM, U 475 3 Université Montpellier1, UFR de médecine, Montpellier, France; 4 CHU Montpellier, Service de Biologie de la Reproduction B, Hôpital Arnaud de Villeneuve, Montpellier, France; 5 Institut de Génétique Humaine, CNRS UPR1142, Montpellier, France; and 6 CHU Montpellier, Service de Gynécologie-Obstétrique B, Hôpital Arnaud de Villeneuve, Montpellier, France
7 To whom correspondence should be addressed at: Research Institute for Biotherapy, HÔpital Saint-Eloi, 80 rue Augustin Fliche, 34295 Montpellier Cedex 5, France. E-mail: devos{at}montp.inserm.fr
8 To whom correspondence should be addressed at: CHU Montpellier, Service de Biologie de la Reproduction B, HÔpital Arnaud de Villeneuve, 371, av. du Doyen Gaston Giraud, 34295 Montpellier Cedex 5. E-mail: s-hamamah{at}chu-montpellier.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgements References |
|---|
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.
Key words: cumulus/germinal cells/microarray/oocytes
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgements References |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgements References |
|---|
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 x 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).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgements References |
|---|
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).
|
|
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.
|
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)-
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.
|
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- 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).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgements References |
|---|
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- 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 |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgements References |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgements References |
|---|
Aaltonen J, Laitinen MP, Vuojolainen K, Jaatinen R, Horelli-Kuitunen N, Seppa L, Louhio H, Tuuri T, Sjoberg J, Butzow R, et al. (1999) Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis. J Clin Endocrinol Metab 84:2744–2750.
Abrieu A, Kahana JA, Wood KW, Cleveland DW. (2000) CENP-E as an essential component of the mitotic checkpoint in vitro. Cell 102:817–826.[CrossRef][Medline]
Bachvarova R, Burns JP, Spiegelman I, Choy J, Chaganti RS. (1982) Morphology and transcriptional activity of mouse oocyte chromosomes. Chromosoma 86:181–196.[Medline]
Burns KH, Owens GE, Ogbonna SC, Nilson JH, Matzuk MM. (2003) Expression profiling analyses of gonadotropin responses and tumor development in the absence of inhibins. Endocrinology 144:4492–4507.
Carr DW, Cutler RE Jr, Cottom JE, Salvador LM, Fraser ID, Scott JD, Hunzicker-Dunn M. (1999) Identification of cAMP-dependent protein kinase holoenzymes in preantral- and preovulatory-follicle-enriched ovaries, and their association with A-kinase-anchoring proteins. Biochem J 344:Pt 2613–623.[CrossRef][ISI][Medline]
Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP. (2000) The human VASA gene is specifically expressed in the germ cell lineage. Proc Natl Acad Sci USA 97:9585–9590.
Cauffman G, Van de Velde H, Liebaers I, Van Steirteghem A. (2005) DAZL expression in human oocytes, preimplantation embryos and embryonic stem cells. Mol Hum Reprod 11:405–411.
Chang HY, Levasseur M, Jones KT. (2004) Degradation of APCcdc20 and APCcdh1 substrates during the second meiotic division in mouse eggs. J Cell Sci 117:6289–6296.
Choi YL, Kim HS, Ahn G. (2000) Immunoexpression of inhibin alpha subunit, inhibin/activin betaA subunit and CD99 in ovarian tumors. Arch Pathol Lab Med 124:563–569.[Medline]
Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC, Langenbach R. (1999) Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1beta. Endocrinology 140:2685–2695.
De La Fuente R, Viveiros MM, Burns KH, Adashi EY, Matzuk MM, Eppig JJ. (2004) 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 275:447–458.[CrossRef][ISI][Medline]
de los Santos MJ, Anderson DJ, Racowsky C, Simon C, Hill JA. (1998) Expression of interleukin-1 system genes in human gametes. Biol Reprod 59:1419–1424.
Dekel N and Beers WH. (1980) Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus. Dev Biol 75:247–254.[CrossRef][ISI][Medline]
Devoto L, Kohen P, Gonzalez RR, Castro O, Retamales I, Vega M, Carvallo P, Christenson LK, Strauss JF III. (2001) Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase. J Clin Endocrinol Metab 86:5633–5639.
Duesbery NS, Choi T, Brown KD, Wood KW, Resau J, Fukasawa K, Cleveland DW, Vande Woude GF. (1997) 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 94:9165–9170.
Eberspaecher U, Becker A, Bringmann P, van der Merwe L, Donner P. (2001) Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries. Cell Tissue Res 303:277–287.[CrossRef][ISI][Medline]
Eisen MB, Spellman PT, Brown PO, Botstein D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95:14863–14868.
Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM. (1999) Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endocrinol 13:1035–1048.
Espey LL and Richards JS. (2002) Temporal and spatial patterns of ovarian gene transcription following an ovulatory dose of gonadotropin in the rat. Biol Reprod 67:1662–1670.
Fair T, Hyttel P, Greve T. (1995) Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Mol Reprod Dev 42:437–442.[CrossRef][ISI][Medline]
Gall L, Ruffini S, Le Bourhis D, Boulesteix C. (2002) Cdc25C expression in meiotically competent and incompetent goat oocytes. Mol Reprod Dev 62:4–12.[CrossRef][ISI][Medline]
Geiman TM, Sankpal UT, Robertson AK, Chen Y, Mazumdar M, Heale JT, Schmiesing JA, Kim W, Yokomori K, Zhao Y, et al. (2004) Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery. Nucleic Acids Res 32:2716–2729.
Giet R, Petretti C, Prigent C. (2005) Aurora kinases, aneuploidy and cancer, a coincidence or a real link? Trends Cell Biol 15:241–250.[CrossRef][ISI][Medline]
Gillio-Meina C, Hui YY, LaVoie HA. (2005) Expression of CCAAT/enhancer binding proteins alpha and beta in the porcine ovary and regulation in primary cultures of granulosa cells. Biol Reprod 72:1194–1204.
Gordon MD, Corless C, Renshaw AA, Beckstead J. (1998) CD99, keratin, and vimentin staining of sex cord-stromal tumors, normal ovary, and testis. Mod Pathol 11:769–773.[ISI]
Grootenhuis AJ, Philipsen HL, de Breet-Grijsbach JT, van Duin M. (1996) Immunocytochemical localization of ZP3 in primordial follicles of rabbit, marmoset, rhesus monkey and human ovaries using antibodies against human ZP3. J Reprod Fertil Suppl 50:43–54.[Medline]
Haffner-Krausz R, Gorivodsky M, Chen Y, Lonai P. (1999) Expression of Fgfr2 in the early mouse embryo indicates its involvement in preimplantation development. Mech Dev 85:167–172.[CrossRef][ISI][Medline]
Hattori N, Fujiwara H, Maeda M, Yoshioka S, Higuchi T, Mori T, Ohishi N, Minami M, Fujii S, Ueda M. (1998) Human large luteal cells in the menstrual cycle and early pregnancy express leukotriene A4 hydrolase. Mol Hum Reprod 4:803–810.
Heikinheimo O, Lanzendorf SE, Baka SG, Gibbons WE. (1995) Cell cycle genes c-mos and cyclin-B1 are expressed in a specific pattern in human oocytes and preimplantation embryos. Hum Reprod 10:699–707.
Hinsch E, Hagele W, van der Ven H, Oehninger S, Schill WB, Hinsch KD. (1998) Immunological identification of zona pellucida 2 (ZP2) protein in human oocytes. Andrologia 30:281–287.[ISI][Medline]
Horowitz E, Zhang Z, Jones BH, Moss SB, Ho C, Wood JR, Wang X, Sammel MD, Strauss JF III. (2005) 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 11:307–317.
Hourvitz A, Widger AE, Filho FL, Chang RJ, Adashi EY, Erickson GF. (2000) Pregnancy-associated plasma protein-A gene expression in human ovaries is restricted to healthy follicles and corpora lutea. J Clin Endocrinol Metab 85:4916–4920.
Huntriss J, Hinkins M, Oliver B, Harris SE, Beazley JC, Rutherford AJ, Gosden RG, Lanzendorf SE, Picton HM. (2004) 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 67:323–336.[CrossRef][ISI][Medline]
Hurwitz A, Ruutiainen-Altman K, Marzella L, Botero L, Dushnik M, Adashi EY. (1996) 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 3:199–208.[CrossRef]
Ingold K, Zumsteg A, Tardivel A, Huard B, Steiner QG, Cachero TG, Qiang F, Gorelik L, Kalled SL, Acha-Orbea H, et al. (2005) Identification of proteoglycans as the APRIL-specific binding partners. J Exp Med 201:1375–1383.
Irizarry RA, Warren D, Spencer F, Kim IF, Biswal S, Frank BC, Gabrielson E, Garcia JG, Geoghegan J, Germino G, et al. (2005) Multiple-laboratory comparison of microarray platforms. Nat Methods 2:345–350.[CrossRef][ISI][Medline]
Jaatinen TA, Penttila TL, Kaipia A, Ekfors T, Parvinen M, Toppari J. (1994) Expression of inhibin alpha, beta A and beta B messenger ribonucleic acids in the normal human ovary and in polycystic ovarian syndrome. J Endocrinol 143:127–137.[Abstract]
Jirawatnotai S, Moons DS, Stocco CO, Franks R, Hales DB, Gibori G, Kiyokawa H. (2003) The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 cooperate to restrict proliferative life span in differentiating ovarian cells. J Biol Chem 278:17021–17027.
Kalous J, Solc P, Baran V, Kubelka M, Schultz RM, Motlik J. (2005) PKB/AKT is involved in resumption of meiosis in mouse oocytes. Biol Cell 98:111–123.
Karstrom-Encrantz L, Runesson E, Bostrom EK, Brannstrom M. (1998) 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 4:1077–1083.
Krebs DL and Hilton DJ. (2000) SOCS: physiological suppressors of cytokine signaling. J Cell Sci 113:Pt 162813–2819.[Abstract]
Kristiansen G, Denkert C, Schluns K, Dahl E, Pilarsky C, Hauptmann S. (2002) CD24 is expressed in ovarian cancer and is a new independent prognostic marker of patient survival. Am J Pathol 161:1215–1221.
Kubota Y, Mimura S, Nishimoto S, Takisawa H, Nojima H. (1995) Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell 81:601–609.[CrossRef][ISI][Medline]
Larsen WJ, Wert SE, Brunner GD. (1986) A dramatic loss of cumulus cell gap junctions is correlated with germinal vesicle breakdown in rat oocytes. Dev Biol 113:517–521.[CrossRef][ISI][Medline]
Le Naour F, Rubinstein E, Jasmin C, Prenant M, Boucheix C. (2000) Severely reduced female fertility in CD9-deficient mice. Science 287:319–321.
Lefievre L, Conner SJ, Salpekar A, Olufowobi O, Ashton P, Pavlovic B, Lenton W, Afnan M, Brewis IA, Monk M, et al. (2004) Four zona pellucida glycoproteins are expressed in the human. Hum Reprod 19:1580–1586.
Leung PC, Cheng CK, Zhu XM. (2003) Multi-factorial role of GnRH-I and GnRH-II in the human ovary. Mol Cell Endocrinol 202:145–153.[ISI][Medline]
Leyens G, Knoops B, Donnay I. (2004) Expression of peroxiredoxins in bovine oocytes and embryos produced in vitro. Mol Reprod Dev 69:243–251.[CrossRef][Medline]
Lincoln AJ, Wickramasinghe D, Stein P, Schultz RM, Palko ME, De Miguel MP, Tessarollo L, Donovan PJ. (2002) Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation. Nat Genet 30:446–449.[CrossRef][ISI][Medline]
Liu K. (2006) Stem cell factor (SCF)-kit mediated phosphatidylinositol 3 (PI3) kinase signaling during mammalian oocyte growth and early follicular development. Front Biosci 11:126–135.[ISI][Medline]
Lyons KM, Pelton RW, Hogan BL. (1989) 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 3:1657–1668.
Magier S, van der Ven HH, Diedrich K, Krebs D. (1990) Significance of cumulus oophorus in in-vitro fertilization and oocyte viability and fertility. Hum Reprod 5:847–852.
Moor RM, Dai Y, Lee C, Fulka J Jr. (1998) Oocyte maturation and embryonic failure. Hum Reprod Update 4:223–236.
Narko K, Saukkonen K, Ketola I, Butzow R, Heikinheimo M, Ristimaki A. (2001) Regulated expression of prostaglandin E(2) receptors EP2 and EP4 in human ovarian granulosa-luteal cells. J Clin Endocrinol Metab 86:1765–1768.
Nishi S, Hoshi N, Kasahara M, Ishibashi T, Fujimoto S. (1999) Existence of human DAZLA protein in the cytoplasm of human oocytes. Mol Hum Reprod 5:495–497.
O’Sullivan MJ, Stamouli A, Thomas EJ, Richardson MC. (1997) Gonadotrophin regulation of production of tissue inhibitor of metalloproteinases-1 by luteinized human granulosa cells: a potential mechanism for luteal rescue. Mol Hum Reprod 3:405–410.
Oksjoki S, Soderstrom M, Vuorio E, Anttila L. (2001) Differential expression patterns of cathepsins B, H, K, L and S in the mouse ovary. Mol Hum Reprod 7:27–34.
Osaki E, Nishina Y, Inazawa J, Copeland NG, Gilbert DJ, Jenkins NA, Ohsugi M, Tezuka T, Yoshida M, Semba K. (1999) Identification of a novel Sry-related gene and its germ cell-specific expression. Nucleic Acids Res 27:2503–2510.
Pal SK, Torry D, Serta R, Crowell RC, Seibel MM, Cooper GM, Kiessling AA. (1994) Expression and potential function of the c-mos proto-oncogene in human eggs. Fertil Steril 61:496–503.[ISI][Medline]
Park OK and Mayo KE. (1991) Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge. Mol Endocrinol 5:967–978.[Abstract]
Paronetto MP, Giorda E, Carsetti R, Rossi P, Geremia R, Sette C. (2004) Functional interaction between p90Rsk2 and Emi1 contributes to the metaphase arrest of mouse oocytes. EMBO J 23:4649–4659.[CrossRef][ISI][Medline]
Platteau P, Smitz J, Albano C, Sorensen P, Arce JC, Devroey P. (2004) Exogenous luteinizing hormone activity may influence the treatment outcome in in vitro fertilization but not in intracytoplasmic sperm injection cycles. Fertil Steril 81:1401–1404.[CrossRef][ISI][Medline]
Prieto I, Tease C, Pezzi N, Buesa JM, Ortega S, Kremer L, Martinez A, Martinez AC, Hulten MA, Barbero JL. (2004) Cohesin component dynamics during meiotic prophase I in mammalian oocytes. Chromosome Res 12:197–213.[CrossRef][ISI][Medline]
Rabinovici J, Spencer SJ, Doldi N, Goldsmith PC, Schwall R, Jaffe RB. (1992) Activin-A as an intraovarian modulator: actions, localization, and regulation of the intact dimer in human ovarian cells. J Clin Invest 89:1528–1536.[ISI][Medline]
Robker RL and Richards JS. (1998) Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1. Mol Endocrinol 12:924–940.
Runesson E, Ivarsson K, Janson PO, Brannstrom M. (2000) Gonadotropin- and cytokine-regulated expression of the chemokine interleukin 8 in the human preovulatory follicle of the menstrual cycle. J Clin Endocrinol Metab 85:4387–4395.
Russell DL, Doyle KM, Ochsner SA, Sandy JD, Richards JS. (2003) Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation. J Biol Chem 278:42330–42339.
Saitou M, Barton SC, Surani MA. (2002) A molecular programme for the specification of germ cell fate in mice. Nature 418:293–300.[CrossRef][Medline]
Salpekar A, Huntriss J, Bolton V, Monk M. (2001) The use of amplified cDNA to investigate the expression of seven imprinted genes in human oocytes and preimplantation embryos. Mol Hum Reprod 7:839–844.
Schatten G, Simerly C, Palmer DK, Margolis RL, Maul G, Andrews BS, Schatten H. (1988) Kinetochore appearance during meiosis, fertilization and mitosis in mouse oocytes and zygotes. Chromosoma 96:341–352.[CrossRef][Medline]
Sirois J, Levy LO, Simmons DL, Richards JS. (1993) 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 268:12199–12206.