1 MISCL (Monash Immunology and Stem Cell Laboratories), Monash University, Wellington Road, Clayton, Victoria, 3800 Australia and 2 Tokyo HART Clinic, 1-22-2 Higashi, Shibuya 150-0011, Japan
3 To whom correspondence should be addressed at: MISCL (Monash Immunology and Stem Cell Laboratories), Level 3 Strip Building 75, Monash University, Wellington Road, Clayton, Victoria 3800, Australia. Email: maria.serafica{at}med.monash.edu.au
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Abstract |
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Key words: apoptosis/human primordial follicle cDNA library/mitochondrial genes/repeat elements/signalling pathways
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Introduction |
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The molecular mechanisms operating to activate or inhibit the initial growth or recruitment of the PF to the next stage, the primary follicle, are unknown (Fortune, 2003; Picton et al., 2003
). Studies on elucidating the mechanisms of PF recruitment have utilized the addition of growth factors and corresponding blocking antibodies using in vitro ovary culture systems of mouse (Durlinger et al., 2002
), rat (Nilsson and Skinner, 2004
), cow (Braw-Tal and Yossefi, 1997
) and baboon (Wandji et al., 1997
), and chorio-allantoic membrane grafts of chick embryos (Cushman et al., 2002
). Models of oocytegranulosa interactions as influenced by the presence of growth factors such as basic fibroblast growth factor and/or leukaemia inhibitory factor in the medium have been proposed (Kezele et al., 2002
; Nilsson et al., 2002
). A complex, bidirectional and coordinated interaction exists between the oocyte and the surrounding granulosa cells in order to ensure synchronous and successful development of both follicular components (Eppig, 2001
).
Fig (Soyal et al., 2000
), a basic helixloophelix transcription factor, has been shown to be essential for formation of PFs and expression of zona pellucida proteins (Liang et al., 1997
). Growth and differentiation factor-9 (gdf-9) and bone morphogenetic protein-15 or bmp-15, which are members of the transforming growth factor (TGF)-
family, are oocyte genes which are expressed beyond the primary follicle stage (Dong et al., 1998
; Carabatsos et al., 1998
; Galloway et al., 2000
). Recently, the transcription factor Foxl2 was shown to be required to maintain granulosa cell function (Schmidt et al., 2004
). Spindlin (Oh et al., 1997
), mater (Tong et al., 2000
) and zar1 (Wu et al., 2003
) are examples of mouse oocyte genes required during the transition from the gamete to the preimplantation stage.
Knowledge of the factors that either activate or inhibit recruitment of oocytes into the growth phase may impact on prolonging the female's reproductive life span, assist in techniques associated with oocyte and/or ovarian tissue cryopreservation of infertile and cancer patients (Oktay et al., 1998) and help improve current or existing in vitro maturation technologies, particularly those that start with immature follicles (Eppig and O'Brien, 1996
; Eppig, 2003
; O'Brien et al., 2003
).
Gene expression studies in human oocytes have been greatly hampered by the lack of available samples and lack of reproducible methods to analyse mRNA expression in single cell samples. The latter difficulty has now been overcome by using PCR-based methods to amplifiy cDNA from samples with <5 ng of total RNA. One such method, the SMART (switching mechanism at the 5' end of the reverse transcript) system that produces tagged ends of amplified cDNA was first used to generate cDNA libraries of human preimplantation embryos (Adjaye et al., 1997, 1999
) in primordial germ cells (Goto et al., 1999
), germinal vesicle (GV) oocytes (Neilson et al., 2000
) and from human GV and MII oocytes (Monk et al., 2001
; Goto et al., 2002
).
Ideally, gene expression libraries at each oocyte growth and developmental stage followed by random sequencing of cDNA clones would give a catalogue of human oocyte genes. Coupling comprehensive libraries with subtractive approaches will enable the identification of stage-specific oocyte genes. Subtractive methods such as differential display were first reported in human GV and MII oocytes (Goto et al., 2002) whereas in mice, suppression subtractive hybridization was used to obtain genes specifically expressed during the mouse MII and during the 8-cell embryo stage (Feng and Schultz, 2003
). cDNA libraries (comprehensive or subtracted) provide a resource for isolating and identifying stage-specific oocyte genes and for printing a human oocyte stage-specific array, thereby facilitating the simultaneous screening of a large number of genes in other oocyte samples by microarray analysis.
In order to obtain a molecular blueprint of human PFs, we generated a human PF cDNA library and analysed 692 clones by DNA sequencing, database and pathway analysis. This is the first detailed report of gene sequences at this earliest stage of oogenesis. Wider applications of these sequence-verified clones to oogenesis gene expression studies are brought forward.
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Materials and methods |
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Isolation of PFs
Human PFs were isolated from the ovaries of a sex-reassigned woman. The whole ovaries were surgically removed from the woman in Canberra, Australia and transported to our laboratory in Melbourne on ice in Dulbecco's modified Eagle's medium (Gibco-BRL, Life Technologies, Grand Island, NY), supplemented with 60 U/ml penicillin and 60 µg/ml streptomycin (Gibco-BRL), on ice. Using a sterile scalpel, pieces of ovarian cortical slices (5 mmx5 mmx1 mm) were obtained from these ovaries. The slices were washed several times in phosphate-buffered saline (PBS) and placed in 10 ml of PBS containing 120 IU of collagenase (Sigma Chemical Co., St Louis, MO) and 14 IU of pancreatic DNase I (Sigma Chemical Co.). The samples were incubated for 48 h at 4°C. Following enzymatic digestion, the PFs were washed several times in PBS and then mechanically isolated by gentle pipetting, using a finely drawn Pasteur pipette. PFs were identified under a stereo microscope as the smallest oocytes surrounded by a single layer of flattened cells. Using this criterion, any possible contamination with primary follicles, if any, was deemed negligible. Fifty PFs were collected in lysis buffer [0.8% Igepal (ICN Pharmaceuticals Inc., Costa Mesa, CA), 1 U/µl of RNase inhibitor (Promega, Australia), 5 mM dithiothreitol (Gibco-BRL)] in a 1.5 ml Eppendorf tube. The sample was snap-frozen in liquid nitrogen and stored at 70°C until RNA extraction.
Preparation of mRNA, cDNA synthesis and PCR amplification of cDNA
Total RNA was extracted from the PF sample using a StrataPrep Absolutely Total RNA Kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. Poly(A)+ RNA was isolated from the total RNA using oligo(dT)-attached magnetic beads (DynaBeads mRNA purification kit, Dynal, Carlton South, Victoria Australia) as previously described (Goto et al., 1999). The isolated mRNA, still attached to the beads, was resuspended in 6 µl of double-distilled water; this minimized the loss of mRNA.
First strand cDNA synthesis followed by cDNA amplification by long distance PCR was performed according to instructions provided in the SMART cDNA Library Construction Kit (Clontech Palo Alto, CA). To prevent distortion of representation of subsets of cDNA molecules within the total cDNA population and to ensure that the double-stranded (ds) cDNA remained in the exponential phase of amplification, the optimal number of PCR cycles was determined by sampling an aliquot of the PCR-amplified sample at 18 cycles and increments of three cycles from there onwards, up to 35 cycles, and running the aliquots on an agarose gel. Care was taken to choose PCR cycles (in this case, 26 PCR cycles) in which the cDNA has not reached saturation by comparing the ethidium bromide intensities of amplified cDNA. The mRNA in 6 µl of double-distilled water was divided into two; one aliquot for a reverse transcriptase reaction (to which the reverse transcriptase enzyme was added) and another for a control tube (no reverse transcriptase was added). A 5 µl aliquot of amplified cDNA (from a total volume of 100 µl) was electrophoresed on a 1.0% ethidium bromide-containing agarose gel to visualize the amount and the size distribution of the cDNA. Gene-specific PCR for a housekeeping gene, -actin, was performed to test the quality of the cDNA preparation.
Construction of the cDNA library
PCR-amplified cDNA was purified using a CHROMA SPIN-400 Column (Clontech) to remove fragments smaller than 0.5 kb in size. The size-fractionated cDNA molecules were then ligated to the arms of a lambda vector, TriplEx2 (Clontech), and the packaging reaction was carried out using Gigapack III Gold Packaging Extract (Stratagene), according to the manufacturer's instruction. The resultant phage was used to transduce Escherichia coli strain XL-1Blue (Stratagene) to produce a titre of 2.5 x 106 plaque-forming units (p.f.u.)/ml. The total volume of the phage lysate was 1.5 ml; therefore, the total number of single independent cDNA clones is 3.75 x 106. A blue/white colony screening using isopropyl-
-D-thiogalactopyranoside (IPTG) and X-Gal showed that 99% of plaques were white (12 x 103 plaques were counted per plate), indicating that 99% of the clones had an insert.
Conversion of recombinant TriplEx2 phage clones to pTriplEx2 plasmid clones
An aliquot of the TriplEx2 phage cDNA library was used to transduce an E.coli strain, BM25.8, to produce 30004000 colonies. The BM25.8 cells possessed Cre recombinase activity, which is capable of converting the loxP-containing
TriplEx2 phage vector to pTriplEx2 plasmid vector (Clontech).
Sequencing of clones and sequence analysis
Plasmid DNA was extracted from randomly picked clones and submitted for DNA sequencing. They were sequenced from the 5' end using a forward vector flanking primer, and ABI-PE Big Dye Terminator Chemistry. Sequencing was performed by the Wellcome Trust DNA Sequencing Facility, located at Prince Henry's Institute of Medical Research (PHIMR), Monash Medical Centre, Melbourne Australia. Sequences were input in ANGIS (Australian National Genomic Information Service, Sydney, New South Wales, Australia for bioinformatic analyses, www.angis.org.au) or directly to the www.ncbi.nlm.nih.gov BLASTN site (non-redundant NCBI database) to determine sequence identity. Functional gene categories were based on GO (Gene Ontology from NCBI) annotations using Gene ID and OMIM for each gene analysed. Where there is no mention of a specific reference citation for a gene in this work, the function and biological process were obtained from NCBI's Gene ID and OMIM descriptions. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database (www.genome.jp/kegg/pathway.html) was used for gene assignation to a signalling pathway. The Pathway Assist Analysis software (Iobion/Ariadne Genomics, version 3.0) was trialled and utilized to build a mitochondrial and a human PF transcription factor pathway. Eleven mitochondrial proteins and 38 transcription factors were imported into this software and the shortest paths between selected nodes was used to build the initial pathway. The transcription factor pathway was modified by selecting proteins of the same group (either a group of positive regulators and negative regulators) and opting for the common targets to these genes. FASTA, MEGABLAST and BLASTN [using the expressed sequence tag (EST) database] searches were performed on those clones which corresponded to human DNA sequences, ESTs, bacterial/phage artificial chromosome (BAC/PAC) clones, RIKEN full insert sequence clones, hypothetical protein-containing clones and clones which showed very low homology to known genes. These clones were screened further for the presence of repeat elements using RepBase (Jurka et al., 1996; Jurka, 1998
).
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Results |
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Repeat elements in eight out of the 84 repeat-containing cDNA clones were located within the coding sequences of known genes and hypothetical proteins. This is exemplified by three Alu cDNA clones, in which coding sequences for the WDR4 gene (WD repeat protein 4 domain), the human ALAD (-aminolaevulinate synthase) gene for phorphobilinogen synthase and the TP53INP1 (tumour protein p53-inducible nuclear protein 1) gene were also located. An HERV70_I repeat sequence overlapped with the coding region of the pro-apoptotic gene BNIP3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein 3) gene. The 194 bp MARNA repeat element overlapped with the coding region of znf395 (zinc finger protein 395); repeat elements MER11B, Alu-Spqxz-LIP_MA2 and LTR12C-HSMAR1 were located, respectively, within the coding regions of three hypothetical genes.
Cellular growth and differentiation genes
Genes shown in Table IV include transcription, translation, energy/metabolism, embryogenesis, membrane receptors, cytoskeleton/extracellular matrix and cell cycle checkpoint genes. Primary response genes, exemplified by transcription factors and proto-oncogenes, are genes that are specifically and rapidly upregulated in response to growth factor stimulation. Their expression is independent of new protein synthesis and requires only the activation of pre-existing transcriptional regulators. A total of 42 genes encoding replication/transcription and 57 genes, consisting of 34 ribosomal and 23 genes, essential to the translation machinery were identified from this PF library.
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Discussion |
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The number of uncharacterized genes in the form of hypothetical proteins is also high. These proteins with no known function corresponded to full-length cDNA transcripts from human fetal brain tissue (Strausberg et al., 2000; Ota et al., 2004
), whereas ESTs from this library were homologous to those found in germ cell tumours, and in various types of human cancers and mouse embryo of different stages (M.Serafica, in preparation).
Only 45 genes were common between the known PF genes obtained in this work and the set of 840 mouse oocyte genes reported to have well-matched human homologues (Stanton and Green, 2001). Likewise, comparison of the human PF genes with the 181 human GV oocyte genes generated by serial analysis of gene expression (SAGE) (Neilson et al., 2000
) showed only 19 genes common between human GV and human PF. Whether this is due to a lack of comprehensiveness or under-representation of genes sampled or this is reflective of true differential stage-specific gene expression between samples remains to be demonstrated.
Nevertheless, based on the above comparisons alone, at least 64 oocyte-specific genes were present in this human PF library. The distinction between oocyte-specific genes and pre-granulosa genes could be resolved using laser capture microdissection followed by either reverse transcription (RT)PCR or a cDNA microarray screen.
Repeat elements
Results of screening the human PF library for repeat elements revealed a high proportion of clones with different types of repeat elements as well as combination of two and three different types of repeats in one clone (Table III). The HERV elements which are members of the long terminal repeat (LTR) class of retroelements as well as the SINE (short interspersed elements) and LINE (long terminal interspersed elements), which belong to the non-LTR class, were identified. SINE elements were represented by the Alu and MIR repeats, while LINE repeats were represented by LI and L3. Depending on the direction of insertion of the repeat element relative to the adjacent gene (whether sense or antisense), a repeat element can enhance gene expression or it can effectively silence it (see various examples given by Mi et al., 2000; Hughes and Coffin, 2001
; Kashkush et al., 2003
; Bannert and Kurth, 2004
). The stage specificity of expression of mouse repeat elements, such as endogenous retroviral element (ERVL), MT-like elements and the ORR1 transposon-like element, from the fully grown oocyte up to the 2-cell stage has been reported (Evsikov et al., 2004
). The MT-like elements were most abundant in the fully grown mouse oocyte (FGO), whereas ERVL and ORR1 elements were most abundant in the 2-cell stage. The IAP class II retroviruses were abundant during the 2-cell stage but scarce in the FGO. Since the stage specificity of expression of these elements also changed with the methylation status, Knowles et al. (2003)
proposed that retroviral elements help shape the stage specificity of gamete and early embryo gene expression. Two other reports of retrotransposons in mouse oocytes and preimplantation embryos were published (Park et al., 2004
; Peaston et al., 2004
). Gene expression by RTPCR of five different retrotransposons (Mu-ERV-L, MT, ORR1, RLTR1B and IAPEz) showed different gene expression patterns, starting from the FGO persisting up to the blastocyst stage. Furthermore, gene expression patterns of six chimeric transcripts (which contained retrotransposons at the 5' end of the gene or EST clone) and the corresponding conventional transcripts were shown to be different, with the chimeric transcripts showing positive expression from the FGO, up to the early 2-cell stage, relative to the corresponding conventional transcript. The use of dsRNAs directed to the mouse MT transposon element resulted in a 43.453% GV stage arrest as well as a decrease in targeted gene expression (Park et al., 2004
). Microinjection of dsRNA for the same repeat element into 1-cell stage and late 2-cell stage mouse embryos revealed a 92.9 and 76.9% cell arrest, respectively.
In the case of human oocytes, LINE-1, HAL1 and MLT1C, repeat elements were identified using differential display between GV and MII oocytes (Goto et al 2002).
Cell growth and differentiation
The battery of primary or early response genes present in the PF (Table IV) indicates that it has the ability to respond to stress of any kind such as pH changes, UV light, polycyclic halogenated hydrocarbons and oxidative stress; the presence of these genes ultimately determines the survival of this arrested oogenesis stage for long periods. Transition into the next growth stage, the primary follicle, is accompanied by an increase in oocyte size, and increased number as well as differentiation of the pre-granulosa cells into a cuboidal shape. Genes associated with negative regulation of cell proliferation and differentiation are likely to be candidates for initial recruitment. Some of these PF genes are CSRP2, NR5A2, SERPINE2 (Bedard et al., 2003), IFITM1 (Table IV), TLE-1 (Liu et al., 1996
) and DUSP1 (Table V). CSRP2 harbours a type of LIM domain motif which defines a zinc-binding domain that is found in a variety of transcriptional regulators, proto-oncogene products and proteins associated with sites of cellsubstratum contact (Weiskirchen et al., 1995
). The CSRP2 gene was found to be differentially regulated in normal versus transformed cells, implicating a role for the CSRP family in control of cell growth and differentiation. The NR5A2 orphan nuclear receptor gene, also called the LRH-1 (liver receptor homologue 1) gene, was reported to induce granulosa cell differentiation by induction of the progesterone biosynthetic pathway (Saxena et al., 2004
), and to be involved in the regulation of human corpus luteum 3
-hydroxysteroid dehydrogenase type II (Peng et al., 2003
) and in regulating the expression of StAR (steroid acute regulatory protein) in human granulosa cells after ovulation (Kim et al., 2004b
). Its role as a downstream target of the Pdx-1 (pancreatic duodenal homeobox 1) regulatory gene cascade during pancreatic development has been reported (Annicotte et al., 2003
). The NR5A2 gene is thus is an example of a PF gene whose expression is important at later stages of folliculogenesis and during embryogenesis. The IFITM1 gene is an interferon-inducible transmembrane protein that can transduce antiproliferative signals as well as promote homotypic adhesion (DeBlandre et al., 1995
) and is involved in mouse germ cell fate specification (Saitou et al., 2002
). The DUSP1 gene plays a pivotal role in the cellular response to oxidative stress and negative regulation of cell proliferation.
Transcriptional repressors
Transcriptional repression is another functional feature of the PF; examples of transcriptional repressors (Table IV) included the co-chaperone prefoldin5 or c-myc-binding protein1 (Mori et al., 1998), CSDA, zinc finger 148 or BERF-1 (Takeuchi et al, 2003
), human I-mfa domain-containing protein (HIC) and SAP18 (sin-3-associated polypeptide). SAP18, for instance, directly interacts with the sin3 component of histone deacetylase, enhancing sin3-mediated transcriptional repression when bound to the promoter (Zhang et al., 1997
). Determining the targets of these repressors will probably reveal the genes that control the recruitment of the PF into a primary follicle.
Embryogenesis genes
Eight genes related to embryogenesis were identified and these included the Wilms tumour1 (WT1) gene which is involved in kidney and gonadal differentiation; neuronal cell adhesion molecule (NRCAM) for development of the central nervous system; ameloblastin gene (AMBN) for tooth and bone mineralization; embryonic ectoderm development protein (EED) which is a member of the Polycomb-group (PcG) family responsible for transcriptional repression; vimentin (VIM), a gene specific for mesenchymal tissue; the hairy/Enhancer of split-related with YRPW motif-like (HEYL) gene for development of the nervous system, somites, heart and craniofacial region; the myoblast determination protein 1 (MYOD1) for muscle differentiation; and the mesoderm developmental candidate 2 gene (MESDC2), whose function is still unknown.
Energy and metabolism genes that were identified included UGP2, AKRB1, SULF2, GALNT10 and GUSB which are involved in carbohydrate metabolism such as interconversion between different forms of carbohydrates and heparan sulfate proteoglycan synthesis, amino acid metabolism (GLUD1, HDC, DPM1 and CPE), purine and pyrimidine synthesis (IMP and DCTD) and haem biosynthesis (ALAD gene for phorphobilinogen synthase). The GATM gene codes for the biosynthesis of creatine, which is a form of storing and transmission of phosphate-bound energy. Enzymes responsible for non-oxidative means of generating ATP include the TALDO1 (transaldolase1) and the lactate dehydrogenase (LDHA) gene.
Signalling pathways present in the human PF
UPP pathway
The UPP provides a complex but tightly regulated pathway of intracellular protein degradation (Ciechanover, 1998; Herschko and Ciechanover, 1998
). In the UPP pathway, proteins targeted for degradation acquire a chain of ubiquitins through the sequential actions of the following enzymes: E1 or ubiquitin-activating enzyme; E2 (ubiquitin-conjugating enzymes) and E3 (ubiquitin-protein ligase). Only ubiquitin-tagged proteins are degraded by the 26S proteasome with the subsequent release of free and recyclable ubiquitins. Proteins involved in cell cycle progression, such as cyclin B (Tokumoto et al., 1997
), cyclin-dependent kinases, polo-like kinase and c-mos, are degraded by the UPP (Peters, 2002
). Apart from cell cycle progression, proteasomes function in differentiation and development, secretory pathways, morphogenesis of neuronal networks and degradation of translational repressor proteins such as the cytoplasmic polyadenylation binding protein or CPEB (Reverte et al., 2001
). One of the structural motifs that target proteins for ubiquitination includes proteins in association with molecular chaperones (Arlander et al., 2003
). The prefoldin 5 (PFDN5) gene acts as a molecular chaperone, by assisting in the correct folding of newly synthesized polypeptides, and can also substitute for the hsp70 chaperone in vitro (Vainberg et al., 1998
). The evolutionarily conserved ring finger protein RNF138 has a zinc ion-binding domain that is involved in protein ubiquitination (Saurin et al., 1996
). Thus, the presence of proteasome components, ubiquitins, E2-conjugating enzyme (UBE2D3) and a deubiquitinating enzyme (USP9X) completes the UPP pathway in the human PF.
The COP9 gene (constitutive, photomorphogenic, Arabidopsis homologue, subunit 7B) is a part of the CSN, which is a highly conserved 450 kDa nuclear protein complex (Deng et al., 2000
) that functions as an important regulator in multiple signalling pathways such as the TGF-
signalling pathway (Kim et al., 2004a
) as well as in transcriptional regulation, endocytosis and cell cycle progression. The homology of each of the eight subunits of the CSN to the eight subunits of the lid subcomplex of the 26S proteasome suggests that the role of CSN in protein degradation is through the UPP (Li and Deng, 2003
). The CSN7 subunit of the Arabidopsis CSN was reported to associate with the eukaryotic translation initiation factor 3, eIF3 (Yahalom et al., 2001
), and with eIF3 and 26S proteasome (Hoareau et al., 2002
).
MAPK and p38/JNK
MAPK10/JNK3A1 is a neuronal-specific form of c-Jun N-terminal kinases (JNKs) that plays regulatory roles in the signalling pathways during neuronal apoptosis. MAPK10/JNK3A1 and PP2CB are members of the JNK/p38 MAP kinase signalling pathway. PAK2, AKAP10 and DUSP1 are members of the MAP kinase signalling pathway. The role of the MAPK kinase pathway in cell cycle regulation is well documented especially during the later stages of oocyte maturation. PRKCN, PRKARI1A and PLCB1 are members of various intracellular signalling cascades, among them Wnt and MAPK signalling pathways. The CSNK2B/phosvitin is part of the Wnt signalling pathway and the cadherin-mediated cell adhesion pathway. Control of cell expansion in many types of stem cells (Kleber and Sommer, 2004), cell lineage decisions and development of the central nervous system are some of the numerous roles ascribed to the Wnt signalling pathway. The TLE1 gene is a frizzled receptor of the Wnt signalling pathway. The HEYL gene, a member of the Delta-Notch signalling pathway, is a basic helixturnhelix transcription factor implicated in cell fate decision and boundary formation (Leimeister et al, 1999
).
Apoptosis signal transduction
Of the 18 hsp genes given in Table V, 10 coded for Hsp90. Hsp90 can suppress tumour necrosis factor- (TNF-
)-induced apoptosis in stable Hsp90-overexpressing murine NIH-3T3 cells by preventing the cleavage of Bid, a pro-apoptotic member of the Bcl family (Zhao and Wang, 2004
). Two other antiapoptotic roles reported for Hsp90 include its binding with Apaf-1 (apoptosis activating factor) (Pandey et al., 2000
) and the binding of Hsp90 with a major antiapoptotic adaptor receptor-interacting protein, resulting in activation of antiapoptotic pathways through NF-
B and MAPK (Lewis et al., 2000
). Hsp70 was reported to prevent both caspase-dependent and caspase-independent cell death functions (Bruey et al, 2000
; Ravagnan et al., 2001
). BNIP3 is a pro-apoptotic member of the Bcl-2 family because it contains a BH3 domain and a transmembrane domain which are associated with pro-apoptotic function. The role of the VDAC2 gene as an anti-apoptotic regulator of the pro-apoptotic BAK gene was reported (Cheng et al., 2003
). Cells deficient only in VDAC2 resulted in enhanced BAK oligomerization and were more susceptible to cell death, but cells overexpressing VDAC2 selectively prevented BAK activation and inhibited the mitochondrial apoptotic pathway. PRKAR1A is an apoptotic inhibitor of the pro-apoptotic BAD gene. Since a PF's fate is either growth or atresia, the presence of a greater number of anti-apoptotic genes or inhibitors of apoptosis relative to pro-apoptotic ones would be favourable for survival and determine recruitment to the next stage. As borne out by the initial pathway analyses done on mitochondrial genes and 38 transcription factors from this library, a mitochondrial pathway involving mitochondrial genes and interactions with insulin, calcium, H2O2 and RAN, a RAS oncogene, were identified. The transcription factor pathway identified for the first time in the human PF underscored a tight balance between cell proliferation and death/apoptosis; this pathway further identified known genes such as MAPK8, BDNF, ESR1, NR3C1, PGR (progesterone receptor 1) and HNRPK, which could interact with human PF transcripts WT1, ATF4 and NR5A2 in either maintaining quiescence or promoting initial recruitment. Experimental approaches, however, are needed to demonstrate and define these interactions.
This set of sequence-verified clones can be used to print a human PF cDNA array for gene expression profiling of stage-specific samples. When isolated and pure populations of stage-specific oocytes and their corresponding granulosa cells are screened using this array, genes expressed specifically by the oocyte and those expressed solely by the granulosa cells will be known. An oogenesis stage-specific array can simultaneously determine and facilitate the relative contribution of retrotransposons to recruitment and or/to the maintenance of the PF stage; this cannot be ascertained using commercial arrays as these do not contain all these repeat elements except one or two types of Alu sequences. Furthermore, the results of this study would greatly augment the molecular parameters currently being used for assessing PF recruitment.
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Acknowledgements |
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Submitted on January 13, 2005; resubmitted on March 16, 2005; accepted on March 22, 2005.
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