doi:10.1093/humrep/dei030
Human Reproduction Vol.20, No.8 pp. 2074–2091, 2005
Advance Access publication April 21, 2005
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 2Tokyo 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. E-mail: [email protected]
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-kB 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
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 mm 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 mm in diameter (Gougeon, 1996). Since the pool of
oocytes is fixed at birth (Zuckerman, 1951), 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., 2004), 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, 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
oocyte– granulosa interactions as influenced by the presence
2074 q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Characterization of a human primordial follicle cDNA library
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).
Figa (Soyal et al., 2000), 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.,
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)-b 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 50 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 sequenceverified clones to oogenesis gene expression studies are
brought forward.
Materials and methods
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 mg/ml streptomycin (GibcoBRL), on ice. Using a sterile scalpel, pieces of ovarian cortical
slices (,5 mm £ 5 mm £ 1 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 48C. 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/ml 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
2708C 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)þ 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 ml 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
2075
M.D.Serafica, T.Goto and A.O.Trounson
not reached saturation by comparing the ethidium bromide intensities of amplified cDNA. The mRNA in 6 ml 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 ml aliquot of amplified cDNA (from a total volume of 100 ml)
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, b-actin, was
performed to test the quality of the cDNA preparation.
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).
Results
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, lTriplEx2 (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 £ 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 £ 106. A blue/white colony screening using isopropyl-b-D thiogalactopyranoside (IPTG) and X-Gal showed that 99% of plaques were white (1 – 2 £ 103 plaques were counted per plate),
indicating that 99% of the clones had an insert.
Conversion of recombinant lTriplEx2 phage clones to pTriplEx2
plasmid clones
An aliquot of the lTriplEx2 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 lox P-containing lTriplEx2 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 50 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.
2076
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., 1989).
Our group (Goto et al., 1999, 2002; M.Serafica, unpublished
results) and others (Adjaye et al., 1997; Neilson et al., 2000)
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,
b-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 Sfi I-digested
vector. By double digestion with Eco RI and Xba I 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.
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
Characterization of a human primordial follicle cDNA library
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 þ 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 b-actin. The presence of the PCR product only in the PF
RT (þ ) lane and its absence in the PF RT (2) and lysis buffer RT (þ) 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.
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.
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.
Figure 2. Insert size of human PF cDNA clones. Twelve randomly chosen cDNA clones were double-digested with restriction enzymes,
Eco RI and Xba I, 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 Eco RI or Xba I site in the
cDNA insert.
2077
M.D.Serafica, T.Goto and A.O.Trounson
Table I. Categories of genes from a human primordial follicle (PF) cDNA
librarya
Category
No. of clonesb
Mitochondrial genes
Repeat elements
Replication, transcription
Ribosomal genes
Translation
Energy metabolism enzymes
Heat shock genes
Cytoskeletal/extracellular matrix
Receptors, ion channel
Protein degradation
Protein kinases/phosphatases
GPCR/GTP-binding proteins
Developmental genes
Apoptosis-related genes
Cell cycle checkpoint genes
Heterogenous genesc
Uncharacterizedd
Totale
87
84
42
34
23
21
18
14
13
11
9
9
8
7
7
20
120
527
a
Categories were based on GO (Gene Ontology), gene ID and OMIM
citations of NCB1.
b
The number refers to the frequency of the gene and not to the number of
unique genes identified.
c
Known genes which do not fall into the major categories listed in this table.
d
Includes clones with homology to hypothetical proteins, human genomic
DNA sequences on phage artificial chromosomes (PACs), bacterial artificial
chromosomes (BACs), RIKEN clones, unknown mRNAs and clones with
,10% homology.
e
The total number of clones analysed was 692 of which 527 readable electropherograms were analysed. A total of 323 out of 527 clones corresponded to
known genes while 204 clones were uncharacterized.
GPCR ¼ G-protein-coupled receptor.
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
(d-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 proapoptotic 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
2078
protein 395); repeat elements MER11B, Alu-SpqxzLIP_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.
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 premade transcripts or early response genes in the human PF.
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, 1998). Inclusion of the
COP9 gene in this pathway is supported by recent reports
Characterization of a human primordial follicle cDNA library
Table II. Mitochondrial genes isolated from the human PF cDNA library
Mitochondrial genes
Description
ND1
Accession no.
NADH dehydrogenase 1 clone IMAGE:4120469
Position 3308–4264 of human mitochondria
ND2
NADH dehydrogenase subunit 2
Position 4471–5514 of human mitochondria
COX1
Cytochrome c oxidase I
Position 5905–7446 of human mitochondria
COX2
Cytochrome c oxidase II clone IMAGE:4041785
Position 7587–8270 of human mitochondria
ATP8
ATP synthase 8
Position 8367–8573 of human mitochondria
ATP6
ATP synthase 6
Position 8528–9206 of human mitochondria
COX3
Cytochrome c oxidase III clone IMAGE:3933642
Position 9208–9988 of human mitochondria
ND4
NADH dehydrogenase 4 clone IMAGE:4041417
Position 10 761–12 138 of human mitochondria
ND5
NADH dehydrogenase 5
Position 12 338–14 149 of human mitochondria
ND6
NADH dehydrogenase 6
Position14 150–14 674 of human mitochondria
CYTB
Cytochrome B
Position 14 748–15 882 of human mitochondria
MRPS31
Mitochondrial ribosomal protein S31
ATP synthase
Human ATP synthase, Hþ transporting mitochondrial F1 complex, b polypeptide
Nuclear genes encoding mitochondrial proteins
TOM20
Human outer membrane receptor Tom20 (TOM20 gene), exon 5,
nuclear gene encoding mitochondrial protein
FDX1
Human ferredoxin I (FDX1), a nuclear gene encoding mitochondrial protein
BNIP3
Human BCL2/adenovirus EIB 19 kDa-interacting protein 3 (BNIP3) nuclear gene
encoding mitochondrial protein
NDUFB1/MNLL
Human NADH dehydrogenase (ubiquinone) 1b subcomplex (7 kDa)
AKAP10
Human A kinase anchor protein 10
MAOB
Human monoamine oxidase B, nuclear gene encoding mitochondrial protein
that the CSN can substitute for the proteasome (Li and Deng,
2003). 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,
2003). 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-kB
signalling pathways were likewise identified.
Discussion
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 b-actin or gapDH were not
randomly sampled with such high frequency. Despite this
BCO09316
NC_001807
AFO14882
NC_001807
NC_001807
No. of clonesa
5
13
12
BCO13391
NC_001807
NC_001807
7
NC_001807
10
BCO13932
NC_001807
BCO14376
NC_001807
NC_001807
7
17
NC_001807
1
NC_001807
10
XM_056489
BC017718
1
1
AF126962
1
XM_040865
NM_00452
1
1
NM004545
NM007202
XM_10261
1
1
1
1
1
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, 1998; Perez et al., 2000) and
repeat elements (Goto et al., 1999) 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
2079
M.D.Serafica, T.Goto and A.O.Trounson
a
Table III. Frequency and chromosome location of repeat elements in human
PF clonesa
Type of repeat elementb
Single type repeat
Alu-J
Alu-Jb
Alu-Jo
Alu-Sc
Alu-Sx
Alu-Y
Alu-Spqxz
Alu-Sxzg
HAL1C
ERVL
HERVH
HERV70_I
LI
L3
LIMC1
LIMC4
LIMEC_5
LIME_ORF2
LIME2
LIMED_5
LIME5
LTR1B
LTR12C
MARNA
MER11B
MER21B
MER45
MER103
MIR
MIR3
GAAAA
(AT)
(AC)
TTA
TTG
THE1C
THE1D
Two repeat combination
Alu-Sz, Alu1
Alu-Jb. LIME_ORF2
Alu-J, MER113
Alu-Spqxz, Alu-J
Alu-Spqxz, LIPMA_2
HERV, Alu-Sxzg
LIMED5, Alu-Sx
LIME2, LIME3A
L1, LIPA4
LIM4B, LIME_ORF2
LIPREC2, LIPB1
LTR32, Alu-Sc
LTR12C, HSMAR1
MER5A, LIME3A
MER28,MER5C
MER45R, Alu-Sc
MER113, Alu-J
THE1B, LIME2
THE1C, THE1D
TTTTG, Alu-Sbo
LIM4B, LIME_ORF2
Triple repeat combination
LTR12, LTR12B, LIMB1
LTR22A, Alu-Jb, MER5A
TIGGR2, MER28,MER5C
Total
2080
No. of clones
containing
the repeat
1
4
3
2
1
1
5
6
Human chromosome
locationc
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
2
1
1
1
21q22.3
7, 8q23, 9, 19
2, 7, 16
9, 22q11
Xq23
6
6, 7, 7q31–32, 9d
7, 9, 22q13, 14,
21q22.2, 22, X,
5
6,13
2, 12p, 12p,
12p13.3– 4.6–10.5
10
8q23
20
11q
NDe
ND
6q21
22q11.2
7q32–34
ND
6
6p24.1– 25.3
8
15q14
5
11
ND
5
10, 20
ND
17
ND
ND
2
2
14
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
22q11
22q11.2
11q13, 11q13f
11q
nd
14
2
22q11.2
9
11q
18
4
4p
14
15
21
11q13, 11q13
15
2
7q31
11q
1
2
4
1
1
1
84
10
15q26.1
15q14
A total of 190 PF clones were screened for repeats using the Repbase database (Jurka et al., 1996; Jurka, 1998). These included 116 clones containing
human genomic and DNA sequences on specific PAC, BAC, RIKEN full
insert sequence clones; human expressed sequence tags and unknown
mRNAs; 30 clones encoding human hypothetical proteins; and 44 clones
containing known human genes.
b
Designations of repeats was based on Jurka et al. (1996) and Jurka (1998):
ERVL ¼ endogenous retroviral element HERV-L; ERVH ¼ endogenous
retroviral element HERV-H; MIR ¼ mammalian wide interspersed repeat;
MIR3 ¼ SINE element associated with L3; LIME5 ¼ 3prime; end of L1
repeat; LIME3A ¼ 30 end of L1 repeat (subfamily LIME3A); LIMB1 ¼ 30
end of L1 repeat (subfamily LIMB1); LIMC1 ¼ 30 end of L1 repeat (subfamily LIMC1); LIMED_5 ¼ LINE1 repetitive element; LIME_ORF2 ¼
LINE1; LIME subfamily LIME_ORF2; LIP_MA2 ¼ LINE1; LIP_MA2 subfamily; LTR32 ¼ LTR from human endogenous virus; LTR12C ¼ LTR
from HERV9; LTR22A ¼ LTR of HERVK22; MER5A ¼ non-autonomous
DNA transposon; medium reiteration frequency repetitive sequence;
MER11B ¼ LTR from HERVK-related endogenous retrovirus HERVK11;
MER45R ¼ hAT-like DNA transposon encoding a protein related to that of
MER69 and Zaphod and ‘activator-like’ transposases in Arabidposis; an
internal deletion product of an autonomous element; MER103 ¼ DNA
transposon; THEID ¼ long terminal repeat subfamily THEID, a retroviruslike MaLR element; HALIC ¼ LINE, L1 family; MARNA ¼ human nonautonomous mariner-like element; TIGGR ¼ human DNA transposons homologous to pogo element found in Drosophila (Smit and Riggs, 1996)
c
Applicable to human DNA and genomic sequences located on PAC, BAC
and RIKEN clones localized to specific human chromosomes.
d
The chromosome location of the fifth Alu-Spxqz containing clone was not
given.
e
ND ¼ not determined.
ff
Represented by two independent clones.
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., 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
Characterization of a human primordial follicle cDNA library
Table IV. Cellular growth and differentiation genes identified from the human PF cDNA library
Gene category
Replication genes
POLE2
PRIM2A
Transcription genes
DHX16
GTF2B
TAF2S
FOG2
CSRP2
SAP18
CSDA
BIF
ATF4
CMAF
NR5A2
NR1H3
WT1
ARNT2
BPTF
HQK
RNAHP
SRP54
RFC2
RFC3
RBBP4
HIFI1A
RBM3
TRIM2
CNOT7
T6BP/TAX1BP1
PDFN5/dMM-1
NDGR1
ZN65
ZNF9
RNF10
MKRN1
BERF-1/Zpf148
Peregrin/BRPF1
PNRC2
NFkB1/ chr6orf4
C40; chr2orf29
CCNH
PCBP2
Ribosomal genes
RPL5
RPL7
RPL7A
RPL10
RPL11
RPL12
RPL15
RPL17
RPL21
RPL22
RPL26
RPL27A
RPL35A
RPL36A
RPL37A þ
RPL65
RPS3A
RPS4X
RPS15a
RPS17
RPS19
RPS23
RPS27
RPS28
MRPS31
Description
Accession no.
Human polymerase (DNA-directed 12)
DNA primase polypeptide 2A
NM_002692
NM_000947
1
1
DEAD box polypeptide16
Human general transcription factor IIB
Human TATA box-binding protein (TBP)-associated factor,
RNA polymerase II, S, 150 kDa
Human friend of GATA2
Cysteine- and glycine-rich protein 2
Human, sin-3-associated polypeptide 18 kDa
Human cold shock domain protein A
Human hepatocytic transcription factor gene alternatively spliced products
Human similar to transcription factor 4 (ATF4)
(tax-responsive enhancer element B67)
Human short form transcription factor C-MAF
Human nuclear receptor subfamily 5 member 2
Human nuclear receptor subfamily 1, group H member 2
Human Wilms tumour 1, transcript varinant A
Human aryl-hydrocarbon receptor nuclear translocator 2
Human BPTF mRNA for bromodomain PHD finger transcription factor
Human gene for RNA-binding protein HQK, exon 8
Human RNA helicase-related protein
Human signal recognition particle 54 kDa
Human replication factor C (activator 1) 2 40 kDa, clone MGC:3665
Human replication factor c (activator 1) 3 (38 kDa)
Human retinoblastoma-binding protein 4
Human hypoxia-inducible factor 1 a subunit
Human RNA-binding motif protein 3
Human tripartite motif-containing 2 mRNA
Human CCR4–NOT transcription complex subunit 7
Human tumour necrosis factor-associated receptor-binding protein (T6BP);
human T cell leukaemia virus type 1-binding protein
Prefoldin5 c-myc-binding protein
n-myc downstream regulated gene 1
Human zinc finger protein 265
Human zinc finger protein 9 (a cellular retroviral nucleic acid-binding protein)
Human ring finger protein 10
Human makorin RING zinc finger protein 1
Mouse zinc finger protein BERF-1 (Zpf148) gene, exons 8 and 9
Bromodomain- and PHD finger-containing 1
Human proline-rich nuclear receptor coactivator 2
Human nuclear factor k binding-activating protein 1
Human clone C40 unknown mRNA; chr2orf29
Cyclin H/p34/CDK-activating kinase
Poly(rC)-binding protein 2
NM003587
XM-032821
NM_006706
1
1
1
XM_005190
NM_001321
U96915
BC015564
AF190464
BC011994
1
1
2
1
1
1
AFO55376
AF049102
XM_006165
NM_000378
NM014862
ABO32251
AB067809
XM_044385
XM_055849
BC002813
XM_038851
XM_018112
XM_050770
XM_047023
XM_018435
XM_044729
AF268075
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
XM_028595
AL132768
XM_032086
XM_052905
XM_035116
AF328796
AK027620
AF374386
AF274303
AF103798
XM_032135
AF236842
1
1
1
1
1
1
1
1
1
1
2
1
1
XM_028341
XM_035494
XM_035105
XM_048415
BCO18970
BCOO8230
XM_041875
BCO17831
BCOO1603
XM_003696
NM_000987
XM_017838
NM_000996
XM_052671
NM_000998
XM_035494
XM_037454
NM_001007
XM_027367
XM_007615
XM_029926
XM_056322
NM_001030.
XM_006026
XM_056489
2
1
2
2
1
1
1
1
1
2
1
2
1
2
2
1
1
1
3
1
1
1
1
1
1
Ribosomal protein 5
Ribosomal protein 7
Ribosomal protein 7A
Ribosomal protein 10
Ribosomal protein 11 clone MGC:19586; IMAGE:4337066
Ribosomal protein 12 clone MGC:9760; IMAGE:3855674
Ribosomal protein 15
Ribosomal protein 17 clone MGC:22482; IMAGE:425133
Ribosomal protein 21
Ribosomal protein 22
Ribosomal protein 26
Ribosomal protein 27A
Ribosomal protein 35A
Ribosomal protein 36a
Ribosomal protein 37a
Ribosomal protein 65
Ribosomal protein S3A
Ribosomal protein S4X; X-linked
Ribosomal protein S15A
Ribosomal protein S17
Ribosomal protein S19
Ribosomal protein S23
Ribosomal protein S27 (metallopanstimulin 1)
Ribosomal protein S28
Mitochondrial ribosomal protein S31
No. of clones
2081
M.D.Serafica, T.Goto and A.O.Trounson
Table IV. Continued
Gene category
Translation genes
IF2
EEF1A1
EIF2B
EEF1G
MARS
ZYGIN II
MTRF1L
NACA
PAIP1
CPSF3
Metabolism genes
DCTD
UGP2
TALDO1
LDHA
AKRB1
MLYCD
KIAA1247/SULF2
PLCB1
PTE-1
GATM
GALNT10
SGPL1
GUSB
GLUD1
DPM1
IMP
PKCI/PKCP1
CPE
HDC
ALAD
Cytoskeletal/ECM genes
ZP2
ACT1, b
ACT1, g
ARHA/RHOH12
TUBB4
TUBB4Q
hSm-LIM
MYPT2
MYH10
COL14A1
Syndecan-2
TMB10
Membrane receptor genes
VDAC2
SLC3A1
CALM1
LAPTM4A
TRAMP/TRAM1
TETRASPAN
JAG1
SARM1
WFDC2
PODXL
IFITM1
SMAP-5
Cell cycle checkpoint genes
BUB3
CDC10
CHK1
SUDD/RIOK3 (yeast)
YWHAQ
2082
Description
Accession no.
No. of clones
Human translation initiation factor IF2 (IF2)
Human similar to eukaryotic translation elongation factor 1a 1
Human eukaryotic initiation factor 2B-1 partial cds
Human similar to eukaryotic translation elongation factor 1g
Human methionine-tRNA synthetase
Human similar to fasciculation and elongation protein z2 (zygin II) (LOC114926)
Human mitochondrial translational release factor 1-like, clone MGC:20252
Human nascent-polypeptide associated complex a
Human polyadenylate-binding protein-interacting protein 1 (PAIP1)
Human similar to cleavage and polyadenylation-specific factor 3,
73 kDa subunit clone MGC:12899
XM_037110
BC009733
U23028
BC015813
BC015011
XM_054855
BC014428
XM_006705
XM_039946
BCO11654
1
10
2
1
1
1
3
2
1
1
Human deoxycytidylate (dCMP) deaminase
Human UDP-glucose pyrophosphorylase 2
Human transaldolase
Lactate dehydrgenase
Human aldo-keto-reductase family 1 member B1
Human malonyl CoA decarboxylase precursor
Human, similar to glucosamine 6-sulphatases (KIAA1247)
Human phosphoinositide-specific phospholipase C-b 1
Human peroxisomal acyl CoA thioesterase 1
Human glycine amidinotransferase (L -arginine:glycine amidinotransferase)
Human putative UDP-N-acetyl-a-D galactosamine::polypeptide
N-acetylgalactosaminyltransferase 10 (DKFZp586H0623)
Human sphingosine-1-phosphate lyase
Human similar to glucuronidase
Human glutamate dehydrogenase 1
Human dolichyl-phosphate mannosyl transferase polypeptide 1
catalytic subunit
Human IMP (inosine monophosphate) dehydrogenase 2 clone MGC:20947
Human protein kinase C inhibitor-2 or protein kinase C-interacting protein
Human carboxypeptidase E
Human histidine decarboxylase
Human d-aminolaevulinate synthase
NM_001921
BCOO2954
BCO10103
XM_050257
AFO97832
AY0101176
NM_015192
XM_029901
BCOO4141
AK023782
1
1
1
1
1
1
2
1
1
1
1
AI128825
XM_018030
XM_042916
XM_030562
1
1
1
1
BCO15567
AF085236
XM_00347
XM_030666
X64467
1
1
1
1
1
Human zona pellucida glycoprotein 2
Human b-actin
Human g-actin
Human ras homologue gene family, member A
Human b tubulin
Human b tubulin 4 pseudogene
Human smooth muscle LIM protein
Human myosin phosphatase target subunit 2
Human, non-muscle myosin heavy polypeptide chain-B
Human extracellular matrix protein collagen type XIV
Human syndecan-2 (heparin sulphate proteoglycan cell-surface
associated proteoglycan)
Human thymosin b10
NM_003460
BC01604
BC015779
XM_047561
NM006086
U83110
U46006
AF324892
M69181
Y11711
AL138878
1
2
2
1
1
2
1
1
1
1
1
XM_002498
1
Human voltage-dependent anion channel 2 in outer mitochondrial membrane
Human solute carrier family 3 (cystine, dibasic and neutral
amino acid transporters, activator of cystine, number 1
Human calmodulin-1 mRNA; phosphorylase kinase, d
Human lysosomal-associated protein transmembrane 4a, clone MGC:4121
Human mRNA for translocating chain-associating membrane protein
Human similar to tetraspan 3 (LOC112640)
Human jagged 1 (Alagille syndrome)
Sterile a and HEAT/Armadillo motif protein
Human WAP four-disulphide core domain 2 or epididymis-specific,
whey acidic protein type
Human, podocalyxin-like
Interferon-inducible transmembrane protein 1/9– 27
Human smooth muscle cell-associated protein-5
XM 032438
XM 030874
1
1
U16850
BC003158
NM 014294
XM_044572
XM_056118
NM_015077
XM_009534
1
1
1
1
1
1
1
XM_004727
BC000897
AB014733
1
1
1
Human BUB3 (budding uninhibited by benzimidazoles) homologue (yeast)
Human mitotic checkpoint component (BUB3)
Human kinetochore protein BUB3
Human CDC10 (cell division cycle 10. Saccharomyces cerevisiae homologue
Human CHK1 (checkpoint, Schizosaccharomyces pombe) homologue protein kinase
Human sudD (suppressor of bimD6, Aspergillus nidulans) homologue/RIO kinase 3
Human tyrosine mono-oxygenase-tryptophan 5-monooxygenase activation protein,
u polypeptide
NM_004725
AF053304
AF081496
NM_001788
XM_006254
NM_003831
NM_0068626
1
2
2
1
1
Characterization of a human primordial follicle cDNA library
Table IV. Continued
Gene category
Description
Accession no.
Embryogenesis genes
WT1
NRCAM
AMBN
EED
VIM
MESDC2
HEYL
MYOD1
Human Wilms tumour 1 (transcript variant A)
Human neuronal cell adhesion molecule
Human ameloblastin gene exons 9 –12; enamel matrix protein
Human embryonic ectoderm development protein
Human vimentin
Human mesoderm development candidate 2
Human hairy/enhancer-of-split related with YRPW motif-like
Human myogenic factor 3 clone MGC:8630/myoblast determination protein 1
NM_000378
NM_005010
AYO09123
XM_056832
XM_056832
XM_051852
XM_044935
BC000353
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 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 50 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., 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.
No. of clones
1
1
1
1
1
1
1
1
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 zincbinding 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.,
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 3bhydroxysteroid 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
2083
M.D.Serafica, T.Goto and A.O.Trounson
Table V. Gene members of signalling pathways from the human PF cDNA library
UPP pathway
Heat shock genes
TRAP1
HSP90
HSPCB
HSP90
HSPD1
HSPA8
DNAJ
HSPC154
HSPC036
PFDN5/MM-1
Ubiquitin and proteasome genes
UBB
UBC
UBCEP80/HUMUBCP
UBE2D3
USP9X/ fat facets Drosophila/DFFRX
STRIN/RNF138
PSMB1
PSMB7
PSMA5
COP9 subunit
MAPK/p38/JNK genes
PRCKN/PRKD3
MAPK10/JNK3A1
PAK2
AKAP10
PRKAR1A
DUSP1/MKP1
PP2CB
PLCB1
GPCR genes
ATG/GTP-binding protein/chr15orf24
GTP-binding protein/Rab6ip1
Ras-related nuclear protein
SAR1/SARA1
GDI-2
RGS2
GNG5
Apoptosis-related genes
TNFAIP1
BNIP3
APG5L/ASP
TP53INP1/p53DINP1/SIP
VDAC2
SGPL1
PBP/RKIP
PRKAR1A
Notch signalling genes
JAG1
HEYL
Wnt signalling genes
CSNK2b/phosvitin
TLE1
NF-kB signalling gene
NF-kB
UPP ¼ ubiquitin– proteasome pathway.
2084
Description
Accession no.
No. of clones
Human heat shock protein 75
Human similar to hsp90.1. a clone IMAGE:3030617
Human similar to hsp90 b clone
Human heart hsp90
Human hsp 60 kDa protein 1
Human heat shock 70 kDa protein 8
DnaJ-like protein
HSPC154 protein, clone MGC:4897
HSPC036
Prefoldin 5/myc-modulator 1
NM_016292
NM_007355
NM_007355
D87666
XM_017472
XM_050982
AF395203
NM_014177
AF125097
XM_028595
1
2
2
6
1
1
2
1
1
1
Ubiquitin B
Ubiquitin C
Ubiquitin carboxyl extension protein; human ubiquitin mRNA 3; end
Human mannosidase, b A lysosomal (MANBA) gene
and ubiquitin-conjugating enzyme E2D3
Ubiquitin-specific protease 9, X-linked,
(fat facet-like Drosophila homologue)
Human ring finger protein 138
Human proteasome (prosome macropain) subunit b type 1
Human proteasome (prosome macropain) subunit b type 7
Human proteasome (prosome, macropain) subunit a type 5
Human constitutive photomorphogenic,
Arabidopsis homologue subunit 7B
XM_037188
XM_055013
S79522/M10939
AF224669
2
1
1
1
NM_004652
1
XM_035408
XM_004535
XM_044811
XM_042737
XM_038331
1
1
1
1
1
Human protein kinase C, nu
Human mitogen-activated protein kinase
10/human JNK3 a1 protein kinase
Human p21 (CDKN1A)-activated kinase-2
Human A kinase anchor protein 10
Human protein kinase, cAMP-dependent, regulatory, type
a (tissue-specific extinguisher 1)
Human dual specificity phosphatase 1/MAP kinase phosphatase 1
Human protein phosphatase 2 catalytic subunit b isoform
Human phosphoinositide-specific phospholipase C-b1
XM_002659
NM_002753/U34820
1
1
NM_002577
NM007202
XM_008409
1
1
1
NM_004417
BCO12028
NM_015192
1
1
1
Human mRNA for putative ATG/GTP-binding protein
Mouse small GTP-binding protein-associated
protein mRNA/Rab6-interacting protein
GTP-binding protein
Human GTP-binding protein SAR1 mRNA
Human GDP dissociation inhibitor 2
Human regulator of G-protein signalling 2, 24 kDa
Human guanine nucleotide-binding protein,
g5 clone MGC:1969 IMAGE:3502879
AF242729
L40894
1
1
AF054183
AYOO8268
NM_001175
NM002923
BC003563
1
1
1
1
3
Human tumour necrosis factor a-induced protein 1 (endothelial) gene
Human BCL2/adenovirus EIB 19 kDa-interacting protein 3 (BNIP3)
nuclear gene encoding mitochondrial protein
Human DNA sequence from clone 134E15 on chr 6q21, contains
Blimp-1, apoptosis-specific protein similar to yeasts
(autophagy 5-like); apoptosis-specific protein
Human p53DINP1 gene (p5-dependent damage-inducible nuclear
protein 1)
Human voltage-dependent anion channel 2
Human sphingosine-1-phosphate lyase
Human prostatic-binding protein/Raf kinase inhibitor protein
Human protein kinase, cAMP-dependent, regulatory,
type a (tissue-specific extinguisher 1)
AY065346
NM_00452
1
1
AL022067
1
AB062056
1
XM_032438/BC012883
XM_005931
NM_002567
XM_008409
1
1
1
1
Human jagged 1 (Alagille syndrome)
Human hairy/enhancer-of-split related with YRPW motif-like
XM_056118
XM_044935
1
1
Human mRNA for phosvitin/casein kinase type II b subunit
Human transducin-like enhancer of split 1
X16937
AL365190
1
1
Human nuclear factor-k-binding-activating protein 1
AF274303
1
Characterization of a human primordial follicle cDNA library
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.
gene plays a pivotal role in the cellular response to oxidative
stress and negative regulation of cell proliferation.
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.
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
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)
2085
M.D.Serafica, T.Goto and A.O.Trounson
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.
family responsible for transcriptional repression; vimentin
(VIM), a gene specific for mesenchymal tissue; the hairy/
Enhancer of split-related with Y RPW motif-like (HEYL)
gene for development of the nervous system, somites, heart
and craniofacial region; the myoblast determination protein 1
2086
(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
Characterization of a human primordial follicle cDNA library
Figure 4. (b) The human PF transcription factor pathway in (a) was expanded by finding common targets for positive regulators of cell proliferation (BDNF, ESR1, CSDA, CSRP1 and TH). In addition to cell proliferation and apoptosis, secretion, assembly, contraction, maturation,
cell survival, death, motility, differentiation plus 16 other cell processes were added to this pathway and 164 regulation interactions were
identified. This pathway shows interactions of ESR1, MAPK8, NR3C1, NR5A2, PGR, ATF4, WT1 and HNRPK with cell processes apart
from proliferation and apoptosis. Five human PF genes (Zpf148, NDRG3, C20orf172, C2orf29 and KIAA0889), shown at the top of the
figure, could not be placed into this pathway.
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.
2087
M.D.Serafica, T.Goto and A.O.Trounson
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 ubiquitintagged 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-b 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
2088
and MAPK signalling pathways. The CSNK2B/phosvitin is
part of the Wnt signalling pathway and the cadherinmediated 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 helix – turn –
helix 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-a (TNF-a)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-kB and MAPK (Lewis
et al., 2000). Hsp70 was reported to prevent both caspasedependent and caspase-independent cell death functions
(Bruey et al, 2000; Ravagnan et al., 2001). BNIP3 is a proapoptotic 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
Characterization of a human primordial follicle cDNA library
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
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
Adjaye J and Monk M (2000) Transcription of homeobox-containing genes
detected in cDNA libraries derived from human unfertilized oocytes and
preimplantation embryos. Mol Hum Reprod 6,707–711.
Adjaye J, Daniels R, Bolton V and Monk M (1997) cDNA libraries from
single human preimplantation embryos. Genomics 46,337– 344.
Adjaye J, Bolton V and Monk M (1999) Developmental expression of
specific genes detected in high quality cDNA libraries from single human
preimplantation embryos. Gene 237,373–383.
Annicotte JS, Fayard E, Swift GH, Selander L, Edlund H, Tanaka T,
Kodama T, Schoonjans K and Auwerx J (2003) Pancreatic –duodenal
homeobox1 regulates expression of liver receptor homolog 1 during pancreas development. J Mol Cell Biol 19,6713–6724.
Arlander SJH, Eapens AK, Vroman BT, McDonald RJ, Toft DO and Karnitz
LM (2003) Hsp90 inhibition depletes Chk1 and sensitizes tumor cells to
replication stress. J Biol Chem 278,52572–52577.
Bannert N and Kurth R (2004) Retroelements and the human genome: new
perspectives on an old relation. Proc Natl Acad Sci USA,101 Epub ahead
of print.
Bedard J, Brule S, Price CA, Silversides DW and Lussier JG (2003) Serine
protease inhibitor-E2 (SERPINE2) is differentially expressed in granulosa
cells of dominant follicle in cattle. Mol Reprod Dev 64,152–165.
Braw-Tal R and Yossefi S (1997) Studies in vivo and in vitro on the
initiation of follicle growth in the bovine ovary. J Reprod Fertil 101,
369–374.
Bruey JM, Ducasse C, Bonniaud P, Ravagnan L, Susin SA, Diaz-Latoud C,
Gurbuxani S, Arrigo A-P, Kroemer G, Solary E et al. (2000) Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat Cell Biol
2,645–652.
Carabatsos MJ, Elvin J, Matzuk MM and Albertini DF (1998) Characterisation of oocyte and follicle development in growth differentiation factor9-deficient mice. Dev Biol 204,373–384.
Cheng EHY, Sheiko TV, Fisher JK, Craigen WJ and Korsmeyer SJ (2003)
VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science
301,513–517.
Ciechanover A (1998) The ubiquitin–proteasome pathway: on protein death
and cell life. EMBO J 17,7151– 7160.
Cushman RA, Wahl CM and Fortune JE (2002) Bovine ovarian cortical
pieces grafted to chick embryonic membranes: a model for studies on the
activation of primordial follicles. Hum Reprod 17,48–54.
Deblandre GA, Marinx OP, Evans SS, Majjaj S, Leo O, Caput D, Huez GA
and Wathelet MG (1995) Expression cloning of an interferon-inducible
17 kDa membrane protein implicated in the control of cell growth. J Biol
Chem 40,23860–23866.
Deng XW, Dubiel W, Wei N, Hofmann K, Mundt K, Colicelli J et al. (2000)
Unified nomenclature for the COP9 signalosome and its subunits: an
essential regulator of development. Trends Genet 16,202–203.
Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N and Matzuk MM
(1998) Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383,531–535.
Durlinger ALL, Gruijters MJG, Kramer P, Karels B, Kumar TR, Matzuk
MM, Rose UM, de Jong FH, Uilenbroek JTJ, Grootegoed JA et al. (2002)
Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 142,4891–4899.
Eppig JJ (2001) Oocyte control of ovarian follicular development and function in mammals. Reproduction 122,829–838.
Eppig JJ (2003) Growth and development of the mammalian oocyte–granulosa cell complex in culture. In Trounson AO and Gosden RG (eds)
Biology and Pathology of the Oocyte: Role in Fertility and Reproductive
Medicine. Cambridge University Press, Cambridge, pp. 273–282.
Eppig JJ and O’Brien MJ (1996) Development in vitro of mouse oocytes
from primordial follicles. Biol Reprod 54,197–207.
Eppig JJ, Wigglesworth K and Pendola FL (2002) The mammalian oocyte
orchestrates the rate of ovarian follicular development. Proc Natl Acad Sci
USA 99,2890–2894.
Evsikov AV, de Vries WN, Peaston AE, Radford EE, Fancher KS, Chen FH,
Blake JA, Bult CJ, Latham KE, Solter D and Knowles BB (2004) Systems
biology of the 2-cell mouse embryo. Cytogenet Genome Res 1–5.
240 –250.
Feng F and Schultz RM (2003) Gene expression in mouse oocytes and preimplantation embryos: use of suppression subtractive hybridization to
identify oocyte and embryo-specific genes. Biol Reprod 68,31–39.
Fortune JE (2003) The early stages of follicular development: activation of
primordial follicles and growth of preantral follicles. Anim Reprod Sci
78,135–163.
Galloway SM, McNatty KP, Cambridge LM, Laitinen MPE, Juengel JL,
Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery G et al.
(2000) Nat Genet 25,279–283.
Goto T, Adjaye J, Rodech CH and Monk M (1999) Identification of genes
expressed in human primordial germ cells at the time of entry of the
female germ line into meiosis. Mol Hum Reprod 5,851–860.
Goto T, Jones GM, Lolatgis N, Pera MF and Trounson AO (2002) Identification and characterization of known and novel transcripts expressed
during the final stages of human oocyte maturation. Mol Reprod Dev 62,
13 –28.
Gougeon A (1996) Regulation of ovarian follicular development in primates:
facts and hypotheses. Endocr Rev 17,121–155.
Gougeon A and Lefevre B (1983) Evolution of the diameters of the largest
healthy and atretic follicles during the human menstrual cycle. J Reprod
Fertil 69,487–502.
Herschko A and Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67,425–479.
Hoareau AK, Bochard V, Rety S and Jalinot P (2002) Association of the
mammalian oncoprotein Int-6 with the three protein complexes eIF3,
COP9 signalosome and 26S proteasome. FEBS Lett 527,15–21.
Hughes JF and Coffin JM (2001) Evidence for genomic rearrangements
mediated by humam endogenous retroviruses during primate evolution.
Nat Genet 29,487–489.
Jansen RP and de Boer K (1998) The bottleneck: mitochondrial imperatives
in oogenesis and ovarian follicular fate. Mol Cell Endocrinol 145,81–88.
Johnson J, Canning J, Kaneko T, Pru JK and Tilly JL (2004) Germline stem
cells and follicle renewal in the postnatal mammalian ovary. Nature
428,145–150.
Jurka J (1998) Repeats in genomic DNA: mining and meaning. Curr Opin
Struct Biol 8,333–337.
Jurka J, Klonowski P, Dagman V and Pelton P (1996) CENSOR—a program
for identification and elimination of repetitive elements from DNA
sequences. Comput Chem 20,119– 122.
Kashkush K, Feldman M and Levy AA (2003) Transcriptional activation of
retrotransposons alters the expression of adjacent genes in wheat. Nat
Genet 33,102–106.
Kezele PR, Nilsson EE and Skinner MK (2002) Insulin but not insulin-like
growth factor promotes the primordial to primary follicle transition. Mol
Cell Endocrinol 192,37–43.
Kim BC, Lee HJ, Park SH, Lee SR, Karpova TS, McNally JG, Felici A, Lee
DK and Kim SJ (2004) Jab1/CSN5, a component of the COP9 signalosome, regulates transforming growth factor b signaling by binding to
smad7 and promoting its degradation. Mol Cell Biol 24,2251–2262.
Kim JW, Peng N, Rainey WE, Carr BR and Attia GR (2004) Liver receptor
homolog-1 regulates the expression of steroidogenic acute regulatory
protein (StAR) in human granulosa cells. J Clin Endocrinol Metab 89,
3042–3047.
Kleber M and Sommer L (2004) Wnt signaling and the regulation of stem
cell function. Curr Opin Cell Biol 16,681–687.
2089
M.D.Serafica, T.Goto and A.O.Trounson
Knowles BB, Evsikov AV, de Vries WN, Peaston AE and Solter D (2003)
Molecular control of the oocyte to embryo transition. Philos Trans R Soc
Lond B 358,1381–1388.
Lee WS, Yoon SJ, Yoon TK, Cha KY, Lee SH, Shimasaki S, Lee S and
Lee KA (2004) Effects of bone morphogenetic protein-7 (BMP-7) on
primordial follicular development in the mouse ovary. Mol Reprod Dev
69,159–163.
Leimeister C, Externbrink A, Klamt B and Gessler M (1999) Hey genes: a
novel subfamily of hairy- and Enhancer of split related genes specifically
expressed during mouse embryogenesis. Mech Dev 85,173–177.
Lewis J, Devin A, Miller A, Lin Y, Rodriguez Y, Neckers L and Liu ZG
(2000) Disruption of Hsp90 results in degradation of the death domain
kinase, receptor-interacting protein (RIP) and blockage of the tumor necrosis factor-induced nuclear factor-kB activation. J Biol Chem 275,
10519–10526.
Li L and Deng XW (2003) The COP9 signalosome: an alternative lid for the
26S proteasome? Trends Cell Biol 13,507–509.
Liang L, Soyal SM and Dean J (1997) Figa, a germ cell-specific transcription factor involved in the coordinate expression of the zona pellucida
genes. Development 124,4939–4947.
Liu Y, Dehni G, Purcell KJ, Sokolow J, Carcangiu ML, Artavanis-Tsakonas
S and Stifani S (1996) Epithelial expression and chromosomal location
of human TLE genes: implications for Notch signaling and neoplasia.
Genomics 31,58–64.
Mi S, Lee X, Li X, Veldman GM, Finnerty H, Racie L, La Vallie E, Tang
XY, Edouard P and Howes S (2000) Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature
403,785–789.
Monk M, Holding C and Goto T (2001) Isolation of novel developmental
genes from human germ cells, oocyte and embryo cDNA by differential
display. Reprod Fertil Dev 13,51–57.
Mori K, Maeda Y, Kitaura H, Taira T, Iguchi-ariga SM and Ariga H (1998)
MM-1, a novel c-myc associating protein that represses transcriptional
activity of c-myc. J Biol Chem 273,29794– 29800.
Neilson L, Andalibi A, Kang D, Coutifaris C, Strauss JF, 3rd, Stanton JA
and Green DP (2000) Molecular phenotype of the human oocyte by PCRSAGE. Genomics 63,13– 24.
Nilsson EE and Skinner MK (2004) Kit ligand and basic fibroblast growth
factor interactions in the induction of ovarian to primary follicle transition.
Mol Cell Endocrinol 214,19–25.
Nilsson E, Parrott JA and Skinner MK (2001) Basic fibroblast growth factor
induces primordial follicle development and initiates folliculogenesis.
Mol Cell Endocrinol 175,123–130.
Nilsson EE, Kezele P and Skinner MK (2002) Leukemia inhibitory factor
(LIF) promotes the primordial to primary follicle transition in rat ovaries.
Mol Cell Endocrinol 188,65–73.
O’Brien MJ, Pendola JK and Eppig JJ (2003) A revised protocol for in vitro
development of mouse oocytes from primordial follicles dramatically
improves their developmental competence. Biol Reprod 68,1682–1686.
Oh B, Hwang SY, Solter D and Knowles BB (1997) Spindlin, a major
maternal transcript expressed in the mouse during the transition from
oocyte to embryo. Development 124,493– 503.
Oktay K, Newton H, Aubard Y, Salha O and Gosden RG (1998) Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology. Fertil Steril 69,1–7.
Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A,
Hayashi K, Sato H et al. (2004) Complete sequencing and characterization
of 21,243 full-length human cDNAs. Nat Genet 36,40–45.
Pandey P, Saleh A, Nakazawa A, Kumar S, Srinivasula SM, Kumar V,
Weichselbaum R, Nalin C, Kumar V and Kharbanda S (2000) Negative
regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90. EMBO J 19,4310–4322.
Park CE, Shin MR, Jeon EY, Lee SH, Cha KY, Kim K, Kim NH and Lee
KA (2004) Oocyte-selective expression of MT transposon-like element,
clone MTi7 and its role in oocyte maturation and development. Mol
Reprod Dev 69,365–374.
Parrott JA and Skinner MK (1999a) Kit ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis. Endocrinology
139,228–235.
Parrott JA and Skinner MK (1999b) Kit ligand actions on ovarian stromal
cells: effects on theca cell recruitment and steroid production. Mol Reprod
Dev 55,55–64.
2090
Peaston AE, Evsikov AV, Graber JH, de Vries WN, Holbrook AE, Solter D
and Knowles BB (2004) Retrotransposons regulate host genes in mouse
oocytes and preimplantation embryos. Dev Cell 7,597–606.
Peng N, Kim JW, Rainey WE, Carr BR and Attia GR (2003) The role of the
orphan receptor, liver receptor homologue-1, in the regulation of human
corpus luteum 3beta-hydroxysteroid dehydrogenase type II. J Clin Endocrinol Metab 88,6020– 6028.
Perez GI, Trbovich AM, Gosden RG and Tilly JL (2000) Mitochondria and
the death of oocytes. Nature 403,500– 501.
Peters JM (2002) The anaphase-promoting complex: proteolysis in mitosis
and beyond. Mol Cell 9,931–943.
Picton HM (2000) Activation of follicle development: the primordial follicle.
Theriogenology 55,1193–1210.
Picton HM, Danfour MA, Harris SE, Chambers EL and Huntriss J (2003)
Growth and maturation of oocytes in vitro. Reproduction (Suppl 61),
445–462.
Rajkovic A, Pangas SA, Ballow D, Suzumori N and Matzuk MM (2004)
NOBOX deficiency disrupts early folliculogenesis and oocytes-specific
gene expression. Science 305,1157–1159.
Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Dauga E, Zamzani N, Mak
T, Jaattela M, Penninger JM, Garrido C and Kroemer G (2001) Heat
shock protein 70 antagonises apoptosis-inducing factor. Nat Cell Biol
3,839–843.
Reverte CG, Ahearn MD and Hake LE (2001) CPEB degradation during
Xenopus oocyte maturation requires a PEST domain and the 26S proteasome. Dev Biol 231,447– 458.
Saitou M, Barton SC and Surani MA (2002) A molecular programme for the
specification of germ cell fate in mice. Nature 418,293–300.
Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
Saurin AJ, Borden KL, Boddy MN and Freemont PS (1996) Does this have a
familiar RING? Trends Biochem Sci 21,208–214.
Saxena D, Safi R, Little-ihrig L and Zeleznik A (2004) Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation. Endocrinology
145,3821– 3829.
Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted Treier AC and Treier
M (2004) The murine winged-helix transcription factor Foxl2 is required
for granulosa cell differentiation and ovary maintenance. Development
131,933– 942.
Smit AFA and Riggs AD (1996) Tiggers and other DNA transposon fossils
in the human genome. Proc Natl Acad Sci USA 93,1443–1448.
Soyal SM, Amleh A and Dean J (2000) Figa, a germ cell-specific transcription factor required for ovarian follicle formation. Development 127,
4645–4654.
Stanton JL and Green DPL (2001) A set of 840 mouse oocyte genes with
well-matched homologues. Mol Hum Reprod 7,521–543.
Strausberg RL, Feingold EA, Grouse LH, Derge JG, Klausner RD, Collins
FS, Wagner L, Shenmen CM, Schuler GD, Altschul SF et al. (2002) Generation and initial analysis of more than 15,000 full-length human and
mouse cDNA sequences. Proc Natl Acad Sci USA 99,16899–16903.
Takayama S, Reed JC and Homma S (2003) Heat shock proteins as regulators of apoptosis. Oncogene 22,9041–9047.
Takeuchi A, Mishina Y, Miyaishi O, Kojima E, Hasegawa T and Isobe K
(2003) Heterozygosity with respect to Zfp148 cause complete loss of fetal
germ cells during mouse embryogenesis. Nat Genet 33,172–176.
Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, Bondy CA, Dean J and
Nelson LM (2000) Mater. A maternal effect gene required for early
embryonic development in mice. Nat Genet 26,267–268.
Tokumoto T, Yamashita M, Tokumoto M, Katsu Y, Horiguchi R, Kajiura H
and Nagayama Y (1997) Initiation of cyclin B degradation by the 26S proteasome upon egg activation. J Cell Biol 138,1313– 1322.
Vainberg IE, Lewis SA, Rommelaere H, Ampe G, Vanderckhove J, Klein
HL and Cowan NJ (1998) Prefoldin, a chaperone that delivers unfolded
proteins to cytosolic chaperonin. Cell 93,863–873.
Wandji SA, Srsen V, Nathanielsz PW, Eppig JJ and Fortune JE (1997)
Initiation of growth of baboon primordial follicles in vitro. Hum Reprod
12,1993– 2001.
Wei N and Deng XW (2003) The COP9 signalosome. Annu Rev Cell Dev
Biol 19,261–286.
Weiskirchen R, Pino JD, Macalma T, Bister K and Beckerle MC (1995) The
cysteine-rich protein family of highly related LIM domain proteins. J Biol
Chem 270,28946 –28954.
Characterization of a human primordial follicle cDNA library
Wu X, Viveiros MM, Eppig JJ, Bai Y, Fitzpatrick SL and Matzuk MM
(2003) Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for
the oocyte-to-embryo transition. Nat Genet 33,187–191.
Yahalom A, Kim TH, Winter E, Karniol B, von Armin AG and Chamovitz
OA (2001) Arabidopsis eIF3e (Int-6) associates with both eIF3c and the
COP9 signalosome subunit CSN7. J Biol Chem 276,334–340.
Zhang Y, Iratni R, Erdjument-Broamage H, Tempest P and Reinberg D
(1997) Histone deacetylases and SAP18, a novel polypeptide, are components of human Sin3 complex. Cell 89,357–364.
Zhao C and Wang E (2004) Heat shock protein 90 suppresses tumor necrosis
factor alpha induced apoptosis by preventing the cleavage of Bid in
NIH3T3 fibroblasts. Cell Signalling 16,313–321.
Zuckerman S (1951) The number of oocytes in the mouse ovary. Recent
Prog Horm Res 6,63–108.
Submitted on January 13, 2005; resubmitted on March 16, 2005; accepted
on March 22, 2005
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