Transcripts from a human primordial follicle cDNA library

Maria D. Serafica1,3, Tetsuya Goto2 and Alan O. Trounson1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Human primordial follicles (PFs) or the oocyte–pre-granulosa complex, constitute the earliest and most immature stage of human oogenesis. The factors, signalling networks and the precise role of the oocyte and the pre-granulosa cells in initiating growth and recruitment from this finite resting pool remain largely unknown at present. METHODS: To obtain a gene resource of this oogenesis stage and thereby determine a molecular blueprint of the human PF, a cDNA library was constructed from 50 isolated human PFs using the phagemid vector pTriplEx2. RESULTS: Sequence analysis showed that 46.67% of these clones corresponded to known genes while 29.48% were uncharacterized genes that included hypothetical proteins, human cDNA clones and novel genes. Bioinformatics analysis revealed a preponderance of mitochondrial genes and repeat elements followed by ribosomal proteins, transcription and translation genes. Transcripts for heat shock proteins, cell cycle, embryogenesis genes and apoptosis genes were identified. Members of the ubiquitin–proteasome pathway, MAPK, p38/JNK, GPCR, Wnt, NF-{kappa}B and notch signalling pathways were identified. A mitochondrial pathway and a transcription factor pathway in the human PF were generated. The gene networks in the transcription factor pathway provided a first glimpse of the balance between proliferation and cell death/apoptosis in this earliest stage of oogenesis. CONCLUSIONS: The abundance and diversity of retroviral elements and transcriptional repressor genes in the human PF suggest these could contribute to the maintainance of this oogenesis stage. The role of these genes in initial recruitment and in subsequent oogenesis stages will be greatly facilitated and elucidated by printing a human PF cDNA array of the sequenced clones and using it for gene profiling.

Key words: apoptosis/human primordial follicle cDNA library/mitochondrial genes/repeat elements/signalling pathways


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human oogenesis is a long (between 40 and 50 years) and protracted process, characterized by an increase in oocyte size, cytoplasmic and nuclear maturation of the oocyte and differentiation of the surrounding granulosa cells. The human primordial follicle (PF) contains a 35 µm dictyate (meioisis 1)-stage-arrested oocyte, surrounded by <10 flattened or squamous pre-granulosa cells, that undergo a period of growth and maturation from 3 to 6 months, culminating in a Graafian follicle whose size ranges from 18 to 25 mm (Gourgeon and Lefevre, 1983). The mature metaphase II (MII) human arrested egg in this Graafian follicle is ~120 µm in diameter (Gougeon, 1996Go). Since the pool of oocytes is fixed at birth (Zuckerman, 1951Go), the reproductive life span of the mammalian female thus ceases upon depletion of this oocyte pool and is signalled by the onset of menopause. This 50-year-old dogma was challenged recently by reports of the presence of proliferative or mitotically dividing germ cells in juvenile and adult mouse ovaries (Johnson et al., 2004Go), implying that a regenerative source of follicles exists in the adult mammalian ovary. While mankind awaits irrefutable proof for this dogma challenge, a mechanism to explain declining oocyte quality and follicle numbers with age still remains one of the unsolved problems of female reproductive biology at present. Identification and elucidation of the genes and signalling pathways present in the PF would give benchmark information as to how this system is able to keep itself in suspended growth for years and what mechanisms are in place to ‘wake’ them up from the ‘sleepy state’. The quality of the oocyte at various stages of growth and maturation can thus be defined at the molecular level using this benchmark information.

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, 2003Go; Picton et al., 2003Go). 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., 2002Go), rat (Nilsson and Skinner, 2004Go), cow (Braw-Tal and Yossefi, 1997Go) and baboon (Wandji et al., 1997Go), and chorio-allantoic membrane grafts of chick embryos (Cushman et al., 2002Go). Models of oocyte–granulosa 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., 2002Go; Nilsson et al., 2002Go). 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, 2001Go).

Fig{alpha} (Soyal et al., 2000Go), a basic helix–loop–helix transcription factor, has been shown to be essential for formation of PFs and expression of zona pellucida proteins (Liang et al., 1997Go). Growth and differentiation factor-9 (gdf-9) and bone morphogenetic protein-15 or bmp-15, which are members of the transforming growth factor (TGF)-{beta} family, are oocyte genes which are expressed beyond the primary follicle stage (Dong et al., 1998Go; Carabatsos et al., 1998Go; Galloway et al., 2000Go). Recently, the transcription factor Foxl2 was shown to be required to maintain granulosa cell function (Schmidt et al., 2004Go). Spindlin (Oh et al., 1997Go), mater (Tong et al., 2000Go) and zar1 (Wu et al., 2003Go) 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., 1998Go) and help improve current or existing in vitro maturation technologies, particularly those that start with immature follicles (Eppig and O'Brien, 1996Go; Eppig, 2003Go; O'Brien et al., 2003Go).

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., 1997Go, 1999Go) in primordial germ cells (Goto et al., 1999Go), germinal vesicle (GV) oocytes (Neilson et al., 2000Go) and from human GV and MII oocytes (Monk et al., 2001Go; Goto et al., 2002Go).

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., 2002Go) 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, 2003Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was covered by Ethics #00031, approved by the Monash Surgical Private Hospital ethics committee, 252–254 Clayton Road, Clayton, Victoria Australia. This committee follows the NHMRC research guidelines and complies with the Infertility (Medical Procedures) Act 1984 and other legal requirements.

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., 1999Go). 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, {beta}-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, {lambda}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-{beta}-D-thiogalactopyranoside (IPTG) and X-Gal showed that 99% of plaques were white (1–2 x 103 plaques were counted per plate), indicating that 99% of the clones had an insert.

Conversion of recombinant {lambda}TriplEx2 phage clones to pTriplEx2 plasmid clones
An aliquot of the {lambda}TriplEx2 phage cDNA library was used to transduce an E.coli strain, BM25.8, to produce 3000–4000 colonies. The BM25.8 cells possessed Cre recombinase activity, which is capable of converting the loxP-containing {lambda}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., 1996Go; Jurka, 1998Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Construction of the human PF expression library
The scarcity of the PF sample and the fact that the origin of the signal for initial recruitment is unknown (it could be the oocyte, the somatic pre-granulosa cells or both) were logical and compelling reasons for constructing a composite library of the PF, instead of separate libraries. Given the minute (pg) amounts of starting mRNA, no normalization could be done such that detection of transcripts was expected to be skewed in favour of highly abundant transcripts (over-representation) and biased against detection of rare messages (under-representation). However, the size distribution of the amplified cDNA from human PFs (0.1–3 kb), as shown in Figure 1a and c, showed that the majority of the mRNA population was more than adequately represented (Sambrook et al., 1989Go). Our group (Goto et al., 1999Go, 2002Go; M.Serafica, unpublished results) and others (Adjaye et al., 1997Go; Neilson et al., 2000Go) have shown that this size distribution was reproducible, indicating that the cDNA from which the library was made was an adequate snapshot of the mRNA species. In order to test the quality of amplified cDNA, a housekeeping gene, {beta}-actin, was used for PCR as shown in Figure 1b. Figure 1c shows the purified size-fractionated cDNAs, which ranged in size from 0.1 to ~3 kb prior to cloning into the SfiI-digested vector. By double digestion with EcoRI and XbaI followed by agarose gel analysis of randomly chosen clones, the size of inserts ranged from 0.2 to 2.5 kb (Figure 2). The identification of two known genes, namely the oocyte extracellular matrix gene, zona pellucida or zp2, gene and the germ cell/oocyte gene, vasa (DEAD box polypeptide 16), from random sequencing of PF clones confirmed the origin of the sample from PFs. BLASTN identity results for these two genes were 99–100%, indicating that no sequencing errors were introduced by the PCR-based method. The same trend was observed for the rest of the sequenced PF clones.



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Figure 1. Generation of PCR-amplified cDNA molecules from isolated human primordial follicles (PFs). (a) A 5% aliquot of the total PCR product was run on an ethidium bromide-containing agarose gel. The+lanes contained RT enzyme and the -lane had no RT enzyme. The size of the amplified cDNA molecules extends up to 3 kb. (b) Gene-specific PCR for {beta}-actin. The presence of the PCR product only in the PF RT (+) lane and its absence in the PF RT (–) and lysis buffer RT (+) lanes confirm that the amplified cDNA preparation originated from mRNA, not from genomic DNA contamination nor from contamination of the reagents used. (c) Size-fractionated cDNA molecules, showing that most of the fragments with sizes <0.5 kb are removed.

 


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Figure 2. Insert size of human PF cDNA clones. Twelve randomly chosen cDNA clones were double-digested with restriction enzymes, EcoRI and XbaI, which excise the cDNA insert with 91 bp of additional vector sequence. The average size of inserts, evaluated from the gel picture, is ~1 kb. The presence of more than one excised fragment (lane 7) indicates the presence of an internal EcoRI or XbaI site in the cDNA insert.

 
Allocation of PF cDNA clones to gene categories
Sequence analysis of 692 clones showed that 323 clones (46.67%) corresponded to known human genes, 204 clones (29.48%) consisted of uncharacterized genes and 165 clones (23.85%) were uninformative (composed of vector-only clones and unreadable electropherograms). The categories of known genes and the number of clones found in each category are shown in Table I. The four classes of known transcripts that were most frequently represented and hence abundantly expressed during the PF stage (expressed as a percentage of known genes) included mitochondrial genes (27.0%), repeat elements (26%), translation and ribosomal genes (17.6%) and transcription genes (12.7%). Uncharacterized genes included clones that showed homology to hypothetical proteins; PAC, BAC and RIKEN clones containing human genomic and DNA sequences on specific chromosomes; ESTs and clones showing <10% homology to known NCBI sequences. In all, a total of 264 unique known genes (Tables II, IV and V) and 204 uncharacterized (Table III) genes were obtained by random sequencing and sequence analysis of this library.


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Table I. Categories of genes from a human primordial follicle (PF) cDNA librarya

 

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Table II. Mitochondrial genes isolated from the human PF cDNA library

 

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Table V. Gene members of signalling pathways from the human PF cDNA library

 

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Table III. Frequency and chromosome location of repeat elements in human PF clonesa

 
Mitochondrial genes
Mitochondria generate ATP for the cell via the oxidative phosphorylation (OXPHOS) pathway. Eleven out of the 13 mitochondria-encoded OXPHOS polypeptides were obtained by random sequencing of PF clones (Table II). NADH:ubiquinone oxidoreductase subunits ND4 and ND2, cytochrome b genes, cyclo-oxygenase (COX)1, COX2 and ATP6 were the most abundant mitochondrial genes sampled. Five mitochondrial genes encoded by the nucleus were likewise identified. A mitochondrial pathway was generated using the Pathway Assist Analysis software (Figure 3). In addition to reinforcing respiration and electron transfer as the cell processes in this organelle, interactions of mitochondrial genes with calcium, insulin receptor II, H2O2 and RAN (a member of the RAS oncogene family) were obtained. For example, insulin may increase the protein content of mitochondria (through its positive effect on cytochrome c oxidase subunit II) by stimulating protein synthesis, and could lead to enhanced respiration.



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Figure 3. Mitochondrial pathway in the human PF, identified by inputting 11 mitochondrial genes (Table II) into Pathway Assist (v 3.0, Ariadne Genomics, USA) software, using the shortest path between selected nodes (genes input). The various interactions of mitochondrial genes such as MTND2, MTCO2 and MCYTB, with insulin and calcium and the inhibitory effect of oxidoreductases on respiration and electron transfer are shown.

 
Retroviral elements from the human PF library
Repeat elements were found in 44% (84 out of 190) of clones screened. The frequency and human chromosome location of the repeat elements are shown in Table III. A total of 60 different repetitive elements were found ranging from Alu sequences and the human endogenous retroviral (HERV) sequence repeats. Eight different types of Alu sequences, three types of HERV repeat sequences, six types of LIME repeats and nine types of medium reiteration frequency repetitive sequences were found. Sixty-one clones had a single type of repeat element; 21 clones had two types of repeat elements, while three clones had three types of repeats. These results show that repeat elements are a distinct feature of the human PF, in terms of abundance and composition.

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 ({delta}-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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Table IV. Cellular growth and differentiation genes identified from the human PF cDNA library

 
A transcription factor pathway in the human PF was created (Figure 4) using Pathway Assist Analysis Software (Ariadne Genomics) by inputting the 39 transcription factors (Table IV, transcription genes). In this pathway, six cell processes, namely proliferation, death, proteolysis, DNA damage recognition, mitogenesis and protein degradation, were identified using 33 proteins (including 15 from the ResNet database), one small molecule, and interactions consisting of binding (13), expression (seven) and regulation (64). From this basic network, it can be seen that these genes tend to converge on proliferation and cell death. Five genes, namely BDNF (brain-derived neutrophic factor), ESR1 (estrogen receptor 1) CSDA (cold shock domain A) (human PF gene, this work), cysteine-rich protein (CSRP)1 and TH, were identified as positively regulating cell proliferation; five genes (TSA, NRC1, NR3C1, NR5A1 and PHF17) and ATF4 (human PF gene, this work) as negative regulators of cell proliferation; and seven genes, (WT1, MAPK8, PCNA, ITGA4, PABPC1, PGR and TRAF6) whose regulatory effects on cell proliferation are unknown. TSA, HNRPK and ATF4 were identified in this pathway as positive regulators of cell death, whereas ESR1 and human PF genes TAX1BP1 and NR5A2 were identified as negative regulators of cell death. The regulatory effects of VHL, BDNF, WT1, PCNA and MAPK8 on cell death are unknown. The basic pathway was modified by finding common targets for the positive regulators of cell proliferation [BDNF ESR1 CSDA, CSRP1 and TH] see Figure 4b. This resulted in identification of 22 more cell processes and highlighted the tight link between cell proliferation and apoptosis; transcription initiation had the lowest connectivity, which seemed to support the idea of pre-made transcripts or early response genes in the human PF.



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Figure 4. (a) Transcription factor pathway in the human PF (built using Pathway Assist Analysis software). Eighteen out 38 transcription factors (Table IV under transcription genes) plus 15 other genes (ResNet Database of the software) were used to generate the pathway. Among the key genes identified were the human PF genes WT1, ATF4, NR5A2, CSDA, C6orf4, RFC2, PNRC2, SAP18 and PCBP2. Other genes that were not randomly sampled from the human PF library but have key roles in proliferation, cell death and DNA damage recognition included MAPK8, BDNF, NR3C1, ESR1 CSRP1, TH and HNRPK.

 
Signalling pathways
Gene members of seven signalling pathways are listed in Table V. Ubiquitins, proteasomal subunits, heat shock proteins (HSPs), co-chaperones and the COP9 signalosome (CSN) 7 subunit genes were grouped together because these genes constitute the ubiquitin–proteasome pathway (UPP) of protein degradation (Ciechanover, 1998Go). Inclusion of the COP9 gene in this pathway is supported by recent reports that the CSN can substitute for the proteasome (Li and Deng, 2003Go). HSPs mediate the evolutionarily conserved cellular stress response and are widely known to act as molecular chaperones assisting other proteins by adopting the correct protein folding or unfolding configuration (Takayama et al, 2003Go). Seven types of hsp genes were found in the hPF library, with hsp90 being the most frequently sampled (Table V). Five kinases and two phosphatase genes of the mitogen-activated protein (MAP) kinase and p38/JNK signalling pathways were identified. Six apoptosis signal transduction genes and seven GTP-binding proteins which constitute the G-protein-coupled receptor (GPCR) protein signalling pathway were identified from the human PF expression library. Gene members of the Wnt, Delta-Notch and NF-{kappa}B signalling pathways were likewise identified.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Preparation of cDNA libraries requires a normalization process in order to obtain an adequate representation of transcripts, i.e. prevent over-representation of the overly abundant transcripts and increase the chances of detecting rare messages. In the case of the human PF library, because of the minute amount and scarcity of samples, such normalization could not be done. If there was a bias towards abundant transcripts such as mitochondria, it is surprising that housekeeping genes such as {beta}-actin or gapDH were not randomly sampled with such high frequency. Despite this inescapable bias, the quality and high fidelity of sequenced clones from this library has enabled us to obtain an initial molecular phenotype of human PFs and, by sequence analysis, gene ontology annotations and initial pathway analysis, has given us functional insights into this so-called quiescent stage of oogenesis. By random sequencing of 692 human PF clones, we identified 264 unique known genes and 204 uncharacterized genes, with mitochondrial genes and repeat elements being the most abundantly sampled and hence most expressed transcripts. Various types and combinations of repetitive sequences, accounting for 26% of genes sampled, were identified. Although this library is a mixture of transcripts constructed from the somatic component (pre-granulosa cells) and the germ cell (oocyte) component, based on what is currently known of this oogenesis stage and the role of mitochondria in the mature MII egg and during preimplantation development, it is highly likely that mitochondrial genes (Jansen and de Boer, 1998Go; Perez et al., 2000Go) and repeat elements (Goto et al., 1999Go) are expressed by the germ cell (oocyte) component and are defining features of the PF. The presence of numerous transcriptional repressors and co-repressors suggests that transcriptional repression is a major mechanism by which the quiescent stage is maintained. The PF stage is characterized further by an active protein synthesis machinery, based on the high number of ribosomal genes found; perhaps the numerous types of chaperone and co-chaperone proteins sampled play a protective/sequestering function for synthesized proteins. The presence of gene members of the UPP and a variety of ring finger proteins may provide for a spatio-temporal mechanism of protein degradation.

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., 2000Go; Ota et al., 2004Go), 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, 2001Go). 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., 2000Go) 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., 2000Go; Hughes and Coffin, 2001Go; Kashkush et al., 2003Go; Bannert and Kurth, 2004Go). 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., 2004Go). 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)Go 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., 2004Go; Peaston et al., 2004Go). Gene expression by RT–PCR 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 43.4–53% GV stage arrest as well as a decrease in targeted gene expression (Park et al., 2004Go). 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 2002Go).

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., 2003Go), IFITM1 (Table IV), TLE-1 (Liu et al., 1996Go) 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 cell–substratum contact (Weiskirchen et al., 1995Go). 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., 2004Go), and to be involved in the regulation of human corpus luteum 3{beta}-hydroxysteroid dehydrogenase type II (Peng et al., 2003Go) and in regulating the expression of StAR (steroid acute regulatory protein) in human granulosa cells after ovulation (Kim et al., 2004bGo). 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., 2003Go). 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., 1995Go) and is involved in mouse germ cell fate specification (Saitou et al., 2002Go). 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., 1998Go), CSDA, zinc finger 148 or BERF-1 (Takeuchi et al, 2003Go), 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., 1997Go). 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, 1998Go; Herschko and Ciechanover, 1998Go). 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., 1997Go), cyclin-dependent kinases, polo-like kinase and c-mos, are degraded by the UPP (Peters, 2002Go). 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., 2001Go). One of the structural motifs that target proteins for ubiquitination includes proteins in association with molecular chaperones (Arlander et al., 2003Go). 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., 1998Go). The evolutionarily conserved ring finger protein RNF138 has a zinc ion-binding domain that is involved in protein ubiquitination (Saurin et al., 1996Go). 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., 2000Go) that functions as an important regulator in multiple signalling pathways such as the TGF-{beta} signalling pathway (Kim et al., 2004aGo) 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, 2003Go). The CSN7 subunit of the Arabidopsis CSN was reported to associate with the eukaryotic translation initiation factor 3, eIF3 (Yahalom et al., 2001Go), and with eIF3 and 26S proteasome (Hoareau et al., 2002Go).

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, 2004Go), 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 helix–turn–helix transcription factor implicated in cell fate decision and boundary formation (Leimeister et al, 1999Go).

Apoptosis signal transduction
Of the 18 hsp genes given in Table V, 10 coded for Hsp90. Hsp90 can suppress tumour necrosis factor-{alpha} (TNF-{alpha})-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, 2004Go). Two other antiapoptotic roles reported for Hsp90 include its binding with Apaf-1 (apoptosis activating factor) (Pandey et al., 2000Go) and the binding of Hsp90 with a major antiapoptotic adaptor receptor-interacting protein, resulting in activation of antiapoptotic pathways through NF-{kappa}B and MAPK (Lewis et al., 2000Go). Hsp70 was reported to prevent both caspase-dependent and caspase-independent cell death functions (Bruey et al, 2000Go; Ravagnan et al., 2001Go). 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., 2003Go). 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This project was funded by Monash IVF Pty Ltd, and supported in part by the National Cooperative Program on Oocyte Quality and Competence, sponsored by the National Institute of Child Health and Human Development and the National Center for Research Resources (U01 HD44778).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 13, 2005; resubmitted on March 16, 2005; accepted on March 22, 2005.





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